WO2004026285A2 - Compositions and methods for treating heart disease - Google Patents

Compositions and methods for treating heart disease Download PDF

Info

Publication number
WO2004026285A2
WO2004026285A2 PCT/CA2003/001387 CA0301387W WO2004026285A2 WO 2004026285 A2 WO2004026285 A2 WO 2004026285A2 CA 0301387 W CA0301387 W CA 0301387W WO 2004026285 A2 WO2004026285 A2 WO 2004026285A2
Authority
WO
WIPO (PCT)
Prior art keywords
pbkgamma
pten
heart
protein
mice
Prior art date
Application number
PCT/CA2003/001387
Other languages
French (fr)
Other versions
WO2004026285A3 (en
Inventor
Michael A. Crackower
Josef M. Penninger
Original Assignee
Amgen Canada Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amgen Canada Inc. filed Critical Amgen Canada Inc.
Priority to CA002499368A priority Critical patent/CA2499368A1/en
Priority to EP03750190A priority patent/EP1539177A2/en
Priority to AU2003269626A priority patent/AU2003269626A1/en
Publication of WO2004026285A2 publication Critical patent/WO2004026285A2/en
Publication of WO2004026285A3 publication Critical patent/WO2004026285A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the invention relates to compositions and methods for treating heart disease by inhibition of PBKgarnma.
  • Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020 (Yusuf et al, 2001). Cardiovascular disease is frequently associated with elevated wall stress as a result of pressure overload such as in hypertension or volume overload seen in valvular disorders. Increased wall stress in the heart triggers a hypertropl ic response (Hunter and Chien, 1999). What is initially a compensatory response, pathological hypertrophy eventually leads to decompensation resulting in left ventricle dilation, myocyte loss, increased interstitial fibrosis and heart failure. While increased wall stress can lead to pathological cardiac hypertrophy, increased demand for cardiac output can also cause a physiological increase in cell size, as commonly seen in endurance athletes (Colan, 1997).
  • PI3K phosphoinositide 3- kinase
  • PI3Ks phosphoinositide-dependent kinases
  • PIP3 phosphoinositide-dependent kinases
  • PI3K ⁇ , ⁇ , ⁇ phosphoinositide-dependent kinases
  • GPCRs G-protein-coupled receptors
  • PI3K signaling has been implicated as a component of cardiac hypertrophy and protection of myocytes from apoptosis mediated by numerous exogenous agonists and stresses (Rabkin et al, 1997; Schluter et al, 1998; Baliga et al, 1999; Kodama et al, 2000).
  • overexpression of constitutively active pi 10a results in increased heart size, whereas cardiac overexpression of dominant negative pi 10a in mice resulted in smaller hearts without affecting heart functions (Shioi et al, 2000).
  • PI3K ⁇ activity is increased upon aortic constriction in mice (Naga Prasad et al, 2000), suggesting that both tyrosine-based and GPCR-linked PI3Ks might play a role in the cardiac hypertrophy response.
  • the tumor suppressor PTEN is a lipid phosphatase that dephosphorylates the D3 position of PIP3 (Maehama and Dixon, 1998). Thus, PTEN lowers the levels of the
  • PI3K product PIP3 within the cells and antagonizes PI3K mediated cellular signaling. It has been recently shown that PTEN can regulate neuronal stem cell proliferation and the cell size of neurons in brain-specifc PTEN mouse mutants similar to patients with Lhermitte-Duclos disease (Backman et al, 2001). Moreover, expression of dominant negative PTEN gene in rat cardiomyocytes in tissue culture results in hypertrophy (Schwartzbauer and Robbins, 2001). Whether PI3Ks and PTEN can indeed control cardiac hypertrophy and heart functions in vivo is not known.
  • the inventors have identified PTEN as a master regulator for heart size and heart function and PDkgamma as a critical regulator of heart muscle function (pumping function) in vivo in the whole mouse (mutant mouse model) and in vitro in single cells. These are completely new and unexpected findings and identify novel pathways to protect from heart failure. Inhibition of PDKgamma and the novel PBKgamma signaling pathway allows maintenance of heart function even in failing hearts. Inhibition of PI3K ⁇ and/or activation of PTEN blocks cardiac hypertrophy.
  • the invention describes a novel and previously unrecognized function for the PI3Kgamma/PTEN pathways in vivo. The functionality was shown in vivo in the whole organism and at the single cell level.
  • mice using multiple mutants and in a heart failure model were used to establish the in vivo role of PDKgamma and PTEN.
  • the invention studied R-TEN-heart muscle specific mutant mice, pi 10 ⁇ ⁇ , dominant-negative /?UOa transgenic mice, and double-mutant mice.
  • B Increased cell sizes of cardiomyocytes isolated from 10 week old mckCRE- PTENA/A mice. Representative images of isolated cardiomyocytes and quantitation of cell sizes are shown. 100 individual cells from three different mice were analyzed per group. ** p ⁇ 0.01 between genetic groups.
  • C D Northern blot analysis and quantitation (mean values +/- SEM) of cardiac hypertrophy markers. GAPDH expression levels were used as a loading control. Results from hearts isolated from 3 individual 10 week old mckCRE-PTENA + and 3 different, age-matched mckCRE-PTENA/A littermate mice are shown.
  • E,F Increased AKT/PKB, GSK3 ⁇ , and p70 S6K phosphorylation in total heart extracts from 10 week old mckCRE-PTENA/A mice.
  • E Western blotting for phosphorylated-AKT/PKB, total AKT/PKB protein, p85, pi lOoc, and pi lO ⁇ protein expression in total heart extracts.
  • F Representative heart sizes and heart/body weight ratios of 10 week old pi 10 ⁇ ' and pllOj 1' littermates. Similar data, i.e, no change in heart size, were obtained at 6 and 12 months of age.
  • A Representative heart sizes of 10 week old wild-type, DN-pll0c, and mckCRE- PTENA/A DN-pllOa littermates.
  • FIG. 1 Single cell contractility and cAMP production
  • A, B Basal contractility of single cardiomyocytes isolated from mckCRE-PTENA A ⁇ wild-type, pi 10y ' , and mckCRE-PTENA/A p H0 ⁇ /" double mutant mice.
  • A representative contractions from single cells are shown.
  • B mean percentage shortening (+/- SEM) of at least 15 different single cells from 3 different hearts per genotype are shown in the presence and absence of thePDK inhibitor LY294002 [30 ⁇ M]. ** p ⁇ 0.01 between groups.
  • Activation of adenylate cyclase via forskolin [25 ⁇ M] increases the levels of cAMP production (mean +/- SEM) in mckCRE-PTENA/A wild-type, and pllOj 1' cardiomyocytes to a similar extent.
  • C Induction of cAMP production in response to the selective ⁇ 2-adrenergic receptor agonist zinterol.
  • Figure 7 a) Homo sapiens phosphoinositide-3 -kinase gamma nucleic acid sequence [SEQ ID NO: 1]; Accession no. AF327656; base count: 950 a 861 c 849 g 750 1 b) Homo sapiens phosphoinositide-3 -kinase gamma amino acid sequence[SEQ ID NO: 2].
  • Figure 8 a) Homo sapiens PTEN nucleic acid sequence[SEQ ID NO: 3]; Accession no. NM_000314; base count: 853 a 742 c 788 g 777 t; b) Homo sapiens PTEN amino acid sequence [SEQ ID NO: 4].
  • FIG. 9 Signaling and cardiac hypertrophy responses pllOf ' and pll Of 1" mice.
  • A) Heart sections from pllOf ' and pi 10 f' " mice treated with a vehicle control or isoproterenol for 7 days.
  • B) Quantitation of heart/body weight and heart weight/tibial length ratios in vehicle and isoproterenol treated mice; n 9 per group. *P ⁇ 0.05; **P ⁇ 0.01 compared with vehicle.
  • C Northern blot analysis and quantitation of cardiac hypertrophy markers mpll0 ⁇ + " andpllO 1 ' mice treated with vehicle (1) or isoproterenol (2) for 7 days. GAPDH expression levels were used as a loading control.
  • D ERK1/2 and p38 phosphorylation in isolated pllOf 1' wApllOf 1' neonatal cardiomyocytes. Cells were (1) left untreated, or stimulated with (2) isoproterenol (lO ⁇ M) and (3) basic fibroblast growth factor (bFGF) (250 ng/ml) for 10 mins. Western blot data are shown.
  • FIG. 10 Loss of pl l O ⁇ protects from interstitial fibrosis and hypertrophy.
  • A) Heart sections from p! 10 ⁇ +/+ and pi 10f' ' mice treated with a vehicle control or isoproterenol for 7 days. Trichrome and picro sirius red (PSR) staining to detect remodeling and interstitial fibrosis are shown.
  • B) Morphometric quantitation of fibrosis in vehicle and isoproterenol treated mice. n 5 per group. *P ⁇ 0.01 compared with vehicle.
  • C) Quantitation of apoptosis in vehicle and isoproterenol treated mice. n 3 per group with 3 sections from each heart. *P ⁇ 0.05 compared with vehicle.
  • FIG. 11 Ventricular ⁇ -adrenergic receptor densities.
  • Protein recovery was similar for pi 10 ⁇ +/" and pll Of' ' ventricles without or with isoproterenol treatment.
  • B) Quantifications of adenylate cycle N and NI (ACN/NI), ⁇ adrenergic ( ⁇ t-AR) and ⁇ 2 -adrenergic ( ⁇ 2 -AR) receptor expression (n 6 per group).
  • Western blots were scanned from film with a laser densitometer (Molecular Dynamics), and quantified using ImageQuant software (Molecular Dynamics). Nalues are mean ⁇ SEM. *P ⁇ 0.05.
  • Modulation of PBKgamma has utility in treating a wide range of heart disease, such as heart attack and heart failure.
  • Blocking PBKgamma protein activity or gene expression increases heart function, for example, by providing increased heart muscle contractility.
  • the present invention includes all uses that relate to the realization of the modulatory properties of PBKgamma including, but not limited to, the development of therapeutic and diagnostic assays and compositions as well as the preparation and/or isolation of other molecules that inhibit PBKgamma.
  • the present invention provides a method of preventing or treating heart disease comprising administering an effective amount of an inhibitor of PBKgamma to an animal in need of such treatment.
  • the invention includes a use of an effective amount of an inhibitor of PBKgamma for preventing or treating heart disease or to prepare a medicament for preventing or treating heart disease.
  • PBKgamma or "PBK ⁇ ” means phosphoinositide 3 -kinase gamma and includes any and all forms of this kinase from any source.
  • the term includes biologically active fragments of PB ⁇ .
  • the PB ⁇ includes the catalytic subunit pi lOgamma (pi lO ⁇ ).
  • an "effective amount" of the inhibitor of PBKgamma of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • the effective amount of the inhibitor of PBKgamma of the invention may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage procedures may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • animal as used herein includes all members of the animal kingdom including mammals such as humans.
  • treatment or treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treating can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • an inhibitor of PBKgamma means any molecule or compound that can inhibit the expression of the PBKgamma gene or that can inhibit the activity of the PBKgamma protein.
  • the inhibitor can be any type of molecule including, but not limited to, proteins (including antibodies), peptides, peptide mimetics, nucleic acids (including DNA, RNA, antisense oligonucleotides, peptide nucleic acids), carbohydrates, organic compounds, small molecules, natural products and library extracts.
  • the inhibitors may be known or novel compounds, including derivatives of the known compounds.
  • Known inhibitors include wortmannin, Ly294002, 2-(4-morpholinyl)-8-phenyl-4H-l- benzopyran-4-one (LY294002) and quercetin (see US patent application no. 20020037276 (Ptasznik et al) and CA 2378650 (Forti)). Derivatives and analogues of these compounds are also useful.
  • the PBKgamma inhibitor is an antibody that binds PBKgamma or an antisense oligonucleotide that inhibits the expression of PBKgamma.
  • RNA that is complimentary to a nucleic acid sequence from a PBKgamma gene can be used in the methods of the present invention to inhibit PBKgamma.
  • the present invention provides a method of preventing or treating heart disease comprising administering an effective amount of an antibody or antisense oligonucleotide to an animal in need thereof.
  • oligonucleotides and antibodies follows.
  • RNA interference techniques such as antisense RNA or siRNA are useful to block gene expression.
  • antisense compounds for inhibition of PI3K expression are described in US patent application no. 20020058638 (Monia et al.).
  • Nucleic acid sequences for RNA inhibition of PBKgamma could be made from [SEQ ID NO:l] or fragments thereof.
  • the term "antisense oligonucleotide” as used herein means a nucleotide sequence that is complimentary to its target.
  • siRNA refers to double stranded RNA (dsRNA) which silences expression of genes having high identity to either of the strands in the RNA duplex (Fire et al. (1998) Nature 391, 806-811). dsRNA directs gene-specific, post-transcriptional silencing in many mammals through mRNA degradation.
  • siRNA may include nucleic acids modified in the same manner as the oligonucleotide nucleic acids described below.
  • oligonucleotide refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages.
  • the term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases.
  • the term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide.
  • the oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol
  • oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.
  • phosphorothioate bonds link all the nucleotides.
  • oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents.
  • An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497). PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro.
  • PNA peptide nucleic acid
  • oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506).
  • Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide.
  • Oligonucleotides may also have sugar mimetics.
  • the nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the nucleic acid molecules of the invention or a fragment thereof may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides.
  • sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
  • the inhibitor of PBKgamma is a PBKgamma specific antibody.
  • Antibodies to PBKgamma may be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S. Patent Nos. RE 32,011; 4,902,614; 4,543,439; and 4,411,993, which are incorporated herein by reference.
  • antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab') 2 ) and recombinantly produced binding partners.
  • the therapeutic methods of the invention can be used to treat any condition wherein it is desirable to modulate PBKgamma expression or activity.
  • Such conditions include heart disease caused by acute coronary syndromes (eg. congestive heart failure, angina, myocardial infarction (heart attack)), cardiac arrythmias (eg. atrial flutter, atrial fibrillation, paroxysmal supraventricular tachycardia) and hypertension (eg. idiopathic hypertrophic subaortic stenosis).
  • the present invention provides a method of preventing or treating heart disease comprising administering an effective amount of an inhibitor of PBKgamma to the recipient animal.
  • the invention includes a use of an effective amount of an inhibitor of PBKgamma to prevent or treat heart disease or to prepare a medicament to prevent or treat heart disease.
  • the above described methods for preventing or treating heart disease using PBKgamma inhibitors may be further enhanced by co-administering other drugs, such as beta-blockers.
  • the present invention provides a method of preventing or treating heart disease comprising administering an effective amount of a competitively inhibiting PBKgamma protein fragment or a nucleic acid sequence encoding a PBKgamma protein fragment to an animal in need thereof.
  • a competitively inhibiting PBKgamma protein includes an inactive full length PBKgamma protein as well as inactive fragments or portions of the protein, such as pl lOgamma, that competitively inhibit binding of PBKgamma to its substrates.
  • Preferred fragments or portions of the protein are those that are sufficient to prevent or treat heart disease.
  • the PBKgamma protein or the nucleic acid encoding the PBKgamma protein can be readily obtained by one of skill in the art.
  • many PBKgamma sequences are available in the GenBank database including the human PBKgamma [SEQ ID NO:2].
  • the PBKgamma protein or nucleic acid may be modified from the known sequences to make it more useful in the methods of the present invention.
  • Competitive inhibitors, such as inactive peptide fragments may be designed based on the pBKgamma, preferably pl lOgamma, sequence.
  • One may also use polypeptides having sequence identity to a fragment of pBKgamma or pi 10.
  • the invention also includes polypeptides which have sequence identity at least about: >20%, >25%, >28%, >30%, >35%, >40%, >50%, >60%, >70%, >80% or >90% more preferably at least about >95%, >99%, to a polypeptides fragment of the invention, such as a fragment of [SEQ ID NO:2].
  • Identity is calculated according to methods known in the art. Sequence identity is most preferably assessed by the BLAST version 2.1 program advanced search (parameters as above; Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool.” J. Mol. Biol.
  • BLAST is a series of programs that are available online at http://www.ncbi.nlm.nih.gov/BLAST.
  • the advanced blast search http ://www.ncbi.nlm.nih. go v/blast/blast.
  • the PBKgamma protein fragment is prepared as a soluble fusion protein.
  • the fusion protein may contain a domain of PBKgamma linked to an immunoglobulin (Ig) Fc Region.
  • Ig immunoglobulin
  • the PBKgamma fusion may be prepared using techniques known in the art. Generally, a DNA sequence encoding the extracellular domain of PBKgamma is linked to a DNA sequence encoding the Fc of the Ig and expressed in an appropriate expression system where the PBKgamma - Fclg fusion protein is produced.
  • the PBKgamma protein may be obtained from known sources or prepared using recombinant DNA techniques. The protein may have any of the known published sequences for PBKgamma.
  • sequences can be obtained from GenBank as described above.
  • the protein may also be modified to contain amino acid substitutions, insertions and/or deletions.
  • conserveed amino acid substitutions involve replacing one or more amino acids of the PBKgamma amino acid sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics.
  • Non-conserved substitutions involve replacing one or more amino acids of the PBKgamma amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.
  • the PBKgamma protein may be modified.
  • disulphide bonds may be formed between two appropriately spaced components having free sulfhydryl groups.
  • the bonds may be formed between side chains of amino acids, non-amino acid components or a combination of the two.
  • the PBKgamma protein or peptides of the present invention may be converted into pharmaceutical salts by reacting with inorganic acids including hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulphonic acid, and tolunesulphonic acids.
  • inorganic acids including hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid, etc.
  • organic acids including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulphonic acid, and
  • the present invention also includes peptide mimetics of the PBKgamma of the invention.
  • Peptide mimetics are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of a peptide, or enhancer or inhibitor of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention.
  • Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic.
  • the mimetics may include isosteric amide bonds, or D- amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states.
  • the mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins.
  • Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.
  • Peptides of the invention may also be used to identify lead compounds for drug development.
  • the structure of the peptides described herein can be readily determined by a number of methods such as NMR and X-ray crystallography. A comparison of the structures of peptides similar in sequence, but differing in the biological activities they elicit in target molecules can provide information about the structure-activity relationship of the target. Information obtained from the examination of structure-activity relationships can be used to design either modified peptides, or other small molecules or lead compounds that can be tested for predicted properties as related to the target molecule. The activity of the lead compounds can be evaluated using assays similar to those described herein. Information about structure-activity relationships may also be obtained from co- crystallization studies.
  • a peptide with a desired activity is crystallized in association with a target molecule, and the X-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison may be used to design compounds expected to possess.
  • the present invention also includes the isolation and/or identification of substances to inhibit PBKgamma expression or activity for treatment of heart disease.
  • substances or PBKgamma modulators may be useful in the above described therapeutic methods.
  • Two examples of PBKgamma modulators include antibodies and antisense molecules which are described in detail above.
  • Other PBKgamma modulators may be identified, for example, using the screening assays described below.
  • Substances that affect PBKgamma activity can be identified based on their ability to bind to PBKgamma.
  • Substances which can bind with the PBKgamma of the invention may be identified by reacting the PBKgamma with a substance which potentially binds to PBKgamma, and assaying for complexes, for free substance, or for non-complexed PBKgamma, or for activation of PBKgamma.
  • a yeast two hybrid assay system may be used to identify proteins which interact with PBKgamma (Fields, S. and Song, O., 1989, Nature, 340:245-247).
  • Systems of analysis which also may be used include ELISA. Accordingly, the invention provides a method of identifying substances which can bind with PBKgamma, comprising the steps of:
  • Conditions which permit the formation of substance and PBKgamma complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
  • the substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • antibody against PBKgamma or the substance, or labeled PBKgamma, or a labeled substance may be utilized.
  • the antibodies, proteins, or substances may be labeled with a detectable substance as described above.
  • the invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of PBKgamma.
  • the invention may be used to assay for a substance that competes for the same binding site of PBKgamma.
  • the invention enables a method for screening candidate compounds for their ability to decrease the activity of a PBKgamma protein.
  • the method comprises providing an assay system for assaying PBKgamma activity, assaying the activity in the presence or absence of the candidate or candidate compound and determining whether the compound has decreased PBKgamma activity.
  • the method further comprises determining whether the compound is suitable for preventing or treating heart disease.
  • the present invention provides a method for identifying a compound that inhibits PBKgamma protein activity or expression comprising: (a) incubating a candidate compound with a PBKgamma protein or a nucleic acid encoding a PBKgamma protein;
  • test substance is capable of increasing heart function, such as heart muscle contractility, and thereby prevent or treat heart disease.
  • the invention enables a method for screening candidate compounds for their ability to decrease expression of a PBKgamma protein.
  • the method comprises putting a cell with a candidate compound, wherein the cell includes a regulatory region of a PBKgamma gene operably joined to a reporter gene coding region, and detecting a change in expression of the reporter gene.
  • the present invention enables culture systems in which cell lines which express the PBKgamma gene, and thus PBKgamma protein products, are incubated with candidate compounds to test their effects on PBKgamma expression.
  • Such culture systems can be used to identify compounds which downregulate PBKgamma expression or its function, through the interaction with other proteins.
  • Such compounds can be selected from protein compounds, chemicals and various drugs that are added to the culture medium. After a period of incubation in the presence of a selected candidate compound(s), the expression of PBKgamma can be examined by quantifying the levels of PBKgamma mRNA using standard Northern blotting procedure to determine any changes in expression as a result of the candidate compound. Cell lines transfected with constructs expressing PBKgamma can also be used to test the function of compounds developed to modify the protein expression.
  • transformed cell lines expressing a normal PBKgamma protein could be mutagenized by the use of mutagenizing agents to produce an altered phenotype in which the role of mutated PBKgamma can be studied in order to study structure/function relationships of the protein products and their physiological effects.
  • the present invention provides a method for identifying a compound that affects the binding of a PBKgamma protein and a PBKgamma binding protein comprising:
  • the invention also includes pharmaceutical compositions containing substances that inhibit PBKgamma inhibitors for use in heart disease as well.
  • Substances that inhibit PBKgamma include substances that inhibit PBKgamma gene expression as well as substances that inhibit PBKgamma activity.
  • Such substances include antisense molecules to PBKgamma, antibodies to PBKgamma as well as other substances or PBKgamma antagonists identified using the screening assays described herein.
  • Such pharmaceutical compositions can be for intralesional, intravenous, topical, rectal, parenteral, local, inhalant or subcutaneous, intradermal, intramuscular, intrathecal, transperitoneal, oral, and intracerebral use.
  • the composition can be in liquid, solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions.
  • compositions of the invention can be intended for administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.
  • the pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the pharmaceutical compositions may additionally contain other agents to prevent or treat heart disease.
  • the pharmaceutical composition for use in preventing or treating heart disease comprises an effective amount of an inhibitor of PBKgamma in admixture with a pharmaceutically acceptable diluent or carrier.
  • the PBKgamma inhibitor is optionally an antisense oligonucleotide to PBKgamma or an antibody that binds to PBKgamma.
  • compositions comprising antisense nucleic acid molecules may be directly introduced into cells or tissues in vivo using delivery vehicles such as retro viral vectors, adeno viral vectors and DNA virus vectors. They may also be introduced into cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. Recombinant molecules may also be delivered in the form of an aerosol or by lavage.
  • the nucleic acid molecules of the invention may also be applied extracellularly such as by direct injection into cells.
  • the pharmaceutical composition for use in preventing or treating heart disease comprises an effective amount of a PBKgamma protein or a nucleic acid encoding a PBKgamma protein in admixture with a pharmaceutically acceptable diluent or carrier.
  • PBKgamma is involved in heart disease allows the detection of conditions involving an increase in PBKgamma activity or expression resulting in heart disease.
  • the present invention provides a method of detecting a condition associated with increased PBKgamma expression or activity comprising assaying a sample for (a) a nucleic acid molecule encoding a PBKgamma protein or a fragment thereof or (b) a PBKgamma protein or a fragment thereof and comparing the amount of PBKgamma nucleic acid or protein detected with a suitable control.
  • Nucleotide probes can be prepared based on the sequence of PBKgamma for use in the detection of nucleotide sequences encoding PBKgamma or fragments thereof in samples, preferably biological samples such as cells, tissues and bodily fluids.
  • the probes can be useful in detecting the presence of a condition associated with PBKgamma or monitoring the progress of such a condition.
  • the present invention provides a method for detecting a nucleic acid molecules encoding PBKgamma comprising contacting the sample with a nucleotide probe capable of hybridizing with the nucleic acid molecule to form a hybridization product, under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.
  • a nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32P, 3H, 14C or the like.
  • detectable substances include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and chemiluminescence.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleic acid to be detected and the amount of nucleic acid available for hybridization.
  • Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleotide probes may be used to detect genes, preferably in human cells, that hybridize to the nucleic acid molecule of the present invention preferably, nucleic acid molecules which hybridize to the nucleic acid molecule encoding PBKgamma under stringent hybridization conditions as described herein.
  • Nucleic acid molecules encoding a PBKgamma protein can be selectively amplified in a sample using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleotide sequence of PBKgamma for use in PCR.
  • a nucleic acid can be amplified from cDNA or genomic DNA using oligonucleotide primers and standard PCR amplification techniques. The amplified nucleic acid can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al, Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL.
  • the PBKgamma protein may be detected in a sample using antibodies that bind to the protein as described in detail above. Accordingly, the present invention provides a method for detecting a PBKgamma protein comprising contacting the sample with an antibody that binds to PBKgamma which is capable of being detected after it becomes bound to the PBKgamma in the sample.
  • Antibodies specifically reactive with PBKgamma, or derivatives thereof, such as enzyme conjugates or labeled derivatives may be used to detect PBKgamma in various biological materials, for example they may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of PBKgamma, and the antibodies.
  • the antibodies may be used to detect and quantify PBKgamma in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.
  • the antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect PBKgamma, to localise it to particular cells and tissues and to specific subcellular locations, and to quantitate the level of expression.
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect PBKgamma.
  • an antibody of the invention may be labelled with a detectable substance and PBKgamma may be localised in tissue based upon the presence of the detectable substance. Examples of detectable substances include various enzymes, fluorescent materials, luminescent materials and radioactive materials.
  • Suitable enzymes include horseradish peroxidase, biotin, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include radioactive iodine 1-125, 1-131 or 3-H.
  • Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualised by electron microscopy.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against PBKgamma.
  • a second antibody having specificity for the antibody reactive against PBKgamma.
  • the antibody having specificity against PBKgamma is a rabbit IgG antibody
  • the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
  • PBKgamma may be localized by autoradiography .
  • the results of autoradiography may be quantitated by determining the density of particles in the autoradiographs by various optical methods, or by counting the grains.
  • the invention includes a method of treating heart disease, such as excessive heart contractility, in a mammal in need thereof by administering an agent that activates PBKgamma or blocks PTEN.
  • RNA compounds, antibodies and other molecules may be used to activate PBKgamma or block PTEN.
  • Such compounds may be prepared as described in this application.
  • PTEN may be blocked by using nucleic acids based on [SEQ ID NO:3] or competitive inhibitors based on [SEQ ID NO:4].
  • the invention also includes the use of an inhibitor of PTEN or an activator of PBKgamma to prepare a medicament to prevent or treat heart disease, such as excessive contractility.
  • the inhibitor of PTEN may comprise inactive PTEN, an inactive fragment of PTEN, an antibody to PTEN or a nucleic acid that inhibits the expression of PTEN, for example by RNA interference.
  • the activator of PBK could include an antibody or other molecule.
  • the invention also includes a pharmaceutical composition for use in preventing or treating heart disease, such as excessive contractility, comprising an inhibitor of PTEN or activator of PIK3 gamma and a carrier.
  • the invention also includes a method for identifying a compound that activates the binding of a PBKgamma protein to its substrate for treatment of heart disease, such as increased contractility, comprising: (a) incubating (i) a candidate compound; (ii) an PBKgamma protein and (iii) an PBKgamma substrate under conditions which permit the binding of PBKgamma protein to the substrate; and
  • the invention also includes a compound identified by this method.
  • the invention also includes a method for identifying a compound that inhibits the binding of a PTEN protein to its substrate for treatment of heart disease, such as increased contractility, comprising: (d) incubating (i) a candidate compound; (ii) an PTEN protein and (iii) an
  • PTEN substrate under conditions which permit the binding of PTEN protein to the substrate
  • the invention also includes a compound identified by this method.
  • the invention also includes the use of an inhibitor of PBK or an activator of PTEN to prepare a medicament to prevent or treat heart disease, such as excessive contractility or hypertrophy.
  • the inhibitor of PBK may comprise inactive PBK, an inactive fragment of PBK, an antibody to PBK or a nucleic acid that inhibits the expression of PBK, for example by RNA interference.
  • the activator of PTEN could include an antibody or other molecule.
  • the invention also includes a pharmaceutical composition for use in preventing or treating heart disease, such as excessive contractility, comprising an inhibitor of PBK or activator of PTEN and a carrier.
  • the invention also includes a method for identifying a compound that activates the binding of a PTEN protein to its substrate for treatment of heart disease, such as hypertrophy, comprising:
  • the invention also includes a compound identified by this method.
  • the invention also includes a method for identifying a compound that inhibits the binding of a PBK protein to its substrate for treatment of heart disease, such as or hypertrophy, comprising:
  • the invention also includes a compound identified by this method.
  • One may also modulate hypertrophy in a mammal in need thereof by administering an agent that modulates PTEN or PBKgamma.
  • the invention includes a method of treating heart disease, such as hypertrophy, in a mammal in need thereof, by administering an agent that activates PTEN or inhibits PBKgamma.
  • Peptide fragments, antibodies, RNA and other compounds described in this application are useful for the above methods of medical treatment.
  • the above diagnostic methods and other methods of the invention described in the application may also be adapted for use with the methods described in this section.
  • cardiac hypertrophy did not result in fibrotic or structural changes in myocyte organization (Figure ID) or perturbations in cardiomyocyte cell death even at 1 year of age showing that loss of PTEN does not result in cardiac decompensation or dilated cardiomyopathy.
  • cardiomyocytes were isolated from adult mice to assess the individual cell size. Loss of PTEN resulted in a marked increase in both the length and width of cardiomyocytes as compared to controls ( Figure 2B). The length-to-width ratio was unchanged in these cells showing that the cell size change was similar to that observed in physiologic hypertrophy (Hunter and Chien, 1999).
  • Pathological cardiac hypertrophy is characterized by a prototypical change in gene expression patterns such as increased ANF, ⁇ MyHC, BNP, and skeletal-actin expression, and a decrease in ⁇ MyHC (Hunter and Chien, 1999).
  • mice The hearts of pl lO ⁇ deficient mice displayed a marked enhancement in contractility as assessed by increased fractional shortening (FS), Ncfc, and peak aortic outflow velocity (PAN) ( Figure 3B,C and Table 1). Hemodynamic measurements confirmed the increased heart contractility in pl lO ⁇ " " mice (Table 1). Telemetry analysis of blood pressure in conscious mice revealed normal blood pressure in pi lO ⁇ " " mice.
  • FS fractional shortening
  • Ncfc peak aortic outflow velocity
  • PI3K ⁇ mediates reduction of heart function in PTE ⁇ -deficient hearts
  • mice deficient for both pi lO ⁇ and PTE ⁇ in the heart were significantly enlarged as compared to wild type and pllO ⁇ "7" mutant mice ( Figure 3 A, Table 1).
  • mice transgenic for heart-specific dominant negative pllO ⁇ have smaller than normal heart size.
  • mice deficient for PTE ⁇ in the hearts and overexpressing the heart specific D ⁇ -pllO ⁇ transgene Hearts of mckCRE-PTE ⁇ A ⁇ ; DN-pl 10a mice displayed a reduction in heart size similar to that seen in single DN-pl 10a transgenic mice (Fig. 4A,B).
  • DN-pl 10a transgene also correlated with a decrease in the basal level of AKT/PKB activation in the absence or presence of a functional PTEN gene (Figure 4C).
  • overexpression of DN-pl 10a on a wild type background affects heart size but has no apparent effect on heart function
  • hearts from mckCRE-PTEN ⁇ ⁇ ; DN-pl 10a displayed a marked decrease in contractility ( Figure 4D, E and Table 1).
  • PBK ⁇ signaling modulates basal cell size of cardiomyocytes while PBK ⁇ signaling controls basal cardiac contractility.
  • PI3Ky modulates baseline cAMP levels in cardiomyocytes
  • PBK ⁇ can bind to GPCR-kinase 2 (GRK2) (Naga).
  • ⁇ -adrenergic signaling may be affected in pi 10 ⁇ _/" hearts, and alterations of PBK activity may affect GRK activity and/or ⁇ -AR expression.
  • GRK activity in both cytosolic and membrane-associated fractions of PTEN-deficient hearts was comparable to that of control hearts ( Figure 6A).
  • Total protein levels of GRK2 were also comparable ( Figure 6A).
  • Radiolabeled ligand binding assays showed that there is also no detectable difference in the density of ⁇ -AR receptors or their affinity for the ⁇ -AR ligand ICYP in the PTEN- or pllO ⁇ -deficient hearts as compared to wild-type mice ( Figure 6B). These data imply that changes in contractility of PTEN and pi lO ⁇ - deficient hearts are not due to alterations in baseline ⁇ -adrenergic receptor levels or the modulation of GRK activity.
  • the G-protein coupled ⁇ 2 -adrenergic receptors are linked to both G s and God G-proteins that can either increase (G s) or decrease (G ⁇ i) cAMP levels and heart muscle contractility (Kuznetsov et al, 1995; Xiao et al, 1999; Rockman et al, 2002).
  • G s G s
  • G ⁇ i G ⁇ i
  • PBK ⁇ can modulate cAMP production following GPCR stimulation
  • neonatal cardiomyocytes from both wild type and pl lO ⁇ " " hearts and stimulated these cells with the ⁇ 2 -AR selective agonist zinterol.
  • the invention shows the roles of PBK-PTEN signaling in cardiac hypertrophy and heart functions in PTEN-heart muscle specific mutant mice, pl lO ⁇ knock-out, dominant-negative pl lO ⁇ transgenic mice, and double-mutant mice.
  • the tyrosine kinase-receptor pl lO ⁇ -PTEN pathway is a critical regulator of cardiac cell size.
  • the PTEN-PBK ⁇ signaling pathway regulates heart muscle contractility via GPCRs. This contractility phenotype is present in single cardiomyocytes and dependent on cAMP signaling.
  • PBK ⁇ regulates cardiac function by negatively regulating cAMP levels and phospholamban phosphorylation upon ⁇ 2 -adrenergic receptor stimulation.
  • the cardiac hypertrophy in PTEN deficient hearts is similar to that in mice overexpressing a constitutively active pi 10a transgene in the heart (Shioi et al, 2000). Like these transgenic mice, the hypertrophy found in PTEN deficient hearts displayed features characteristic of physiological hypertrophy, such as increase in both the length and width of the myocytes, no fibrotic changes and no decompensation into dilated cardiomyopathy (Hunter and Chien, 1999). The reversal of heart size in the PTEN deficient hearts that expressed a dominant negative PBK ⁇ transgene indicates that it is this isoform of PBK that modulates basal cell size in cardiomyocytes.
  • any other PBK isoform can compensate for the loss of PBK ⁇ function in vivo. It should be pointed out that it is possible that this transgene interferes with not only pl lO ⁇ , but also pl lO ⁇ and pl lO ⁇ as all of these require p85 activity which is likely sequestered as a result of the transgene expression. Nevertheless, there is a differential action of class la and class lb PBKs as it is unlikely that this transgene interferes with pllO ⁇ function. This is further supported by the absence of any observable alteration in cardiac contractility in mice expressing the DN-pl 10a transgene.
  • PBK signaling has been implicated in cell size regulation in other tissues and organisms such as drosophila (Scanga et al, 2000). Mice deficient for p70 S6K have reduced body size due to a reduction in cell size (Shima et al, 1998). Recently, brain specific disruptions of PTEN have been reported which result in the increase in neuronal cell size (Backman et al, 2001). Moreover, transgenic expression of activated AKT/PKB in mouse heart leads to a hypertrophic phenotype and this phenotype can be inhibited with a DN-pl 10a transgene (Shioi et al, 2002; Matsui et al, 2002).
  • PBK ⁇ has no role in the homeostatic control of cardiomyocyte cell size.
  • GPCR agonists used to induce PI3K-dependent cardiomyocyte hypertrophy in vitro are GPCR agonists (Rabkin et al, 1997; Schluter et al, 1999).
  • PBK ⁇ is activated in pressure overload mduced hypertrophy in vivo (Naga Prasad et al, 2000), implying a role for PBK ⁇ in agonist-induced cardiac hypertrophy.
  • PTEN deficient hearts showed cardiac hypertrophy and surprisingly also displayed a dramatic decrease in cardiac contractility. Conversely, PBK ⁇ deficient hearts exhibited an increase in cardiac contractility.
  • PBK ⁇ -PTEN signaling controls heart muscle function in vivo.
  • PBK ⁇ can modulate contractility in the absence of exogenous agonists. These contractility changes are at least in part mediated by alteration in cAMP levels.
  • Hearts deficient in both PTEN and PBK ⁇ also displayed increased cardiac contractility showing that increased PIP3 generated via PBK ⁇ causes the decrease in cardiac contractility seen in the PTEN deficient hearts.
  • the different receptors recruit different downstream targets for the same phospholipids.
  • ⁇ 2 adrenergic receptors are coupled to both Gas and G ⁇ i signaling (Rockman et al., 2002). The opposing effects of these two G protein subunits result in no global net effect on cardiac contractility or cAMP production upon receptor stimulation (Kuznetsov et al, 1995; Xiao et al, 1999).
  • Cardiac hypertrophy is associated with a prototypical alteration in gene expression profiles.
  • PBK In models of cardiomyocyte hypertrophy in culture, PBK has been shown to play a role in the onset of these alterations in gene expression.
  • mRNA levels of hypertrophy markers To determine if loss of pl lOgamma alters the changes in gene expression seen in isoproterenol induced cardiac hypertrophy, we assed the mRNA levels of hypertrophy markers. Isoproterenol has been shown to induce significant alterations in gene expression albeit blunted in comparison to other models of hypertrophy. Consistently, we observed a prototypical increase in ANF and BNP expression in control hearts. We also observed a slight decrease in a-myosin heavy chain expression.
  • PBKalpha modulates basal cell size in cardiomyocytes while loss of PBKgamma results in increased cardiac contractility with no effect on basal cell size.
  • GPCR agonists have been used for the induction of hypertrophy suggesting that PBKgamma the isoform linked to GPCR signaling, may play a role in agonist induced cardiac hypertrophy.
  • the increased cardiac contractility seen in PBKgamma deficient hearts suggest a potential role for PBKgamma inhibition in the treatment of heart failure.
  • PBKgamma deficient mice have an attenuated hypertrophic response to isoproterenol induced cardiac hypertrophy in vivo and that PBKgamma deficient mice retain their increase in pump function following this chronic beta-adrenergic receptor stimulation.
  • Control hearts treated with isoproterenol displayed an increase in cardiac hypertrophy and a dilation of the left ventricle consistent with the onset of dilated cardiomyopathy.
  • the architecture of pl lOgamma deficient hearts remained largely unchanged.
  • PBKgamma may not be required for all forms of cardiac hypertrophy but possibly may play a critical role in hypertrophy induced through GPCR mediated pathways.
  • Our data here suggests that this hypertrophy may be in part mediated by activation of PKB/AKT pathways.
  • overexpression of an activated form of AKT could induce cardiac hypertrophy in vivo.
  • PBKgamma is essential for ERK activation in some systems.
  • Transgenic mouse models with heart specific increases in ERK activation have been shown to lead to a physiologic hypertrophy.
  • a MAP kinase mediated pathway may also play a role in the hypertrophy observed here.
  • the invention shows that inhibition of PBKgamma improves function in a model of heart failure.
  • Table 3 shows this result. Mice were treated with isoproterenol chronically for 7 days. In control +/- animals heart function is reduced, but in -/- heart function is maintained.
  • mice The generation and genotyping of the PTEN-floxed, mckCRE, pllO ⁇ , and DN-pl 10a mice have been previously described (Suzuki et al, 2001; Bruning et al, 1998; Sasaki et al, 2000; Shioi et al, 2000; Hirsch et al, 2000). Only littermate mice were used as controls. Mice were bred and maintained following institutional guidelines.
  • Protein and mRNA expression analyses were carried out as described (Crackower et al, 2002) using antibodies to PTEN (Cascade), pllO ⁇ , pl lO ⁇ , pl lO ⁇ (Santa Cruz), p85 (Upstate Biotechnologies), phospho- AKT/PKB (S473), AKT/PKB, phospho-GSK3 ⁇ , GSK3 ⁇ , phospho-p70 S6K , phospho- ERKl/2, ERKl/2, phospho-phospholamban (Serinel ⁇ ), phospholamban (Cell Signaling Technology), and GAPDH (Research Diagnostics).
  • lysates were incubated with anti-GRK2/3 Abs (Upstate Biotechnologies) conjugated with Sepharose beads for 16 hours at 4°C. Immunoprecipitates were separated using SDS-PAGE gel electrophoresis, transferred onto membranes and immunoblotted.
  • Heart morphometry, echocardiography, and hemodynamic measurements were carried out as described (Crackower et al, 2002). Catecholamines were extracted from the plasma of male mice using alumina, eluted and then assayed using HPLC (Waters Associates Inc., Milford, MA, USA) coupled with electrochemical detection (ESA Inc., Bedford, MA, USA). The intra-assay and inter-assay coefficient of variation were ⁇ 5% and ⁇ 10%, respectively.
  • Cardiomyocyte purification Adult ventricular myocyte were isolated as described (Sah et al, 2002). Cell size was measured using Openlab 2.2.5 software (Improvision Ltd). Ventricular myocytes were placed in a Plexiglas chamber and continuously perfused with oxygenated Tyrodes buffer (137mM NaCI, 5.4mM KC1, lOmM glucose, 1 OmM HEPES, 0.5mM NaH 2 PO 4 , lmM MgCl 2 , 1.2mM CaCl 2 , and pH 7.4) at 2.5ml/min at 36°C.
  • Tyrodes buffer 137mM NaCI, 5.4mM KC1, lOmM glucose, 1 OmM HEPES, 0.5mM NaH 2 PO 4 , lmM MgCl 2 , 1.2mM CaCl 2 , and pH 7.4
  • Neonatal ventricular cardiac myocytes were isolated from 1-3 day old mice in CBFHH buffer (137mM NaCI, 5.36mM KC1, 0.81mM MgSO 4 -7H 2 O, 5.55mM dextrose, 0.44mM KH 2 PO 4 , 0.34mM Na 2 HPO 4 -7H 2 O, 20 ⁇ M Hepes, pH 7.4) as described (Steinberg et al, 1991). Experiments were carried out 48-72 hrs after plating.
  • Cardiomyocytes were stimulated at 1 Hz with a Grass S44 stimulator (pulse duration 3 ms; 15-20 volts) and a video edge detector (Crescent Electronics) was used to track myocyte contractions at 240Hz. Steady state contractions were recorded at 1kHz following a 4 mins equilibrium period using a Phillips 800 camera system (240Hz) and Felix acquisition software (Photon Technologies Inc.). Cardiomyocyte length, percent fractional shortening, shortening rate (+dL/dT), and relaxation rate (— dL/dT) were determined at baseline.
  • Myocytes were incubation with LY294002 (30 ⁇ M) (Calbiochem) and Rp-cAMPS (25 ⁇ M) (Sigma/RBI) for 10 minutes and recordings were made during continuous perfusion of the drug dissolved in Tyrode.
  • phosphodiesterase activity was inhibited by pre-treating the cells with theophylline (lOOmM) for 30 min prior to stimulation.
  • Wortmannin treatment (lOOnM) was initiated at the time of theophylline addition.
  • Zinterol (Bristol-Myers Squibb) stimulation was carried out at RT for 10 min.
  • Forskolin (Sigma) was used at 25mM.
  • Isoproterenol (Sigma) stimulation was carried out at RT for 10 min, following 5 min. pretreatment of ICI 118,551 (Sigma/RBI) (10 "7 M).
  • cAMP was measured by RIA according to the supplier's protocol (Amersham).
  • GRK activity was measured by rhodopsin phosphorylation as described (Louden et al., 2000). Hearts were homogenized in 20mM Tris pH 7.3, 0.8mM MgCl 2 , 2mM EDTA. Membrane fractions were isolated by centrifuging at 40,000g for 30min at 4°C. 300 ⁇ g and 20 ⁇ g of protein were assayed for cytosolic and membrane fractions, respectively.
  • b-AR density and affinity for its ligand ICYP (3-Iodocyanopindolol [ 125 I] ) were determined by equilibrium radio-ligand binding assays on sarcolemmal membranes prepared from ventricular myocardium as described (Steinberg et al., 1995). Briefly, assays were performed with 30mg membrane protein and ICYP (5-250pM) in the absence or presence of O.lmM unlabeled propranolol to determine non-specific binding in a final volume of 1ml for 60min at 37°C. ICYP bound to membranes was separated from free, unbound ICYP by rapid vacuum filtration over glass-fiber filters (German A E).
  • mice PI lOgamma deficient mice have previously been described. Hypertrophy was induced by chronic infusion of isoproterenol (sigma) for 7 days at a dose of 15mg/Kg/day using alzet osmotic minipumps. Mice were aneastitized with O2/isoflourane and the pumps were inserted subcutaneously at the level of the scapula. The incision was closed and mice were allowed to recover. Pumps were removed 24 hrs. prior to echocardiographic analysis.
  • R ⁇ A was prepared form hearts using trizol ( ). 20 mg of R A was resolved on a 0.8% formamide gel, blotted to nylon membranes (Amersham), and probed with cD ⁇ A probes for A ⁇ F, B ⁇ P, ⁇ MHC, and GAPDH. AKT kinase activity was measured from whole hearts according to manufacturer's protocols (Upstate).
  • PBK ⁇ is critical for the induction of hypertrophy, fibrosis, and cardiac dysfunction function in response to ⁇ -adrenergic receptor stimulation in vivo.
  • PBK ⁇ is a novel therapeutic target for the treatment of decreased cardiac function in heart failure.
  • isoproterenol a ⁇ -adrenergic receptor agonist
  • PBK ⁇ isoform plays no role in the downregulation of beta-adrenergic receptors following chronic beta-adrenergic stimulation.
  • mice e . pllOf' ' mice have previously been described (Sasaki ete al., 2000). Hypertrophy and heart failure were induced by chronic infusion of isoproterenol (Sigma) for 7 days at a dose of 15mg/kg/day using Alzet® osmotic mini-pumps. Pumps were removed 24h prior to echocardiography and hemodynamic measurements. All experiments were performed in accordance to institutional guidelines. Only littermate male mice of 10-12 weeks of age were used. Heart morphometry, TUNEL assay, echocardiography, and hemodynamic measurements. For heart morphometry, hearts were arrested with KC1, fixed with 10% buffered formalin, and embedded in paraffin. Myocardial interstitial fibrosis was determined as collagen volume fraction (PSR-stained sections) using color-subtractive computer assisted image analysis (Image Processing Tool Kit version 2.5). In situ
  • Akt/PKB kinase activity was measured using a commercial in vitro kinase assay kit according to the manufactures protocol (Upstate Biotechnologies) (Sasaki et al. 2000).
  • Ventricular cardiomyocytes were isolated as described (Crackower et al. 2002). Experiments were carried out 48-72 hrs after plating and removal of fibroblasts.
  • ERKl/2 phosphorylation, p38 phosphorylation, and ERKl/2 protein levels were determined by Western blotting (Cell Signaling Technology).
  • Beta-adrenergic receptor densities Mice were anaesthetized with isoflurane/O 2 , hearts were removed and ventricular tissue excised and frozen. Samples were processed and light sarcolemmae were not treated (-) or treated with PNG-F (+) and subjected to SDS-PAGE as previously described (Rybin et al. 2000).
  • mice To show the role of PBK ⁇ in GPCR-induced cardiac hypertrophy and cardiomyopathy, we chronically infused littermate ⁇ iiOy 7" (knockout) and pll0 ⁇ +/' (control) mice with the ⁇ -adrenergic receptor agonist, isoproterenol. Control mice displayed a marked increase in heart size (Fig. 9A), heart/body weight and heart/tibial length ratios (Fig. 9B) and left ventricular mass and wall thickness (Table 4). In isoproterenol-infused i/0y " " mice, the hypertrophic response still occurred but was markedly attenuated (Fig. 9 A and B) (Table 4).
  • Cardiomyocyte cell death was comparable between isoproterenol-treated pll Of' " and control mice (Fig. IOC). Importantly, measurements of cardiomyocyte sizes in cross-sections confirmed impaired cardiomyocyte hypertrophy i pi 10 f' ' ice (Fig. 10D). There were no differential changes in ⁇ l- or ⁇ 2-adrenergic receptor expression in membrane fractions from pll Of' ' and pi 1 Of' ' hearts at baseline and in response to chronic in vivo isoproterenol stimulation (Fig. 11A and B). Expression levels of the major cardiac adenylate cyclase isoforms V and VI, (ACV/VI), were comparable between pi 1 ⁇ " and pllOf ' " hearts (Fig. HA and B).
  • pll Of' ' mice display a resistance to the effects of isoproterenol on cardiac structure and function.
  • An increase in cardiac function may serve to improve myocardial performance in models of dilated cardiomyopathy and we confirmed that pllOf' ' hearts maintained their hypercontractility following chronic adrenergic stimulation.
  • the induction of several hypertrophy markers in response to isoproterenol was not affected by the loss of pll 0 ⁇ which shows that pllO ⁇ is not required for alteration in fetal gene expression seen in myocardial hypertrophy and that these genetic changes can be uncoupled from the hypertrophic process.
  • Akt/ protein kinase B (PKB) is a major molecular target of the PBK signaling pathway involved in cell hypertrophy in multiple systems, and the mitogen- activated protein kinase, ERKl/2 also plays a central role in cardiac hypertrophy.
  • PKA protein kinase B
  • mice showed that ⁇ - adrenergic receptor stimulation induced in vivo activation of Akt/PKB and in vitro activation ERKl/2.
  • PBK ⁇ is the essential PBK isoform required for Akt/PKB and ERKl/2 activation in cardiomyocytes.
  • pressure- overload hypertrophy there is also activation of Akt/PKB via the PBK ⁇ isoform.
  • Akt/PKB and ERKl/2 pathways in pll Of' " mice which are clearly antiapoptotic pathways, apoptosis was not increased in the pll Of' " mice which is likely related to the antiapoptotic effects mediated by enhanced ⁇ 2 - adrenergic receptor signaling inpllOf' ' cardiomyocytes.
  • PBK also has a role in GPCR signaling in isolated cardiac fibroblasts (Kim et al., 2002), the reduced interstitial fibrosis in the heart may be the direct consequence of attenuated ⁇ -adrenergic receptor-mediated stimulation of cardiac fibroblasts in the pllOf' " hearts.
  • PBKg might be involved in ⁇ -adrenergic receptor down-regulation (internalization).
  • internalization the loss of PBK ⁇ has no apparent effect on the down- modulation of ⁇ -adrenergic receptors in response to chronic adrenergic stimulation.
  • PBK isoforms in the downregualtion of beta-adrenergic receptors and there exists the distinct possibility that receptor desensitization could be differentially affected i.e. functional responses are different.
  • the PI3K5 has an important role to maintain heart function under the pathologic condition of chronic adrenergic stimulation and our results represent the first genetic evidence for a therapeutic role of the modulation of PBK ⁇ signaling in heart failure.
  • mice "p ⁇ 0.01 vs wildtype mice.
  • Bpm heart beats per minute
  • AW anterior wall thickness
  • LVEDD left ventricle end diastolic dimension
  • LVESD left ventricle end systolic dimension
  • LVM left ventricular mass
  • %FS percent fractional shortening
  • Vcfc Velocity of circumferential fiber shortening
  • PAV peak aortic outflow velocity.
  • dP/dT max maximum 1st derivative of the change in left ventricular pressure/time
  • dP/dT min minimum 1st derivative of the change in left ventricular pressure/time
  • Phosphoinositide-dependent protein kinase 1 phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr. Biol. 8, 69-81.
  • Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature. 417, 822-828.
  • the tumor suppressor, PTE ⁇ /MMAC1 dephosphorylates the lipid second messenger, phophatidylinositol 3,4,5-triphosphate. J. Biol. Chem. 273, 13375-13378.
  • the tumor suppressor gene PTEN can regulate cardiac hypertrophy and survival. J. Biol. Chem. 276, 35786-35793.
  • Thrombin modulates phosphoinositide metabolism, cytosolic calcium, and impulse initiation in the heart. Circ. Res. 68, 1216-1229

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Biochemistry (AREA)
  • Environmental Sciences (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. We show that cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double mutant mice revealed that the cardiac hypertrophy and the contractility defects can be genetically uncoupled. PI3KϜ mediates the alteration in cell size while PI3KϜ acts as a negative regulator of cardiac contractility. Mechanistically, PI3KϜ inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3KϜ pathway in the modulation of heart muscle contractility.

Description

COMPOSITIONS AND METHODS FOR TREATING HEART DISEASE
FIELD OF THE INVENTION
The invention relates to compositions and methods for treating heart disease by inhibition of PBKgarnma.
BACKGROUND OF THE INVENTION
Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020 (Yusuf et al, 2001). Cardiovascular disease is frequently associated with elevated wall stress as a result of pressure overload such as in hypertension or volume overload seen in valvular disorders. Increased wall stress in the heart triggers a hypertropl ic response (Hunter and Chien, 1999). What is initially a compensatory response, pathological hypertrophy eventually leads to decompensation resulting in left ventricle dilation, myocyte loss, increased interstitial fibrosis and heart failure. While increased wall stress can lead to pathological cardiac hypertrophy, increased demand for cardiac output can also cause a physiological increase in cell size, as commonly seen in endurance athletes (Colan, 1997).
In flies and mammals, hypertrophic responses can be initiated via phosphoinositide 3- kinase (PI3K) signaling pathways (Leevers et al, 1996; Shioi et al, 2000). PI3Ks constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, adhesion, cell size, and protection from apoptosis (Toker and Cantley, 1997; Stephens et al, 1993). These responses result from the activation of membrane trafficking proteins and enzymes such as the phosphoinositide-dependent kinases (PDKs), PKB/Akt, or S6 kinases by the key second messenger PIP3 (Franke et al, 1997; Alessi et al, 1998; Downward, 1998). Four different type I PI3Ks have been described three of which (PI3Kα, β, δ) are activated by receptor tyrosine kinase pathways. PI3Kγ is activated by the βγ subunit of G-proteins and acts downstream of G-protein-coupled receptors (GPCRs) (Toker and Cantley, 1997). Recent genetic evidence in haematopoietic cells indicates that PI3Kγ is the sole PI3K that couples to GPCRs (Sasaki et al, 2000; Hirsch et al, 2000; Li et al, 2000).
In isolated cardiomyocytes, PI3K signaling has been implicated as a component of cardiac hypertrophy and protection of myocytes from apoptosis mediated by numerous exogenous agonists and stresses (Rabkin et al, 1997; Schluter et al, 1998; Baliga et al, 1999; Kodama et al, 2000). In transgenic mice, overexpression of constitutively active pi 10a results in increased heart size, whereas cardiac overexpression of dominant negative pi 10a in mice resulted in smaller hearts without affecting heart functions (Shioi et al, 2000). Furthermore, PI3Kγ activity is increased upon aortic constriction in mice (Naga Prasad et al, 2000), suggesting that both tyrosine-based and GPCR-linked PI3Ks might play a role in the cardiac hypertrophy response.
The tumor suppressor PTEN is a lipid phosphatase that dephosphorylates the D3 position of PIP3 (Maehama and Dixon, 1998). Thus, PTEN lowers the levels of the
1 PI3K product PIP3 within the cells and antagonizes PI3K mediated cellular signaling. It has been recently shown that PTEN can regulate neuronal stem cell proliferation and the cell size of neurons in brain-specifc PTEN mouse mutants similar to patients with Lhermitte-Duclos disease (Backman et al, 2001). Moreover, expression of dominant negative PTEN gene in rat cardiomyocytes in tissue culture results in hypertrophy (Schwartzbauer and Robbins, 2001). Whether PI3Ks and PTEN can indeed control cardiac hypertrophy and heart functions in vivo is not known.
Summary of the Invention
The inventors have identified PTEN as a master regulator for heart size and heart function and PDkgamma as a critical regulator of heart muscle function (pumping function) in vivo in the whole mouse (mutant mouse model) and in vitro in single cells. These are completely new and unexpected findings and identify novel pathways to protect from heart failure. Inhibition of PDKgamma and the novel PBKgamma signaling pathway allows maintenance of heart function even in failing hearts. Inhibition of PI3Kγ and/or activation of PTEN blocks cardiac hypertrophy. The invention describes a novel and previously unrecognized function for the PI3Kgamma/PTEN pathways in vivo. The functionality was shown in vivo in the whole organism and at the single cell level. Gene targeting in mice using multiple mutants and in a heart failure model was used to establish the in vivo role of PDKgamma and PTEN. The invention studied R-TEN-heart muscle specific mutant mice, pi 10γ ~, dominant-negative /?UOa transgenic mice, and double-mutant mice. Two independent PI3K signaling pathways exist in cardiomyocytes that can be genetically uncoupled. While the tyrosine kinase-receptor pl lOα-PTΕΝ pathway is a critical regulator of cardiac cell size, the GPCR-linked PI3Kγ-PTΕΝ signaling pathway modulates heart muscle contractility.
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention will be described in relation to the drawings in which:
Figure 1. Cardiac Hypertrophy in PTEN-mutant hearts
A, Western blot analysis of proteins from different tissues of mckCRE-PTENA + and mckCRE-PTENA/A mice.
B, Western blot analysis for pllO , pllOβ, pl lOγ, and p85 expression levels in total heart lysates from 2 representative mckCRE-PTENA + and two mckCRE-PTENA A mice.
C, Representative heart sizes of 10 week old mckCRE-PTENA/+ and mckCRE- PTENA/A littermates.
D, Heart sections from 10 week (upper and middle panels) and 1 year (lower panels) old mckCRE-PTENA/+ and mckCRE-PTENA/A mice. The middle and lower panels show higher magnifications to detect interstitial fϊbrosis (trichrome). LV = left ventricle; RV = right ventricle.
Figure 2. Cardiac Hypertrophy and PI3K activation
A, Quantitation of heart/body weight ratios from 10 week old mckCRE-PTENA/+ (n=8) and mckCRE-PTENA/A (n=9) and 12 month old mckCRE-PTENA/+ (n=6) and mckCRE-PTENA/A (n=6) littermate mice. B, Increased cell sizes of cardiomyocytes isolated from 10 week old mckCRE- PTENA/A mice. Representative images of isolated cardiomyocytes and quantitation of cell sizes are shown. 100 individual cells from three different mice were analyzed per group. ** p < 0.01 between genetic groups. C,D Northern blot analysis and quantitation (mean values +/- SEM) of cardiac hypertrophy markers. GAPDH expression levels were used as a loading control. Results from hearts isolated from 3 individual 10 week old mckCRE-PTENA + and 3 different, age-matched mckCRE-PTENA/A littermate mice are shown.
E,F Increased AKT/PKB, GSK3β, and p70S6K phosphorylation in total heart extracts from 10 week old mckCRE-PTENA/A mice. Western blot of the expression of phospho-AKT/PKB, phospho-GSK3β, phospho-p70S6K, phospho-ERKl/2, and their respective loading controls. Densitometric quantitation of phospho-AKT/PKB and phospho-GSK3β levels relative to total cellular AKT/PKB and GSK3β. Mean values +/- SEM are representative of 3 independent experiments. ** p < 0.01 between genetic groups.
Figure 3. Modulation of cardiac contractility in PTEN and pllOγ mutant hearts
A, Left ventricular mass (LNM; mean values +/- SEM) in hearts from 10 week old mckCRE-PTENA/A (n=8), wild-type (n=7), pllO (n=7), and mckCRE-PTENA/A pllOγ ' (n=8) mice. ** p < 0.01 between genetic groups.
B, Percent fractional shorting (%FS) and velocity of circumferential fibre shortening (Ncfs) in 10 week old mckCRE-PTENA A} wild-type, pi 10 f and mckCRE-PTENA/A pllOγ " double mutant mice. Mean values +/- SEM were determined by echocardiography. ** p < 0.01 between groups. C, M-mode echocardiographic images of contracting hearts in 10 week old mckCRE- PTENA A, Wiid-type, pi 1 Of'', and mckCRE-PTENA/A pllOγ/~ mice. Note that contracting hearts in mckCRE-PTENA/ApHOγ " double mutants are similar to that of pllO mice. D. Western blot analysis for pi 10γ expression in control and pi 10γ deficient isolated cardiomyoctes. Note the smaller non-specific band.
E, Western blotting for phosphorylated-AKT/PKB, total AKT/PKB protein, p85, pi lOoc, and pi lOβ protein expression in total heart extracts. F, Representative heart sizes and heart/body weight ratios of 10 week old pi 10^' and pllOj1' littermates. Similar data, i.e, no change in heart size, were obtained at 6 and 12 months of age.
G, Western blot analysis of AKT/PKB phosphorylation and total AKT/PKB protein levels in hearts from individual pi 10y' mckCRE-PTENA/A a d mckCRE-PTENA/A pllOγ ' double mutant mice.
Figure 4. Heart sizes and functions in mckCRE-PTENΛ/Δ DN- pllOa mice
A, Representative heart sizes of 10 week old wild-type, DN-pll0c, and mckCRE- PTENA/A DN-pllOa littermates. B, Left ventricular mass (LNM; mean values +/- SEM) in hearts from 10 week old mckCRE-PTENA/A (n=8), wild-type (n=6), DN-pl 10a(n=4), and mckCRE-PTENA/A DN-pl 10a double transgenic (n=4). ** p < 0.01 between genetic groups.
C, Western blot analysis of AKT/PKB phosphorylation and total AKT/PKB protein levels in hearts from wild type (lane 1), DN-pllOa single transgenic (lane 2), and mckCRE-PTENA/A DN-pl 10a double transgenic (lane 3) mice.
D, M-mode echocardiographic images of contracting hearts in 10 weeks old wild type, mckCRE-PTENA A, and mckCRE-PTENA/A DN-pl 10a double transgenic mice.
E, Percent fractional shorting (%FS) and velocity of circumferential fibre shortening (Ncfs) in 10 weeks old mckCRE-PTENA/A (n=8), wild-type (n=6), DN-pl 10a (n=4), and mckCRE-PTENA/A DN-pl 10 αdouble transgenic (n=4) mice. Nalues (mean +/- SEM) were determined by echocardiography. ** p < 0.01 between groups.
Figure 5. Single cell contractility and cAMP production A, B, Basal contractility of single cardiomyocytes isolated from mckCRE-PTENA Aι wild-type, pi 10y ' , and mckCRE-PTENA/A pH0γ/" double mutant mice. In A, representative contractions from single cells are shown. In B, mean percentage shortening (+/- SEM) of at least 15 different single cells from 3 different hearts per genotype are shown in the presence and absence of thePDK inhibitor LY294002 [30μM]. ** p < 0.01 between groups.
C, Basal cAMP levels (mean +/- SEM) in purified cardiomyocytes from mckCRE- PTENA At wild-type, and pllOγ'' mice (n = 4 for each genotype). Samples were either left untreated (-) or treated with wortmannin (wort, +). * p < 0.05 between groups.
D, Activation of adenylate cyclase via forskolin [25μM] increases the levels of cAMP production (mean +/- SEM) in mckCRE-PTENA/A wild-type, and pllOj1' cardiomyocytes to a similar extent.
E, Inhibition of cAMP dependent functions via Rp-cAMPS [25 μM] reduces cell shorting (contractility) in single wild-type andpllOγ ' cardiomyocytes. Mean values
(+/- SEM) of percent shortening in the presence of Rp-cAMPS (left panel) and percent decrease in contractility using Rp-cAMPS are shown. ** p < 0.01 between groups.
Figure 6. PI3Kγ and β-adrenergic signaling
A, GRK kinase activities from fractionated heart lysates from individual mckCRE- PTENA + and mckCRE-PTENA/A mice were determined using in vitro kinase assays and rhodopsin as substrate. Since GRKs are recruited to membranes, data from cytosolic and membrane fractions are shown. C = control using recombinant GRK2. The lower panel shows a Western blot of total GRK2/3 expression levels in the hearts. B, β-AR densities were determined using ICYP as a ligand as described in Experimental Procedures (n = 7 for wild type; n = 5 for mckCRE-PTENA A and n = 5 fox pi 10^. C, Induction of cAMP production in response to the selective β2-adrenergic receptor agonist zinterol. Note that zinterol has no detectable effect on β2-AR induced cAMP levels in wild type cardiomyocytes (n = 5) but significantly increases cAMP levels in pllOf1" cardiomyocytes (n = 5). * p < 0.05 between groups. D, Induction of cAMP production in response to βl-adrenergic receptor stimulation. Both wild type (n = 4) and pi 1 Of1' cardiomyocytes (n = 4) respond equally to isoproterenol. β2-AR were blocked with pretreatment of ICI 118,551.
E, Western blot analysis of phosphorylated phospholamban (P-PLB) and total phospholamban (PLB) in wild-type and pi 10 '~ cardiomyocytes in response to different concentrations of the selective β2-AR agonist zinterol. C = control. Note the increased phosphorylation of PLB in unstimulated pllOf1' cardiomyocytes that mirrors increased basal cAMP levels in these cells. One representative experiment is shown.
Figure 7. a) Homo sapiens phosphoinositide-3 -kinase gamma nucleic acid sequence [SEQ ID NO: 1]; Accession no. AF327656; base count: 950 a 861 c 849 g 750 1 b) Homo sapiens phosphoinositide-3 -kinase gamma amino acid sequence[SEQ ID NO: 2].
Figure 8. a) Homo sapiens PTEN nucleic acid sequence[SEQ ID NO: 3]; Accession no. NM_000314; base count: 853 a 742 c 788 g 777 t; b) Homo sapiens PTEN amino acid sequence [SEQ ID NO: 4].
Figure 9. Signaling and cardiac hypertrophy responses pllOf' and pll Of1" mice. A) Heart sections from pllOf' and pi 10 f'" mice treated with a vehicle control or isoproterenol for 7 days. B) Quantitation of heart/body weight and heart weight/tibial length ratios in vehicle and isoproterenol treated mice; n=9 per group. *P<0.05; **P<0.01 compared with vehicle. C) Northern blot analysis and quantitation of cardiac hypertrophy markers mpll0γ+ " andpllO1 ' mice treated with vehicle (1) or isoproterenol (2) for 7 days. GAPDH expression levels were used as a loading control. It should be noted that the baseline levels of the indicated markers were comparable between p!10γ+ " and pllOf1' hearts. D) ERK1/2 and p38 phosphorylation in isolated pllOf1' wApllOf1' neonatal cardiomyocytes. Cells were (1) left untreated, or stimulated with (2) isoproterenol (lOμM) and (3) basic fibroblast growth factor (bFGF) (250 ng/ml) for 10 mins. Western blot data are shown. E) Impaired Akt/PKB activation in total heart extracts from pll0γ+/" and pll Of'' mice treated with vehicle control or isoproterenol for 7 days; n=5 per group. *P< 0.01 between all groups. Nalues are mean±SEM.
Figure 10. Loss of pl l Oγ protects from interstitial fibrosis and hypertrophy. A) Heart sections from p! 10γ+/+ and pi 10f'' mice treated with a vehicle control or isoproterenol for 7 days. Trichrome and picro sirius red (PSR) staining to detect remodeling and interstitial fibrosis are shown. B) Morphometric quantitation of fibrosis in vehicle and isoproterenol treated mice. n=5 per group. *P<0.01 compared with vehicle. C) Quantitation of apoptosis in vehicle and isoproterenol treated mice. n=3 per group with 3 sections from each heart. *P<0.05 compared with vehicle. D) Quantitation of cardiomyocyte size based on cross-sectional area in vehicle and isoproterenol treated mice. n=3 per group with 3 sections from each heart. *P<0.05; * *P<0.01 compared with vehicle. Nalues are mean±SEM.
Figure 11. Ventricular β-adrenergic receptor densities. A) Immunoblots on purified sarcolemmal preparations (PM). PMs were enriched in the specific markers such as Νa-K ATPase, as well as caveolin-1 and caveolin-3 (not shown). Immunoblot analysis was with anti-type N/NI adenylyl cyclase (upper panel, 20 mg protein loaded), anti-βt-AR (middle panel, 20 μg protein loaded) or anti-β2-AR (bottom panel, 100 mg protein loaded) antibodies or following antibody preblocking with antigen (+ Antigen). Protein recovery was similar for pi 10γ+/" and pll Of'' ventricles without or with isoproterenol treatment. B) Quantifications of adenylate cycle N and NI (ACN/NI), β adrenergic (βt-AR) and β2-adrenergic (β2-AR) receptor expression (n=6 per group). Western blots were scanned from film with a laser densitometer (Molecular Dynamics), and quantified using ImageQuant software (Molecular Dynamics). Nalues are mean±SEM. *P< 0.05.
DETAILED DESCRIPTION OF THE INVENTION
Modulation of PBKgamma has utility in treating a wide range of heart disease, such as heart attack and heart failure. Blocking PBKgamma protein activity or gene expression increases heart function, for example, by providing increased heart muscle contractility. Accordingly, the present invention includes all uses that relate to the realization of the modulatory properties of PBKgamma including, but not limited to, the development of therapeutic and diagnostic assays and compositions as well as the preparation and/or isolation of other molecules that inhibit PBKgamma.
I. Therapeutic Methods
(a) Preventing or Treating Heart Disease with small molecules, oligonucleotides or antibodies
In one aspect, the present invention provides a method of preventing or treating heart disease comprising administering an effective amount of an inhibitor of PBKgamma to an animal in need of such treatment. The invention includes a use of an effective amount of an inhibitor of PBKgamma for preventing or treating heart disease or to prepare a medicament for preventing or treating heart disease.
The term "PBKgamma" or "PBKγ" means phosphoinositide 3 -kinase gamma and includes any and all forms of this kinase from any source. The term includes biologically active fragments of PBγ. In a preferred embodiment, the PBγ includes the catalytic subunit pi lOgamma (pi lOγ).
Administration of an "effective amount" of the inhibitor of PBKgamma of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. The effective amount of the inhibitor of PBKgamma of the invention may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The term "animal" as used herein includes all members of the animal kingdom including mammals such as humans.
The term "treatment or treating" as used herein means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treating" can also mean prolonging survival as compared to expected survival if not receiving treatment.
The term "an inhibitor of PBKgamma" means any molecule or compound that can inhibit the expression of the PBKgamma gene or that can inhibit the activity of the PBKgamma protein. The inhibitor can be any type of molecule including, but not limited to, proteins (including antibodies), peptides, peptide mimetics, nucleic acids (including DNA, RNA, antisense oligonucleotides, peptide nucleic acids), carbohydrates, organic compounds, small molecules, natural products and library extracts. The inhibitors may be known or novel compounds, including derivatives of the known compounds.
Known inhibitors include wortmannin, Ly294002, 2-(4-morpholinyl)-8-phenyl-4H-l- benzopyran-4-one (LY294002) and quercetin (see US patent application no. 20020037276 (Ptasznik et al) and CA 2378650 (Forti)). Derivatives and analogues of these compounds are also useful.
In another embodiment, the PBKgamma inhibitor is an antibody that binds PBKgamma or an antisense oligonucleotide that inhibits the expression of PBKgamma. RNA that is complimentary to a nucleic acid sequence from a PBKgamma gene can be used in the methods of the present invention to inhibit PBKgamma.
Consequently, the present invention provides a method of preventing or treating heart disease comprising administering an effective amount of an antibody or antisense oligonucleotide to an animal in need thereof. Detailed discussion of oligonucleotides and antibodies follows.
RNA interference
RNA interference techniques, such as antisense RNA or siRNA are useful to block gene expression. Examples of antisense compounds for inhibition of PI3K expression are described in US patent application no. 20020058638 (Monia et al.). Nucleic acid sequences for RNA inhibition of PBKgamma could be made from [SEQ ID NO:l] or fragments thereof. The term "antisense oligonucleotide" as used herein means a nucleotide sequence that is complimentary to its target. siRNA, refers to double stranded RNA (dsRNA) which silences expression of genes having high identity to either of the strands in the RNA duplex (Fire et al. (1998) Nature 391, 806-811). dsRNA directs gene-specific, post-transcriptional silencing in many mammals through mRNA degradation. siRNA may include nucleic acids modified in the same manner as the oligonucleotide nucleic acids described below.
The term "oligonucleotide" refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases. The term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide.
The oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
Other oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. For example, the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates. In an embodiment of the invention there are phosphorothioate bonds links between the four to six 3'- terminus bases. In another embodiment phosphorothioate bonds link all the nucleotides.
The oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents. An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497). PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506). Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide. Oligonucleotides may also have sugar mimetics.
The nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. The nucleic acid molecules of the invention or a fragment thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides. The sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
Antibodies
In another embodiment, the inhibitor of PBKgamma is a PBKgamma specific antibody. Antibodies to PBKgamma may be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S. Patent Nos. RE 32,011; 4,902,614; 4,543,439; and 4,411,993, which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference). Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab')2) and recombinantly produced binding partners.
The therapeutic methods of the invention can be used to treat any condition wherein it is desirable to modulate PBKgamma expression or activity. Such conditions include heart disease caused by acute coronary syndromes (eg. congestive heart failure, angina, myocardial infarction (heart attack)), cardiac arrythmias (eg. atrial flutter, atrial fibrillation, paroxysmal supraventricular tachycardia) and hypertension (eg. idiopathic hypertrophic subaortic stenosis).
In one embodiment, the present invention provides a method of preventing or treating heart disease comprising administering an effective amount of an inhibitor of PBKgamma to the recipient animal. The invention includes a use of an effective amount of an inhibitor of PBKgamma to prevent or treat heart disease or to prepare a medicament to prevent or treat heart disease. The above described methods for preventing or treating heart disease using PBKgamma inhibitors may be further enhanced by co-administering other drugs, such as beta-blockers.
(b) Preventing or treating heart disease with competitively inhibiting protein fragments
In another aspect, the present invention provides a method of preventing or treating heart disease comprising administering an effective amount of a competitively inhibiting PBKgamma protein fragment or a nucleic acid sequence encoding a PBKgamma protein fragment to an animal in need thereof. The term "competitively inhibiting PBKgamma protein" includes an inactive full length PBKgamma protein as well as inactive fragments or portions of the protein, such as pl lOgamma, that competitively inhibit binding of PBKgamma to its substrates. Preferred fragments or portions of the protein are those that are sufficient to prevent or treat heart disease. The PBKgamma protein or the nucleic acid encoding the PBKgamma protein can be readily obtained by one of skill in the art. For example, many PBKgamma sequences are available in the GenBank database including the human PBKgamma [SEQ ID NO:2]. The PBKgamma protein or nucleic acid may be modified from the known sequences to make it more useful in the methods of the present invention. Competitive inhibitors, such as inactive peptide fragments, may be designed based on the pBKgamma, preferably pl lOgamma, sequence. One may also use polypeptides having sequence identity to a fragment of pBKgamma or pi 10. The invention also includes polypeptides which have sequence identity at least about: >20%, >25%, >28%, >30%, >35%, >40%, >50%, >60%, >70%, >80% or >90% more preferably at least about >95%, >99%, to a polypeptides fragment of the invention, such as a fragment of [SEQ ID NO:2]. Identity is calculated according to methods known in the art. Sequence identity is most preferably assessed by the BLAST version 2.1 program advanced search (parameters as above; Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403_410). BLAST is a series of programs that are available online at http://www.ncbi.nlm.nih.gov/BLAST. The advanced blast search (http ://www.ncbi.nlm.nih. go v/blast/blast. cgi? Jform= 1 is set to default parameters, (ie Matrix BLOSUM62; Gap existence cost 11 ; Per residue gap cost 1; Lambda ratio 0.85 default).
In one embodiment, the PBKgamma protein fragment is prepared as a soluble fusion protein. The fusion protein may contain a domain of PBKgamma linked to an immunoglobulin (Ig) Fc Region. The PBKgamma fusion may be prepared using techniques known in the art. Generally, a DNA sequence encoding the extracellular domain of PBKgamma is linked to a DNA sequence encoding the Fc of the Ig and expressed in an appropriate expression system where the PBKgamma - Fclg fusion protein is produced. The PBKgamma protein may be obtained from known sources or prepared using recombinant DNA techniques. The protein may have any of the known published sequences for PBKgamma. For example, the sequences can be obtained from GenBank as described above. The protein may also be modified to contain amino acid substitutions, insertions and/or deletions. Conserved amino acid substitutions involve replacing one or more amino acids of the PBKgamma amino acid sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics. Non-conserved substitutions involve replacing one or more amino acids of the PBKgamma amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.
The PBKgamma protein may be modified. For example, disulphide bonds may be formed between two appropriately spaced components having free sulfhydryl groups. The bonds may be formed between side chains of amino acids, non-amino acid components or a combination of the two. In addition, the PBKgamma protein or peptides of the present invention may be converted into pharmaceutical salts by reacting with inorganic acids including hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulphonic acid, and tolunesulphonic acids.
The present invention also includes peptide mimetics of the PBKgamma of the invention. "Peptide mimetics" are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of a peptide, or enhancer or inhibitor of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention.
Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D- amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.
Peptides of the invention may also be used to identify lead compounds for drug development. The structure of the peptides described herein can be readily determined by a number of methods such as NMR and X-ray crystallography. A comparison of the structures of peptides similar in sequence, but differing in the biological activities they elicit in target molecules can provide information about the structure-activity relationship of the target. Information obtained from the examination of structure-activity relationships can be used to design either modified peptides, or other small molecules or lead compounds that can be tested for predicted properties as related to the target molecule. The activity of the lead compounds can be evaluated using assays similar to those described herein. Information about structure-activity relationships may also be obtained from co- crystallization studies. In these studies, a peptide with a desired activity is crystallized in association with a target molecule, and the X-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison may be used to design compounds expected to possess.
II. Screening Assays
The present invention also includes the isolation and/or identification of substances to inhibit PBKgamma expression or activity for treatment of heart disease. Such substances or PBKgamma modulators may be useful in the above described therapeutic methods. Two examples of PBKgamma modulators include antibodies and antisense molecules which are described in detail above. Other PBKgamma modulators may be identified, for example, using the screening assays described below.
(a) Substances that Bind PBKgamma and are Useful to Treat Heart Disease
Substances that affect PBKgamma activity can be identified based on their ability to bind to PBKgamma.
Substances which can bind with the PBKgamma of the invention may be identified by reacting the PBKgamma with a substance which potentially binds to PBKgamma, and assaying for complexes, for free substance, or for non-complexed PBKgamma, or for activation of PBKgamma. In particular, a yeast two hybrid assay system may be used to identify proteins which interact with PBKgamma (Fields, S. and Song, O., 1989, Nature, 340:245-247). Systems of analysis which also may be used include ELISA. Accordingly, the invention provides a method of identifying substances which can bind with PBKgamma, comprising the steps of:
(a) reacting PBKgamma and a test substance, under conditions which allow for formation of a complex between the PBKgamma and the test substance, (b) assaying for complexes of PBKgamma and the test substance, for free substance or for non complexed PBKgamma, wherein the presence of complexes indicates that the test substance is capable of binding PBKgamma; and (c) determining whether the test substance is capable of increasing heart function, such as heart muscle contractility, and thereby prevent or treat heart disease.
Conditions which permit the formation of substance and PBKgamma complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
The substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against PBKgamma or the substance, or labeled PBKgamma, or a labeled substance may be utilized. The antibodies, proteins, or substances may be labeled with a detectable substance as described above.
The invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of PBKgamma. Thus, the invention may be used to assay for a substance that competes for the same binding site of PBKgamma.
(b) Drug Screening Methods
In accordance with one embodiment, the invention enables a method for screening candidate compounds for their ability to decrease the activity of a PBKgamma protein. The method comprises providing an assay system for assaying PBKgamma activity, assaying the activity in the presence or absence of the candidate or candidate compound and determining whether the compound has decreased PBKgamma activity. The method further comprises determining whether the compound is suitable for preventing or treating heart disease.
Accordingly, the present invention provides a method for identifying a compound that inhibits PBKgamma protein activity or expression comprising: (a) incubating a candidate compound with a PBKgamma protein or a nucleic acid encoding a PBKgamma protein;
(b) determining an amount of PBKgamma protein activity or expression and comparing with a control (i.e. in the absence of the test substance), wherein a change in the PBKgamma protein activity or expression as compared to the control indicates that the candidate compound has an effect on PBKgamma protein activity or expression; and
(c) determining whether the test substance is capable of increasing heart function, such as heart muscle contractility, and thereby prevent or treat heart disease.
In accordance with a further embodiment, the invention enables a method for screening candidate compounds for their ability to decrease expression of a PBKgamma protein. The method comprises putting a cell with a candidate compound, wherein the cell includes a regulatory region of a PBKgamma gene operably joined to a reporter gene coding region, and detecting a change in expression of the reporter gene.
In one embodiment, the present invention enables culture systems in which cell lines which express the PBKgamma gene, and thus PBKgamma protein products, are incubated with candidate compounds to test their effects on PBKgamma expression. Such culture systems can be used to identify compounds which downregulate PBKgamma expression or its function, through the interaction with other proteins.
Such compounds can be selected from protein compounds, chemicals and various drugs that are added to the culture medium. After a period of incubation in the presence of a selected candidate compound(s), the expression of PBKgamma can be examined by quantifying the levels of PBKgamma mRNA using standard Northern blotting procedure to determine any changes in expression as a result of the candidate compound. Cell lines transfected with constructs expressing PBKgamma can also be used to test the function of compounds developed to modify the protein expression. In addition, transformed cell lines expressing a normal PBKgamma protein could be mutagenized by the use of mutagenizing agents to produce an altered phenotype in which the role of mutated PBKgamma can be studied in order to study structure/function relationships of the protein products and their physiological effects.
Accordingly, the present invention provides a method for identifying a compound that affects the binding of a PBKgamma protein and a PBKgamma binding protein comprising:
(a) incubating (i) a candidate compound; (ii) a PBKgamma protein and (iii) a PBKgamma binding protein under conditions which permit the binding of PBKgamma protein to the PBKgamma binding protein; and (b) assaying for complexes of PBKgamma protein and the PBKgamma binding protein and comparing to a control (i.e. in the absence of the test substance), wherein a reduction of complexes indicates that the compound has an effect on the binding of the PBKgamma protein to a PBKgamma binding protein; (c) determining whether the test substance is capable of increasing heart function, such as heart muscle contractility, and thereby prevent or treat heart disease.
All testing for novel drug development is well suited to defined cell culture systems which can be manipulated to express PBKgamma and study the result of PBKgamma protein modulation. Animal models are also important for testing novel drugs and thus may also be used to identify any potentially useful compound affecting PBKgamma expression and activity and thus physiological function.
III. Compositions
The invention also includes pharmaceutical compositions containing substances that inhibit PBKgamma inhibitors for use in heart disease as well. Substances that inhibit PBKgamma include substances that inhibit PBKgamma gene expression as well as substances that inhibit PBKgamma activity. Such substances include antisense molecules to PBKgamma, antibodies to PBKgamma as well as other substances or PBKgamma antagonists identified using the screening assays described herein. Such pharmaceutical compositions can be for intralesional, intravenous, topical, rectal, parenteral, local, inhalant or subcutaneous, intradermal, intramuscular, intrathecal, transperitoneal, oral, and intracerebral use. The composition can be in liquid, solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions.
The pharmaceutical compositions of the invention can be intended for administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration. The pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
On this basis, the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The pharmaceutical compositions may additionally contain other agents to prevent or treat heart disease.
In one embodiment, the pharmaceutical composition for use in preventing or treating heart disease comprises an effective amount of an inhibitor of PBKgamma in admixture with a pharmaceutically acceptable diluent or carrier. The PBKgamma inhibitor is optionally an antisense oligonucleotide to PBKgamma or an antibody that binds to PBKgamma.
Pharmaceutical compositions comprising antisense nucleic acid molecules may be directly introduced into cells or tissues in vivo using delivery vehicles such as retro viral vectors, adeno viral vectors and DNA virus vectors. They may also be introduced into cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. Recombinant molecules may also be delivered in the form of an aerosol or by lavage. The nucleic acid molecules of the invention may also be applied extracellularly such as by direct injection into cells.
In another aspect, the pharmaceutical composition for use in preventing or treating heart disease comprises an effective amount of a PBKgamma protein or a nucleic acid encoding a PBKgamma protein in admixture with a pharmaceutically acceptable diluent or carrier.
IV. Diagnostic Assays
The finding that PBKgamma is involved in heart disease allows the detection of conditions involving an increase in PBKgamma activity or expression resulting in heart disease.
Accordingly, the present invention provides a method of detecting a condition associated with increased PBKgamma expression or activity comprising assaying a sample for (a) a nucleic acid molecule encoding a PBKgamma protein or a fragment thereof or (b) a PBKgamma protein or a fragment thereof and comparing the amount of PBKgamma nucleic acid or protein detected with a suitable control.
(a) Nucleic acid molecules
Nucleotide probes can be prepared based on the sequence of PBKgamma for use in the detection of nucleotide sequences encoding PBKgamma or fragments thereof in samples, preferably biological samples such as cells, tissues and bodily fluids. The probes can be useful in detecting the presence of a condition associated with PBKgamma or monitoring the progress of such a condition. Accordingly, the present invention provides a method for detecting a nucleic acid molecules encoding PBKgamma comprising contacting the sample with a nucleotide probe capable of hybridizing with the nucleic acid molecule to form a hybridization product, under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.
A nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32P, 3H, 14C or the like. Other detectable substances which may be used include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and chemiluminescence. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleic acid to be detected and the amount of nucleic acid available for hybridization. Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The nucleotide probes may be used to detect genes, preferably in human cells, that hybridize to the nucleic acid molecule of the present invention preferably, nucleic acid molecules which hybridize to the nucleic acid molecule encoding PBKgamma under stringent hybridization conditions as described herein.
Nucleic acid molecules encoding a PBKgamma protein can be selectively amplified in a sample using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleotide sequence of PBKgamma for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using oligonucleotide primers and standard PCR amplification techniques. The amplified nucleic acid can be cloned into an appropriate vector and characterized by DNA sequence analysis. cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al, Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FL).
(b) Proteins
The PBKgamma protein may be detected in a sample using antibodies that bind to the protein as described in detail above. Accordingly, the present invention provides a method for detecting a PBKgamma protein comprising contacting the sample with an antibody that binds to PBKgamma which is capable of being detected after it becomes bound to the PBKgamma in the sample. Antibodies specifically reactive with PBKgamma, or derivatives thereof, such as enzyme conjugates or labeled derivatives, may be used to detect PBKgamma in various biological materials, for example they may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of PBKgamma, and the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination and histochemical tests. Thus, the antibodies may be used to detect and quantify PBKgamma in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.
In particular, the antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect PBKgamma, to localise it to particular cells and tissues and to specific subcellular locations, and to quantitate the level of expression. Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect PBKgamma. Generally, an antibody of the invention may be labelled with a detectable substance and PBKgamma may be localised in tissue based upon the presence of the detectable substance. Examples of detectable substances include various enzymes, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, biotin, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include radioactive iodine 1-125, 1-131 or 3-H. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualised by electron microscopy.
Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against PBKgamma. By way of example, if the antibody having specificity against PBKgamma is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
Where a radioactive label is used as a detectable substance, PBKgamma may be localized by autoradiography . The results of autoradiography may be quantitated by determining the density of particles in the autoradiographs by various optical methods, or by counting the grains.
Other Modulation of PBKgamma and PTEN
One may also activate PBKgamma or block PTEN for excessive heart contractility. The invention includes a method of treating heart disease, such as excessive heart contractility, in a mammal in need thereof by administering an agent that activates PBKgamma or blocks PTEN. RNA compounds, antibodies and other molecules may be used to activate PBKgamma or block PTEN. Such compounds may be prepared as described in this application. For example, PTEN may be blocked by using nucleic acids based on [SEQ ID NO:3] or competitive inhibitors based on [SEQ ID NO:4].
The invention also includes the use of an inhibitor of PTEN or an activator of PBKgamma to prepare a medicament to prevent or treat heart disease, such as excessive contractility. The inhibitor of PTEN may comprise inactive PTEN, an inactive fragment of PTEN, an antibody to PTEN or a nucleic acid that inhibits the expression of PTEN, for example by RNA interference. The activator of PBK could include an antibody or other molecule. The invention also includes a pharmaceutical composition for use in preventing or treating heart disease, such as excessive contractility, comprising an inhibitor of PTEN or activator of PIK3 gamma and a carrier.
The invention also includes a method for identifying a compound that activates the binding of a PBKgamma protein to its substrate for treatment of heart disease, such as increased contractility, comprising: (a) incubating (i) a candidate compound; (ii) an PBKgamma protein and (iii) an PBKgamma substrate under conditions which permit the binding of PBKgamma protein to the substrate; and
(b) assaying for complexes of PBKgamma protein and the substrate or metabolites thereby produced and comparing to a control, wherein an increase of complexes or metabolites indicates that the candidate compound has an effect on the binding of the PBKgamma protein to the substrate;
(c) determining whether the candidate compound is useful for treatment of heart disease.
The invention also includes a compound identified by this method.
The invention also includes a method for identifying a compound that inhibits the binding of a PTEN protein to its substrate for treatment of heart disease, such as increased contractility, comprising: (d) incubating (i) a candidate compound; (ii) an PTEN protein and (iii) an
PTEN substrate under conditions which permit the binding of PTEN protein to the substrate; and
(e) assaying for complexes of PTEN protein and the substrate or metabolites thereby produced and comparing to a control, wherein a reduction of complexes or metabolites indicates that the candidate compound has an effect on the binding of the PTEN protein to the substrate;
(f) determining whether the candidate compound is useful for treatment of heart disease, such as increased contractility. The invention also includes a compound identified by this method.
Inhibition of PBK and/or activation of PTEN blocks cardiac hypertrophy. The invention also includes the use of an inhibitor of PBK or an activator of PTEN to prepare a medicament to prevent or treat heart disease, such as excessive contractility or hypertrophy. The inhibitor of PBK may comprise inactive PBK, an inactive fragment of PBK, an antibody to PBK or a nucleic acid that inhibits the expression of PBK, for example by RNA interference. The activator of PTEN could include an antibody or other molecule. The invention also includes a pharmaceutical composition for use in preventing or treating heart disease, such as excessive contractility, comprising an inhibitor of PBK or activator of PTEN and a carrier. The invention also includes a method for identifying a compound that activates the binding of a PTEN protein to its substrate for treatment of heart disease, such as hypertrophy, comprising:
(g) incubating (i) a candidate compound; (ii) a PTEN protein and (iii) a PTEN substrate under conditions which permit the binding of PTEN protein to the substrate; and (h) assaying for complexes of PTEN protein and the substrate or metabolites thereby produced and comparing to a control, wherein an increase of complexes or metabolites indicates that the candidate compound has an effect on the binding of the PTEN protein to the substrate; (i) determining whether the candidate compound is useful for treatment of heart disease, such as hypertrophy.
The invention also includes a compound identified by this method.
The invention also includes a method for identifying a compound that inhibits the binding of a PBK protein to its substrate for treatment of heart disease, such as or hypertrophy, comprising:
(j) incubating (i) a candidate compound; (ii) a PBK protein and (iii) a PBK substrate under conditions which permit the binding of PBK protein to the substrate; and (k) assaying for complexes of PBK protein and the substrate or metabolites thereby produced and comparing to a control, wherein a reduction of complexes or metabolites indicates that the candidate compound has an effect on the binding of the PBK protein to the substrate; (1) determining whether the candidate compound is useful for treatment of heart disease, such hypertrophy.
The invention also includes a compound identified by this method. One may also modulate hypertrophy in a mammal in need thereof by administering an agent that modulates PTEN or PBKgamma. The invention includes a method of treating heart disease, such as hypertrophy, in a mammal in need thereof, by administering an agent that activates PTEN or inhibits PBKgamma. Peptide fragments, antibodies, RNA and other compounds described in this application are useful for the above methods of medical treatment. The above diagnostic methods and other methods of the invention described in the application may also be adapted for use with the methods described in this section.
EXAMPLES
Example 1
Experimental Results
Generation of cardiac muscle specific PTEN knockout mice
Inactivation of PTEN in all cells results in early embryonic lethality between day 6.5 to 9.5 (Suzuki et al, 1998; Di Cristofano et al, 1998). We therefore used a tissue specific gene targeting approach to mutate PTEN in all cardiomyocytes and skeletal muscle cells. Mice that have exon 4 and 5 of PTEN flanked by loxP sites by homologous recombination (PTENflox) (Suzuki et al, 2001), were mated with a CRE deleter line that expresses the CRE transgene under the control of the muscle creatinine kinase promoter (mckCRE) (Bruning et al, 1998). We generated homozygous mutant mice that express CRE and are floxed at both PTEN alleles (mckCRE-PTEN^), heterozygous mice that express CRE but are only floxed at one PTEN allele (mckCRE-PTEN^), and control mice that do not express mckCRE but have both PTEN alleles floxed (PTENflox flox). No phenotypic differences between mckCRE-PTEN^ and pχENflox/flox genotypes were observed. Western blotting showed a significant reduction in the amount of PTEN protein in both the heart as well as skeletal muscle, but no other tissues, of mckCRE-PTEN^ mice (Figure 1A). The presence of residual PTEN protein in mckCRE-PTEN^ mice is due to PTEN expression in cardiac non-myocyte cell types such as endothelial cells or fibroblasts. Hearts from mckCRE-PTEN^ mice showed no alteration in the expression of PBKα, PBKβ, PBKγ catalytic subunits nor changes in p85α expression (Figure IB).
Spontaneous cardiac hypertrophy in PTEN-deficient hearts
Analysis of skeletal muscle from mckCRE-PTENΔ/Δ mice showed no gross abnormalities or any overt changes in cell size compared to control mckCRE-PTENΔ + or pτ/ENflox flox littermates. Overall body weights were comparable between all three genotypes at all ages analyzed (10 wks, 6 months and 12 months). Moreover, there was no indication of diabetes and blood glucose levels did not differ between mckCRE-PTEN^, mckCRE-PTEN^, and PTENflox flox littermates.
Whereas skeletal muscle appeared normal, loss of PTEN in cardiac muscle resulted in greatly increased heart sizes in the mutant mckCRE-PTENΔ Amice (Figure 1C, D). Concordantly, there was a significant increase in heart/body weight ratios in the PTEN-deficient hearts indicative of cardiac hypertrophy at 10 weeks and 12 months of age (Figure 2A). The increased heart size was already detectable in newborn mckCRE-PTENΔ/Δmice. Histologically, the increase in heart size was observed throughout the heart (Figure ID). Interestingly, cardiac hypertrophy did not result in fibrotic or structural changes in myocyte organization (Figure ID) or perturbations in cardiomyocyte cell death even at 1 year of age showing that loss of PTEN does not result in cardiac decompensation or dilated cardiomyopathy.
To further show the cardiac hypertrophy in PTEN-deficient hearts, cardiomyocytes were isolated from adult mice to assess the individual cell size. Loss of PTEN resulted in a marked increase in both the length and width of cardiomyocytes as compared to controls (Figure 2B). The length-to-width ratio was unchanged in these cells showing that the cell size change was similar to that observed in physiologic hypertrophy (Hunter and Chien, 1999). Pathological cardiac hypertrophy is characterized by a prototypical change in gene expression patterns such as increased ANF, βMyHC, BNP, and skeletal-actin expression, and a decrease in αMyHC (Hunter and Chien, 1999). However, no significant alterations in the expression profile of these hypertrophic genes were observed with the exception of an increase in βMyHC (Figure 2C, D). Thus, loss of PTEN in the myocardium induces hypertrophy without the typical pathological changes in the gene expression profiles or cardiac decompensation.
Activation of PI3K signaling in PTEN-deficient hearts
Since PTEN antagonizes the action of PBK, we showed that the loss of PTEN in the hearts results in the activation of downstream targets of PBK signaling (Downward, 1998). Loss of PTEN resulted in a significant increase in the basal phosphorylation state of AKT/PKB (Figure 2E, F). In addition, we found an increase in the phosphorylation of GSK3β and p70S6 , both downstream targets of PBK signaling (Figure 2E, F). No apparent changes in the basal activation of ERK1 ERK2 were observed between the mutant mckCRE-PTEN^mice and their mckCRE-PTENΔ + and PTEN+ + littermates (Figure 2E, F). Loss of PTEN in cardiomyocytes results in increased PBK signaling leading to activation and phosphorylation of multiple PBK target molecules.
Loss of PTEN in the heart results in decreased cardiac contractility
To further show characterize the role of PTEN in mutant mckCRE-PTEN1^ mice, we analyzed the hearts using echocardiography. Consistent with the physiological cardiac hypertrophy, echocardiography of PTEN-deficient hearts revealed an increase in the anterior wall thickness and an increase of the left ventricle mass (LNM) at all ages analyzed (Figure 3A and Table 1). Moreover, the change in end diastolic dimension of the left ventricle (LNEDD) reflected this overall enlargement of PTEΝ-deficient heart. No difference in heart rate was detected in the PTEN-deficient hearts (Table 1).
Surprisingly, assessment of cardiac function by echocardiography revealed that mutant mckCRE-PTEN^mice exhibited a dramatic reduction in cardiac contractility characterized by decreased fractional shortening (FS), decreased velocity of circumferential fibre shortening (Ncfc) and reduced peak aortic outflow velocity (PAN) (Figure 3B,C and Table 1). To confirm the echocardiographic alterations, functional invasive hemodynamic measurements were performed. Our hemodynamic measurements showed that both dP/dT-max and dP/dT-min were markedly reduced in the PTEN-mutant mice (Table 1), again indicating severe impairment of contractile heart function. Despite marked reductions in contractility at 10 weeks, there was no further decrease in the function of the PTEN-deficient hearts analyzed at 12 months of age (Table 1). In addition, there was no evidence of dilation, wall thinning or tissue fibrosis in older animals (Figure ID, Table 1). PTEN controls cardiomyocyte size and heart muscle contractility. Increased contractility in PI3Kγ mutant mice
The decrease in cardiac contractility found in the PTEN-mutant hearts suggested that increased PBK signaling serves not only to increase cell size in the heart but to also suppress basal heart function. Neither the pl lOα dominant negative nor pl lOα constitutively active heart specific transgenic animals showed any alteration in heart function (Shioi et al, 2000) suggesting that the effect on contractility is mediated by PBK isoforms other than PBKα. Several GPCR signaling pathways, including adrenergic receptors (Rockman et al, 2002), are important mediators of cardiac function. Decreased heart function in PTEN mutant mice can be mediated by PBKγ. We observed pllOγ protein expression in total heart extracts of mice (Figure IB) and in isolated cardiomyocytes (Figure 3D). Loss of pllOγ expression does not alter the expression levels of p85, pl lOα, and pl lOβ (Figure 3E). Moreover, p85 associated PBK activity in cardiomyocytes was comparable between p 110γ" " and wild type littermates using in vitro lipid kinase assays (not shown). There was also no apparent difference in the expression or phosphorylation of AKT/PKB (Figure 3E). Mice deficient for pi lOγ were found to have no alteration in heart size or left ventricle mass (LNM) and displayed normal heart rates (Figure 3 A, F and Table 1). No structural alterations were observed in the hearts of pi 10γ" " mice using histological analyses and there were no changes in the gene expression profiles of the cardiac hypertrophy markers ANF, βMyHC, BNP, skeletal-actin, and MyHC. Thus, whereas loss of PTEN results in cardiac hypertrophy, P13Kγ has no role in the control of basal cardiomyocyte size.
The hearts of pl lOγ deficient mice displayed a marked enhancement in contractility as assessed by increased fractional shortening (FS), Ncfc, and peak aortic outflow velocity (PAN) (Figure 3B,C and Table 1). Hemodynamic measurements confirmed the increased heart contractility in pl lOγ" " mice (Table 1). Telemetry analysis of blood pressure in conscious mice revealed normal blood pressure in pi lOγ" " mice. In addition, plasma epinephrine and norepinephrin levels were not altered in pl lOγ" " mice compared to controls (norepinephrin (59.63 ± 9.99 in wild type (n=6) versus 61.28 ± 8.82 in pl lOγ"7" mice (n=6), and epinephrin (4.65 ± 0.80 in wild type (n=6) versus 4.28 ± 0.55 in pi 10γ_/" mice (ιv=6)).
To show the long term effects of this increase in cardiac function in pllOγ deficient mice, we analyzed heart function by echocardiography in 1 year old mice. Similar to that observed in younger mice, there remained an increase in cardiac contractility (fractional shortening (48.49 ± 0.76 in wild type (n=6) vs. 55.16 ± 0.33 in pl lOγ'" mice (n=6), and Ncfc (8.65 ± 0.20 in wild type (n=6) versus 10.86 ± 0.17 in pllOγ'" mice (n=6)). To ensure that the increase in cardiac contractility observed in the pi lOγ was due to the disruption of the gene and not an aberrant gene targeting event, we analyzed heart function in a second independently generated mouse line deficient for pl lOγ (Hirsch et al, 2000). All alterations in heart functions analyzed were similar between the two independent pl lOγ deficient mouse lines. Specifically, the second pi lOγ mouse line (Hirsch et al, 2000) had increased contractility as assessed by fractional shortening (48.07 ± 0.68 in wild type (n=6) vs. 56.47 ± 0.87 in pl lOγ"'" mice (n=6), and Ncfc (8.62 ± 0.16 in wild type (n=6) versus 11.35 ± 0.28 in pl lOγ'" mice (n=6)). Moreover, hypercontractility persisted (Ncfc: 8.65 ± 0.2 in wild type versus 10.86 ± 0.17 in pl lOγ" mice; n= 6; pO.Ol) and there was no evidence of cardiac hypertrophy or increased myocardial fibrosis in 1 year old mice. Thus, loss of PBKγ results in a marked increase in cardiac contractility.
PI3Kγ mediates reduction of heart function in PTEΝ-deficient hearts
The alterations in heart muscle contractility in pi lOγ- and PTEΝ-mutant hearts were exactly opposite, that is loss of PTEΝ resulted in decreased heart functions whereas loss of pi lOγ leads to a marked enhancement in cardiac contractility. To show that the role of PTEΝ in regulating cell size and heart function could be genetically uncoupled, we generated mice deficient for both pi lOγ and PTEΝ in the heart. Hearts from mckCRE-PTEN pllOγ7" double mutant mice were significantly enlarged as compared to wild type and pllOγ"7" mutant mice (Figure 3 A, Table 1). The extent of cardiac hypertrophy in mckCRE-PTEN^ pi 10γ" _ double mutant mice was similar to that observed for hearts from mckCRE-PTENΛ Δsingle mutants (Figure 3 A). Histologically, hearts from mckCRE-PTEN^ pi lOγ" " double mutant mice were also similar to that of mckCRE-PTENΔ A single mutant mice. Importantly, similar to PTEN-deficient hearts, AKT/PKB was hyperphosphorylated in the hearts of mckCRE-PTEN^pllOγ " double mutant mice (Figure 3G). Thus, loss of PBKγ in PTEN-deficient hearts has no affect on the increased AKT/PKB phosphorylation and does not rescue the cardiac hypertrophy phenotype.
However, unlike PTEN-deficient hearts, mckCRE-PTENΔ ΔpllOγ"/" double mutant hearts were found to be hypercontractile, as assessed by increased fractional shortening (FS), Ncfc, and peak aortic outflow velocity (PAN) (Figure 3B,C, Table 1). These improved heart functions were similar to that of pi lOγ single mutant hearts. The defect in cardiac contractility seen in the PTEΝ-deficient hearts is dependent on the PBKγ signaling pathway. Importantly, PTEΝ regulated hypertrophy can be genetically uncoupled from the cardiac contractility changes mediated by PTEΝ- PBKγ.
Reversal of cardiac hypertrophy in PTEΝ-deficient hearts by a dominant-negative pllOα transgene
The increase in cell size found in PTEΝ-deficient hearts strongly resembles that seen in transgenic mice for heart specific constitutively active pllOα that couples to receptor tyrosine kinases (Shioi et al, 2000). Furthermore, mice transgenic for heart- specific dominant negative pllOα (DΝ-pllOa) have smaller than normal heart size. We therefore generated mice deficient for PTEΝ in the hearts and overexpressing the heart specific DΝ-pllOα transgene. Hearts of mckCRE-PTEΝA Δ; DN-pl 10a mice displayed a reduction in heart size similar to that seen in single DN-pl 10a transgenic mice (Fig. 4A,B). The presence of the DN-pl 10a transgene also correlated with a decrease in the basal level of AKT/PKB activation in the absence or presence of a functional PTEN gene (Figure 4C). However, whereas overexpression of DN-pl 10a on a wild type background affects heart size but has no apparent effect on heart function, hearts from mckCRE-PTENΛ Δ ; DN-pl 10a displayed a marked decrease in contractility (Figure 4D, E and Table 1). PBKα signaling modulates basal cell size of cardiomyocytes while PBKγ signaling controls basal cardiac contractility.
PI3Kγ and PTEN control contractility in single cells
To further analyse these in vivo contractility defects, we showed that loss of PTEN and PBKγ can affect contractility of a single adult cardiomyocyte in vitro. In agreement with both the in vivo echocardiographic and hemodynamic data, it was found that PTEN deficient cardiomyocytes display a reduction in contractility as determined by a reduction in percent cell shortening and positive and negative dL/dTs as compared to wild type cardiomycoytes (Figure 5A,B; Table 2). Conversely, pl lOγ deficient myocytes displayed an increase in contractility (Figure 5A,B; Table 2). Cardiomyocytes deficient for both PTEN and pi lOγ display increased contractility despite the hypertrophy present in these cells (Figure 5A,B; Table 2). Moreover, treatment of wild type myocytes with the PBK inhibitor LY294002 lead to a significant increase in cardiomyocyte contractility. In pllOγ deficient cardiomyocytes addition of LY294002 had no effect on the already increased contractility (Figure 5B; Table 2). These in vitro data show that independent of exogenous agonists PTEN and PBKγ control basal cardiomyocyte contractility within a single cell.
Other inhibitors described in this application also trigger the same result as that obtained in the cells (KO cells) with wortmannin and LY294002.
PI3Ky modulates baseline cAMP levels in cardiomyocytes
One intriguing aspect of the in vitro contractility data is that there was a significant increase in the rate of relaxation in pi lOγ deficient cardiomyocytes compared to control cells despite a marked increase in peak contraction (Table 2). Cardiac relaxation is modulated via cAMP dependent pathways, where cAMP activates PKA which then inhibits phospholamban (PLB) leading to increased sarcaplasmic reticulum calcium ATPase (SERCA) activity (Brittsan and Kranias, 2000). Moreover, it has previously been shown in vascular smooth muscle cells that pharmacological inhibition of PBK can lead to increases in cAMP levels (Komalavilas et al, 2001). To show how alterations in cAMP levels can be contributing to the phenotype observed here, we measured baseline cAMP levels in isolated myocytes. In unstimulated PTEN-deficient cardiomyocytes, cAMP levels were significantly reduced (Figure 5C). Conversely, an increase in cAMP levels was detected in isolated cardiomyocytes deficient for pi lOγ (Figure 5C). Treatment of PTEN-deficient and wild-type cardiomyocytes with the PBK inhibitor Wortmannin resulted in a marked increase of cAMP to levels observed in pi lOγ deficient cells (Figure 5C). Induction of cAMP production in response to the adenylate cyclase agonist forskolin was comparable among all genotypes analyzed (Figure 5D) showing that the basal machinery for cAMP production is operational in all genotypes. PBKγ and PTEN modulate cAMP production in cardiomyocytes.
To show how the alterations in cAMP levels contribute to altered contractility, we measured contractility in single cells following blockade of cAMP-dependent activation of downstream targets using Rp-cAMP. In wild type adult mouse cardiomyocytes, treatment with Rp-cAMP lead to a marked decrease in contractility
(Figure 5E; Table 2). Importantly, treatment of pl lOγ deficient cardiomyocytes with the cAMP blocker lead to a greater reduction in contractility as compared to wild type cells such that the difference in the resultant contractility of wild type and pl lOγ deficient cells treated with Rp-cAMP was greatly reduced compared to untreated cells
(Figure 5E; Table 2). In addition, the rate of relaxation in pl lOγ deficient cardiomyocytes treated with Rp-cAMP was reduced to wildtype levels (Table 2).
Alterations in baseline cAMP levels contribute to the increased contractility in pi lOγ" '" hearts.
PI3Kγ and β-adrenergic signaling
It has been previously shown that PBKγ can bind to GPCR-kinase 2 (GRK2) (Naga
Prasad et al, 2001a), a kinase that mediates desensitization of GPCRs like β- adrenergic receptors (β-AR) (Lefkowitz, 1998). Thus, β-adrenergic signaling may be affected in pi 10γ_/" hearts, and alterations of PBK activity may affect GRK activity and/or β-AR expression. Using an in vitro kinase assay, GRK activity in both cytosolic and membrane-associated fractions of PTEN-deficient hearts was comparable to that of control hearts (Figure 6A). Total protein levels of GRK2 were also comparable (Figure 6A). Radiolabeled ligand binding assays showed that there is also no detectable difference in the density of β-AR receptors or their affinity for the β-AR ligand ICYP in the PTEN- or pllOγ-deficient hearts as compared to wild-type mice (Figure 6B). These data imply that changes in contractility of PTEN and pi lOγ- deficient hearts are not due to alterations in baseline β-adrenergic receptor levels or the modulation of GRK activity. The G-protein coupled β2-adrenergic receptors (β2-AR) are linked to both G s and God G-proteins that can either increase (G s) or decrease (Gαi) cAMP levels and heart muscle contractility (Kuznetsov et al, 1995; Xiao et al, 1999; Rockman et al, 2002). To determine if PBKγ can modulate cAMP production following GPCR stimulation, we purified neonatal cardiomyocytes from both wild type and pl lOγ" " hearts and stimulated these cells with the β2-AR selective agonist zinterol. As described previosuly for adult rat myocytes (Kuznetsov et al, 1995), stimulation of the β2-AR had no affect on cAMP levels in wild type mouse cardiomyoctes. By contrast, selective stimulation of the β2-AR in pl lOγ" " cardiomyocytes resulted in a marked increase in cAMP levels (Figure 6C). Conversely, stimulation of β1-AR receptors resulted in a similar increase in cAMP levels in both wild type and pl lOγ-deficient cardiomyocytes demonstrating selectivity for the action of PBKγ on cAMP production (Figure 6D).
In cardiomyocytes, increased cAMP levels result in enhanced contractility via activation of PKA and subsequent phosphorylation of phospholamban (PLB) in the sarcoplasmic reticulum (Brittsan and Kranias, 2000). Similar to cAMP production, no alteration of PLB phosphorylation could be detected in wild type cells upon stimulation with the β2-AR agonist zinterol (Figure 6E). However, when pi 10 f cardiomyocytes were stimulated with the same β2-AR agonist, a dose dependent increase in PLB phosphorylation was observed. Thus, in addition to its role in baseline cardiac contractility, loss of pll 0γ expression releases the inhibition of the β2-AR thereby permitting receptor-induced cAMP production and subsequent PLB- phosphorylation.
The invention shows the roles of PBK-PTEN signaling in cardiac hypertrophy and heart functions in PTEN-heart muscle specific mutant mice, pl lOγ knock-out, dominant-negative pl lOα transgenic mice, and double-mutant mice. The tyrosine kinase-receptor pl lOα-PTEN pathway is a critical regulator of cardiac cell size. The PTEN-PBKγ signaling pathway regulates heart muscle contractility via GPCRs. This contractility phenotype is present in single cardiomyocytes and dependent on cAMP signaling. PBKγ regulates cardiac function by negatively regulating cAMP levels and phospholamban phosphorylation upon β2-adrenergic receptor stimulation. These data show that the tumor suppressor PTEN has an important in vivo role in GPCR signaling and that PTEN and PBKγ signaling control heart function.
PTEN and heart muscle size
The cardiac hypertrophy in PTEN deficient hearts is similar to that in mice overexpressing a constitutively active pi 10a transgene in the heart (Shioi et al, 2000). Like these transgenic mice, the hypertrophy found in PTEN deficient hearts displayed features characteristic of physiological hypertrophy, such as increase in both the length and width of the myocytes, no fibrotic changes and no decompensation into dilated cardiomyopathy (Hunter and Chien, 1999). The reversal of heart size in the PTEN deficient hearts that expressed a dominant negative PBKα transgene indicates that it is this isoform of PBK that modulates basal cell size in cardiomyocytes. Moreover, it does not appear that any other PBK isoform can compensate for the loss of PBKα function in vivo. It should be pointed out that it is possible that this transgene interferes with not only pl lOα, but also pl lOβ and pl lOδ as all of these require p85 activity which is likely sequestered as a result of the transgene expression. Nevertheless, there is a differential action of class la and class lb PBKs as it is unlikely that this transgene interferes with pllOγ function. This is further supported by the absence of any observable alteration in cardiac contractility in mice expressing the DN-pl 10a transgene. Previously, PBK signaling has been implicated in cell size regulation in other tissues and organisms such as drosophila (Scanga et al, 2000). Mice deficient for p70S6K have reduced body size due to a reduction in cell size (Shima et al, 1998). Recently, brain specific disruptions of PTEN have been reported which result in the increase in neuronal cell size (Backman et al, 2001). Moreover, transgenic expression of activated AKT/PKB in mouse heart leads to a hypertrophic phenotype and this phenotype can be inhibited with a DN-pl 10a transgene (Shioi et al, 2002; Matsui et al, 2002). This is consistent with our data on the differential modulation of AKT/PKB phosphorylation in PTEN but not PBKγ mutant hearts. Thus, it appears that AKT/PKB and p70S6K signaling plays an important role in the regulation of basal cardiomyocyte size in vivo.
Our results show that PBKγ has no role in the homeostatic control of cardiomyocyte cell size. Yet many agonists used to induce PI3K-dependent cardiomyocyte hypertrophy in vitro are GPCR agonists (Rabkin et al, 1997; Schluter et al, 1999). Moreover, PBKγ is activated in pressure overload mduced hypertrophy in vivo (Naga Prasad et al, 2000), implying a role for PBKγ in agonist-induced cardiac hypertrophy. We determine the role of PBKγ in agonist-induced hypertrophy in vivo following stimulation of GPCRs.
Regulation of heart muscle contractility by PI3Kγ and PTEN
PTEN deficient hearts showed cardiac hypertrophy and surprisingly also displayed a dramatic decrease in cardiac contractility. Conversely, PBKγ deficient hearts exhibited an increase in cardiac contractility. PBKγ-PTEN signaling controls heart muscle function in vivo. PBKγ can modulate contractility in the absence of exogenous agonists. These contractility changes are at least in part mediated by alteration in cAMP levels.
Most likely the phenotypes observed here are due to the lipid kinase/phosphatase activity of these enzymes. Hearts deficient in both PTEN and PBKγ also displayed increased cardiac contractility showing that increased PIP3 generated via PBKγ causes the decrease in cardiac contractility seen in the PTEN deficient hearts. Moreover, the different receptors recruit different downstream targets for the same phospholipids. β2 adrenergic receptors are coupled to both Gas and Gαi signaling (Rockman et al., 2002). The opposing effects of these two G protein subunits result in no global net effect on cardiac contractility or cAMP production upon receptor stimulation (Kuznetsov et al, 1995; Xiao et al, 1999). It has been shown, however, that if the Gαi subunit is inhibited, a Gas response and an increase in myocyte contractility ensues (Xiao et al, 1999). Our results indicate that loss of PBKγ in heart muscle cells relieves an inhibition resulting in cAMP production and PLB phosphorylation upon β2 AR agonist stimulation consistent with an inhibition of Gαi. In this scenario, loss of PBKγ would result in a lack of Gαi cAMP inhibition and a subsequent increase in cAMP levels and PLB phosphorylation resulting in the inhibition of PLB and increased SERCA function effectively enhancing contractility (Zvaritch et al, 2000; Frank and Kranias, 2000). Additional molecular mechanisms controlling cAMP levels and PLB phosphorylation in a PTEN-PBKγ dependent manner cannot be excluded.
In heart failure, βl-AR densities decrease leading to a greater prominence of β2 adrenergic signaling (Naga Prasad et al, 2001b). Furthermore, it has been shown that increased PLB activity is a critical regulator of decreased contractility in dilated cardiomyopathy (Minamisawa et al, 1999). It is likely that if PBKγ is upregulated in heart failure, and, it is known that pi lOγ expression can be induced in pressure overload hypertrophy (Naga Prasad et al, 2000). In the presence of increased PBKg, b2 adrenergic signaling leads to a reduction in cardiac contractility. Inhibition of PBKg will lead to better cardiac contractility in heart failure. To further study remodeling in the hearts histological analysis was carried out. In control hearts treatment with isoproterenol lead to disorganization of the myocytes and a significant increase in cardiac fibrosis. Quantification of interstitial fibrosis showed a significant increase in collagen deposition in isoproterenol treated control hearts. This increased fibrosis and disorganization is consistent with the decompensation of the hypertrophic myocardium and the progression to dilated cardiomyopathy. In contrast to control hearts, pl lOgamma deficient hearts diplayed no increase in cardiac fibrosis and no alteration in myocyte organization.
Cardiac hypertrophy is associated with a prototypical alteration in gene expression profiles. In models of cardiomyocyte hypertrophy in culture, PBK has been shown to play a role in the onset of these alterations in gene expression. To determine if loss of pl lOgamma alters the changes in gene expression seen in isoproterenol induced cardiac hypertrophy, we assed the mRNA levels of hypertrophy markers. Isoproterenol has been shown to induce significant alterations in gene expression albeit blunted in comparison to other models of hypertrophy. Consistently, we observed a prototypical increase in ANF and BNP expression in control hearts. We also observed a slight decrease in a-myosin heavy chain expression. In pi lOgamma deficient hearts a similar alteration in expression profiles was found compared to control hearts, despite the attenuation of hypertrophy. This would suggest that pl lOgamma may not be required for alteration in expression profile seen in cardiac hypertrophy, and that alterations in gene expression profiles can be uncoupled from cardiac hypertrophy.
The uncoupling of expression profiles from cardiac hypertrophy further suggests that these alterations in gene expression profile may not be sufficient to induce cardiac hypertrophy. Previously it has been shown that increase in endogenous activation of PKB/AKT is associated with increases in cardiomyocyte size, and overexpression of a constitutively active form of PKB/AKT could induce an increase in heart size. PKB/AKT is a major downstream effector of PBK signaling, suggesting that AKT activation may play a role in the cardiac hypertrophy observed here. To determine if AKT activation was associated with the isoproterenol induced cardiac hypertrophy, we assessed AKT activation using an in vitro kinase assay. In control hearts, chronic stimulation with isoproterenol led to a significant increase in AKT kinase activity. However, in pl lOgamma deficient hearts, chronic isoproterenol infusion did not increase endogenous levels of AKT activity. This data correlates well with the observed attenuation of cardiac hypertrophy seen in PBKgamma deficient hearts and suggests that AKT activation may play a critical role in the hypertrophic response to isoproterenol infusion. Inhibition of PBK gamma improves function in a model of heart failure
A role for PBK in cardiomyocyte hypertrophy has been demonstrated upon agonist stimulation in cultured cells. Previously we have shown that PBKalpha modulates basal cell size in cardiomyocytes while loss of PBKgamma results in increased cardiac contractility with no effect on basal cell size. However, in many instances where PBK signaling has been implicated in cardiac hypertrophy, GPCR agonists have been used for the induction of hypertrophy suggesting that PBKgamma the isoform linked to GPCR signaling, may play a role in agonist induced cardiac hypertrophy. Moreover, the increased cardiac contractility seen in PBKgamma deficient hearts suggest a potential role for PBKgamma inhibition in the treatment of heart failure. To show the role for PBKgamma in agonist induced hypertrophy and cardiomyopathy, we induced cardiac hypertrophy and subsequent impairment of pump function by chronic infusion of isoproterenol, a beta-adrenergic agonist in PBKgamma deficient mice. Here we show that PBKgamma deficient mice have an attenuated hypertrophic response to isoproterenol induced cardiac hypertrophy in vivo and that PBKgamma deficient mice retain their increase in pump function following this chronic beta-adrenergic receptor stimulation. These data highlight the in vivo role of PBKgamma in hypertrophy, and show that PBKgamma is a novel therapeutic target for the treatment of decreased pump function in heart failure. Control hearts treated with isoproterenol displayed an increase in cardiac hypertrophy and a dilation of the left ventricle consistent with the onset of dilated cardiomyopathy. In contrast the architecture of pl lOgamma deficient hearts remained largely unchanged. Previously we have shown that these pi lOgamma deficient hearts display an increase in cardiac hypertrophy. It has been suggested that this increase in pump function may serve to improve cardiac performance in models of dilated cardiomyopathy. We assessed cardiac function in control and PBKgamma deficient hearts with and without the induction of hypertrophy using echocardiography. In control hearts the chronic infusion of isoproterenol lead to a significant reduction in systolic heart function. Both fractional shortening and velocity of circumferential fiber shortening were significantly reduced in these hearts. This data is consistent with the increased interstitial fibrosis and dilation of the left ventricle observed in these hearts, and strongly supports the onset on dilated cardiomyopathy in these hearts. On the other hand, chronic isoproterenol stimulation of pi lOgamma deficient hearts did not significantly reduce cardiac function and these hearts retained an increase pump function relative to untreated control hearts. We have shown here that PBKgamma plays a role in isoprorternol induced cardiac hypertrophy. It has been previously shown that PBKg is activated in pressure overload hypertrophy but that this increase in activity was not required for the hypertrophic response. This suggests that PBKgamma may not be required for all forms of cardiac hypertrophy but possibly may play a critical role in hypertrophy induced through GPCR mediated pathways. Our data here suggests that this hypertrophy may be in part mediated by activation of PKB/AKT pathways. Recently it has been shown that overexpression of an activated form of AKT could induce cardiac hypertrophy in vivo. It has also been shown that PBKgamma is essential for ERK activation in some systems. Transgenic mouse models with heart specific increases in ERK activation have been shown to lead to a physiologic hypertrophy. Thus a MAP kinase mediated pathway may also play a role in the hypertrophy observed here. Nevertheless we were unable to detect alterations in phospho-MAPK in isoproterenol induced hypertrophic hearts. Thus the role of MAPK activation in our model of hypertrophy remains unclear. In control hearts isoproternol infusion led to a decrease in cardiac function and the onset of cardiomyopathy. In pl lOgamma deficient hearts, however, displayed a resistance to any overt effects on pump function induced by isoproterenol. This data represents the first evidence for a potential therapeutic role of modulation of PBK signaling in heart failure. We have shown that the increase in cardiac contractility in PBKgamma deficient hearts is mediated by an increase in phospholamban inhibition. It has been shown that modulation of PLB activity either directly or indirectly can rescue numerous models of heart failure. Currently there is a great interest in determining the validity of PLB modulation as a therapeutic target for heart failure indications as a way to safely improve pump function, an aspect of heart failure not sufficiently attenuated by current therapeutics. While the assessment of the effect PBKgamma inhibition has in other genetic and acquired models of heart failure is required it is interesting to speculate that specific inhibitors of PBKgamma may prove to be useful therapeutics for heart failure indications. Importantly, however, it will be necessary to alter PLB activity following the onset of heart failure, as opposed to prior to phenotypic onset to properly assess its potential as a therapeutic modality. The invention shows that inhibition of PBKgamma improves function in a model of heart failure. Table 3 below shows this result. Mice were treated with isoproterenol chronically for 7 days. In control +/- animals heart function is reduced, but in -/- heart function is maintained.
Experimental Procedures
Mutant mice. The generation and genotyping of the PTEN-floxed, mckCRE, pllOγ, and DN-pl 10a mice have been previously described (Suzuki et al, 2001; Bruning et al, 1998; Sasaki et al, 2000; Shioi et al, 2000; Hirsch et al, 2000). Only littermate mice were used as controls. Mice were bred and maintained following institutional guidelines.
Protein and mRNA expression analyses. Northern and Western blotting were carried out as described (Crackower et al, 2002) using antibodies to PTEN (Cascade), pllOα, pl lOβ, pl lOγ (Santa Cruz), p85 (Upstate Biotechnologies), phospho- AKT/PKB (S473), AKT/PKB, phospho-GSK3β, GSK3β, phospho-p70S6K, phospho- ERKl/2, ERKl/2, phospho-phospholamban (Serinelό), phospholamban (Cell Signaling Technology), and GAPDH (Research Diagnostics). For immunoprecipitations, lysates were incubated with anti-GRK2/3 Abs (Upstate Biotechnologies) conjugated with Sepharose beads for 16 hours at 4°C. Immunoprecipitates were separated using SDS-PAGE gel electrophoresis, transferred onto membranes and immunoblotted.
Heart morphometry, echocardiography, and hemodynamic measurements. For heart morphometry, hearts were arrested with KCL, fixed with 10% buffered formalin and embedded in paraffin. Fibrosis was analyzed using trichrome staining and quantitative morphometry using color-subtractive computer assisted image analysis. Echocardiographic assessments and invasive hemodynamic measurements were carried out as described (Crackower et al, 2002). Catecholamines were extracted from the plasma of male mice using alumina, eluted and then assayed using HPLC (Waters Associates Inc., Milford, MA, USA) coupled with electrochemical detection (ESA Inc., Bedford, MA, USA). The intra-assay and inter-assay coefficient of variation were <5% and <10%, respectively.
Cardiomyocyte purification. Adult ventricular myocyte were isolated as described (Sah et al, 2002). Cell size was measured using Openlab 2.2.5 software (Improvision Ltd). Ventricular myocytes were placed in a Plexiglas chamber and continuously perfused with oxygenated Tyrodes buffer (137mM NaCI, 5.4mM KC1, lOmM glucose, 1 OmM HEPES, 0.5mM NaH2PO4, lmM MgCl2, 1.2mM CaCl2, and pH 7.4) at 2.5ml/min at 36°C. Neonatal ventricular cardiac myocytes were isolated from 1-3 day old mice in CBFHH buffer (137mM NaCI, 5.36mM KC1, 0.81mM MgSO4-7H2O, 5.55mM dextrose, 0.44mM KH2PO4, 0.34mM Na2HPO4-7H2O, 20μM Hepes, pH 7.4) as described (Steinberg et al, 1991). Experiments were carried out 48-72 hrs after plating.
Contractility assays and cAMP measurements. Cardiomyocytes were stimulated at 1 Hz with a Grass S44 stimulator (pulse duration 3 ms; 15-20 volts) and a video edge detector (Crescent Electronics) was used to track myocyte contractions at 240Hz. Steady state contractions were recorded at 1kHz following a 4 mins equilibrium period using a Phillips 800 camera system (240Hz) and Felix acquisition software (Photon Technologies Inc.). Cardiomyocyte length, percent fractional shortening, shortening rate (+dL/dT), and relaxation rate (— dL/dT) were determined at baseline. Myocytes were incubation with LY294002 (30μM) (Calbiochem) and Rp-cAMPS (25μM) (Sigma/RBI) for 10 minutes and recordings were made during continuous perfusion of the drug dissolved in Tyrode. In all experiments phosphodiesterase activity was inhibited by pre-treating the cells with theophylline (lOOmM) for 30 min prior to stimulation. Wortmannin treatment (lOOnM) was initiated at the time of theophylline addition. Zinterol (Bristol-Myers Squibb) stimulation was carried out at RT for 10 min. Forskolin (Sigma) was used at 25mM. Isoproterenol (Sigma) stimulation was carried out at RT for 10 min, following 5 min. pretreatment of ICI 118,551 (Sigma/RBI) (10"7M). cAMP was measured by RIA according to the supplier's protocol (Amersham).
GRK activity and b-Adrenergic Receptor binding. GRK activity was measured by rhodopsin phosphorylation as described (Louden et al., 2000). Hearts were homogenized in 20mM Tris pH 7.3, 0.8mM MgCl2, 2mM EDTA. Membrane fractions were isolated by centrifuging at 40,000g for 30min at 4°C. 300μg and 20μg of protein were assayed for cytosolic and membrane fractions, respectively. b-AR density and affinity for its ligand ICYP (3-Iodocyanopindolol [125I] ) were determined by equilibrium radio-ligand binding assays on sarcolemmal membranes prepared from ventricular myocardium as described (Steinberg et al., 1995). Briefly, assays were performed with 30mg membrane protein and ICYP (5-250pM) in the absence or presence of O.lmM unlabeled propranolol to determine non-specific binding in a final volume of 1ml for 60min at 37°C. ICYP bound to membranes was separated from free, unbound ICYP by rapid vacuum filtration over glass-fiber filters (German A E).
Histology Methods
Mice. PI lOgamma deficient mice have previously been described. Hypertrophy was induced by chronic infusion of isoproterenol (sigma) for 7 days at a dose of 15mg/Kg/day using alzet osmotic minipumps. Mice were aneastitized with O2/isoflourane and the pumps were inserted subcutaneously at the level of the scapula. The incision was closed and mice were allowed to recover. Pumps were removed 24 hrs. prior to echocardiographic analysis.
Heart morphometry, echocardiography, hemodynamics and blood pressure measurements For heart morphometry, hearts were arrested with KCL and fixed with 10% buffered formalin, and subsequently embedded in paraffin. Myocardial interstitial fibrosis was determined using trichrome stained sections and quantitative morphometry using color-subtractive computer assisted image analysis (Image Processing Tool Kit version 2.5). Echocardiographic assessments were performed as described (Wickenden et al, 1999). Briefly, mice were anesthetized with isoflourane/oxygen and examined by transthoracic echocardiography using a Acuson® Sequoia C256 equipped with a 15MHz linear transducer. (FS) was re calculated as: FS = [(EDD - ESD)/EDD] x 100. Ncfc was calculated as FS/ejection time corrected for heart rate.
Protein Kinase and mRΝA expression analyses
Total RΝA was prepared form hearts using trizol ( ). 20 mg of R A was resolved on a 0.8% formamide gel, blotted to nylon membranes (Amersham), and probed with cDΝA probes for AΝF, BΝP, αMHC, and GAPDH. AKT kinase activity was measured from whole hearts according to manufacturer's protocols (Upstate).
Example 2
PBKγ is critical for the induction of hypertrophy, fibrosis, and cardiac dysfunction function in response to β-adrenergic receptor stimulation in vivo. Thus, PBKγ is a novel therapeutic target for the treatment of decreased cardiac function in heart failure. We infused isoproterenol, a β-adrenergic receptor agonist, into PBKγ deficient mice. Compared to controls, isoproterenol infusion in PBKγ deficient mice resulted in an attenuated cardiac hypertrophic response and markedly reduced interstitial fibrosis. Intriguingly, chronic β-adrenergic receptor stimulation triggered impaired heart functions in wild type mice, whereas PBKγ deficient mice retained their increased heart function and did not develop heart failure. The lack of PBKγ attenuated the activation of Akt/PKB and ERKl/2 signaling pathways in cardiac myocytes in response to isoproterenol. Betal- and beta2-adrenergic receptor densities were decreased by similar amounts in PBKγ deficient and control mice showing that
PBKγ isoform plays no role in the downregulation of beta-adrenergic receptors following chronic beta-adrenergic stimulation.
Methods
Mic e . pllOf'' mice have previously been described (Sasaki ete al., 2000). Hypertrophy and heart failure were induced by chronic infusion of isoproterenol (Sigma) for 7 days at a dose of 15mg/kg/day using Alzet® osmotic mini-pumps. Pumps were removed 24h prior to echocardiography and hemodynamic measurements. All experiments were performed in accordance to institutional guidelines. Only littermate male mice of 10-12 weeks of age were used. Heart morphometry, TUNEL assay, echocardiography, and hemodynamic measurements. For heart morphometry, hearts were arrested with KC1, fixed with 10% buffered formalin, and embedded in paraffin. Myocardial interstitial fibrosis was determined as collagen volume fraction (PSR-stained sections) using color-subtractive computer assisted image analysis (Image Processing Tool Kit version 2.5). In situ
Q DNA fragmentation was labeled using the TUNEL assay (ApopTag Plus kit) (Oncor,
Gaithersburg, MD). Echocardiographic assessments and invasive hemodynamic measurements were carried out as described. (Crackower et al. 2002).
Signaling, mRNA and Western Blot analyses. Total RNA was prepared form hearts using Trizol, resolved on a 0.8% formamide gel, blotted to nylon membranes
(Amersham), and probed with cDNA probes for ANF, BNP, aMHC, and GAPDH.
Akt/PKB kinase activity was measured using a commercial in vitro kinase assay kit according to the manufactures protocol (Upstate Biotechnologies) (Sasaki et al. 2000).
Ventricular cardiomyocytes were isolated as described (Crackower et al. 2002). Experiments were carried out 48-72 hrs after plating and removal of fibroblasts.
ERKl/2 phosphorylation, p38 phosphorylation, and ERKl/2 protein levels were determined by Western blotting (Cell Signaling Technology).
Beta-adrenergic receptor densities. Mice were anaesthetized with isoflurane/O2, hearts were removed and ventricular tissue excised and frozen. Samples were processed and light sarcolemmae were not treated (-) or treated with PNG-F (+) and subjected to SDS-PAGE as previously described (Rybin et al. 2000).
Results
To show the role of PBKγ in GPCR-induced cardiac hypertrophy and cardiomyopathy, we chronically infused littermate ^iiOy7" (knockout) and pll0γ+/' (control) mice with the β-adrenergic receptor agonist, isoproterenol. Control mice displayed a marked increase in heart size (Fig. 9A), heart/body weight and heart/tibial length ratios (Fig. 9B) and left ventricular mass and wall thickness (Table 4). In isoproterenol-infused i/0y" " mice, the hypertrophic response still occurred but was markedly attenuated (Fig. 9 A and B) (Table 4). Cardiac hypertrophy is associated with prototypical alterations in gene expression (Hunter et al., 1999). We therefore assessed the mRNA levels of hypertrophy markers in hearts of p!10g+/" and pllOf'' mice chronically stimulated with isoproterenol. Intriguingly, despite the attenuation of hypertrophy in^iiøy^'hearts, we observed a similar alteration in expression profiles in ANF, BNP and α-myosin heavy chain compared to pllO'f "hearts (Fig. 9C). It was unclear whether PBKγ is essential for GPCR-signaling in cardiomyocytes (Rockman et al. 2002; Naga Prasad et al. 2002). We therefore stimulated purified ventricular cardiomyocytes from pi 10 f' and pllOf'' littermates with the GPCR- agonist, isoproterenol, or the tyrosine kinase-based receptor agonist, basic fibroblast growth factor (bFGF) (Fig. 9D). Whereas bFGF-induced ERKl/2 activation occurred normally, isoproterenol-induced ERKl/2 phosphorylation was completely abolished ixi pllOf ' cardiomyocytes. Similarly, pllOf'" hearts, chronic isoproterenol infusion did not increase endogenous Akt/protein kinase B (PKB) activity (Fig. 9E). By contrast, activation of the stress kinase p38 appeared to be normal in both pllOf" and pll Of'" cardiomyocytes (Fig. 9D). Given the marked differences in signaling and hypertrophic responses, we showed the functional changes in heart function in mice infused with isoproterenol following withdrawal of the drug. Chronic infusion of isoproterenol into pllθ '" mice lead to a significant reduction in heart functions as assessed by markedly decreased fractional shortening, velocity of circumferential fiber shortening and peak aortic velocity (Table 4). Invasive hemodynamic measurements showed increased left ventricular end-diastolic pressure, and decreases in ±dP/dtmaxand blood pressure in pllθ '~ animals (Table 4), again indicating severe impairment of heart function. By contrast, chronic isoproterenol stimulation of pll Of'' mice caused a markedly smaller reduction in cardiac function and blood pressures. Moreover, these hearts retained an increased pump function relative to untreated control hearts (Table 4).
Decompensation of the hypertrophic myocardium and the progression to cardiomyopathy is accompanied by increased fibrosis and disorganization of myocytes. We therefore carried out histological analysis to further study the cardiac remodeling response. In control hearts treatment with isoproterenol resulted in disorganization of the myocytes and a marked increase in cardiac fibrosis (Fig. 10A). Quantification of interstitial fibrosis showed a significant increase in collagen deposition in the hearts of isoproterenol-treated control mice (Fig. 10B). By contrast, pi 10 f'" mice displayed no significant increase in cardiac fibrosis or alteration in myocyte organization (Fig. 10A,B). Cardiomyocyte cell death was comparable between isoproterenol-treated pll Of'" and control mice (Fig. IOC). Importantly, measurements of cardiomyocyte sizes in cross-sections confirmed impaired cardiomyocyte hypertrophy i pi 10 f'' ice (Fig. 10D). There were no differential changes in βl- or β2-adrenergic receptor expression in membrane fractions from pll Of'' and pi 1 Of'' hearts at baseline and in response to chronic in vivo isoproterenol stimulation (Fig. 11A and B). Expression levels of the major cardiac adenylate cyclase isoforms V and VI, (ACV/VI), were comparable between pi 1 θ "and pllOf '" hearts (Fig. HA and B).
Excessive catecholamines and adrenergic stimulation has been clearly linked to cardiac hypertrophy and disease. Indeed, altered signaling via the G-protein coupled β-adrenergic receptors have been implicated in heart failure in humans (Rockman et al, 2002; Hajjar and MacRae, 2002; Scheuer, 1999; Brede et al., 2002). Loss of pllOγ leads to an increase in cardiomyocyte contractility. Our data show that PBKγ, the PBK-isoform linked to GPCRs, is critical for the induction of myocardial hypertrophy, interstitial fibrosis, and cardiac dysfunction in response to β-adrenergic receptor stimulation in vivo. Importantly, pll Of'' mice display a resistance to the effects of isoproterenol on cardiac structure and function. An increase in cardiac function may serve to improve myocardial performance in models of dilated cardiomyopathy and we confirmed that pllOf'' hearts maintained their hypercontractility following chronic adrenergic stimulation. However, the induction of several hypertrophy markers in response to isoproterenol was not affected by the loss of pll 0γ which shows that pllOγ is not required for alteration in fetal gene expression seen in myocardial hypertrophy and that these genetic changes can be uncoupled from the hypertrophic process.
Our data show that chronic activation of β-adrenergic signaling results in markedly decreased heart function and the onset of cardiomyopathy in pi lθ ~'~ mice, loss of pllOγ renders animals resistant to increased interstitial fibrosis and cardiac dysfunction. Akt/ protein kinase B (PKB) is a major molecular target of the PBK signaling pathway involved in cell hypertrophy in multiple systems, and the mitogen- activated protein kinase, ERKl/2 also plays a central role in cardiac hypertrophy. Genetic evidence in hematopoietic cells show that PBKγ is the sole PBK that couples to GPCRs. Importantly, infusion of isoproterenol into/ 7707+/"mice showed that β- adrenergic receptor stimulation induced in vivo activation of Akt/PKB and in vitro activation ERKl/2. These genetic data demonstrate for the first time that following GPCR stimulation, PBKγ is the essential PBK isoform required for Akt/PKB and ERKl/2 activation in cardiomyocytes. In other models of heart disease (pressure- overload hypertrophy), there is also activation of Akt/PKB via the PBKγ isoform. Despite the lack of activation of Akt/PKB and ERKl/2 pathways in pll Of'" mice, which are clearly antiapoptotic pathways, apoptosis was not increased in the pll Of'" mice which is likely related to the antiapoptotic effects mediated by enhanced β2- adrenergic receptor signaling inpllOf'' cardiomyocytes. Since PBK also has a role in GPCR signaling in isolated cardiac fibroblasts (Kim et al., 2002), the reduced interstitial fibrosis in the heart may be the direct consequence of attenuated β-adrenergic receptor-mediated stimulation of cardiac fibroblasts in the pllOf'" hearts. PBKg might be involved in β-adrenergic receptor down-regulation (internalization). However, the loss of PBKγ has no apparent effect on the down- modulation of β-adrenergic receptors in response to chronic adrenergic stimulation. However, we cannot rule out the role of other PBK isoforms in the downregualtion of beta-adrenergic receptors and there exists the distinct possibility that receptor desensitization could be differentially affected i.e. functional responses are different. In summary, we have shown that the PI3K5 has an important role to maintain heart function under the pathologic condition of chronic adrenergic stimulation and our results represent the first genetic evidence for a therapeutic role of the modulation of PBKγ signaling in heart failure.
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Table 1. Heart functions of mutant mice
10 weeks 12 months mckCRE EN"* mckCRE-PTEN"* mckCRE mckCRE mckCRE mckCRE-PT -PTEN"* p110γ+ DN-p110a DN-p110a -PTEN"* -PTEN"* -PTEN"* p110y- (wlldtype) n=7 n=7 n=6 n=6 n=7 n=8 n=4 n=4
Heart Rale, bpm 516 1 13 545 ± 19 545 ± 9 529 ± 18 486 ± 25 535 ± 11 522 ± 7 533 ± 7 AW, mm 0.67 ± 0.03 0.74 ± 0.03 ** 0.70 ± 0.03 0.84 ± 0.02 ** 0.70 ±0.02 0.8010.01** 0.61 ±0.01* 0.57 ±0.03*
LVEDD. mm .00 ± 0.07 4.35 ± 0.15 * . 4.10 ± 0.05 4.32 ± 0.15 3.99 ± 0.11 4.12 ± 0.04* 3.87 ± 0.09 3.85 ± 0.14 LVESD, mm 2.20 ± 0.0 3.11 10.14 ** 2.24 ± 0.05 2.9210.15* 1.74 ± 0.06** 1.94 ± 0.06* 2.10 ±0.07 2.57 ±0.07** LVM, mg 97.39 ± 2.82 134.04 ± 2.64 ** 103.57 ± 4.87 142.93 ± 4.59 * 92.22 ± β.75 128.87 ± 2.59* 83.18 ±1.23* 83.5511.42* % FS 44.9 ± 0.68 28.39 ± 1.90 ** 44.50 ± 1.28 32.86 ± 1.26 ** 56.50 ± 0.95** 52.96 ± 1.39** 45.62 ± 0.82 33.26 10.80**
Vcfc, circ s 8.71 ± 0.10 5.26 ± 0.30 ** 9.23 ±0.18 6.41 10.23 ** 11.1510.34** 10.78 ± 0.36* 9.39 ±0.22 6.S1 10.21** PAV, m/s 0.940 ± 0.024 0.887 ± 0.023 * 0.826 ± 0.029 0.697 10.019 * 1.017 ± 0.052** 1.116 ± 0.070** 0.886 ± 0.044 0.830 ± 0.024 dP/dT-max + 6045 ± 148 + 4077 ± 243** + 92021473** dP/dT-min - 5274 ±251 - 3731 ± 158** - 8036 ±500**
*p < 0.05 vs wildtype mice.
"p < 0.01 vs wildtype mice.
Bpm = heart beats per minute; AW = anterior wall thickness; LVEDD = left ventricle end diastolic dimension; LVESD = left ventricle end systolic dimension; LVM = left ventricular mass; %FS = percent fractional shortening; Vcfc = Velocity of circumferential fiber shortening; PAV = peak aortic outflow velocity. dP/dT max = maximum 1st derivative of the change in left ventricular pressure/time; dP/dT min = minimum 1st derivative of the change in left ventricular pressure/time
Table 2. Contractility of isolated single cardiomyocytes
Wildtype mckCRE p110γ+ mckCRE-PTEN"* Wildtype p110γ* Wildtype p110γ+ -PTEN"* pHOγ-t- (LY294002) (LY294002) (Rp-CAMPs) (Rp-'CAMPs)
(30μM) (30μM) (25μM) (25μM)
RestiπgLength.μm 121.7 ± 3.1 146.7 ±2.9 ** 123.61 3.2 147.312.4 ** 123.812.1 124.1 13.4 120.612.6 122.1 12.2
% Shortening 11.4 ± 051 7.1 ± 0.43 ** 17.4 ± 1.1 ** 16.611.2 ** 15.610.97** 16.511.3** 9.6810.37** 12.1 ±0.41t
+dUdT, μm s 339 ± 17.2 224 ± 14.3 ** 797145** 7231 2 ** 503122.5** 717153** 278112.3** 496121.31
-dL/dT, μm/s 358 1 18.1 242 ± 16.4 ** 960 ± 56 " 889158 ** 521 125.8** 912163** 289117.2 ** 579131.6 f
TPS, ms 55.3 ± 3.2 494 1 3.8 48.7 12.2 50.212.4 50.3 ± 2.9 53.313.1 54.712.8 53.1 12.6
T90R, ms 71.9 ± 3.6 76.714.5 54.712.8 ** 51.312.6 ** 50.1 12.3** 50.512.5** 76.913.2 68.813.7 f
**p < 0.01 vs wildtype cells. t p< 0.01 vs untreated p1 lOγ7-
Mean ± SEM are shown; n=15 cells from 3 hearts for each group. +dLtdT max = maximum 1st derivative of the change in cell length/time;
-dP/dT = minimum 1st derivative of the change in cell length /time; TPS=time to peak shortening; T90R=time to 90% relaxation.
Table 3. Heart ftwetiorts of mckC
Figure imgf000055_0001
isoproterenol Isoproterenol HjO (15mg/r!g/day/7days) H20 (■15mg Kg/day/7clays)
Bod Vtfeight. g 29.60 ±1.02 29.4510.50 28.76 10.64 29.21 ±0.50
Hsart Vufeight/θodyVtfeight, mg/g 4.6210.11 6.6410.35 4.6910.18 5.3910.15
Hart Rate, bpm 495112 485117 436 ±26 492 17
Aτteriorwall thickness, mm 0.69 ±0.02 0.7610.02 0.7010.02 0.7110.03
LVEDD, rum 3.98 ±0.12 4.32 ±O.iO 3,99 10.11 4.1410.10
LVESD, mm 2,22 ±0.09 2.9410.13 1.7 10.06 1.8310.09
%F$ 44.3411.16 31.2211.48 56.50 ±0.05 53.43 11.51
PAV £75 ±0.032 79910.034 1.017 ±0.052 816 10.014
VCFC 8.26 ±0.20 6.0410.41 11.1513.34 10.5210.38
LV ASS 93.25 ±4.91 131.1011.43 82.22 ±3.76 111.6511.41
LVB 3.16 ±0.18 4.7810.03 3.20 ±0,20 3.65 ±0.08
*p < 0.05 mckCRE-PTEN"* vs mckCRE-PTEN" mice.
"p < 081 mckCRE-PTEN" v mckCRE-PTEN«πϊce.
Bpm = heart beats per πinute; AW= anterior mall thickness; LVEDD = left lentride end diastdic drrension; LVESD = left ventricle end systolic dirensi n; ISM- calculated left ventricular mass; ΪFS = percert fractional shortening; Vsfc = Velocity of circumferential fiber shortening; PAV= peak aortic outflow velocity.
Table 4. Echocardiographic and hemodynamic parameters in littermate p110γ +/" and p770y "A mice following chronic β-adrenergic recepto stimulation
p110γ+/' pHOf ' ' + pllOf' +Iso p110γ/'+ Iso
Figure imgf000056_0001
÷Vehicle Vehicle
HR (bpm) 521+13.5 503+14.8 491+17.2 513+9.3
AW (mm) 0.693+0.019 0.696+0.015 0.879±0.02A 0.733+0.017
PW (mm) 0.687±0.016 0.706±0.022 0.884±0.021A 0.742±0.018
LVEDD (mm) 3.98+0.11 4.03+0.12 4.15±0.08B 3.96+0.1
LVESD (mm) 2.16+0.09 1.73±0.06 2.89±0.07A 1.95±0.09
LVM/BW (mg/g) 3.16±0.18 3.2±0.17 4.79+0.13A 3.65±0.11ϋ
FS (%) 46.7+1.2 57.8±1.3B 29.6+1.9A 50.3+1 A
PAV (cm/s) 87.5+3.2 98.9±5.1 68.6±3.4A 92.5±2.9
VCFc (circ/s) 8.26+0.27 11.3±0.42B 5.43±0.31A 10.3±0.38
SBP (mmHg) 108.3+2.9 112+3.6 75.6±3.3A 107.8±3.2
MABP (mmHg) 83.4±3 85.4±2.2 53.2±2.4A 81.4±2.9
LVEDP (mmHg) 5.84±0.3 5.65±0.44 10.9±0.86A 6.55+0.58
+dP/dtmax +5995±120 +8984±241B +3142±294A +7963±257u (mmHg/sec)
-dP/dtmin -5487+128 -8093±257B -2706+218A -6987+216G (mmHg/sec) n=8 for all groups; values are mean±SEM; p<0.01 compared with all other groups, p<0.01 compared with Control+Vehicle group; ; cp <0.05 compared wit p110γ "+Vehicle group; HR=Heart Rate; AW, PW=Anterior and Posterior Left Ventricular Wall Thickness; LVEDD, LVESD=Left Ventricular End Diastolic an Systolic Diameter; LVM=Left Ventricular Mass; BW=Body Weight; FS=Fractional Shortening; PAV=Peak Aortic Velocity; VCFc=Velocity of Circumferentia Shortening corrected for Heart Rate; SBP=Systolic Blood Pressure; MABP=Mean Arterial Blood Pressure; LVEDP=Left Ventricular End-Diastolic Pressure +dP/dtmax and -dP/dtmin=maximum and minimum first derivative of the change in left ventricular pressure; Iso = Isoproterenol.
REFERENCES
Alessi, D.R., Kozlowski, M.T., Weng, Q.P., Morrice, N., and Avruch, J. (1998). 3- Phosphoinositide-dependent protein kinase 1 (PDKl) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr. Biol. 8, 69-81.
Backman, S.A., Stambolic, V., Suzuki, A., Haight, J., Elia, A., Pretorius, J., Tsao, M.S., Shannon, P., Bolon, B., Ivy, G.O., and Mak, T. . (2001). Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte- Duclos disease. Nat. Genet. 29, 396-403
Baliga, R.R., Pimental, D.R., Zhao, Y.Y., Simmons, W.W., Marchionni, M.A., Sawyer, D.B., Kelly, R.A. (1999). NRG-1-induced cardiomyocyte hypertrophy. Role of PI-3 -kinase, p70(S6K), and MEK-MAPK-RSK. Am. J. Physiol. 277(5 Pt 2), H2026-H2037.
Bondeva, T., Pirola, L., Bulgarelli-Leva, G., Rubio, I., etzker, R., and Wymann, M.P. (1998). Bifurcation of lipid and protein kinase signals of PBKgamma to the protein kinases PKB and MAPK. Science 282, 293-296.
Brede M, Wiesmann F, Jahns R, Hadamek K, Arnolt C, Neubauer S, Lohse MJ, Hein L. Feedback inhibition of catecholamine release by two different alpha2-adrenoceptor subtypes prevents progression of heart failure. Circulation. 2002;106:2491-6.
Brittsan, A.G., and Kranias, E.G. (2000). Phospholamban and cardiac contractile function. J. Mol. Cell. Cardiol. 32, 2131-2139.
Bruning, J.C., Michael, M.D., Winnay, J.N., Hayashi, T., Horsch, D., Accili, D., Goodyear, L.J., Kahn, C.R. (1998). A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol. Cell 2, 559-569. Bueno OF, Molkentin JD. Involvement of extracellular signal-regulated kinases 1/2 in cardiac hypertrophy and cell death. Circ Res. 2002;91:776-81.
Chruscinski, A.J., Rol rer, D.K., Schauble, E., Desai, K.H., Bernstein, D., and Kobilka, B.K. (1999). Targeted disruption of the beta2 adrenergic receptor gene. J. Biol. Chem. 274, 16694-16700.
Colan, S.D. (1997). Mechanics of left ventricular systolic and diastolic function in physiologic hypertrophy of the athlete's heart. Cardiol. Clin. 15, 355-372
Crackower, M.A., Sarao, R., Oudit, G.Y., Yagil, C, Kozieradzki, I., Scanga, S.E., Oliveira-dos-Santos, A.J., da Costa, J., Zhang, L., Pei, Y., Scholey, J., Ferrario, CM., Manoukian, A.S., Chappell, M.C., Backx, P.H., Yagil, Y., and Penninger, J.M. (2002). Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature. 417, 822-828.
Crackower MA, Oudit GY, Kozieradzki I, Sarao R, Sun H, Sasaki T, Hirsch E, Suzuki A, Shioi T, Irie-Sasaki J, Sah R, Cheng HY, Rybin NO, Lembo G, Fratta L, Oliveira-dos-Santos AJ, Benovic JL, Kahn CR, Izumo S, Steinberg SF, Wymann MP, Backx PH, Penninger JM. Regulation of myocardial contractility and cell size by distinct PI3K-PTEΝ signaling pathways. Cell. 2002;110:737-49.
Di Cristofano, A., Pesce, B., Cordon-Cardo, C, and Pandolfi, P.P. (1998). Pten is essential for embryonic development and tumour suppression. Nat. Genet. 19, 348- 355.
Downward, J. (1998). Mechanisms and consequences of activation of protein kinase B/Akt. Curr. Opin. Cell Biol. 10, 262-267.
Frank, K., and Kranias, E.G. (2000). Phospholamban and cardiac contractility. Arm. Med. 32,572-578. Franke, T.F., Kaplan, D.R., Cantley, L.C., and Toker, A. (1997). Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275, 665-668.
Hajjar RJ, MacRae CA. Adrenergic-receptor polymorphisms and heart failure. N Engl J Med. 2002;347:1196-9.
Hirsch, E., Katanaev, V.L., Garlanda, C, Azzolino, O., Pirola, L., Silengo, L., Sozzani, S., Mantovani, A., Altruda, F., and Wymann, M.P. (2000). Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science 287, 1049-1053.
Hunter, J.J., and Chien, KR. (1999). Signaling pathways for cardiac hypertrophy and failure N. Engl. J. Med. 341, 1276-1283.
Kim J, Eckhart AD, Eguchi S, Koch WJ. Beta-adrenergic receptor-mediated DNA synthesis in cardiac fibroblasts is dependent on transactivation of the epidermal growth factor receptor and subsequent activation of extracellular signal-regulated kinases. J Biol Chem. 2002;277:32116-23.
Koch, W.J., Rocknian, H.A., Samama, P., Hamilton, R.A., Bond, R.A., Milano, C.A., and Lefkowitz, RJ. (1995). Cardiac function in mice overexpressing the beta- adrenergic receptor kinase or abeta ARK inhibitor. Science 268, 1350-1353.
Kodama, H., Fukuda, K., Pan, J., Sano, M., Takahashi, T., Kato, T., Makino, S., Manabe, T., Murata, M., and Ogawa, S. (2000). Significance of ERK cascade compared with JAK/STAT and PI3-K pathway in gpl30-mediated cardiac hypertrophy. Am. J. Physiol. Heart Circ. Physiol. 279, H1635-H1644.
Komalavilas, P., Mehta, S., Wingard, C.J., Dransfield, D.T., Bhalla, J., Woodrum, J.E., Molinaro, J.R., and Brophy, CM. (2001). PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways. J. Appl. Physiol. 91, 1819-1827 Kuschel, M., Zhou, Y.Y., Cheng, H., Zhang, S.J., Chen, Y., Lakatta, E.G., and Xiao, R.P. (1999). G(i) protein-mediated functional compartmentalization of cardiac beta(2)-adrenergic signaling. J. Biol. Chem. 274, 22048-22052.
Kuznetsov, N., Pak, E., Robinson, R.B., and Steinberg, S.F. (1995). Beta 2-adrenergic receptor actions in neonatal and adult rat ventricular myocytes. Circ. Res. 76, 40-52.
Leevers, S.J., Weinkove, D., MacDougall. L.K., Hafen, E., and Waterfield, M.D. (1996). The Drosophila phosphoinositide 3-kinase Dpi 10 promotes cell growth. EMBO J. 15, 6584-6594.
Li, Z., Jiang, H., Xie, W., Zhang, Z., Smrcka, AN., and Wu, D. (2000). Roles of PLC-beta2 and -beta3 and PBKgamma in chemoattractant-mediated signal transduction. Science 287, 1046-1049.
Lefkowitz, R. J. (1998) G Protein-coupled Receptors III. new roles for receptor kinanses and b-arrestins in receptor signalling and desensitization. J. Biol. Chem. 273, 18677-18680.
Maehama, T., and Dixon, J.E. (1998). The tumor suppressor, PTEΝ/MMAC1, dephosphorylates the lipid second messenger, phophatidylinositol 3,4,5-triphosphate. J. Biol. Chem. 273, 13375-13378.
Matsui, T., Li, L., Wu, J.C, Cook, S.A., Νagoshi, T., Picard, M.H., Liao, R., and Rosenzweig, A. (2002). Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J Biol Chem [epub ahead of print]
Milligan, G., and White, J.H. (2001). Protein-protein interactions at G-protein- coupled receptors. Trends Pharmacol. Sci. 22, 513-518. Minamisawa, S., Hoshijima, M., Chu, G., Ward, C.A., Frank, K., Gu, Y., Martone, M.E., Wang, Y., Ross, J. Jr., Kranias, E.G., Giles, W.R., and Chien, K.R. (1999). Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell 99, 313-322.
Naga Prasad, S.V., Esposito, G., Mao, L., Koch, W.J., and Rockman, H.A. (2000). Gbetagamma-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy. J. Biol. Chem. 275, 4693-4698.
Naga Prasad, S.V., Barak, L.S., Rapacciuolo, A., Caron, M.G., and Rockman, H.A. (2001a). Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration. J. Biol. Chem. 276", 18953-18959.
Naga Prasad, S.N., Nienaber, J., and Rockman, H.A. (2001b) Beta-adrenergic axis and heart disease. Trends Genet. 17, S44-S49.
Naga Prasad SV, Laporte SA, Chamberlain D, Caron MG, Barak L, Rockman HA. Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex. J Cell Biol. 2002;158:563-75.
Rabkin, S.W., Goutsouliak, V., and Kong, J.Y. (1997). Angiotensin II induces activation of phosphatidylinositol 3-kinase in cardiomyocytes. J. Hypertens. 15, 891- 899.
Rockman, H.A., Koch, W.J., and Lefkowitz, RJ. (2002). Seven-transmembrane- spanning receptors and heart function. Nature 415, 206-212.
Rohrer, D.K., Desai, K.H., Jasper, J.R., Stevens, M.E., Regula, D.P. Jr, Barsh, G.S., Bernstein, D., and Kobilka, B.K. (1996). Targeted disruption of the mouse betal- adrenergic receptor gene: developmental and cardiovascular effects. Proc. Natl. Acad. Sci. U S A. 93, 7375-7380. Rol rer, D.K., Chruscinski, A., Schauble, E.H., Bernstein, D., and Kobilka, B.K. (1999). Cardiovascular and metabolic alterations in mice lacking both betal- and beta2-adrenergic receptors. J. Biol. Chem. 274, 16701-16708.
Rommel, C, Bodine, S.C., Clarke, B.A., Rossmanm R., Nunez, L., Stitt, T.N., Yancopoulos, G.D., and Glass, D.J. (2001). Mediation of IGF- 1 -induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat. Cell Biol. 3, 1009-1013.
Rybin NO, Xu X, Lisanti MP, Steinberg SF. Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway. J Biol Chem. 2000;275:41447-57.
Sah, R., Oudit, G.Y., Nguyen, T.T., Lim, H.W., Wickenden, A.D., Wilson, G.J., Molkentin, J.D., and Backx, P.H. (2002). Inhibition of calcineurin and sarcolemmal Ca2+ influx protects cardiac morphology and ventricular function in K(v)4.2N transgenic mice. Circulation 105, 1850-1856.
Sasaki, T., Irie-Sasaki, J., Jones, R.G., Oliveira-dos-Santos, A.J., Stanford, W.L., Bolon, B., Wakeham, A., Itie, A., Bouchard, D., Kozieradzki, I., Joza, N., Mak, T.W., Ohashi, P.S., Suzuki, A., and Penninger, J.M. (2000). Function of PBKgamma in thymocyte development, T cell activation, and neutrophil migration. Science 287, 1040-1046.
Scanga, S.E., Ruel, L., Binari, R.C, Snow, B., Stambolic, N., Bouchard, D., Peters, M., Calvieri, B„ Mak, T.W., Woodgett, J.R., and Manoukian, A.S. (2000). The conserved PB'K/PTEΝ/Akt signaling pathway regulates both cell size and survival in Drosophila. Oncogene 19, 3971-3977.
Scheuer J. Catecholamines in cardiac hypertrophy. Am J Cardiol. 1999;83:70H-74H. Schluter, K.D., Simm, A., Schafe,r M., Taimor, G., and Piper, H.M. (1999). Early response kinase and PI 3-kinase activation in adult cardiomyocytes and their role in hypertrophy. Am. J. Physiol. 276, H1655-H1663.
Schwartzbauer, G., and Robbins, J. (2001). The tumor suppressor gene PTEN can regulate cardiac hypertrophy and survival. J. Biol. Chem. 276, 35786-35793.
Shima, H., Pende, M., Chen, Y., Fumagalli, S., Thomas, G., and Kozma, S.C (1998). Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J. 17, 6649-6659.
Shioi, T., Kang, P.M., Douglas, P.S., Hampe, J., Yballe, CM., Lawitts, J., Cantley, L.C, and Izumo, S. (2000). The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J. 19, 2537-2548.
Shioi, T., McMullen, J.R., Kang, P.M., Douglas, P.S., Obata, T., Franke, T.F., Cantley, L.C, and Izumo, S. (2002). Akt/protein kinase B promotes organ growth in transgenic mice. Mol. Cell. Biol. 22, 2799-2809
Steinberg, S.F., Robinson, R.B., Lieberman, H.B., Stern, D.M., and Rosen, M.R. (1991). Thrombin modulates phosphoinositide metabolism, cytosolic calcium, and impulse initiation in the heart. Circ. Res. 68, 1216-1229
Steinberg, S.F., Zhang, H., Pak, E., Pagnotta, G., and Boyden, P.A. (1995). Characteristics of the β- Adrenergic Receptor Complex in the Epicardial Border Zone of the 5-Day Infarcted Canine Heart. Circulation 9, 2824-2833.
Stephens, L., Eguinoa, A., Corey, S., Jackson, T., and Hawkins, P.T. (1993) Receptor stimulated accumulation of phosphatidylinositol (3,4,5)-trisphosphate by G-protein mediated pathways in human myeloid derived cells. EMBO J. 12, 2265-2273. Suzuki, A., de la Pompa, J.L., Stambolic, N., Elia, A.J., Sasaki, T., del Barco Barrantes, I., Ho, A., Wakeham, A., Itie, A., Khoo, W., Fukumoto, M., and Mak, T.W. (1998). High cancer susceptibility and embryonic lethality associated with mutation of the PTEΝ tumor suppressor gene in mice. Curr. Biol. 8, 1169-1178.
Suzuki, A., Yamaguchi, M.T., Ohteki, T., Sasaki, T. Kaisho, T., Kimura, Y., Yoshida, R., Wakeham, A., Higuchi, T., Fukumoto, M., Tsubata, T., Ohashi, P.S., Koyasu, S., Penninger, J.M., Νakano, T., and Mak, T.W. (2001). T cell-specific loss of Pten leads to defects in central and peripheral tolerance. Immunity 14, 523-534
Tamura, M., Gu, J., Matsumoto, K., Aota, S., Parsons, R., and Yamada, K.M. (1998). Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEΝ. Science 280, 1614-1617.
Toker, A., and Cantley, L.C. (1997). Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 357, 673-676.
Yusuf, S., Reddy, S., Ounpuu, S., and Anand, S. (2001). Global burden of cardiovascular diseases Part I: General considerations, the epidemiologic transition, risk factors, and impact of urbanization. Circulation 104, 2746-2753.
Xiao, R.P., Avdonin, P., Zhou, Y.Y., Cheng, H., Akhter, S.A., Eschenhagen, T., Lefkowitz, R.J., Koch, W.J., and Lakatta, E.G. (1999). Coupling of beta2- adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ. Res. 84, 43-52.
Zvaritch, E. et al. The transgenic expression of highly inhibitory monomeric forms of phospholamban in mouse heart impairs cardiac contractility. J. Biol. Chem. 275, 14985-14991 (2000).

Claims

Claims:
1. A use of an inhibitor of PBKgamma to prepare a medicament to prevent or treat heart disease.
2. A use according to claims 1-6 wherein the inhibitor of PBKgamma comprises inactive P13Kgamma, an inactive fragment of P13Kgamma, an antibody to PBKgamma or an antisense oligonucleotide that inhibits the expression of PBKgamma.
3. A use according to claim 1, wherein the inhibitor of PBKgamma is selected from the group consisting of wortmannin, Ly294002, 2-(4-morpholinyl)-8-phenyl-4H-l- benzopyran-4-one (LY294002) and quercetin and derivatives or analogues of the foregoing.
4. A method of medical treatment of heart disease in a mammal in need thereof, comprising administering to the mammal an inhibitor of PBKgamma.
5. The method of claim 4, wherein the inhibitor of PBKgamma is inactive P13Kgamma, an inactive fragment of P13Kgamma, an antibody to PBKgamma or an antisense oligonucleotide that inhibits the expression of PBKgamma.
6. The method of claim 4, wherein the inhibitor of PBKgamma is selected from the group consisting of wortmannin, Ly294002, 2-(4-morpholinyl)-8-ρlιenyl-4H-l- benzopyran-4-one (LY294002) and quercetin and derivatives or analogues of the foregoing.
7. The use or method of any one of claims 1-6, wherein the heart disease is selected from the group consisting of acute coronary syndromes, cardiac arrythmias and hypertension.
8. The use or method of claim 7, wherein the heart disease is selected from the group consisting of: congestive heart failure, angina, myocardial infarction (heart attack) atrial flutter, atrial fibrillation, paroxysmal supraventricular tachycardia and idiopathic hypertrophic subaortic stenosis.
9. A pharmaceutical composition for use in preventing or treating heart disease comprising an inhibitor of PBKgamma and a carrier.
10. A method for identifying a compound that inhibits the binding of a PBKgamma protein to its substrate for treatment of heart disease comprising: a) incubating (i) a candidate compound; (ii) an PBKgamma protein and (iii) an PBKgamma substrate under conditions which permit the binding of PBKgamma protein to the substrate; and b) assaying for complexes of PBKgamma protein and the substrate or metabolites thereby produced and comparing to a control, wherein a reduction of complexes or metabolites indicates that the candidate compound has an effect on the binding of the PBKgamma protein to the substrate; c) determining whether the candidate compound is useful for treatment of heart disease.
11. A compound identified according to claim 10.
PCT/CA2003/001387 2002-09-19 2003-09-19 Compositions and methods for treating heart disease WO2004026285A2 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002499368A CA2499368A1 (en) 2002-09-19 2003-09-19 Compositions and methods for treating heart disease
EP03750190A EP1539177A2 (en) 2002-09-19 2003-09-19 Compositions and methods for treating heart disease
AU2003269626A AU2003269626A1 (en) 2002-09-19 2003-09-19 Compositions and methods for treating heart disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA002400254A CA2400254A1 (en) 2002-09-19 2002-09-19 Compositions and methods for treating heart disease
CA2,400,254 2002-09-19

Publications (2)

Publication Number Publication Date
WO2004026285A2 true WO2004026285A2 (en) 2004-04-01
WO2004026285A3 WO2004026285A3 (en) 2004-07-29

Family

ID=32000082

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2003/001387 WO2004026285A2 (en) 2002-09-19 2003-09-19 Compositions and methods for treating heart disease

Country Status (4)

Country Link
EP (1) EP1539177A2 (en)
AU (1) AU2003269626A1 (en)
CA (1) CA2400254A1 (en)
WO (1) WO2004026285A2 (en)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8138195B2 (en) 2000-04-25 2012-03-20 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
US8440677B2 (en) 2009-03-24 2013-05-14 Gilead Calistoga Llc Atropisomers of 2-purinyl-3-tolyl-quinazolinone derivatives and methods of use
US8546409B2 (en) 2009-04-20 2013-10-01 Gilead Calistoga Llc Methods of treatment for solid tumors
USRE44599E1 (en) 2004-05-13 2013-11-12 Icos Corporation Quinazolinones as inhibitors of human phosphatidylinositol 3-kinase delta
US8691829B2 (en) 2009-07-21 2014-04-08 Gilead Calistoga Llc Treatment of liver disorders with PI3K inhibitors
US8865730B2 (en) 2012-03-05 2014-10-21 Gilead Calistoga Llc Polymorphic forms of (S)-2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one
US9238070B2 (en) 2008-11-13 2016-01-19 Gilead Calistoga Llc Therapies for hematologic malignancies
WO2016103176A1 (en) * 2014-12-24 2016-06-30 Kither Biotech S.R.L. Novel pi3k gamma inhibitor peptide for treatment of respiratory system diseases
US9492449B2 (en) 2008-11-13 2016-11-15 Gilead Calistoga Llc Therapies for hematologic malignancies
US9567337B2 (en) 2013-12-20 2017-02-14 Gilead Calistoga Llc Process methods for phosphatidylinositol 3-kinase inhibitors
US9708327B2 (en) 2013-12-20 2017-07-18 Gilead Calistoga Llc Polymorphic forms of a hydrochloride salt of (S)-2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one
US11021467B2 (en) 2014-06-13 2021-06-01 Gilead Sciences, Inc. Phosphatidylinositol 3-kinase inhibitors

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000003746A2 (en) * 1998-07-14 2000-01-27 The Brigham And Women's Hospital, Inc. Upregulation of type iii endothelial cell nitric oxide synthase by agents that disrupt actin cytoskeletal organization

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000003746A2 (en) * 1998-07-14 2000-01-27 The Brigham And Women's Hospital, Inc. Upregulation of type iii endothelial cell nitric oxide synthase by agents that disrupt actin cytoskeletal organization

Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8653077B2 (en) 2000-04-25 2014-02-18 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
US10695349B2 (en) 2000-04-25 2020-06-30 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
US10398695B2 (en) 2000-04-25 2019-09-03 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
US10010550B2 (en) 2000-04-25 2018-07-03 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
US9487772B2 (en) 2000-04-25 2016-11-08 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
US8138195B2 (en) 2000-04-25 2012-03-20 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
US8637533B2 (en) 2000-04-25 2014-01-28 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
US8779131B2 (en) 2004-05-13 2014-07-15 Icos Corporation 6-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one as an inhibitor of human phosphatidylinositol 3-kinase delta
US8586597B2 (en) 2004-05-13 2013-11-19 Icos Corporation 6-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)ethyl]-3H-quinazolin-4-one as an inhibitor of human phosphatidylinositol 3-kinase delta
USRE44638E1 (en) 2004-05-13 2013-12-10 Icos Corporation Quinazolinones as inhibitors of human phosphatidylinositol 3-kinase delta
US10906907B2 (en) 2004-05-13 2021-02-02 Icos Corporation Tert-butyl (s)-(1-(5-fluoro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)propyl)carbamate precursor of a quinazolinone inhibitor of human phosphatidylinositol 3-kinase delta and a process for preparing thereof
US8980901B2 (en) 2004-05-13 2015-03-17 Icos Corporation 5-fluoro-3-phenyl-2[1-(9H-purin-6-ylamino)propyl]-3H-quinazolin-4-one and 6-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)ethyl]-3H-quinazolin-4-one as inhibitors of human phosphatidylinositol 3-kinase delta
US8993583B2 (en) 2004-05-13 2015-03-31 Icos Corporation 5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)propyl]-3H-quinazolin-4-one and 6-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)ethyl]-3H-quinazolin-4-one as inhibitors of human phosphatidylinositol 3-kinase delta
US9149477B2 (en) 2004-05-13 2015-10-06 Icos Corporation 5-fluoro-3-phenyl-2-[1-(9h-purin-6-ylamino)propyl]-3h-quinazolin-4-one as an inhibitor of human phosphatidylinositol 3-kinase delta
US10336756B2 (en) 2004-05-13 2019-07-02 Icos Corporation (S)-2-(1-aminopropyl)-5-fluoro-3-phenylquinazolin-4(3H)-one precursor of a quinazolinone as inhibitor of human phosphatidylinositol 3-kinase delta
USRE44599E1 (en) 2004-05-13 2013-11-12 Icos Corporation Quinazolinones as inhibitors of human phosphatidylinositol 3-kinase delta
US9238070B2 (en) 2008-11-13 2016-01-19 Gilead Calistoga Llc Therapies for hematologic malignancies
US10154998B2 (en) 2008-11-13 2018-12-18 Gilead Calistoga Llc Therapies for hematologic malignancies
US9492449B2 (en) 2008-11-13 2016-11-15 Gilead Calistoga Llc Therapies for hematologic malignancies
US8440677B2 (en) 2009-03-24 2013-05-14 Gilead Calistoga Llc Atropisomers of 2-purinyl-3-tolyl-quinazolinone derivatives and methods of use
US8546409B2 (en) 2009-04-20 2013-10-01 Gilead Calistoga Llc Methods of treatment for solid tumors
US8691829B2 (en) 2009-07-21 2014-04-08 Gilead Calistoga Llc Treatment of liver disorders with PI3K inhibitors
US8865730B2 (en) 2012-03-05 2014-10-21 Gilead Calistoga Llc Polymorphic forms of (S)-2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one
US9469643B2 (en) 2012-03-05 2016-10-18 Gilead Calistoga, LLC. Polymorphic forms of (S)-2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one
US10730879B2 (en) 2012-03-05 2020-08-04 Gilead Calistoga Llc Polymorphic forms of (S)-2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one
US10954199B2 (en) 2013-12-20 2021-03-23 Gilead Sciences, Inc. Process methods for phosphatidylinositol 3-kinase inhibitors
US10059677B2 (en) 2013-12-20 2018-08-28 Gilead Calistoga Llc Process for preparing phosphatidylinositol 3-kinase inhibitors and intermediates thereof
US10047060B2 (en) 2013-12-20 2018-08-14 Gilead Calistoga Llc Process methods for phosphatidylinositol 3-kinase inhibitors
US10414737B2 (en) 2013-12-20 2019-09-17 Gilead Sciences, Inc. Process methods for phosphatidylinositol 3-kinase inhibitors
US10442805B2 (en) 2013-12-20 2019-10-15 Gilead Calistoga Llc Polymorphic forms of a hydrochloride salt of (S)-2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one
US9708327B2 (en) 2013-12-20 2017-07-18 Gilead Calistoga Llc Polymorphic forms of a hydrochloride salt of (S)-2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one
US9567337B2 (en) 2013-12-20 2017-02-14 Gilead Calistoga Llc Process methods for phosphatidylinositol 3-kinase inhibitors
US11021467B2 (en) 2014-06-13 2021-06-01 Gilead Sciences, Inc. Phosphatidylinositol 3-kinase inhibitors
WO2016103176A1 (en) * 2014-12-24 2016-06-30 Kither Biotech S.R.L. Novel pi3k gamma inhibitor peptide for treatment of respiratory system diseases
US10730921B2 (en) 2014-12-24 2020-08-04 Kither Biotech S.R.L. PI3Kγ inhibitor peptide for treatment of respiratory system diseases
US10421794B2 (en) 2014-12-24 2019-09-24 Kither Biotech Srl PI3K gamma inhibitor peptide for treatment of respiratory system diseases
CN107406488A (en) * 2014-12-24 2017-11-28 凯思生物技术公司 For treating the new PI3K gamma inhibitors peptide of respiratory disease
CN113563451A (en) * 2014-12-24 2021-10-29 凯思生物技术公司 Novel PI3K gamma inhibitor peptides for the treatment of respiratory diseases
US11352400B2 (en) 2014-12-24 2022-06-07 Kither Biotech Srl PI3K gamma inhibitor peptide for treatment of respiratory system diseases

Also Published As

Publication number Publication date
CA2400254A1 (en) 2004-03-19
EP1539177A2 (en) 2005-06-15
AU2003269626A1 (en) 2004-04-08
WO2004026285A3 (en) 2004-07-29

Similar Documents

Publication Publication Date Title
Crackower et al. Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways
Zhang et al. Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis
Kim et al. Divergent regulation of Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats.
Li et al. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex
US7189802B2 (en) Integrin-linked kinase and its uses
Li et al. β1 integrin gene excision in the adult murine cardiac myocyte causes defective mechanical and signaling responses
Cha et al. PICOT is a critical regulator of cardiac hypertrophy and cardiomyocyte contractility
EP1539177A2 (en) Compositions and methods for treating heart disease
Shi et al. Disruption of ROCK1 gene attenuates cardiac dilation and improves contractile function in pathological cardiac hypertrophy
Mende et al. Dilated cardiomyopathy in two transgenic mouse lines expressing activated G protein αq: lack of correlation between phospholipase C activation and the phenotype
Schaeffer et al. Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice
WO2005027902A1 (en) MODULATORS OF Cdk9 AS A THERAPEUTIC TARGET IN CARDIAC HYPERTROPHY
US6703215B2 (en) Inhibitors of the inositol polyphosphate 5-phosphatase SHIP2 molecule
US20080248032A1 (en) Compositions and methods for protection against cardiac and/or central nervous system tissue injury by inhibiting sphingosine-1-phosphate lyase
US20040242461A1 (en) Modulators of telomere stability
JP2002516670A (en) Methods for altering protein activity of the PKA signaling pathway
CA2499368A1 (en) Compositions and methods for treating heart disease
JP2007505628A (en) Modulation of cardiac contractility and heart failure tendency
WO2007137414A1 (en) Overexpression of integrin linked kinase (ilk) to improve post-infarct remodeling
US20140234321A1 (en) Hunk, a snf1-related kinase essential for mammary tumor metastasis
US20060153806A1 (en) Proteins involved in the regulation of energy homeostasis
US7794956B2 (en) Gab1 involvement in glucose homeostasis regulation by hepatocytes
Simpson The α 1-Adrenergic Receptors: Lessons From Knockouts
US20050180959A1 (en) Kinases involved in the regulation of energy homeostasis
JP2006502744A (en) Modulation of S6 kinase activity for obesity treatment

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2003269626

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2499368

Country of ref document: CA

Ref document number: 2003750190

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2004536720

Country of ref document: JP

WWP Wipo information: published in national office

Ref document number: 2003750190

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2003750190

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: JP