WO2006071603A9 - Protection de la phosphodiesterase 4d dans le complexe du recepteur de la ryanodine contre une insuffisance cardiaque - Google Patents

Protection de la phosphodiesterase 4d dans le complexe du recepteur de la ryanodine contre une insuffisance cardiaque

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Publication number
WO2006071603A9
WO2006071603A9 PCT/US2005/045914 US2005045914W WO2006071603A9 WO 2006071603 A9 WO2006071603 A9 WO 2006071603A9 US 2005045914 W US2005045914 W US 2005045914W WO 2006071603 A9 WO2006071603 A9 WO 2006071603A9
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WIPO (PCT)
Prior art keywords
pde
ryr2
receptor
agent
pde4d
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PCT/US2005/045914
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English (en)
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WO2006071603A3 (fr
WO2006071603A2 (fr
Inventor
Andrew R Marks
Stephan E Lehnart
Xander H T Wehrens
Steven Reiken
Marco Conti
Catherine S L Jin
Original Assignee
Univ Columbia
Andrew R Marks
Stephan E Lehnart
Xander H T Wehrens
Steven Reiken
Marco Conti
Catherine S L Jin
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Application filed by Univ Columbia, Andrew R Marks, Stephan E Lehnart, Xander H T Wehrens, Steven Reiken, Marco Conti, Catherine S L Jin filed Critical Univ Columbia
Priority to CA002591924A priority Critical patent/CA2591924A1/fr
Publication of WO2006071603A2 publication Critical patent/WO2006071603A2/fr
Publication of WO2006071603A3 publication Critical patent/WO2006071603A3/fr
Publication of WO2006071603A9 publication Critical patent/WO2006071603A9/fr

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    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04053—3',5'-Cyclic-AMP phosphodiesterase (3.1.4.53)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel compositions and methods to treat and prevent disorders and diseases associated with the RyR receptors that regulate calcium channel functioning in cells.
  • the sarcoplasmic reticulum is a structure in cells that functions, among other things, as a specialized intracellular calcium (Ca 2+ ) store. Channels in the SR called ryanodine receptors (RyRs) open and close to regulate the release of Ca 2+ from the SR into the intracellular cytoplasm of the cell. Release of Ca 2+ into the cytoplasm from the SR increases cytoplasmic Ca 2+ concentration.
  • Open probability (Po) of the RyR receptor refers to the likelihood that the RyR channel is open at any given moment, and therefore capable of releasing Ca 2+ into the cytoplasm from the SR.
  • RyRl is found predominantly in skeletal muscle as well as other tissues
  • RyR2 is found predominantly in the heart as well as other tissues
  • RyR3 is found in the brain as well as other tissues.
  • the RyR channels are formed by four RyR polypeptides in association with four FK506 binding proteins (FKBPs), specifically FKBP12 (calstabinl) and FKBP12.6 (calstabin2).
  • FKBPs FK506 binding proteins
  • Calstabinl binds to RyRl
  • calstabin2 binds to RyR2
  • calstabinl binds to RyR3.
  • the FKBP proteins (calstabinl and calstabin2) bind to the RyR channel (one molecule per RyR subunit), stabilize RyR-channel functioning, and facilitate coupled gating between neighboring RyR channels, thereby preventing abnormal activation of the channel during the channel's closed state.
  • PKA protein kinase A
  • Dissociation of calstabin from RyR causes increased open probability of RyR, and therefore increased Ca 2+ release from the SR into the intracellular cytoplasm.
  • Ca 2+ release from the SR in skeletal muscle cells and heart cells is a key physiological mechanism that controls muscle performance, because increased concentration of Ca 2+ in the intracellular cytoplasm causes contraction of the muscle.
  • Excitation-contraction (EC) coupling in skeletal muscles involves electrical depolarization of the plasma membrane in the transverse tubule (T-tubule), which activates voltage-gated L-type Ca 2+ channels (LTCCs).
  • LTCCs trigger Ca 2+ release from the SR through physical interaction with RyRl .
  • the resulting increase in cytoplasmic Ca + concentration induces actin-myosin interaction and muscle contraction.
  • intracellular Ca 2+ is pumped back into the SR via SR Ca 2+ - ATPase pumps (SERCAs), which is regulated by phospholamban (PLB) depending on the muscle fiber type.
  • SERCAs SR Ca 2+ - ATPase pumps
  • RyR2 is the major Ca 2+ - release channel required for EC coupling and muscle contraction.
  • depolarization of the cardiac-muscle cell membrane during phase zero of the action potential activates voltage-gated Ca 2+ channels.
  • Ca 2+ influx through the open voltage-gated channels in turn initiates Ca 2+ release from the SR via RyR2.
  • This process is known as Ca 2+ -induced Ca 2+ release.
  • the RyR2 -mediated, Ca 2+ -induced Ca 2+ release then activates the contractile proteins in the cardiac cell, resulting in cardiac muscle contraction.
  • Phosphorylation of cardiac RyR2 by PKA is an important part of the "fight or flight” response that increases cardiac EC coupling gain by augmenting the amount of Ca + released for a given trigger.
  • This signaling pathway provides a mechanism by which activation of the sympathetic nervous system, in response to stress, results in increased cardiac output.
  • PKA phosphorylation of RyR2 increases the open probability of the channel by dissociating calstabin2 (FKBP 12.6) from the channel complex. This, in turn, increases the sensitivity of RyR2 to Ca 2+ -dependent activation.
  • heart failure remains an important cause of mortality in Western countries.
  • An important hallmark of heart failure is reduced myocardial contractility.
  • contractile abnormalities result, in part, from alterations in the signaling pathway that allows the cardiac action potential to trigger Ca 2+ release via RyR2 channels and muscle contraction.
  • the amplitude of the whole-cell Ca transient is decreased and the duration prolonged.
  • Atrial fibrillation is the most common cardiac arrhythmia in humans, and represents a major cause of morbidity and mortality.
  • Structural and electrical remodeling including shortening of atrial refractoriness, loss of rate-related adaptation of refractoriness, and shortening of the wavelength of re-entrant wavelets - accompany sustained tachycardia. This remodeling is likely important in the development, maintenance and progression of atrial fibrillation. Studies suggest that calcium handling plays a role in electrical remodeling in atrial fibrillation.
  • SCD Sudden cardiac death
  • Catecholaminergic polymorphic ventricular tachycardia is an inherited disorder in individuals with structurally normal hearts. It is characterized by stress-induced ventricular tachycardia - a lethal arrhythmia that causes SCD. In subjects with CPVT, physical exertion and/or stress induce bidirectional and/or polymorphic ventricular tachycardias that lead to SCD even in the absence of detectable structural heart disease. CPVT is predominantly inherited in an autosomal-dominant fashion. Individuals with CPVT have ventricular arrhythmias when subjected to exercise, but do not develop arrhythmias at rest. Studies have identified mutations in the human RyR2 gene, on chromosome Iq42-q43, in individuals with CPVT.
  • Failing hearts are characterized by a maladaptive response that includes chronic hyperadrenergic stimulation.
  • chronic beta-adrenergic stimulation is associated with the activation of beta-adrenergic receptors in the heart, which, through coupling with G-proteins, activate adenylyl cyclase and thereby increase intracellular cAMP concentration.
  • cAMP activates cAMP-dependent PElA, which has been shown to induce hyperphosphorylation of RyR2.
  • chronic heart failure is a chronic hyperadrenergic state which results in several pathologic consequences, including PKA hyperphosphorylation of RyR2.
  • PKA-hyperphosphorylated RyR2 is very sensitive to low-level Ca 2+ stimulation, and this manifests itself as a diastolic SR Ca 2+ leak through the PKA hyperphosphorylated RyR2 channel.
  • a subpopulation of RyR2 that are particularly "leaky” can release SR Ca 2+ during the resting phase of the cardiac cycle, diastole. This results in depolarizations of the cardiomyocyte membrane known as delayed after-depolarizations (DADs), which are known to trigger fatal ventricular cardiac arrhythmias.
  • DADs delayed after-depolarizations
  • Cardiac arrhythmias are known to be associated with diastolic SR Ca 2+ leaks in patients with CPVT mutations in their RyR2 and otherwise structurally-normal hearts. In these cases, the most common mechanism for induction and maintenance of ventricular tachycardia is abnormal automaticity.
  • One form of abnormal automaticity known as triggered arrhythmia, is associated with aberrant release of SR Ca 2+ , which initiates DADs.
  • DADs are abnormal depolarizations in cardiomyocytes that occur after repolarization of a cardiac action potential.
  • the molecular basis for the abnormal SR Ca 2+ release that results in DADs has not been fully elucidated. However, DADs are known to be blocked by ryanodine, providing evidence that RyR2 plays a key role in the pathogenesis of this aberrant Ca 2+ release.
  • co-pending U.S. Patent Application No. 09/288,606 discusses a method for regulating contraction of a subject's heart by administering a compound which regulates PKA phosphorylation of an RyR2 receptor and specifically decreases PKA phosphorylation.
  • Co-pending U.S. Patent Application No. 10/608,723 also discusses a method for treating and prophylaxis for atrial tachyarrhythmia and exercise and stress-induced arrhythmias by administration of an agent which inhibits PKA phosphorylation of RyR2.
  • Phosphodiesterases control the temporal and spatial dynamics of the second messenger 3 ',5' cyclic adenosine monophosphate (cAMP), allowing for highly localized cAMP gradients in cells (Zaccolo and Pozzan, 2002). Localization of PDEs in close proximity to cAMP-dependent protein kinase A (PKA) is thought to control access of cAMP to the regulatory kinase subunit (Conti et al., 2003 and Houslay and Adams, 2003). PKA phosphorylation of proteins mediates a wide variety of signals, including those generated during activation of the sympathetic nervous system (SNS) as part of the "fight or flight" response. On the other hand, chronic activation of the SNS is a characteristic finding in heart failure, and acute stimulation of the SNS has been linked to triggered arrhythmias associated with sudden cardiac death.
  • SNS sympathetic nervous system
  • PDE4 phosphodiesterase 4
  • T transverse
  • SR tubule/sarcoplasmic reticulum
  • AKAPs muscle A-kinase anchoring proteins
  • PDEs in cardiac muscle are complexed with proteins that mediate signals from SNS, including ⁇ -adrenergic receptors and ⁇ -arrestin (Mongillo et al., 2004, Perry et al., 2002 and Xiang et al., 2005).
  • the PDE superfamily is subgrouped into 11 families that include at least
  • PDE4D cAMP specific (Conti et al., 2003).
  • PDE4D1-9 nine variants (PDE4D1-9) with identical catalytic domains and carboxyl termini and unique amino termini important for subcellular localization.
  • PDE4D3 binds to the targeting protein mAKAP via its unique N-terminal region, creating a mAKAP -PKA-PDE4D3 signaling module (Dodge et al., 2001 and Tasken et al., 2001) in which PKA phosphorylation increases PDE4D3 activity approximately 2-fold (Carlisle Michel et al., 2004 and Sette and Conti, 1996).
  • mAKAP colocalizes with the ryanodine receptor (RyR2)/calcium-release channel in cardiac muscle (Ruehr et al., 2003 and Yang et al., 1998), where it is part of the RyR2 macromolecular signaling complex (Marx et al., 2000 and Wehrens et al., 2003).
  • RyR2 ryanodine receptor
  • PKA-PDE signaling has been identified as a therapeutic target in several major diseases (Conti et al., 2003). Inhibitors of the PDE4 family are under development for asthma, chronic obstructive lung disease (COPD), cognitive disorders including Alzheimer's disease, and stroke (Gong et al., 2004, Gretarsdottir et al., 2003 and Vignola, 2004). However, nonspecific PDE inhibition with theophylline, commonly used to treat asthma and COPD, and trials using PDE3 inhibition to treat heart failure have demonstrated increased mortality due to cardiac arrhythmias (Barnes, 2003 and Packer et al., 1991).
  • RyR2 channels were PKA hyperphosphorylated and exhibited a "leaky” phenotype in PDE4D-deficient mice, similar to RyR2 defects previously observed by the inventors in patients with heart failure and sudden cardiac death (SCD) (Marx et al., 2000 and Wehrens et al., 2003). In failing human hearts, PDE4D3 levels were reduced in the RyR2 complex. Moreover, mice with PDE4D deficiency exhibited accelerated progression of heart failure following myocardial infarction associated with RyR2 channels that were PKA hyperphosphorylated and exhibited a "leaky” phenotype.
  • SCD sudden cardiac death
  • the inventors now show, for the first time that that PDE4D plays a protective role in the heart against heart failure and arrhythmias.
  • the inventors also demonstrate herein that PDE deficiency is associated with a severe cardiac phenotype consisting of heart failure and lethal cardiac arrhythmias.
  • PDE4D3 deficiency in the RyR2 complex contributes to PKA hypersphosporylation of RyR2 in human and animal hearts, and that PDE4D3 activity provides an important negative feedback mechanism to limit ⁇ -AR-dependent PKA phosphorylation of RyR2-Ser2808.
  • the present invention generally provides compositions useful for treating or preventing a ryanodine receptor associated disorder comprising: a phosphodiesterase (PDE)-associated agent; and optionally a pharmaceutically acceptable carrier.
  • the PDE-associated agent may be a PDE protein, a nucleic acid encoding a PDE protein, a member of a PDE signal- transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the PDE-associated agent is PDE4D protein or a nucleic acid encoding a PDE4D protein.
  • the ryanodine receptor associated disorder may be a RyR2-l, RyR2 or RyR3 associated disorder.
  • kits for use in delivering a PDE- associated agent to a ryanodine receptor complex in a subject comprising a PDE- associated agent, optionally with a pharmaceutically acceptable carrier and a catheter.
  • the invention provides methods for treating or preventing a ryanodine receptor associated disorder in a subject comprising augmenting PDE in a ryanodine receptor complex of the subject.
  • PDE is augmented in the ryanodine receptor complex by contacting the ryanodine receptor complex with a PDE-associated agent.
  • the PDE-associated agent is selected from the group consisting of a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal-transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the nucleic acid is operatively linked to an inducible promoter.
  • the phosphodiesterase is PDE4D including PDE4D3.
  • the subject of the present invention may be any animal, including amphibians, birds, fish mammals, and marsupials, but is preferably a mammal, including but not necessarily limited to a mouse, rat, cat, dog, horse, monkey cow or pig. In an preferred embodiment, the subject is human.
  • the ryanodine receptor associated disorder is a RyRl receptor associated disorder.
  • the ryanodine receptor associated disorder is a RyR2 receptor associated disorder.
  • the ryanodine receptor associated disorder is an RyR3 receptor associated disorder.
  • the ryanodine receptor associated disorder may be a cardiac disorder and/or disease including, but not limited to, an irregular heartbeat disorder or disease; exercise-induced irregular heart beat disorder or disease; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure.
  • the irregular heartbeat disorders and diseases and exercise-induced irregular heartbeat disorders and diseases may include, but are not necessarily limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catechlaminergic polymorphic ventricular tachycardia (CPTV); and exercise-induced variants thereof.
  • the ryanodine receptor associated disorder is a skeletal muscular disorder and/or disease, including but not limited to skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence.
  • the ryanodine receptor associated disorder is a cognitive disorder and/or disease including, but not necessarily limited to Alzheimer's Disease, dementia, forms of memory loss, and age- dependent memory loss.
  • the ryanodine receptor associated disorder is a malignant hyperthermia, diabetes, or sudden infant death syndrome.
  • the present invention additionally provides methods for regulating PKA phosphorylation of a ryanodine receptor comprising contacting the ryanodine receptor complex with an agent that modulates the level of PDE in the ryanodine receptor complex, wherein contacting the ryanodine receptor complex with an agent that increases the level of PDE in the complex results in a reduction of PKA phosporylation of the ryanodine receptor, and contacting the ryanodine receptor complex with an agent that decreases the level of PDE in the complex results in an increase of PKA phosporylation of the ryanodine receptor.
  • the agent is selected from the group consisting of a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal-transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the phosphodiesterase is PDE4D or PDE4D3.
  • the ryanodine receptor is a
  • the ryanodine receptor is a RyR2 receptor. In a further embodiment, the ryanodine receptor is an RyR3 receptor.
  • Also provided by the present invention are methods for decreasing PKA phosphorylation of a ryanodine receptor by contacting the ryanodine receptor complex with an agent that increases the level of PDE in the ryanodine receptor complex.
  • the receptor is hyperphosphorylated prior to contacting the ryanodine receptor complex with the agent.
  • the agent is a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal-transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the phosphodiesterase is PDE4D or PDE4D3.
  • the ryanodine receptor is a RyRl receptor. In another embodiment , the ryanodine receptor is a RyR2 associated receptor. In a further embodiment, the ryanodine receptor is a RyR3 associated receptor.
  • the present invention additionally provides methods for regulating Ca release and reuptake in the sarcoplasmic reticulum of a cell comprising contacting a ryanodine receptor complex of the cell with an agent that modulates the level of PDE, wherein contacting the ryanodine receptor complex with an agent that increases the level of PDE results in a reduction of Ca release from and reuptake into the sarcoplasmic reticulum and contacting the ryanodine receptor complex with an agent that decreases the level of PDE in the complex results in an increase of Ca 2+ release from and reuptake into the sarcoplasmic reticulum.
  • the agent is selected from the group consisting of a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal- transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the phosphodiesterase is PDE4D or PDE4D3.
  • the ryanodine receptor is a RyRl receptor. In another embodiment, the ryanodine receptor is a RyR2 receptor. In a further embodiment, the ryanodine receptor is a RyR3 receptor.
  • the present invention further provides methods for decreasing Ca 2+ release and reuptake in the sarcoplasmic reticulum of a cell comprising contacting a ryanodine receptor complex of the cell with an agent that increases the level of PDE.
  • the receptor is hyperphosphorylated prior to contacting the ryanodine receptor complex with the agent.
  • the agent is selected from the group consisting of a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal-transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the phosphodiesterase is PDE4D, including PDE4D3.
  • the ryanodine receptor is a RyRl receptor. In another embodiment, the ryanodine receptor is a RyR2 receptor. In a further embodiment, the ryanodine receptor is a RyR3 receptor.
  • FIG. 1 PDE4D Deficiency Promotes Age-Related Cardiomyopathy. *p ⁇
  • C Age-dependent decrease in ejection fraction (EF) in PDE4D ⁇ / ⁇ mice (open bar, wt; filled bar, PDE4D ⁇ 7 ⁇ ).
  • D Reduced cardiac contractility (dP/dt/P id ) in PDE4D ⁇ / ⁇ mice at 3, 9, and 15 months of age (open squares, wt; filled squares, PDE4D "7" ).
  • E Histology showing dilated cardiomyopathy in PDE4D-deficient mouse hearts.
  • FIG. 2 Normal cAMP and ⁇ -Adrenergic-Receptor Levels in PDE4D "7"
  • FIG. 3 shows Age-Dependent Alterations in RyR2-Channel Complex
  • Channel openings are upward; full openings are 4 pA; closed state is indicated by “c.”
  • FIG. 4 PDE4D3 Is a Component of the RyR2 Ca 2+ -Release-Channel
  • FIG. 5 Reduced PDE4D3 in the RyR2 Complex in Human Heart Failure.
  • FIG. 6 Cardiac Arrhythmias due to PDE4D3 Inhibition Are Suppressed in
  • FIG. 7 PDE4D3 Deficiency Promotes HF Progression.
  • A LVEDD increased in PDE4D +/ ⁇ (black squares) compared to wt (open squares) mice before (control, CO) and 14 and 28 days after myocardial infarction (MI) (*p ⁇ 0.05 versus wt).
  • PDE4D3 activity provides an important negative feedback mechanism to limit ⁇ AR-dependent PKA phosphorylation of RyR2-Ser 2809 .
  • PDE4D3 regulates local PKA activity and channel activation at RyR2-Ser 2809 and prevents excess accumulation of cAMP and uncontrolled PKA activation.
  • PDE4D3 deficiency contributes to RyR2 PKA hyperphosphorylation, calstabin2 (FKBP 12.6) depletion and hyperactive, "leaky" RyR2 channels.
  • the present invention provides novel compounds and methods for treating and preventing disorders and diseases associated with the RyR receptors that regulate calcium channel functioning in cells.
  • the present invention encompasses and provides compositions useful for treating or preventing a ryanodine receptor associated disorder comprising a phosphodiesterase (PDE)-associated agent; and optionally a pharmaceutically acceptable carrier.
  • the PDE-associated agent may be a PDE protein, a nucleic acid encoding a PDE protein, a member of a PDE signal- transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the PDE-associated agent is PDE4D protein or a nucleic acid encoding a PDE4D protein.
  • the ryanodine receptor associated disorder may be a RyR2-l, RyR2 or RyR3 associated disorder.
  • disorders and diseases associated with the RyR receptors means disorders and diseases that can be treated and/or prevented by modulating PDE in the RyR receptor complex in cells.
  • “Ryanodine receptor associated disorders” and/or “Disorders and diseases associated with the RyR receptors” include, without limitation, cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome.
  • Cardiac disorder and diseases include, but are not limited to, irregular heartbeat disorders and diseases; exercise-induced irregular heartbeat disorders and diseases; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure.
  • Irregular heartbeat disorders and diseases include and exercise-induced irregular heartbeat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catecholaminergic polymorphic ventricular tachycardia (CPVT); and exercise-induced variants thereof.
  • Skeletal muscular disorder and diseases include, but are not limited to, skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence.
  • Cognitive disorders and diseases include, but are not limited to, Alzheimer's Disease, forms of memory loss, and age-dependent memory loss.
  • RyR includes RyRl , RyR2, and RyR3, and also includes an "RyR protein” and an "RyR analogue.”
  • An "RyR analogue” is a functional variant of the RyR protein, having RyR biological activity, that has 60% or greater amino-acid-sequence homology with the RyR protein.
  • the RyR of the present invention are unphosphorylated, phosphorylated (e.g., by PKA), or hyperphosphorylated (e.g., by PKA).
  • RyR biological activity refers to the activity of a protein or peptide that demonstrates an ability to associate physically with, or bind with, FKBP12 (calstabinl) in the case of RyRl and RyR3, and FKBP12.6 (calstabin2) in the case of RyR2 (i.e., binding of approximately two fold or, approximately five fold, above the background binding of a negative control), under the conditions of the assays described herein.
  • RyR complex refers to the RyR macromolecular signaling complex as described herein.
  • the present invention provides methods for treating or preventing a ryanodine receptor associated disorder in a subject comprising augmenting PDE in a ryanodine receptor complex of the subject.
  • PDE is augmented in the ryanodine receptor complex by contacting the ryanodine receptor complex with a PDE-associated agent.
  • the PDE-associated agent is selected from the group consisting of a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal-transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the nucleic acid is operatively linked to an inducible promoter.
  • the phosphodiesterase is PDE4D including PDE4D3.
  • the subject of the present invention may be an in vitro or in vivo system and any animal, including amphibians, birds, fish mammals, and marsupials, but is preferably a mammal, including but not necessarily limited to a mouse, rat, cat, dog, horse, monkey cow or pig. In a preferred embodiment of the invention, the subject is human.
  • the ryanodine receptor associated disorder is a RyRl receptor associated disorder.
  • the ryanodine receptor associated disorder is a RyR2 receptor associated disorder.
  • the ryanodine receptor associated disorder is an RyR3 receptor associated disorder.
  • the cells of a subject include striated muscle cells.
  • a striated muscle is a muscle in which the repeating units (sarcomeres) of the contractile myofibrils are arranged in registry throughout the cell, resulting in transverse or oblique striations that are observed at the level of a light microscope.
  • Examples of striated muscle cells include, without limitation, voluntary (skeletal) muscle cells and cardiac muscle cells.
  • the cell used in the method of the present invention is a human cardiac muscle cell.
  • the term "cardiac muscle cell” includes cardiac muscle fibers, such as those found in the myocardium of the heart.
  • Cardiac muscle fibers are composed of chains of contiguous heart-muscle cells, or cardiomyocytes, joined end to end at intercalated disks. These disks possess two kinds of cell junctions: expanded desmosomes extending along their transverse portions, and gap junctions, the largest of which lie along their longitudinal portions.
  • the ryanodine receptor associated disorder may be a cardiac disorder and/or disease including, but not limited to, an irregular heartbeat disorder or disease; exercise-induced irregular heart beat disorder or disease; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure.
  • the irregular heartbeat disorders and diseases and exercise-induced irregular heartbeat disorders and diseases may include, but are not necessarily limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catechlaminergic polymorphic ventricular tachycardia (CPTV); and exercise-induced variants thereof.
  • the ryanodine receptor associated disorder is a skeletal muscular disorder and/or disease, including but not limited to skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence.
  • the ryanodine receptor associated disorder is a cognitive disorder and/or disease including, but not necessarily limited to Alzheimer's Disease, dementia, forms of memory loss, and age- dependent memory loss.
  • the ryanodine receptor associated disorder is a malignant hyperthermia, diabetes, or sudden infant death syndrome.
  • an effective amount or “pharmaceutically effective amount” refers to any amount of an agent which, when administered to a subject suffering from a disorder against which the agent is effective, causes reduction, remission or regression or prevents recurrence of the disorder.
  • “Prophylactically effective amount” refers to any amount of an agent which, when administered to a subject prone to suffer from a disorder, inhibits the onset of the disorder.
  • the present invention additionally provides methods for regulating PKA phosphorylation of a ryanodine receptor comprising contacting the ryanodine receptor complex with an agent that modulates the level of PDE in the ryanodine receptor complex, wherein contacting the ryanodine receptor complex with an agent that increases the level of PDE in the complex results in a reduction of PKA phosporylation of the ryanodine receptor, and contacting the ryanodine receptor complex with an agent that decreases the level of PDE in the complex results in an increase of PKA phosporylation of the ryanodine receptor,
  • the agent is selected from the group consisting of a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal-transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the phosphodiesterase is PDE4D or PDE4D
  • the ryanodine receptor is a
  • the ryanodine receptor is a RyR2 receptor. In a further embodiment, the ryanodine receptor is an RyR3 receptor.
  • PKA phosphorylation refers to a reaction in which a phosphate group is substituted for a hydroxyl group by the enzyme protein kinase A (PKA).
  • kits for use in delivering a PDE- associated agent to a ryanodine receptor complex in a subject comprising a PDE- associated agent, optionally with a pharmaceutically acceptable carrier and a catheter.
  • Also provided by the present invention are methods for decreasing PKA phosphorylation of a ryanodine receptor by contacting the ryanodine receptor complex with an agent that increases the level of PDE in the ryanodine receptor complex.
  • the receptor is hyperphosphorylated prior to contacting the ryanodine receptor complex with the agent.
  • the agent is a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal-transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the phosphodiesterase is PDE4D or PDE4D3.
  • the ryanodine receptor is a RyRl receptor. In another embodiment , the ryanodine receptor is a RyR2 associated receptor. In a further embodiment, the ryanodine receptor is a RyR3 associated receptor.
  • the present invention additionally provides methods for regulating Ca 2+ release and reuptake in the sarcoplasmic reticulum of a cell comprising contacting a ryanodine receptor complex of the cell with an agent that modulates the level of PDE, wherein contacting the ryanodine receptor complex with an agent that increases the level of PDE results in a reduction Of Ca 2+ release from and reuptake into the sarcoplasmic reticulum and contacting the ryanodine receptor complex with an agent that decreases the level of PDE in the complex results in an increase of Ca 2+ release from and reuptake into the sarcoplasmic reticulum.
  • the agent is selected from the group consisting of a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal- transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the phosphodiesterase is PDE4D or PDE4D3.
  • the ryanodine receptor is a RyRl receptor. In another embodiment, the ryanodine receptor is a RyR2 receptor. In a further embodiment, the ryanodine receptor is a RyR3 receptor.
  • the present invention further provides methods for decreasing Ca 2+ release and reuptake in the sarcoplasmic reticulum of a cell comprising contacting a ryanodine receptor complex of the cell with an agent that increases the level of PDE.
  • the receptor is hyperphosphorylated prior to contacting the ryanodine receptor complex with the agent.
  • the agent is selected from the group consisting of a PDE protein, a nucleic acid encoding a PDE, a member of a PDE signal-transduction pathway, and a modulator of a member of a PDE signal transduction pathway.
  • the phosphodiesterase is PDE4D, including PDE4D3.
  • the ryanodine receptor is a RyRl receptor. In another embodiment, the ryanodine receptor is a RyR2 receptor. In a further embodiment, the ryanodine receptor is a RyR3 receptor.
  • PDEs are enzyme proteins, found in certain cells, which hydrolyze phosphodiester bonds. PDEs regulate the local concentration of 3', 5' cyclic adenosine monophosphate (cAMP) within cells. In the heart, PDE4 contributes to the regulation of c AMP levels in cardiac myocytes.
  • the PDE superfamily is subgrouped into 11 families tht include at least 20 genes and 50 unique isoforms.
  • the PDE4D gene encodes nnine variants (PDE4D1-9) with identical catalytic domains and carboxyl termini and unique amino termini important for subcellular localization. In a preferred embodiment of the present invention, the PDE is PDE4D3.
  • PDE includes both a “PDE protein” and a “PDE analogue”.
  • protein shall include a protein, protein domain, polypeptide, or peptide, and any fragment or variant thereof having protein function.
  • the variants preferably have greater than about 75% homology with the naturally-occurring protein sequence, more preferably have greater than about 80% homology, even more preferably have greater than about 85% homology, and most preferably, have greater than about 90% homology with the protein sequence. In some embodiments, the homology may be as high as about 93-95%, 98%, or 99%.
  • These variants may be substitutional, insertional, or deletional variants.
  • the variants may also be chemically-modified derivatives: proteins which have been subjected to chemical modification, but which retain the biological characteristics of the naturally-occurring protein.
  • PDE analogue is a functional variant of the PDE protein, having PDE biological activity, that has 60% or greater (preferably, 70% or greater) amino-acid-sequence homology with the PDE protein.
  • PDE biological activity refers to the activity of a protein or peptide that demonstrates an ability to hydrolyze cAMP, as described herein.
  • PDE may be augmented or increased in a cell or subcellular compartment, or more particularly, in a ryanodine receptor complex (RyR complex) of a cell by activating, facilitating, inducing, or stimulating one or more functions, activities, or effects (e.g., downstream effects of the PDE in the PDE signal transduction pathway) of PDE in a cell or in a RyR of a cell, particularly those that result in promotion of heart-tissue generation, or by increasing the amount, expression, or level of PDE in the cells.
  • RyR complex ryanodine receptor complex
  • one or more PDE functions, activities, effects, expression, and levels in a cell, subcellular compartment or RyR complex may be augmented by targeting PDE directly, or by targeting PDE indirectly, via an enzyme or other endogenous molecule that regulates or modulates the functions, activities, effects, expression, and/or levels of PDE in the cell.
  • PDE expression may also be augmented by engineering the PDE gene so that PDE is expressed on an inducible promoter. In such a case, PDE expression would be sustained in the presence of a suitable inducing agent, but would shut down once the supply of inducer was depleted, thereby bringing about a decrease in the amount or level of PDE in the cell.
  • PDE also may be augmented in a cell or RyR complex by activating, facilitating, inducing, or stimulating the functions, activities, effects, expression, and levels of endogenous PDE, or by introduction of an exogenous PDE, particularly where the PDE is under the control of a strong promoter.
  • the functions, activities, effects, expression, and/or levels of PDE are augmented in a cells or RyR complex by an amount effective to promote hydrolyzation of c AMP. This amount may be readily determined by the skilled artisan, based upon known procedures, including analysis of titration curves established in vivo, methods disclosed herein, and techniques known to one of skill in the art.
  • the functions, activities, effects, expression, and/or levels of PDE in a cell, subcellular compartment or RyR complex are preferably augmented by contacting the cells (i.e., treating the cells) with a PDE- associated agent.
  • a PDE-associated agent shall include a protein, polypeptide, peptide, nucleic acid (including DNA, RNA, and an antisense oligonucleotide), antibody (monoclonal and polyclonal), Fab fragment, F(ab')2 fragment, molecule, compound, antibiotic, drug, and any combinations thereof, and may be an agent reactive with PDE or a member of a PDE signal transduction pathway.
  • a Fab fragment is a univalent antigen-binding fragment of an antibody, which is produced by papain digestion.
  • a F(ab')2 fragment is a divalent antigen-binding fragment of an antibody, which is produced by pepsin digestion.
  • PDE-associated agent includes a PDE protein, including an exogenous PDE protein; a PDE nucleic acid (i, e., a nucleic acid encoding a PDE); a member of a PDE signal-transduction pathway (including upstream and downstream effectors and activators, in either protein or nucleic acid form); and a modulator (e.g., inhibitor, activator, antagonist, or agonist) of a member of the PDE signal transduction pathway or system (i.e., a modulator which affects the expression, activity, function, and/or effect of a member of the PDE signal-transduction pathway), in either protein or nucleic acid form, including a modulator of PDE expression.
  • a "member of a PDE signal-transduction pathway” includes a downstream effector or an upstream regulator of PDE in cells.
  • RyR complex may be augmented by contacting the cells or RyR complex with a small molecule or protein mimetic that stimulates PDE activity and/or that is reactive with PDE or a member of a PDE signal transduction pathway.
  • the level of PDE in a cell or RyR complex may be augmented by directly or indirectly causing, inducing, or stimulating the upregulation of PDE expression within a subject. Accordingly, in one embodiment of the present invention, activity of PDE is increased in a subject by administering to the subject a modulator of PDE expression.
  • the PDE-associated agent is a protein.
  • proteins for use in the present invention include, without limitation, PDE proteins, members of the PDE signal-transduction pathway (including upstream and downstream effector and activator polypeptides), modulators (e.g., inhibitors, activators, antagonists, or agonists) of a member of the PDE signal-transduction pathway/system, PDE-associated antibodies (e.g., IgA, IgD, IgE, IgG, IgM, single-chain antibodies, and Fab' fragments, such as scFv) that are capable of binding and inhibiting a negative regulator of the PDE signal-transduction pathway, and PDE-associated ligands (e.g., a ligand for a member of the PDE signal-transduction pathway, and derivatives thereof).
  • the PDE-associated protein is PDE4D protein.
  • the protein of the present invention is an antibody
  • the protein is preferably a mammalian antibody (e.g., a human antibody) or a chimeric antibody (e.g., a humanized antibody). More preferably, the antibody is a human or humanized antibody.
  • the term "humanized antibody” refers to a genetically-engineered antibody in which the minimum portion of an animal antibody (e.g., an antibody of a mouse, rat, pig, goat, or chicken) that is generally essential for its specific functions is "fused" onto a human antibody.
  • a humanized antibody is 1-25%, preferably 5- 10%, animal; the remainder is human. Humanized antibodies usually initiate minimal or no response in the human immune system.
  • the antibody is a single-chain antibody.
  • the single-chain antibody is a human or humanized single-chain antibody.
  • the antibody is a murine antibody.
  • the PDE-associated agent of the present invention may also be a nucleic acid.
  • a "nucleic acid” or “polynucleotide” includes a nucleic acid, an oligonucleotide, a nucleotide, a polynucleotide, and any fragment or variant thereof.
  • the nucleic acid or polynucleotide may be double-stranded, single-stranded, or triple-stranded DNA or RNA (including cDNA), or a DNA-RNA hybrid of genetic or synthetic origin, wherein the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides and any combination of bases, including, but not limited to, adenine, thymine, cytosine, guanine, uracil, inosine, and xanthine hypoxanthine.
  • the nucleic acid or polynucleotide may be combined with a carbohydrate, lipid, protein, or other materials. In one embodiment of the present invention, the nucleic acid encodes PDE4D protein.
  • nucleic acid refers, herein, to a nucleic acid molecule which is completely complementary to another nucleic acid, or which will hybridize to the other nucleic acid under conditions of high stringency.
  • High-stringency conditions are known in the art (see e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor: Cold Spring Harbor Laboratory, 1989) and Ausubel et al., eds., Current Protocols in Molecular Biology (New York, NY: John Wiley & Sons, Inc., 2001)).
  • Stringent conditions are sequence-dependent, and may vary depending upon the circumstances.
  • cDNA refers to an isolated DNA polynucleotide or nucleic acid molecule, or any fragment, derivative, or complement thereof. It may be double-stranded, single-stranded, or triple-stranded, it may have originated recombinantly or synthetically, and it may represent coding and/or noncoding 5' and/or 3' sequences.
  • the nucleic acid agent of the present invention may be a plasmid.
  • a plasmid may comprise a nucleic acid sequence encoding PDE or another PDE-associated protein, although it is to be understood that other types of nucleic acid agents, such as recombinant viral vectors, may also be used for the purposes of the present invention.
  • the nucleic acid e.g., plasmid
  • the nucleic acid encodes at least one PDE-associated protein.
  • the nucleic acid encodes PDE4D protein.
  • plasmid refers generally to circular double- stranded DNA, which is not bound to a chromosome.
  • the DNA for example, may be a chromosomal or episomal-derived plasmid.
  • the plasmid of the present invention may optionally contain a terminator of transcription, a promoter, and/or a discrete series of restriction-endonuclease recognition sites, located between the promoter and the terminator.
  • a polynucleotide insert of interest e.g., one encoding a PDE- associated protein
  • the promoter may be its native promoter or a host-derived promoter.
  • the promoter may also be a tissue-specific promoter, such as a cardiomyocyte-specific promoter or other heart- tissue-specific promoter.
  • the promoter may further be a regulatable promoter, which may be turned off when the expression of the gene is no longer desired. Examples of promoters for use in the present invention include the actin promoter and viral promoters. Other suitable promoters will be known to the skilled artisan.
  • the nucleic acid encodes or comprises at least one gene-silencing cassette, wherein the cassette is capable of silencing the expression of genes that negatively affect the PDE signal- transduction pathway/system.
  • a gene may be silenced at a number of stages, including, without limitation, pre-transcription silencing, transcription silencing, translation silencing, post-transcription silencing, and post- translation silencing.
  • the gene-silencing cassette encodes or comprises a post-transcription gene-silencing composition, such as antisense RNA or RNAi. Both antisense RNA and RNAi may be produced in vitro, in vivo, ex vivo, or in situ.
  • the PDE-associated agent of the present invention may be an antisense RNA.
  • Antisense RNA is an RNA molecule with a sequence complementary to a specific RNA transcript, or niRNA, whose binding prevents further processing of the transcript or translation of the mRNA.
  • Antisense molecules may be generated, synthetically or recombinantly, with a nucleic-acid vector expressing an antisense gene- silencing cassette.
  • Such antisense molecules may be single-stranded RNAs or DNAs, with lengths as short as 15-20 bases or as long as a sequence complementary to the entire mRNA. RNA molecules are sensitive to nucleases.
  • an antisense deoxyoligonucleotide may be synthesized as a phosphorothioate, in which one of the nonbridging oxygens surrounding the phosphate group of the deoxynucleotide is replaced with a sulfur atom (Stein et al., Oligodeoxynucleotides as inhibitors of gene expression: a review, Cancer Res., 48:2659-68, 1998).
  • Antisense molecules designed to bind to the entire mRNA may be made by inserting cDNA into an expression plasmid in the opposite or antisense orientation. Antisense molecules may also function by preventing translation initiation factors from binding near the 5' cap site of the mRNA, or by interfering with interaction of the mRNA and ribosomes (see e.g., U.S. Patent No. 6,448,080, Antisense modulation of WRN expression; U.S. Patent Application No. 2003/0018993, Methods of gene silencing using inverted repeat sequences; U.S.
  • Patent Application No., 2003/0017549 Methods and compositions for expressing polynucleotides specifically in smooth muscle cells in vivo; Tavian et al. 5 Stable expression of antisense urokinase mRNA inhibits the proliferation and invasion of human hepatocellular carcinoma cells, Cancer Gene Ther., 10:112-20, 2003; Maxwell and Rivera, Proline oxidase induces apoptosis in tumor cells and its expression is absent or reduced in renal carcinoma, J. Biol. Chem., 278:9784-89, 2003; Ghosh et al., Role of superoxide dismutase in survival of Leishmania within the macrophage, Biochem. J., 369:447-52, 2003; and Zhang et al., An anti-sense construct of full-length ATM cDNA imposes a radiosensitive phenotype on normal cells, Oncogene, 17:811-8, 1998).
  • Oligonucleotides antisense to a member of the PDE signal-transduction pathway/system may be designed based on the nucleotide sequence of the member of interest. For example, a partial sequence of the nucleotide sequence of interest (generally, 15-20 base pairs), or a variation sequence thereof, may be selected for the design of an antisense oligonucleotide. This portion of the nucleotide sequence may be within the 5' domain.
  • a nucleotide sequence complementary to the selected partial sequence of the gene of interest, or the selected variation sequence then may be chemically synthesized using one of a variety of techniques known to those skilled in the art, including, without limitation, automated synthesis of oligonucleotides having sequences which correspond to a partial sequence of the nucleotide sequence of interest, or a variation sequence thereof, using commercially-available oligonucleotide synthesizers, such as the Applied Biosystems Model 392 DNA/RNA synthesizer.
  • oligonucleotide synthesizers such as the Applied Biosystems Model 392 DNA/RNA synthesizer.
  • the antisense oligonucleotide may be contacted with a cells or RyR receptor complex, and the levels of PDE expression or activity in the cells or RyR complex may be determined using standard techniques, such as Western-blot analysis and immunostaining.
  • the antisense oligonucleotide may be delivered to a cell or RyR complex using a liposome vehicle, then the levels of PDE expression or activity in the cells may be determined using standard techniques, such as Western-blot analysis and inununostaining.
  • the oligonucleotide could be an appropriate PDE-associated agent for use in modulating PDE in cells.
  • oligonucleotides antisense to a member of the PDE signal-transduction pathway/system may be linked to another agent, such as a drug or a ribozyme, in order to increase the effectiveness of treatments using PDE-associated agents and/or to increase the efficacy of targeting.
  • antisense oligonucleotides may be prepared using modified bases (e.g., a phosphorothioate), as discussed above, to make the oligonucleotides more stable and better able to withstand degradation.
  • the PDE-associated agent of the present invention also may be an interfering RNA, or RNAi, including PDE small interfering RNA (siRNA).
  • RNAi refers to a double-stranded RNA (dsRNA) duplex of any length, with or without single-strand overhangs, wherein at least one strand, putatively the antisense strand, is homologous to the target mRNA to be degraded.
  • a "double-stranded RNA" molecule includes any RNA molecule, fragment, or segment containing two strands forming an RNA duplex, notwithstanding the presence of single- stranded overhangs of unpaired nucleotides.
  • a double- stranded RNA molecule includes single-stranded RNA molecules forming functional stem-loop structures, such that they thereby form the structural equivalent of an RNA duplex with single-strand overhangs.
  • the double-stranded RNA molecule of the present invention may be very large, comprising thousands of nucleotides; preferably, however, it is small, in the range of 21-25 nucleotides.
  • the RNAi of the present invention comprises a double-stranded RNA duplex of at least 19 nucleotides.
  • RNAi is produced in vivo by an expression vector containing a gene-silencing cassette coding for RNAi.
  • RNAi is produced in vitro, synthetically or recombinantly, and transferred into the microorganism using standard molecular-biology techniques.
  • RNAi RNA interference in eukaryotic pathogens, Trends Microbial., 11:37-43, 2003;
  • Nikolaev et al. Pare: A Cytoplasmic Anchor for p53, Cell, 112:29-40, 2003; Wilda et al., Killing of leukemic cells with a BCR/ABL fusion gene RNA interference (RNAi), Oncogene, 21:5716-24,2002; Escobar et al., RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis, Proc.
  • RNAi BCR/ABL fusion gene RNA interference
  • the plasmid is an expression plasmid.
  • the expression plasmid may contain sites for transcription initiation, termination, and, optionally, in the transcribed region, a ribosome-binding site for translation.
  • the coding portions of the mature transcripts expressed by the plasmid may include a translationinitiating codon at the beginning, and a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • the PDE-associated gene to be expressed from the expression plasmid may be under the specific regulatory control of certain types of promoters.
  • these promoters are constitutive promoters. Genes under the control of these constitutive promoters will be expressed continually.
  • the promoters are inducible promoters. Genes under the control of these inducible promoters will be expressed only upon the presence of an inducer molecule or the absence of an inhibitor molecule, thereby providing a method to turn off expression of the gene when it is not desired.
  • the promoters are cell-type- specific promoters or tissue-specific (e.g., heart-tissue-specific) promoters. Genes under the control of cell-type-specific promoters will be expressed only in certain cell types, preferably only in cardiomyocytes.
  • the PDE-associated agent is a modulator (e.g., inhibitor, activator, antagonist, or agonist) of PDE expression/activity, including a modulator of a member of the PDE signal-transduction pathway/system.
  • the modulator of the present invention may be a protein, polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab')2 fragment, molecule, compound, antibiotic, or drug, including an agent reactive with PDE, and an agent that induces or upregulates PDE expression or activity.
  • Modulators of PDE or a member of the PDE signal-transduction pathway/ system may be identified using a simple screening assay. For example, to screen for candidate modulators of PDE, cells may be plated onto microtiter plates, then contacted with a library of drugs. Any resulting increase in, or upregulation of, PDE expression then may be detected directly or indirectly using a luminescence reporter, nucleic acid hybridization, and/or immunological techniques known in the art, including an ELISA. Additional modulators of PDE expression may be identified using screening procedures well known in the art or disclosed herein.
  • the modulator of PDE expression may be linked to another agent, or administered in combination with another agent, such as a drug or a ribozyme, in order to increase the effectiveness of treatments using PDE-associated agents and/or increase the efficacy of targeting.
  • additional PDE-associated agents may be identified using screening procedures well known in the art, and methods described herein.
  • the present invention contemplates the use of proteins and protein analogues generated by synthesis of polypeptides in vitro, e.g., by chemical means or in vitro translation of mRNA.
  • PDE may be synthesized by methods commonly known to one skilled in the art (Modern Techniques of Peptide and Amino Acid Analysis (New York: John Wiley & Sons, 1981); Bodansky, M., Principles of Peptide Synthesis (New York: Springer- Verlag New York, Inc., 1984)).
  • amino acid sequences examples include, but are not limited to, solid-phase peptide synthesis, solution-method peptide synthesis, and synthesis using any of the commercially-available peptide synthesizers.
  • the amino acid sequences of the present invention may contain coupling agents and protecting groups, which are used in the synthesis of protein sequences, and which are well known to one of skill in the art.
  • PDE in cells may be modulated, and cells may be contacted with a PDE-associated agent (e.g., by introducing a PDE-associated agent directly into the cells) including stem cells containing a PDE-associated agent either in vitro, or in vivo in a subject.
  • a PDE-associated agent e.g., by introducing a PDE-associated agent directly into the cells
  • stem cells containing a PDE-associated agent either in vitro, or in vivo in a subject.
  • the agent may be added directly to the cell-culture medium.
  • a PDE-associated agent may be contacted with cells in vivo in a subject, by introducing the agent into the subject (e.g., by introducing the agent directly into cells of the subject) and/or administering the agent to the subject.
  • the subject may be any animal, including amphibians, birds, fish, mammals, and marsupials, but is preferably a mammal (e.g., a human; a domestic animal, such as a cat, dog, monkey, mouse, and rat; or a commercial animal, such as a cow or pig).
  • a mammal e.g., a human; a domestic animal, such as a cat, dog, monkey, mouse, and rat; or a commercial animal, such as a cow or pig.
  • the subject is a human.
  • the PDE-associated agent of the present invention may be contacted with a cell or RyR complex, either in vitro, or in vivo (including in situ) in a subject, by known techniques used for the introduction and administration of proteins, nucleic acids, and other drugs.
  • methods for contacting the cells with (i.e., treating the cells with) a PDE-associated agent include, without limitation, absorption, electroporation, immersion, injection (including microinjection), introduction, liposome delivery, stem cell fusion (including embryonic stem cell fusion), transduction, transfection, transfusion, vectors, and other protein-delivery and nucleic-acid-delivery vehicles and methods.
  • the agent When the cells are localized to a particular portion of a subject, it may be desirable to introduce the agent directly to the cells, by injection or by some other means (e.g., by introducing the agent into the blood or another body fluid).
  • contacting is accomplished via a catheter inserted directly into the subject's heart tissue.
  • a catheter would be useful in achieving targeted delivery of the agent to heart tissue cells.
  • Targeted delivery is especially appropriate for cardiomyocytes, which are joined by intercalated disks. These disks should allow passage of the agent from one cardiomyocyte to adjoining cardiomyocytes, thereby aiding in the distribution of the agent throughout the heart tissue.
  • a PDE-associated agent is a protein
  • it may be introduced into a cell or RyR complex directly, in accordance with conventional techniques and methods disclosed herein.
  • a protein agent may be introduced into a cell or RyR complex indirectly, by introducing into the cells a nucleic acid encoding the agent, in a manner permitting expression of the protein agent.
  • the PDE-associated agent may be introduced into cells, in vitro or in vivo, using conventional procedures known in the art, including, without limitation, electroporation, DEAF dextran transfection, calcium phosphate transfection, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, in vivo gene therapy, ex vivo gene therapy, viral vectors, and naked DNA transfer, or any combination thereof.
  • Recombinant viral vectors suitable for gene therapy include, but are not limited to, vectors derived from the genomes of such viruses as retrovirus, HSV, adenovirus, adeno-associated virus, Semilild Forest virus, cytomegalovirus, lentivirus, and vaccinia virus.
  • the amount of nucleic acid to be used in the method of the present invention is an amount sufficient to express an amount of protein effective to promote PDE level. These amounts may be readily determined by the skilled artisan. It is also within the confines of the present invention to use an ex vivo approach, wherein a nucleic acid encoding a protein agent is introduced into suitable a cell or RyR complex in vitro, using conventional procedures, to achieve expression of the protein agent in the cells. Cells expressing protein agent are then introduced into a subject to promote PDE activity in vivo.
  • a PDE-associated agent including stem cells containing the agent, may be administered to a human or animal subject by known procedures, including, without limitation, oral administration, parenteral administration, transdermal administration, and by way of a catheter.
  • the agent may be administered parenterally, by intracranial, intraspinal, intrathecal, or subcutaneous injection.
  • the agent of the present invention also may be administered to a subject in accordance with any of the above-described methods for effecting in vivo contact between a cell or RyR complex and PDE-associated agents.
  • a formulation comprising a PDE-associated agent may be presented as capsules, tablets, powders, granules, or as a suspension.
  • the formulation may have conventional additives, such as lactose, mannitol, cornstarch, or potato starch.
  • the formulation also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, cornstarch, or gelatins.
  • the formulation may be presented with disintegrators, such as cornstarch, potato starch, or sodium carboxymethylcellulose.
  • the formulation also may be presented with dibasic calcium phosphate anhydrous or sodium starch glycolate.
  • the formulation may be presented with lubricants, such as talc or magnesium stearate.
  • a PDE- associated agent may be combined with a sterile aqueous solution that is preferably isotonic with the blood of the subject.
  • a formulation may be prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile.
  • physiologically-compatible substances such as sodium chloride, glycine, and the like
  • the formulation may be presented in unit or multi-dose containers, such as sealed ampoules or vials.
  • the formulation may be delivered by any mode of injection, including, without limitation, epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous, or sublingual, or by way of a catheter.
  • an agent may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like, which increase the permeability of the skin to the agent, and permit the agent to penetrate through the skin and into the bloodstream.
  • skin penetration enhancers such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like, which increase the permeability of the skin to the agent, and permit the agent to penetrate through the skin and into the bloodstream.
  • the agent/enhancer composition also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which may be dissolved in solvent, such as methylene chloride, evaporated to the desired viscosity, and then applied to backing material to provide a patch.
  • a polymeric substance such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like
  • solvent such as methylene chloride
  • the method of the present invention may also be used either to treat a RyR receptor associated disorder or disease in vivo in a subject, or to prevent a RyR receptor associated disorder or disease in vivo in a subject.
  • augmented PDE in a cell or RyR complex has the ability protect the heart against heart failure and catecholaminergic arrhythmias by regulating local PKA activity and channel activation at RyR2-Ser 2809 , and preventing excess accumulation of c AMP and uncontrolled PKA activation.
  • the present invention also provides a therapeutic composition comprising a PDE-associated agent and, optionally, a pharmaceutically-acceptable carrier.
  • the PDE-associated agent may include a PDE protein or nucleic acid, a PDE-associated protein, a PDE-associated nucleic acid, a member of the PDE signal - transduction pathway (including upstream and downstream effectors and activators, in protein or nucleic acid form), and a modulator (e.g., inhibitor, activator, antagonist, or agonist) of a member of the PDE signal-transduction pathway/system (i.e., a modulator which affects the expression and/or activity of PDE or a member of the PDE signal transduction pathway).
  • a modulator e.g., inhibitor, activator, antagonist, or agonist
  • the pharmaceutically-acceptable carrier must be "acceptable” in the sense of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.
  • the pharmaceutically-acceptable carrier employed herein is selected from various organic or inorganic materials that are used as materials for pharmaceutical formulations, and which may be incorporated as analgesic agents, buffers, binders, disintegrants, diluents, emulsifiers, excipients, extenders, glidants, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles, and viscosity-increasing agents.
  • pharmaceutical additives such as antioxidants, aromatics, colorants, flavor- improving agents, preservatives, and sweeteners
  • pharmaceutical additives such as antioxidants, aromatics, colorants, flavor- improving agents, preservatives, and sweeteners
  • acceptable pharmaceutical carriers include, without limitation, carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc, and water, among others.
  • Formulations of the therapeutic composition of the present invention may be prepared by methods well-known in the pharmaceutical arts.
  • a PDE- associated agent may be brought into association with a carrier or diluent, as a suspension or solution.
  • one or more accessory ingredients e.g., buffers, flavoring agents, surface active agents, and the like
  • the choice of carrier will depend upon the route of administration.
  • the PDE-associated agent is provided in an amount that is effective to hydrolyze cAMP in a cell or RyR complex in a subject to whom the therapeutic composition is administered. This amount may be readily determined by the skilled artisan.
  • the PDE-associated agent is a protein that is expressed in a target heart tissue cell using an expression construct.
  • Expression of the protein may be controlled by methods known in the art, including the use of attenuators, downregulators, inhibitors, and other molecules known to inhibit protein expression.
  • attenuators, downregulators, inhibitors, and other molecules known to inhibit protein expression By way of example, where the therapeutic composition of the present invention is administered to a subject, such that the composition expresses a PDE- associated protein in the subject, this expression may be shut off in vivo by subsequently administering to the subject an attenuator, downregulator, inhibitor, or other molecule that will inhibit expression of the exogenous molecule.
  • Control of expression of the PDE- associated protein is also advantageous, in that it allows one to turn off the expression of the protein when desired, thereby minimizing any harmful side-effects in a subject to whom the composition is administered. Continuous expression of such a protein, beyond an appropriate time limit, may harm the subject. For example, a significant interference with a PDE signal transduction pathway may cause neoplasia or apoptosis.
  • the therapeutic composition of the present invention may further comprise a vehicle for assisting in the delivery of the composition that target specific cells, including but not limited to heart tissue cells or skeletal muscle cells.
  • a vehicle for assisting in the delivery of the composition that target specific cells, including but not limited to heart tissue cells or skeletal muscle cells.
  • a variety of biological delivery systems e.g., antibodies, bacteria, liposomes, and viral vectors
  • drugs, genes, immunostimulators, pro-drug converting enzymes, radiochemicals, and other therapeutic agents to the vicinity of target cells (see e.g., Ng et al., An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells, Proc. Natl. Acad. Sci.
  • U.S. Patent No. 6,491,905 provides a prokaryotic cell stably carrying a vector that includes a DNA sequence encoding a purine nucleotide phosphorylase or hydrolase, and the use of such a cell, together with a purine pro-drug, to treat tumors.
  • the vehicle is a liposome.
  • Liposomal vesicles may be prepared by various methods known in the art, and liposome compositions may be prepared using any one of a variety of conventional techniques for liposome preparation known to those skilled in the art. Examples of such methods and techniques include, without limitation, chelate dialysis, extrusion (with or without freeze- thaw), French press, homogenization, microemulsification, reverse phase evaporation, simple freeze-thaw, solvent dialysis, solvent infusion, solvent vaporization, sonication, and spontaneous formation. Preparation of the liposomes may be carried out in a solution, such as an aqueous saline solution, aqueous phosphate buffer solution, or sterile water. Liposome compositions also may be prepared by various processes involving shaking or vortexing.
  • the therapeutic composition of the present invention may be incorporated into the layers of a liposome, or enclosed within the interior of the liposome.
  • the liposome containing the composition then may be fused cell, in accordance with known methods of fusion of liposomes to cell membranes, such that the composition protein is delivered into the membrane of the cell or into the interior of the cell, as the case may be.
  • the present invention also provides a kit for use in delivering a PDE- associated agent to a cells, cell subcompartment or RyR complex in a subject.
  • the kit comprises a therapeutic composition and a catheter.
  • the therapeutic composition may comprise a PDE-associated agent; optionally, a pharmaceutically- acceptable carrier; and, optionally, a liposome,- viral vector, or other vehicle.
  • the pACN plasmid was a backbone vector containing a cassette (ACN) with genes for neomycin resistance, Cre recombinase and a testes-specific promoter (tACE), flanked by loxP sites.
  • ACN a cassette
  • tACE testes-specific promoter
  • the promoter tACE initiates expression of Cre recombinase only during spermatogenesis, resulting in excision of the ACN cassette.
  • the 3' targeting arm consisting of the 2463 bps upstream of the Eco RI site, was obtained by PCR of genomic mouse DNA.
  • PDE4D ⁇ / - mice were generated and genotyped as described (Jin et al.,
  • RyR2-S2808A knockin mice generated using homologous recombination as described immediately above, exhibited normal cardiac structure and function, and no PKA phosphorylation of RyR2 was detected using a kinasing reaction with [ ⁇ - 32 P]ATP or with a phosphoepitope-specific antibody that detects PKA-phosphorylated RyR2.
  • EXAMPLE 2 TRANSTHORACIC ECHOCARDIOGRAPHY AND IN VIVO HEMODYNAMIC ANALYSES ON MICE
  • mice were anesthetized with 1.0-1.5% isoflurane in Oz and placed on a 37°C heating pad. Hearts were visualized parastemally along the short axis to obtain 2-D images and M-mode tracings of the anterior wall, left ventricular cavity, and posterior wall. Left ventricular dimensions and function were measured in triplicate from different cardiac cycles for the number of animals indicated.
  • PDE4D ⁇ / ⁇ and age-and-litter-matched wild-type mice (4 to 5 months old) were anesthetized with 1.5% isoflurane and ventilated with a small-rodent respirator (Harvard Apparatus).
  • a left thoracotomy was performed, and the left anterior descending artery (LAD) was ligated proximally with an 8-0 suture as described (Wehrens et al., 2005).
  • RyR2 channels were immunoprecipitated and immunoblotted as previously described (Marx et al., 2000) .
  • RyR2 channels were immunoprecipitated from 100 ⁇ g of human or murine cardiac homogenates using anti-RyR antibody (Jayaraman et al., 1992) in 0.5 ml of RIP A buffer (50 mM Tris-HCl buffer), pH 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na 3 VO 4 , 0.25% Triton-XIOO, and protease inhibitor mix (Roche) overnight at 4°C.
  • RIP A buffer 50 mM Tris-HCl buffer
  • pH 7.4 pH 7.4
  • NaCl 5.0 mM NaF
  • 1.0 mM Na 3 VO 4 0.25% Triton-XIOO
  • protease inhibitor mix (Roche) overnight at 4°C.
  • the use of human tissues was approved by the Institutional Review Board of Columbia-Pres
  • Immunoblots were developed with an enhanced chemiluminescence system using primary antibodies against RyR (5029; 1:3,000) (Jayaraman et al., 1992), PDE splice- variant 4D3 (1: 1 ,000) (Reiken et al, 2003b) calstabin2 (I: 1 ,000) (Wehrens et al., 2003), PKA catalytic subunit (1:1 ,000), PPI (1 : 1 ,000) and PP2A (1 :1 ,000) (Transduction Labs, Lexington, KY) (Marx et al., 2000).
  • Phosphodiesterase (PDE) activity was measured using selective binding of PDE
  • Cardiomyocytes were infected with recombinant adenoviruses expressing CFP attached to the PKA regulatory subunit (RII-CFP) and YFP attached to the PKA catalytic subunit (C-YFP) as described previously (Warrier et al., 2005). Simultaneous infection of mouse cardiomyocytes occurred at a multiplicity of infection of 50 to 100 for each virus. Cells expressing approximately equal amounts of CFP and YFP as evidenced by fluorescence at 48-72 hrs, were used for intracellular cAMP imaging by FRET.
  • RII-CFP PKA regulatory subunit
  • C-YFP PKA catalytic subunit
  • Imaging was performed with an inverted microscope (Olympus 1X70) equipped with a 4OX water immersion objective (1.3 NA, Olympus) and a CCD camera (Hamamatsu, Orca ER) as described previously (Warrier et al., 2005). Fluorescence images were acquired using 2x2 binning and analyzed using Simple PCI imaging software (Compix Inc.) and changes in cAMP concentrations at the Z-line containing RyR2 complexes were defined as the relative changes in the intensity of CFP and YFP measured at the Z-lines within a region of interest. Isoproterenol bitartrate (Iso; Sigma RBI) was prepared as stock solution and applied by rapid perfusion.
  • Isoproterenol bitartrate Iso; Sigma RBI
  • PKA phosphorylation of PDE4D3 was assessed using a kinasing reaction on PDE4D3 immunoprecipitated from 100 ⁇ g of human cardiac homogenates.
  • PDE4D3 was immunoprecipitated from 100 ⁇ g of human cardiac homogenates with anti-PDE4D3 antibody (5 ⁇ g/ml) in 0.5 ml of RIPA buffer overnight at 4 0 C.
  • RyR2 single channels were recorded in planar lipid bilayers as previously described (Marx et al., 2000). Symmetrical solutions used were (in mM) trans-HEPES 250 and Ca(OH) 2 53 (pH 7.35) and cis-HEPES 250, Tris 125, EGTA 1.0, and CaCl 2 0.5 (pH 7.35). Free Ca concentrations were calculated by CHELATOR software. At the conclusion of each experiment, ryanodine (5 ⁇ M) or ruthenium red (20 ⁇ M) was applied to confirm RyR2-channel identity.
  • RyR2 was immunoprecipitated from 250 ⁇ g of mouse heart homogenate. PKA phosphorylation of RyR2 was initiated with 5 ⁇ l of 100 ⁇ M Mg-ATP (for autoradiography, the Mg-ATP contained 10% [ 32 P]- ⁇ ATP (NEN Life Sciences, Boston, MA) in kinase buffer (8 ⁇ M MgCI 2 , 10 mM EGTA, and 50 mM Tris/piperazine- N,N'-bis(2ethanesulfonic acid), pH 6.8). The reaction was terminated after 8 min at room temperature with 5 ⁇ l of stop solution (4% SDS and 0.25 M DTT). Samples were heated to 95 DC and size-fractionated on 6% SDS-PAGE.
  • PDE4D ⁇ ⁇ mice had reduced ejection fractions (EF) and cardiac contractility (dP/dt)/P; d , documented by cardiac catheterization ( Figures 1C and ID). Histologic examination of PDE4D ⁇ 7 ⁇ hearts confirmed that the 15-month-old hearts were dilated, with no other structural abnormalities ( Figure IE). These data show that PDE4D deficiency is associated with progressive cardiac dysfunction consistent with a dilated cardiomyopathy similar to that seen in patients with chronic heart failure.
  • the SR Ca 2+ - release channel, RyR2 has been shown to be PKA hyperphosphorylated in heart failure (Antos et al, 2001, Marx et al., 2000, Reiken et al., 2003a, Reiken et al., 2003b, Yano et al., 2000 and Yano et al., 2003), although this finding has been challenged by others (Jiang et al., 2002).
  • RyR2 in hearts from PDE4D-deficient mice the inventors examined the single-channel properties of RyR2 in planar lipid bilayers. Compared to channels from wt mice, RyR2 from PDE4D mice exhibited significantly increased open probability (Po) and frequency of openings (Fo) and decreased mean open and closed times when channels were examined under conditions that mimic diastole in the heart (cytosolic (cis) [Ca 2+ ] 150 nM) ( Figures 3F and 3G).
  • PDE4D1-9 PDE4D splice variants
  • PDE4D splice variants were identified by RT-PCR in heart (data not shown), and PDE4D3, PDE4D8, and PDE4D9 expression in the heart was demonstrated using isoform-specific antibodies ( Figure 4B), confirming that these are the major PDE4D isoforms expressed in heart muscle (Richter et al., 2005).
  • PDE4D3 was also associated with RyR2 from human hearts ( Figure 5A).
  • the inventors also crossed the PDE4D +/ ⁇ mice with RyR2-S2808A mice to investigate the specific role of PKA hyperphosphorylation of RyR2 in the development of cardiac dysfunction in PDE4D + ⁇ mice.
  • RyR2-S2808A mice harbor RyR2 that cannot be PKA phosphorylated ( Figure 7D).
  • Prevention of PKA hyperphosphorylation improved cardiac function in PDE4D + ⁇ mice subjected to MI ( Figures 7A and 7C, green line and bars).
  • PDE4D phosphodiesterase
  • PDE4 inhibitors are being tested in clinical trials to treat common chronic diseases including Alzheimer's disease (Gong et al., 2004), asthma, and COPD (Giembycz, 2002), it is important to understand the consequences of long-term inhibition of PDE4 activity in the heart, where PDE4 activity plays a major role in regulating cAMP-dependent signals (Perry et al., 2002, Verde et al., 1999 and Xiang et al., 2005).
  • PDE4 is a localized regulator of ⁇ -adrenergic receptor ( ⁇ 2-AR) signaling in cardiomyocytes (Baillie et al., 2003, Mongillo et al., 2004, Perry et al., 2002 and Xiang et al., 2005).
  • ⁇ 2-AR ⁇ -adrenergic receptor
  • PDE4D3 can be targeted to specific compartments including the cardiomyocyte Z line via the targeting protein mAKAP (Carlisle Michel et al., 2004, Dodge et al., 2001, Sette and Conti, 1996 and Yang et al., 1998).
  • PDE4D3 may regulate local PKlA activity and channel activation via phosphorylation of RyR2-Ser2808, thereby preventing excess accumulation of cAMP (Zaccolo and Pozzan, 2002) and uncontrolled PKA-mediated activation of the channel.
  • loss of negative feedback due to PDE4D3 deficiency in the RyR2 complex likely contributes to RyR2 PKA hyperphosphorylation; calstabin2 depletion; and hyperactive, "leaky” RyR2 channels (Marx et al., 2000 and Pieske et al., 1999).
  • PDE4 inhibitors could increase the risk of cardiac arrhythmias due to "leaky” RyR2 channels as observed in individuals with genetic forms of sudden cardiac death linked to RyR2 mutations (Lehnart et al., 2004 and Wehrens et al., 2003) and in patients with heart failure.
  • the present study shows that phosphodiesterase (PDE4D) deficiency is associated with a severe cardiac phenotype consisting of heart failure and lethal cardiac arrhythmias.
  • PDE4D phosphodiesterase
  • PDE inhibition has been associated with increased mortality in patients with heart failure (Packer et al., 1991), arrhythmias, and sudden cardiac death (Bittar and Friedman, 1991 and Suissa et al., 1996), although the mechanism has been unknown.
  • PDE4 inhibitors are being tested in clinical trials to treat common chronic diseases including Alzheimer's disease (Gong et al., 2004), asthma, and COPD (Giembycz, 2002), it is important to understand the consequences of long-term inhibition of PDE4 activity in the heart, where PDE4 activity plays a major role in regulating cAMP-dependent signals (Perry et al., 2002, Verde et al., 1999 and Xiang et al., 2005).
  • PDE4 is a localized regulator of ⁇ -adrenergic receptor ( ⁇ 2-AR) signaling in cardiomyocytes (Baillie et al., 2003, Mongillo et al., 2004, Perry et al., 2002 and Xiang et al., 2005).
  • ⁇ 2-AR ⁇ -adrenergic receptor
  • PDE4D3 can be targeted to specific compartments including the cardiomyocyte Z line via the targeting protein mAKAP (Carlisle Michel et al., 2004, Dodge et al., 2001, Sette and Conti, 1996 and Yang et al., 1998).
  • PDE4D3 in the regulation of RyR2, the major intracellular Ca 2+ -release channel in the heart.
  • PDE4D3 activity provides an important negative-feedback mechanism to limit ⁇ -AR-dependent PKA phosphorylation of RyR2-Ser2808.
  • PDE4D3 may regulate local PKA activity and channel activation via phosphorylation of RyR2-Ser2808, thereby preventing excess accumulation of cAMP (Zaccolo and Pozzan, 2002) and uncontrolled PKA-mediated activation of the channel.
  • Beta 1- and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium coupling of both receptor subtypes to muscle contraction and selective beta I-receptor down-regulation in heart failure. Circ. Res. 59, 297-309 (1986).
  • mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J. 20, 1921-30, (2001).
  • PDE4 cAMP phosphodiesterases modular enzymes that orchestrate signaling cross-talk, desensitization and compartmentalization. Biochem. J. 370, 1-8 (2003).
  • Beta-blockers restore calcium release channel function and improve cardiac muscle performance in human heart failure. Circulation 107, 2459-66 (2003).
  • the ratPDE3/IVd phosphodiesterase gene codes for multiple proteins differentially activated by cAMP-dependent protein kinase. J. Biol. Chem. 269, 18271-4 (1994).
  • AKAP 100 A-kinase anchoring protein 100 (AKAP 100) is localized in multiple subcellular compartments in the adult rat heart. J. Cell. Biol. 142, 511-22 (1998).

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Abstract

La présente invention concerne des compositions utilisées dans le traitement et la prévention de troubles associés au récepteur de la ryanodine, lesdites compositions renfermant un agent lié à la phosphodiestérase (PDE) et un excipient acceptable pharmaceutiquement. Cette invention a aussi pour objet des méthodes de traitement ou de prévention de troubles associés au récepteur de la ryanodine, y compris, des maladies et des troubles cardiaques, des maladies et des troubles musculaires du squelette, des maladies et des troubles cognitifs, une hyperthermie maligne, des diabètes et le syndrome de mort subite du nourrisson. Ladite invention a, également, trait à des méthodes de régulation de la phosphorylation PKA d'un récepteur de la ryanodine, ainsi qu'à des méthodes de régulation de la libération et de la réabsorption de Ca+2 dans des cellules. L'invention concerne, enfin, des kits à utiliser dans l'administration d'un agent associé à PDE au niveau de cellules cardiaques chez un sujet, lesdits kits contenant la composition susmentionnée et un cathéter.
PCT/US2005/045914 2004-12-16 2005-12-15 Protection de la phosphodiesterase 4d dans le complexe du recepteur de la ryanodine contre une insuffisance cardiaque WO2006071603A2 (fr)

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