WO2007028117A2 - P8 as a marker for heart failure - Google Patents

Abstract

The present invention is related to the diagnosis and treatment of p8-related conditions, particularly heart failure.

Description

p8 AS A MARKER FOR HEART FAILURE

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. provisional application 60/714,067, which was filed on September 2, 2005, the content of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is related to the diagnosis and treatment of p8-related conditions, particularly heart failure.

BACKGROUND

The increased prevalence of obesity, hypertension, diabetes, and atherosclerosis, as well as an aging population, have led to a rising incidence of heart failure. Congestive heart failure (CHF) is characterized by the failure of the heart to pump blood at sufficient flow rates to meet the metabolic demands of tissues, especially the demand for oxygen. As heart failure progresses, portions of a patient's heart are remodeled with resulting differences in the heart's size and shape and the thickness of the heart wall. For example, a damaged left ventricle may have some localized thinning and stretching of a portion of the myocardium. The thinned portion of the myocardium often is functionally impaired, and other portions of the myocardium attempt to compensate. As a result, the other portions of the myocardium may expand so that the stroke volume of the ventricle is maintained notwithstanding the impaired zone of the myocardium. Such expansion may cause the left ventricle to assume a somewhat spherical shape.

Cardiac remodeling often subjects the heart wall to increased wall tension or stress, which further impairs the heart's functional performance. Often, the heart wall will dilate further in order to compensate for the impairment caused by such increased stress. Thus, an undesirable cycle can result in which dilation leads to further dilation and greater functional impairment.

Historically, congestive heart failure has been managed with a variety of drugs. Devices have also been used to improve cardiac output. For example, left ventricular assist devices (VADs) help the heart to pump blood. Multi-chamber pacing has also been employed to optimally synchronize the beating of the heart chambers to improve cardiac output. Various skeletal muscles, such as the latissimus dorsi, have been used to assist ventricular pumping. Researchers and cardiac surgeons have also experimented with prosthetic "girdles" disposed around the heart. Although some of these approaches have helped, there remains a need in the art for methods of preventing and treating CHF.

SUMMARY

The present invention is based, in part, on our discovery that p8, a small basic helix-loop- helix (HLH) protein induced by pro-inflammatory stimuli, is induced in myocardial samples obtained from humans with heart failure. Accordingly, in one aspect, the invention features methods of detecting a cardiac anomaly or the impending occurrence thereof by assessing the expression or activity of ρ8, including any naturally-occurring variant thereof {e.g., a splice variant or other variant). The anomaly can be any undesirable change in the architecture of the heart, including a change in the heart's size or shape {e.g., hypertrophy hi one or more regions) or in the function of the heart (e.g., reduced output (e.g., reduced stroke volume)). The anomaly can also be manifest as a change at the cellular level (e.g., as cardiomyocyte hypertrophy or extracellular matrix remodeling). The anomaly can occur as the result of heart disease or a surgical procedure (e.g., following transplantation of a heart from a donor to a recipient). Thus, patients who have heart disease as a result of either a disorder or surgical procedure are amenable to the methods described herein. When these methods are performed as diagnostic procedures on, for example, a human patient, they can be followed by an appropriate and appropriately aggressive treatment (i.e., the level of p8 expression or activity can be taken into consideration when deciding upon the nature of the treatment to be provided).

The expression or activity of p8 can be assessed once or repeatedly over the course of time. For example, a diagnostic test can be repeated at certain intervals in time (e.g., about once a year) or upon the occurrence or reoccurrence of a sign or symptom of heart failure. The expression or activity of p8 can also be monitored at one or more points in tune following the application of a treatment. For example, the expression or activity of p8 can be assessed at one or more points in time (e.g., daily, weekly or monthly) following the initiation of therapy with a medication or VAD. A decrease in expression or activity of p8 would indicate or reflect the effectiveness of the therapy; the greater the decrease (or the nearer the level (or range) to a normal level (or range), the more effective the therapy. The expression or activity of p8 can be compared relative to a reference standard (e.g., a range of normal expression and/or activity levels obtained from a population of patients who do not have heart disease) or compared to a level or levels of p8 expression obtained from the patient being treated prior to the time they were suspected of having heart disease. As in making an initial diagnosis, p8 can be assessed together with other markers, images of the heart, or signs and symptoms of the underlying anomaly in order to facilitate decisions regarding, or conclusions concerning, the effectiveness of a therapeutic regime. The therapeutic regime one wishes to monitor can begin or the therapeutic compound(s) can be administered before, during, or after any event that stimulates the expression or activity of p8.

The sample one uses to assess the expression or activity of p8 can be a small sample of material obtained from cardiac muscle. The sample can be obtained in the course of performing another procedure or it may be performed independently using minimally invasive techniques. p8 expression can also be detected in other samples, such as blood samples. While the invention is not limited to the use of samples of any particular type, p8-expressing cells maybe shed from the heart and present in the circulation, particularly as they become necrotic.

The therapeutic regime can be one that is carried out using a commercially available composition or device (e.g., a known treatment for heart failure) or one that is carried out in a screening or experimental setting to test compositions and devices. Thus, the expression or activity of p8 can be assessed in the context of identifying a new therapeutic regime or therapeutic composition (or combinations thereof). For example, one can determine whether a therapeutic regime or therapeutic composition (or a combination thereof) modulates (e.g., inhibits or promotes) an increase in the expression or activity of p8 that would otherwise occur or be expected to occur. The screening assay can be carried out in vitro or in cell culture. For example, a test compound (whether or not known to have a beneficial impact on heart failure) can be exposed to cardiomyocytes and/or cardiofibroblasts; to cell lines derived from these cell types; or to intact pieces of cardiac muscle or whole hearts. One would then assess the expression or activity of p8, which can be done, for example, with methods known and used in the art for assessing gene or protein expression or for detecting protein activity (some of the known activities of p8 are described further below). The levels of expression or activity can be compared to a reference standard or to a control sample (e.g., a sample that is treated identically except that it is exposed to no test compound or to test compound known to be inactive). Alternatively, or in addition, the screening assays can be carried out in vivo. For example, compounds can be screened in the context of an animal model of chronic heart failure, such as those described by Muders and Eisner (Pharmacol. Res. 41:605-612, 2000). Alternatively, or in addition, hypertrophy assays such as those described in the references cited in our studies below can be used.

We use the terms "compound" and "agent" to refer broadly to any potential therapeutic agent. Compounds that can be screened include nucleic acids, peptides and proteins, antibodies, and small molecules (e.g, small organic compounds). These types of agents are available to those of ordinary skill in the art and may be screened individually or in libraries or subsets thereof. For example, a commercially available library of antibodies or small molecules can be randomly divided and tested; subsets that produce a favorable response (e.g., a reduction in the expression or activity of p8 or an attenuation of p8 elevation in response to a stimulating event), can be further divided until the active compound(s) are identified.

In the methods described above, the therapeutic regime can begin or the therapeutic compound(s) can be administered before, during, or after any event that stimulates the expression or activity of p8. The event can be, for example, induction of, or exposure to, one or more stressors that lead to atrophy or a proinflammatory stimulus (nutrient deprivation and other stimuli are further described in the report of our studies that follows, and these events can be used in the methods of the invention to screen test compounds; agents can also be assessed in the context of endothelin- or β-adrenergic agonist-induced cardiomyocyte hypertrophy). In any of the methods of the invention, whether diagnostic, therapeutic, or carried out in the process of screening potential therapeutic compounds, p8 can be assessed along with any other known gene or protein (e.g., an inflammatory protein) that has been linked to hypertrophy, ventricular remodeling, or any other cardiac anomaly (see FIG. 1).

We have shown that p8 is required for endothelin- and β-adrenergic agonist-induced cardiomyocyte hypertrophy. It is also required for TNF-stimulated induction of two matrix metalloproteases (MMPs) — MMP9 and MMPl 3 — in cardiac fibroblasts. These MMPs are linked to general inflammation and are associated with adverse ventricular remodeling in heart failure. In a stimulus-dependent manner, p8 associates with chromatin containing c-Jun and with the cardiomyocyte atrial natriuretic factor (anj) promoter and the cardiac fibroblast mmp9 and mmpl3 promoters (established AP-I effectors). Any of these activities can be assessed as indicators of p8 activity. p8 is also induced strongly in the failing human heart by a process reversed upon therapeutic intervention. Our results identify an unexpectedly broad involvement for p8 in key cellular events linked to the development and progression of heart failure.

Accordingly, the invention features methods of treating a cardiac anomaly by impeding the expression or activity of p8. This can be accomplished by administering, to a patient who is experiencing a cardiac anomaly or who is a risk thereof, a small nucleic acid molecule or other agent that inhibits the expression of p8 or an antibody or other agent that specifically binds and/or inhibits the activity of p8. These agents are described further below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a proposed model for the regulation of p8 expression and function in cardiomyocytes and cardiac fibroblasts.

FIG. 2A is a representation of a p8-encoding nucleic acid sequence (SEQ ID NO:1). Underlined sequence is targeted by siRNA SEQ ID NO:3 (see Table 1).

FIG. 2B is representation of a nucleic acid sequence (SEQ ID NO:2) encoding a p8 splice variant having an additional 10 amino acids before the bHLH region. Underlined sequence is targeted by the siRNA having the sequence set forth in SEQ ID NO:3 (see Table 1).

DETAILED DESCRIPTION

The progression to heart failure involves an initial phase of pathologic cardiomyocyte hypertrophy, which develops as a consequence of excess hemodynamic work load and may be triggered by the β-adrenergic agents angiotensin-II and/or endothelin. Pathologic cardiomyocyte hypertrophy is followed by left ventricular decompensation, which is characterized by cardiomyocyte loss and interstitial fibrosis. These are direct contributors to adverse ventricular remodeling. Ultimately, the contractile properties of the heart are compromised, and heart failure results (Hunter and Chien, N. Engl. J. Med. 341:1276-1283, 1999; Kang and Izumo, Trends MoI. Med. 9:177-182, 2003; McKinsey and Olson, J. Clin. Invest. 115:538-546, 2005). The molecular components and cellular events required for heart failure remain incompletely understood, and few genes have been linked to both pathologic hypertrophy of cardiomyocytes and matrix remodeling (Hunter and Chien, N. Engl. J. Med. 341:1276-1283, 1999; Kang and Izumo, Trends MoI. Med. 9:177-182, 2003; McKinsey and Olson, J. CHn. Invest. 115:538-546, 2005; Dorn and Force, J. Clin. Invest. 115:527-537, 2005). Post-myocardial infarction, in addition to hypertrophy of surviving cardiomyocytes, remodeling of the extracellular matrix occurs, particularly within the territory of the infarct, as lost myocytes are replaced by fibrous tissue (Lindsey et al, Am. J. Physiol. Heart Circ. Physiol. (doi:10.1152/ajpheart.00457.2005) 2004). Key to this remodeling process is the production and release of matrix metalloproteases (MMPs) from both resident cells (especially cardiac fibroblasts) and infiltrating leukocytes. Inflammatory cytokine production (especially TNF, IL-I, and IL-6 family members) by these cells is believed to be the major trigger for induction of MMP expression.

Studies employing broad spectrum inhibitors of MMPs have shown that TNF-stimulated upregulation of the expression of MMPs is a central factor leading to left ventricular dilation post-myocardial infarction, an event that increases a patient's risk of heart failure (Yarbrough et al, J. Thome. Cardiovasc. Surg. 125:602-610, 2003; Mukherjee et al, Circulation 107:618- 625, 2003; Rohde et al, Circulation 99:3063-3070, 1999). Studies of mice with a targeted deletion of mmp9 clearly implicate this factor in not only left ventricular dilation, but also in inhibition of neoangiogenesis post-infarct (Heymans et al, Nat. Med. 5:1135-1142, 1999; Ducharme et al, J. Clin. Invest. 106:55-62, 2000; Spinale Circ. Res. 90:520-530, 2002; Bradham et al, Am. J. Physiol. Heart Circ. Physiol. 282:H1288-H1295, 2002; Lindsey et al, Am. J. Physiol. Heart Circ. Physiol . (doi:10.1152/ajpheart.00457.2005), 2005). Other studies suggest MMP 13 may also be important in late progression of remodeling (Wilson et al, Circulation 107:2857-2863, 2003).

The p8 gene is also called candidate of metastasis- 1, coml, nuclear protein 1, and nuprl. p8 encodes an 8-kDa nuclear bHLH protein strongly induced in a mouse model of acute pancreatitis and implicated in several diverse functions including transcriptional regulation, cell cycle control, stress responses, and diabetic renal hypertrophy (Mallo et al, J. Biol Chem. 272:32360-32369, 1997; Ree et al, Cancer Res. 59:4675-4680, 1999; Su et al, Clin. Cancer Res. 7:309-313, 2001 ; Vasseur et al, EMBO J. Rep. 3:165-170, 2002; Hoffmeister et al, J. Biol. Chem. 277:22314-22319, 2002; Goruppi et al, EMBOJ. 21:5427-5436, 2002) as well as apoptotic regulation (Carracedo et al, Cancer Cell 9:301-312, 2006; Malicet et al, Proc. Natl Acad. Sci. USA 103:2671-2676, 2006). In the Examples presented below, we show that p8 is a transcriptional regulator critical to two key cellular events in heart failure: cardiomyocyte hypertrophy and cardiac fibroblast matrix metalloprotease (MMP) expression. The compositions and methods described herein can be used in the diagnosis, monitoring, or treatment of a patient who has, or who is at risk of developing, a cardiac anomaly, as occurs, for example, in the event of heart failure. When the compositions are administered (or the methods are carried out) when there is little or no evidence of the anomaly, the compositions and methods can help prevent an anomaly from developing or from developing as rapidly as it otherwise would. Also provided are methods of screening for agents that inhibit the expression or activity of p8.

Patients amenable to treatment: Any patient at risk for a heart condition, such as heart failure, is a candidate for treatment with a p8 inhibitor described herein. For example, a patient suitable for treatment can be obese or hypertensive, have low HDL (high density lipoprotein) cholesterol levels, or suffer from diabetes (type I or type II) or atherosclerosis. An individual with a genetic disposition or a family history of heart disease or heart failure is also suitable for treatment with a p8 inhibitor. A patient who has previously experienced a cardiac infarction, a heart valve disease (such as a disease due to past rheumatic fever), endocarditis, myocarditis, or a cardiomyopathy is also a candidate. A patient having a physical anamoly to the heart tissue is also a candidate. For example, the patient can have an irregular heartbeat, a left ventrical with a somewhat spherical shape (which can be indicative of congestive heart failure), or a congenital heart defect. A human who engages in behavior that places him/her at high risk for heart failure, such as smoking or physical inactivity, is also a candidate for treatment with a p8 inhibitor.

A patient identified as having or being at risk for having a heart condition can be administered a therapeutic regime that includes a pharmaceutical composition containing a p8 inhibitor {e.g., any pharmaceutical composition described herein). The patient can additionally be prescribed a traditional therapy for treatment of the condition. For example, a patient identified as having or at risk for having heart failure can be prescribed a medication, such as a diuretic, an angiotensin antagonist, beta blocker, digoxin, aldosterone antagonist or inotrope. Alternatively or in addition, the patient can also be directed to modify lifestyle habits (e.g., to quit smoking, or to modify the diet to lower sodium and cholesterol intake).

Inhibitors ofp8: A p8 inhibitor can affect activity by interfering with protein activity (e.g., binding to the protein) or stability (e.g., degradation). For example, an inhibitor can be an antibody or a peptide {e.g., a short interfering peptide), or a small molecule {e.g., a small organic compound). Alternatively, a p8 inhibitor can interfere with transcription or mRNA translation. For example, the p8 inhibitor can be a small interfering RNA (siRNA), antisense RNA, or ribozyme that interferes with translation. The p8 inhibitor can also be an siRNA-encoding vector {e.g., a viral vector).

This invention features compounds, compositions {e.g., pharmaceutical compositions and kits), and methods useful for modulating p8 gene expression using short interfering nucleic acid (siNA) molecules. This invention also includes compounds, compositions, and methods useful for modulating the expression and/or activity of other genes involved in pathways of p8 gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the compositions can include, and the methods can be practiced with, small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules that modulate {e.g., inhibit) the expression of p8. Exemplary siRNAs for inhibition of p8 are shown in Table 1.

Table 1. Exemplary siRNAs targeting human p8

The sequence targeted by SEQ ID NO:3 is shown in FIGs. 2A and 2B. The sequence targeted by SEQ ID NO:4 is present in a p8 variant. An siRNA sequence that targets a rodent p8 is 5'-GGACGTACCAAGAGAGAAGCT-S' (SEQ ID NO:23).

An siNA can be unmodified or chemically-modified. An siNA can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating p8 gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

Antibody based inhibitors: An antibody that specifically binds p8 can be a useful inhibitor of p8. A fragment of an antibody, such as an antigen-binding fragment, can also be a p8 inhibitor. The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term "antibody" refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat et ah, Sequences of Proteins of Immunological Interest. Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; and Chothia et ah, J. MoI. Biol. 196:901- 917, 1987, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from ammo-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.

An anti-p8 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CHl , CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.

As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids) are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes {e.g., gamma (encoding about 330 amino acids)).

The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen (e.g., a p8 polypeptide or fragment thereof). Examples of antigen-binding fragments of an anti-p8 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, Nature 341:544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see Bird et al, Science TA^AllAlβ, 1988; and Huston et al, Proc. Natl. Acad. ScL USA 85:5879-5883, 1988). Such single chain antibodies are also encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

An anti-p8 antibody can be a polyclonal antibody (see, e.g., Goruppi and Kyriakis, Jour. Biol. Chem. 279:20950-20958, 2004; and Abeam, Inc., Cambridge, MA). Alternatively, an anti- p8 antibody can be a monoclonal antibody. The antibody can be recombinantly produced, for example, such as by phage display or by combinatorial methods.

An anti-p8 antibody can be a fully human antibody. For example, the antibody can be made in a mouse that has been genetically engineered to produce an antibody from a human immunoglobulin sequence. An anti-p8 antibody can also be a non-human antibody, such as a rodent (mouse or rat), goat, rabbit, primate (e.g., monkey), or camel antibody. Methods of producing antibodies are known in the art.

An anti-p8 antibody can be one in which the variable region, or a portion thereof, such as the CDR's, are generated in a non-human organism, such as a rat or mouse. Chimeric, CDR- grafted, and humanized antibodies can be used in the methods herein to detect p8 and/or inhibit a p8 protein. Antibodies generated in a non-human organism, such as a rat or mouse, and then modified, such as in the variable framework or constant region, to decrease antigenicity in a human are also within the invention.

A CDR of an anti-p8 antibody can be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to a mutant BL- polypeptide or a fragment thereof.

Pharmaceutical formulations: A pharmaceutical composition for use in therapeutic applications can include at least one type of p8 inhibitor, such as an siNA, an antibody, a polypeptide, or a small molecule. A pharmaceutical composition featured in the invention can include a single type of p8 inhibitor or multiple types. For example, a pharmaceutical composition can include 2, 3, 4 or more different siRNA molecules, each having a different nucleotide sequence but all targeting ρ8 RNA. Alternatively, a pharmaceutical composition can include one or more siRNAs that target p8 and one or more siRNAs that target a gene that is in the same pathway as p8 (e.g., the TNF gene) for treatment of a heart condition, such as heart failure.

A p8 inhibitor for use in a pharmaceutical composition can be mixed with a sterile, pharmaceutically acceptable diluent (such as normal saline). As noted below, and as known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives. The p8 inhibitor can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is typically selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formularly).

Within the scope of the invention is use of a composition as described herein in the preparation of a medicament and use of a composition as described herein in the preparation of a medicament for the treatment of a cardiac anomaly.

A pharmaceutical composition featured in the invention (e.g., a composition containing a p8 inhibitor) is formulated to be compatible with its intended route of administration. Examples of routes of administration include intraperitoneal, intramuscular, subcutaneous, and intravenous administration. It is expected that the intravenous route will be preferred. A p8 inhibitor can also be administered through a pump, catheter, patch or other device directly to the target tissue, such as to the heart.

Pharmaceutical compositions containing p8 inhibitors expressly exclude extremely heterogeneous mixtures, such as libraries (e.g., combinatorial or compound libraries, including those that contain synthetic and/or natural products, and custom analog libraries, which may contain compounds based on a common scaffold). Such libraries can include hundreds or thousands of distinct compounds or random pools thereof. Whether or not commercially available, such libraries are excluded from the meaning of a pharmaceutical composition.

Effective Dose: Pharmaceutical compositions containing p8 inhibitors can be administered on multiple occasions and at varying concentrations. It is well known in the medical arts that dosages for any one patient depend on many factors, including the general health, sex, weight, body surface area, and age of the patient, as well as the particular compound to be administered, the time and route of administration, and other drugs being administered concurrently. Dosages for a p8 inhibitor will vary depending on the structure of the inhibitor (e.g., nucleic acid, polypeptide, antibody, or small molecule) and the route of administration.

Toxicity and therapeutic efficacy of the compositions disclosed herein can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD₅₀/ED₅₀. Polypeptides or other compounds that exhibit large therapeutic indices are preferred. Data obtained from the cell culture assays and further animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (that is, the concentration of the p8 inhibitor which achieves a half-maximal inhibition of p8 activity, e.g., p8 mRNA or protein levels, or expression of a downstream marker gene, such as matrix metalloproteina.se 9 (mmp9) or mmplS) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The amount of p8 inhibitor hi a therapeutic dose is selected as an amount that causes an improvement in a symptom of human having a heart condition (e.g., suffering from heart failure) without significant, adverse side effects. Such amount can vary depending on the patient and the severity of the disease or condition.

An optimal dose for a particular pharmaceutical composition can be ascertained by standard studies involving observation of patient response. Effective treatment of heart failure, for example, can be monitored by evaluating symptoms that include an improvement in heart muscle function resulting in improved blood flow; an improvement in exercise tolerance as indicated by the ability to exercise for longer periods of time without shortness of breath; decreased dyspnea; and decreased fluid retention as indicated by decreased swelling (edema) (e.g., in the legs, feet and ankles) and/or weight loss.

An effective dose of a pharmaceutical composition containing a p8 inhibitor does not have to cure a heart condition in a human. An improvement in symptoms is sufficient to qualify a dose as an "effective dose".

Generally, administration of a pharmaceutical composition containing a p8 inhibitor facilitates an intended purpose for both prophylaxis and treatment without undesirable side effects, such as toxicity, irritation or allergic response. Although individual needs may vary, the determination of optimal ranges for effective amounts of formulations is within the skill of the art. Human doses can readily be extrapolated from animal studies (Katocs et ah, Chapter 27 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990). Generally, the dosage required to provide an effective amount of a formulation will vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s) (Nies et al, Chapter 3, In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).

A pharmaceutical composition containing a p8 inhibitor can be used to prevent a heart condition, such as heart failure, in a human at risk for developing such condition. When the terms "prevent," "preventing," "prevention", or "prophylaxis" are used herein in connection with a given treatment for a given condition, they mean that the treated patient either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than he/she would have absent the treatment. These terms are not limited solely to a situation in which the patient experiences no aspect of the condition whatsover. For example, a treatment will be said to have "prevented" the condition if it is given during exposure of a patient to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the patient's experiencing fewer and/or milder symptoms of the condition than otherwise expected. A treatment can "prevent" a heart condition by resulting in the patient's displaying only mild overt symptoms of the condition; it does not imply that there must have been no development of the condition whatsoever. p8 as a diagnostic agent: Detection or monitoring of p8 levels can be used as a diagnostic agent. For example, detection of elevated levels of p8 can indicate a cardiac anamoly, such as cardiomyocyte hypertrophy or an extracellular matrix remodeling of a heart tissue, each of which can be a predictor of heart failure. p8 can be detected by immunohistochemistry techniques performed, for example, on cardiac tissue isolated from a patient, or by measurement of p8 polypeptide or polypeptide fragments in a fluid sample (e.g., a blood, urine or saliva sample) isolated from a patient. Upon a determination that p8 levels are elevated, the patient can be placed on a therapeutic regime, which can include one or more of a pharmaceutical composition containing a p8 inhibitor; medications including diuretics, angiotensin antagonists, beta blockers, digoxin, aldosterone antagonists or inotropes; and instructions to modify lifestyle habits (e.g., instructions to quit smoking, or to modify diet to lower sodium and cholesterol intake). Kits: A pharmaceutical or diagnostic composition containing a p8 inhibitor can be provided in solution, such as in a sterile aqueous solution, or the composition can be packaged in a lyophilized form. Kits containing the compositions can include solubilizing reagents, such as sterile water or buffers and/or reagents for diluting a solution and/or otherwise adjusting the properties of a solution in preparation for an intended use. For example, a kit containing a p8 inhibitor for diagnostic use can include buffers and reagents for detecting p8 in a tissue or fluid sample obtained from a patient.

The compositions containing a p8 inhibitor can be packaged in a variety of suitable containers. For example, a composition can be contained in a bottle, vial, or syringe, composed of a material such as glass or plastic. Optionally, a pharmaceutical composition can be packaged in an individual dosage form, such as in an ampoule, syringe, or blister pack. Containers can be air tight and/or waterproof, and can be labeled for use, such as for a treatment of a heart condition.

A kit can also include informational material that can be descriptive, instructional, marketing or other material that relates to methods (e.g., the diagnostic or treatment methods) suitable for use with a p8 inhibitor. For example, a kit containing a p8 inhibitor for treatment of a human can include informational material that describes patients suitable to receive the compositions included in the kit (e.g., patients diagnosed as having or being at risk for having heart failure), and how and at what dosage is suitable for administration according to the patient's symptoms and/or diagnosis. The instructional material can also describe suitable adjustments to dosage according to such variables as the patient's age and weight. In another example, a kit containing a p8 inhibitor for diagnostic purposes can include informational material that describes how to perform the assay and further how to interpret the results of the assay.

The informational material of the kits is not limited in its form. In many cases, the informational material is provided in printed matter, such as in printed text, drawings, and/or photographs. The instructional material can be in the form of a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. The informational material can include contact information, such as a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about an immunotherapeutic composition and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.

Screening assays. Methods of identifying a p8-modulating agent are also provided. The methods are useful for identifying any form of modulating agent, including nucleic acid, protein and small molecule agents. A screening method can be performed by providing a cell that expresses biologically active p8, exposing the cell to a test agent (e.g., a nucleic acid, polypeptide, or small molecule), and determining whether the test compound inhibits the expression or activity of p8. A compound that inhibits the expression or activity of p8 is identified as a p8-modulating agent.

Cells useful for the screening methods featured in the invention include mammalian cells, such as human, rodent (e.g., mouse or rat), rabbit, and primate cells. The cells can be derived from heart tissue, and can be, for example cardiomyocytes or cardiac fibroblasts.

A collection of test nucleic acid inhibitors (e.g. , siNAs) can be designed by targeting various sequences of a p8 target nucleic acid (e.g. , the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO:2), including splice variants of p8. To design such a collection, a gene walk can be performed such that every possible sequence of p8 is targeted by a siNA. Software programs, such as that available on-line at "siRNA at Whitehead," can be used to focus the test library on specific candidate sequences.

Libraries that encode or contain candidate compounds are available to those of ordinary skill in the art through charitable sources (e.g., ChemBridge Corporation (San Diego, CA) (which provides useful information about chemical libraries on the worldwide web)) and commercial suppliers. Sources include Asinex (Moscow, Russia); Bionet (Camelford, England); ChemDiv (San Diego, California); Comgenex (Budapest, Hungary); Enamine (Kiev, Ukraine); IF Lab (Ukraine); Interbioscreen (Moscow, Russia); Maybridge (Tintagel, UK); Specs (The Netherlands); Timtec (Newark, DE); and Vitas-M Lab (Moscow, Russia).

Assays to determining whether a test compound inhibits the expression or activity of p8, include assays to measure p8 protein and mRNA levels, or assays that detect expression levels of genes in the p8 protein expression pathway. For example, downregulation of mmp9 or mmpl3 protein or mRNA levels can indicate downregulation of the p8. Assays for monitoring gene expression are numerous and well-known in the field of cell and molecular biology, and include, for example, Western analysis or immunocytochemistry to measure p8 protein.levels, and Northern analysis or RT-PCR to measure p8 mRNA levels. Exemplary assays are described in, e.g., Ausubel et ah, eds., Protocols in Molecular Biology.

The invention is further illustrated by the following example, which should not be construed as further limiting.

EXAMPLE

General methods

Human tissue: Biopsies of non-failing left ventricular (LV) heart were obtained at autopsy from individuals with no evidence of cardiac disease. Failing human myocardial samples were obtained consecutively from the hearts of patients who had undergone heart transplantation because of severe heart failure consequent to LV systolic dysfunction. Myocardial samples were obtained first during placement of a ventricular assist device (VAD) and subsequently at the time of heart transplant, after VAD support (Patten et ah, J. Am. Coll. Cardiol. 45:1419-1424, 2005).

This protocol was approved by the Institutional Review Board for Human Studies at Tufts-New England Medical Center.

Cells and treatments, immunofluorescence and hypertrophy assays, siRNA-dependent RNAi: Primary neonatal rat LV cardiomyocytes and fibroblasts were isolated, cultured, and assayed for hypertrophy as described (Choukroun et ah, J. Clin. Invest. 102:1311-1320, 1998). 293 and U2OS cells were cultured in DMEM supplemented with 10% FBS. TNF, PE, ET-I, MG132, Lactacystin, LY294002, U0126, SB203580 and JNK inhibitor were from Calbiochem. Transfection methods, immunofluorescence assays, pCMV Myc-tagged Ub and pcDNA His5- (human) p8 have been previously described (Goruppi and Kyriakis, J. Biol. Chem. 279:20950- 20958, 2004). The siRNA and RNAi for rat ^S have been reported previously (Goruppi et ah, EMBOJ. 21:5427-5436, 2002; Goruppi and Kyriakis, J. Biol. Chem. 279:20950-20958, 2004).

RT-PCR and Northern Blot analysis: Total RNA was isolated from cardiomyocytes and cardiac fibroblasts as described (Goruppi et ah, EMBO J. 21:5427-5436, 2002). RT-PCR was performed using the Titan One Tube™ RT-PCR kit (Roche), β-actin or gapdh served as internal controls. Primer sequences were as follows (5'—> 3'): p8 forward

ATGGCCACCTTCCCACCAGC (SEQ ID NO:5); p8 reverse TCAGCGCCGTGCCCCTCGCT (SEQ ID NO:6); mmp-9 forward AAGGATGGTCTACTGGCA (SEQ ID NO:7); mmp-9 reverse AGAGATTCTCACTGGGGC (SEQ ID NO:8); mmp-13 forward CCTGGGATTTCCAAAAGAGGT (SEQ ID NO:9); mmp-13 reverse TAACACCACAATAAGGAATTT (SEQ ID NO: 10); on/forward

CCATATTGGAGCAAATCCTG (SEQ ID NO: 11); an/reverse CGGCATCTTCTCCTCCAGG (SEQ ID NO:12); $-actin forward CCAAGGCCAACCGCGAGAAGATGAC (SEQ ID NO:13); β-actin reverse AGGGTACATGGTGGTGCCGCCAGAC (SEQ ID NO: 14). Northern analysis of p8 expression was performed as previously described (Goruppi et al, EMBO J. 21:5427-5436, 2002).

ChIP: Cardiomyocytes were serum starved and treated with PE as indicated. Cardiac fibroblasts were serum starved and treated with vehicle or TNF for 15 hours. 293 cells were kept in 10% FBS until harvest. Chromatin immunoprecipitation (ChIP) was performed as described (Chadee et al, J. Biol. Chem. 274:24914-24920, 1999) using the EZ ChIP™ kit (Upstate). Endogenous p8 or c-Jun was immunoprecipitated from soluble chromatin as described (Goruppi and Kyriakis, J. Biol Chem. 279:20950-20958, 2004). For c-Jun ChIP, the antibody used was from Santa Cruz (H-79). For ChIP of transfected p8, pcDNA, His-tagged p8 was expressed in 293 cells. Recombinant p8 was isolated on Ni-NTA resin (Qiagen). Co-purified DNA was amplified using the following primers: (two primer sets, for the distal and proximal AP-I sites implicated in regulation of cardiac mmp9 expression; Sato and Sieki, Oncogene 8:395-405, 1993; Heymans et al, Nat. Med. 5:1135-1142, 1999; Spinale, Circ. Res. 90:520-530, 2002; Bradham et al, Am. J. Physiol Heart Circ. Physiol 282:H1288-H1295, 2002; Ma et al, MoI Cell. Biol. 24:5496-5509, 2004) - dAP-1 and pAP-1, respectively, were used to amplify segments of the cardiac fibroblast mmp-9 promoter): dAP-1 mmp-9 forward TGTCCCCTTTACTGCCCTGA (SEQ ID NO: 15); dAP-1 mmp-9 reverse ACTCCAGGCTCTGTCCTCTT (SEQ ID NO: 16); pAP-1 mmp-9 forward TGACCCCTGAGTCAGCACTT (SEQ ID NO: 17); pAP-1 mmp-9 reverse CTGCCAGAGGCTCATGGTGA (SEQ ID NO:18); mmp-13 forward CTCAAATTCTACCACAAACC (SEQ ID NO:19); mmp-13 reverse GAAGGCAGCCAGGACCCCTG (SEQ ID NO:20). For the αn/promoter, the following primers were used: ^/forward: GGCCAGAGGTCCACCCACGA (SEQ ID NO:21); anf reverse: CCAGACCCTCAGCTGCAAGA (SEQ ID NO:22). In parallel experiments, the chromatin containing recombinant p8 isolated from untransfected and transfected 293 cells was analyzed by immunoblotting with anti Jun and p8 antibodies after the chemical reversal of the cross-linker.

In-gel zymography: Metalloprotease (MMP) activity was assayed as described (Siwik et al, Circ. Res. 86:1259-1265, 2000).

Immunoblotting and protein immunoprecipitations and pull-downs: p8 antibodies and Western blotting have been described (Goruppi et al, EMBO J. 21:5427-5436, 2002; Goruppi and Kyriakis, J. Biol. Chem. 279:20950-20958, 2004). Antibodies for P-ERK, P-JNK, P-S6, P- Akt, P-MAPK substrate and total Jun were obtained from Cell Signaling Technologies. Antibodies for total Akt, ERK, p70 S6K, SGK and GFP were from Santa Cruz, and anti-actin antibody was from Sigma. For kinase inhibitor studies, cells were pre-treated either with vehicle, SB203580 (20 μM), JNK inhibitor peptide (10 μM), LY294002 (20 μM) or U0126 (10 μM) for 30 minutes. Cells were then stimulated with 30 ng/ml TNF, 100 nM ET-I or 100 μM PE for various times. Cell lysates were then analyzed by immunoblotting.

For ubiquitination and SUMOylation studies, 293 cells were transfected with the indicated plasmids (pcDNA-p8-His-tagged, pCMVMyc-Ub, pGFP-SUMOl) and treated for 3 hours with 30 ng/ml of TNF before addition of MG132 (10 μM) or Lactacystin (10 μM) for an additional 16 hours. Cells were then lysed in 8 M urea, 100 mM Tris, pH 8; and p8 protein was isolated (Goruppi and Kyriakis, J. Biol. Chem. 279:20950-20958, 2004). Quantification of immunoblots was performed using ImageJ™ software (http://rsb.info.nih.gov/ij) on an Apple OSX™ platform. Sumoylated p8 was detected with anti GFP antibodies.

PE and ET-I induce cardiomyocyte p8 mRNA and p8 protein. p8 is required for PE- and ET-I -stimulated cardiomyocyte hypertrophy: ET-I and PE couple to G-protein-coupled receptors (GPCRs) and can trigger cardiomyocyte hypertrophy in vivo and in tissue culture (Choukroun et al, J. Clin. Invest. 102:1311-1320, 1998; Choukroun et al, J. Clin. Invest. 104:391-398, 1999; Dorn and Force, J. Clin. Invest. 115:527-537, 2005). ET-I and PE stimulate a strong induction of cardiomyocyte endogenous p8 mRNA andp8 protein first detected at 1 hour and reaching a maximum by 4 hours. By contrast, the PE-stimulated appearance of p8 protein is first seen by 3 hours of stimulation with protein levels remaining elevated for at least 24 hours. Immunocytochemical analysis reveals that PE stimulates the accumulation of endogenous p8 protein in the nucleus. p8 protein levels are modestly reduced upon inhibition of either ERK with U0126, a specific inhibitor of MAPK/ERK-kinase-1 (MEKl), a direct upstream activator of ERK (Davies et al, Biochem. J. 351:95-105, 2000; Kyriakis, Mammalian MAP kinase pathways. In Protein Kinase Functions, Woodgett JR (Ed) pp 40-156, Oxford, Oxford University Press, 2000), or inhibition of PI-3-kinase with the specific pharmacologic agent LY294002 (Davies et al, Biochem. J. 351:95-105, 2000). Thus, in cardiomyocytes, the accumulation of p8 protein in response to GPCR agonists involves primarily an enhancement of p8 transcription accompanied by a relatively more modest ERK- and PI-3-kinase-dependent stabilization of the p8 polypeptide.

We find that PE- and ET-1-induced p8 is required for both PE and ET-I -stimulated hypertrophy. We used RNA interference (RNAi) mediated by short interfering RNA (siRNA) to assess the consequences of depletion of endogenous p8 on PE or ET-I -stimulated cardiomyocyte hypertrophy. Species specific (rat), but not human p8 siRNA completely abolished detectable neonatal rat cardiomyocyte p8 mRNA induced by ET-I and p8 protein induced by PE.

Increased protein synthesis (detected as an increase in the incorporation of 3H-leucine into acid-insoluble material) and the induction of the embryonic gene atrial natriuretic factor (anf) are two characteristic features of cardiomyocyte hypertrophy (Choukroun et al, J. CHn. Invest. 102:1311-1320, 1998; 1999; Hunter and Chien, N. Engl J. Med. 341:1276-1283, 1999; Kang and Izumo, Trends MoI. Med. 9:177-182, 2003). We find that both processes require p8. Thus, either PE- or ET-I -stimulated neonatal rat cardiomyocyte protein synthesis requires p8, and can be blunted with rat-specific, but not nonspecific control (human p8 sequence) p8 siRΝAs. Likewise, PE-stimulated induction of neonatal rat cardiomyocyte αnfrequires p8 and can be abrogated completely by specific p8 siRΝA. Consistent with previous observations (Wang and Proud, Circ. Res. £1:821-829, 2002) indicating that PE-stimulated cardiomyocyte protein synthesis is, at least in part, ERK-dependent, and with our finding that PE-stimulated induction of p8 protein requires ERK, we observe that inhibition of MEK/ERK signaling also suppresses PE-stimulated cardiomyocyte hypertrophy. p8 associates with chromatin that contains the anf promoter. p8 localizes to the nucleus where it may modulate transcription (Hoffmeister et al, J. Biol. Chem. 277:22314-22319, 2002). We performed ChIP experiments to begin to identify the chromatin domains with which p8 associates. Endogenous p8 was immunoprecipitated from cardiomyocytes before and after treatment with PE. The immunoprecipitates were subjected to PCR with primers derived from an enhancer element in the αλzfpromoter, which contains a canonical AP-I site. Coincident with PE induction of p8 protein expression (first detectable after 3 hours stimulation), we find that, in contrast to samples from untreated controls, starting at 3 hours of PE treatment and continuing for 24 hours of PE treatment, immunoprecipitates of endogenous ρ8 from cardiomyocyte chromatin contain substantial chromatin from the αw/promoter AP-I enhancer region. Induction of anf is mediated by AP-I (Rosenzweig et al. , Circulation 83:1256- 1265, 1991; von Harsdorf et al, J. Clin. Invest. 100:1294-1304, 1997). We used ChIP to assess the kinetics of PE-stimulated p8 and c-Jun (an AP-I component) binding to the αn/promoter, and compared this to PE induction of anf as measured by RT-PCR. PE stimulated induction of p8 protein is first seen at 3 hours. Starting at 3 hours of stimulation and reaching a maximum at 6 hours, both p8 and c-Jun bind to chromatin that contains the same canonical AP-I element. At 6 hours, when p8 and c-Jun binding are maximal, PE-stimulated αnf expression is first seen. p8 binding to the αn/^"promoter, as well as induction of αnf, continue for up to 24 hours.

Results

TNF induces cardiac fibroblast p8, andp8 is required for TNF induction of cardiac fibroblast MMP9 and MMPl 3: Our results indicate that, in primary cultures of cardiac fibroblasts, p8 is strongly and rapidly induced by TNF, with the accumulation of p8 protein preceding the appearance of p8 rnRNA. Thus, we stimulated primary cardiac fibroblasts with TNF and subjected the extracts to immunoblotting with anti-p8, anti-phospho-JNK and anti-Akt (gel loading control). TNF induces a rapid increase in JNK activation (phospho-JNK immunoreactivity) first detectable at 5 minutes of stimulation and reaching a maximum at 15 minutes. TNF stimulates a rise in p8 protein levels, which follows closely the increase in JNK activation. By contrast, p8 mRNA is not detectable until 1 hour of TNF stimulation. TNF stimulates the accumulation of endogenous p8 polypeptide in the nuclei of primary cardiac fibroblasts. Thus, it is likely that the rapid increase in p8 protein incurred by TNF is due to stabilization of the p8 polypeptide. Consistent with this, TNF stabilizes recombinant p8 protein expressed in transfected cells. Similarly, the proteasome inhibitor MGl 32 also stabilizes transfected p8. These results fit with our previous findings (Goruppi and Kyriakis, J. Biol. Chem. 279:20950-20958, 2004) that p8 protein levels can be regulated by the ubiquitin proteasome.

The TNF-stimulated accumulation of p8 polypeptide requires the JNK and ERK pathways, but not the p38 MAPK pathway. Thus, JNK inhibitor- 1 , a highly specific cell permeable peptide inhibitor of JNK based on the JNK binding domain of JNK interacting protein- 1 (JIPl) (Dickens et al, 1997), reduced both basal and TNF-stimulated endogenous cardiac fibroblast p8 protein levels, as did U0126. SB203580, a specific inhibitor of p38 (Davies et al, Biochem. J. 351 :95-104, 2000), was without effect. We did not observe TNF activation of cardiac fibroblast Akt, making it unlikely that Akt contributes significantly to TNF induction of p8 mRNA or p8 protein. The inhibitors exerted highly similar if not identical effects on p8 expressed from transfected plasmids, and the inhibitors produced the expected effects on MAPK signaling in situ. This again implicates the JNK and ERKl /2 pathways in p8 protein stabilization. Finally, consistent with a role for JNK and EJAK in p8 stabilization, transfected p8 was stabilized upon co-expression with upstream signaling kinases that recruit ERK1/2 (B-Raf), JNK (MAPK- kinase-7 [MKK7], MAPK/ERK-kinase-1 [MEKKl], or apoptosis signal-regulating kinase-1 [ASKl]) or both (mixed lineage kinase-3 [MLK3]) (Kyriakis, supra, 2000; Kyriakis and Avruch, Physiol. Rev. 81:807-869, 2001). An exception was MEKK3, which, upon overexpression, activates all known MAPKs (Kyriakis and Avruch, Physiol. Rev. 81:807-869, 2001). However, in the heart, MEKK3 likely functions more prominently in the regulation of p38 (Yang et al, Nat. Genet. 24:309-313, 2000). Thus, under the conditions employed, MEKK3 maynothave generated a sufficient ERK1/2 or JNK activation signal to stabilize p8. Seram/glucocorticoid- inducible kinase-1 (SGKl), which plays no known role in ERK or JNK signaling (Lang and Cohen, Science's STKE on the worldwide web at stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/108/43.7, 2001), failed to stabilize p8. Thus, whereas PE and ET-I induction of cardiomyocyte p8 likely proceeds largely through increased transcription, TNF induction of p8 involves an initial JNK/ERK-dependent stabilization of the protein followed by an increase in mRNA.

The rapid TNF-stimulated stabilization of p8 protein, coupled with the stabilization of p8 by inhibitors of the proteasome prompted us to ask if TNF could affect the level of p8 ubiquitination and degradation. To test this, we transfected 293 cells with Myc-tagged ubiquitin (Ub), plus either vector or His-tagged p8. Cells were then treated either with vehicle, TNF, lactacystin or both. Recombinant p8 was isolated and immunoblotted with anti p8 or, to detect Ub-p8, anti-Myc. We found that TNF and Lactacystin alone lead to comparably striking increases in p8 protein levels. Although the amount of p8 isolated from cells increased with TNF treatment, the level of Myc-Ub immunoreactivity in these same samples decreased in a TNFdependent manner even if the cells were treated with lactacystin. Thus, the stoichiometry of p8 ubiquitination decreases with TNF stimulation. Similar results were obtained when another proteasome inhibitor, MGl 32, was used. Small ubiquitin-like modifier-1 (SUMOl) is one of three SUMO group proteins that share with Ub a similar secondary structure motif, the ubiquitin superfold (Welchman et al.,NaL Rev. MoI. Cell. Biol. 6:599-609, 2005). Covalent modification with SUMO can have wide-ranging effects on target proteins including, regulation of protein- protein interactions, subcellular localization and antagonism of ubiquitination (Seeler and Dejean, Nat. Rev. MoI. Cell. Biol. 4:690-699, 2003; Welchman et al, Nat. Rev. MoI. Cell. Biol. 6:599-609, 2005; Ulrich, Trends Cell Biol. 15:525-534, 2005). Coincident with the TNFdependent increase in p8 polypeptide levels, and the decrease in p8 ubiquitination, we observed a TNF-stimulated increase in p8 SUMOylation (with SUMOl). Thus, cotransfection of cells with p8 and SUMOl (green fluorescent protein — GFP-tagged) results in a small degree of p8 SUMOylation, which, along with total p8 polypeptide, is increased with TNF. Treatment with MGl 32 stimulates a large increase in p8 protein levels. In the presence of both. TNF and MG- 132 there is a further, more modest increase in total p8 protein accompanied by a striking increase in p8 SUMOylation. This increase in SUMOylation is still observed if the levels of total p8 protein in samples from TNF plus MG132-treated cells are diluted such that the level of p8 present is equivalent to that in samples from cells treated with MGl 32 alone. Thus, TNF stimulates p8 SUMOylation. Although the p8 polypeptide contains many Ser/Thr residues, use of antibodies that detect phosphorylation of MAPK/proline-directed kinase phosphoacceptor sites detected no TNF-stimulated MAPK phosphorylation of p8. p8 contains no consensus Akt phosphorylation sites, and similar experiments using antibodies that detect phosphorylation catalyzed by Akt revealed no Akt phosphorylation of p8. Thus, TNF stimulates a reduction in the stoichiometry of p8 ubiquitination that accompanies increased p8 protein levels. Inasmuch as p8 is apparently neither an Akt nor a MAPK substrate, stabilization arising via direct phosphorylation of p8 by these kinases in vivo is unlikely. Instead, these pathways may act either to promote deubiquitination or inhibit ubiquitination (perhaps via SUMOylation),with reduced p8 ubiquitination enhancing p8 levels by preventing p8 degradation by the proteasome. We used siRNA-mediated RNAi to evaluate the role of p8 in TNF stimulation of cardiac fibroblast MMP expression. TNF induction of rat cardiac fibroblast p8 mRNA was unaffected by a human-specific p8 siRNA. However, a rat-specific siRNA completely abolished detectable TNF induction of p8 mRNA and p8 protein.

Our RNAi findings indicate that p8 is required for TNF-stimulated expression oimmp9 and mmpl3. Thus, silencing of primary rat cardiac fibroblast p8 with a specific siRNA completely inhibited TNF-stimulated induction of mmp9 and m?npl3 mRNA. Zymogram analysis indicated that silencing of primary rat cardiac fibroblast p8 siRNA also completely inhibited TNF-stimulated elaboration of functional MMP9 protein. Likewise, transfection of recombinant p8 into HeLa or U2OS cells resulted in an elevation in basal and TNF-stimulated MMP9 activity coincident with TNF stabilization of the recombinant p8 protein. p8 associates with chromatin that contains the mmp9 and mmpl 3 promoters as well as with chromatin that contains the AP-I component c-Jun: The ability of p8 to associate with the αra/promoter and to affect PE-stimulated αra/transcription combined with the requirement for p8 for induction of mmp9 and mmpl 3 prompted us to investigate if p8 associated with chromatin that contained the mmp9 or mmpl 3 promoters. We immunoprecipitated endogenous p8 from cardiac fibroblast chromatin and performed PCR on the co-purified DNA. Use of either of two primers designed to flank consensus AP-I sites in the mmp9 promoter (Sato and Sieki, 1993) identified both AP-I -containing regions of the endogenous mmp9 promoter in the anti p8 ChIP. Of note, within 24 hours of treatment, TNF stimulates a dramatic increase in the amount of endogenous p8 associated with the mmp9 promoter.

Moreover, ChIP isolates of recombinant p8 (expressed in 293 cells) contain the mmp9 and mmpl 3 promoters, as detected by PCR, indicating thatpδ is capable of associating with the mmp9 and mmpl 3 promoters in different cell types. The induction by TNF of mmp9 and mmpl 3 is mediated in part by the activator protein- 1 (AP-I) transcription factor (Sato and Sieki, Oncogene 8:395-405, 1993; Spinale, Ore. Res. 90:520-530, 2002; Rm<&am et al.,Am. J. Physiol. Heart Circ. Physiol. 282:H1288-H1295, 2002; Shin et al, Biochim. Biophys. Acta. 1589:311-316, 2002; Ma et al., MoI. Cell Biol. 24:5496-5509, 2004). Consistent with this, we also observe that recombinant p8 associates with chromatin that contains the AP-I component c-Jun. Taken together, these results indicate that endogenous and recombinant p8 associate with chromatin, hi this capacity, p8 serves as a required component in PE induction of cardiomyocyte an/ ^'and TNF-induction of cardiac fibroblast oϊmmp9 and mmpl 3, the expression of which is important to heart disease (Rhode et al, Circulation 99:3063-3070, 1999; Heymans et al, Nat. Med. 5:1135-1142, 1999; Ducharme et al, J. CHn. Invest. 106:55-62, 2000; Spinale, Circ. Res. 90:520-530, 2002; Bradham et al, Am. J. Physiol. Heart Circ. Physiol. 282:H1288-H1295, 2002; Yarbrough et al., J. Thorac. Cardiovasc. Surg. 125:602-610, 2003; Mukherjee et al, Circulation 107:618-625, 2003; Dorn and Force, J. Clin. Invest. 115:527-537, 2005).

Our results indicate that p8 is required for mmp9 induction by TNF. However, these assays were performed after 24 hours of TNF stimulation. By contrast, TNF stimulates maximal p8 protein levels by 15 minutes, with significant nuclear accumulation of p8 by 30 minutes. We used ChIP to explore the kinetics of endogenous p8 and c-Jun binding to cardiac fibroblast chromatin containing the dAP-1 consensus AP-I binding site of the mnιp9 promoter. Consistent with the rapid TNF-stimulated accumulation of p8 protein in the nucleus, at 30 minutes of TNF stimulation, binding of p8 to chromatin containing the mmp9 promoter is first detectable. Binding reaches a maximum at 3 hours and is sustained for 24 hours. TNF-stimulated c-Jun binding to chromatin containing the mmp9 promoter is slower, first detected at 1 hour and reaching a maximum at 3 hours. As with p8 binding, c-Jun binding is sustained for 24 hours. TNF-stimulated induction of mmp9 mRNA, detected by RT-PCR, is first observed at 3 hours, a time when both p8 and c-Jun are associated with chromatin containing the mmp9 promoter. mmp9 expression is sustained for 24 hours. These results fit with the idea that p8 is a regulator of TNF-stimulated endogenous mmp9 transcription.

Consistent with the idea that p8 and AP-I collaborate to induce mmp9, we find that high efficiency transfection of either c-fos, c-jun oτp8 into U2OS cells stimulates expression of endogenous mmp9. Coexpression of p8 with either c-fos or c-jun stimulates the expression of mmp9 to a degree greater than that incurred by each of these constructs alone. p8 protein is strongly induced in human heart failure by a process reversed with therapeutic intervention: p8 protein is dramatically induced in the failing human heart. We obtained myocardial samples from three individuals (at autopsy) without heart failure (non-failing controls) and from three individuals with severe heart failure secondary to left ventricular systolic dysfunction. Two myocardial samples were taken from the heart failure patients: the first obtained at the time of VAD implantation and the second at the time of heart transplant, after mechanical unloading with VAD therapy. The samples were subjected to immunoblotting with an anti p8 antibody. p8 protein levels were low to undetectable in the non failing heart controls. By contrast, myocardial samples taken from the heart failure patients showed a striking increase in p8 protein levels. Interestingly, VAD therapy significantly reduced p8 protein levels, suggesting that mechanical unloading of the failing heart coincides with a reduction in p8 levels.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a small nucleic acid molecule that specifically inhibits the expression of p8.

2. The pharmaceutical composition of claim 1, wherein the small nucleic acid molecule is a short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) molecule.

3. The pharmaceutical composition of claim 2, wherein the siRNA is a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a p8 RNA via RNA interference (RNAi), wherein (a) each strand of said siNA molecule is about 19 to about 23 nucleotides in length and (b) one strand of said siNA molecule comprises a nucleotide sequence having sufficient complementarity to said p8 RNA for the siNA molecule to direct cleavage of the p8 RNA via RNAi.

4. The pharmaceutical composition of claim 3, wherein the siNA molecule comprises no ribonucleotides.

5. The pharmaceutical composition of claim 3, wherein the siNA molecule comprises one or more ribonucleotides.

6. A pharmaceutical composition comprising an antibody that specifically binds and inhibits the activity of p8.

7. The pharmaceutical composition of claim 6, wherein the antibody is an immunoglobulin of the G type (IgG).

8. The pharmaceutical composition of claim 6, wherein the antibody is a single chain antibody.

9. A method of detecting, in a patient, a cardiac anomaly or the impending occurrence thereof, the method comprising: providing a tissue sample from the patient; and assessing the level of expression or activity of p8 in the sample, wherein an elevated level of expression or activity indicates that the patient is experiencing a cardiac anomaly or the impending occurrence thereof.

10. The method of claim 9, wherein the cardiac anomaly is an undesirable change in the size or shape of the heart; cardiomyocyte hypertrophy; or extracellular matrix remodeling.

11. The method of claim 9 or claim 10, wherein the patient is a human.

12. The method of any of claims 9-11, wherein, upon finding that the expression or activity of p8 is elevated, initiating a therapeutic regime.

13. The method of claim 12, wherein the therapeutic regime comprises administering the pharmaceutical composition of any of claims 1-8.

14. A method of identifying a p8-modulating agent, the method comprising:

(a) providing a cell that expresses biologically active p8;

(b) exposing the cell to a test compound; and

(c) determining whether the test compound inhibits the expression or activity of p8, wherein a compound that inhibits the expression or activity of p8 is a p8-modulating agent.

15. The method of claim 14, wherein the agent is a nucleic acid, a protein, or a small organic compound.

16. A method of treating a patient who has, or who is at risk of developing, a cardiac anomaly, the method comprising:

(a) identifying a patient who has or may have a cardiac anomaly; and

(b) administering to the patient an agent mat inhibits the expression or activity of p8.

17. The method of claim 16, wherein the agent is within the pharmaceutical composition of any of claims 1-8.

18. Use of a composition as described herein in the preparation of a medicament.

19. Use of a composition as described herein in the preparation of a medicament for the treatment of a cardiac anomaly.