WO2001096398A2 - Uncoupling proteins as targets for the treatment of heart failure - Google Patents

Abstract

The present invention provides methods of identifying an agent effective for the treatment of heart failure by modulating the expression or activity of uncoupling proteins (UCP). The present invention also provides compositions and methods for treating heart failure.

Description

TITLE: Uncoupling Proteins as Targets for the Treatment of Heart Failure

Related Application

This application claims the benefit of U.S. Provisional Application No. 60/211,536, filed June 15, 2000, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of identifying agents effective for treating heart failure and methods of treating heart failure. More specifically, the agents are identified by determining whether they modulate the expression or activity of an uncoupling protein.

BACKGROUND

Heart failure affects approximately 2 to 3 million Americans, and 400,000 new cases are diagnosed each year. Heart failure is slightly more common among men than women and is twice as common among African Americans as whites. The term "heart failure" suggests a sudden and complete stop of heart activity, but actually, the heart does not suddenly stop. Rather, heart failure usually develops slowly, often over years, as the heart gradually loses its pumping ability and works less efficiently. Some people may not become aware of their condition until symptoms appear years after their heart began its decline. The term "congestive heart failure" is often used to describe all patients with heart failure. However, congestion (the buildup of fluid) is just one feature of the condition and does not occur in all patients.

There are two main categories of heart failure: systolic heart failure and diastolic heart failure. Systolic heart failure occurs when the heart's ability to contract decreases. The heart cannot pump with enough force to push a sufficient amount of blood into the circulation. Blood coming into the heart from the lungs may back up and cause fluid to leak into the lungs, a condition known as pulmonary congestion. Diastolic heart failure, on the other hand, occurs when the heart has a problem relaxing. The heart cannot properly fill with blood because the muscle has become stiff, losing its ability to relax. This form of heart failure may lead to fluid accumulation, especially in the feet, ankles, and legs. Some patients may have lung congestion.

Congestive heart failure (CHF) is not a specific disease, but rather a compilation of signs and symptoms, all of which are caused by an inability of the heart to appropriately increase cardiac output during exertion. The cardiac diseases associated with symptoms of congestive failure include dilated cadiomyopathy, restrictive/constrictive cardiomyopathy, and hypertrophyic cardiomyopathy.

Although patients with all three types of cardiomyopathy are characterized with the classical symptoms of shortness of breath, edema, weakness, lethargy, and sometimes coughing, it is clear that the vast majority of patients presenting heart failure have dilated cardiomyopathy. Therefore, congestive heart failure is generally considered equivalent to dilated cardiomyopathy.

Dilated cardiomyopathy patients have typical symptoms that are caused by both systolic as well as diastolic dysfunction, although the systolic dysfunction clearly predominates. In approximately half of the patients with dilated cardiomyopathy, the cause of the heart dysfunction is ischemic heart disease due to coronary atherosclerosis. That is, patients have had either a single myocardial infarction or multiple myocardial infarctions and the resultant scarring and remodeling has resulted in the development of a dilated and hypofunctional heart. In the remaining patients, the disease is referred to as idiopathic dilated cardiomyopathy as the causative agent remains undefined.

Although modest differences exist between the patient with idiopathic (IDC) and ischemic (ISC) heart failure, they both share an abysmal prognosis and excessive morbidity and mortality. Indeed, patients with congestive heart failure have a one year survival of nearly 70% and a five year survival of only 20% after referral to a tertiary heart failure center. Morbidity is also significant as the average heart failure patient is hospitalized approximately two times each year with an average length of stay of greater than five days. Approximately half of all patients with congestive failure die suddenly—presumably due to a ventricular arrhythmia and sudden death, while the remaining patients die of worsening congestive failure.

Treatment and Therapy

Since the primary abnormality in CHF is marked systolic dysfunction, investigators presumed that an inotropic agent, i.e., a drug that increases cardiac contractility, would benefit patients with heart failure. At present, only one oral and three intravenous inotropic agents are approved for treatment of heart failure in the U.S. Digoxin is the only oral inotrope shown in a large randomized, double-blind and placebo controlled trial to have a neutral effect on survival in heart failure. However, there are concerns that it might have deleterious effects in some subgroups. Milrinone, amrinone, and dobutamine are beneficial in the acute therapy of congestive heart failure. However, chronic therapy with oral milrinone or amrinone is associated with a marked increase in mortality. Similarly, chronic therapy with dobutamine has also been associated with increased mortality.

Another approach to the therapy of patients with congestive heart failure is based on the recognition that patients with congestive heart failure expressed a group of neurohormonal substances whose plasma concentrations could be inversely associated with morbidity and mortality in large populations of patients with CHF. These neurohormonal agents all share a common finding: when given in vivo or in vitro they can initiate an aladaptive remodeling of the heart and in some cases are cardiotoxic. Additionally, in experimental CHF models, they delay or attenuate the development of the heart failure phenotype. The first neurohormonal agent to successfully serve as a therapeutic target was angiotensin II, a potent vasoconstrictor and activator of aldosterone. Studies have shown a direct relationship between increasing levels of angiotensin II (and/or renin) and cardiovascular mortality. Interestingly, transgenic mice that over-express angiotensinogen and therefore display elevated levels of angiotensin II demonstrate hypertension, do not have a phenotype consistent with congestive heart failure. Thus, it may be their effects on activating the bradykinin pathway or even an anti-adrenergic effect that is responsible for the beneficial effects of angiotensin converting enzyme (ACE) inhibitors. Although ACE inhibitors have become a mainstay of therapy in patients with CHF and have been shown to be cost effective for long-term therapy, their overall impact on CHF has actually been very modest, approximately a 18 to 22 percent decrease in mortality over four years. ACE inhibitors appear to slow the progression towards end-stage heart failure in patients.

Over the past decade, investigators have identified a group of maladaptive changes that appear to be ubiquitous in the failing human heart. These include: 1) a decrease in beta adrenergic receptor density; 2) an increase in adrenergic drive; 3) uncoupling of the beta 2-adrenergic receptor from cyclic AMP stimulation; 4) adrenergic insensitivity; and 5) enhanced function of the inhibitory guanine nucleotide regulatory protein. Thus, it appears that the beta-adrenergic receptor may be associated with congestive heart failure. In a large multi-center trial of the beta-blocker metoprolol, no difference in survival in patients receiving active drug as versus those receiving placebo is reported. However, there is a significant benefit when assessing the combined end-point of either death or the need for cardiac transplantation. As would be expected with an adrenergic blocker, the use of metoprolol is associated with a significant up-regulation of myocardial beta-adrenergic receptors. More recently, a group of clinical trials have assessed the use of carvedilol, a novel beta-adrenergic antagonist having vasodilator properties. Although the survival benefits of carvedilol is greater than those seen with the ACE inhibitors, metaanalysis of the U.S. trials included only 50 events. Therefore, the exact benefits on survival remain undefined. Beta-blockers are not easy to use in patients with CHF, as they require careful up-titration and cannot be utilized as rescue therapy. Carvedilol, for example, cannot be used for rescue therapy as it requires careful up-titration over a minimum of several weeks.

Overexpression of beta- Adrenergic Receptor Gene and CHF

The beta-adrenergic receptor signal transduction pathway is critical for rapid adjustments to increased cardiovascular demand (e.g., during exercise). In the face of chronic stimulation of this pathway, as occurs in the pathogenesis of heart failure, beta-adrenergic receptor stimulation may become maladaptive. Under these conditions, elevation of circulating catecholamines and depletion of cardiac tissue stores of norepinephrine occur in the failing heart, resulting in desensitization. Whether or not stimulation or inhibition of the beta-adrenergic receptor signaling pathway is beneficial in heart failure is controversial.

Natner et al. (1999, Am. J. Cardiol., 83, 80H-85H) address this question by specifically overexpressing a component of the beta-adrenergic receptor signaling pathway in a transgenic mouse heart. They characterized young and old adult mice with overexpressed cardiac Gsα which couples the beta-adrenergic receptor to adenylyl cyclase. In younger animals, beta-adrenergic receptor stimulation results in an augmented heart rate and cardiac contractility. Over the life of the animal, however, a picture of cardiomyopathy develops. The result is a dilated heart with a large amount of fibrosis and myocyte hypertrophy, degeneration atrophy, and apoptosis. Conversely, chronic beta-adrenergic receptor blockade prevents the development of cardiomyopathy. These experiments support the point of view that chronic beta-adrenergic stimulation during the development of heart failure is deleterious and that protecting the heart with chronic beta-adrenergic receptor blockade is salutary.

Uncoupling Proteins

Uncoupling protein (UCP) has been known to uncouple oxidative phosphorylation by moving protons across the mitochondrial inner membrane toward the mitochondrial atrix (Fleury et al, (1997) Nat. Genet. 15, 269-272). In the past, it was thought that UCP was expressed only in mammalian brown and white adipose tissue, since UCP1, the first uncoupling protein identified, is expressed only in these tissues. However, recently, uncoupling proteins expressed in other tissues and species have been reported. In 1995, a plant uncoupling protein was discovered and later sequenced (Vercesi et al (1995) Nature, 375-24; Laloi et al, (1997) Nature 389, 135). In 1997, UCP2 and UCP3 were cloned (Fleury et al, (1997) Nat. Genet. 15, 269-272; Boss et al, (1997) FEBS Lett. 408, 39-42; Vidal-Puig et al (1997) Biochem. Biophys Res. Commun. 235, 79-82). UCP-3 is expressed in skeletal muscle, whereas UCP-2 is expressed ubiquitously in mammalian tissues. Expression of UCP2 is relatively high in heart tissues (Fleury et al, (1997), supra; Boss et al, (1997), supra). Additionally, UCP4 has recently been described as a brain-specific UCP (Mao et al. (1999) FEBS Lett. 443-326-330).

The expression of UCP2 during the development of the heart has been investigated by Van Der Lee et al. (2000, FASEB, 14, 495-502). Van Der Lee et al. (supra) report that UCP2 mRNA levels increased threefold after birth when plasma fatty acid levels rise and cardiac fatty acid utilization increases. In adult heart, UCP2 mRNA levels are approximately five times higher than before birth. Additionally, Van Der Lee et al. (supra) show that in hypertrophied hearts of aorta-banded rats, expression of UCP2 remained unchanged compared to hearts of sham-operated animals. It is known that cardiac fatty acid utilization decreases during cardiac hypertrophy (Kagaya et al, (1990) Circulation, 81, 1353-1361). Thus, fatty acid may play a role in the activity of uncoupling proteins.

Although Van Der Lee et al. (supra) provides an animal model for hypertrophied heart, the model is an acute hypertrophied heart model, since pressure is placed on the aorta. Accordingly, the model is not applicable to chronic heart failure and the role of uncoupling proteins in congestive heart failure has not been previously elucidated. SUMMARY OF THE INVENTION

The present invention is based on the discovery that UCPs are overexpressed in a heart failure animal model.

The present invention provides methods of identifying an agent effective for the treatment of heart failure comprising administering the agent, and monitoring expression or activity of an uncoupling protein (UCP) to determine whether the agent modulates the activity or expression of UCP. An agent that modulates the activity or expression of UCP is effective for treating heart failure. In one embodiment, the method comprises administering the agent to in vitro cells. Preferably, the cells are selected from the group consisting of H9C2 cells, hepatocytes, and neonatal cardiomyocytes. In another embodiment, the method comprises administering the agent to host cells engineered to express an UCP. In an alternate embodiment, the method comprises administering the agent to a Gsα transgenic animal, preferably a transgenic mouse.

The present invention includes monitoring the expression or activity of an UCP selected from the group consisting of UCP- 1 , UCP-2, and UCP-3.

In a preferred embodiment, the agent is an inhibitor or inducer of UCP expression or activity. Preferably, the agent is an inhibitor selected from the group consisting of a purine nucleotide, an UCP antibody, and an antisense molecule of an UCP. Preferably, the agent is an inducer selected from the group consisting of a fatty acid and a fatty acid- activated transcription factor.

Also contemplated are agents that modulate the concentration of cAMP. Such agents include but are not limited to phosphodiesterase inhibitors, forskolin, and inhibitors and stimulators of adenylate cyclase.

The present invention also provides a method of treating heart failure comprising administering an agent that modulates expression or activity of an UCP. Preferably, the agent modulates the expression or activity of an UCP selected from the group consisting of UCP-1, UCP-2, and UCP-3. The present invention also contemplates treatment of the underlying causes of heart failure, including but are not limited to, congestive heart failure, cardiomyopathy, and ischemic heart disease.

Another aspect of the present invention involves a method of diagnosing heart failure comprising detecting the expression level of an UCP. In one embodiment the expression level of an UCP is detected using a nucleic acid probe that detects an UCP transcript. In another embodiment the expression level of an UCP is detected using an antibody.

DETAILED DESCRIPTION OF THE INVENTION 1. General Description

The present invention is based on the discovery that an UCP is overexpressed in Gsα transgenic mouse. The Gsα transgenic mouse, which exhibits symptoms of heart failure similar to that found in human patients is a useful animal model for studying heart failure.

As used herein and described in Section E, the term "heart failure" refers to a wide variety of underlying disease states, including among others, congestive heart failure, cardiomyopathy, and ischemic heart disease.

The present invention is also based on the discovery that a modulator of UCP expression or activity is effective in the treatment of heart failure. The present invention provides methods for identifying agents that modulate the expression or activity of UCP comprising administering the agent and monitoring the expression or activity of UCP. The present invention also provides methods for treating heart failure comprising administering an agent that modulates the expression or activity of an UCP. The present invention is also based in part on the finding that overexpression of a

UCP is associated with heart failure. The present invention provides method of diagnosing heart failure, comprising detecting the level of expression of an UCP. 2. Specific Embodiments

A. Gsα Transgenic Mouse as an Animal Model for Heart Failure Over the past several years, several genetic mouse models of heart failure have been produced. The inventors of the instant application have engineered a transgenic mouse which overexpresses the alpha subunit of the stimulatory G protein, termed Gsα (Gaudin et al, (1995) J. Clin. Invest., 95(4), 1676-83). This mouse exhibits a remarkably enhanced sensitivity to catecholamines, specifically in the heart. This is exhibited by a leftward shift of both the chronotropic and inotropic dose response curves for isoproterenol. Thus, at any given level of β-adrenergic receptor occupancy by an agonist, there is a greater activation of adenylyl cyclase and other downstream signaling pathways. In this mouse model, the concentration of Gsα is enhanced about fourfold, accounting for the enhanced catecholamine sensitivity. Over the first six months of life, these animals exhibit an enhanced contractile response to catecholamines. Their basal heart rates are markedly elevated as well. However, at about eight to nine months of age, there is increasing myocyte cytoplasmic and nuclear degeneration and cell loss, increased positive terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining characteristic of apoptosis (Geng et al, (1999) Circ. Res. 84, 34-42), myocardial interstitial and replacement fibrosis, myocyte hypertrophy, cardiac dilation, depressed left ventricular (LV) function, arrhythmias, and sudden death. All of these findings are consistent with, and characteristic of, the human picture of congestive heart failure (Iwase et al, (1996) Circ. Res. 78, 517-524.; Iwase et al, (1997) Am. J. Physiol. 272, H585-H589). Therefore, there must exist underlying genetic changes in the Gsα mouse that occurred over time that resulted in these deleterious effects. In order to investigate heart failure, the inventors have used the Gsα mouse as the animal model. The present invention is based in part on obtaining gene expression patterns over the life of the Gsα animal and comparing them to its wild type litter-mate control at each time point. The gene profile patterns at three months, six months, nine months, twelve mohths, fifteen months and twenty months of age, in the Gsα animal are compared to the wild type litter-mate.

B. Expression of UCP2 in Gsα Mouse and Other UCPs The present invention is based in part on the unexpected finding that UCP2 is twofold to five-fold overexpressed in the Gsα mouse, from three months to fifteen months of age. UCP2 is one of several isoforms, (homologous proteins) of uncoupling proteins including UCP1, UCP3, UCP4, and UCP2. UCP1, the first of these to be identified, is expressed in brown and white adipose tissue, particularly in rodents. Its content is thought to be regulated by the β3 -adrenergic receptor, specifically through a cyclic AMP response element. UCP1 uncouples substrate utilization in the mitochondria from ATP generation; specifically, its expression promotes dissipation of the proton gradient across the mitochondria that is the responsible driving force for ATP production. It is thought to play an important role in energy metabolism and has been implicated in states of obesity. Unlike UCP 1 , UCP2 is widely expressed in a variety of tissues, and UCP3 is more restricted to skeletal muscle. It is known that UCP2 is expressed in the heart, in cardiomyocytes. Upregulation of UCP2 in the heart of the Gsα transgenic mouse, may play a direct role in the development of cardiomyopathy in this mouse model.

C. Modulators of UCP Expression or Activity

The present invention is based in part on the finding that modulators of UCP expression or activity are effective in treating heart failure. As used herein, modulators of UCP expression or activity include anything that regulates or adjusts the expression or activity of an UCP and therefore include both inhibitors and inducers of expression or activity of UCP. Inhibitors of UCP expression or activity include but are not limited to purine nucleotides, antibodies that bind UCP2, and antisense molecules. A preferred example of a purine nucleotide is ATP. Inducers of UPC expression and activity include but are not limited to fatty acids and fatty acid-activated transcription factors. Preferred examples of fatty acids are palmitic oil and oleic oil, and preferred example of fatty acid- activated transcription factors include peroxisome proliferator activated receptor (PPAR)α.

Yoshitomi et al. (1999, Biochem. J. 340, 397-404) teach the UCP2 gene has a cyclic AMP response element in its promoter region. It is known that cyclic AMP generation is turned on in the Gsα mouse, particularly in response to β-adrenergic receptor occupancy. Therefore, agents that modulate the concentration of cyclic AMP would be expected to modulate the expression of UCP2. In one embodiment, preferred agents that modulate UCP2 expression include but are not limited to phosphodiesterase inhibitors, forskolin, and inhibitors and stimulators of adenylate cyclase. In another embodiment, preferred agents that stimulates expression of UCP2 include but are not limited to cyclic AMP analogs such as dibutyryl-cAMP and 8-Br-cAMP. It is also contemplated that other UCPs encoded by genes comprising a cyclic AMP response element in its promoter region can be modulated by the same preferred agents.

Nan Der Lee et al. (2000, supra) disclose that stimulation of neonatal cardiomyocytes with triiodothyronine increases UCP2 mRΝA levels. Accordingly, triiodothyronine is also contemplated as a modulator for UCP expression or activity to be used in the claimed methods.

D. Methods for Identifying an Agent Effective for the Treatment of Heart Failure Whole Cell Assays

The present invention provides methods of identifying agents effective for the treatment of heart failure or disease. The method comprises administering a test agent and monitering the expression or activity of an UCP to determine whether the agent is effective for treating heart failure. An effective agent for treating heart failure modulates the expression or activity of an UCP.

As used herein, the term "test agent" refers to any molecule that is to be tested. The term encompasses inducers and inhibitors.

In one embodiment, the method is performed using cells that naturally express UCPs, preferably embryonic rat heart derived H9C2 cells, hepatocytes, or cardiomyoctes. In another embodiment, the method is performed using a eukaryotic host cell engineered to express a desired UCP, i.e. transfected with the nucleic acid encoding the desired UCP. Examples of eukaryotic host cells include mammalian cells, insect cells, plant cells, or fungal cells. Preferably, the eukaryotic host cells are mammalian cells and yeast cells.

Examples of mammalian host cells include but are not limited to Chinese hamster ovary cells (CHO), 3T3 cells (derived from Swiss, Balb-c or NIH mice) , COS-1 cells, COS-7 cells, CV-1 cells, HeLa cells, L-929 cells, BHK cells, and HaK cells. Preferably, the mammalian host cells are COS-1, COS-7, HEK, and 3T3. The selection of suitable mammalian host cells and methods for fransfection, culture, amplification, and product production are known in the art.

Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. The preferred yeast cell is Saccharomyces cerevisiae. In an alternate embodiment, the method is performed using prokaryotic cells. The

UCP is expressed in bacteria and reconstituted into liposomes to measure activity. Various prokaryotic cells are known in the art. Preferred prokaryotic cells include various strains of E. coli such as HB101, DH5α, DH10, and MC10161.

Methods of transformation or fransfection of a desired nucleic acid into host cells are well known. General techniques for introducing nucleic acid into a host cell, for example, are disclosed in Sambrook et al., Molecular Cloning: A laboratory Manual, Second Edition (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press) 1989.

The activity of an UCP can be monitored using fluorescent probes to measure the potential across the mitochondrial membrane or using probes to measure the flux activity of an uncoupling protein. This method is routinely used to study the activity of UCP, see for example, Jaburek et al, (1999) J. Biol. Chem. 274, 26003-26007; Simonyan et al, (1998), FEBS Letters 436, 81-84; and Korshunov et al, (1998) FEBS Letters 435, 215- 218. Expression of UCP can be detected by northern blot analysis and quantitated by densitomen . The amount of UCP mRNAs can also be quantitated by reverse transcription-competitive PCR (RT-competitive PCR; Auboeuf et al, (1997) Anal. Biochem., 245, 141-148) or any other nucleic acid detection mean. The content of the expressed protein can be detected by Western analysis or radioimmunoassay (RIA) using an appropriate antibody.

High Throughput Assays

The present invention also provides high throughput assays for identifying agents effective in treating heart failure. In one embodiment, assays are performed using native cell types such as cardiocytes or liver cells. The test agent is administered to cardiocytes or liver cells. The UCP is extracted from the native cell and an antibody to the protein is used to measure its level to determine whether the agent modulates the expression of UCP. An agent that modulates the expression of UCP is effective in treating heart failure. In another embodiment, high throughput assays are developed for identifying agents that modulate the transcriptional activity of the UCP gene. This requires a nucleic acid construct comprising the promoter of the gene of an UCP containing all the important cis-acting elements which drive tissue specific expression and regulation, coupled to a marker protein such as β-galactosidase, luciferase, placental alkaline phosphatase, or others well known in the art. The assay comprises transfecting the construct into a host cell, adding a test agent to the cell, and measuring the amount of marker protein. The level of the marker protein directly reflects the transcriptional activity of the UCP gene.

Assays Using Gsa Transgenic Mouse

The present invention also provides methods of identifying agents effective in treating heart failure using the Gsα transgenic mouse. The method comprises administering the test agent to the Gsα transgenic mouse and monitoring the expression or activity of an UCP to determine whether the agent is effective for treating heart failure. The test agent can be administered in the drinking water at a concentration from about 0.2 to 0.8 g/L and preferably about 0.5 g/L. The agent can also be administered parenterally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or transdermally at a dosage of 1 μg to 200 mg/kg/day. To determine whether the test agent modulates UCP expression or activity, the expression level of UCP in transgenic Gsα mice can be compared with that of control transgenic mice. Other methods for determining whether the test agent modulates UCP expression include but are not limited to age of death, echocardiography (e.g. ejection fraction), measuring heart rate, measuring arterial pressure, and histology of the heart, including assessment of cellular apoptosis, necrosis, and fibrosis of the heart. The results from the treated transgenic mice can be compared to a control transgenic mice. The expression profile of UCP is also obtained for the treated and the control transgenic mice for comparison. RIA, Western analysis, and ³¹P-NMR spectroscopy of the heart can be used to assess the energy status of the heart which could be altered by UCP modulators. Finally, cardiocytes can be isolated from the heart and their UCP activities assessed using methods described previously.

E. Pharmaceutical Compositions and Methods of Delivery As used herein, the term "heart failure" is meant an abnormality of cardiac function where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues. Heart failure includes a wide range of disease states such as congestive heart failure, myocardial infarction, tachyarrhythmia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, and myocarditis. Heart failure can be caused by any number of factors, including ischemic, congenital, rheumatic, or idiopathic forms. Chronic cardiac hypertrophy is a significantly diseased state which is a precursor to congestive heart failure and cardiac arrest.

As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. The object is to prevent or slow down (lessen) hypertrophy. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented. The hypertrophy may be from any cause including but not limited to congenital, viral, idiopathic, cardiotrophic, or myotrophic causes, or as a result of ischemia or ischemic insults such as myocardial infarction, or pressure or volume overload. The treatment may also be administered to those with liver failure. Typically, the treatment is performed to stop or slow the progression of hypertrophy, especially after heart damage, such as from ischemia, has occurred. Preferably, for treatment of myocardial infarctions, the agent(s) is given immediately after the myocardial infarction, to prevent or lessen hypertrophy and/or cellular damage or death. The present invention provides composition comprising an agent that is effective to treat heart failure and routes for administering the composition to the patient. An amount of an agent that is effective for treating heart failure is administered to the patient. Typically, an effective dosage of an agent for a 50 kg to 100 kg human being is in the range of about 0.1 mg to 3000 mg per day in single or divided doses, and preferably about one mg to about 1000 mg per day in single or divided doses. More preferably, a dosage range is 10 mg to 100 mg per day in single or divided doses.

The amount and timing of the agent administered will be dependent upon the subject being treated, on the severity of the affliction, on the manner of administration and upon the judgment of the prescribing physician. Thus, due to patient to patient variability, the dosages given above are intended to be a guideline. The physician may titrate doses of the agent to achieve the treatment, e.g., congestive heart failure improvement, that the physician considers appropriate for the patient. In considering the degree of treatment desired, the physician must balance a variety of factors such as age of the patient, presence of preexisting disease, as well as presence of other diseases. In one embodiment of the invention, the agent may be administered alone or in combination with other agents such as but not limited to digitalis, thiazide diuretics, other diuretics and ACE inhibitors.

In another embodiment, the agent is administered in the form of a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to a composition comprising an agent together with a pharmaceutically acceptable carrier or diluent. A pharmaceutical composition of the present invention is directed to a composition suitable for the treatment of heart failure. A pharmaceutically acceptable carrier includes, but is not limited to, physiological saline, ringers, phosphate buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, anti-oxidants, colorants, and diluents. Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not canceled or inhibited to such an extent that treatment is ineffective. Thus, the agent can be administered individually or together in any conventional oral, parenteral, rectal or transdermal dosage form. In a preferred embodiment, the agent may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, oils (e.g., peanut oil, sesame oil) and various organic solvents. The pharmaceutical compositions formed by combining an agent and pharmaceutically acceptable carriers can then be readily administered in a variety of dosage forms such as tablets, powders, lozenges, emulsions, oil soft gels, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus, for purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch, methylcellulose, alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof.

Also contemplated is parenteral administration in which solutions containing the agent in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art. It is also contemplated that for purposes of transdermal (e.g., topical) administration, dilute sterile, aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, are prepared.

Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art. For examples of methods of preparing pharmaceutical compositions, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th Edition (1990).

Preferably, pharmaceutical compositions according to this invention may contain 0.1%-95% of the agent. In any event, the composition or formulation to be administered will contain a quantity of the agent in an amount effective to treat the disease/condition of the subject being treated, e.g., heart failure.

F. Methods of Delivering Nucleic Acid Molecules In Vivo UCP modulators or agents effective for treating heart failure that are not nucleic acid molecules can be delivered to target sites in patients as discussed above. UCP modulators and agents effective for treating heart failure that are nucleic acid molecules can be delivered to target sites in patients as discussed below. Gene therapy is a method for delivering functionally active therapeutic or other forms of genes into targeted cells. Initial efforts of gene transfer into somatic tissues have relied on indirect means, such as ex vivo gene therapy, wherein target cells are removed from the body, transfected or infected with vectors carrying recombinant genes, and re-implanted into the body. Techniques currently used to transfer DNA in vitro into cells include calcium phosphate-DNA precipitation, DEAE-Dextran fransfection, electroporation, liposome mediated DNA transfer or transduction with recombinant viral vectors. These transfection protocols have been used to transfer DNA into different cell types including epithelial cells (U.S. Pat. No. 4,868,116; Morgan et al, 1987), endothehal cells (WO89/05345), hepatocytes (Ledley et al. , 1987; Wilson et al. , 1990) fibroblasts (Rosenberg et al, 1988; U.S. Pat. No. 4,963,489), lymphocytes (U.S. Pat. No. 5,399,346; Blaese et al, 1995) and hematopoietic stem cells (Lim et al, 1989; U.S. Pat. No. 5,399,346).

Direct in vivo gene transfer has been carried out with formulations of DNA trapped in liposomes (Ledley et al, 1987), or in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al, 1983), and with DNA coupled to a polylysine-glycoprotein carrier complex. In addition, "gene guns" have been used for gene delivery into cells (Australian Patent No. 9068389). Lastly, naked DNA, or DNA associated with liposomes, can be formulated in liquid carrier solutions for injection into interstitial spaces for transfer of DNA into cells (WO90/11092).

Viral vectors are often the most efficient gene therapy system, and recombinant replication-defective viral vectors have been used to transduce (i.e., infect) cells both ex vivo and in vivo. Such vectors include retroviral, adenovirus and adeno-associated and herpes viral vectors. Accordingly, in one embodiment, an UCP modulator, such as an UCP antisense molecule, or a nucleic acid molecule effective for treating heart failure can be subloned into an appropriate vector and transferred into a cell or tissue by gene transfer techniques discussed above.

In another embodiment, an UCP modulator, such as an UCP antisense molecule, or a nucleic acid molecule effective for treating heart failure can be provided to the cell or tissue using a fransfection facilitating composition, such as cationic liposomes containing desired polynucleotide.

G. Diagnostic Probes The upregulation of UCP2 in Gsα mouse suggests that UCP2 and its homologs can be used as diagnostic marker for detecting heart failure. An antibody that binds to UCP can be used as a probe to detect and quantitate the expression of the UCP in a biological sample to determine whether the UCP is over expressed. Likewise, the level of expression of the nucleic acid encoding an UCP in a biological sample can be quantitated to determine whether the UCP is overexpressed. Methods of using antibody to detect and to quantitate a specific protein include but not limited to Western analysis and RIA and are well known to the skilled artisan (see for example Sivitz et al, (1999) Endocrinology 140, 1511-9). Measuring the level of expression of a nucleic acid is also well known in the art. Preferably, the expression level of a nucleic acid is quantitated by northern blot analysis (Yoshitomi et al, (1999) Biochem. J. 340, 397-404), by reverse transcription- competitive PCR (RT-competitive PCR; Millet et al, (1997) J. Clin. Invest., 100, 2665- 2670), or by DNA microarray technology (Voehringer et al, (2000) Proc. Natl. Acad. Sci., 97, 2680-2685).

In light of the foregoing general discussion, the specific examples presented below are illustrative only and are not intended to limit the scope of the invention. Other generic and specific configurations will be apparent to those persons skilled in the art.

EXAMPLES

Example 1 Overexpression of UCP2 in Gsα Transgenic Mouse

Differential Display Methods (Shimkets et al., (1999) Nat. Biotechnol. 17(8): 798- 803) were used to analyze cDNA which had been generated from both Gsα and wild type litter-mates. This analysis showed that UCP-2 mRNA was elevated in the Gs-alpha animals at all stages of development (up to fifteen months of age) on average 2 to 3 times over wild type litter-mates.

Example 2 Methods of Identifying an Agent Effective for Treating Heart Failure using Gsα

Transgenic Mice

Animals.

Twenty-one transgenic (TG) mice (9.4 + 0.1 months old) and 24 age-matched wild-type (WT) litter-mates (9.5 + 0.1 months old) of either sex from the same genetic background as the TG mice are studied. Briefly, the transgene consists of a rat α-myosin heavy chain promoter linked to a Gs minigene coding for the short isoform of Gs (Gaudin et al. (1995) J. Clin. Invest. 95, 1676-1683). Animals used in the present study are maintained in accordance with guidelines from the National Institutes of Health (National Institutes of Health, Office of Science and Health Reports. 1996. Guide for care and use of laboratory animals. Publication no. 83-23. Office of Science and Health Reports, Department of Health and Human Services. Bethesda, MD.).

Treatment of Animals with Test Agent TG and WT mice are randomly assigned to either a test agent treated group

(TG, n = 9; WT, n = 14) or an untreated group (TG, n = 12; WT, n = 10). A test agent is administered in the drinking water at a concentration of 0.5 g/L, or tap water was given to the untreated group. Water with freshly added drug is changed 3 times weekly, and mice are treated for 6-7 months.

Echocardiography

Echocardiographic studies are performed in 19 TG mice (9 test agent-treated and 10 untreated, 14.4 ± 0.1 months old) and 24 WT controls (14 test agent-treated and 10 untreated, 14.8 ± 0.2 months old), hi addition, echocardiographic studies are performed in 10 younger TG mice (8.8 ± 0.1 months old). After determination of body weight, mice are anesthetized with ketamine (0.065 mg/g), acepromazine (0.002 mg/g), and xylazine (0.013 mg/g) injected intraperitoneally. The procedure for echocardiography has been described in detail in Iwase et al, (1996.) Circ. Res., 78, 517-524 and Iwase et al, (1997) Am. J. Physiol., 272, H585-H589.

Heart Rate.

Heart rate is measured using telemetry techniques in the conscious, unrestrained state in 16 TG mice (9 test agent-treated and 7 untreated, 15.6 + 0.1 months old) and 23 WT mice (14 test agent-treated and 9 untreated, 15.9 ± 0.1 months old). Mice are anesthetized as already described, and a telemetry transducer (TA10EA-F20; Data Science Co., St. Paul, Minnesota, USA) is implanted in the peritoneal cavity with paired electrodes placed subcutaneously over the thorax (chest bipolar electrocardiogram leads) (Uechi et al, (1998) Circ. Res. 82, 416-423). Experiments are initiated 3-5 days after recovery from surgical instrumentation. Mice with implanted telemetry devices are housed in individual cages with free access to food and water, and are exposed to 12-hour light/ 12-hour dark cycles.

Arterial pressure

Arterial pressure is measured in 16 TG mice (9 test agent treated and 7 untreated, 16.1 ± 0.1 months old) and 23 WT mice (14 propranolol-treated and 9 untreated, 16.3 + 0.1 months old). The mice are anesthetized as already described, and arterial blood pressures are measured by a 1.4F micromanometer catheter (Millar Instruments Inc., Houston, Texas, USA), which is inserted into the carotid artery. Northern-blot Analysis

Total RNA is extracted by RNAzol B, as described in Yoshitomi et al. (1999, supra). Total RNA (15 μg) is applied per lane on 1.2% agarose gel containing 2.2 M formaldehyde, and electrophoresis is carried out. RNA is then transferred onto nylon membranes (GeneScreen Plus, DuPont, Boston, MA, U.S.A.) for 20 h and crosslinked by UV irradiation. It is confirmed that total RNA is loaded equally for all lanes by hybridization of human 28S probe (Clontech). The probes for detecting UCPs are generated with PCT methodology and labelled by random priming in the presence of [α- ³²P]dCTP by BcaBEST random-priming kit (Takara). Hybridization is performed at 68°C for 2-5 h in ExpressHyb Hybridization solution (Clontech), followed by washing twice for 10-30 min at room temperature with 2 x SSC (where 1 x SSC is 0.15 M NaCl / 0.015 M sodium citrate) / 0.05% SDS, twice for 15 min at 50°C with 2 x SSC/ 0.05 % SDS, and finally twice for 5-30 min at 50°C with 0.1 x SSC / 0.1% SDS. The filter is exposed to X-ray film (Konica, Tokyo, Japan) at -80°C for 5-48 h, and each band intensity is quantified with a bioimaging analyser, BAS2000 system (Fuji Photofilm, Tokyo, Japan).

Histology

Histological studies are conducted in hearts from 21 older TG mice (15.7 + 0.2 months old), 23 older WT mice (16.3 ± 0.1 months old), and 10 untreated younger TG mice (8.9 ± 0.1 months old). The heart is perfused with a brief saline wash followed by paraformaldehyde or formalin via a 21 -gauge needle inserted directly into the LV apex. All hearts are fixed by perfusion fixation with 3% paraformaldehyde, except for the hearts of 5 untreated TG mice that died prematurely, and are fixed with 10% buffered formalin. All animals are anesthetized as already described. The heart is dissected to remove the atria and right ventricular free wall, and each portion is weighed. Fixed tissues are dehydrated, embedded in paraffin, sectioned at 6-μm thickness, and stained with hematoxylin and eosin, Gomori's aldehyde fuchsin trichrome, and picric acid/sirius red. Heart sections are also dehydrated and embedded in glycol methacrylate, sectioned at 1-μm thickness, and stained with methylene blue-basic fuchsin for light-microscopic examination. Methacrylate sections are also stained with silver-gold (Accustain; Sigma Diagnostics, St. Louis, Missouri, USA) for basement membrane to outline cardiac myocytes for cross-sectional area measurement.

Myocardial connective tissue is quantitatively analyzed on a cross-section of LV obtained mid-distance from base to apex and stained with picric acid sirius red. Images are obtained from a Sony CCD video camera (Sony Electronics Inc., Park Ridge, New Jersey, USA) attached to a Nikon E800 microscope (Nikon Inc. Melville, New York, USA) with a x60 objective, and are analyzed with MetaMorph image analysis software (Universal Imaging Corp., West Chester, Pennsylvania, USA). Myocyte cross-sectional area is measured from images captured from silver-stained 1-μm-thick methacrylate sections obtained mid-distance from base to apex. Suitable cross-sections are defined as having nearly circular capillary profiles and circular-to-oval myocyte sections. No correction for oblique sectioning is made. The outline of 100-200 myocytes is traced in the LV of each animal, using the MetaMorph image system software to determine myocyte cross-sectional area. The mean area is calculated for the LV in each animal, and the group mean is calculated for each region and group.

DNA fragmentation is detected in situ by using TUNEL (dUTP nick end-labeling) on paraffin sections of the mouse hearts from TG and WT mice, as described previously (Geng et al, (1999) Circ. Res. 84, 34-42). After the TUNEL procedure, the slides are washed in PBS, mounted in a Vector DAPI medium, and observed under a fluorescence microscope. The mean number of myocyte nuclei per x40 field in LV regions is determined by manual counting of DAPI-stained nuclei with ultraviolet excitation. At the same magnification, a minimum of 20 fields in the LV regions of each heart are examined for TUNEL-positive myocytes. All morphometric measurements are performed by at least 2 independent individuals in a blinded manner. Example 3

Measurement of UCP Activity Using Whole Cells

Expression of UCP in E. coli Nucleic acids encoding UCP open reading frame are amplified by PCR and inserted into the Ndel and Notl sites of the pET21a vector (Novagen). DNA sequencing of the constructs is used to confirm that the encoded proteins have an amino acid sequence identical to the corresponding wild-type UCP. Plasmids are transformed into the bacterial strain BL21 (Novagen). Transformed cells are grown at 30°C to A600 = 0.6 and then induced with 1 mM isopropyl~D-thiogalactopyranoside at 30°C for 6 h. Cells from a 700-ml culture are lysed in a French press in 20 ml of lysis buffer (10 mM Tris, pH 7, 1 mM EDTA, 1 mM dithiothreitol); the lysate is centrifuged at 27,000 x g for 15 min; and the pellet is resuspended in 20 ml of lysis buffer and centrifuged at 1000 x g for 3 min. One ml aliquots of the supernatant are centrifuged at 14,000 x g for 15 min in a microcentrifuge, and the resulting pelleted inclusion bodies are stored frozen at 70 °C. The nucleic acids encoding UCPs can also be expressed in mammalian and yeast host cells for production of the encoded protein. Mammalian and yeast host cells are known to the skilled artisan and are described earlier.

Extraction of UCP from Inclusion Bodies

Solubilization of E. coli inclusion bodies is previously described in Jaburek et al, (1999 J. Biol. Chem. 274, 26003-26007). The pelleted inclusion bodies (about 2 mg of protein) are suspended and washed three times in wash buffer (tetraethylammonium (TEA+) salts of 0.15 M phosphate, 25 mM EDTA, 1 mM ATP, and 1 mM dithiothreitol, pH 7.8). The final pellet is solubilized in 0.4 ml of 50 mM TEA-TES, pH 7.2, containing 1.5% sodium lauroylsarcosinate (SLS). The extract is supplemented with 10 mg/ml asolectin and 3% octylpentaoxyethylene (C8E5 detergent) and then dialyzed for 15 h against 3 x 400 ml of extraction buffer (TEA+ salts of 50 mM TES and 1 mM EDTA, pH 7.2) to remove SLS. In the first two dialysis periods (1 and 13 h), the extraction buffer is supplemented with 1 mM dithiothreitol and 0.03% sodium azide. These are removed from the final dialysis (1 h). Aliquots of the dialyzed extract, containing about 0.2 mg of protein, are stored at 20 °C.

Reconstitution of Uncoupling Proteins into Liposomes

UCPs are reconstituted as described in Jaburek et al, (1999 J. Biol. Chem. 274, 26003-26007). Egg yolk phosphatidylcholine or soybean phospholipids are supplemented with cardiolipin (2 mg/ml), dried, and stored under nitrogen. Internal medium (TEA+ salts of TES (30 mM), SO₄ (80 mM), and EDTA (1 mM), pH 7.2) is added to give a final concentration of 40 mg of phospholipid/ml of proteoliposome stock. The mixture is vortexed and sonicated to clarity in a bath sonicator, and detergent (10% C8E5), protein extract, and fluorescent probe are added. The final mixture (1.1 ml) is applied onto 2 ml of Bio-Bead SM-2 (Bio-Rad) column to remove the detergent. After 2 h of incubation, the column is centrifuged, and the resulting proteoliposomes are applied onto a new 2-ml Bio-Bead column, incubated for 30 min, and centrifuged. The formed vesicles (1 ml) are passed through a Sephadex G-25-300 column to remove external probe.

Fluorescence Measurements of Ion Fluxes

Ion flux in proteoliposomes is measured using ion-specific fluorescent probes and an SLM Aminco 8000C spectrofluorometer. Measurements of H* fluxes are obtained from changes in 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence due to quenching by the anion of TES buffer (Orosz et al, (1993) Anal. Biochem. 210, 1-15). Measurements of K+ fluxes, reflecting the movement of ionic charge across the membrane, are obtained from changes in potassium-binding benzofuran isophthalate fluorescence (Garlid et al, (1995) Methods Enzymol. 266, 331-348; Jeek et al, (1990) J. Biol. Chem. 265, 10522-10526 ). Internal and external media contain K⁺ or TEA⁺ salts of TES buffer (30 mM), SO₄ (80 mM), and EDTA (1 mM), pH 7.2. Each proteoliposome preparation is individually calibrated for fluorescent probe response, and its internal volume was estimated from the volume of distribution of the fluorescent probe (Garlid et α/.,(1995) Methods Enzymol. 266, 331-348 ). An agent to be tested for inhibiting or inducing UPC activity is added for measurement of H⁺ fluxes. It should be understood that the foregoing discussion and examples merely present a detailed description of certain preferred embodiments. It therefore should be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention. All journal articles, other references, patents, and patent applications that are identified in this patent application are incorporated by reference in their entirety.

Claims

1. A method of identifying an agent effective for the treatment of heart failure comprising, (a) administering the agent; and,

(b) monitoring expression or activity of an uncoupling protein (UCP) to determine whether the agent is effective in treating heart failure.

2. The method of claim 1, wherein the agent is administered to in vitro cells.

3. The method of claim 2, wherein the cells are H9C2 cells, hepatocytes, or neonatal cardiomyocytes.

4. The method of claim 2, wherein the cells are host cells engineered to express an UPC.

5. The method of claim 1 , wherein the agent is administered to a GSα transgenic animal.

6. The method of claim 1, wherein step (b) comprises monitoring the expression of an UCP.

7. The method of claim 1, wherein step (b) comprises monitoring the activity of an UCP.

8. The method of claim 1, wherein step (b) comprises monitoring the expression or activity of an UCP selected from the group consisting of UCP- 1, UCP-2, and UCP-3.

9. The method of claim 1 , wherein the agent is an inhibitor or an inducer of UCP expression or activity.

10. The method of claim 9, wherein the inhibitor is selected from the group consisting of a purine nucleotide, an UCP antibody, and an antisense molecule of an UCP.

11. The method of claim 9, wherein the inducer is selected from the group consisting of a fatty acid and a fatty acid-activated transcription factor.

12. The method of claim 1 , wherein the agent modulates the concentration of cAMP.

13. The method of claim 12, wherein the agent is selected from the group consisting of phosphodiasterase inhibitors, forskolin, and inhibitors and stimulators of adenylate cyclase.

14. A method of treating heart failure comprising administering an agent that modulates UCP expression or activity.

15. The method of claim 14, wherein the agent modulates the expression or activity of an UCP selected from the group consisting of UCP-1, UCP-2, and UCP-3.

16. The method of claim 14, wherein the agent modulates the expression of an UCP.

17. The method of claim 14, wherein the agent modulates the activity of an UCP.

18. The method of claim 14, wherein heart failure is selected from the group consisting of congestive heart failure, cardiomyopathy, and ischemic heart disease.

19. A method of diagnosing heart failure comprising detecting the expression level of UCP.

20. The method of claim 19, wherein the expression level of UCP is detected using a UCP nucleic acid probe.

21. The method of claim 19, wherein the expression level of UCP is detected using an antibody against an UCP.