WO2008028065A2

WO2008028065A2 - Sirt activation in managing heart failure

Info

Publication number: WO2008028065A2
Application number: PCT/US2007/077280
Authority: WO
Inventors: Mahesh Gupta
Original assignee: University of Chicago
Current assignee: University of Chicago
Priority date: 2006-08-31
Filing date: 2007-08-30
Publication date: 2008-03-06
Anticipated expiration: 2009-02-28
Also published as: WO2008028065A3

Abstract

Activation of a member of a class III histone deacetylase (e.g., SIRT1 and SIRT3) inhibits or blocks most of the events associated with heart failure (e.g., cardiomyocyte hypertrophy, cell death, loss of α-myosin isoform shift and fetal gene activation) and protects myocytes from hypertrophy agonist-mediated cell-death. SIRT1 and SIRT3 activation favors α-MHC expression and alters the myosin-isoform switch from β-MHC to α-MHC during cardiac hypertrophy. SIRT1 and SIRT3 activation negatively regulates cardiac hypertrophy and cell-phenotype, and these characteristics of SIRTs are therapeutically valuable for the treatment of heart failure.

Description

SIRT ACTIVATION IN MANAGING HEART FAILURE

INVENTOR: MAHESH GUPTA

[0001]

BACKGROUND

[0002] Activation of Sirtuins (SIRT) that belong to class III histone deacetylases is a target for designing new therapeutic strategies for the management of heart failure.

[0003] Heart failure is an end-stage pathological condition of virtually all heart diseases. It is usually characterized by cardiac myocyte hypertrophy, myosin- isoform-shift, myocyte cell-death, interestial fibrosis and finally chamber-dilation and pump failure. Several mechanisms and targets have been identified but, none so far could block/reverse all these changes.

[0004] Mammalian cardiac myocytes are mostly terminally differentiated cells.

They lose their ability to divide soon after birth, but undergo hypertrophic growth in response to a variety of stress stimuli. In conditions of sustained increase of workload, which could arise from cardiovascular disorders such as hypertension, valve dysfunction, myocardial infarction, arrhythmias and mutations of cardiac contractile proteins, the adult heart typically becomes enlarged because of cardiomyocyte hypertrophy. During cardiac hypertrophy myocytes not only increase in size, but they also add additional sarcomeres and induce expression of a group of genes, which are usually expressed during fetal heart development. These genes include activation of β-myosin heavy chain (MHC), skeletal-α-actin, atrial natriuretic factor and the repression of α-MHC and SR-CaATPase. These changes may be initially salutary for an overloaded heart; however, prolonged hypertrophy leads to myocyte cell-death and dysfunction of viable myocytes, which eventually results in heart failure. A growing body of evidence obtained from patients and animal models of cardiac hypertrophy has indicated that inhibition of cardiac hypertrophy and blockade of myosin-isoform shift, particularly prevention of α- MHC loss, may be beneficial to preserve the heart function and prevent it from going into failure. However, very little is known to date about the mechanism(s) through which negative regulation of cardiac hypertrophy and a change in muscle phenotype could be achieved, without having undesired effects on cell-survival. [0005] Sirtuins (SIRT) belong to class-Ill group of histone deacetylases (HDACs).

They are homologous to the yeast Sir2 gene, which has been implicated in chromatin silencing, cell-survival and aging. So far, seven different human SIRT isoforms (SIRTl -7) have been identified (human SIRTl is orthologue of mouse Sir2α). SIRTl, SIRT6 and SIRT7 are primarily localized in the nucleus, whereas SIRT2 is in the cytoplasm and SIRT3, SIRT4 and SIRT5 are present in mitochondria. Unlike the class-I and II HDACs, the deacetylase activity of SIRTs (Sir2) is absolutely dependent upon the availability of cell NAD content. They catalyze a unique reaction in which the NAD is broken into nicotinamide and ADP-ribose moiety. The acetyl group that is removed from the substrate is transferred to ADP-ribose moiety, giving rise to formation of O-acetyl- ADP- ribose, a novel metabolite. Gene knock-out studies have documented a critical role of Sir2α in mouse embryogenesis. The Sir2α null homozygous pups are smaller and die before maturation; whereas heterozygous littermates develop gross defects in many organs including the heart.

[0006] SIRTs are considered nuclear sensors of redox-signaling. In stress conditions a change in the metabolic state of the cell (NAD/NADH ratio) alters the deacetylase activity of SIRTs, and this has the potential to influence the cell fate. Increased cellular NAD content elevates the SIRT deacetylase activity, whereas high nicotinamide and NADH levels act as inhibitors of Sirtuins. Recently, SIRTs have attracted significant attention because of their role in genetic control of aging. In yeast, the life span has been shown to be shortened by a null mutation in the Sir2 gene, and it is extended by the presence of an extra copy. Likewise, over- expression of Sir2 orthologues increased life span (>50%) of other species, including worms, and flies, indicating that Sir2 controls longevity of the organism.

[0007] SIRTl has been shown to participate in controlling glucose and fat metabolism and is implicated in the calorie-restriction mediated cell-survival. In addition to histones, SIRTl has been shown to deacetylate many other proteins, including proteins participating in cell division and apoptosis, such as p53, E2F, Ku70; Bax, FOXOs, NF-kB, p300 and PCAF; factors controlling muscle-specific gene expression, such as MyoD; as well as general transcription factors, such TAF168, which participates in rRNA transcription. In the brain, over activation of SIRTl has been shown to prevent axonopathy and neuronal degeneration. SUMMARY

[0008] Methods and compositions to manage heart failure by blocking cardiac hypertrophy and myosin isoform shift through activation of a Sirtuin (SIRT) are described. Activation of a member of the Sirtuin group of histone deacetylases (e.g., Sir2α or SIRTl and SIRT3) prevents/blocks many of the changes associated with a hypertrophied/failing heart. Use of myocytes and fibroblast cultures as well as animal models of hypertrophy demonstrated that myocyte hypertrophy, cell- death, interstitial fibrosis and myosin-isoform-shift were blocked by SIRTl/Sir2-α or SIRT3 activation. Activation of SIRTl and SIRT3 functions as a negative regulator of cardiac hypertrophy and increases α-myosin heavy chain (α-MHC) expression.

[0009] Activation of SIRTl, a member of class III histone deacetylases, results in

MHC isoform shift from β- to α-myosin heavy chain (α-MHC). SIRTl activation favors the α-MHC expression by preserving or increasing the α-MHC levels and that minimize the loss of cardiac myocytes contractility during heart failure. Activation of SIRTl, therefore minimizes the loss of α-MHC during heart failure.

[00010] Activation of SIRTl or SIRT3 reduces or blocks myocyte hypertrophy, which is beneficial for an overloaded (failing) heart during heart failure. Activation of SIRTl and/or SIRT3, therefore, minimizes the decompensation response during heart failure.

[00011] Activation of SIRTl reduces proliferation of fibroblasts and interstitial fibrosis and collagen deposition during heart failure.

[00012] Activation of SIRTl and/or SIRT3 increases the number of viable myocytes and decreases the number of dysfunctional myocytes during heart failure. SIRTl and/or SIRT3 activation thus reduces cell-death of hypertrophied myocytes.

[00013] The effects of SIRTl and/or S IRT3 activation on responsiveness of cardiomyocytes to hypertrophy agonists are described. Primary cultures of neonatal rat heart myocytes were stimulated with a series of hypertrophy agonists, e.g. phenylephrine, angiotensin-II and serum. The hypertrophy response of myocytes was determined by enhanced protein synthesis, sarcomere organization and the induction of fetal gene expression.

[00014] The SIRTl effect on myocyte growth was examined by infecting cells with adenovirus vectors, expressing either wild-type SIRTl or a mutant (See Example 2), lacking deacetylase activity. Agonist treatment resulted in enhanced myocyte protein content, highly organized structures of sarcomeres with increased myocyte size and induction of fetal gene expression; i.e. up-regulation of ANF and β-MHC and down-regulation of α-MHC expression. This agonist-mediated hypertrophy response of cardiomyoctes was blocked by over-expression of the wild-type SIRTl, but not by its dominant negative mutant.

[00015] SIRTl activation plays a role in the induction of cardiac α-MHC and suppression of β-MHC levels, a desired phenotype anticipated to restore myocyte contractility during hypertrophy. Activation of SIRTl also prevents loss of α-MHC in hypothyroid animals, where it is otherwise drastically reduced, thus suggesting that SIRTl plays a major role in α-MHC expression. These results demonstrate that SIRTl activation functions as a negative regulator of cardiomyocte hypertrophy, promotes myocytes cell-survival and shifts myosin isoform in favor of α-MHC expression. Regulation of SIRTl has therapeutic potential for the management of heart failure.

[00016] A pharmaceutical composition for reducing cardiac hypertrophy includes activated class III histone deacetylase. The class III histone deacetylases include Sirtuin 1 (SIRT1/Sir2α) and SIRT3. The SIRTs are activated by, for example, a small molecule or by direct gene expression or by promoter upregulation. Pharmaceutical compositions including SIRTs are used for treatment of various aspects of heart failure. Pharmaceutical compositions to active SIRTs may be administered orally, for example, in foods or pills.

[00017] Reducing myosin-heavy-chain (MHC) isoform shift from predominantly β- to α-MHC isoform during cardiac failure is accomplished by activating a sirtuin histone deacetylase.

[00018] Activated a sirtuin 1 (SIRTl) histone deacetylase reduces a fetal-gene activation during cardiac hypertrophy. Fetal-gene activation includes a gene selected from a group of ANF, β-MHC and CARP.

[00019] PARPl (poly ADP-ribose polymerase- 1) is activated during oxidative stress and DNA damage, leading to cardiomyocyte cell-death during heart failure. Activation of SIRT suppresses PARPl activity and blocks this type of myocyte cell-death and protects heart from going into failure.

[00020] A method to identify an agent for activating SIRT during heart failure includes the steps of: (a) providing a candidate agent to a cardiomyocyte cell culture that has been induced for cardiac hypertrophy;

(b) monitoring the activation of SIRT and/or by measuring acetylation status of p53, a SIRT target; and

(c) identifying the candidate agent as the agent for activating SIRTl based on the reduction of the cardiac hypertrophy phenotype.

[00021] Over-expression of SIRTs is used to treat various aspects of heart failure.

Gene therapy is a way to deliver extra copies of genes encoding SIRTs to target tissues to produce higher levels of SIRTs than endogenously expressed.

BRIEF DESCRIPTION OF DRAWINGS

[00022] FIG. 1 shows that SIRTl activation blocks agonist-mediated enhanced protein synthesis. (A) Primary cultures of cardiomyocytes grown in serum-free medium were treated with a SIRTl activator, resveratrol (50OnM), inhibitor sirtinol (30μM) or PE (20μM) for 48 hours. The expression of SIRTl was measured by the western-blot analysis using SIRTl specific antibody. (B) Cardiomyocytes were stimulated with PE (20μM) in presence or absence of resveratrol (50OnM) or sirtinol (30μM). Third day following PE stimulation cells were harvested and the incorporation of 3H-leucine into total cell protein was determined and normalized with DNA content of the cells. Values are mean ± SE of four separate experiments. (C) Cardiomyoytes plated in serum-free media were transduced with adenovirus vectors expressing either wild-type (wt) or mutated (mut) SIRTl . Twelve hours following viral infection, cells were stimulated with hypertrophy agonists PE (20μM), Ang (10OnM) or FBS (5%) for 72 hours. Afterwards cells were harvested and 3H-leucine incorporation into total cell protein was measured and expressed as per unit DNA content. Values are mean ± SE of three separate experiments. *P<0.01 compared to cells infected with the SIRTl mutant vector.

[00023] FIG. 2 demonstrates the effects of SIRTl activation on S6 protein synthesis. Cardiomyocytes transduced with SIRTl wild-type or mutant adenovirus vectors were stimulated with different hypertrophy agonists, as in FIG.1. After seventy two hours of stimulation, cells were harvested, the expression level of S6 protein (32Kda) was determined by western-blot analysis and quantified by measuring the relative intensity of bands using an image analyzer. For a loading control, the top portion of the gel was cut out before transfer to membrane and stained with comassie-blue dye (Com Blue). Values are presented as mean±SE of four independent experiments.

[00024] FIG. 3 shows the effect of SIRTl activation on the sarcomere organization.

(A) Cardiomyocytes grown on a cover-slip were infected with SIRTl adenovirus vectors followed by treatment with PE (20μM), Ang (10OnM) or FBS (5%). Seventy two hours following treatment with hypertrophy agonists, cells were stained for α-actinin using actinin-specific antibody, conjugated with FITC to reveal sarcomere structures. The agonist-mediated sarcomere organization was blocked by over-expression of the wild-type SIRTl. (B) For a reference control, the expression level of Flag-SIRT following viral infection of myocytes was also determined by staining cells with a FITC conjugated FLAG antibody. Position of nuclei was determined by DAPI staining. The photo shows abundant expression of SIRTl in the nucleus, however, a small portion of SIRTl was also found expressed in the cytoplasm. (C) The deacetylase activity of wild-type and mutant SIRTl was confirmed by western analysis using an acetylated H3 (histone) specific antibody. Pictures shown are representative of at least 20-30 slides stained with specific antibodies.

[00025] FIG. 4 demonstrates that SIRTl activation suppresses the agonist-induced

ANF expression: (A) Primary cultures of cardiomyocytes infected with SIRTl wild-type, SIRTl mutant or a mock (backbone) adenoviral vectors were treated with PE (20μM). After 48 hours of treatment cells were harvested and ANF mRNA and protein was measured by Northern and cytoblot analyses, respectively. Values presented are mean ±SE of 4-6 separate experiments. (B) Cardiomyocytes grown on a cover slip were infected with SIRTl wild-type or the mutant vector. Twelve hours following viral infection cells were treated with different hypertrophy agonists (PE 20μM, Ang 10OnM or FBS 5%), as shown above each panel. After an additional 48 hours of treatment ANF expression was detected by indirect imumnofluorescence using ANF-specific antibody. Hypertrophy agonist induced perinuclear expression of ANF was reduced by over-expression of wild-type SIRTl.

[00026] FIG. 5 presents evidence for the effect of SIRTl activation on the expression of MHC transcripts. (A&B) Cardiomyocytes infected with SIRTl wild type or mutated adenovirus vectors were stimulated with PE (20μM). Forty eight hours following PE treatment cells were harvested and RNA was analyzed by Northern-blot analysis using transcript specific probes. GAPDH transcript level was used as a reference control. Values in the bar diagram represents mean ±SE of 5-7 independent experiments. * P<0.05 significantly different from untreated controls. (C&D) RNA obtained from control (no hypertrophy) cadiomyocytes infected with adenovirus vectors for three days was analyzed by the Northern analysis. Band intensity of 28S and 18S ribosomal RNA was used as a loading control. Values are mean ±SE of three separate experiments. *p<0.05, significantly lower from cells infected with SIRTl -mutant vector. Note that SIRTl wild-type over-expression reduced the endogenous β-MHC mRNA levels. (E &F) siRNA- mediated silencing of SIRTl was carried out by transfecting cardiomyocytes with pSUPER RNAi SIRTl system. Cells transfected with scrambled oligos (control- siRNA) were used as a negative control. Four days following transfection with oligos, cells were harvested and RNA analyzed for the expression level of α and β- MHC mRNA transcripts. In the parallel experiments SIRTl protein levels were determined by western analysis. Tracings shown are representatives of three separate experiments. (G) Quantification of MHC levels following knocking down the SIRTl deacetylase by siRNA. Values are mean ± SE of three separate experiments. *P<0.01 significantly different from controls. Note: repression of endogenous α-MHC mRNA levels in cells where SIRTl levels were knocked down.

[00027] FIG. 6 demonstrates that resveratrol, an SIRTl activator suppresses MHC isoform shift in hypothyroid animals. (A) Adult male mice were fed with a PTU (6-poly-2-thiouracil) diet or PTU diet containing resveratrol (0.067%) for 8 weeks. At the end of experiment animals were sacrificed and myosin isoforms were separated by SDS-PAGE. A longer exposure of lane 3 is shown at the right side of the gel (blot 3'). (B) Quantitative representation of MHC isoform in different groups. Note that mice fed with PTU diet expressed 100% β-MHC (lane 2); however, animals fed with PTU plus resveratrol diet expressed considerably lower β-MHC levels (75%) and noticeable amount of α-MHC levels (-25%) (lane 3). (C) In the same hearts SIR2α/SIRTl levels were determined by the western-blot analysis. Blots shown are representative of five separate experiments.

[00028] FIG. 7 shows that transgenic mice over expressing SIRTl wild-type are protected from loss of α-MHC during hyopothyroid state. Transgenic mice overexpressing SIRTl in the heart were fed with PTU diet for 8 weeks. The non- transgenic animals (Non-Tg) were used as positive controls. At the end of the experiment, animals were sacrificed, hearts removed and MHC isoforms separated by SDS-PAGE analysis. Expression level of SIRTl in mice hearts was determined by the Western analysis (middle panel). Tracings shown are representative of 10 different animals analyzed in each group, with consistent results. Note, that α- MHC almost disappeared in PTU-treated non-transgenic animals, but it was significantly expressed in SIRTl -transgenic mice (SIRTl-Tg).

[00029] FIG. 8 shows that SIRTl activation blocks PE-mediated induction of fetal cardiac gene promoters. Cardiomyocytes were infected with viral vectors expressing wild-type or mutant SIRTl. The second day following viral transduction, cells were transfected with different promoter/luciferase reporter plasmids, as given above each bar diagram. Cells were either left untreated or stimulated with PE (20μM). Following 48 hours of PE treatment protein extract were prepared and assayed for luciferase activity. Values are normalized with the protein content in the assay reaction. PE treatment increased CARP, ANF as well as β-MHC promoter activity, but not the SV40 promoter. Over-expression of SIRTl prevented the PE mediated induction of fetal gene promoter activity. Values are mean ± SE of 4-7 different experiments.

[00030] FIG. 9 demonstrates that SIRTl over-expression protects myocytes from

PARP-I mediated cell-death. Cardiomyocytes were infected with SIRTl adenovirus vectors, 12 hours later they were either left untreated or treated with Ang (lμM). Following forty eight hours of treatment, cells were harvested and protein extract was analyzed for PARP activity either, (A) by western-blot analysis using anti-poly-ADP-ribose (PAR) antibody or (B) by using a commercially available PARP-assay kit. (C & D) Cardiomyocytes transduced with adenoviral vectors were treated with Ang (lOμM) or with a free-radical generating mixture of H₂O₂ and FeSO₄ (H₂O₂; O.lmM each). Forty eight hours after treatment cells were stained with Hoechst 33345 (blue) and propidium iodide (red) dyes. Reduced red color in SIRTl .wt infected cells reflects increased cell-survival. Quantification of cell-death was done FACS analysis. Values are mean ± SE of 7-10 independent experiments. * PO.01 significantly different from cells infected with SIRTl mutant vector. [00031] FIG. 10 demonstrates that anti-hypertrophy effect of SIRTl activation is accentuated by PARP-inhibition. (A) Cardiomyocytes were pretreated with a PARP-inhibitor, 3-aminobenzamide (3AB; 2 and 4mM) either alone or together with 50OnM of resveratrol (Res). Next morning cells were stimulated with PE (20μM) and 3H-leucine incorporation was determined 72 hours later. (B) In a parallel set of plates SIRTl levels were measured by the Western-blot analysis. (C) Quantitative representation of SIRTl levels in myocytes following Res and 3AB treatments. Bar represents mean ± SE values of three experiments. * P<0.01 significantly different from controls.

DETAILED DESCRIPTION

[00032] Activation of a sirtuin (SIRTl) resulted in reducing cardiac myocyte hypertrophy and favoring the α-myosin isoform expression during heart failure. SIRTl activation prevented the agonist-mediated enhanced protein synthesis, sarcomere organization, induction of fetal gene program as well as myocyte cell death. Moreover, SIRTl was indicated to have a role in the regulation of two cardiac MHC isoforms expression in the heart. These data for the first-time demonstrate that a genetic determinant of longevity, the SIRTl deacetylase, has a negative regulatory potential to control cardiac myocyte hypertrophy and up- regulate the α-MHC expression.

[00033] Histone deacetylases (HDACs) are divided into three main classes based on their structural similarities and their requirement of co-factor for the deacetylase activity. Members of class-I may have pro-hypertrophic activity, while class-II members are generally considered to be anti-hypertrophy. Several lines of evidence described herein using classic hallmarks of cardiac hypertrophy, have demonstrated that SIRTl activation is highly effective in blocking cardiomyocyte hypertrophy. Sirtuins, as the name stands, "silent information regulator", are generally considered to be repressors of gene transcription. The silencing function of sirtuins is mediated by different multiprotein complexes at different gene regulatory steps. Some of the gene -repression effects of SIRTs are mediated by deacetylation of histones, leading to chromatin condensation and repression of gene transcription. [00034] Phenylephrine-treatment (PE) of cardiomyocytes induces the activity of histone-acetylases, p300/CBP; and over activation of p300 produces cardiac hypertrophy. The amount of PE capable of inducing hypertrophy reduces the expression level of endogenous SIRTl, and the blockade of PE-mediated hypertrophy response could be seen only with the over-expression of wild-type SIRTl . The mutant SIRTl, lacking the deacetylase activity, was devoid of anti hypertrophy effects, indicating that the negative hypertrophy effects of SIRTl are indeed carried out by its ability to deacetylate the target proteins. In the protein synthesis experiments in control (un-stimulated) cells, SIRTl activation, either by treating cells with an activator (e.g., resveratrol) or by over expressing with the deacetylase, increased the total cell protein content. This effect was surprising, since SIRTl activation was capable of blocking the agonist mediated hypertrophy. To distinguish between these two contrasting results, the expression of other hypertrophy markers were analyzed, e.g. ANF and β-MHC levels in un-stimulated cells. However the expression levels of ANF and β-MHC were not elevated by SIRTl activation. Based on these results, in un-stimulated cells, mild increase of protein content by SIRTl activation does not reflect myocyte hypertrophy, since no other hypertrophy marker could be seen activated in these cells. In serum free cell-culture conditions (as utilized herein) myocytes are essentially in a stressed condition, and SIRTl activation by relieving this stress provides a positive support to cells of well being, which is imitated as mild increase of protein accumulation. Under microscope also these serum-starved cells expressing SIRTl appeared healthy and made better connection with each other, as opposed to control cells infected with the vector backbone.

[00035] Data obtained from promoter reporter gene assays indicated that SIRTl could also block hypertrophy and fetal-gene activation by interfering with some transcription factors that are responsive to PE and not to the basic transcription machinery, as constitutively active SV40- promoter was found without response. The most profound effect of SIRTl activation was seen on the ANF promoter. This effect was also confirmed by suppression of ANF secretion by SIRTl over- expression, consistent with a previous report where nicotinamide, an inhibitor of SIRTl, was shown to up-regulate the ANF transcript levels. In addition to histones, SIRTl has been shown to deacetylate many other transcription factors and modify their gene activation potential. NF -kB is also a target of SIRTl deacetylase. SIRTl deacetylates the Rel/p65 subunit of NF-kB and suppresses the Nf-kB dependent gene activation. Skeletal muscle-specific transcription factor, MyoD has been shown to be a target of SIRTl deacetylase, and deacetylation of MyoD suppresses its myogenic potential. Although, so far, no cardiac specific factor has been shown to be a target of SIRTl , acetylation of GAT A4 (a cardiac myogenic factor) by p300 is reported to induce fetal gene expression and cardiomyocyte hypertrophy. Some of the negative hypertrophy effects of SIRTl in cardiac myocytes may be attributed to the SIRTl -dependent deacetylation of signaling molecule(s) and/or cardiac-specific factor(s) that are yet to be identified.

[00036] SIRTl activation exerted an opposite effect on the expression of two cardiac MHC isoforms. SIRTl repressed the expression of β-MHC, but had a positive regulatory effect on the expression of cardiac α-MHC transcripts. It was surprising to see that the activation of a class III HDAC (SIRTl) stimulates the α- MHC expression. Previous studies carried out with a class-I & II HDAC inhibitor, TSA has documented that inhibition of HDAC activity up-regulates the expression of this transcript. Thus, different classes of HDACs have different effects on MHC isoform expression.

[00037] Ku70 is a SIRTl deacetylase target. We have recently found that Ku70 is also targeted by SIRT3 deacetylase. In failing hearts, Ku70 levels are increased by several fold, and that has been implicated as an underlying mechanism of α-MHC repression during heart failure. Ku70 is deacetylated by SIRTl as well as SIRT3, and this post-translational modification enhances the protein-binding ability of Ku70 to partner proteins, such as Bax, leading to retention of Bax into cytoplasm and thus inhibiting the pro-apoptotic activity of Bax. In a similar fashion, in cardiac myocytes, Ku70 upon deacetylation by SIRT1/SIRT3 may bind to other partner proteins and that in turn eliminates its negative regulatory effect on the α- MHC gene promoter.

[00038] SIRTs are considered a nuclear sensor of the metabolic and oxidative states of the cells. A change in cell NAD/NADH ratio exerts a major effect on the expression level of SIRTs. Previous studies carried out for the analysis of cardiac MHC expression have documented that a change in metabolic state of the animal, occurring with the high fructose diet and semi starvation, leads to a profound effect on MHC isoform expression. SIRTs may be the down-stream target conferring the metabolism-dependent regulation of the two cardiac MHC isoforms expression. [00039] Activation of PARP-I in failing hearts causes depletion of cellular NAD content and that subsequently leads to repression of SIRTl levels. SIRTl and PARP have many common features. Both use NAD as a substrate for their enzymatic reactions, and modify the activity of many of their common targets, including, P53, Ku70, P300, PCAF and NF-kb. While SIRTl mediated deacetylation in general suppresses the gene activation potential of these targets, poly (ADP)-ribosylation by PARPl augments their activity. Therefore, in order for SIRTl to effectively block the effect of these targets it may be necessary for the cell to control the activity of their activator, PARP-I. SIRTl activation was capable of blocking the enzymatic activity of PARP-I . PARP-mediated depletion of NAD levels could be prevented by over activation of SIRTl. Inhibition of PARPl by gene-dysfunction protects myocytes against hypertrophy response. SIRTl may also protect cells by upholding the activity of cell-survival factors such as FOXOl . A recent report indicates that SIRTl activation overrides the phosphorylation-dependent nuclear exclusion of FOXOl and promotes the nuclear retention of the factor, leading to enhanced transcription activity of FOXOl . SIRTl potentiates the FOXOl -mediated transcription of the antioxidant gene, MnSOD, and that, in part, may account for the protective effects of SIRTl against oxidative stress mediated cell-death. An anti-hypertrophy role of FOXO group of factors has also been demonstrated in primary cultures of cardiac myocytes. SIRTl activation protects myocytes by either or both mechanisms, by repressing the activity of pro-death pathways and/or by promoting the activity of survival signals. [00040] Activation of SIRTl and SIRT3 deacetylases is a novel strategy to prevent cardiac hypertrophy rather than the inhibition of class I and II HDACs by trichostatin A (TSA). TSA has been shown to have a very narrow widow of therapeutic index and some of the negative hypertrophy effects of TSA are still suspected to be compounded with its cell toxicity. In contrast, SIRTl is considered a cell survival factor and it has been shown to prevent degeneration of non- proliferating cells such neurons, and implicated for the treatment of Alzheimer disease. SIRTl and SIRT3 deacetylase as a therapeutic targets and have the therapeutic potential for the management of cardiac hypertrophy and heart failure. EXAMPLES

[00041] The following examples are for illustrative purposes only and are not intended to limit the scope of the disclosure.

[00042] Example 1: Inhibition of cardiomyocyte hypertrophy by SIRTl deacetylase activation. Decreased levels of SIRTl deacetylase were identified during heart failures. The role of SIRTl in cardiac hypertrophy and the effect of SIRTl activation on the rate of protein synthesis were examined. Enhanced protein synthesis resulting in increased total cellular protein is considered a hallmark of cardiomyocyte hypertrophy. To assay change in protein synthesis primary cultures of neonatal rat heart myocytes were treated with a hypertrophy agonist (PE) and incorporation of 3H-leucine into total cell protein was measured. The effect of SIRTl activation on protein synthesis was measured by treating cells with resveratrol and sirtinol, a most potent activator and inhibitor of SIRTl, respectively (FIG. IA). Treatment of cells with resveratrol (Res) slightly, but significantly, induced protein accumulation and made better connections among myocytes, which resulted in independently beating islands of cells in the culture plate; while the sirtinol treatment had the opposite effect. When myocytes were stimulated with the hypertrophy agonist, PE, a marked increase (2 fold) in protein accumulation was observed, compared to controls, which was associated with reduced levels of SIRTl (FIG. IA). This PE-mediated hypertrophy response of cardiomyocytes was completely blocked by pretreatment of cells with resveratrol, but not with sirtinol (FIG. IB). The efficacy of resveratrol and sirtinol to alter SIRTl levels was validated by western-blot analysis (FIG. IA). These results indicated that SIRTl activation inhibits agonist-mediated cardiac myocyte hypertrophy.

[00043] Example 2: Direct activation of SIRTl deacetylase by adenoviral vectors. Direct effects of SIRTl stimulation on hypertrophy were analyzed using adenoviral vectors. Adenovirus vectors were utilized expressing either wild-type SIRTl or mutant SIRTl, having the H355Y mutation. This mutation has been reported to destroy the deacetylase activity of the SIRTl. For these experiments cardiomyocytes were treated with a series of hypertrophy agonists (PE, Ang and FBS) and the effect of SIRTl activation was examined by infecting cells with wild-type and mutant vectors. As shown in FIG. 1C, stimulation of cells with hypertrophy agonists PE, Ang or FBS increased the total protein synthesis of myocytes by 2-3 fold compared to untreated controls. When cells were made to overexpress the wild-type SIRTl, this agonist- mediated increased protein synthesis was prevented, but, not when mutant SIRTl was overexpressed. Accumulation of a specific protein, ribosomal subunit 6 protein (S6P), was measured by quantitative western blot analysis. Accumulation of this particular protein is often used to determine cardiomyocyte hypertrophy. As shown in FIG. 2, stimulation of cells with PE increased the expression level of S6P by 4 fold, which was totally blocked by wild-type SIRTl over-expression, but not by the SIRTl mutant. Comparable results were obtained when cardiomyocytes were stimulated with two other hypertrophy agonists, Ang and FBS. To further substantiate this anti-hypertrophy potential of SIRTl, cardiomyocytes were stained for α-actinin, a molecule located at the Z-disc of the sarcomere and often utilized to examine sarcomere organization. The sarcomeres of the control cardiomyocytes were disorganized; however, stimulation of cells with hypertrophy agonists (PE, ANG and FBS) resulted into highly organized structures of sarcomeres with increased size of cardiomyocytes. When cells were overexpressed with SIRTl, this agonist mediated sarcomere organization was blocked and cells appeared smaller as compared to cells over expressing the mutant SIRTl (FIG. 3A). To ensure that these effects of SIRTl activation were carried out by its enzymatic activity, the expression pattern and the deacetylase activity of both the wild-type and mutant vectors was validated. As shown in FIG. 3B, cardiomyocytes infected with the wild-type vector robustly expressed SIRTl primarily in the cell nucleus, although a small fraction of deacetylase was also seen expressed in the cytoplasm. A similar expression pattern was seen by infecting cells with the mutant ad-vector. To evaluate the enzymatic activity of these vectors, the acetylation status of histone3 (H3), a SIRTl target was monitored by the western blot analysis. As shown in FIG. 3C, acetylation of H3 was markedly reduced by wild-type SIRTl over- expression, but not by its dominant negative mutant; thus, confirming the deacetylase activity of these vectors. These results indicated that the deacetylase activity of SIRTl is necessary for its negative hypertrophy effects. Example 3: Suppression of fetal gene program by SIRTl activation.

To further assess the ability of SIRTl activation to block cardiac hypertrophy the effect of deacetylase on the expression of ANF was examined, a secreted peptide from cardiac myocytes that is known as a most sensitive marker of hypertrophy. Cardiomyoctes were infected with SIRTl -wt and mutant vectors and then stimulated with PE. Forty-eight hours following treatment, cells were harvested and the ANF mRNA and protein expression was assayed by northern and cytoblot analyses, respectively. As shown in FIG. 4A, PE stimulation of cells increased the ANF expression (both mRNA and protein) by almost 4 fold. A similar increase of ANF expression was observed in PE-treated cells infected with a negative ad- vector (mock) or with the vector expressing SIRTl mutant. However, in cells where SIRTl -wt was overexpressed no significant induction of ANF was observed following PE stimulation, suggesting that SIRTl deacetylase has the potential to block the agonist-mediated up-regulation of ANF expression in cardiomyocytes (FIG. 4A). ANF expression can also be examined by immuno-staining of cardiac myocytes with ANF specific antibody. ANF expression was visualized by immuno-staining of cells. As shown in FIG. 4B, stimulation of cells with PE, Ang and FBS led to prominent expression of ANF, as seen by secreted peptides localized mostly in the perinuclear cytoplasm, consistent with the observed induction of ANF mRNA and protein by PE stimulation of cardiomyocytes (FIG. 4A). This agonist-induced ANF immuno-staining was markedly diminished when SIRTl. wt was overexpressed, although some residual ANF expression could still be seen. Example 4: Role of SIRTl activation in preserving the α-myosin heavy chain levels during heart failure. In addition to ANF induction a shift in myosin isoform expression from α-MHC to β-MHC is also considered a hallmark of cardiac hypertrophy. To further evaluate the ability of SIRTl to block hypertrophy, the role of this deacetylase on the expression levels of α/β-MHC mRNAs was examined. As shown in FIG. 5, SIRTl over-expression prevented the induction of β-MHC mRNA expression in response to PE, and instead it triggered the expression of α-MHC mRNA, which is normally down-regulated in hypertrophied myocytes (FIG. 5A & B). The effect of SIRTl activation was determined on the expression level of MHC isoforms in control (un-stimulated) myocytes. As shown in FIG. 5 C & D, SIRTl activation not only prevented the hypertrophy agonist mediated MHC isofrom shift from β- to α-MHC, but it significantly reduced the expression of β-MHC and slightly provoked the expression of α-MHC transcripts in un-stimulated control cells (FIG. 5D). Since the effect of SIRTl over-expression was only marginal on the α-MHC transcript, the role of endogenous SIRTl gene on these MHC isoforms was examined. For this purpose the SIRTl levels of cardiac myocytes were decreased by siRNA and then the expression levels of both MHC isoforms was determined. As shown in FIG. 5 E, F &G, cellular levels of α-MHC mRNA were significantly reduced by knocking down SIRTl levels by siRNA, whereas, no appreciable change was observed in the expression level of β-MHC transcripts, indicating that SIRTl may have a direct role in regulation of the two cardiac MHC isoforms. These effects of SIRTl are again mediated by its enzymatic activity, as a single point mutation that destroyed the catalytic activity of the deacetylase was found ineffective in changing the expression level of either MHC isoforms. Example 5: In vivo SIRTl activation up-regulates the α-myosin heavy chain levels in animal models. The observed effect of SIRTl on the expression levels of α-MHC mRNA was surprising and unexpected, as no other HDAC isoform has been shown, so far, to have a similar effect. To determine whether this effect of SIRTl can be recapitulated in in vivo, the effect of SIRTl induction was examined in hypothyroid mice. Animals were made hypothyroid by feeding them with a PTU-rich diet for 8 weeks. This treatment resulted in lack of circulating ligands and a general hypothyroid phenotype, which included shift in MHC isoforms expression from predominantly α- to near complete β-MHC isoform. These animals provided an in vivo model to test any detectable increase in α-MHC levels following SIRTl induction. Accordingly, hypothyroid mice were fed with a SIRT activator, resveratrol-rich diet for 8 weeks. Afterwards animals were sacrificed, the heart removed and MHC isoforms separated by SDS-PAGE. As shown in FIG. 6, control euthyroid mice mostly expressed α-MHC isoform, while β-MHC was less than 10%. The hypothyroid mice on PTU diet expressed predominantly β-MHC (100%), and α-MHC was reduced to almost non-detectable levels (FIG. 6, lane 2). However, when hypothyroid animals were fed with a resveratrol-rich diet β-MHC levels were significantly reduced and a considerable amount of α-MHC isoform (25%) could be detected (FIG. 6 A &B, lane 3). In these animals, the circulating T3 and T4 levels and cardiac SIRTl levels were measured. SIRTl levels were increased in animals fed with a resveratrol-rich diet (FIG. 6C); however, no change was observed in circulating T3 and T4 ligands. These results were also confirmed by using transgenic mice, over expressing SIRTl in the heart. When these mice were made hypothyroid by feeding them with PTU diet, a significant amount of α-MHC level was found expressed, which was undetectable in non-transgenic animals (FIG. 7). Together, these results strongly support that SIRTl (Sir2α) deacetylase plays a prominent role in the regulation of two cardiac MHC isoforms.

[00047] Example 6: The effect of SIRTl on the promoter activity of genes which are responsive to hypertrophy agonists. Induction of a fetal gene program during hypertrophy has been documented to be due to alterations in the promoter activity of the respective genes. To understand the underlying mechanism, whereby SIRTl -activation blocked the induction of fetal-gene program, the effect of SIRTl on the promoter activity of different genes which are known to be responsive to hypertrophy agonists was examined. The promoter activity of ANF, β-MHC as well as CARP (Cardiac ankyrin repeat protein), an early marker of cardiac myogenic differentiation, which is highly expressed during hypertrophy was analyzed. Cardiac myocytes were transfected with the promoter-luciferase reporter plasmids and the responsiveness of the promoter to PE was examined with or without over-expression of SIRTl vectors. As shown in FIG. 8, stimulation of cells with PE induced the promoter activity of ANF, β-MHC as well as CARP by 3-4 fold. Over-expression of SIRTl. wt completely blocked the PE-mediated activation of the promoter activity of these genes, but not by over expressing the dominant negative mutant. To test that this was not a general effect of SIRTl activation on the promoters, the effect of the deacetylase on a constitutively active SV40 promoter was analyzed. Results indicated that the suppression of the promoter activity by SIRTl was specific to hypertrophy agonist responsive promoters, and not to a constitutively active promoter. These results, thus, demonstrated that the SIRTl deacetylase prevents induction of fetal gene program by interfering with the agonist mediated activation of the fetal gene promoter activity.

[00048] Example 7: SIRTl activation protects cardiomyocytes by suppressing the enzymatic activity of PARP-I: The effect of SIRTl on the activity of poly (ADP) polymerase- 1, a chromatin bound enzyme activated by cell-oxidative stress was examined. Previous studies by the inventor have shown that Ang mediated cardiomyocyte hypertrophy and cell death is associated with marked induction of PARP-I. PARP-I has been implicated also in aortic banding mediated pressure- overload hypertrophy. To determine whether the protective effect of SIRT-I could be due to its ability to interfere with the enzymatic activity of PARP-I, cardiomyocytes were over-expressed with SIRTl and treated with Ang-II. Forty eight hours following treatment cells were harvested and PARP-I activity was monitored by measuring poly ADP-ribosylation of cellular proteins by western analysis and by utilizing a commercially available PARP-assay kit. As shown in FIG. 9 A & B, stimulation of cells with Ang-II increased the PARP activity by 4-5 fold as reflected by increased poly ADP-ribosylation of total cellular proteins. However, when cells were over-expressed with wild-type SIRTl this Ang-II induced PARP activity was markedly attenuated, suggesting that SIRTl interferes with the enzymatic activity of PARP-I. To further substantiate these results, the effect of SIRTl on Ang-II and H2O2 (known activator of PARPl) mediated cell- death was examined. Cardiomyocytes infected with SIRTl vectors were treated with Ang (lOμM) or a mixture OfH₂O₂ and FeSO₄ (0.1 mM each). On the third day following treatments cells were stained with Hoechst and propidium-iodide dyes and cell viability was determined by FACS analysis. As shown in FIG. 9 C & D, over-expression of wild-type SIRTl, but not the mutant, protected cardiomyocytes from Ang as well as from free-radical generating mixture OfH₂O₂ and FeSO₄. Similarly, SIRT3 over-expression also prevented cardiomyocyte cell death induced by oxidative stress. These results demonstrated that some of the protective effects of SIRTs may, in part, be mediated by its ability to antagonize the enzymatic activity of PARP 1. Example 8: PARP inhibition accentuates the anti-hypertrophy effects of SIRTl activation. Previous studies from the inventor's lab have shown that PARP inhibition activates endogenous SIRTl levels and protects cardiomyocytes from agonist-mediated hypertrophy. To demonstrate whether PARP-inhibition could modify the negative hypertrophy effects of SIRTl, the effect of a PARP inhibitor, 3-aminobenzamide (3AB) was examined either alone or together with the SIRTl activator, resveratrol on PE-mediated cardiac hypertrophy. Cardiomyocytes were pretreated overnight with two different concentrations of 3AB (2 and 4mM) and next morning cells were stimulated with PE. As shown in FIG. 1OA, the concentration of 3AB (4mM) that reduced the PARP activity by 50% also blocked the agonist-mediated accumulation of cellular proteins, thus implicating that inhibition of PARP prevented agonist mediated cardiomyocyte hypertrophy. When 3AB was combined with resveratol, a further reduction of PE- mediated hypertrophy was observed, and this was associated with induction of endogenous SIRTl levels. These results, together, provide strong evidence for a role of PARP-inhibition in the negative regulation of cardiac hypertrophy by SIRTl activation.

[00050] Example 9: Activation of the longevity factor, SIRTl deacetylase blocks the phenylephrine-induced cardiomyocyte hypertrophy. SIRTl is a member of class-Ill histone deacetylases. It has been shown to participate in a wide array of cellular functions including, gene silencing, cell-growth, apoptosis and aging. Histone acetylases (p300/CBP) have been shown to participate in the development of cardiomyocyte hypertrophy. SIRTl deacetylase may function as a negative regulator of cardiac hypertrophy.

[00051] Primary cultures of neonatal rat heart myocytes, maintained in serum-free media, were stimulated with phenylephrine (PE, 20μM) to induce hypertrophy. Seventy two hours following PE-stimulation, cells were harvested and hypertrophy was determined by monitoring protein-synthesis, sarcomere organization and induction of fetal genes (ANF, βMHC and sk-α-actin) expression. To examine the effect of SIRTl on myocyte growth, cells were infected with adenovirus vectors expressing either wild-type or mutant SIRTl, having the H355Y mutation that destroys the catalytic activity of the deacetylase. PE-stimulation enhanced the myocyte protein content by almost two fold and resulted into highly organized structures of sarcomeres with increased myocyte size. This PE-mediated hypertrophy response of myocytes was completely blocked by over-expression of the SIRTl as well as by SIRT3 deacetylase, but not the mutant vector. Similarly, SIRTl or SIRT3 activation prevented the PE-mediated induction of fetal gene transcripts of ANF, βMHC and sk-α-actin, thus, indicating a negative hypertrophy role of SIRTl and SIRT3 activation. To understand the mechanism behind this anti-hypertrophy effect of SIRTl, its effect on the ANF promoter/luciferase reporter gene activity was analyzed by the transient transfection analysis. PE- treatment induced the ANF promoter activity by almost 4 folds in control cells; however, in cells where SIRTl was over-expressed, no reporter gene induction could be observed. Also a shorter fragment of the ANF promoter that was insensitive to PE-treatment was unresponsive to SIRTl activation, suggesting that SIRTl interferes with the activity of a PE-responsive transcription factor that regulates the ANF promoter activity during hypertrophy. SIRTl deacetylase is a negative regulator of agonist-mediated cardiomyocyte hypertrophy.

[00052] Example 10: Activation of SIRT3 deacetylase blocks the agonist mediated cardiomyocyte hypertrophy and cell-death.

[00053] Background: SIRT3 is a member of class-Ill histone deacetylases. It has been shown to participate in wide array of cellular functions including, energy metabolism, cell-growth and apoptosis. SIRT3 is primarily located in the mitochondria of the cell and it has been implicated in calorie-restriction mediated prolonged cell-survival.

[00054] Methods and results: To determine the role of SIRT3 in hypertrophic growth of cardiomyocytes, primary cultures of neonatal rat cardiac myocytes, maintained in serum-free media were utilized. Cells were stimulated with phenylephrine (PE, 20μM) to induce hypertrophy or with H2O2 to induce cell- death. Expression of endogenous SIRT3 was examined by immunostaining of cells. In control (unstimulated cells) SIRT3 was expressed at very low level in the cell nucleus. Following stimulation with H2O2 or PE a robust expression of SIRT3 was observed in mitochondrial compartment of cardiomyoctes. SIRT3 levels were also measured in in vivo models of cardiac hypertrophy (pressure overload hypertrophy) and increased expression of this deacetylase was found in hypertrophied hearts. To examine the effect of SIRT3 on the myocyte hypertrophy, cells were infected with adenovirus vectors expressing wild-type SIRT3 or the mutant lacking deacetylase activity. Hypertrophy of cardiomycytes was determined by measuring 3H-leucine incorporation into total cell protein and by analyzing induction of fetal genes (ANF, sk-α-actin and MHC isoform shift from α-MHC to β-MHC). PE stimulation enhanced myocyte protein content by almost two fold. This agonist-mediated protein accumulation was completely blocked in cells over expressing SIRT3 deacetylase, but not the mutant vector. The sarcomeres of the control myocytes were disorganized; however, stimulation with PE resulted into highly organized structures of sarcomeres with increased myocyte size. When cells were over-expressed with SIRT3, the PE-induced sarcomere organization was prevented and cells appeared smaller and shrank, compared to cells over expressing the mutant vector. Analyses of fetal genes indicated that SIRT3 over-expression prevented MHC isoform shift and induction of ANF and skeletal α-actin transcripts. The effect of SIRT3 on an α-MHC gene promoter was analyzed and SIRT3 found to be capable of inducing the promoter activity of this gene, indicating that SIRT3 has direct effect on transcription regulation of αMHC gene expression. These results demonstrate that activation of SIRT3 provides new therapeutic opportunities to manage heart failure.

MATERIALS AND METHODS

[00055] Plasmids and Vectors: The promoter/luciferase reporter plasmids, containing -450bps of β-MHC and -600bps of CARP promoters were furnished by Dr. A. Stewart (Department of Biochemistry, University of Ottawa Heart Institute, Ottawa, Canada). The ANF/ luciferase reporter plasmid containing 638bps of ANF promoter was a gift from Dr E. Svensson (Department of Medicine, University of Chicago, Chicago, IL). Replication defective adenovirus vectors expressing flag- tagged wild-type SIRTl and H355A SIRTl mutant were constructed using the pAd-Easy system. For myocyte transduction, typically cultures were incubated with adenovirus at a multiplicity of infection of 20-50 for 12 hours. Under these conditions transduction efficiency was >90%. siRNA mediated silencing of Sir2 was carried out by transfection of pSUPER RNAi system (VEC-PBS-0003/0004) with incorporating 20 nucleotides of Sir2α (gaagttgacctcctcattgt).

[00056] Cell culture and transfection: Primary cultures of cardiac myocytes were prepared from 2 day old neonatal rat hearts as described previously. Myocytes were initially grown in Dulbecco's Modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum and 5 mg/ml of each penicillin and streptomycin (Invitogen) for 48 hours, afterwards cells were maintained in serum-free medium. Enrichment of cardiac myocytes in cultures was examined by immunostaining for sarcomeric α-actinin. For induction of hypertrophy, myocytes were treated with phenylephrine (PE, 20μM), Angiotensin- II (Ang, 10OnM) or fetal bovine serum (FBS, 5%) for 48 hours, unless indicated otherwise. For promoter/reporter gene analysis, typically IxIO⁵ myocytes/well were transfected 48 h after plating with plasmids using lipofectamine reagent (Invitrogen). Transfected cells were incubated in serum-free DMEM for 18 hrs followed by transduction with SIRTl wt or SIRTl -mutant (H355A) adenoviruses vectors for an additional 12 hrs and then cells were stimulated with PE. After 48 hours of stimulation with PE cells were harvested, cell lysate prepared and assayed for luciferase activity using the luciferase assay system (Promega), β-galactosidase and protein content.

[00057] [³HlLeucine Incorporation: Cardiomyocytes were transduced with adenovirus vectors or treated with resveratrol (50OnM) or sirtinol (30μM) overnight prior to stimulation with hypertrophy agonists. Immediately following treatment with agonists, cells were incubated with [³H]leucine (1.0 mCi/ml, 167 Ci/mmol Sp. Activity, (Amersham Biosciences) in leucine-free DMEM medium (Invitrogen) for 72hrs. To precipitate proteins, cells were washed with PBS and then incubated in 10% trichloroacetic acid. The resultant pellets were solublized in NaOH (0.25 N), lysates were diluted with one-sixth volume of scintillation fluid and counted in a scintillation counter. Values were normalized with DNA content, which was measured by using Quant-iT picogreen dsDNA assay kit (Invitrogen).

[00058] Immunostaining of cells: Cardiomyocytes were plated on laminin-coated glass cover slipsin 12-well dishes (1x10⁵ cells/well). Cells were infected with Ad- SIRTl wt or mutant adenovirus followed by treatment with hypertrophy agonists as indicated. Forty eight hours after treatment with agonists, cells were fixed with formaldehyde (3.7%) in PBS, permeabilized (0.1%, Triton X-100), and blocked with 10% Goat serum diluted in PBS and then incubated with primary antibodies for sarcomeric α-actinin (mouse monoclonal, 1 :200 dilution, Sigma, catalog no.A- 7811) or atrial natriuretic factor (ANF) (rabbit polyclonal, 1 :100 dilution, Peninsula Laboratories, catalog no.T-4014). Cover slips were washed five times in PBS and incubated with fluorescein-conjugated secondary antibodies (1 :200 dilution, Sigma catalog no.F-2012). Finally cover slips were washed three times with PBS, one time with water, and mounted on glass slides using Vectashield mounting medium (Vector Laboratories). Cells were visualized with a fluorescence microscope and images were captured using a Zeiss Axioplan PXL cooled charged coupled device camera (Roper, Tucson, AZ).

[00059] PARP assay: PARP activity was measured using Universal colorimetric

PARP-assay kit of Trevigen, Inc, (4672-096K), according to manufacturer's protocol. Briefly, the PARP enzyme activity of cell-lysate was estimated based on the incorporation of biotinylated poly ADP-ribose units onto histone proteins coated in a 96-well plate, provided with the kit. The values were calculated from a standard curve generated by using known amounts of PARP enzyme and normalized to the protein content. [00060] Western and Cytoblot analyses: After the period of treatments indicated, cells were washed twice with PBS, cell lysate was prepared in RIPA buffer (Ix PBS, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, lOmg/ml phenymethylsulfonyl fluride, 10OmM sodium orthovanadate, and protease inhibitors). Protein concentration was measured using a BioRad protein assay reagent. Typically, lOOμg of protein sample (unless otherwise specified) was heated at 65⁰C for 5 minutes and resolved by 8% SDS-PAGE. Western blot analysis was performed according to the standard procedures. The primary antibodies used for the analysis were as follows: Mouse polyclonal antibody ribosomal S6 protein antibody from Cell Signaling (catalog no. 2317), mouse monoclonal poly ADP-ribose (PAR) antibody from Alexis Biochemicals (ALX- 804-220), rabbit anti-Sir2α antibody from Upstate (07-131), rabbit polyclonal anti- α-tubulin and goat anti GAPDH antibody from Santa Cruz (sc20357). Primary antibodies were typically used at 1:1000 dilution and secondary (HRP-conjugated) anti-rabbit or anti-mouse antibodies at 1 :3000 dilution were used. For cytoblot assay, after indicated period of transduction and treatment, cardiomyocytes were washed twice with PBS, fixed with formaldehyde (4%) in PBS (30 min), permeabilized with Triton-X (0.1%) in PBS (lOmin) and blocked with BSA (1%) in PBS (2 h). Cells were incubated with PBS containing BSA (1%) and normal goat serum (1%) and primary antibody against ANF (rabbit polyclonal, 1 :100 dilution, Peninsula Laboratories, catalog no.T-4014) for 1 h. Cells were washed twice with PBS containing BSA (1%) and incubated with HRP-conjugated anti- rabbit secondary antibody (1 :1000 dilution). Cells were washed twice with PBS containing BSA (1%) and one time with PBS alone. Luminol was added to cells and luminescence was detected using a UniRead 800 Universal Microplate Reader.

[00061] Measurement of myocyte cell death: Myocytes were transduced with adenovirus vectors for overnight followed by treatment with Ang (lOμM) or with a free-radical generating mixture of H2O2 and FeSO4 (0.ImM). Forty eight hours following treatment cells were washed three times with PBS and then stained with two DNA binding dyes, Hoechst 33342 and propidium iodide, according to manufacturer's protocol (Molecular probes). Hoechst readily penetrates cell- membrane and stains nuclei of all (both live and dead) cells. Propidium iodide which does not penetrate cell-membrane stains nuclei of dead cells only. To determine the extent of cell-death in both adherent and detached populations of cells, fluorescence activated cell sorting (FACS) analysis was performed. For this analysis, cells were trypsinized, and the enzyme was inactivated by addition of an equal volume of fetal bovine serum. Cells were collected by centrifugation, and a single cell suspension of myocytes was prepared in PBS and stained with both Hoechst 33345 and propidium iodide for 5 min. Cells that detached spontaneously were also collected and included in the analysis. Cells were washed twice, resuspended in PBS, and analyzed by FACS analysis. Cell debris was gated out using forward scatter versus side scatter and propidium iodide versus Hoechst33345 fluorescence was analyzed by gating propidium iodide negative and propidium iodide positive cells.

[00062] Myosin isoform separation; Adult male mice weighing 30-40 gm were fed ad-libitum with PTU (6-poly-2-thiouracil) rich (0.15%) diet or PTU diet containing reseveratrol (0.067%) for 8 weeks. Transgenic mice over expressing SIRTl in the heart were also made hypothyroid by feeding them with PTU-rich diet. Age and weight matched adult male mice were used as euthyroid controls. At the end of experiment animals were anesthetized with a bolus injection (IM) of pentobarbital sodium and the heart rapidly excised and processed for MHC isoform separation. Myosin heavy chain isoforms were separated by gel electrophoresis essentially as described previously. Gels were scanned and quantitated with UVP GDS 8000 Bioimaging system. All animal protocols were approved by the University of Chicago Animal Care and Use Committee.

[00063] RNA analysis; Total RNA was extracted from control and treated cardiomyocytes with Trizol reagent (Invitrogen) according to the method provided by the manufacturer. Northern blot analysis was performed with ANF and GAPDH cDNA probes and synthetic oligonucleotide probes complementary to the unique 3'-untranslated sequences of the rat α- and β-MHC mRNA. Sequences of the single stranded oligonucteotide probes are rat α-MHC: 5' GTG GGA TAG CAA CAG CGA GGC 3' and rat β-MHC: 5' GGT CTC AGG GCT TCA CAG GC 3'.

[00064] Scanning Densitometry and Statistical analysis; Autoradiograms were scanned using Scion Image for Windows analysis software, based on NIH image for Macintosh by Wayne Rasband (National Institute of Health, Bethesda, MD). Signal intensity was adjusted for background density of the blot. Student paired t- test was utilized to determine statistical significance between two groups. [00065] Gene Therapy Using Viral Vectors: Specific viral vectors for use in gene transfer systems as described herein are now well established and include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, and herpes simplex viral vectors. Additionally, viral vectors may be administered in combination with transient immunosuppressive or immunomodulatory therapies. [00066] In other embodiments, viral serotypes, e.g., the general adenovirus types 2 and

5 (Ad2 and Ad5, respectively) may be administered, possibly on an alternating dosage schedule where multiple treatments will be administered. Specific dosage regimens may be administered: over the course of several days, when an immune response against the viral vector is anticipated, or both. In non-limiting examples of specific embodiments, Ad5-based viral vectors may be used on day 1, Ad2 -based viral vectors may be used on day 2, or vice versa.

[00067] Administration of the viral particles comprising viral vectors described herein can be via any of the accepted modes of administration for such viral particles well known by a person of ordinary skill in the art. For example, the viral particles may be administered by systemic or local administration, including oral, nasal, parenteral, transdermal, topical, intraocular, intrabronchial, intraperitoneal, intravenous, subcutaneous, and intramuscular administration, or by direct injection into cells, tissues, organs, or tumors. The adenoviral particles/vectors may be formulated in any art-accepted formulation well known to a person of ordinary skill in the art. [00068] Administration of Chemical Formulations (Including Agents to Activate

SIRTs): Administration of the compositions disclosed herein may be via any route known to be effective by the physician of ordinary skill. Peripheral, parenteral administrations are suitable. Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe. Peripheral parenteral routes include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration. Intravenous, intramuscular, and subcutaneous routes of administration of the compositions disclosed herein are suitable. For parenteral administration, the peptides disclosed herein can be combined with phosphate buffered saline (PBS) or any suitable pyrogen-free pharmaceutical grade buffer that meets FDA standard for human subject administration. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, 20^th Edition, A. R. Gennaro (Williams and Wilkins, Baltimore, MD, 2000) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Solutions or suspensions of the compositions described herein can also include a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; chelating agents, such as EDTA; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. A parenteral preparation of the compositions can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic, in accordance with standard practice in the field. The compositions disclosed herein can be stored as a lyophilized sterile powder in vials containing for reconstitution and the unreconstituted product may be stored at -20⁰C.

Claims

CLAIMS:

1. A pharmaceutical composition used to reduce cardiomyocyte hypertrophy, the composition comprising an agent that activates a class III histone deacetylase.

2. The composition of claim 1, wherein the class III histone deacetylase is sirtuin (SIRT).

3. The composition of claim 1, wherein the agent that activates SIRT is a small molecule.

4. The composition of claim 1, wherein the agent that activates SIRT is produced by direct gene expression.

5. The composition of claim 1, wherein the agent that activates SIRT is promoter upregulation.

6. Use of sirtuin 1 (SIRTl) and/or sirtuin 3 (SIRT3) histone deacetylase to up- regulate the α-MHC isoform and reduce the levels of β-MHC isoform during heart failure, to prevent MHC isoform shift during cardiac failure.

7. Use of sirtuin 1 (SIRTl) and/or sirtuin 3 (SIRT3) histone deacetylase to reduce fetal-gene activation during cardiac hypertrophy.

8. The use of claim 7, wherein the fetal-gene activation comprises a gene selected from the group consisting of ANF/BNP, β-MHC and CARP.

9. Use of the sirtuins 1 (SIRTl) and/or sirtuin 3 (SIRT3) histone decatylase, to protect cardiomyocytes from oxidative-stress mediated cell-death that leads to dilation of the ventricle and ultimately results into heart failure.

10. Use of SIRTl and SIRT3 to protect cells against hypertrophy agonist -mediated cardiomyocyte cell death.

11. The use of claim 10 where the agonist is selected from the group consisting of Ang, PE, catecholamines and mechanical-stretch.

12. Use of SIRTl and/or SIRT3, either alone or in combination with a cardioprotective agent selected from the group consisting of beta-blockers, angiotensin converting enzyme inhibitors, PARP-inhibtors, anti-oxidents, and class I & II HDACs modulator to reduce cardiac hypertrophy or collagen deposition (fibrosis), fetal gene activation, myocyte cell-death and ventricular dilation and preventing or reversing cardiac remodeling during haemodynamic overloads that results in heart failure.

13. The uses of claims 6-12 wherein SIRTl and SIRT3 are activated or overexpressed.

14. A composition to identify an agent for activating SIRTl and/or SIRT3 during heart failure, the method comprising:

(a) providing a candidate agent to cardiomyocyte cell cultures that have been induced for cardiac hypertrophy;

(b) monitoring the activation of SIRTl and SIRT3 by Western-blot analysis and by measuring acetylation status of SIRTl and SIRT3 target proteins; and

(c) identifying the candidate agent as the agent for activating SIRTl and SIRT3 based on the reduction of protein accumulation and suppression of cardiac hypertrophy phenotype.