Activation of Autophagic Flux Blunts Cardiac Ischemia/Reperfusion Injury.

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Pharmacological priming of adipose-derived stem cells promotes myocardial repair.

Adipose-derived stem cells (ADSCs) have myocardial regeneration potential, and transplantation of these cells following myocardial infarction (MI) in animal models leads to modest improvements in cardiac function. We hypothesized that pharmacological priming of pre-transplanted ADSCs would further improve left ventricular functional recovery after MI. We previously identified a compound from a family of 3,5-disubstituted isoxazoles, ISX1, capable of activating an Nkx2-5-driven promoter construct. Here, using ADSCs, we found that ISX1 (20 mM, 4 days) triggered a robust, dose-dependent, fourfold increase in Nkx2-5 expression, an early marker of cardiac myocyte differentiation and increased ADSC viability in vitro. Co-culturing neonatal cardiomyocytes with ISX1-treated ADSCs increased early and late cardiac gene expression. Whereas ISX1 promoted ADSC differentiation toward a cardiogenic lineage, it did not elicit their complete differentiation or their differentiation into mature adipocytes, osteoblasts, or chondrocytes, suggesting that re-programming is cardiomyocyte specific. Cardiac transplantation of ADSCs improved left ventricular functional recovery following MI, a response which was significantly augmented by transplantation of ISX1- pretreated cells. Moreover, ISX1-treated and transplanted ADSCs engrafted and were detectable in the myocardium 3 weeks following MI, albeit at relatively small numbers. ISX1 treatment increased histone acetyltransferase (HAT) activity in ADSCs, which was associated with histone 3 and histone 4 acetylation. Finally, hearts transplanted with ISX1-treated ADSCs manifested significant increases in neovascularization, which may account for the improved cardiac function. These findings suggest that a strategy of drug-facilitated initiation of myocyte differentiation enhances exogenously transplanted ADSC persistence in vivo, and consequent tissue neovascularization, to improve cardiac function.

Dimethyl α-ketoglutarate inhibits maladaptive autophagy in pressure overload-induced cardiomyopathy.

It has been a longstanding problem to identify specific and efficient pharmacological modulators of autophagy. Recently, we found that depletion of acetyl-coenzyme A (AcCoA) induced autophagic flux, while manipulations designed to increase cytosolic AcCoA efficiently inhibited autophagy. Thus, the cell permeant ester dimethyl α-ketoglutarate (DMKG) increased the cytosolic concentration of α-ketoglutarate, which was converted into AcCoA through a pathway relying on either of the 2 isocitrate dehydrogenase isoforms (IDH1 or IDH2), as well as on ACLY (ATP citrate lyase). DMKG inhibited autophagy in an IDH1-, IDH2- and ACLY-dependent fashion in vitro, in cultured human cells. Moreover, DMKG efficiently prevented autophagy induced by starvation in vivo, in mice. Autophagy plays a maladaptive role in the dilated cardiomyopathy induced by pressure overload, meaning that genetic inhibition of autophagy by heterozygous knockout of Becn1 suppresses the pathological remodeling of heart muscle responding to hemodynamic stress. Repeated administration of DMKG prevents autophagy in heart muscle responding to thoracic aortic constriction (TAC) and simultaneously abolishes all pathological and functional correlates of dilated cardiomyopathy: hypertrophy of cardiomyocytes, fibrosis, dilation of the left ventricle, and reduced contractile performance. These findings indicate that DMKG may be used for therapeutic autophagy inhibition.

Regulation of autophagy by cytosolic acetyl-coenzyme A.

Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy.

Histone deacetylase inhibition blunts ischemia/reperfusion injury by inducing cardiomyocyte autophagy.

BACKGROUND: Reperfusion accounts for a substantial fraction of the myocardial injury occurring with ischemic heart disease. Yet, no standard therapies are available targeting reperfusion injury. Here, we tested the hypothesis that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor approved for cancer treatment by the US Food and Drug Administration, will blunt reperfusion injury. METHODS AND RESULTS: Twenty-one rabbits were randomly assigned to 3 groups: (1) vehicle control, (2) SAHA pretreatment (1 day before and at surgery), and (3) SAHA treatment at the time of reperfusion only. Each arm was subjected to ischemia/reperfusion surgery (30 minutes coronary ligation, 24 hours reperfusion). In addition, cultured neonatal and adult rat ventricular cardiomyocytes were subjected to simulated ischemia/reperfusion to probe mechanism. SAHA reduced infarct size and partially rescued systolic function when administered either before surgery (pretreatment) or solely at the time of reperfusion. SAHA plasma concentrations were similar to those achieved in patients with cancer. In the infarct border zone, SAHA increased autophagic flux, assayed in both rabbit myocardium and in mice harboring an RFP-GFP-LC3 transgene. In cultured myocytes subjected to simulated ischemia/reperfusion, SAHA pretreatment reduced cell death by 40%. This reduction in cell death correlated with increased autophagic activity in SAHA-treated cells. RNAi-mediated knockdown of ATG7 and ATG5, essential autophagy proteins, abolished SAHA's cardioprotective effects. CONCLUSIONS: The US Food and Drug Administration-approved anticancer histone deacetylase inhibitor, SAHA, reduces myocardial infarct size in a large animal model, even when delivered in the clinically relevant context of reperfusion. The cardioprotective effects of SAHA during ischemia/reperfusion occur, at least in part, through the induction of autophagic flux.

C/EBP transcription factors mediate epicardial activation during heart development and injury.

The epicardium encapsulates the heart and functions as a source of multipotent progenitor cells and paracrine factors essential for cardiac development and repair. Injury of the adult heart results in reactivation of a developmental gene program in the epicardium, but the transcriptional basis of epicardial gene expression has not been delineated. We established a mouse embryonic heart organ culture and gene expression system that facilitated the identification of epicardial enhancers activated during heart development and injury. Epicardial activation of these enhancers depends on a combinatorial transcriptional code centered on CCAAT/enhancer binding protein (C/EBP) transcription factors. Disruption of C/EBP signaling in the adult epicardium reduced injury-induced neutrophil infiltration and improved cardiac function. These findings reveal a transcriptional basis for epicardial activation and heart injury, providing a platform for enhancing cardiac regeneration.

Histone deacetylase (HDAC) inhibitors attenuate cardiac hypertrophy by suppressing autophagy.

Histone deacetylases (HDACs) regulate cardiac plasticity; however, their molecular targets are unknown. As autophagy contributes to pathological cardiac remodeling, we hypothesized that HDAC inhibitors target autophagy. The prototypical HDAC inhibitor (HDACi), trichostatin A (TSA), attenuated both load- and agonist-induced hypertrophic growth and abolished the associated activation of autophagy. Phenylephrine (PE)-triggered hypertrophy and autophagy in cultured cardiomyocytes were each blocked by a panel of structurally distinct HDAC inhibitors. RNAi-mediated knockdown of either Atg5 or Beclin 1, two essential autophagy effectors, was similarly capable of suppressing ligand-induced autophagy and myocyte growth. RNAi experiments uncovered the class I isoforms HDAC1 and HDAC2 as required for the autophagic response. To test the functional requirement of autophagic activation, we studied mice that overexpress Beclin 1 in cardiomyocytes. In these animals with a fourfold amplified autophagic response to TAC, TSA abolished TAC-induced increases in autophagy and blunted load-induced hypertrophy. Finally, we subjected animals with preexisting hypertrophy to HDACi, finding that ventricular mass reverted to near-normal levels and ventricular function normalized completely. Together, these data implicate autophagy as an obligatory element in pathological cardiac remodeling and point to HDAC1/2 as required effectors. Also, these data reveal autophagy as a previously unknown target of HDAC inhibitor therapy.

Cardiac autophagy is a maladaptive response to hemodynamic stress.

Cardiac hypertrophy is a major predictor of heart failure and a prevalent disorder with high mortality. Little is known, however, regarding mechanisms governing the transition from stable cardiac hypertrophy to decompensated heart failure. Here, we tested the role of autophagy, a conserved pathway mediating bulk degradation of long-lived proteins and cellular organelles that can lead to cell death. To quantify autophagic activity, we engineered a line of "autophagy reporter" mice and confirmed that cardiomyocyte autophagy can be induced by short-term nutrient deprivation in vivo. Pressure overload induced by aortic banding induced heart failure and greatly increased cardiac autophagy. Load-induced autophagic activity peaked at 48 hours and remained significantly elevated for at least 3 weeks. In addition, autophagic activity was not spatially homogeneous but rather was seen at particularly high levels in basal septum. Heterozygous disruption of the gene coding for Beclin 1, a protein required for early autophagosome formation, decreased cardiomyocyte autophagy and diminished pathological remodeling induced by severe pressure stress. Conversely, Beclin 1 overexpression heightened autophagic activity and accentuated pathological remodeling. Taken together, these findings implicate autophagy in the pathogenesis of load-induced heart failure and suggest it may be a target for novel therapeutic intervention.

Suppression of class I and II histone deacetylases blunts pressure-overload cardiac hypertrophy.

BACKGROUND: Recent work has demonstrated the importance of chromatin remodeling, especially histone acetylation, in the control of gene expression in the heart. In cell culture models of cardiac hypertrophy, pharmacological suppression of histone deacetylases (HDACs) can either blunt or amplify cell growth. Thus, HDAC inhibitors hold promise as potential therapeutic agents in hypertrophic heart disease. METHODS AND RESULTS: In the present investigation, we studied 2 broad-spectrum HDAC inhibitors in a physiologically relevant banding model of hypertrophy, observing dose-responsive suppression of ventricular growth that was well tolerated in terms of both clinical outcome and cardiac performance measures. In both short-term (3-week) and long-term (9-week) trials, cardiomyocyte growth was blocked by HDAC inhibition, with no evidence of cell death or apoptosis. Fibrotic change was diminished in hearts treated with HDAC inhibitors, and collagen synthesis in isolated cardiac fibroblasts was blocked. Preservation of systolic function in the setting of blunted hypertrophic growth was documented by echocardiography and by invasive pressure measurements. The hypertrophy-associated switch of adult and fetal isoforms of myosin heavy chain expression was attenuated, which likely contributed to the observed preservation of systolic function in HDAC inhibitor-treated hearts. CONCLUSIONS: Together, these data suggest that HDAC inhibition is a viable therapeutic strategy that holds promise in the treatment of load-induced heart disease.