trichostatin-a and Myocardial-Infarction

Histone deacetylases (HDACs) play a critical role in the regulation of gene transcription, cardiac development, and diseases. The aim of this study was to investigate whether the inhibition of HDACs improves cardiac remodeling and its underlying mechanisms in a mouse myocardial infarction (MI) model.. The HDAC inhibitor trichostatin A (TSA, 0.1 mg/kg/day) was administered via daily intraperitoneal injections for 8 consecutive weeks after MI in C57/BL mice. Echocardiography and tissue histopathology were used to assess cardiac function. Cultured neonatal rat cardiac fibroblasts (NRCFs) were subjected to simulated hypoxia in vitro. Autophagic flux was measured using the tandem fluorescent mCherry-GFP-LC3 assay. Western blot was used to detect autophagic biomarkers.. After 8 weeks, the inhibition of HDACs in vivo resulted in improved cardiac remodeling and hence better ventricular function. MI was associated with increased LC3-II expression and the accumulation of autophagy adaptor protein p62, indicating impaired autophagic flux, which was reversed by TSA treatment. Cultured NRCFs exhibited increased cell death after simulated hypoxia in vitro. Increased cell death was associated with markedly increased numbers of autophagosomes but not autolysosomes, as assessed by punctate dual fluorescent mCherry-green fluorescent protein tandem-tagged light chain-3 expression, indicating that hypoxia resulted in impaired autophagic flux. Importantly, TSA treatment reversed hypoxia-induced impaired autophagic flux and led to a 40% decrease in cell death. This was accompanied by improved mitochondrial membrane potential. The beneficial effects of TSA therapy were abolished by RNAi intervention targeting LAMP2; likewise, in vivo delivery of chloroquine abolished the TSA-mediated cardioprotective effects.. Our results provide evidence that the HDAC inhibitor TSA prevents cardiac remodeling after MI and is dependent on restoring autophagosome processing of cardiac fibroblasts.

Topics: Animals; Apoptosis; Autophagosomes; Autophagy; Cell Hypoxia; Fibroblasts; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Lysosomal-Associated Membrane Protein 2; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Myocardial Infarction; Myocytes, Cardiac; Rats; RNA Interference; RNA, Small Interfering; Ventricular Remodeling

Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression through remodeling of chromatin structures. However, the molecular mechanisms involved in this event remain unknown. In this study, we sought to examine whether HDAC inhibition-mediated protective effects involved HDAC4 sumoylation, degradation, and the proteasome pathway. Isolated neonatal mouse ventricular myocytes (NMVM) and H9c2 cardiomyoblasts were subjected to 48 h of hypoxia (H) (1% O2 ) and 2 h of reoxygenation (R). Treatment of cardiomyocytes with trichostatin A (TSA) attenuated H/R-elicited injury, as indicated by a reduction of lactate dehydrogenase (LDH) leakage, an increase in cell viability, and decrease in apoptotic positive cardiomyocytes. MG132, a potent proteasome pathway inhibitor, abrogated TSA-induced protective effects, which was associated with the accumulation of ubiquitinated HDAC4. NMVM transduced with adenoviral HDAC4 led to an exaggeration of H/R-induced injury. TSA treatment resulted in a decrease in HDAC4 in cardiomyocytes infected with adenoviral HDAC4, and HDAC4-induced injury was attenuated by TSA. HDAC inhibition resulted in a significant reduction in reactive oxygen species (ROS) in cardiomyoblasts exposed to H/R, which was attenuated by blockade of the proteasome pathway. Cardiomyoblasts carrying wild type and sumoylation mutation (K559R) were established to examine effects of HDAC4 sumoylation and ubiquitination on H/R injury. Disruption of HDAC4 sumoylation brought about HDAC4 accumulation and impairment of HDAC4 ubiquitination in association with enhanced susceptibility of cardiomyoblasts to H/R. Taken together, these results demonstrated that HDAC inhibition stimulates proteasome dependent degradation of HDAC4, which is associated with HDAC4 sumoylation to induce these protective effects.

Topics: Animals; Animals, Newborn; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Hypoxia; Mice; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Proteolysis; Reactive Oxygen Species

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.. 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.. 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.

Topics: Animals; Animals, Genetically Modified; Apoptosis; Autophagy; Cells, Cultured; Disease Models, Animal; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rabbits; Rats; Rats, Sprague-Dawley; Vorinostat

We and others have demonstrated that HDAC inhibition protects the heart against myocardial injury. It is known that Akt-1 and MAP kinase play an essential role in modulation of myocardial protection and cardiac preconditioning. Our recent observations have shown that Akt-1 was activated in post-myocardial infarction following HDAC inhibition. However, it remains unknown whether MKK3 and Akt-1 are involved in HDAC inhibition-induced myocardial protection in acute myocardial ischemia and reperfusion injury. We sought to investigate whether the genetic disruption of Akt-1 and MKK3 eliminate cardioprotection elicited by HDAC inhibition and whether Akt-1 is associated with MKK3 to ultimately achieve protective effects. Adult wild type and MKK3⁻/⁻, Akt-1⁻/⁻ mice received intraperitoneal injections of trichostatin A (0.1 mg/kg), a potent inhibitor of HDACs. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after twenty four hours to elicit pharmacologic preconditioning. Left ventricular function was measured, and infarct size was determined. Acetylation and phosphorylation of MKK3 were detected and disruption of Akt-1 abolished both acetylation and phosphorylation of MKK3. HDAC inhibition produces an improvement in left ventricular functional recovery, but these effects were abrogated by disruption of either Akt-1 or MKK3. Disruption of Akt-1 or MKK3 abolished the effects of HDAC inhibition-induced reduction of infarct size. Trichostatin A treatment resulted in an increase in MKK3 phosphorylation or acetylation in myocardium. Taken together, these results indicate that stimulation of the MKK3 and Akt-1 pathway is a novel approach to HDAC inhibition -induced cardioprotection.

Topics: Acetylation; Animals; Blotting, Western; Fluorescent Antibody Technique; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Immunoenzyme Techniques; Immunoprecipitation; Male; MAP Kinase Kinase 3; Mice; Mice, Knockout; Myocardial Infarction; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Ventricular Function, Left

We have previously shown that the inhibition of histone deacetylases (HDACs) protects the heart against acute myocardial ischemia and reperfusion injury. We also demonstrated that HDAC inhibition stimulates myogenesis and angiogenesis in a cultured embryonic stem cell model. We investigate whether in vivo inhibition of HDAC preserves cardiac performance and prevents cardiac remodeling in mouse myocardial infarction (MI) through the stimulation of endogenous regeneration. MI was created by ligation of the left descending artery. Animals were divided into three groups: 1) sham group, animals that underwent thoracotomy without MI; 2) MI, animals that underwent MI; and 3) MI + trichostatin A (TSA), MI animals that received a daily intraperitoneal injection of TSA. In addition, infarcted mice received a daily intraperitoneal injection of TSA (0.1 mg/kg), a selective HDAC inhibitor. 5-Bromo-2-deoxyuridine (50 mg/kg) was delivered every other day to pulse-chase label in vivo endogenous cardiac replication. Eight weeks later, the MI hearts showed a reduction in ventricular contractility. HDAC inhibition increased the improvement of myocardial functional recovery after MI, which was associated with the prevention of myocardial remodeling and reduction of myocardial and serum tumor necrosis factor α. HDAC inhibition enhanced the formation of new myocytes and microvessels, which was consistent with the robust increase in proliferation and cytokinesis in the MI hearts. An increase in angiogenic response was demonstrated in MI hearts receiving TSA treatment. It is noteworthy that TSA treatment significantly inhibited HDAC activity and increased phosphorylation of Akt-1, but decreased active caspase 3. Taken together, our results indicate that HDAC inhibition preserves cardiac performance and mitigates myocardial remodeling through stimulating cardiac endogenous regeneration.

Topics: Animals; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Male; Mice; Mice, Inbred ICR; Muscle Development; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Neovascularization, Physiologic; Ventricular Remodeling

Trichostatin A (TSA) is a potent histone deacetylase inhibitor and widely used as a promising anticancer agent. Recently, a novel insight for TSA has been shown to protect the heart from ischemia/reperfusion (I/R) injury in mice, but the underlying mechanism remains unclear. The purpose of this study is to investigate whether TSA can influence endoplasmic reticulum stress (ERS) and whether its cardioprotective effect is mediated by inhibiting myocardial ERS-induced apoptosis in rats.. Male Wistar rats were used and pretreated with saline or TSA (0.05, 0.1 and 0.2 mg·kg(-1)) once daily i.p. for 5 days. I/R model was established by occlusion/release of the left anterior descending coronary artery.. TSA significantly reduced myocardial infarct size and plasma activities of lactate dehydrogenase and creatine kinase in a dose-dependent manner in rats. Accompanied by the reduced injury, TSA also markedly reduced I/R-induced myocardial apoptosis (30 min/24 h) by the TUNEL assay. In addition, increased expression of glucose-regulated protein 78 (an ERS marker) by Western blot showed the effects of TSA on ERS. Induction of C/EBP homologous protein (CHOP), a critical mediator for ERS-induced apoptosis, was attenuated by TSA after reperfusion for 6 h and 24 h.. Our findings showed that inhibition of histone deacetylase ameliorated I/R-induced myocardial injury in vivo and for the first time provided the evidence that suppression of CHOP expression and attenuation of the CHOP-induced apoptosis may contribute to the cardioprotection of TSA against myocardial I/R injury.

Topics: Animals; Apoptosis; Endoplasmic Reticulum Stress; Hydroxamic Acids; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Rats; Rats, Wistar; Transcription Factor CHOP

Histone deacetylases (HDACs) play a critical role in the regulation of gene transcription, cardiac development, and diseases. The aim of this study was to test whether inhibition of HDACs induces myocardial repair and cardiac function restoration through c-kit signaling in mouse myocardial infarction models. Myocardial infarction in wild type Kit(+/+) and Kit(W)/Kit(W-v) mice was created following thoracotomy by applying permanent ligation to the left anterior descending artery. The HDAC inhibitor, trichostatin A (TSA, 0.1 mg/kg), was intraperitoneally injected daily for a consecutive 8 weeks after myocardial infarction. 5-Bromo-2-deoxyuridine (BrdU, 50 mg/kg) was intraperitoneally delivered every other day to pulse-chase label in vivo endogenous cardiac replication. Eight weeks later, inhibition of HDACs in vivo resulted in an improvement in ventricular functional recovery and the prevention of myocardial remodeling in Kit(+/+) mice, which was eliminated in Kit(W)/Kit(W-v) mice. HDAC inhibition promoted cardiac repairs and neovascularization in the infarcted myocardium, which were absent in Kit(W)/Kit(W-v) mice. Re-introduction of TSA-treated wild type c-kit(+) CSCs into Kit(W)/Kit(W-v) myocardial infarction heart restored myocardial functional improvement and cardiac repair. To further validate that HDAC inhibition stimulates c-kit(+) cardiac stem cells (CSCs) to facilitate myocardial repair, GFP(+) c-kit(+) CSCs were preconditioned with TSA (50 nmol/liter) for 24 h and re-introduced into infarcted hearts for 2 weeks. Preconditioning of c-kit(+) CSCs via HDAC inhibition with trichostatin A significantly increased c-kit(+) CSC-derived myocytes and microvessels and enhanced functional recovery in myocardial infarction hearts in vivo. Our results provide evidence that HDAC inhibition promotes myocardial repair and prevents cardiac remodeling, which is dependent upon c-kit signaling.

Topics: Animals; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Male; Mice; Mice, Mutant Strains; Muscle Proteins; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Neovascularization, Physiologic; Proto-Oncogene Proteins c-kit; Regeneration; Time Factors

To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice.. We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells.. Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells.

Topics: Animals; Azacitidine; Bone Marrow Cells; Cell Differentiation; Decitabine; Endothelial Cells; Epigenesis, Genetic; Gene Expression Regulation; Heart; Hydroxamic Acids; Inflammation; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocytes, Cardiac; Neovascularization, Physiologic; Pluripotent Stem Cells; Stem Cell Transplantation; Stem Cells; Ventricular Dysfunction, Left

We have recently demonstrated that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia-reperfusion (I/R) injury. The mechanism by which HDAC inhibition confers myocardial protection remains unknown. The purpose of this study is to investigate whether the disruption of NF-kappaB p50 would eliminate the protective effects of HDAC inhibition. Wild-type and NF-kappaB p50-deficient mice were treated with trichostatin A (TSA; 0.1 mg/kg ip), a potent inhibitor of HDACs. Twenty-four hours later, the hearts were perfused in Langendorff model and subjected to 30 min of ischemia and 30 min of reperfusion. Inhibition of HDACs by TSA in wild-type mice produced marked improvements in left ventricular end-diastolic pressure, left ventricular rate pressure product, and the reduction of infarct size compared with non-TSA-treated group. TSA-induced cardioprotection in wild-type animals was absent with genetic deletion of NF-kappaB p50 subunit. Notably, Western blot displayed a significant increase in nuclear NF-kappaB p50 and the immunoprecipitation demonstrated a remarkable acetylation of NF-kappaB p50 at lysine residues following HDAC inhibition. EMSA exhibited a subsequent increase in NF-kappaB DNA binding activity. Luciferase assay demonstrated an activation of NF-kappaB by HDAC inhibition. The pretreatment of H9c2 cardiomyoblasts with TSA (50 nmol/l) decreased cell necrosis and increased in cell viability in simulated ischemia. The resistance of H9c2 cardiomyoblasts to simulated ischemia by HDAC inhibition was eliminated by genetic knockdown of NF-kappaB p50 with transfection of NF-kappaB p50 short interfering RNA but not scrambled short interfering RNA. These results suggest that NF-kappaB p50 acetylation and activation play a pivotal role in HDAC inhibition-induced cardioprotection.

Topics: Acetylation; Animals; Cells, Cultured; Disease Models, Animal; DNA; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Male; Mice; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Necrosis; NF-kappa B p50 Subunit; RNA, Small Interfering; Ventricular Function, Left

Recent evidence has demonstrated the importance of histone deacetylases (HDAC) in the control of hypertrophic responses in the heart. However, it remains unknown whether inhibition of HDACs plays a role in myocardial ischemia and reperfusion (I/R) injury. We hypothesize that HDAC inhibition triggers preconditioning-like effects against I/R injury.. Isolated mouse hearts were perfused with 3 cycles of 5-minute infusion and 5-minute washout of 50 nM of trichostatin A (TSA), a potent inhibitor of HDACs to mimic early pharmacologic preconditioning. This was followed by 30 min of ischemia and 30 min of reperfusion. In addition, mice were treated with saline or TSA (0.1 mg/kg, i.p.) to investigate delayed pharmacologic preconditioning. Twenty-four hours later, the hearts were subjected to I/R. Ventricular function and infarct size were measured, and HDAC 3, 4 and 5 were assessed by Western blot and immunofluorescence. HDAC and p38 mitogen-activated protein kinase activities were determined. TSA produced marked improvements in post-ischemic ventricular function recovery and a reduction in infarct size in both early and delayed preconditioning. Cardioprotection elicited by TSA was abrogated by SB203580, an inhibitor of p38. HDAC 3, 4 and 5 proteins were detected in mouse myocardium. TSA treatments resulted in a significant inhibition of HDAC activity. HDAC inhibition caused a dramatic increase in phosphorylation of p38 and p38 activity. Notably, HDAC inhibition also resulted in remarkable acetylation of p38 at lysine residues.. These results suggest that inhibition of HDACs triggers pharmacologic preconditioning to protect the ischemic heart, which involves p38 activation.

Topics: Animals; Disease Models, Animal; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Imidazoles; Ischemic Preconditioning, Myocardial; Male; Mice; Mice, Inbred ICR; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; p38 Mitogen-Activated Protein Kinases; Pyridines; Signal Transduction; Ventricular Function, Left