Abstract 18078: Hdac4 Degradation Involves Hdac Inhibition-Induced Protective Effects in Hypoxia in Mouse Cardiomyocytes

Abstract

Background: Histone deacetylases (HDACs) have been recently demonstrated to play critical roles in modulating myocardial protection and cardiomyocyte survivals in response to stress. However, the molecular mechanism(s) of specific HDACs attributable for the regulation of myocyte survivals are poorly understood. Objective: Our goal was to investigate whether HDAC inhibition elicits a protective effect through specific HDAC4, and whether HDAC4 ubiquitination involves induction of cellular protective effect in mouse cardiomyocytes exposed to hypoxia/re-oxygenation. Methods: Neonatal mouse cardiomyocytes (1-3 day old) were isolated and then subjected to 48 hours of hypoxia (1% oxygen) followed 2 hours of reoxygenation. Prior to hypoxia cardiomyocytes were infected with adenoviral HDAC4 and adenoviral GFP. Cardiomyocytes were preconditioned with trichostatin A (TSA, 50nmol/L) to block HDAC activity, whereas MG132 (0.2 μmol/L), a selective proteasome inhibitor, was utilized to inhibit the proteasome pathway. The cytotoxicities were estimated by detection of lactate dehydrogenase (LDH) and myocyte viabilities were measured by MTT assays. Western blot was employed to assess HDAC4 degradation. RT-PCR was performed to analyze HDAC4 mRNA. Results: Inhibition of HDACs with TSA enhanced cardiomyocyte viabilities and reduced the LDH leakages of cardiomycytes during hypoxia/reoxygenation. Pretreatment of myocytes with MG132 abolished the protective effects of TSA. Infection of myocytes with adv-HDAC4 mitigated HDAC inhibition-induced protection. Co-immunoprecipitation assay and Western blot indicate the HDAC4 uniquitination following TSA treatments. TSA specifically resulted in the reduction of HDAC4 protein, but not mRNA, which was blocked by MG132. Conclusion: HDAC inhibition protects cardiomyocytes against hypoxic stress, which depends on HDAC4 and HDAC4 ubiquitination.