Abstract 10439: Deacetylation of Histone H2AX by Sirt1 Regulates DNA Damage Response and Protects Cardiomyocytes Against Doxorubicin-Induced Injury

Abstract

Introduction: Accumulated DNA damage has been implicated in doxorubicin (DOX)-induced cardiotoxicity. We recently found that deletion of SIRT1, a histone deacetylase, in the cardiomyocyte worsens DOX-induced cardiac injury in mice. However, its molecular mechanism remains unclear. Ser139 phosphorylation of histone H2AX catalyzed by ATM (mutated in ataxia-telangiectasia) at the sites of DNA damage mediates DNA repair. Hypothesis: Deacetylation of H2AX by SIRT1 mediates DNA damage response and protects cardiomyocytes from DOX-induced injury. Methods and Results: Wild-type (WT) and tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout (SIRT1 cKO) mice received DOX (4 IP injections of 5 mg/kg/week) or a vehicle. Immunoblotting showed that myocardial phospho-Ser139 H2AX level was increased by 1.6-fold in WT at 1 week after final DOX, but such a response was absent in SIRT1 cKO. Ser1981 phosphorylation of ATM by DOX were comparable in WT and SIRT1 cKO. The increase in TUNEL-positive nuclei by DOX was greater in SIRT1 cKO (0.13 to 0.38%) than WT (0.07 to 0.19%), suggesting higher DNA damage in SIRT1 cKO. In H9c2 cardiomyocytes, SIRT1 knockdown (KD) also abolished Ser139 phosphorylation of H2AX by DOX without changing phospho-ATM levels. Increases in DNA damage evaluated by the comet assay and cleavage of caspase-3 by DOX were also enhanced by SIRT1 KD. Immunostaining for acetyl-Lys5 H2AX in the heart sections revealed that acetyl-Lys5 H2AX levels were increased in SIRT1 cKO by 58% compared with WT. In H9c2 cells, acetyl-Lys5 H2AX level was also increased by SIRT1 KD and reduced by expression of wild-type SIRT1. In COS7 cells, a mutant in which Lys5 was substituted to glutamine (K5Q H2AX), a mimic of acetylated Lys5, showed attenuated Ser139 phosphorylation of H2AX by DOX. DOX-induced cleavage of caspase-3 was enhanced in H9c2 cells expressing K5Q H2AX as well as S139A H2AX, that cannot be phosphorylated at Ser139, compared with cells expressing WT H2AX. Conclusions: Aberrant Lys5 acetylation of H2AX via SIRT1 suppression interferes Ser139 phosphorylation, leading to accumulation of damaged DNA and promotion of the apoptotic response. Such regulation of the DNA damage response underlies protection by SIRT1 against DOX-induced cardiotoxicity.