Introduction: We recently found that deletion of SIRT1, a histone deacetylase, in the cardiomyocyte worsens doxorubicin (DOX)-induced cardiac injury in mice. However, its molecular mechanism remains unclear. We hypothesized that SIRT1 protects cardiomyocytes from DOX-induced injury by mediating DNA damage response (DDR) via deacetylation of H2AX, a critical mediator of DDR.
Methods and Results: In H9c2 cardiomyocytes, treatment with Dox increased cleaved caspase-3 level, which was enhanced by knockdown (KD) of SIRT1. The comet assay showed that SIRT1 KD also increased the degree of DNA damage induced by Dox. Immunoblotting revealed that treatment of cells with Dox increased levels of Ser139 phosphorylation of H2AX, indicating the DDR by Dox. SIRT1 KD abolished this response. Immunostaining showed that acetyl-Lys5 H2AX level was 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. In H9c2 cells, DOX-induced cleavage of caspase-3 was enhanced in 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 apoptotic cell death. Such regulation may underlie protection by SIRT1 against DOX-induced cardiotoxicity.