Abstract 1824: The Longevity Factor, SIRT3 Deacetylase Protects Cardiomyocytes From Stress-mediated Cell Death By Deacetylation of Ku-70
SIRT3 is a member of Class III HDACs. It has been implicated in regulation of chromatin remodeling, genetic control of aging, and calorie restriction-mediated longevity of organisms ranging from yeast to humans. This study was designed to examine the role of SIRT3 in cardiomyocytes under stress. Primary cultures of neonatal rat heart myocytes were grown in different stress conditions and the expression of SIRT3 was analyzed by western analysis. We found that cells grown in serum-free media had 2- to 3-fold higher level of SIRT3 expression, compared to controls maintained in serum containing medium. To determine the function of SIRT3 in stress conditions, cardiomyocytes were infected with adenovirus vectors expressing SIRT3 wild-type or the mutant, having no deacetylase activity. Viral-infected cells were then treated with MNNG (500 μM), a genotoxic stress inducing agent, or H2O2 (500 μM), an oxidative stress inducing agent for 48 hours. We found that the MNNG or H2O2 treatment killed almost 60% of cells expressing the mutant-SIRT3; however, the SIRT3 wild-type expressing cells were mostly protected. To delineate the mechanism of cell-protection by SIRT3, we searched for its partner proteins. By co-immunoprecipitation (Co-IP) and protein-binding experiments we found that SIRT3 physically binds to Ku70 and keeps it in the deacetylated form. Ku70 is known to protect cells by binding to pro-apoptotic protein Bax. To examine whether SIRT3 was capable of promoting Ku70-Bax interaction in vivo, HeLa cells were transfected with plasmids synthesizing Bax, Ku70 and/or SIRT3. We found that cells expressing SIRT3 wild-type, Bax and Ku70 together had a significantly lower rate of cell death, compared to those expressing Bax and Ku70 alone. In contrast, cells expressing the mutant-SIRT3 were not resistant to Bax-mediated cell death even when Bax and Ku70 were combined. Co-IP experiments with lysates of these transfectants indicated that Bax was readily pull-down with Ku70 from SIRT3 wild-type expressing cells, but not from the mutant cells, thus suggesting that SIRT3 enhances Ku70-Bax interaction. These data demonstrated that SIRT3 is a stress responsive factor in cardiomyocytes that protects cells by deacetylating Ku70 and hence promoting Ku70/Bax association.