Abstract 365: Activation of a Class-III Histone Deacetylase, SIRT1, Prevents the Oxidative-stress Mediated Cell-death by Suppressing the Activity of Poly (ADP) Ribose Polymerase-1
Background: Poly (ADP) ribose polymerase-1 (PARP-1) and SIRT1 deacetylase have many common features. Both are NAD-dependent chromatin bound enzymes, play a pivotal role in DNA-repair, cell-cycle progression, apoptosis and aging. Previous studies from this lab have shown that increased PARP-1 activity during heart failure induces myocyte cell-death by NAD-depletion and reduced SIRT1 deacetylase activity. This study was undertaken to examine whether PARP-mediated cell-death could be prevented by SIRT1 activation.
Methods and Results: Primary cultures of neonatal rat heart myocytes, grown in serum-free media, were treated with cell-death inducing dose of angiotensin-II (Ang-II, 20μM) or a free-radical generating mixture of H2O2 and FeSO4 (0.1mM each, abbreviated as H2O2). For a positive control cells were treated with MNNG (100μM), a DNA-damaging agent, known to robustly activate PARP-1. Forty eight hours following treatment, cells were harvested and cellular levels of NAD, poly (ADP) ribosylated nuclear proteins and acetylated p53 were measured. Cell death was measured by Hoechst and propidium-iodide staining followed by FACS analysis. Stimulation of cells with Ang-II or H2O2 induced massive cardiomyocyte cell-death (80%), which was associated with increased levels of poly ADP-ribosylated proteins, acetylated p53 and reduced NAD levels. Similar changes were observed in positive control cells treated with MNNG. This type of cell-death was protected by pretreatment with a PARP-inhibitor, 3-aminobenzamide, thus confirming a role of PARP-1 activation in this form of cell-death. To examine the effect of SIRT1 deacetylase, cells were first infected with adenovirus vectors expressing either wild-type or mutant SIRT1, and then treated with the cell-death inducing agent. Over expression of SIRT1 wild-type, but not the mutant, completely blocked the Ang-II, H2O2 as well as MNNG mediated cell-death, which was associated with reduced protein poly ADP-ribosylation, p53 acetylation and depletion of NAD levels; thus, suggesting suppression of the PARP-1 enzymatic activity by SIRT1 activation.
Conclusion: These data reveals a novel mechanism of SIRT1-mediated cell protection, which is, in part, contributed by reduced enzymatic activity of PARP-1.