Abstract 862: Ser517:-phosphorylation Of Sirt1 Is Required For Its Pi3k/akt-mediated Nuclear Translocation And Cardiomyocyte Protection Against Oxidant Stress In Failing Hearts.
[Purpose] We recently found that SIRT1, a protein deacetylase, shuttles between the nucleus and cytoplasm. In this study, we examined the role of nuclear SIRT1 in cardiomyocyte protection against oxidant stress and involvement of PI3K/Akt in the nuclear translocation.
[Methods and Results] First, the critical intracellular location of SIRT1 for its anti-apoptotic function was examined. C2C12 cells were transfected with wild-type SIRT1 (WT) or SIRT1 with site-directed mutations in the nuclear localizing signal (mtNLS) and exposed to antimycin A (AA), an oxidative stressor. AA-induced apoptosis was suppressed in WT-transfected cells expressing SIRT1 in the nuclei compared with that in mtNLS-transfected cells expressing SIRT1 in the cytoplasm (TUNEL-positive cells = 4.4±0.7% vs. 34.6±8.0%). AA-induced apoptosis and also angiotensin II-(angII)-induced apoptosis in neonatal rat cardiomyocytes (NRCM) were suppressed by resveratrol, a SIRT1 activator. This protective effect of resveratrol was attenuated by transfection of SIRT1-siRNA but not by transfection of control siRNA. Next, we assessed the role of PI3K/Akt in nuclear translocation of SIRT1. SIRT1 in NRCM was localized in both the nucleus and cytoplasm under baseline conditions, and IGF-1 induced its nuclear translocation. This effect of IGF-1 was suppressed by LY294002 (LY), a PI3K inhibitor. Deletion mutagenesis study showed that LY-induced nuclear exclusion was observed for SIRT1[223–540] but not for SIRT1[223– 489]. Replacement of serine517 with alanine (S517A) increased cytoplasmic SIRT1, and S517A showed attenuated nuclear translocation in response to IGF-1, indicating that serine517 is the target site of PI3K/Akt. Finally, to confirm heart failure-associated SIRT1 translocation in vivo, myocardial infarction was induced in WKY rats. The number of ventricular cardiomyocytes with nuclear SIRT1 at 4 weeks after infarction was significantly larger than that in sham-operated hearts (10.2±2.9% vs. 0.7±0.2%).
[Conclusion] The results suggest that phosphorylation of SIRT1 at Ser517 by PI3K/Akt is involved in nuclear translocation of SIRT1, which contributes to cardiomyocyte protection from oxidant stress-mediated injury in failing hearts.