Exogenous NAD(+) supplementation protects H9c2 cardiac myoblasts against hypoxia/reoxygenation injury via Sirt1-p53 pathway.

Nicotinamide adenine dinucleotide (NAD(+) ) not only transfers electrons in mitochondrial respiration, but also acts as an indispensable cosubstrate for Sirt1, the class III histone/nonhistone deacetylase. However, NAD(+) is depleted in myocardial ischemia/reperfusion (IR) injury. The objective of this study was to investigate the role of exogenous NAD(+) supplementation in hypoxia/reoxygenation (HR)-stressed H9c2 cardiac myoblasts. Firstly, the effects of distinct treating time points and doses of NAD(+) supplementation on the viability of HR-stressed H9c2 cells were detected. Secondly, intracellular NAD(+) levels in HR-stressed H9c2 cells at various extracellular NAD(+) concentrations were determined. Thirdly, the role of NAD(+) supplementation in HR-induced cell apoptosis and its relevance to Sirtuin 1-p53 pathway were investigated. Exogenous NAD(+) supplementation elevated intracellular NAD(+) level and reduced HR-induced cell death in both time- and concentration-dependent manners. It appeared that NAD(+) supplementation exerted the greatest protection when extracellular concentration ranged from 500 to 1000 μm and when NAD(+) was added immediately after reoxygenation began. NAD(+) replenishment restored Sirt1 activity, reduced the acetylation level of p53 (Lys373 & 382), and attenuated cell apoptosis in HR-stressed H9c2 cells, whereas inhibition of Sirt1 activity alleviated the effects of NAD(+) replenishment. These results indicated that exogenous NAD(+) supplementation attenuated HR-induced cell apoptosis, which was at least partly mediated by restoring Sirt1 activity and subsequently inhibiting p53 activity via deacetylating p53 at lysine 373 and 382.