Exogenous NAD + supplementation protects H 9c2 cardiac myoblasts against hypoxia/reoxygenation injury via S irt1‐p53 pathway

Nicotinamide adenine dinucleotide ( NAD + ) not only transfers electrons in mitochondrial respiration, but also acts as an indispensable cosubstrate for S irt1, 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 H 9c2 cardiac myoblasts. Firstly, the effects of distinct treating time points and doses of NAD + supplementation on the viability of HR ‐stressed H 9c2 cells were detected. Secondly, intracellular NAD + levels in HR ‐stressed H 9c2 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 μ m and when NAD + was added immediately after reoxygenation began. NAD + replenishment restored S irt1 activity, reduced the acetylation level of p53 ( L ys373 & 382), and attenuated cell apoptosis in HR ‐stressed H 9c2 cells, whereas inhibition of S irt1 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 S irt1 activity and subsequently inhibiting p53 activity via deacetylating p53 at lysine 373 and 382.