Exogenous NAD~+Supplementation Protects H9c2Cardiac Myoblasts Against Hypoxia/Reoxygenation Injury Via Sirt1-p53Pathway

ObjectiveNicotinamide adenine dinucleotide(NAD~+) not only transfers electronsin mitochondrial respiration, but also acts as an indispensable co-substratefor Sirt1, the cardioprotective class III histone/nonhistone deacetylase.However, NAD~+is depleted in myocardial ischemia/reperfusion (IR) injury.This study aimed to investigate the role of exogenous NAD~+supplementation in the viability of hypoxia/reoxygenation(HR)-stressedH9c2cells, in HR-induced apoptosis and its relevance to Sirt1.MethodsPart1After48h of cell culture in DMEM/F-12medium supplemented with10%inactivated fetal bovine serum(FBS), cells were incubated in “ischemiabuffer”(no sugar and no FBS) under hypoxic condition(1%oxygen) for4hours followed by being incubated in complete culture medium undernormoxic condition for4hours to establish hypoxia/reoxygenationmodel(HR). To determine the time-effect relationship of NAD~+ supplementation in HR-stressed H9c2cells, NAD~+(500μmol/L) was addedat respective time points(2h before hypoxia, immediately after hypoxiabegan,2h after hypoxia began, immediately after reoxygenation began and2h after oxygenation began) into distinct culture media. To determine thedose-effect relationship of NAD~+supplementation in HR-stressed H9c2cells, NAD~+was added at various extracellular concentrations (0,50,100,500,1000and5000μmol/L) immediately after reoxygenation begin. A cck-8kit was utilized to detect viability of HR-stressed H9c2cells.Part2After48h of cell culture in DMEM/F-12medium supplemented with10%inactivated FBS, H9c2cells were randomly divided into six groups:CON, HR, NAD, NADHR, ENHR and EHR. Group CON: H9c2cells wereincubated under normal condition for8hours. Group HR: H9c2cellssuffered from hypoxia/reoxygenation injury. Group NAD: H9c2cells wereincubated under normal condition for8hours with NAD~+(500μmol/L)treatment at the end of the fourth hour. Group NADHR: H9c2cells sufferedfrom hypoxia/reoxygenation injury with NAD~+(500μmol/L) treatment at thebeginning of reoxygenation. Group ENHR: H9c2cells suffered fromhypoxia/reoxygenation injury with NAD~+(500μmol/L) and EX527(1μmol/L)treatment at the beginning of reoxygenation. Group EHR: H9c2cellssuffered from hypoxia/reoxygenation injury with EX527(1μmol/L)treatment at the beginning of reoxygenation. After the indicated treatment, cell apoptosis was detected with TUNEL analysis and DAPI nucleus staining.The expressions of BAX and Sirt1in H9c2cells was determined withWestern Blot, as well as the expressions of p53and ac-p53(lys373&382) inthe nucleus. Sirt1deacetylation activity in the nucleus was determined withthe Sirt1Deacetylase Fluorometric Assay Kit.ResultsPart1There was a mild and insignificant improvement in viability ofHR-stressed H9c2cells when NAD~+was added2hours before hypoxia.Significant improvement in cell viability in HR-stressed cells was observedwhen NAD~+was treated at other time points. NAD~+replenishment inducedthe greatest improvement in viability of HR-stressed H9c2cells when NAD~+was added immediately after reoxygenation began. There was a certainextent of improvement in cell viability (though insignificant) whenextracellular NAD~+concentration was50μmol/L. However, along withextracellular NAD~+concentration increasing, improvement in cell viabilitywas significantly observed. NAD~+replenishment induced the greatestimprovement in viability of HR-stressed H9c2cells when NAD~+concentration ranged from500to1000μmol/L in the culture medium.Part2The variance of cell apoptotic ratio and BAX expression:Compared with group CON, the apoptotic ratio of H9c2cells in group HR was elevated significantly, as well as the expression of pro-apoptoticprotein BAX. Compared with group HR, the apoptotic ratio of H9c2cells ingroup NADHR was decreased significantly, as well as the expression ofpro-apoptotic protein BAX. However, Compared with group NADHR, theapoptotic ratio of H9c2cells in group ENHR was elevated significantlyagain, as well as the expression of pro-apoptotic protein BAX.The variance of Sirt1activity and Sirt1expression:Compared with group CON, Sirt1activity in the nucleus in group HRwas decreased significantly. Compared with group HR, a marked elevationin Sirt1activity was detected in group NADHR. However, Compared withgroup NADHR, Sirt1activity in group ENHR was decreased substantiallyagain. There were no differences in Sirt1expression between groups.The variance of ac-p53(lys373&382)/p53:Compared with group CON, the expression ratio ofac-p53(lys373&382) to p53in group HR was elevated significantly.Compared with group HR, the expression ratio of ac-p53(lys373&382) top53in group NADHR was markedly declined. However, Compared withgroup NADHR, the expression ratio of ac-p53(lys373&382) to p53in groupENHR was substantially elevated again.ConclusionExogenous supplementation of NAD~+to H9c2cells attenuatedHR-induced cell death in both time-and dose-dependent manners. It exerts the greatest effect when NAD~+is added immediately after reoxygenationbegins and the optimal extracellular NAD~+concentration is500-1000μmol/L. Exogenous NAD~+supplementation attenuatesHR-induced cell apoptosis, which is at least partly mediated by restoringSirt1activity in the nucleus and subsequently inhibiting p53activity viadeacetylating p53at lysine373and382.