Cardiac positive inotropic effect of NADPH in heart

• 1、Department of Pharmacy,Soochow University,Suzhou,Jiangsu 215123

Abstract：Aim: Our previous study has demonstrated the protective effects of NADPH against myocardial ischemia/reperfusion(I/R) injury. Here, we investigate exougeous NADPH\'s effects on heart failure and the underlying mechanisms. Methods: Endogenous serum NADPH in normal people and clinical patients with heart failure was measured.Isolated frog heart perfusion model, isoproterenol (ISO)-induced heart failure model and transverse aortic constriction (TAC)-induced heart failure model were established. Myocardial contractility, cardiac output and heart rate were measured in frog heart. Heart weight index, left ventricular index and cardiomyocyte area were examined. Ejection fraction and fraction shortening were measured by echocardiography. AKT and ERK1/2 proteins were detected by Western blot. Mitochondrial morphology was observed using transmission electron microscopy. Oxidative stress indicators such as malondialdehyde (MDA), glutathione (GSH) and hydrogen peroxide (H2O2) were detected. ATP content was also detected. Western blot was used to detect the expression of SIRT3, SIRT1, AcSOD, acetylation of whole proteins, and phosphorylation of AMP-dependent protein kinase (AMPK) and liver kinase B1(LKB1). Acetylation of LKB1 and succinic dehydrogenase A (SDHA) and ATP synthase F1 subunit (ATP5A1) were downstreams of SIRT3, were detected by immunoprecipitation. Results:A decline of endogeous serum NADPH was found in patients with heart failure compared to normal people. NADPH, NAD, and ATP had positive inotropic effects in frog heart. The ATP receptor antagonist suramin partially abolished the NADPH\'s positive inotropic action, while the NAD inhibitor 3-AP could not cancel NADPH\'s action. In the ISO model, NADPH decreased heart weight index and left ventricular index. The improvement of NAD on heart weight index was less than that of NADPH at the same molar dose. Exogenous NADPH increased total NADPH level in both serum and heart. In the TAC model, NADPH decreased heart weight index and left ventricular index, and down-regulated phosphorylation of AKT and ERK1/2. NADPH reduced oxidative stress and increases ATP production. Under transmission electron microscopy, NADPH improved the mitochondrial damage and decreased the mitochondrial area. NADPH increased the expression of SIRT3 protein, and deacetylated LKB1, activating the LKB1-AMPK signaling pathway. Also, NADPH might down-regulate acetylation of SOD2 to resist oxidative stress. NADPH might deacetylate ATP5A1 and SDHA to maintain mitochondrial function. Conclusion: Exogenous NADPH showed positive inotropic effect on isolated heart and played a protective role on heart failure through activation of SIRT3 and thus improvement of oxidative stress and energy metabolism.

Keywords： Pharmacology NADPH heart failure inotropic effect SIRT3 acetylation ROS energy metabolism