Abstract 411: Mitochondrial NAD(H) Signaling in Cardiac Physiology and Pathology

Abstract

The heart is a high energy-consuming organ and mitochondrial dysfunction is widely observed in heart failure. Previously, we reported that imbalanced NADH/NAD+ homeostasis in cardiomyocyte is responsible for stress-induced mitochondrial protein hyper-acetylation and accelerated progression to heart failure, which can be rescued by supplementing NAD+ precursor. The goals of this study are to develop novel fluorescent biomarkers and directly monitor mitochondrial NAD(H) redox regulation under pathophysiological conditions in adult cardiomyocytes. We constructed adenoviruses containing mitochondria-targeted NAD+ biosensor and NADH/NAD+ ratio biosensor (SoNar). We validated their mitochondrial targeting and ability to monitor acute changes in NAD+ and NADH/NAD+ ratio in adult cardiomyocytes. Pyruvate (1 mM) acutely increased mitochondrial NAD+ level and NADH/NAD+ ratio consistent with the notion that stimulating mitochondrial metabolism expanded NAD+ pool and promoted the reduction of NAD+ to NADH, which exceeded NADH consumption. In contrast, glucose (10 mM) slightly increased NADH/NAD+ ratio and palmitate had no acute effect suggesting that metabolism is only mildly stimulated and the NAD+ pool remains stable. However, palmitate treatment for 24 hr did decrease NAD+ and increase NADH/NAD+ ratio in mitochondria suggesting that the effects of physiological substrates on mitochondrial NAD(H) redox status are slow, accumulative or indirect through modulating other mitochondrial functions. Elevating cytosolic calcium with caffeine increased mitochondrial NAD+ level and decreased NADH/NAD+ ratio suggesting accelerated NADH to NAD+ turnover. Calcium has been shown to boost both energy production and utilization, but its overall impact on energy homeostasis is not clear. Our results indicate that acute increase in cytosolic calcium may have a more profound effect on energy utilization. Finally, ischemia reperfusion induced acute fluctuations in NADH and NADH/NAD+ ratio. Taken together, these results provide the first glimpse into the real-time modulation of compartmentalized NAD(H) redox state and new clues as to how mitochondrial redox is tightly controlled by mitochondrial metabolism in adult cardiomyocytes.