Reverse-Mode of the Mitochondrial Transhydrogenase Consumes NADPH and Provokes Oxidative Stress in Response to Elevated Cardiac Workload

Mitochondrial production of reactive oxygen species (ROS) contributes to the progression of heart failure, but the mechanisms of ROS generation are incompletely resolved. Superoxide (.O2−) is generated at the electron transport chain, dismutated to H2O2 and eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (NNT) is highly expressed in the heart and catalyzes the reaction NADH+NADP+ to NADPH+NAD+. Since this reaction is coupled to the proton-motive force, it is perceived that the NNT prevents ROS production by regenerating mitochondrial NADPH. The exact role of the NNT in cardiomyocyte biology, however, has never been assessed. We took advantage of a loss-of-function mutation in the Nnt gene in C57BL/6J, but not C57BL/6N mice. In isolated cardiac myocytes exposed to a physiological increase in workload, β-adrenergic stimulation led to mitochondrial Ca2+-induced Krebs cycle activation with NADH regeneration, providing a substrate for the forward mode NNT reaction to regenerate NADPH. In contrast, under Ca2+-free conditions in isolated mitochondria, acceleration of NADH-coupled respiration by ADP favoured the reverse-mode NNT reaction, regenerating NADH by oxidizing NADPH. Accordingly, in response to an elevated workload in isolated hearts, the NADPH-coupled antioxidants glutathione and peroxiredoxin were oxidized through reverse-mode NNT reaction. This resulted in elevated mitochondrial formation of H2O2 in vivo after thoraco-aortic constriction (TAC) for 6 weeks. In NNT-deficient C57BL/6J mice, TAC-induced oxidative stress in vivo, cardiac fibrosis, left ventricular dysfunction and early mortality were ameliorated. Furthermore, scavenging mitochondrial ROS with the peptide SS-31 in vivo reduced TAC-induced mortality in C57BL/6N mice to levels observed in C57BL/6J mice. In conclusion, we believe that we discovered the mechanism how an inadequate increase in cardiac workload produces mitochondrial oxidative stress that leads to maladaptive cardiac remodelling and cardiac decompensation.