Mitochondrial Transhydrogenase: Yin and Yang of Antioxidative Capacity in Cardiac Myocytes

Mitochondrial production of reactive oxygen species (ROS) contributes to the pathogenesis of various diseases and aging. Superoxide (•O2-) is generated as by-product of the electron transport chain (ETC), rapidly dismutated to H2O2 and eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (Nnt) catalyzes the reaction NADH+NADP+→NADPH+NAD+, which is coupled to the proton motive force across the inner mitochondrial membrane (ΔµH). Thus, the Nnt is considered to play a key role in regenerating NADPH and maintaining mitochondrial antioxidant capacity. Recently, a loss-of-function mutation in the Nnt gene was discovered in C57BL/6J (J-) but not C57BL/6N (N-) mice, rendering this strain glucose-intolerant due to increased ROS production in pancreatic islet cells.Here, we analyze the role of Nnt in cardiac mitochondria of N- and J-mice by applying various techniques including fluorescence imaging, patch-clamping and EPR spin-trap measurements on isolated mitochondria or cardiac myocytes. In the absence of ADP and Ca2+, •O2- and H2O2 formation were comparable in energized mitochondria from J- and N-mice. Accelerating NADH-coupled respiration with ADP or uncoupler oxidized both NADH and NADPH in N-mice, but only NADH in J-mice, indicating that Nnt mediates NADPH oxidation through its reverse reaction when NADH is consumed by the ETC. This was associated with lower H2O2 and •O2- formation in uncoupled mitochondria from Nnt-deficient J-mice. In intact myocytes, however, an increase in work (β-adrenergic stimulation, 5 Hz stimulation frequency) was associated with similarly increased cytosolic and mitochondrial [Ca2+] and accelerated NADH regeneration by the Krebs cycle. Under these conditions, lack of NADPH regeneration via Nnt in the forward mode provoked increased H2O2 formation in J- vs. N-mouse mitochondria.We conclude that in cardiac mitochondria, the Nnt either prevents or promotes ROS production, depending on the energetic state of the cell.