NADPH supply and the contribution of NAD(P)⁺ transhydrogenase (NNT) to H₂O₂ balance in skeletal muscle mitochondria

H₂O₂ is endogenously generated and its removal in the matrix of skeletal muscle mitochondria (SMM) is dependent on NADPH likely provided by NAD(P)⁺ transhydrogenase (NNT) and isocitrate dehydrogenase (IDH2). Importantly, NNT activity is linked to mitochondrial protonmotive force. Here, we demonstrate the presence of NNT function in detergent-solubilized and intact functional SMM isolated from rats and wild type (Nnt^+/+) mice, but not in SMM from congenic mice carrying a mutated NNT gene (Nnt^-/-). Further comparisons between SMM from both Nnt mouse genotypes revealed that the NADPH supplied by NNT supports up to 600 pmol/mg/min of H₂O₂ removal under selected conditions. Surprisingly, SMM from Nnt^-/- mice removed exogenous H₂O₂ at wild-type levels and exhibited a maintained or even decreased net emission of endogenous H₂O₂ when substrates that support Krebs cycle reactions were present (e.g., pyruvate plus malate or palmitoylcarnitine plus malate). These results may be explained by a compensation for the lack of NNT, since the total activities of concurrent NADP⁺-reducing enzymes (IDH2, malic enzymes and glutamate dehydrogenase) were ~70% elevated in Nnt^-/- mice. Importantly, respiratory rates were similar between SMM from both Nnt genotypes despite differing NNT contributions to H₂O₂ removal and their implications for an evolving concept in the literature are discussed. We concluded that NNT is capable of meaningfully sustaining NADPH-dependent H₂O₂ removal in intact SMM. Nonetheless, if the available substrates favor non-NNT sources of NADPH, the H₂O₂ removal by SMM is maintained in Nnt^-/- mice SMM.