Dehydrogenase-linked metabolic markers of changes in the mitochondrial redox status

Abstract

Mitochondria are essential to normal cellular function due to their production of adenosine triphosphate (ATP), which serves as the primary energy source for most biochemical and physiological processes. Dysfunction of the mitochondrion is commonly characterised by impaired oxidative phosphorylation (OXPHOS) and altered redox status. Redox status regulates a plethora of biological processes, including cellular signalling pathways, regulation of antioxidant defence systems and metabolic pathways, therefore maintenance of it is essential for normal cellular function. Metabolic investigations conducted in the past consistently indicated that a disruption in redox status contributes to cellular adaptations observed in both patients with mitochondrial diseases and animal models. However, due to the challenges in directly measuring NAD+/NADH ratios in cells and tissues, indirect measurements are primarily utilised, particularly the ratios between the substrate and product pairs of dehydrogenase-catalysed reactions. To validate whether these metabolite ratios accurately reflect changes in redox status, it is necessary to compare them with direct measurements of NAD+/NADH ratios. Therefore, this study aimed to determine whether dehydrogenase-linked metabolite ratios can serve as valid indicators of mitochondrial redox state status (NAD+/NADH ratio) at different stages of mitochondrial dysfunction. This aim was achieved through the generation of SoNar- and mtLbNox-expressing cell models, followed by investigating the relationship between m.3243A>G heteroplasmy, dehydrogenase-linked metabolite ratios and NAD+/NADH balance, as well as the evaluation of mtLbNox expression on these factors. The results demonstrate that increasing m.3243A>G heteroplasmy induces compartment-specific changes in NAD+ levels and variable changes in metabolite ratios, while mtLbNox intervention confirmed that pyruvate/lactate, oxaloacetate/malate, and 2-ketoisocaproate/2hydroxyisocaproate ratios consistently reflected changes in redox state. Other metabolite ratios did not respond proportionally to altered redox state and therefore require further investigation. The results of this study demonstrate that while dehydrogenase-linked metabolite ratios can reflect aspects of redox-associated metabolic adaptation, their relationship with redox state is complex, compartment-dependent and not consistent across all pathways.