Chemogenetic manipulation of mitochondrial hydrogen peroxide reveals distinct muscle-wasting phenotypes in skeletal muscle

Background: Muscle-wasting conditions pose a major health burden, affecting millions worldwide, with no effective treatment available. Characterized by progressive muscle mass and strength loss, these conditions increase the risk of cardiometabolic diseases and mortality. Elevated mitochondrial reactive oxygen species (ROS) are a common feature across various forms of muscle wasting; however, the causal role of mitochondrial-specific ROS in muscle atrophy remains debated due to methodological limitations. This study examined the dose-dependent effects of in vivo chemogenetic hydrogen peroxide (H2O2) production targeted to the mitochondrial matrix on skeletal muscle wasting. Methods: C57Bl/6N mice were unilaterally transfected with an adeno-associated virus expressing recombinant yeast D-amino acid oxidase (mtDAAO) in the gastrocnemius muscle. D-alanine, the mtDAAO substrate, was provided ad libitum in drinking water at different dosages (0.2, 0.4, or 1M for 1, 4, or 8 days), with a subgroup recovering for 7 or 14 days after 8 days at 0.4M. Muscle samples were analyzed using immunoblotting and histochemistry. Results: Chemogenetic mtH2O2 generation induced a dosage-dependent increase in lipid oxidation (4-HNE), from 5% at 0.2M for 4 days to >93% at higher doses. The lowest 0.2M dose caused ∼36% atrophy after 4 days, with mild ubiquitination (∼28%) and reduced protein synthesis (∼24%) after 1 day. At 0.4M, atrophy (∼32%) was observed at 4 days, with increased ubiquitination and upregulation of the denervation marker NCAM. At 0.4M for 8 days, overt degeneration and regeneration occurred.
Conclusion: Mitochondrial H2O2 production induces at least three muscle-wasting phenotypes, with distinct molecular signatures resembling disuse, denervation, and degeneration/repair. This model provides a tool for studying muscle-wasting mechanisms and the dosage-dependent role of mitochondrial H2O2 in these pathologies.