1. Inducible and reversible SOD2 knockdown in mouse skeletal muscle drives impaired pyruvate oxidation and reduced metabolic flexibility.
- Author
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Ostrom, Ethan L., Stuppard, Rudy, Mattson-Hughes, Aurora, and Marcinek, David J.
- Subjects
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PYRUVATE dehydrogenase kinase , *TYPE 2 diabetes , *SKELETAL muscle , *MUSCLE aging , *MUSCLE fatigue , *RESPIRATION , *MUSCLE contraction - Abstract
Skeletal muscle mitochondrial dysfunction is a key characteristic of aging muscle and contributes to age related diseases such as sarcopenia, frailty, and type 2 diabetes. Mitochondrial oxidative stress has been implicated as a driving factor in these age-related diseases, however whether it is a cause, or a consequence of mitochondrial dysfunction remains to be determined. The development of flexible genetic models is an important tool to test the mechanistic role of mitochondrial oxidative stress on skeletal muscle metabolic dysfunction. We characterize a new model of inducible and reversible mitochondrial redox stress using a tetracycline controlled skeletal muscle specific short hairpin RNA targeted to superoxide dismutase 2 (iSOD2). Methods: iSOD2 KD and control (CON) animals were administered doxycycline for 3- or 12- weeks and followed for up to 24 weeks and mitochondrial respiration and muscle contraction were measured to define the time course of SOD2 KD and muscle functional changes and recovery. Maximum knockdown of SOD2 protein occurred by 6 weeks and recovered by 24 weeks after DOX treatment. Mitochondrial aconitase activity and maximum mitochondrial respiration declined in KD muscle by 12 weeks and recovered by 24 weeks. There were no significant differences in antioxidant or mitochondrial biogenesis genes between groups. Twelve-week KD showed a small, but significant decrease in muscle fatigue resistance. The primary phenotype was reduced metabolic flexibility characterized by impaired pyruvate driven respiration when other substrates are present. The pyruvate dehydrogenase kinase inhibitor dichloroacetate partially restored pyruvate driven respiration, while the thiol reductant DTT did not. We use a model of inducible and reversible skeletal muscle SOD2 knockdown to demonstrate that elevated matrix superoxide reversibly impairs mitochondrial substrate flexibility characterized by impaired pyruvate oxidation. Despite the bioenergetic effect, the limited change in gene expression suggests that the elevated redox stress in this model is confined to the mitochondrial matrix. [Display omitted] • SOD2 knockdown and recovery is achieved by using a shRNA targeted to SOD2 mRNA controlled by a TET-ON System. • SOD2 KD is induced by administering doxycycline (DOX) in the drinking water. • Mitochondrial functional decline and recovery follows the time course of SOD2 protein decline and recovery. • Sustained SOD2 KD drives impaired pyruvate respiration in skeletal muscle mitochondria in the presence of other substrates. [ABSTRACT FROM AUTHOR]
- Published
- 2025
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