ABSTRACT: Mitochondrial dysfunction caused by mitochondrial (mtDNA) deletions have been associated with skeletal muscle atrophy and myofiber loss. However, whether such defects occurring in myofibers cause sarcopenia is unclear. Also, the contribution of mtDNA alterations in muscle stem cells (MuSCs) to sarcopenia remain to be investigated. We expressed a dominant-negative variant of the mitochondrial helicase, which induces mtDNA alterations, specifically in differentiated myofibers (K320Eskm mice) and MuSCs (K320Emsc mice), respectively, and investigated their impact on muscle structure and function by immunohistochemistry, analysis of mtDNA and respiratory chain content, muscle transcriptome and functional tests. K320Eskm mice at 24 months of age had higher levels of mtDNA deletions compared to controls in soleus (SOL, 0.07673 % vs 0.00015 %, p=0.0167), extensor digitorium longi (EDL, 0.0649 vs 0.000925, p= 0.0015) and gastrocnemius (GAS, 0.09353 vs 0.000425, p=0.0004). K320Eskm mice revealed a progressive increase in the proportion of cytochrome c oxidase deficient fibers (COX-) in cross sections, reaching a maximum of 3.03%, 4.36%, 13.58% and 17.08% in EDL, SOL, tibialis anterior (TA) and GAS, respectively. However, mice did not show accelerated loss of muscle mass, muscle strength or physical performance. Histological analyses revealed ragged red fibers but also stimulated regeneration, indicating activation of MuSCs. RNAseq demonstrated enhanced expression of genes associated with protein synthesis, but also degradation, as well as muscle fiber differentiation and cell proliferation. In contrast, 7 days after destruction by cardiotoxin, regenerating TA of K320Emsc mice showed 30% of COX- fibers. Notably, regenerated muscle showed dystrophic changes, increased fibrosis (2.5% vs 1.6%, p=0.0003), increased abundance of fat cells (2.76% vs 0.23%, p=0.0144) and reduced muscle mass (regenerated TA: 40.0mg vs 60.2mg, p=0.0171). In contrast to muscles from K320Eskm mice, freshly isolated MuSCs from aged K320Emsc mice were completely devoid of mtDNA alterations. However, after passaging, mtDNA copy number as well as respiratory chain subunits and p62 levels gradually decreased.Taken together, accumulation of large-scale mtDNA alterations in myofibers alone is not sufficient to cause sarcopenia. Expression of K320E is tolerated in quiescent MuSCs, but progressively leads to mtDNA and respiratory chain depletion upon activation, in vivo and in vitro, possibly caused by an increased mitochondrial removal. Altogether, our results suggest that the accumulation of mtDNA alterations in myofibers activates regeneration during aging, which leads to sarcopenia if such alterations have expanded in MuSCs as well.
