Lower Mitochondrial Genome Turnover and Greater Mutation Frequency in Skeletal Muscle of Aged Compared to Adult OKC-HET Rats

Mitochondrial genomic integrity, which is supported by processes such as replication, repair, and turnover, is critical in supporting metabolism and organismal health. Accumulation of mitochondrial DNA (mtDNA) mutations contributes to mitochondrial and whole-tissue dysfunction, which lead to chronic diseases including sarcopenia. The underlying mechanisms that promote generation and age-related accumulation of mtDNA mutations are unclear. mtDNA mutants may have a replicative advantage over wild-type mtDNA that propagates the accumulation of mtDNA mutations with age. However, there is limited in vivo data regarding effects of age on mtDNA replication, particularly in skeletal muscle. We hypothesized mtDNA synthesis rates would increase with age as mutation burden increased in skeletal muscle in both male and female rats. Methods: We measured mtDNA synthesis over 14 days using the stable isotope tracer deuterium oxide (D2O) and assessed mtDNA copy number and mutation deletion frequency using digital PCR in quadriceps muscle of 9- and 26 month-old (mo) male and female OKC-HET rats, which have heterogenous mtDNA backgrounds. Results: There were no differences in mtDNA copy number between sexes or ages. However, 26 month-od rats had lower rates of mtDNA synthesis compared to 9 month-old rats (9 mo: 0.509 ± 0.009 %/day, 26 month: 0.371 ± 0.048 %/day; p=0.0024) and greater mtDNA half-lives (9 mo: 132 ± 1.79 days, 216 ± 25.63 days; p=0.0009). Concomitant with lower mtDNA synthesis, 26 month-old rats had greater (p=0.003) mtDNA deletion mutation frequency (2.023e-004 ± 7.18e-005) than 9 month-old rats (9.310e-005 ± 2.404e-005). 26 month-old female rats (1.309e-004 ± 2.386e-005) had a lower (p=0.008) mutation burden than male rats (2.74e-004 ± 4.484e-005). Conclusion: Contrary to our hypothesis, mtDNA synthesis declined with age as mutation burden increased in the quadriceps. Because mtDNA copy number was not different between ages, while synthesis decreased, mtDNA turnover declined with age. Altogether, these results suggest lower mtDNA turnover rates may contribute to age-related mtDNA mutation burden. Future studies should test how modulating mtDNA turnover affects mutation frequency and mitochondrial function.