Age-induced changes in skeletal muscle mitochondrial DNA synthesis, quantity, and quality in genetically unique rats

Abstract

Mitochondrial genomic integrity is a key element of physiological processes and health. Changes in the half-life of the mitochondrial genome are implicated in the generation and accumulation of age-induced mitochondrial DNA (mtDNA) mutations, which are implicated in skeletal muscle aging and sarcopenia. There are conflicting data on the half-life of mtDNA, and there is limited information on how aging affects half-life in skeletal muscle. We hypothesized that skeletal muscle mtDNA synthesis rates would decrease with age in both female and male rats concomitant with changes in mtDNA integrity reflected in mtDNA copy number and mutation frequency. We measured mitochondrial genome half-life using stable isotope labeling over a period of 14 days and assessed mtDNA copy number and deletion mutation frequency using digital PCR in the quadriceps muscle of 9-month-old and 26-month-old male and female OKC-HET rats. We found a significant age-related increase in mtDNA half-life, from 132 days at 9 months to 216 days at 26 months of age in OKC-HET quadriceps. Concomitant with the increase in mtDNA half-life, we found an age-related increase in mtDNA deletion mutation frequency in both male and female rats. Notably, 26-month-old female rats had a lower mutation frequency than male rats, and there were no changes in mtDNA copy number with sex, age, or mitochondrial genotype. These data reveal several key findings: (1) mtDNA turnover in rat skeletal muscle decreases with age, (2) mtDNA half-lives in skeletal muscle are approximately an order of magnitude longer than what is reported for other tissues, and (3) muscle mtDNA turnover differs significantly from the turnover of other mitochondrial macromolecules including components of the mitochondrial nucleoid. These findings provide insight into the factors driving age-induced mtDNA mutation accumulation, which contribute to losses of mitochondrial genomic integrity and may play a role in skeletal muscle dysfunction.

Keywords: Aging; Deuterium oxide; Mitochondrial DNA; Mutation; Rats; Skeletal muscle.

Fig. 1

OKC-HET rat model breeding scheme and experimental design. A Breeding scheme and mtDNA genotype for each cross in the OKC-HET rat model. BN, Brown-Norway; F344, Fischer 344; WKY, Wistar-Kyoto; LEW, Lewis. B Experimental design for mtDNA FSR and mtDNA molecular measures

Fig. 2

Nuclear DNA contamination affects measured D₂O enrichment of enriched mtDNA. Over a wide range of purity levels, there is a weak correlation between mtDNA purity and measured mtDNA fraction new (A). Using only samples with a purity of greater than 85% mtDNA by mass, no relationship was observed between purity and mtDNA fraction new (B). Data were analyzed as a simple linear regression

Fig. 3

mtDNA half-life is longer in aged OKC-HET rats. mtDNA FSR (A) is slower and half-life (B) is longer in 26-month-old OKC-HET rats compared to 9-month-old counterparts. N = 4–7 for each age and sex. The statistical method used was two-way ANOVA with rat age and sex as factors

Fig. 4

Quadriceps mtDNA copy number does not change with age in OKC-HET rats. Quadriceps mtDNA copy number is not different with sex or with age at 9 or 26 months in the OKC-HET rat. N = 19–20 for each age and sex. The statistical method used was a two-way ANOVA with rat age and sex as factors

Fig. 5

Correlation between quadriceps mtDNA copy number and other muscle masses. Quadriceps mtDNA copy number is related to other hindlimb muscle masses in 26-month-old male OKC-HET only. N = 18–19 for each age and muscle. Abbreviations: GA, gastrocnemius; PLA, plantaris; SOL, soleus; TA, tibialis anterior; EDL, extensor digitorum longus. Data were analyzed as simple linear regressions

Fig. 6

mtDNA deletion mutation frequency is greater in aged OKC-HET rats. mtDNA deletion mutation frequency on a logarithmic scale is greater in male compared to female OKC-HET rats and is greater in 26-month-old compared to 9-month-old rats. N = 19–20 for each age and sex. The statistical method used was two-way ANOVA with rat age and sex as factors