Isolation of mitochondrial DNA to investigate mitochondrial genome turnover in skeletal muscle

Authors

Kathryn Spry, Loyola Marymount UniversityFollow
Morgan DanielFollow
Clover D. Crenshaw, Loyola Marymount UniversityFollow
Robert Musci, Loyola Marymount UniversityFollow

Abstract

The quality of mitochondrial function is an essential part of cellular health. However, mutations occur within the cell during the metabolic processes of replication, repair, respiration and turnover. A build up of these mutations can cause mitochondrial dysfunction, which in turn leads to cell, tissue and eventually organismal dysfunction. Disease such as sarcopenia and cardiovascular disease are prime examples of the results of this process. Thus, better understanding mitochondrial DNA (mtDNA) turnover rates could contribute to a decreased risk of disease. PURPOSE: The purpose of this study was to 1) outline a procedure to isolate pure mtDNA from frozen skeletal muscle of the gastrocnemius in mice and 2) evaluate the mtDNA turnover fluctuations by differing the labeling period in 3 mo and 19 mo C57BI6/J male mice. METHODS: Using the stable isotope racer deuterium oxide, mtDNA synthesis was measured over 14 days to evaluate the mtDNA copy number and mutation deletion frequency. This was accomplished using digital PCR in the quadriceps muscle of 9 mo and 26 mo male and female OKC-HET rats. There was no nuclear DNA (nDNA) detectable by PCR, so the mtDNA isolation procedure was successful as it consistently yielded a purity greater than 95% mtDNA. Any sample with a purity below 85% mtDNA was excluded from analysis. RESULTS: Contamination of nDNA affects measured D₂O enrichment of mtDNA. Over a wide range of purity levels, there is a direct, linear relationship between mtDNA purity and measured mtDNA fraction. Time course analysis reveals that 3 mo and 19 mo mice have different rates of mtDNA turnover (r²>0.7 for each curve, p=0.007). 19 mo have a lower plateau indicating a smaller portion of fluxuating mtDNA. However, mtDNA half-life in the gastrocnemius remains the same between 3 mo and 19 mo male mice. MtDNA is slower and the half-life is longer in 26 mo than 9 mo OKC-HET rats. CONCLUSION: The procedure was capable of detecting deoxyribose using GCMS by successfully isolating mtDNA that was free of nDNA (in quantities

Recommended Citation

Spry, Kathryn; Daniel, Morgan; Crenshaw, Clover D.; and Musci, Robert (2025) "Isolation of mitochondrial DNA to investigate mitochondrial genome turnover in skeletal muscle," International Journal of Exercise Science: Conference Proceedings: Vol. 14: Iss. 5, Article 88.
Available at: https://digitalcommons.wku.edu/ijesab/vol14/iss5/88