A stable isotope method for measuring mitochondrial DNA synthesis in vivo in rodents and humans

Mitochondrial mass is affected by internal (physiologic) and external (i.e. pharmacologic) factors. However, the nature and regulation of mitochondrial biogenesis remains largely unknown. To begin to investigate this area, a stable isotope technique was developed for measuring mitochondrial DNA (mtDNA) synthesis in vivo using 2H2O, that can be potentially applied to humans.; Several results of potential importance emerged from the studies described here. The normal turnover (replacement) rate of mitochondria from rat cardiac and skeletal muscle was established. Comparison of labeling studies from young male rats and mature female rats served as models for measuring mtDNA synthesis in settings that included somatic growth and mtDNA replacement independent of growth. Die-away curves from de-labeling studies produced similar kinetics as label incorporation curves. MtDNA from human platelets produced a label incorporation curve over 10 weeks, reaching a plateau at approximately 5% enrichment by week 5.; Mitochondrial biogenesis was studied in response to a training stimulus in female Sprague Dawley rats. The response of mtDNA synthesis to treadmill training for 1--6 weeks was compared to sedentary controls. MtDNA synthesis and absolute biogenesis from trained rats increased by 2-fold in cardiac and skeletal muscle. Cytochrome c oxidase content increased 4-fold in trained rats compared to sedentary controls. Body weights were not significantly different between training and control groups.; Mitochondrial DNA synthesis responses to antiretroviral therapy with nucleoside reverse transcriptse inhibitors (NRTI' S) in rodents and humans were also measured. Neither body weight or food intake was affected by NRTI therapy in rodents. MtDNA synthesis decreased after 4 weeks of NRTI therapy compared to controls. Cytochrome C oxidase levels were decreased almost 2-fold in NRTI rats compared to controls. Fractional synthesis of platelet mtDNA synthesis from human subjects on antiretroviral therapy were decreased approximately 50% compared to values from normal healthy human controls.; In conclusion, this method for measuring mtDNA synthesis may provide insights into various effects on mitochondrial biogenesis of numerous factors including, inherited mitochondrial genetic disorders and testing of drugs with putative effects on mitochondria. Increased mtDNA proliferation observed in treadmill trained rats may prove useful as an objective biomarker of tissue oxidative demand and oxidative capacity. Values of mtDNA proliferation observed in NRTI-treated rats and humans may be used as a measure of mitochondrial toxicity of therapeutic drugs.