Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

Abstract

Key points• Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects.• The most commonly used markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I-V protein, and complex I-IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres).• Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content.• mtDNA was found to be a poor biomarker of mitochondrial content.• Complex IV activity was closely associated with mitochondrial oxidative phosphorylation capacity.Abstract Skeletal muscle mitochondrial content varies extensively between human subjects. Biochemical measures of mitochondrial proteins, enzyme activities and lipids are often used as markers of mitochondrial content and muscle oxidative capacity (OXPHOS). The purpose of this study was to determine how closely associated these commonly used biochemical measures are to muscle mitochondrial content and OXPHOS. Sixteen young healthy male subjects were recruited for this study. Subjects completed a graded exercise test to determine maximal oxygen uptake (V O 2 peak ) and muscle biopsies were obtained from the vastus lateralis. Mitochondrial content was determined using transmission electron microscopy imaging and OXPHOS was determined as the maximal coupled respiration in permeabilized fibres. Biomarkers of interest were citrate synthase (CS) activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I-V protein content, and complex I-IV activity. Spearman correlation coefficient tests and Lin's concordance tests were applied to assess the absolute and relative association between the markers and mitochondrial content or OXPHOS. Subjects had a large range ofV O 2 peak (range 29.9-71.6 ml min −1 kg −1 ) and mitochondrial content (4-15% of cell volume). Cardiolipin content showed the strongest association with mitochondrial content followed by CS and complex I activities. mtDNA was not related to mitochondrial content. Complex IV activity showed the strongest association with muscle oxidative capacity followed by complex II activity. We conclude that cardiolipin content, and CS and complex I activities are the biomarkers that exhibit the strongest association with mitochondrial content, while complex IV activity is strongly associated with OXPHOS capacity in human skeletal muscle.

Thermal acclimation and subspecies-specific effects on heart and brain mitochondrial performance in a eurythermal teleost (Fundulus heteroclitus)

“…We estimated mitochondrial quantity by measuring heart and brain CS activity (Fig. 8) (Larsen et al, 2012). Thermal acclimation exerted limited effects on CS activity when compared with our mitochondrial respiration data, and subspecies effects were the opposite in the heart (Figs 2-7).…”

“…We approximated changes in heart and brain mitochondrial quantity following thermal acclimation by measuring changes in citrate synthase (CS) activity (Srere, 1969;Larsen et al, 2012) in a different subset of fish than those used for mitochondrial assays. Tissues were frozen in liquid N 2 and stored at −80°C until they were ready to be assayed.…”

Variation in Metabolic Rate among Individuals Is Related to Tissue-Specific Differences in Mitochondrial Leak Respiration

“…Finally, cytochrome c oxidase (COX) activity per mass of tissue was measured in the presence of complex I inhibitor (rotenone) and complex III inhibitor (antimycin A) and after addition of ascorbate and N,N,N 0 ,N 0 -tetramethyl-p-phenylenediamine dihydrochloride (see appendix for full details of the titration protocol, oxygraph calibration, oxygen flux, and substrate concentrations). COX, an inner mitochondrial membrane enzyme involved in the electron transport chain, is a marker of mitochondrial content and is highly correlated to mitochondrial respiratory capacity (Larsen et al 2012). Oxygen consumption at each step in the protocol was averaged over several minutes after stabilization of the flux, and the mass-specific mitochondrial respiration rate was expressed in picomoles of oxygen per second per milligram wet weight of tissue (Pesta and Gnaiger 2012).…”

Exercise Intolerance and Myoglobinuria Associated with a Novel Maternally Inherited MT-ND1 Mutation

“…Serial frozen 10 mm sections of muscle tissue were stained with hematoxylin and eosin (H&E), G€ om€ ori trichrome, COX/ SDH, and ATPase. Mitochondrial enzyme activity in all muscle biopsies was measured at 37 C as previously described (Larsen et al 2012). …”