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

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 (VO2peak) 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 of VO2peak (range 29.9–71.6ml 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.

Figures

Figure 1. Representative electron microscopy images and western blots of complex I–V

Images and blots are from the two subjects with the lowest mitochondrial content (A) and second highest mitochondrial content (B). C, representative image used for measuring cristae surface area. A and B are ×20,000 magnification; scale bars are 1 μm. C is ×80,000 magnification and scale bar is 200 nm. Arrows shows examples of outer membrane and cristae.

Figure 2. Fibre respiration

Oxygen consumption (pmol s−1 (mg ww)−1) in permeabilized fibres. GM3: glutamate + malate + ADP-Mg; GM3u: glutamate + malate + ADP-Mg + FCCP; GMS3: glutamate + malate + ADP-Mg + succinate; GMS3u: glutamate + malate + ADP-Mg + succinate + FCCP; Oct3: l-malate + octanoyl-l-carnitine + ADP-Mg; Oct3u: malate + octanoyl-l-carnitine + ADP-Mg + FCCP; TMPD: ADP-Mg + cytochrome c+ antimycin A + ascorbate +N,N,N′,N′-tetramethyl-1,4-benzenediamine dihydrochloride; PGM3: pyruvate + malate + glutamate + ADP-Mg; PGMS3: pyruvate + glutamate + malate + ADP-Mg + succinate; PGMSOct3: pyruvate + glutamate + malate + ADP-Mg + succinate + octanoyl-l-carnitine. Numbers denote the statistical rank using one-way ANOVA with repeated measures, i.e. values denoted with the number 3 are significantly lower than values denoted with the number 2 and significantly higher than the values denoted with the number 4. *P= 0.06 compared with GM3u. Values are mean ± SEM.

Figure 3. Correlation plots

Correlation between the relative variation of the mitochondrial content and the six biomarkers (A–F) of mitochondrial content that showed the highest Lin's concordance coefficient (Rc) and two biomarkers of certain interest (G and H). TMPD + ascorbate are redox substrates feeding electrons directly to complex IV. Continuous lines represent the perfect linear fit (slope = 1) and dashed lines represent the actual linear fit. The linear fit was only shown when significance was present.