Intrinsic aerobic capacity correlates with greater inherent mitochondrial oxidative and H2O2 emission capacities without major shifts in myosin heavy chain isoform

Abstract

Exercise capacity and performance strongly associate with metabolic and biophysical characteristics of skeletal muscle, factors that also relate to overall disease risk. Despite its importance, the exact mechanistic features that connect aerobic metabolism with health status are unknown. To explore this, we applied artificial selection of rats for intrinsic (i.e., untrained) aerobic treadmill running to generate strains of low- and high-capacity runners (LCR and HCR, respectively), subsequently shown to diverge for disease risk. Concurrent breeding of LCR and HCR per generation allows the lines to serve as reciprocal controls for unknown environmental changes. Here we provide the first direct evidence in mitochondria isolated from skeletal muscle that intrinsic mitochondrial capacity is higher in HCR rats. Maximal phosphorylating respiration was ~40% greater in HCR mitochondria, independent of substrate and without altered proton leak or major changes in protein levels or muscle fiber type, consistent with altered control of phosphorylating respiration. Unexpectedly, H(2)O(2) emission was ~20% higher in HCR mitochondria, due to greater reduction of more harmful reactive oxygen species to H(2)O(2); indeed, oxidative modification of mitochondrial proteins was lower. When the higher mitochondrial yield was considered, phosphorylating respiration and H(2)O(2) emission were 70-80% greater in HCR muscle. Greater capacity of HCR muscle for work and H(2)O(2) signaling may result in enhanced and more immediate cellular repair, possibly explaining lowered disease risks.

Fig. 1.

Whole body characteristics of low and high capacity runners (LCR and HCR, respectively). A: for phenotyping, rats ran on a treadmill 3 times until exhaustion, on 3 separate days, 1 day apart. An improvement in running performance across session was evident in HCR only; ***P < 0.001, two-way ANOVA, Bonferroni post hoc test, vs. HCR trial 1. For each trial, P < 0.001, HCR vs. LCR; n = 12/group. Some error bars are within the symbols. B: body and organ weights. gWAT, gonadal white adipose tissue. Right: organ weights/body weight. **P = 0.01; ***P < 0.001, unpaired Student's t-test; n = 12/group. ○: LCR; ●: HCR. Values are means ± SE.

Fig. 2.

Increased maximal phosphorylating O₂ consumption in skeletal muscle mitochondria from HCR. A: mitochondrial content, as yield of mitochondria from the isolation procedure, per wet muscle weight. ***P < 0.001, unpaired Student's t-test; n = 9/group. B: maximal phosphorylating (state 3) and nonphosphorylating (state 4) respiration with pyruvate/malate (5 mM/2.5 mM; n = 6/group) or palmitoyl-l-carnitine/malate (20 μM/1 mM; n = 4/group) as substrates. RCR, respiratory control ratio (state 3/state 4); SDHA, succinate dehydrogenase subunit A; COX-I, cyclooxygenase-I.**P = 0.02; ***P = 0.005: unpaired Student's t-test. In all cases, 0.2 mg mitochondrial protein/ml were used. C: expression of electron transport chain proteins and of the adenine nucleotide translocase (ANT). In all cases, 20 μg of mitochondrial protein/lane were used. Left: representative blots; right: quantification by densitometry, relative to Ponceau stained blot or complex I (39-kDa protein). *P = 0.05, unpaired t-test; n = 6/group. Values are means ± SE.

Fig. 3.

Higher H₂O₂ emission in skeletal muscle mitochondria from HCR. A: H₂O₂ emission from mitochondria oxidizing palmitoyl-l-carnitine (Pcarn; 18 μM + 5 μM antimycin), or pyruvate/malate (P/M, 5 mM/2.5 mM) + 5 μM rotenone, or succinate alone (10 mM). Values are means ± SE; n = 6 rats/group. **P = 0.03, unpaired t-test. In all cases, 0.3 mg mitochondrial protein/ml were used. Rates were calculated from the linear portion of the curves. B: expression of antioxidant proteins in mitochondria. Top: representative Western blots; bottom: values are means ± SE; n = 6 rats/group, relative to complex I (39-kDA protein). In all cases, 20 μg of mitochondrial protein/lane were used. SOD2, superoxide dismutase2, UCP3: uncoupling protein 3; Gpx4, glutathione peroxidase4; Hex II, hexokinase II; a.u., arbitrary units. **P = 0.03, unpaired t-test. C: proton leak kinetics. Values are means ± SE; n = 7 rats/group. Oxygen consumption (y-axis) and proton motive force (x-axis), measured in parallel under the same conditions (37°C; 0.2 mg protein/ml, 5 μM rotenone, 0.4 μg/ml nigericin, 10 mM succinate, and 1 mM malonate titrations). D: oxidative stress, measured as the extent of 4-hydroxynonenal (4-HNE) protein modification by Western blot. Left: representative blot; right: quantification. **P < 0.01.

Fig. 4.

Myosin heavy chain isoform proportion and fiber size in skeletal muscle from LCR and HCR. A: representative histology. Images were taken at the same magnification and illumination. B: proportion of myosin heavy chain isoforms. *P = 0.05; **P = 0.03; ***P = 0.007, unpaired Student's t-test. C: fiber size for selected fiber types and muscles. **P = 0.03; ***P = 0.008, unpaired Student's t-test. Values are means ± SE; n = 5–6 rats/group.