Resistance Training Alters Mitochondrial Function in Muscle
Eleven young (26 ± 5 yr) healthy men participated in the current study. Basic subject characteristics before and after RET are presented in Table 1. Total body mass increased by 1.9 ± 2 kg after 12 wk of RET (P < 0.01). Body mass index also increased significantly with RET (P < 0.01). Subjects gained a significant amount (2.5 ± 1.4 kg) of fat-free mass with RET (P < 0.001), whereas absolute fat mass was not significantly different before and after RET. Subsequently, there was a significant increase in the relative proportion of fat-free mass (P < 0.05) and a significant decrease in the relative proportion of fat mass (P < 0.05) after RET. Muscle thickness data before and after RET are presented in Table 1. RET significantly increased muscle thickness of the VL by 12% (P < 0.001). Although habitual physical activity and dietary habits were not determined in the current study, it is likely that participants increased their dietary caloric and nitrogen intake, as indicated by the increase in body mass and fat-free mass after RET.
Muscle strength data before and after RET are presented in Table 2. RET significantly increased the isometric peak torque of both the quadriceps (P < 0.001) and biceps femoris (P < 0.001) muscle groups of the leg. Isokinetic peak torque of the quadriceps muscles also increased with RET (P < 0.001). The increase in isokinetic peak torque of the biceps femoris muscle group after RET did not reach statistical significance.
Mitochondrial respiratory capacity per milligram wet weight of muscle tissue was increased by RET (Fig. 1). State 2 leak respiration (LI) was increased twofold by RET (10.7 ± 1.5 vs 21.8 ± 3.2 pmol·s·mg; P < 0.05). Similarly, coupled state 3 respiration with electron input from complex I (PI) increased twofold after RET (30.2 ± 2.8 vs 58.9 ± 3.5 pmol·s·mg; P < 0.001). Maximal coupled (oxphos) respiration with electron input from both complex I and II of the electron transport chain (PI+II) increased 1.4-fold after RET (54.5 ± 6.5 vs 75.6 ± 5.8 pmol·s·mg; P < 0.05). The addition of cyt C had little effect on respiratory flux, suggesting that the muscle biopsy procedure and the preparation of permeabilized myofiber bundles did no damage to the outer mitochondrial membrane. Addition of 10 μM of cyt C increased respiration by 2.1% ± 1.5% on average before RET and 1.6% ± 1.0% on average after RET. Maximal uncoupled mitochondrial respiration, a marker of mitochondrial respiratory capacity, was achieved by titration of the ionophore CCCP. RET significantly increased electron transfer system capacity (E) by 65% (64.0 ± 5.1 vs 104.4 ± 9.8 pmol·s·mg; P < 0.001).
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Figure 1.
Skeletal muscle mitochondrial respiration. Skeletal muscle mitochondrial respiration in MIR05 buffer alone (basal), after the addition of pyruvate, octanoyl-L-carnitine, glutamate and malate (LI), ADP (PI), succinate (PI+II), cyt C, and CCCP (E), before (circles) and after (triangles) 12 wk of RET. L and P denote leak and phosphorylating respiratory states, respectively. I and II indicate electron transfer through complex I and complex II of the electron transport chain, respectively. The absence in a respiratory response to cyt C indicates that mitochondria within myofiber bundles were viable. *P < 0.05. **P < 0.01. ***P < 0.001.
Skeletal muscle CS activity, a marker of oxidative capacity, is presented in Figure 2A. RET tended to increase CS activity in the skeletal muscle (56.1 ± 4.5 vs 65.8 ± 5.4 (μmol·g·min/(mg protein)). However, this 17% increase was not statistically significant. CS activity was not correlated with maximal coupled mitochondrial respiration (P) before (Fig. 2B) or after (Fig. 2C) RET. Maximal respiratory capacity (E) correlated with maximal coupled respiration (P) both before (R = 0.96, P < 0.001) (Fig. 2D) and after (R = 0.92, P < 0.001) (Fig. 2E) RET.
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Figure 2.
Skeletal muscle oxidative capacity. Skeletal muscle CS activity (A) before (black bars) and after (gray bars) RET. There was no correlation between CS and PI+II before (B) or after (C) RET. There was a significant correlation between PI+II and E before (D) and after (E) RET.
Skeletal muscle mitochondrial respiration values were normalized to maximal uncoupled respiration (E) to control for mitochondrial respiratory capacity (Fig. 3A). Leak respiration with complex I substrates (LI) normalized to E was not altered by RET (0.17 ± 0.02 vs 0.22 ± 0.03). Coupled respiration supported by complex I (PI) was significantly increased by RET when normalized to E (0.48 ± 0.04 vs 0.60 ± 0.04, P < 0.05). Coupled respiration supported by both complex I and II was not altered by RET when normalized to E (0.83 ± 0.05 vs 0.73 ± 0.03). The RCR for ADP was not different after RET (3.2 ± 0.4 vs 3.5 ± 0.6) (Fig. 3B). The substrate control ratio (SCR) for succinate was significantly reduced after RET (1.8 ± 0.1 vs 1.3 ± 0.1; P < 0.001) (Fig. 3C).
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Figure 3.
Specific mitochondrial respiration and coupling control. Skeletal muscle mitochondrial respiration normalized to respiratory capacity (E) before and after RET (A). B, The RCR for ADP calculated as PI/LI. C, The SCR for succinate calculated as PI+II/PI. *P <0.05. ***P <0.001.
The protein abundances of complex I to IV of the electron transport chain and ATP synthase (complex V) are presented in Figure 4. Complex II, III, and IV protein expression was not different after RET. Similarly, complex V protein expression was unaltered by RET. The protein expression of complex I of the electron transport chain (NADH oxidase) increased by 11% with RET (P = 0.054).
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Figure 4.
Altered mitochondrial gene expression and protein abundance after RET. A, Mitochondrial respiratory chain protein levels determined in cytoplasmic skeletal muscle lysates before (black bars) and after (gray bars) 12 wk of RET. I, II, III, IV, and V represent complex I (NADH oxidase), complex II (succinate dehydrogenase), complex III (cytochrome oxidoreductase, complex IV (cytochrome C oxidase), and complex V (ATP synthase) of the respiratory chain. B, Representative blot images of the human heart mitochondria isolate internal control and a pre- and postsample (loaded in duplicate). C, Changes in RNA levels of COX18, COX4I1, MFN1, NAMPT, NDRG2, PGC1α, and TFAM before and after RET. *P <0.05.
The mRNA expression of COX18, MFN1, NDRG2, peroxisome proliferator-activated receptor gamma coactivator (PGC1α), and TFAM was unaltered by RET, whereas the mRNA expression of COX4I1 and NAMPT was significantly increased after RET (P < 0.05) (Fig. 4C).
Results
Subject Characteristics
Eleven young (26 ± 5 yr) healthy men participated in the current study. Basic subject characteristics before and after RET are presented in Table 1. Total body mass increased by 1.9 ± 2 kg after 12 wk of RET (P < 0.01). Body mass index also increased significantly with RET (P < 0.01). Subjects gained a significant amount (2.5 ± 1.4 kg) of fat-free mass with RET (P < 0.001), whereas absolute fat mass was not significantly different before and after RET. Subsequently, there was a significant increase in the relative proportion of fat-free mass (P < 0.05) and a significant decrease in the relative proportion of fat mass (P < 0.05) after RET. Muscle thickness data before and after RET are presented in Table 1. RET significantly increased muscle thickness of the VL by 12% (P < 0.001). Although habitual physical activity and dietary habits were not determined in the current study, it is likely that participants increased their dietary caloric and nitrogen intake, as indicated by the increase in body mass and fat-free mass after RET.
Muscle Strength
Muscle strength data before and after RET are presented in Table 2. RET significantly increased the isometric peak torque of both the quadriceps (P < 0.001) and biceps femoris (P < 0.001) muscle groups of the leg. Isokinetic peak torque of the quadriceps muscles also increased with RET (P < 0.001). The increase in isokinetic peak torque of the biceps femoris muscle group after RET did not reach statistical significance.
Skeletal Muscle Mitochondrial Respiration
Mitochondrial respiratory capacity per milligram wet weight of muscle tissue was increased by RET (Fig. 1). State 2 leak respiration (LI) was increased twofold by RET (10.7 ± 1.5 vs 21.8 ± 3.2 pmol·s·mg; P < 0.05). Similarly, coupled state 3 respiration with electron input from complex I (PI) increased twofold after RET (30.2 ± 2.8 vs 58.9 ± 3.5 pmol·s·mg; P < 0.001). Maximal coupled (oxphos) respiration with electron input from both complex I and II of the electron transport chain (PI+II) increased 1.4-fold after RET (54.5 ± 6.5 vs 75.6 ± 5.8 pmol·s·mg; P < 0.05). The addition of cyt C had little effect on respiratory flux, suggesting that the muscle biopsy procedure and the preparation of permeabilized myofiber bundles did no damage to the outer mitochondrial membrane. Addition of 10 μM of cyt C increased respiration by 2.1% ± 1.5% on average before RET and 1.6% ± 1.0% on average after RET. Maximal uncoupled mitochondrial respiration, a marker of mitochondrial respiratory capacity, was achieved by titration of the ionophore CCCP. RET significantly increased electron transfer system capacity (E) by 65% (64.0 ± 5.1 vs 104.4 ± 9.8 pmol·s·mg; P < 0.001).
(Enlarge Image)
Figure 1.
Skeletal muscle mitochondrial respiration. Skeletal muscle mitochondrial respiration in MIR05 buffer alone (basal), after the addition of pyruvate, octanoyl-L-carnitine, glutamate and malate (LI), ADP (PI), succinate (PI+II), cyt C, and CCCP (E), before (circles) and after (triangles) 12 wk of RET. L and P denote leak and phosphorylating respiratory states, respectively. I and II indicate electron transfer through complex I and complex II of the electron transport chain, respectively. The absence in a respiratory response to cyt C indicates that mitochondria within myofiber bundles were viable. *P < 0.05. **P < 0.01. ***P < 0.001.
Skeletal Muscle Oxidative Capacity
Skeletal muscle CS activity, a marker of oxidative capacity, is presented in Figure 2A. RET tended to increase CS activity in the skeletal muscle (56.1 ± 4.5 vs 65.8 ± 5.4 (μmol·g·min/(mg protein)). However, this 17% increase was not statistically significant. CS activity was not correlated with maximal coupled mitochondrial respiration (P) before (Fig. 2B) or after (Fig. 2C) RET. Maximal respiratory capacity (E) correlated with maximal coupled respiration (P) both before (R = 0.96, P < 0.001) (Fig. 2D) and after (R = 0.92, P < 0.001) (Fig. 2E) RET.
(Enlarge Image)
Figure 2.
Skeletal muscle oxidative capacity. Skeletal muscle CS activity (A) before (black bars) and after (gray bars) RET. There was no correlation between CS and PI+II before (B) or after (C) RET. There was a significant correlation between PI+II and E before (D) and after (E) RET.
Skeletal Muscle Mitochondrial Function
Skeletal muscle mitochondrial respiration values were normalized to maximal uncoupled respiration (E) to control for mitochondrial respiratory capacity (Fig. 3A). Leak respiration with complex I substrates (LI) normalized to E was not altered by RET (0.17 ± 0.02 vs 0.22 ± 0.03). Coupled respiration supported by complex I (PI) was significantly increased by RET when normalized to E (0.48 ± 0.04 vs 0.60 ± 0.04, P < 0.05). Coupled respiration supported by both complex I and II was not altered by RET when normalized to E (0.83 ± 0.05 vs 0.73 ± 0.03). The RCR for ADP was not different after RET (3.2 ± 0.4 vs 3.5 ± 0.6) (Fig. 3B). The substrate control ratio (SCR) for succinate was significantly reduced after RET (1.8 ± 0.1 vs 1.3 ± 0.1; P < 0.001) (Fig. 3C).
(Enlarge Image)
Figure 3.
Specific mitochondrial respiration and coupling control. Skeletal muscle mitochondrial respiration normalized to respiratory capacity (E) before and after RET (A). B, The RCR for ADP calculated as PI/LI. C, The SCR for succinate calculated as PI+II/PI. *P <0.05. ***P <0.001.
Electron Transport Chain Protein Expression
The protein abundances of complex I to IV of the electron transport chain and ATP synthase (complex V) are presented in Figure 4. Complex II, III, and IV protein expression was not different after RET. Similarly, complex V protein expression was unaltered by RET. The protein expression of complex I of the electron transport chain (NADH oxidase) increased by 11% with RET (P = 0.054).
(Enlarge Image)
Figure 4.
Altered mitochondrial gene expression and protein abundance after RET. A, Mitochondrial respiratory chain protein levels determined in cytoplasmic skeletal muscle lysates before (black bars) and after (gray bars) 12 wk of RET. I, II, III, IV, and V represent complex I (NADH oxidase), complex II (succinate dehydrogenase), complex III (cytochrome oxidoreductase, complex IV (cytochrome C oxidase), and complex V (ATP synthase) of the respiratory chain. B, Representative blot images of the human heart mitochondria isolate internal control and a pre- and postsample (loaded in duplicate). C, Changes in RNA levels of COX18, COX4I1, MFN1, NAMPT, NDRG2, PGC1α, and TFAM before and after RET. *P <0.05.
Select Mitochondrial mRNA Expression
The mRNA expression of COX18, MFN1, NDRG2, peroxisome proliferator-activated receptor gamma coactivator (PGC1α), and TFAM was unaltered by RET, whereas the mRNA expression of COX4I1 and NAMPT was significantly increased after RET (P < 0.05) (Fig. 4C).
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