Fatigue Resistance and Mitochondrial Adaptations to Isometric Interval Training in Dystrophin-Deficient Muscle: Role of Contractile Load

Abstract

In normal mouse skeletal muscles, interval training (IT)-mimicking neuromuscular electrical stimulation enhances muscle fatigue resistance and mitochondrial content, with greater gains observed at high (100 Hz stimulation, IT100) compared to low (20 Hz stimulation, IT20) contractile load. In this study, we compared the effects of repeated IT100 and IT20 on fatigue resistance and mitochondrial adaptations in young male mdx52 mice (4- to 6-week-old), an animal model for Duchenne muscular dystrophy. Plantar flexor muscles were stimulated in vivo using supramaximal electrical stimulation to induce isometric contractions every other day for 4 weeks (a total of 15 sessions). In non-trained muscles of mdx52 mice, decreased fatigue resistance was associated with reduced citrate synthase activity, lower peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) protein expression, and diminished levels of mitochondrial respiratory chain complex II, and an increased percentage of Evans Blue dye-positive areas. IT100, but not IT20, markedly improved fatigue resistance and restored all these alterations in mdx52 mice. Furthermore, an acute session of IT100, but not IT20, led to increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and elevated mRNA levels of PGC-1α, which were blocked by the p38 MAPK inhibitor SB203580. These findings suggest that contractile load is a key determinant of isometric IT-induced improvements in fatigue resistance, even in dystrophin-deficient muscles, potentially through a p38 MAPK/PGC-1α-mediated increase in mitochondrial content.

Keywords: contractile load; fatigue resistance; isometric interval training; mitochondria; muscular dystrophy.

FIGURE 1

Schematic overview of the experimental design. (A) In Experiment (Exp) 1, fatigue resistance and intracellular events were evaluated in WT and mdx52 mice with and without interval training (IT) at low contractile load (20 Hz stimulation, IT20) or high contractile load (100 Hz stimulation, IT100). IT was performed using electrical stimulation every other day for a total of 15 sessions. In Exp 2, cellular signaling underlying IT‐induced physiological adaptations was investigated after an acute single bout of IT20 or IT100 in mdx52 mice. In Exp 3, the mdx52 mice underwent a single bout of IT100 with or without the administration of the p38 MAPK inhibitor SB203580. Typical torque traces during an IT20 (B) and IT100 (C) session. The mean peak torque (D; n = 720 per group) and torque‐time integral (volume) (E, n = 120 per group) were calculated across all training sets and sessions for all mice. Data are presented as individual values and mean ± SD. Unpaired t‐test was performed. ^# p < 0.05 versus WT.

FIGURE 2

Isometric interval training with high, but not low, contractile load improves fatigue resistance in dystrophin deficient muscle. (A–D) Representative torque recording during the in vivo fatigue protocol (70 Hz, 350 ms tetani every 3 s) of the plantar flexor muscles from WT and mdx52 mice. The mdx52 mouse underwent isometric interval training (IT) with either low (IT20) or high (IT100) contractile load. (E) Mean (±SD) relative tetanic torque during fatiguing stimulation. The torque in the first tetanus was set to 100% for each muscle. Data are presented as mean ± SD for eight muscles per group. A two‐way repeated measures ANOVA with Tukey post hoc test was performed. *p < 0.05 versus WT, ^# p < 0.05 versus mdx52, ^‡ p < 0.05 versus mdx52+IT20. (F) Maximum isometric torque. Data are presented as mean ± SD for eight muscles per group. A Kruskal–Wallis one‐way ANOVA was used on ranks. *p < 0.05 versus WT.

FIGURE 3

Isometric interval training with high, but not low, contractile load abolishes EBD positive fibers in dystrophin‐deficient muscles. Representative images of transverse sections of medial gastrocnemius muscles stained for hematoxylin and eosin (H&E) and Evans Blue dye (EBD) of gastrocnemius muscles in WT and mdx52 mice. The mdx52 mouse underwent isometric interval training with either high (IT100) or low contractile load (IT20) (A). Scale bars 1 mm. The percentage of the EBD‐positive area over the total cross‐sectional muscle area (B). Data show mean ± SD for eight muscles per group. A Kruskal–Wallis one‐way ANOVA was used on ranks. *p < 0.05 versus WT, ^# p < 0.05 versus mdx52, ^‡ p < 0.05 versus mdx52+IT20.

FIGURE 4

Effects of isometric interval training with high, but not low, contractile load on the levels of proteins involved in membrane integrity and repair. Representative Stain‐Free (S‐F) images and western blots illustrating the levels of utrophin (A), integrin α7B (C), integrin β1D (E), β‐dystroglycan (DG) (G), α‐sarcoglycan (SG) (I), and dysferlin (K) in gastrocnemius muscles in WT and mdx52 mice. The mdx52 mouse underwent isometric interval training with either high (IT100) or low contractile load (IT20). The levels of utrophin (B), integrin α7B (D), integrin β1D (F), β‐DG (H), α‐SG (J), and dysferlin (L) were normalized to total protein in S‐F images. Data show mean ± SD for 6–eight muscles per group. One‐way ANOVA with Tukey post hoc test was performed. *p < 0.05 versus WT, ^# p < 0.05 versus mdx52.

FIGURE 5

Isometric interval training with high, but not low, contractile load increases mitochondrial content and respiratory complexes in dystrophin‐deficient muscles. (A) Blots showing electrophoretically separated myosin heavy chain (MyHC) isoforms in the gastrocnemius muscles of each group. (B) Distribution of MyHC isoforms. Citrate synthase (CS) activity (C) in plantaris muscles from WT and mdx52 mice. The mdx52 mouse underwent isometric interval training with either high (IT100) or low contractile load (IT20). Representative Stain‐Free (S‐F) images and western blots illustrating the levels of peroxisome proliferator activated receptor γ coactivator 1 alpha (PGC‐1α) (D), complex I (CI) subunit (NDUFB8), CII subunit (SDHB), CIII subunit (UQCRC), CIV subunit (MTC01), CV subunit (ATP5) (F) in gastrocnemius muscles. The levels of PGC‐1α (E) and CI‐V (G) were normalized to total protein in S‐F images. Data show mean ± SD for six to eight muscles per group. One‐way ANOVA with Tukey post hoc test was performed, except for the distribution of the MyHC isoforms, where a Kruskal–Wallis one‐way ANOVA was used on ranks. *p < 0.05 versus WT, ^# p < 0.05 versus mdx52, ^‡ p < 0.05 versus mdx52+IT20.

FIGURE 6

The phosphorylation levels of MAPK are not chronically elevated after isometric interval training with high and low contractile loads. Representative Stain‐Free (S‐F) images and western blots of total and phosphorylated p38 MAPK Thr180/Tyr182 (A), ERK1/2 Thr202/Tyr204 (C), and JNK Thr183/Tyr185 (E) of gastrocnemius muscles in WT and mdx52 mice. The mdx52 mouse underwent isometric interval training with either high (IT100) or low contractile load (IT20). The phosphorylation levels of p38 MAPK Thr180/Tyr182 (B), ERK1/2 Thr202/Tyr204 (D), and JNK Thr183/Tyr185 (F) relative to total protein content. Data show mean ± SD for six to seven muscles per group. One‐way ANOVA with Tukey post hoc test was performed. *p < 0.05 versus WT, ^# p < 0.05 versus mdx52, ^‡ p < 0.05 versus mdx52+IT20.

FIGURE 7

An acute bout of isometric interval training with high, but not low, contractile load increases the phosphorylation levels of p38 MAPK. Representative Stain‐Free (S‐F) images and western blots of total and phosphorylated p38 MAPK Thr180/Tyr182 (A), ERK1/2 Thr202/Tyr204 (C), and JNK Thr183/Tyr185 (E), and AMPK Thr172 (G) of gastrocnemius muscles in WT and mdx52 mice. The mdx52 mouse underwent a single bout of isometric interval training with either high (IT100) or low contractile load (IT20) (Exp 2). The phosphorylation levels of p38 MAPK Thr180/Tyr182 (B), ERK1/2 Thr202/Tyr204 (D), and JNK Thr183/Tyr185 (F), and AMPK Thr172 (H) relative to total protein content. Data show mean ± SD for six muscles per group. One‐way ANOVA with Tukey post hoc test was performed, except for the phosphorylation levels of ERK1/2 Thr202/Tyr204 relative to total protein content, where a Kruskal–Wallis one‐way ANOVA was used on ranks. *p < 0.05 versus WT, ^# p < 0.05 versus mdx52.

FIGURE 8

p38 MAPK inhibitor prevents the increase in PGC‐1α mRNA induced by an acute bout of isometric interval training with high contractile load. Representative western blots of total and phosphorylated p38 MAPK Thr180/Tyr182 (A) of gastrocnemius muscles in WT and mdx52 mice. The mdx52 mouse underwent a single bout of isometric interval training with high contractile load (IT100) with or without the administration of SB203580, a p38 MAPK inhibitor (Exp 3). The phosphorylation levels of p38 MAPK Thr180/Tyr182 relative to total protein content (B). The mRNA levels of PGC‐1α (C). Data show mean ± SD for six muscles per group. Two‐way ANOVA with Tukey post hoc test was performed. *p < 0.05 versus mdx52, ^# p < 0.05 versus mdx52+SB203580, ^‡ p < 0.05 versus mdx52+IT100.