Voluntary running protects against neuromuscular dysfunction following hindlimb ischemia-reperfusion in mice

Abstract

Ischemia-reperfusion (IR) due to temporary restriction of blood flow causes tissue/organ damages under various disease conditions, including stroke, myocardial infarction, trauma, and orthopedic surgery. In the limbs, IR injury to motor nerves and muscle fibers causes reduced mobility and quality of life. Endurance exercise training has been shown to increase tissue resistance to numerous pathological insults. To elucidate the impact of endurance exercise training on IR injury in skeletal muscle, sedentary and exercise-trained mice (5 wk of voluntary running) were subjected to ischemia by unilateral application of a rubber band tourniquet above the femur for 1 h, followed by reperfusion. IR caused significant muscle injury and denervation at neuromuscular junction (NMJ) as early as 3 h after tourniquet release as well as depressed muscle strength and neuromuscular transmission in sedentary mice. Despite similar degrees of muscle atrophy and oxidative stress, exercise-trained mice had significantly reduced muscle injury and denervation at NMJ with improved regeneration and functional recovery following IR. Together, these data suggest that endurance exercise training preserves motor nerve and myofiber structure and function from IR injury and promote functional regeneration. NEW & NOTEWORTHY This work provides the first evidence that preemptive voluntary wheel running reduces neuromuscular dysfunction following ischemia-reperfusion injury in skeletal muscle. These findings may alter clinical practices in which a tourniquet is used to modulate blood flow.

Keywords: endurance exercise training; ischemia reperfusion; mitochondria; motor nerve; neuromuscular junction; oxidative stress; skeletal muscle.

Fig. 1.

Long-term voluntary running preserves neuromuscular function following ischemia-reperfusion (IR). To determine whether endurance exercise training provides protection against IR injury-mediated loss of neuromuscular function, sedentary (Sed) and exercise-trained (Ex) mice were subjected to IR, followed by measurements of muscle weight and muscle and nerve function 7 days after IR. A: peak isometric torque elicited by direct muscle stimulation before and during recovery from IR, and only statistical differences between Sed and Ex are indicated. B: peak isometric torque of plantar flexors elicited by nerve stimulation before and during recovery from IR. For simplicity, only statistical differences between sedentary and exercise are indicated. C: gastrocnemius muscle wet weight (mg) normalized to tibia length (mm) to account for differences in body size. D: torque-frequency relationship of muscle contractions elicited by direct muscle stimulation 7 days following IR. E: force-frequency relationship of muscle contractions elicited by nerve stimulation 7 days following IR (n = 6). Data are represented as means ± SD. *P < 0.05 and ***P < 0.001; n = 6.

Fig. 2.

Long-term voluntary running does not attenuate ischemia-reperfusion (IR)-induced oxidative stress in myofibers and motor nerve. We measured mitochondrial and whole cell markers of oxidative stress in sedentary (Sed) and exercise-trained (Ex) mice following IR. A: representative immunoblots and quantification of expression of antioxidant proteins superoxide dismutase (SOD)1, SOD2, and SOD3 and catalase normalized by actin in skeletal muscle (n = 5). B: representative immunoblot images and quantification of expression of antioxidant proteins SOD1, SOD2, and SOD3 and catalase normalized by actin in sciatic nerve (n = 5). C: representative confocal images and quantification of MitoTimer red:green ratio in skeletal muscle 3 h after IR. Scale, 25 μm (n = 4–7). D: representative confocal images and quantification of MitoTimer red/green ratio in sciatic nerve 3 h after IR. Scale, 25 μm (n = 4–7). E: representative immunoblot images and quantification of 4-hydroxynoneal (4-HNE) in skeletal muscle (n = 6). F: representative immunoblot images and quantification of 4-HNE in sciatic nerve (n = 6). Data are represented as means ± SD. *P < 0.05; **P < 0.01.

Fig. 3.

Long-term voluntary running renders myofibers resistant to ischemia-reperfusion (IR). Morphological and biochemical evaluations of muscle damage were conducted following IR. A: representative images of hematoxylin and eosin (H & E)-stained transverse sections of skeletal muscle 3 h after IR. Scale bar, 50 μm. B: serum creatine kinase activity 3 h after IR. C: representative images of H & E-stained transverse sections of skeletal muscle 7 days after IR. Scale bar, 50 μm. D: %total myofibers with centralized nuclei. Data are represented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001; n = 6. Sed, sedentary; Ex, exercise trained.

Fig. 4.

Long-term voluntary running attenuates denervation of skeletal muscle following ischemia-reperfusion (IR). To elucidate the consequence of endurance exercise training on skeletal muscle innervation, muscles were collected from sedentary (Sed) and exercise-trained (Ex) mice follwing IR, and innervation at neuromuscular junction (NMJ) and muscle denervation were measured using immunofluorescent techniques. A: representative confocal images of presynaptic motor neurons identified by Tuj1 (green) and postsynaptic acetylcholine receptors detected with α-bungarotoxin (red) and quantification of denervated NMJs 3 h after injury. Scale bars, 20 (top) and 5 μm (bottom), respectively (*P < 0.05 and ***P < 0.001; n = 8). B: representative confocal images of transverse sections of plantaris muscle expressing cytosolic Ncam (red) and laminin (green) and DAPI staining (blue) and quantification of %cytosolic Ncam⁺ myofibers 7 days following IR. Mice in the Sed group had a trend of increase toward significance (P = 0.053). Scale bar, 100 µm (n = 5). Data are represented as means ± SD.