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The regeneration of injured muscles is facilitated by intermittent heat stress. The 72-kDa heat shock protein (HSP72), the level of which is increased by heat stress, is likely involved in this effect, but the precise mechanism remains unclear. This study was conducted to investigate the localization and role(s) of HSP72 in the regenerating muscles in heat-stressed rats using immunohistochemistry. Heat stress was applied by immersion of the rat lower body into hot water (42C, 30 min, every other day) following injection of bupivacaine into the soleus muscles. After 1 week, we found that HSP72 was expressed at high levels not only in the surviving myofibers but also in the blood vessels of the regenerating muscles in heated rats. In addition, leukocytes, possibly granulocytes, expressing cluster of differentiation 43 within the blood capillaries surrounding the regenerating myofibers also highly expressed HSP72. In contrast, marked expression of HSP72 was not observed in the intact or regenerating muscles without heat stress. These results suggest that heat-stress-induced HSP72 within the myofibers, blood vessels, and circulating leukocytes may play important roles in enhancing regeneration of injured muscles by heat stress. Our findings would be useful to investigate cell-specific role(s) of HSP72 during skeletal muscle regeneration.

The effects of 3 months of spaceflight (SF), hindlimb suspension, or exposure to 2G on the characteristics of neck muscle in mice were studied. Three 8-week-old male C57BL/10J wild-type mice were exposed to microgravity on the International Space Station in mouse drawer system (MDS) project, although only one mouse returned to the Earth alive. Housing of mice in a small MDS cage (11.6 × 9.8-cm and 8.4-cm height) and/or in a regular vivarium cage was also performed as the ground controls. Furthermore, ground-based hindlimb suspension and 2G exposure by using animal centrifuge (n = 5 each group) were performed. SF-related shift of fiber phenotype from type I to II and atrophy of type I fibers were noted. Shift of fiber phenotype was related to downregulation of mitochondrial proteins and upregulation of glycolytic proteins, suggesting a shift from oxidative to glycolytic metabolism. The responses of proteins related to calcium handling, myofibrillar structure, and heat stress were also closely related to the shift of muscular properties toward fast-twitch type. Surprisingly, responses of proteins to 2G exposure and hindlimb suspension were similar to SF, although the shift of fiber types and atrophy were not statistically significant. These phenomena may be related to the behavior of mice that the relaxed posture without lifting their head up was maintained after about 2 weeks. It was suggested that inhibition of normal muscular activities associated with gravitational unloading causes significant changes in the protein expression related to metabolic and/or morphological properties in mouse neck muscle.

Effect of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.

Effects of gravitational loading or unloading on the growth-associated increase in the cross-sectional area and length of fibers, as well as the total fiber number, in soleus muscle were studied in rats. Furthermore, the roles of satellite cells and myonuclei in growth of these properties were also investigated. The hindlimb unloading by tail suspension was performed in newborn rats from postnatal day 4 to month 3 with or without 3-mo reloading. The morphological properties were measured in whole muscle and/or single fibers sampled from tendon to tendon. Growth-associated increases of soleus weight and fiber cross-sectional area in the unloaded group were approximately 68% and 69% less than the age-matched controls. However, the increases of number and length of fibers were not influenced by unloading. Growth-related increases of the number of quiescent satellite cells and myonuclei were inhibited by unloading. And the growth-related decrease of mitotically active satellite cells, seen even in controls (20%, P > 0.05), was also stimulated (80%). The increase of myonuclei during 3-mo unloading was only 40 times vs. 92 times in controls. Inhibited increase of myonuclear number was not related to apoptosis. The size of myonuclear domain in the unloaded group was less and that of single nuclei, which was decreased by growth, was larger than controls. However, all of these parameters, inhibited by unloading, were increased toward the control levels generally by reloading. It is suggested that the satellite cell-related stimulation in response to gravitational loading plays an essential role in the cross-sectional growth of soleus muscle fibers.