Heat stress facilitates the regeneration of injured skeletal muscle in rats

J Orthop Sci (2007) 12:74–82 DOI 10.1007/s00776-006-1083-0

Original article Heat stress facilitates the regeneration of injured skeletal muscle in rats Atsushi Kojima1,2, Katsumasa Goto2,3, Shigeta Morioka1,2, Toshihito Naito1,2, Tatsuo Akema2, Hiroto Fujiya4, Takao Sugiura5, Yoshinobu Ohira6, Moroe Beppu1, Haruhito Aoki1, and Toshitada Yoshioka2,7 1

Department of Orthopaedic Surgery, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki 216-8511, Japan Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Japan 3 Laboratory of Physiology, Toyohashi Sozo University, Toyohashi, Japan 4 Department of Sports Medicine, St. Marianna University School of Medicine, Kawasaki, Japan 5 Faculty of Education, Yamaguchi University, Yamaguchi, Japan 6 Graduate School of Medicine, Osaka University, Osaka, Japan 7 Hirosaki Gakuin University, Hirosaki, Japan 2

Abstract Background. Skeletal muscle stem cells, so-called muscle satellite cells, are responsible for the repair and the regeneration of adult skeletal muscle tissues. Heat stress can facilitate the proliferation and the differentiation of myoblasts in vitro and can enhance their proliferative potential, which may stimulate the regrowth of atrophied skeletal muscle. The purpose of this study was to investigate the effect of heat stress on the regeneration of skeletal muscle injury induced by cardiotoxin. Methods. Male Wistar rats, aged 7 weeks, were randomly divided into six groups: a nonheated control group that received a physiological saline injection, a group heat stressed before physiological saline injection, a group heat stressed after physiological saline injection, a group injected with cardiotoxin without heat stress, a group heat stressed before cardiotoxin injection, and a group heat stressed after cardiotoxin injection (25 in each group). To initiate muscle injury and regeneration, 0.5 ml of 10 µM cardiotoxin was injected into the left tibialis anterior muscle. Conscious rats in some groups were exposed to environmental heat stress (41°C for 60 min) in a heat chamber 24 h before or immediately after cardiotoxin or physiological saline injection. The heating protocol in the present study causes an increase in the colonic temperature to 41°C. The left tibialis anterior muscles were dissected 1, 3, 7, 14, and 28 days after injection of cardiotoxin or physiological saline. Results. The wet weight and water content of muscles increased 1 day after cardiotoxin injection regardless of the application of heat stress, but normalized after 7–14 days. The muscle protein content in control rats had increased 7 days after heat stress. Although the muscle protein content decreased on cardiotoxin injection, heat stress caused a significant recovery in protein level. Expression of heat shock protein 72 (HSP72) and the number of Pax7-positive nuclei decreased after cardiotoxin injection but increased on the application of heat stress in both normal control and cardiotoxin-injected groups.

Offprint requests to: A. Kojima Received: June 8, 2006 / Accepted: September 25, 2006

Conclusions. Heat stress stimulated not only the proliferation of satellite cells but also protein synthesis during the regeneration of injured skeletal muscle. It is thus strongly suggested that the heating of injured skeletal muscle may facilitate recovery. There was no direct relationship between the level of HSP72 expression and muscle protein content, suggesting that HSP72 expression may not be the key signal for protein synthesis in the necrosis–regeneration process.

Introduction Skeletal muscle exhibits a great capacity for regeneration after various injuries and diseases. Skeletal muscle stem cells, so-called muscle satellite cells, are responsible for the repair and the regeneration of adult skeletal muscle tissues.1,2 Histopathological analysis of injured muscle has clearly shown increased numbers of muscle satellite cells, and these cells differentiate into myotubes and myofibers.3 It has been considered that satellite cells are in the G0 phase of the cell cycle under noninjured conditions, and that the cells leave the G0 stage, proliferate, and fuse to form multinucleated myotubes when they are activated in response to injury.1,4–7 The newly formed myotubes subsequently replace the damaged muscle fibers.1,4–6 Although it is clear that muscle satellite cells play a critical role in the formation of new myofibers during the regeneration process,2,4–6,8,9 the detailed mechanism responsible for skeletal muscle regeneration is still unclear. Recently, it has been reported that heat stress has several effects on the properties of skeletal muscles. Heat stress can attenuate unloading-related muscle atrophy10 and can enhance the resistance to ischemic injury.11,12 These phenomena induced by heat stress are partially related to the increase in the expressions of heat shock proteins (HSPs), which act as molecular

A. Kojima et al.: Heat stress and muscle regeneration

chaperones.13 The synthesis of muscular protein may also be stimulated by heat stress.14–17 Heat stress can facilitate the proliferation and the differentiation of myoblasts in vitro18,19 and can enhance their proliferative potential, which may stimulate the regrowth of atrophied skeletal muscle.14 In general, the application of heat stress on injured muscle tissues (hyperthermia treatment) is contradictory. However, if heat stress application can facilitate the proliferation and the differentiation of muscle satellite cells, the application of heat stress may facilitate muscle regeneration. Therefore, the current study was performed to investigate the effects of heat stress on the regeneration process of skeletal muscle after drug-induced injury in vivo. The evidence obtained in this study suggests that the regeneration process can be facilitated by the application of heat stress to injured skeletal muscle. Materials and methods Animals and grouping All experimental procedures were conducted in accordance with the Japanese and American Physiological Society’s Guide for the Care and Use of Laboratory Animals. The study was also approved by the Animal Use Committee at St. Marianna University School of Medicine. Male Wistar rats, aged 7 weeks, were randomly divided into six groups: (1) nonheated controls undergoing physiological saline (PS) injection (NC), (2) heat stressed before PS injection (BC), (3) heat stressed after PS injection (AC), (4) cardiotoxin (CTX) injected without heat stress (NX), (5) heat stressed before CTX injection (BX), and (6) heat stressed after CTX injection (AX, n = 25 in each group). Two or three rats were housed in a cage 55 × 38 cm and 34 cm high in an animal room maintained at 23° ± 1°C (mean ± SEM) and at around 50% humidity with a 12 : 12-h light : dark cycle. Solid food and water were provided ad libitum. Initiation of muscle injury and regeneration To induce a necrosis–regeneration cycle, 0.5 ml of CTX (10 µM in PS, Sigma, St. Louis, MO, USA) from Naja naja atra venom was injected into the proximal, midbelly, and distal region (0.16–0.17 ml in each) of the left tibialis anterior (TA) muscle of rats, except for those in the NC, BC, and AC groups, using a 27-gauge needle20,21 under anesthesia with the rats breathing an isofluraneoxygen-nitrous oxide gas mixture (forane). This procedure for the initiation of necrosis–regeneration was performed carefully to avoid damage to the nerves and blood vessels.21 The same volume of PS was injected similarly into the left TA of rats in the NC, BC, and AC groups.

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Exposure to heat stress The BC, AC, BX, and AX groups were exposed to environmental heat stress (41°C for 60 min) in a heat chamber 24 h before (BC and BX groups) or immediately after PS or CTX injection (AC and AX groups), after the rats had completely recovered from the anesthesia. The heating protocol in the present study caused an increase in colonic temperature up to 41°C and induced HSP72 expression in rat skeletal muscle.14,16,17 After 60 min of heating, rats of all groups were housed in the animal room maintained at 23° ± 1°C and about 55% humidity, as explained above. Sampling Venous blood (∼5 ml) was withdrawn from the jugular vein under anesthesia with sodium pentobarbital (5 mg/ 100 g body weight, i.p.) 1, 3, 7, 14, and 28 days after PS or CTX injection. The blood was centrifuged at 1500 g for 10 min (4°C) and the serum was saved. TA muscle was also dissected from the left limb. The muscles were rapidly weighed and stretched longitudinally to an optimum length and frozen in isopentane cooled by liquid nitrogen. The samples were stored at −80°C until analysis. Serum creatine kinase activity Serum creatine kinase (CK) activity was determined by the Ultraviolet Spectrophotometry (UV) method (at 37°C), as standardized by the Japan Society of Clinical Chemistry, and was expressed in international units per ml (IU/ml). Histological and immunohistochemical analyses Frozen TA muscles were cut cross-sectionally into two portions at the midbelly region. Serial transverse cryosections (8 µm thick) of the midbelly region of the proximal side were cut at −20°C and mounted on glass slides. The sections were air dried and stained to analyze the histological stages and the profiles of Pax7-positive nuclei (muscle satellite cells) by using hematoxylin and eosin (H&E) staining and by the standard immunohistochemical technique, respectively. Monoclonal anti-Pax7 antibody (undiluted tissue culture supernatant of hybridoma cells obtained from the Developmental Studies Hybridoma Bank, Iowa, IA, USA) was used for the detection of muscle satellite cells.2,8,9,22,23 Cross sections were fixed with 4% paraformaldehyde for 15 min, and then were postfixed in ice-cold methanol for 15 min. After washing three times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (TPBS), sections were blocked for

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30 min by using a blocking reagent (1% Roche Blocking Reagent, Roche Diagnostic, Penzberg, Germany). Then, samples were incubated with the primary antibodies for Pax7 and rabbit polyclonal anti-laminin (Z0097, DakoCytomation, Glostrup, Denmark; diluted 1 : 500 in PBS) for 36 h at 4°C. After washing with TPBS, sections were also incubated with the second primary antibodies for Cy3-conjugated anti-mouse IgG1 (Jackson Immuno Research, West Grove, PA, USA; diluted 1 : 500 in PBS) and with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Sigma; diluted 1 : 500 in PBS) for 90 min at room temperature. After washing with TPBS, nuclei were stained by 10-min incubation in a solution of 2,4-diamidino-2phenylindole dihydrochloride n-hydrate (Dapi, 0.5 mg/ ml; Sigma) in TPBS at room temperature prior to the final wash. A cover glass was then placed above the section using Vector Shield (Vector Laboratories, Burlingame, CA, USA).

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Electrophoresis To determine the levels of HSP72 expression in the muscle, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting was performed by using the techniques described earlier.14–18 Briefly, the remaining portions of the homogenate samples, which were prepared for the determination of muscle protein content, were solubilized in SDS sample buffer [30% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, 2.3% (w/v) SDS, 62.5 mM Tris-HCl, 0.05% (w/v) bromophenol blue, pH 6.8] and 1 mg of protein was loaded on a 10% SDSpolyacrylamide gel. The electrophoresis was carried out at a constant current of 20 mA for 60 min according to the method of Laemmli.24 A Bio-Rad Precision marker was applied to both sides of the 14 lanes as an internal control for the transfer process or electrophoresis. Western blotting

Imaging of muscular sections and analyses The images of muscle sections were incorporated into a personal computer (AxioVision 3.1.2.1, Carl Zeiss Japan, Tokyo, Japan) by using a microscope (Axioskop2 plus with AxioCam HR, Carl Zeiss Japan). The percentage of Pax7-positive nuclei located within the laminin-positive basal membrane relative to the total number of Dapi-positive nuclei in the whole transverse section of approximately 2500 muscle fibers was calculated.

Muscle protein and water content The protein and water contents were determined in the midbelly region of the distal side. Muscle protein content was determined by using the techniques described earlier.14,17 Briefly, the muscle was homogenized in 10 volumes of isolation buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.1 mM EDTA, pH 7.6), and completely solubilized by alkali treatment with one volume of 2 N NaOH at 37°C for 30 min. Protein concentration in the homogenates was determined by using a protein assay kit (Bio-Rad, Hercules, CA, USA) and bovine serum albumin (Sigma) as the standard. Total protein content in whole muscle was also calculated. The remaining portion of the distal muscle was used for analysis of water content as described earlier.14,17 Frozen muscles, which were weighed, were placed in a freeze dryer (−45°C) under vacuum for between 48 and 72 h.10 Muscle tissues were then reweighed after 48 and 72 h. The results showed that water was lost completely within 48 h. The water content was expressed as the total content in whole muscle.

We investigated the expression of HSP72 (HSP70 inducible) in each muscle. Following SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (0.2 µm pore size, Bio-Rad) using a mini trans-blot cell (Bio-Rad) at a constant voltage of 100 V for 60 min at 4°C. After the transfer, PVDF membranes were blocked for 1 h using a blocking buffer (5% skimmed milk in Tris-buffered saline, pH 7.5). Then, the membranes were incubated for 1 h with a polyclonal antibody for HSP72 (SPA-812; StressGen, Victoria, BC, Canada) and then reacted with a secondary antibody (goat anti-rabbit immunoglobulin G conjugate to alkaline phosphatase, Sigma) for 2 h. The membranes were subsequently reacted with bromochloroindolyl phosphate-nitroblue tetrazolium substrate. The bands from immunoblots were quantified using computerized densitometry (SCION Image, Scion corporation, Federick, MA, USA). Standard curves were constructed to ensure linearity.14,17,18 Statistical analyses All values were expressed as the mean ± SEM. We performed statistical tests among the NC, NX, BX, and AX groups to evaluate the effects of CTX injection with or without heat stress. To establish the effects of heat stress alone, we compared values among the NC, BC, and AC groups. Statistical significance was analyzed by using the analysis of variance followed by the Bonferroni/Dunn test. Statistical significance was set at P < 0.05.

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Results Body weight and serum CK activity No significant changes in body weight in response to CTX injection and/or heat stress were observed (data not shown). Serum CK activity increased on CTX injection (Fig. 1). The peak serum CK activity was observed at day 14 in the CTX-injected NX, BX, and AX groups. However, statistically significant elevation above the control level was induced only in group NX. The CK activity in the NX group was higher than that in the NC group at day 1 and 14 (P < 0.05). The level normalized after 28 days. Muscle wet weights

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group AX at day 14 was significantly lower than that in the NC group (P < 0.05). After day 7, the absolute weights in the NX and BX groups gradually increased again toward the level in group NC (P > 0.05), although the relative weights were unchanged. In group AX, the mean absolute and relative weights increased toward the control level after day 14, although this was not significant. The absolute weight in group NX at day 28 was significantly less than the age-matched group NC (P < 0.05). Muscle protein content The total protein content of whole muscle in the control group, NC, gradually increased during the experimental period and the mean levels at day 14 and 28 were

The absolute muscle weights in the control groups with or without heat stress increased gradually, and the weights relative to body weight were stable during the 28-day experimental period (Fig. 2). Intramuscular injection of CTX caused an acute increase in muscle weight, regardless of the application of heat stress. The absolute (Fig. 2A) and relative weights (Fig. 2B) in groups NX, BX, and AX at day 1 were significantly greater than in the NC group (P < 0.05). The muscle weights then gradually decreased until day 7 in the NX and BX groups to a level below that of the NC group. The absolute weight in group AX slightly increased further at day 3, although the relative weight remained stable. However, both absolute and relative weights decreased up to day 14 (P < 0.05). The absolute weight of

Fig. 1. Changes in serum creatine kinase (CK) activity after cardiotoxin (CTX) or physiological saline (PS) injection with or without the application of heat stress. NC, nonheated control with PS injection; BC, heat stressed before PS injection; AC, heat stressed after PS injection; NX, CTX injected without heat stress; BX, heat stressed before CTX injection; and AX, heat stressed after CTX injection. *P < 0.05, §P < 0.05: significantly different compared with the values of the NC group on day 14 and day 1, respectively. Values are mean ± SEM, n = 5/group for each day

Fig. 2. Changes in the absolute muscle wet weight (A) and the muscle wet weight relative to body weight (B) after cardiotoxin or physiological saline injection with or without heat stress. *P < 0.05, §P < 0.05: significantly different compared with the values of the NC group each day and on day 1, respectively. Values are mean ± SEM. n = 5/group for each day

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Fig. 3. Changes in whole muscle protein content after CTX or PS injection with or without heat stress. *P < 0.05, †P < 0.05: significantly different compared with the values of the NC and NX groups, respectively, each day. §P < 0.05: significantly different compared with the value of the NC group on day 1. Values are mean ± SEM. n = 5/group for each day

significantly greater than that in group NC at day 1 (Fig. 3). Heat stress alone caused a further increase in protein content. Significant elevation in group BC and AC versus the level of group NC at day 1 and the age-matched control, NC, was noted even at day 7 (P < 0.05). In contrast, muscle protein content was decreased by CTX injection. The protein content in group NX and BX at day 7 was significantly less than that in group NC at day 1 and 7 (P < 0.05). The total protein content of muscle in the group that underwent CTX injection without heat stress (NX) was stable during the rest of the experimental period. However, the protein content in the groups in which heating was applied before or after CTX injection gradually increased thereafter and reached the control level at day 28, although the mean levels of group BX and AX at day 14 were still lower than the agematched group NC (P < 0.05). The mean protein level in group NX at day 28 was significantly less than that in any other group (P < 0.05). Muscle water content Total muscle water content in the normal control group, NC, gradually increased (Fig. 4) following the growth of muscle mass shown in Fig. 2A. Heat stress alone or injection of PS did not influence the water content. The levels in groups NC and BC at day 28 were significantly greater than that in group NC at day 1 (P < 0.05). Water content at day 1 increased on injection of CTX with or without heat stress (P < 0.05). The water content in group NX and BX gradually decreased until day 7 or 14 (P < 0.05). Although the mean level in group AX fur-

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Fig. 4. Changes in whole muscle water content after CTX or PS injection with or without heat stress. *P < 0.05, §P < 0.05: significantly different compared with the values of the NC group each day and on day 1, respectively. Values are mean ± SEM. n = 5/group for each day

ther increased slightly at day 3 (P > 0.05), the water content also decreased up to day 14 (P < 0.05). During the remaining 14 days of the experimental period, the water content in all groups insignificantly increased toward the control level. HSP72 expression HSP72 expression in the control group, NC, remained stable during the experimental period (Fig. 5). Heating alone caused an insignificant increase of HSP72 expression of group BC and AC even on day 1 (versus group NC). The mean levels in both groups further increased at day 3 (P < 0.05) compared with NC at day 1 and 3 and gradually decreased toward the nonheated control level during the rest of the experimental period. In contrast, muscle injury without heat stress (NX) or with heat stress prior to CTX injection (BX) caused an insignificant decrease in HSP72 expression at day 1. However, the level of HSP72 expression in rats with application of heat stress after CTX injection (AX) remained unchanged at day 1. The mean HSP72 expression in groups NX, BX, and AX, then gradually increased and reached its highest level on day 7, at which point the mean level of group AX was significantly greater than the level of group NC at day 1 and day 7 (P < 0.05). The level of HSP72 expression in the AX group generally stayed at a higher level compared with other groups, even though the mean level tended to decrease toward the control value (P > 0.05). The mean value of group AX at day 14 was greater than that in group NC at day 1 and 14 (P < 0.05).

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Fig. 6. Changes in the relative population of Pax7-positive nuclei after CTX or PS injection with or without heat stress. *P < 0.05, †P < 0.05: significantly different compared with the values of the NC and NX groups, respectively, each day. §P < 0.05: significantly different compared with the value of the NC group on day 1. Values are mean ± SEM. n = 5/group for each day

Fig. 5. Representative patterns (A) and the mean (± SEM) levels of HSP72 expression (B) in response to CTX or PS injection with or without heat stress. OD, optical density. *P < 0.05, §P < 0.05: significantly different compared with the values of the NC group each day and on day 1, respectively. n = 5/ group for each day

Pax7-positive nuclei Using double immunostaining for anti-Pax7 and antilaminin with additional nuclear staining (Dapi) of cryosections, Pax7-postive nuclei were counted in each muscle sample. The percentage of Pax7-positive nuclei among the total nuclei in group NC was stable throughout the experimental period (Fig. 6). Three days after the application of heat stress, the number of Pax7positive nuclei was increased. The mean level in both the BC and AC groups was significantly greater than that of group NC at day 1 and 3 (P < 0.05). However, the level in both groups gradually decreased toward the nonheated control level thereafter. The population of Pax7-positive nuclei decreased initially (day 1) after CTX injection, especially the mean level in group NX and AX was significantly less than that in group NC (P < 0.05). However, a significant increase in Pax7-positive nuclei was observed in the CTXinjected NX, BX, and AX groups at day 3 (P < 0.05). The magnitude of the increase was greater in the heat-

stressed groups, BX and AX, and the mean level in group AX was also significantly greater than that in the age-matched group NX (P < 0.05). The mean value in these groups was even significantly greater than that in the age-matched control group NC (P < 0.05). These levels gradually decreased toward the control level thereafter, although the level in group BX remained unchanged up to day 7. However, the number of Pax-7 positive nuclei in the groups with CTX injection and heat stress at day 7 (group BX and AX) was significantly higher than that in the nonheated normal control, NC (P < 0.05). Histological analyses Images of H&E staining showed that cross sections of TA muscles in CTX-injected rats had substantial fiber damage and edema at day 1 (Fig. 7). On day 3, many mononucleated cells had infiltrated the necrotic area. At day 7, mononucleated cells still actively infiltrated the necrotic area, but newly regenerating myotubes (myofibers) having central nuclei and with small diameters were observed in the NX, BX, and AX groups. In the heated groups (BX and AX groups), however, the inflammatory response was reduced and many newly formed myotubes (myofibers) were observed compared with the nonheated NX group, suggesting that the regenerative process was facilitated by the application of heat stress. The numbers of infiltrating cells were reduced 14 days after injury and the diameter of regenerating muscle fibers had become considerably larger, especially in the heated BX and AX groups. Regenera-

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A. Kojima et al.: Heat stress and muscle regeneration

Fig. 7. Transverse cryosections of the midbelly region of rat tibialis anterior muscle stained with hematoxylin and eosin. Bar 50 µm

tion in the heat-stressed BX and AX groups was almost complete 28 days after CTX injection; however, the size of fibers in the nonheated NX group was small and regeneration was still in progress.

Discussion To our knowledge, this is the first report showing that the regeneration of injured skeletal muscle is stimulated by the application of heat stress. In general, the effect of heat stress on injured muscle tissues is considered to be contradictory because of the induction of heatingrelated acute inflammation. In the present study, however, heat stress activated the proliferative potential of satellite cells during the regeneration of injured TA muscle in rats. Recovery from CTX-injection-related necrosis and decreased protein content were facilitated by the application of heat stress. HSP72 HSP72 is known as a molecular chaperon13,25 and is sensitive to various cellular stresses, such as heat stress, toxins, radiation, and oxidants.11,14,18,25 A previous study suggested that HSP72 may be a key factor in muscle protein synthesis.18 The present study also showed that the expression of HSP72 was upregulated by the application of heat stress alone. In contrast, HSP72 expression was downregulated by the injection of CTX. Among

the groups with muscle injury, the level of HSP72 expression was higher when heat stress was applied after CTX injection (AX) than in the groups undergoing heat stress before CTX injection (BX) or undergoing no heat stress (NX). The level of HSP72 expression in group AX at day 1 was identical to that in the normal control. This phenomenon suggests that the injury-related decrease of HSP72 expression may be inhibited by heat application after CTX injection. There was no significant difference in HSP72 expression between the BX and NX groups throughout the experimental period, suggesting that the application of heat stress prior to the induction of muscle injury was not beneficial to the expression of HSP72. Although the heat-stress-related increase in protein content of the group with muscle injury was delayed, the level of whole muscle protein in the groups with or without CTX injection (group AX, BC, and AC) was increased when the HSP72 expression was stimulated by heat stress. However, it is unclear why the recovery of protein content from the injury-related decrease was stimulated even in group BX without significant elevation of HSP72 expression. There was no relationship in the time course of changes between the level of HSP72 expression and muscle protein content in the experimental groups. These results suggest that HSP72 expression may not be the key signal for protein synthesis in the necrosis–regenerating process. Further study is needed to investigate the functional role of HSP72 in protein synthesis.

A. Kojima et al.: Heat stress and muscle regeneration

It has been suggested that HSP72 has a cytoprotective effect against several stressors, such as metals, oxidants, and drugs.26 HSP72 expression in muscle without CTX injection was upregulated 24 h after heat stress in the BC and AC groups. According to the results on muscle weight and protein content, and also from the histochemical analyses, however, it is still unclear whether the upregulation of HSP72 has a cytoprotective effect against cytotoxic drugs such as CTX. On the other hand, recovery from the CTX-injection-related decrease in muscle protein content was facilitated by heat stress (in both BX and AX groups) compared with the group not undergoing heat stress (NX), suggesting that the beneficial effect of heating was not directly related to the timing of heat stress application. Heat stress application itself, regardless of the HSP72 expression, may be associated with the stimulation of protein synthesis. Muscle satellite cells It has been reported that muscle satellite cells are also responsible for postnatal growth and regrowth, as well as the regeneration of skeletal muscle.2,8 It is generally accepted that the paired-box gene Pax7 is the specific indicator for both quiescent and mitotic active and proliferating muscle satellite cells.2,8,27 Since homozygous Pax7 mutant mice completely lack muscle satellite cells, it has been considered that Pax7 expression is essential at the top of the molecular hierarchy controlling the differentiation to satellite cells.9 The present study clearly indicated that the population of Pax7-positive nuclei (satellite cells) decreased 1 day after CTX injection, but increased within 3 days. A large induction of the Pax7 gene, analyzed by using semiquantitative reverse transcription-PCR, was also observed 3 days after CTX-induced muscle injury in another study28 Following freeze-crush-related or CTXinjection-related skeletal muscle damage, the number of Pax7-positive satellite cells increased, suggesting an increase of satellite cell-derived myoblasts in the injured muscle and an upregulation of Pax7 gene expression in proliferating myoblasts.29 Oustanina et al.29 also reported that muscle regeneration is impaired in Pax7knockout (−/−) adult mice. Further, a correlation between the number of satellite cells and the ability of muscle regeneration was observed. Therefore, an increase in the number of muscle satellite cells may be an essential factor for muscle regeneration. In the present study, heating increased the relative number of Pax7-positive nuclei and the muscle protein content, which were decreased after CTX injection. The number of Pax7-positive nuclei in heat-stressed groups (BX and AX groups) reached levels that were approximately twice the age-matched normal control at day 3

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and gradually decreased to the control level thereafter. The muscle protein content in the heat-stressed BX and AX groups also increased to levels approximately twice that of the age-matched group that also underwent CTX injection (NX) at day 28. However, such a large increase of protein content was not induced if heat stress was not applied, even though the CTX-injection-related decrease in the percentage of Pax7-positive nuclei increased to the control level within 3 days. The protein content, which decreased following CTX injection, remained low if heat was not applied. Therefore, the data obtained from the current study clearly indicate that heat stress, which caused an increase in Pax7-positive satellite cells and muscle protein content even in normal control rats, played an essential role in the regeneration of injured muscle. A previous study showed that heat stress enhanced the proliferative potential of skeletal muscle and induced muscle hypertrophy.17 In the present study, heat stress also induced an increase in the number of Pax7positive nuclei in noninjured skeletal muscle. The application of heat stress may act as an extracellular stimulus for the facilitation of proliferation and the differentiation of Pax7-positive satellite cells, suggesting that the morphological properties of muscle are regulated by heat-stress-related metabolic factor(s). On the other hand, mechanical load-dependent regulation of satellite cell numbers, which influence the number of myonuclei and the size of muscle fibers, was also recently reported by Wang et al.30 Serum CK activity In general, serum CK activity is used as an indicator of damage in several tissues. The elevation of serum CK activity, observed in the present study, and the morphological images of muscle cross sections indicate that severe muscle damage was induced by CTX injection. The peak value of serum CK activity was observed 14 days after the injection of CTX. However, the histological images of H&E-stained cryosections indicate that the damage (necrosis) in muscle tissues was typically observed 3–7 days after CTX injection and newly regenerating myotubes (myofibers) were observed at day 14. Further, the timing of the responses of muscle weight, protein content, and the level of HSP72 expression obtained in the present study was not closely associated with the level of CK activity. It is not clear why the responses of these parameters are different from the level of serum CK activity. Further, it is clear that the CTX-injection-related increase in the leakage of CK into the blood circulation was partially, at least, prevented by the application of heat stress, although the mechanism responsible for this phenomenon is unclear.

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Conclusion Heat stress stimulated not only the proliferation of satellite cells but also protein synthesis during the regeneration of injured skeletal muscle. There was no direct relationship between the level of HSP72 expression and muscle protein content, suggesting that HSP72 expression may not be the key signal for protein synthesis in the necrosis–regeneration process. It was also strongly suggested that heating injured skeletal muscle may facilitate recovery. In the present study, muscle injury was induced by CTX injection, because CTX-induced muscle injury is often utilized as one of the most reproducible experimental methods.2 Although this direct cause of muscle damage is not necessarily the same as that in sport-related injuries, the data suggest that heat stress may be a useful tool for promotion of muscular mass and force generation in patients during rehabilitation. Acknowledgments. The authors thank Dr. H. Tanaka from the Department of Developmental Neurobiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, for supplying the Pax7 antibody. This study was supported, in part, by Grants-in-Aid for Scientific Research (C, 17500444, KG), (C, 15500453, TY), (A, 18200042, TY ) and (A, 15200049, YO) from the Japan Society for the Promotion of Science, and by Research Grant (16B-2) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare (YO).

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