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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil; and 2Cardiovascular Institute and Department of Physiology, Loyola University of Chicago, Maywood, Illinois
Submitted 9 August 2005 ; accepted in final form 8 November 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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regeneration; radicicol; transgenic mouse; myoprotection
Although the process of the skeletal muscle damage and subsequent regeneration has been explored carefully at the structural level for many years, the identification and characterization of molecular players involved are ongoing. Accordingly, a family of proteins known as heat shock proteins (HSPs) might be important players in skeletal muscle response to injury and subsequent regeneration, because these proteins play a key role in cytoprotection (4, 25).
The inducible 70-kDa heat shock protein (HSP70) is the most strictly stress-inducible member of the HSP family and therefore is regarded as a marker for cell stress (2). Studies have demonstrated that the HSP70 is involved in diversified cellular functions, which include being part of the ubiquitin-proteasome system, chaperoning proteins into degradation pathways, binding of newly synthesized amino acid chains on ribosomes, and maintaining translocation-competent folding of endoplasmic reticulum and mitochondrial precursor proteins in the cytosol (32). Functional studies have shown that HSP70 plays a pivotal role in cardiac protection against ischemia in vivo and in vitro (4). In addition, HSP70 is induced by certain stressors in skeletal muscle, including physical and chemical factors as well as metabolic challenges (19) such as exercise-induced physiological and biochemical changes (20).
A series of compounds able to either disrupt the chaperone complex or inhibit HSP90 are available to address the biological role of HSPs. These compounds include the benzoquinone ansamycin antibiotics geldanamycin and herbimycin A (7, 29) and recently a more powerful compound, the macrocyclic antifungal antibiotic radicicol (13). Ultimately, the compounds activate HSP expression by binding to the 90-kDa HSP (HSP90), which has been implicated in the regulation of heat shock factor 1 (HSF1). HSF1 is responsible for the transcriptional activation of the heat shock genes. It has been shown that radicicol strongly induces HSP expression and subsequently ischemic protection in neonatal rat cardiomyocytes (13).
Because HSP70 has been recognized as a tissue protector, and because overexpression of HSP70 in transgenic mice protects cardiomyocytes against ischemia-reperfusion injury (21) and skeletal muscles against a lengthening contraction damage model and age-related muscle dysfunction (22), we hypothesized that the HSP70 is involved in skeletal muscle protection against damage and regeneration. Therefore, we aimed to investigate the role of HSP70 in histological, immunohistochemical, and molecular aspects of skeletal muscle damage and regeneration of cryolesioned HSP70-overexpressing transgenic mice. Furthermore, we evaluated the effect of the HSP inducer radicicol immediately after cryolesioning in skeletal muscle of wild-type mice.
Our results suggest that the HSP70 protects skeletal muscle against injury and that radicicol might be useful as a therapeutic myoprotective agent, because both HSP70-overexpressing transgenic mice and wild-type mice treated with radicicol have minimized myonecrosis induced by cryolesioning.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Animals.
The transgenic mice overexpressing HSP70 were previously generated and have been described in detail elsewhere (21, 24). In brief, the rat HSP70 was cloned into pCAGGS, a vector known to produce high transgenic expression using a human cytomegalovirus enhancer upstream of the chicken 
-actin promoter intron. Standard methods were used to generate founders, which were screened using Southern blot analysis, and breed them to homozygosity. Male HSP70-overexpressing transgenic mice (n = 27) and wild-type CB6F1 mice (n = 35; 3 mo old) weighing 22 ± 2.5 g and 28.5 ± 2.3 g, respectively, were housed in standard plastic cages in an animal room under controlled environmental conditions and maintained on standard food and water ad libitum.
Experimental design.
In all animals used in the present study, except those from the radicicol control group, one soleus and one tibialis anterior (TA) muscle were cryolesioned (left leg), and the contralateral soleus and TA (right hindlimb) muscles were used as controls. The animals were assigned to eight groups; that is, three groups of both HSP70 mice and CB6F1 mice were cryolesioned and analyzed 1, 10, and 21 days after lesioning (n = 9 each), one group (n = 4) was treated with radicicol immediately (
2 min) after cryolesioning and evaluated after 1 day, and another group (n = 4) was treated with radicicol alone for 1 day. The animals were anesthetized, and the soleus and TA muscles to be lesioned were surgically exposed.
Cryolesioning consisted of one freeze-thaw cycle of the muscles in situ. Freezing was performed by precisely applying the flat end of metal precooled in liquid nitrogen to the surface of the muscles and maintaining it in this position for 10 s (1 x 9 mm and 2 x 9 mm in soleus and TA muscles, respectively). The wounds were closed with polyamide threads (6-0 sutures) after the muscles had thawed, and thereafter the animals were maintained for several hours on a warm plate (37°C) to prevent hypothermia.
Drug treatment.
In four wild-type mice, radicicol (diluted 1:1 with ethanol; Sigma, St. Louis, MO) was injected intramuscularly into soleus and TA muscles from the left hindlimb (0.05 mg/20 g body wt) immediately (2 min) after cryolesioning. The right hindlimb was injected with vehicle only. The other experimental group was treated with radicicol alone.
Histology. Mice were euthanized, the left and right soleus and TA muscles were removed and weighed, and half of each muscle was immediately frozen in hypercooled isopentane and stored in liquid nitrogen. Frozen muscles were cut using a cryostat (IEC Digital Minotome; GMI, Ramsey, MN) to generate 10-µm-thick entire muscle cross sections. The sections were collected on gelatin-coated glass slides, dried for 1 h at room temperature, and stored at –20°C. The other half of each muscle was used for immunoblot analysis or enzyme assay preparation.
Unfixed entire histological cross sections were stained with aqueous toluidine blue-borax solution (both 1% wt/vol) to reveal the general morphology (28, 34). The cross sections of the cryolesioned muscles were analyzed and digitized (see Figs. 2–4) only at the site of injury, thus avoiding the muscle fibers that were not affected by cryolesioning.
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Antibodies used for immunostaining and/or Western blot analysis. The primary antibodies used for immunostaining were 1) mouse anti-myosin heavy chain (anti-MHC) type II MAb, clone MY-32 (1:1,000 dilution; catalog no. M4276, Sigma); 2) mouse anti-skeletal MHC type I MAb, clone NOQ7.5.4D (1:4,000 dilution, catalog no. M8421; Sigma); 3) rabbit anti-neural cell adhesion molecule (NCAM) affinity-purified PAb (2.5 µg/ml, catalog no. AB5032; Chemicon International, Temecula, CA); and 4) mouse anti-developmental/neonatal MHC MAb (1:20 dilution MHCd/n, RNMy2/9D2, catalog no. VPM664; Novocastra, Newcastle upon Tyne, UK).
The corresponding secondary antibodies used for immunostaining were 1) goat anti-mouse IgG-FITC (1:50 dilution, catalog no. SC-2010; Santa Cruz Biotechnology, Santa Cruz, CA); 2) goat anti-mouse IgG-FITC (1:50 dilution, catalog no. SC-2010; Santa Cruz Biotechnology); 3) rhodamine red goat anti-rabbit IgG (1:50 dilution, catalog no. Rb394; Molecular Probes, Eugene, OR); 4) mouse IgG conjugated to horseradish peroxidase (1:20 dilution, catalog no. PK-6102, Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA).
The primary antibodies used for Western blot analysis were 1) HSP70 PAb (1:2,000 dilution) raised in the rabbit against a synthetic peptide as previously described (23) and 2) mouse anti-actin MAb clone C-4 (1:5,000 dilution, catalog no. 69100; MP Biomedicals, Irvine, CA). The corresponding secondary antibodies used for Western blot analysis were 1) goat anti-rabbit IgG peroxidase-conjugated PAb (1:5,000 dilution; catalog no. PI-1000; Vector Laboratories) and 2) goat anti-mouse IgG horseradish peroxidase-conjugated PAb (1:5,000 dilution, catalog no. 31430; Pierce Biotechnology, Rockford, IL).
Immunostaining. The cross sections of muscles used for immunostaining against all antibodies except MHCd/n were fixed with 4% paraformaldehyde in 0.2 M phosphate buffer (PB) for 10 min at room temperature, blocked with 0.1 M glycine in PBS for 5 min, and permeabilized in 0.2% Triton X-100-PBS for 10 min. The slides were incubated with a solution containing the primary antibody, 3% normal goat serum, and 0.3% Triton X-100-0.1 M PB overnight in a moisture chamber (4°C). After washing the slides with 0.1 M PB (3 times for 10 min each), we added a solution containing the secondary antibody and 0.3% Triton X-100-0.1 M PB for 2 h in a dark room. The slides were washed in 0.1 M PB (3 times for 10 min each) and incubated with a 3,3'-diaminobenzidine substrate kit for peroxidase (catalog no. SK-4100; Vector Laboratories). After being washed for 5 min in PBS, the slides were mounted with Vectashield mounting medium for fluorescence with 4,6-diamidino-2-phenylindole (catalog no. H-1200; Vector Laboratories) applied to coverslips.
Unfixed muscle cross sections were immunostained against MHCd/n MAb using the Vectastain Elite ABC kit according to the manufacturer's recommendations. These sections were then counterstained with hematoxylin.
Observations were made by obtaining photomicrographs of the stained sections using a fluorescence microscope (804388; Nikon, Japan) equipped with FITC, rhodamine, and fura filters or using the Radiance 2000 confocal microscopy system equipped with an inverted microscope (Eclipse TE 300; Nikon) and a scanning system (Bio-Rad Laboratories, Hercules, CA). FITC fluorochrome was excited using an argon laser (488 nm), and rhodamine fluorochrome was excited using a He/Ne laser (543 nm). A x40 magnification oil-immersion lens objective was used.
Western blot analysis. Cellular protein extracts were prepared from the muscle tissue of both control and transgenic mice. The level of HSP70 was quantified using Western blot analysis as previously described (13) with a specific antibody to HSP70. Protein samples were fractionated for Western blot analysis on an 8% SDS-PAGE gel and electrotransferred onto nitrocellulose membrane using a submersion electrotransfer apparatus (Bio-Rad Laboratories). The nitrocellulose blots were reacted with PAb that binds specifically to HSP70. Blots were then reacted with an anti-rabbit IgG biotin-streptavidin horseradish peroxidase-conjugated antibody and developed using an ECL developing kit (catalog no. 34080; Pierce Biotechnology).
Determination of antioxidant enzyme activity. To measure levels of glutathione peroxidase (GPX), frozen skeletal muscle tissues from both cryolesioned and control animals were thawed on ice. Tissues were soaked in 1x PBS with 0.16 mg/ml heparin for 1 min. The tissue was removed, pulverized in liquid nitrogen, and transferred to a microfuge tube.
Samples for GPX activity were homogenized using a Dounce homogenizer with 10 µl/mg homogenization buffer (in mM: 50 Tris·HCl, 5 EDTA, and 1 DTT, pH 7.5). They were then spun at 10,000 g for 15 min at 4°C. Supernatants were transferred to new tubes and stored at –80°C until use. An assay was performed according to the GPX assay kit instructions (catalog no. 703102; Cayman Chemical, Ann Arbor, MI). Values were calculated according to the extinction coefficient of NADPH in solution and adjusted for path length as detailed by the manufacturer.
Quantitative and morphometric analysis. The quantitative and morphometric analysis were evaluated using a digitizing unit connected to computer software (Image-Pro Plus; MediaCybernetics, Silver Spring, MD).
To assess the incidence of muscle fiber types I and II and their respective cross-sectional areas (CSAs), a total of 500 fibers (soleus) and 1,000 fibers (TA) per muscle in each group were counted, classified, and measured after immunostaining with antibodies against MHC types I and II in the soleus and TA muscles, respectively. Three to four cross sections of the soleus and TA muscles from different animals were analyzed in all groups.
The number of soleus myofibers that demonstrated necrosis was expressed as the mean percentage of 500 counted fibers per muscle (total of 1,500 fibers/group) and, for the TA muscle, as the mean percentage of 1,000 counted fibers per muscle (total of 3,000 fibers/group). Three cross sections of the soleus and TA muscles from different animals were analyzed in all groups.
As previously described (9), the frequency of NCAM per muscle were expressed as a percentage of the ratio of the number of NCAM-positive SCs to the number of fibers associated or not associated with SCs. The MHCd/n-positive area was quantified and expressed as the percentage of whole muscle CSA (11). Three whole muscle cross sections from different animals in each group were used for both NCAM and MHCd/n analyses.
The soleus and TA muscle fiber CSAs, after different regeneration times, were obtained from a total of 1,000 and 2,000 fibers, respectively. In the cryolesioned groups, the CSA measurements were obtained only in the regions that previously had been injured. Approximately three or four cross sections of the soleus and TA muscles from different animals were analyzed in all groups.
The data regarding muscle weight and fiber CSAs after different regeneration times are presented as relative induction increase the better to show alterations over time and to avoid the influence of initial differences in muscle weight and fiber CSAs between wild-type and HSP70-overexpressing mice (Table 2).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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23%, soleus and TA muscle weights by 26.5% and 19.5%, respectively, and fiber-type CSA from soleus and TA muscles by 15% (MHC type I; soleus), 32.5% (MHC type II; soleus) and 14.5% (MHC type II; TA), respectively, compared with the body and muscle weights and CSAs of wild-type mice (Table 1). Our immunostaining results revealed that both soleus and TA muscles from HSP70-overexpressing mice did not have changes in MHC type I or II abundance of muscle fiber type I and II compared with muscles from wild-type mice (Table 1).
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80% and 60%, respectively) (Table 2), and these weights returned to the control values at 10 and 21 days after cryolesioning. TA muscle weights from wild-type mice also significantly increased 1 day postlesioning (30%) (Table 2), whereas no change in TA weight was found in HSP70-overexpressing mice 1 day postlesioning. At 10 days postlesioning, TA muscle weights from only HSP70-overexpressing mice had decreased (30%) (Table 2). At 21 days postlesioning, TA muscle weights from both wild-type and HSP70-overexpressing mice did not have significant differences compared with their controls.
The fiber CSA was also measured at 10 and 21 days postlesioning, and the results indicate that, as expected, soleus muscles from wild-type mice had a greater decrease in fiber CSA than those from HSP70-overexpressing mice at 10 days (55% and 20%, respectively) (Table 2) and 21 days after cryolesioning (20% and 5%, respectively) (Table 2). The fiber CSAs of TA muscles from wild-type mice decreased (40%) (Table 2) at 10 days postlesioning and nearly reached the control values at 21 days postlesioning (Table 2). In contrast, the TA muscles from HSP70-overexpressing mice did not have significant alterations in fiber CSA (Table 2) at all time points evaluated.
Histological cross sections of soleus and TA muscles from nonlesioned mice and 1, 10, and 21 days after cryolesioning were stained with toluidine blue and microscopically analyzed. In line with a normal tissue structure, the intact control muscles from wild-type and HSP70-overexpressing mice exhibited fibers with polygonal shape and peripheral nuclei (Figs. 2, A and B, and 3, A and B).
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The soleus and TA muscles examined at 10 days of regeneration from both wild-type and HSP70-overexpressing mice had cells in different stages of regeneration with the presence of inflammatory cell infiltration, basophilic regenerating cells, and centrally nucleated cells of various sizes (Figs. 2, E and F, and 3, E and F). However, the muscles from wild-type mice had more inflammatory processes and neighboring cells with smaller CSAs compared with muscles from HSP70-overexpressing mice, which is shown in TA muscle (Fig. 3E).
At 21 days after cryolesioning, the soleus and TA muscles from wild-type and HSP70-overexpressing mice were mostly regenerated as highlighted by the presence of classic signals of regeneration (18), i.e., cells with centralized nuclei and split fibers (Figs. 2, G and H, and 3, G and H).
GPX enzymatic activity was determined in homogenates obtained from TA muscles of wild-type and HSP70-overexpressing mice to assess the antioxidant defense against free radicals produced in tissue damage. At 1 day after cryolesioning, the GPX activity similarly increased significantly in TA muscles from both wild-type and HSP70-overexpressing transgenic mice (2.4- and 1.7-fold, respectively) (Fig. 5). GPX activity remained elevated in the muscles from both wild-type and HSP70-overexpressing mice up to 21 days after cryolesioning (10 days, 2.1- and 2.3-fold, respectively; 21 days, 2.1- and 1.7-fold, respectively) (Fig. 5). In addition, GPX activity in TA muscles from HSP70-overexpressing mice was significantly higher than in TA muscles from wild-type mice 10 days postinjury (1.4-fold) (Fig. 5).
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TA muscle cross sections were immunostained with antibody against NCAM to detect and count the SCs at 1, 10, and 21 days after cryolesioning. As expected, the amount of NCAM-positive SCs in normal intact muscles from wild-type mice was low (TA, 0.3%) (Fig. 6) (6).The HSP70-overexpressing mice also had a basal number of SCs similar to that of wild-type mice (Fig. 6). At 1 and 10 days after cryolesioning, the number of SCs was increased in TA muscles from wild-type mice (7.6- and 4.3-fold, respectively) (Fig. 6). Although the muscles from HSP70-overexpressing mice followed the same trend as muscles from wild-type mice with regard to the increase in SCs at 1 and 10 days postlesioning, this amount was significant only at 10 days (5-fold) (Fig. 6). At 21 days of regeneration, the number of SCs noticeably had returned to control values in TA muscles (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Initially, we confirmed that the transgenic mice used in the present study in fact were overexpressing HSP70 (Fig. 1, A and B). Furthermore, we show a slight induction of HSP70 in cryolesioned skeletal muscle at 1 day postlesioning and a substantial induction at 10 days postlesioning. Also, treatment of wild-type mouse skeletal muscle with radicicol causes a marked increase in expression of HSP70 at 1 day after radicicol administration (Fig. 1C). Therefore, although cryolesioning does result in a slow increase in HSP70, administration of radicicol results in a rapid increase in HSP70 expression. In addition, we have shown that in the basal state (no lesioning), the body and muscle weights and fiber CSAs of both soleus and TA muscles (representative of slow- and fast-twitch muscles, respectively) were significantly lower in HSP70-overexpressing mice compared with wild-type mice (Table 1). These results are in line with a previous study (22) that showed that HSP70-overexpressing transgenic mice had 10% reduced body weight, 20% reduced muscle weight, and 16% reduced fiber CSAs compared with the body and muscle weights and fiber CSAs of wild-type mice. On the other hand, the fiber-type proportions were unaltered in both soleus and TA muscles from HSP70-transgenic mice compared with controls, indicating that the phenotype of the skeletal muscle fiber types is unaffected by overexpression of HSP70.
Cryolesioning is a model that provides an excellent opportunity to assess both the protective nature of the skeletal muscle against injury as well as its regenerative capacity. This model (36, 37) is well recognized to induce necrosis, and subsequently regeneration, in a well-delineated area of skeletal muscles (15, 26). In the present study, soleus and TA muscle cross sections were evaluated at 1, 10, and 21 days after cryolesioning. These time points were chosen because they represent well the structurally and functionally distinct phases of skeletal muscle adaptations to injury, including intense necrosis and edema, evident regeneration, and restoration of the structural features of skeletal muscle normal morphology (15, 26).
The molecular mechanisms underlying tissue protection against a severe injury such as cryolesioning is currently unclear. It is well known that sarcolemma disruption followed by a rise in intracellular Ca2+ concentration and proteolysis are key events in skeletal muscle fiber degeneration (6) and that the expression of heat-shock genes occurs in response to any stress that produces denatured proteins, including a Ca2+-mediated injury (18). Therefore, it is likely that denatured sarcolemma proteins initiate a heat shock response after cryolesioning, possibly by binding HSP90, which results in the dissociation of HSP90 from HSF proteins (1). Under basal conditions, HSP90 binds to and suppresses HSF activation (39). The dissociation of HSP90 from HSFs can lead to the phosphorylation and subsequent activation of HSFs. The activated HSFs then form trimers (38) that bind to the promoters of HSP genes to stimulate transcription (27). Consequently, the levels of HSP70 mRNA and protein are increased in stressed cells. Once synthesized, HSP70 binds to denatured proteins and attempts to restore their tertiary structure and enzymatic activity in a cycle driven by ATP hydrolysis (12). In addition, cryolesioning-induced HSPs could be involved in the inflammatory process during skeletal muscle injury, because it has been shown that they participate in cytokine signaling and cytokine gene expression and enhance antigen presentation to T lymphocytes (30).
At 1 day after cryolesioning, the soleus and TA muscles from HSP70-overexpressing mice clearly had minimized lesioning signals compared with the muscles from wild-type mice, which shows that the overexpression of HSP70 attenuated skeletal muscle damage induced by cryolesioning (Figs. 2 and 3). Skeletal muscle protection in HSP70-overexpressing mice is indicated by sparing injured TA muscle from weight loss (Table 2). Accordingly, a dramatic decrease in necrosis at 1 day after injury was observed in soleus and TA muscles from HSP70-transgenic mice compared with wild-type mice (Table 3). In addition, at 10 days postlesioning, the decrease in fiber CSA is attenuated in the soleus and TA muscles from HSP70-overexpressing animals relative to that in wild-type animals (Table 2). These results are in line with those described in a previous study (22) that showed that the overexpression of HSP70 in transgenic adult and old mice is able to significantly minimize the force deficit in muscles submitted to a lengthening contraction damage compared with muscles from wild-type mice, suggesting that HSP70 also contributes to functional preservation of skeletal muscle in response to damage. Nonetheless, future studies, particularly at the single skeletal muscle fiber level, are required to investigate whether HSP70 protects and/or preserves contractile and/or metabolic functions after injury. At that point, although limited histological analysis had been performed, it was completely unclear how the regenerative response would be influenced by HSP70 overexpression. In the present study, it is clear that the skeletal muscle regenerative process was less recruited. Thus overexpression of HSP70 appears to provide protection rather than induce improved regeneration.
To assess the relative contributions of the antioxidant defense system, we examined GPX activity after cryolesioning. GPX is an important enzyme involved in the glutathione-glutathione disulfide cycle to remove types of reactive oxygen species, more specifically the H2O2 and organic hydroperoxides, which are produced in the cytosol and mitochondria of damaged cells (33). We observed that GPX activity was elevated 1 day after cryolesioning and was maintained during the entire experimental procedure (up to 21 days) in both wild-type and HSP70-overexpressing mice (Fig. 5), and furthermore that muscles from HSP70-overexpressing mice had significantly higher GPX activity 10 days after cryolesioning compared with muscles from wild-type mice. This finding suggests that overexpression of HSP70 exerts a positive effect (increased expression and/or activity) on GPX, which protects the skeletal muscle against injury.
The results obtained in the present study prompted us to investigate the effect of a pharmacological HSP inducer, radicicol, on injured skeletal muscles. In line with our results in HSP70-overexpressing mice, radicicol, which quickly increases the expression of HSP70, significantly reduced the cryolesioning-induced damage at 1 day (Fig. 4 and Table 3).
One interpretation on the data described above is that elevated levels of HSPs might have a sparing effect on skeletal muscle from injury without having any significant effect on skeletal muscle regeneration per se. In fact, this hypothesis is in line with the molecular effects of HSPs, i.e., restoration of tertiary structure of denatured proteins and maintenance of cell structure and function (12). On the other hand, HSPs may modulate the skeletal muscle regeneration process after injury. Therefore, we aimed to investigate the regenerative capacity of cryolesioned skeletal muscles from HSP70-overexpressing mice. To better assess the effect of HSP70 in the regenerating process, we examined the expression of NCAM and MHCd/n.
It is noteworthy that no differences in histological features, i.e., analysis of muscles stained with toluidine blue and/or acid phosphatase and of fiber CSA, were detected between soleus and TA muscles from HSP70-transgenic mice, demonstrating that the important role of HSP70 in skeletal muscle takes place independently of fiber type. Therefore, in the subsequent analysis (focused on skeletal muscle regenerative capacity, i.e., SC number and MHCd/n immunodetection), we evaluated only TA muscle.
NCAM is a SC marker commonly used to study skeletal muscle plasticity (9, 10). We found that elevated amounts of NCAM-positive SCs are raised at 1 and 10 days after cryolesioning in TA muscle from wild-type mice (Fig. 6). Indeed, previous work demonstrated that the number of muscle cells expressing NCAM is increased at both 2 and 11 days of regeneration after denervation (31). Furthermore, it has been demonstrated that SCs become activated within 6 h of injury and in response to locally released growth factors from injured myofibers and macrophages, and also that SCs proliferate extensively within 2–3 days of cardiotoxin-induced injury (14). Our results clearly show a lower number of SCs in skeletal muscles of HSP70-overexpressing mice 1 day postinjury in TA muscle (Fig. 6). This finding suggests that because skeletal muscles from HSP70-transgenic mice are partially spared from cryolesioning-induced damage, they require less activation of SCs to repair the skeletal muscle profile. This interpretation leads to the idea that HSP70-overexpressing mice are protected against injury and have a normal regenerative capacity.
To further examine the regenerative response of HSP70-cryolesioned skeletal muscle, MHCd/n immunoexpression was examined. MHCd/n is known to be strongly upregulated after skeletal muscle injury in adult animals (11, 16). In our study, MHCd/n expression was detected only at 10 days after cryolesioning in TA muscle (Fig. 7), which is in agreement with a previous study that demonstrated MHCd/n gene induction at 10 days postcryolesioning (26). Also, it has been shown that MHCd/n expression is progressively increased to a peak on day 7 postinjury and is no longer detected on day 14 (11). The 10-day analysis of MHCd/n immunoexpression in our study revealed that the muscles from HSP70-transgenic mice have comparatively fewer regenerating cells, contributing to the idea that the decreased damage associated with HSP70 expression subsequently necessitates fewer regenerating cells.
Although signals of regeneration are diminished in injured skeletal muscles of HSP70-overexpressing mice, whether HSPs modulate the regenerating capacity in skeletal muscle cannot be ascertained at the present time. It is important to highlight that wild-type and HSP70-overexpressing muscles had different degrees of necrosis after injury in our experimental model; therefore, it is difficult to determine whether the regenerative response after injury was in fact changed. Future studies designed to examine injuries of progressive intensities in wild-type and HSP70-overexpressing animals should be performed to compare the regenerative responses under a similar damage state.
In summary, our present results suggest that the expression of HSP70, whether by transgenesis or pharmacological induction, strongly attenuates skeletal muscle injury after cryolesioning and therefore protects skeletal muscle in the early stages after damage. In addition, this study is the first to provide histological evidence for the role of radicicol as a potential therapeutic agent to be used in the treatment of skeletal muscle injury. Nonetheless, functional studies need to be performed to deepen knowledge of HSPs as tissue protectors and also to highlight the potential benefits of radicicol treatment.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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