Regeneration Of Injured Skeletal Muscle Research Articles

Treatment of skeletal muscle injury: a review.

Skeletal muscle injuries are the most common sports-related injuries and present a challenge in primary care and sports medicine. Most types of muscle injuries would follow three stages: the acute inflammatory and degenerative phase, the repair phase and the remodeling phase. Present conservative treatment includes RICE (rest, ice, compression, elevation), nonsteroidal anti-inflammatory drugs (NSAIDs) and physical therapy. However, if use improper, NSAIDs may suppress an essential inflammatory phase in the healing of injured skeletal muscle. Furthermore, it remains controversial whether or not they have adverse effects on the healing process or on the tensile strength. However, several growth factors might promote the regeneration of injured skeletal muscle, many novel treatments have involved on enhancing complete functional recovery. Exogenous growth factors have been shown to regulate satellite cell proliferation, differentiation and fusion in myotubes in vivo and in vitro, TGF-β1 antagonists behave as inhibitors of TGF-β1. They prevent collagen deposition and block formation of muscle fibrosis, so that a complete functional recovery can be achieved.

Effects of IL‐6 receptor inhibition and/or heat treatment on the regeneration of injured mouse skeletal muscle

Effects of inhibited interleukin‐6 (IL‐6) signal using the rat anti‐mouse IL6 receptor (MR16‐1) with or without heat treatment on the regeneration of injured skeletal muscle was studied in mice. 10‐week‐old C57BL/6JJc1 mice were divided into 5 groups: (1) normal controls, (2) cardiotoxin injection (CTX), (3) administration of MR16‐1 after CTX injection (CTX‐MR), (4) heat treatment after CTX injection (CTX‐H), and (5) administration of MR16‐1 plus heat treatment after CTX injection (CTX‐MR‐H). The CTX (10 μl) was injected into the right tibialis anterior (TA) muscle. MR16‐1 (2 mg) was injected i.p immediately after CTX injection. For heat treatment, the conscious mice were placed in a heat chamber (42 °C) for 60 min 1, 3, and 5 days after CTX injection. Muscles were sampled 7 days after these treatments and cross‐sectional analyses were performed. Regenerating myofibers with centrally located nuclei were noted 7 days after CTX injection. Immunohistochemical analyses revealed that desmin‐ and dystrophin‐positive (+) cells were increased in CTX‐MR and CTX‐MR‐H groups. Further, a number of myogenin + cells were seen in the damaged myofibers of CTX‐H group. These results suggest that blockage of IL‐6 signal and heat treatment facilitate the regeneration of skeletal muscle. This study was supported by Grant‐in‐Aid for Scientific Research S (19100009) from the Japan Society for the Promotion of Science.

Administration of granulocyte colony-stimulating factor facilitates the regenerative process of injured mice skeletal muscle via the activation of Akt/GSK3αβ signals

Effects of administration of granulocyte colony-stimulating factor (G-CSF) on the regeneration of injured mammalian skeletal muscles were studied in male C57BL/6J mice. Muscle injury was induced by injection of cardiotoxin (CTX) into tibialis anterior muscles bilaterally. G-CSF was administrated for 8 consecutive days from 3 days before and 5 days after the injection. Significant decreases of wet weight and protein content were noted in the necrotic muscle with CTX injection. A large number of the regenerating fibers having central nucleus were observed 7 days after the injection. The regeneration of injured muscle was further facilitated by the G-CSF treatment. Population of Pax7-positive nuclei was increased by the G-CSF treatment at day 7. Phospho-Akt and phospho-glycogen synthase kinase 3alphabeta (GSK3alphabeta) signals were also activated by G-CSF-administrated group during the regenerative process. It was suggested that G-CSF treatment may facilitate the regeneration of injured skeletal muscles via the activation of Akt/GSK3alphabeta signals.

Functional Overloading Facilitates the Regeneration of Injured Soleus Muscles in Mice

The effect of functional overloading on the regenerating process of injured skeletal muscle was investigated in 10-week-old male mice (C57BL/6J). Functional overloading on soleus of both hindlimbs was performed by cutting the distal tendons of plantaris and gastrocnemius muscles for 2 weeks before cardiotoxin (CTX) injection as the preconditioning and also during 10 weeks of recovery. To activate the necrosis-regeneration cycle, 0.1 ml of 10-microM CTX was injected into soleus muscle. The mean values of absolute muscle weight and the percentage of Pax7-positive nuclei in soleus were increased by the preconditioning. These values, as well as total muscle protein content, in the group with CTX injection plus overloading were larger than in the group with CTX injection alone. Fibers with central nucleus were noted in the group with CTX injection with or without overloading. The rate of disappearance of fibers having central nucleus during recovery was stimulated by overloading. Histological analyses revealed that the regeneration of injured soleus muscle with overloading proceeded more rapidly than the muscle without overloading. These results, in combination with previous lines of evidence, strongly suggest that functional overloading may facilitate the regeneration of injured skeletal muscles.

Heat stress facilitates the regeneration of injured skeletal muscle in rats

BackgroundSkeletal 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. MethodsMale 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, 0.5ml of 10μM 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 24h 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. ResultsThe 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 cardiotox-in-injected groups. ConclusionsHeat 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.

Chemokine receptor CCR2 involvement in skeletal muscle regeneration

Chemokines, signaling through the CCR2 receptor, are highly expressed in injured skeletal muscle. Their target specificity depends on the cellular expression of the specific receptors. Here we demonstrate that, in freeze-injured muscle, CCR2 co-localized with Mac-3, a marker of activated macrophages as well as with myogenin, a marker of activated muscle precursor cells. The degeneration/regeneration process in skeletal muscle of CCR2-/- and wild-type mice was not significantly different at day 3. However in contrast to the regenerated muscle of the wild-type mice, the muscle from CCR2-/- mice was characterized by impaired regeneration, inflammation, and fibrotic response at day 14, increased fat infiltration, fibrosis, and calcification at day 21, and impaired strength recovery until at least 28 days post-injury. Consistently, the increased expression of Mac-1 and TNF-alpha was prolonged in the injured muscle of CCR2-/- mice. The expression pattern of the myogenic factors MyoD and myogenin was similar for both types of mice, while NCAM, which is associated with the initiation of fusion of muscle precursor cells, was more increased in the injured muscle of CCR2-/- mice. In conclusion, the study delineates that signaling through CCR2 is involved in muscle precursor cell activities necessary for complete and rapid regeneration of injured skeletal muscle.

Leukemia inhibitory factor blocks early differentiation of skeletal muscle cells by activating ERK

Leukemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 family and has been shown to stimulate regeneration of injured skeletal muscle. Although LIF has been shown to stimulate muscle cell proliferation, its precise role in differentiation is unclear. Thus, we examined the effect of LIF on the differentiation of cultured C2C12 myoblast cells. In this study, we used both non-glycosylated LIF expressed in bacteria and glycosylated LIF secreted from NIH3T3 cells infected with Ad-LIF. Both non-glycosylated and glycosylated LIF blocked differentiation of myoblasts as measured by expression of myosin heavy chain and myotube formation. Treatment of myoblasts with LIF induced phosphorylation of ERK, and the LIF-induced inhibitory effect on myogenesis was blocked by pretreatment with U0126, a specific MEK inhibitor, and transient transfection with dominant negative (DN)-MEK1. In contrast, although LIF activated STAT3, the LIF-induced repression of the MCK transcriptional activity was not reversed by pretreatment with AG490, a specific Jak kinase inhibitor or transient transfection with DN-STAT3. Additionally, LIF exhibited its inhibitory effect on myogenesis only when cells were treated at earlier than 12 h after inducing differentiation. Taken together, these results suggest that LIF strongly inhibited early myogenic differentiation though activation of the ERK signaling pathway and its effect is irrespective of glycosylation.

Purinoceptor expression in regenerating skeletal muscle in the mdx mouse model of muscular dystrophy and in satellite cell cultures.

ATP is an important extracellular signaling molecule mediating its effects by activation of P2X and P2Y receptors. P2 receptors are expressed during muscle development, and recent findings demonstrate that ATP can regulate myoblast proliferation and differentiation in vitro. However, the role of purinergic signaling during regeneration of injured skeletal muscle has not been investigated. To examine this process in a clinically relevant system, we used the mouse model of muscular dystrophy (mdx), in which muscle degeneration is rapidly followed by regeneration. The latter process, in vivo muscle regeneration, was the focus of this study, and to study the cellular mechanisms involved in it, a parallel study on normal rat skeletal myoblast cultures was conducted. Using immunohistochemistry, RT-PCR, and electrophysiology, we investigated the expression of the P2X1-7 receptor subtypes and the P2Y1,2,4,6 receptors. Experiments in vitro and in vivo demonstrated the sequential expression of the P2X5, P2Y1, and P2X2 receptors during the process of muscle regeneration. The P2X5 and P2Y1 receptors were expressed first on activated satellite cells, and the P2Y1 receptor was also expressed on infiltrating immune cells. Subsequent P2X2 receptor expression on newly formed myotubes showed significant colocalization with AChRs, suggesting a role in regulation of muscle innervation. Thus, this study provides the first evidence for a role for purinergic signaling in muscle regeneration and raises the possibility of new therapeutic strategies in the treatment of muscle disease.

In vivo activation of STAT3 signaling in satellite cells and myofibers in regenerating rat skeletal muscles.

Although growth factors and cytokines play critical roles in skeletal muscle regeneration, intracellular signaling molecules that are activated by these factors in regenerating muscles have been not elucidated. Several lines of evidence suggest that leukemia inhibitory factor (LIF) is an important cytokine for the proliferation and survival of myoblasts in vitro and acceleration of skeletal muscle regeneration. To elucidate the role of LIF signaling in regenerative responses of skeletal muscles, we examined the spatial and temporal activation patterns of an LIF-associated signaling molecule, the signal transducer and activator transcription 3 (STAT3) proteins in regenerating rat skeletal muscles induced by crush injury. At the early stage of regeneration, activated STAT3 proteins were first detected in the nuclei of activated satellite cells and then continued to be activated in proliferating myoblasts expressing both PCNA and MyoD proteins. When muscle regeneration progressed, STAT3 signaling was no longer activated in differentiated myoblasts and myotubes. In addition, activation of STAT3 was also detected in myonuclei within intact sarcolemmas of surviving myofibers that did not show signs of necrosis. These findings suggest that activation of STAT3 signaling is an important molecular event that induces the successful regeneration of injured skeletal muscles.

Possible role for the c-ski gene in the proliferation of myogenic cells in regenerating skeletal muscles of rats.

Skeletal muscle regeneration after injury involves various processes, such as infiltration by inflammatory cells, the proliferation of satellite cells and fusion to myotubes. The c-ski nuclear protein has been implicated in the control of cell proliferation and/or terminal differentiation in the growth of skeletal muscle. However, there have been no reports concerning the involution of c-ski in the regeneration of injured skeletal muscle in mammals. A possible role for c-ski in the proliferation of myogenic cells in rat skeletal muscle during regeneration has been investigated with the assistance of in vitro experiments with L6 skeletal muscle cells. The expression levels of c-ski mRNA in regenerating tissues increased to approximately threefold that of intact tissues at 2 days after injury and decreased to normal levels at 2 weeks after injury. Many mononuclear cells among the Ski-positive cells expressed desmin and proliferating cell nuclear antigen, indicating that Ski-producing cells include the proliferating myogenic cells. The proliferation of L6 cells was significantly retarded by expression of the antisense ski gene. The results of the present study reveal that the c-ski gene plays an important role in the proliferation of myogenic cells in the regeneration of injured skeletal muscle.

Effects of calcium phosphate bioceramics on skeletal muscle cells.

With advances in ceramics technology, calcium phosphate bioceramics have been applied as bone substitutes. The effects of implants on bony tissue have been investigated. The effects upon adjacent skeletal muscles have not been determined. The focus of this work is to elucidate the biological effects of various calcium phosphate bioceramics on skeletal muscles. Four different kinds of powder of calcium phosphate biomaterials including beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA), beta-dicalcium pyrophosphate (beta-DCP) and sintered beta-dicalcium pyrophosphate (SDCP), were tested by myoblast cell cultures. The results were analyzed by cell count, cell morphology and concentration of transforming growth factor beta 1 (TGF-beta 1) in culture medium. The cell population and TGF-beta 1 concentration of the control sample increased persistently as the time of culture increased. The changes in cell population and TGF-beta 1 concentration in culture medium of the beta-TCP and HA were quite low in the first 3 days of culture, then increased gradually toward the seventh day. The changes in cell population and TGF-beta 1 concentration in culture medium of the silica, beta-DCP, and SDCP were quite similar. They were lower during the first day of culture but increased and reached that of the control medium after 7 days' culture. Most cells on B-TCP and HA diminished in size with radially spread, long pseudopods. We conclude that HA and beta-TCP are thought to have an inhibitory effect on growth of the myoblasts. The HA and beta-TCP may interfere with the repair and regeneration of injured skeletal muscle after orthopedic surgery.

Changes in the forms of the components of the troponin complex during regeneration of injured skeletal muscle.

Using the immunoperoxidase technique, antibodies to the fast components of the troponin complex stained all regenerating cells after localized alcohol injury to rat skeletal muscle. Antibodies to slow troponin components stained only some of these cells. About 6 weeks after injury with the nerve intact, the fast and slow forms of the troponin components were located in different cells. During the later stages of regeneration, staining for myosin ATPase correlated with the staining with antibodies to fast and slow troponin components. A similar staining pattern was also observed in the early stages of regeneration of muscle denervated at the time of injury. In this case, antibodies to fast skeletal muscle troponin components continued to stain all the cells 10 weeks after injury. Injured denervated muscle cells stained equally dark by myosin ATPase after preincubation at pH 9.4 over this period. None of the regenerating myotubes in denervated muscle stained for myosin ATPase after preincubation at pH 4.3.