Gravitational unloading inhibits the regenerative potential of atrophied soleus muscle in mice

Aim: The present study was performed to investigate the influence of unloading on the regeneration of atrophied and injured skeletal muscle. Methods: Male mice (C57BL/6J), aged 8 weeks, were used. Cardiotoxin (CTX) was injected into soleus muscles bilaterally. Gravitational unloading on soleus muscle was performed by hind limb suspension for 2 weeks before and additionally 6 weeks after CTX injection in one group. Soleus muscles in the remaining groups were loaded keeping the mice in the cages and were dissected 14, 28 and 42 days after the injection. Results: Recovery of the wet weight and protein content of soleus in the CTX‐injected group was inhibited by unloading. Increase in satellite cell number, induced by CTX injection and loading, was also inhibited by unloading. Disappearance of infiltration of mononucleated cells into the necrotic area was also delayed. This phenomenon suggests that regeneration, which is indicated by the appearance of fibres with central nuclei, was inhibited by unloading. Conclusion: Results suggested that loading plays an important role in the activation of the regenerating potential of injured skeletal muscle.     

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 3αβ (GSK3αβ) 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/GSK3αβ signals.     

Autologous bone marrow-derived cells enhance muscle strength following skeletal muscle crush injury in rats

Insufficient post-traumatic skeletal muscle regeneration with consecutive functional deficiency continues to be a serious problem in orthopedic and trauma surgery. Transplantation of autologous muscle precursor cells has shown encouraging results in muscle trauma treatment but is associated with significant donor site morbidity. In contrast to this, bone marrow-derived (BMD) cells can be obtained without any functional deficit by puncture. The goal of this study was to examine whether regular muscle regeneration can be improved by local application of autologous BMD cells in a rat model of blunt skeletal muscle trauma. One week after standardized open blunt crush injury to the left soleus muscle, 10(6) autologous BMD cells were injected into the traumatized muscle of male Sprague Dawley rats. Rats of the control group received saline solution as treatment. Three weeks after application, the fast twitch and tetanic contraction capacity of the soleus muscles was measured bilaterally by stimulating the sciatic nerves. Contraction forces of injured soleus muscles in control animals recovered to 39 +/- 10% (tetanic) and 59 +/- 12% (fast twitch) of the contralateral noninjured soleus muscles (p < 0.001). In contrast, autologous BMD cell injection significantly restored contractile forces to 53 +/- 8% (tetanic) and 72 +/- 13% (fast twitch) compared to those observed in contralateral noninjured soleus muscles. Thus, muscle function was significantly increased by BMD cell treatment (tetanic, p = 0.014; fast twitch, p = 0.05). In conclusion, autologous BMD cell grafting leads to an increase in contraction force, 14% in tetanic and 13% in fast twitch stimulation, demonstrating its potential to improve functional outcome after skeletal muscle crush injury.     

Suppression of Myostatin Stimulates Regenerative Potential of Injured Antigravitational Soleus Muscle in Mice under Unloading Condition

Effects of myostatin (MSTN)-suppression on the regeneration of injured skeletal muscle under unloading condition were investigated by using transgenic mice expressing a dominant-negative form of MSTN (MSTN-DN). Both MSTN-DN and wild-type (WT) mice were subjected to continuous hindlimb suspension (HS) for 6 weeks. Cardiotoxin (CTX) was injected into left soleus muscle under anesthesia 2 weeks after the initiation of HS. Then, the soleus muscles were excised following 6-week HS (4 weeks after CTX-injection). CTX-injection caused to reduce the soleus fiber cross-sectional area (CSA) in WT mice under both unloading and weight-bearing conditions, but not in MSTN-DN mice. Under unloading condition, CTX-injected muscle weight and fiber CSA in MSTN-DN mice were significantly higher than those in WT mice. CTX-injected muscle had many damaged and regenerating fibers having central nuclei in both WT and MSTN-DN mice. Significant increase in the population of Pax7-positive nuclei in CTX-injected muscle was observed in MSTN-DN mice, but not in WT mice. Evidences indicate that the suppression of MSTN cause to increase the regenerative potential of injured soleus muscle via the increase in the population of muscle satellite cells regardless of unloading conditions.     

Activity-induced fiber regeneration in rat soleus muscle

In an attempt to understand why muscle recovery is limited following atrophy due to limb immobilization, satellite cell activity and muscle fiber regeneration were analyzed in rat soleus muscles. Adult rat hindlimbs were immobilized in plaster casts for a period of two to ten weeks. Soleus muscles were examined by electron microscopy for evidence of fiber degeneration or regeneration, and to quantify satellite cell nuclei. Immunocytochemical localization of embryonic myosin was used to identify regenerating myofibers. Soleus muscle wet weight to body weight ratios for the casted muscles significantly decreased over the 10-week immobilization period. The casted muscles displayed ultrastructural evidence of minor fiber damage, including myofibrillar atrophy, Z-disc disruption, and abnormal triadic junctions. No ultrastructural evidence of regeneration was seen in the casted animals. The number of satellite cells in the casted muscles significantly decreased from 6.4% to 3. 3% by eight to 10 weeks of immobilization. Approximately 1.0% of extrafusal fibers in the control soleus muscles appeared to be regenerating since they expressed embryonic myosin and were of a small diameter, while in casted muscles, only 0.1% of the fibers were embryonic myosin-positive. Following release from immobilization, a reappearance of embryonic myosin-positive fibers was noted within four days of renewed activity. In contrast to control muscles, embryonic myosin-positive fibers in the recovery muscles included both small and large diameter fibers. Subtle changes in functional activity influence muscle damage and subsequent myofiber regeneration. Reduced activity reduces muscle fiber regeneration, while increased activity, as seen by increased hindlimb weight bearing and return to normal activity following immobilization, increase regenerating fibers and also the expression of embryonic myosin in adult fibers.     

Hindlimb suspension reduces muscle regeneration

Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2′-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P<0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P<0.05) BrdU labeling in uninjured soleus muscles compared to weightbearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P>0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers.     

Activity‐induced fiber regeneration in rat soleus muscle

In an attempt to understand why muscle recovery is limited following atrophy due to limb immobilization, satellite cell activity and muscle fiber regeneration were analyzed in rat soleus muscles. Adult rat hindlimbs were immobilized in plaster casts for a period of two to ten weeks. Soleus muscles were examined by electron microscopy for evidence of fiber degeneration or regeneration, and to quantify satellite cell nuclei. Immunocytochemical localization of embryonic myosin was used to identify regenerating myofibers. Soleus muscle wet weight to body weight ratios for the casted muscles significantly decreased over the 10‐week immobilization period. The casted muscles displayed ultrastructural evidence of minor fiber damage, including myofibrillar atrophy, Z‐disc disruption, and abnormal triadic junctions. No ultrastructural evidence of regeneration was seen in the casted animals. The number of satellite cells in the casted muscles significantly decreased from 6.4% to 3.3% by eight to 10 weeks of immobilization. Approximately 1.0% of extrafusal fibers in the control soleus muscles appeared to be regenerating since they expressed embryonic myosin and were of a small diameter, while in casted muscles, only 0.1% of the fibers were embryonic myosin‐positive. Following release from immobilization, a reappearance of embryonic myosin‐positive fibers was noted within four days of renewed activity. In contrast to control muscles, embryonic myosin‐positive fibers in the recovery muscles included both small and large diameter fibers. Subtle changes in functional activity influence muscle damage and subsequent myofiber regeneration. Reduced activity reduces muscle fiber regeneration, while increased activity, as seen by increased hindlimb weight bearing and return to normal activity following immobilization, increase regenerating fibers and also the expression of embryonic myosin in adult fibers. Anat Rec 258:176–185, 2000. © 2000 Wiley‐Liss, Inc.     

Chronic stimulation accelerates functional recovery of immobilized soleus muscles of the rabbit

Unilateral immobilization of rabbit hindlimbs was carried out for 2 weeks with an ankle angle of 90 deg. This was followed either by natural recovery for a further 2 or 4 weeks, or by chronic electrical stimulation of the soleus nerve during 2 weeks recovery using a low-frequency pattern of activation. Immobilization caused gross degeneration and functional disruption of soleus. Mean fibre area was reduced by 60% compared with unoperated controls, twitch and tetanic tensions by 90% and there was speeding of the rising phase of contraction. Natural recovery for 2 weeks had little effect; some regeneration occurred but muscle fibres remained atrophied and immature as indicated by the histochemical expression of both fast and slow myosin. Function continued to be grossly impaired, twitch and tetanic tensions were 66 and 79% reduced and the time-to-peak twitch tension 31% faster than controls. Prolonging the natural recovery period to 4 weeks increased muscle fibre area to 60% of normal, and increased the acquisition of mature staining properties but most functional characteristics remained impaired. Stimulated muscles had normal fibre areas, mature appearance and functional improvements which matched and in most cases exceeded those seen after natural recovery for 4 weeks. Thus chronic low-frequency stimulation of soleus muscles accelerates recovery of structure and function following degenerative immobilization atrophy and may represent an important therapeutic aid in patient rehabilitation.     

Changes in muscle T 2 relaxation properties following spinal cord injury and locomotor training

Magnetic resonance (MR) is frequently used to study structural and biochemical properties of skeletal muscle. Changes in proton transverse relaxation (T ₂) properties have been used to study muscle cellular damage, as well as muscle activation during exercise protocols. In this study, we implemented MR imaging to characterize the T ₂ relaxation properties of rat hindlimb muscles following spinal cord injury (SCI) and locomotor training. After moderate midthoracic contusion SCI, Sprague–Dawley rats were assigned to either treadmill training, cycle training or an untrained group. T ₂ weighted images were obtained and mean muscle T ₂ times were calculated in the tibialis anterior, soleus, and gastrocnemius (GAS) muscles at pre-injury as well as at 1, 2, 4, 8, and 12 weeks post-injury. Following SCI, hindlimb muscles in untrained animals showed a significant increase in muscle T ₂, with the most dramatic shift (+5.46 ms) observed in soleus muscle at 1 week post-SCI. Subsequently, all muscle groups showed a spontaneous recovery in muscle T ₂ with normalized T ₂ values in the GAS and tibilias anterior muscles at 4 weeks and the soleus at 12 weeks post-SCI. Both training paradigms, treadmill and cycling training, accelerated the recovery of soleus muscle T ₂. As a result, soleus muscle T ₂ recovered back to pre-injury values within 3 weeks of training in both training groups. Finally, in vitro histological assessments of rat skeletal muscles demonstrated that there was no apparent muscle injury in any of the muscles studied at 1 week post-SCI.     

Electron microscopic and autoradiographic characterization of hindlimb muscle regeneration in the mdx mouse

The pattern of postnatal growth and development of skeletal muscle in mdx mice was studied by light and transmission electron microscopy and by autoradiography and was compared with that in their normal age-matched controls at 4 and 32 weeks of age. The muscle weights of both the extensor digitorum longus (EDL) and soleus muscles of mdx mice were significantly greater than those in control mice at both ages. Body weights of male and female mdx mice were also increased over controls up to 12 weeks of age. At 4 weeks, both the EDL and soleus muscles exhibited focal areas of degeneration, necrosis, and regeneration of centrally nucleated extrafusal fibers resulting in a wide range of fiber sizes. By 32 weeks, the majority of fibers in both muscles were centrally nucleated, and focal areas of recent regeneration were observed. By electron microscopy, the course of macrophage infiltration into areas of degenerating fibers and the ongoing regeneration of myofibers within redundant cylinders of external lamina were noted. This pattern was frequent in 4-week-old mdx muscles and was present to a lesser degree at 32 weeks. A notable lack of both adipose tissue infiltration and fibrotic change in the endomysium were observed in muscles at both ages. Autoradiograms of muscles from 4-week-old mdx mice injected with tritiated thymidine showed an increased proportion of labeled sublaminal nuclei at 24 and 48 hours after injection compared to controls. At 32 weeks of age, labeling of nuclei in muscles of mdx mice was also greater than in controls, but was reduced compared to muscle labeling in 4-week-old mdx mice. The observed features of mdx muscle tissue suggest that this animal model is more applicable to the study of regeneration dynamics than to Duchenne-type human muscular dystrophy.     

Nerve growth in botulinum toxin poisoned muscles

The rate of growth of motor axon sprouts in muscles fully paralysed with botulinum toxin was studied using the zinc iodide—osmium tetroxide stain. In the mouse soleus and peroneus muscles, the proportions of endplates with nerve terminal sprouts rose to almost 100% in two to three weeks. In both muscles, the initial rate of terminal sprout growth was rather slow—about 3 μm per day. Two to three weeks after the injection the rate had risen to about 15 μm per day. Substantial numbers of polyneuronally innervated muscle fibres could be demonstrated in the soleus, but not the peroneus, by twelve days after the injection. The soleus remained polyneuronally innervated over a year after the injection.The time course of the tension recovery by the blocked soleus nerve was compared with that of the tension development by a fibular nerve implanted into a fully paralysed or completely denervated soleus muscle. The rate of tension recovery by the fibular nerve was the same whether the soleus was denervated or simply blocked. After an initial delay, the recovery of tension by the paralysed soleus nerve followed a course very similar to that of the implanted nerves.The implanted nerve did not affect the initial terminal sprouting of the paralysed soleus endplates. However, after three weeks, sprouts from soleus nerve endplates on many fibular innervated muscle fibres had disappeared. The implanted nerve did not affect the sprouting of soleus endplates on the muscle fibres which it did not innervate. The recovery of tension by the soleus nerves to muscles innervated by implanted nerves was reduced.It is concluded (1) that recovery from paralysis after botulinum toxin poisoning is slow because formation of new extrajunctional synapses is slow; (2) that motor nerve terminals are more readily induced to sprout by changes occurring on their own muscle fibres than by inactivity induced changes in muscle fibres elsewhere in the same muscle; (3) that paralysis of the mouse soleus renders the muscle as receptive to innervation extrajunctionally as does denervation.     

Satellite cells and myonuclei in long-term denervated rat muscles

Background: The percentage of satellite cells rapidly decreases in aneurally regenerating soleus muscles of rat. Also denervation of intact muscles causes fiber loss and regeneration, but the fate of satellite cells is unknown; myonuclei have been suggested to undergo changes resembling those in apoptotic cells. Methods: Rat soleus and extensor digitorum longus (EDL) muscles were denervated at birth or at age 5 weeks and investigated after periods of up to 38 weeks. At least 400 myonuclei in each muscle were assessed by electron microscopy, and satellite cell nuclei were counted. In sity nick translation and tailing were performed after 30 weeks denervation in order to demonstrate DNA breaks associated with apoptosis. Results: Myotubes indicating regeneration were prominent in the adult denervated soleus and deep layers of EDL muscles after 7 weeks and in the superficial parts of EDL muscle after 16 weeks. The percentage of satellite cell nuclei slowly decreased to less than one fifth of normal after 20–30 weeks. Almost all satellite cells had vanished 10 weeks after neonatal denervation. Degenerating myonuclei in adult, but not in neonatally denervated muscles, remotely resembled apoptotic nuclei of lymphocytes, but no evidence of DNA breaks was found. Conclusion: Denervation of rat skeletal muscles causes, in addition to fiber atrophy, loss of fibers with subsequent regeneration. Proliferation of satellite cells under aneural conditions may lead to exhaustion of the satellite cell pool. This process is more rapid in growing than in adult muscles. Myonuclei in denervated muscles do not show DNA breaks which can be demonstrated by in situ nick translation. © 1995 Wiley-Liss, Inc.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [¹⁴C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [¹⁴C]-palmitic acid into the phospholipid moieties.     

Effects of hindlimb suspension on contractile properties of young and old rat muscles and the impact of electrical stimulation on the recovery process

The purpose of this study was to investigate the effects of hindlimb suspension (HS) on contractile properties of skeletal muscles of young and old rats and to determine the impact of electrical stimulation (ES) on the quality and degree of recovery of these muscles. After 21 days of HS, young soleus (SOL) muscle became faster, but there was no impact on young extensor digitorum longus (EDL) muscle. Twitch tension (Pt) decreased 61% in young and 70% in old SOL muscles. Specific tetanic tension (Po) decreased 53% in young and 64% in old SOL muscles, but again there was no impact on EDL muscle. After a 14-day period of recovery, contraction time (CT), half-relaxation time (RT1/2), Pt and Po returned to control group values in both young and old SOL muscles. Measurements of the contractile properties of young and old skeletal rat muscles showed ES sometimes to be beneficial but also sometimes to be harmful. A 14-day period of recovery, with or without ES, seemed sufficient for many variables to return to control group values.     

Nerve-dependent recovery of metabolic pathways in regenerating soleus muscles

Summary The metabolic recovery potential of muscle was studied in regenerating soleus muscles of young adult rats. Degeneration was induced by subfascial injection of a myotoxic snake venom. After regeneration for selected periods up to 2 weeks, samples of whole muscle were analysed for hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), lactate dehydrogenase (EC 1.1.11.27), adenylokinase (EC 2.7.4.3), creatine kinase (EC 2.7.3.2), malate dehydrogenase (EC 1.1.11.37), citrate synthase (ED 4.1.3.7) and -hydroxyacyl CoA dehydrogenase (EC 1.1.1.35). Lactate dehydrogenase, adenylokinase, malate dehydrogenase and -hydroxyacyl CoA dehydrogenase were also measured in individual fibres of muscle regenerating up to 4 weeks. We found that in the presence of nerve there was complete recovery of muscle metabolic capacity. However, there were differences in the rate of recovery of the activity of enzymes belonging to different energy-generating pathways. Lactate dehydrogenase, an enzyme representing glycolytic metabolism, reached normal activity immediately upon myofibre formation, only 3 days after venom injection, while oxidative enzymes required a week or more to reach normal activity levels. The delay in oxidative enzyme recovery coincided with physiological parameters of reinnervation. Therefore, to further test the role of nerve on the metabolic recovery process, muscle regeneration was studied following venom-induced degeneration coupled with denervation. In the absence of innervation, most enzymes failed to recover to normal activity levels. Lactate dehydrogenase was the only enzyme to achieve normal levels, and it did so as rapidly as in innervated-regenerating soleus muscles. The remainder of the glycotytic enzymes and the high energy phosphate enzymes recovered only partially. Oxidative enzymes showed no recovery and were severely reduced in the absence of reinnervation. Thus, it appears that enzymes of oxidative metabolism are more dependent upon innervation than enzymes of glycolytic metabolism for full expression in regenerating soleus muscle.     

Relationship between skin blood flow and sweating rate in prepubertal boys and young men

We have examined the effect of male sexual hormones on the regeneration of skeletal muscles. Degeneration/regeneration of the left soleus and extensor digitorum longus muscles (EDL) of Wistar male rats was induced by an injection of snake venom (2 microg, Notechis scutatus scutatus). During the muscle regeneration (25 days), rats were treated with either oil (CON), nandrolone (NAN), NAN combined with exercise (NAN + EXE) or were castrated (CAS). Muscle growth and myosin heavy chain (MyHC) isoform content of regenerating muscles were studied. Castration altered the concentrations of MyHC in venom-treated EDL (P < 0.01) and soleus (P < 0.05). NAN increased the mass (P < 0.01) of regenerating soleus and decreased the relative amount of fast MyHC protein (% of total, P < 0.05). The effect of NAN + EXE on the fast MyHC proteins of venom-treated soleus was opposite (P < 0.05). NAN and NAN + EXE were without effect on the regenerating EDL (P > 0.05). In conclusion, it is possible that male sexual hormones play a role in the growth (synthesis of contractile proteins) of regenerating muscles in rat. In addition, contrary to NAN + EXE, NAN could be beneficial to soleus regeneration.     

Physiological properties and pattern of innervation of regenerated muscles in the rat

The regeneration of fast and slow muscles was compared following "mincing" and replacement into their own or alien muscle bed. At intervals varying from 2 to 9 weeks the tension developed by the regenerated muscles was assessed and compared to that developed by the muscles from the contralateral unoperated side. This parameter was then taken as an indication of recovery. The regenerated muscles never developed more than half of the tension of the control muscles. Muscles regenerated in the bed of extensor digitorum longus became fast-twitch muscles and muscles regenerated in the bed of soleus became slow-twitch muscles, no matter whether they originated from an extensor digitorum longus or soleus "mince". The regeneration of the muscle tissue in the place of extensor digitorum longus developed better than in the place of soleus. The pattern of innervation of the regenerated muscles was analysed using a combined cholinesterase silver stain. Many of the regenerated fibres had more than one end plate and some end plates more than one axon terminal. These results show that in adult animals muscle redevelopment can occur, but only to a limited extent. Moreover, on reinnervation of regenerated muscle fibres the axons do not assume their original pattern of innervation.     

Thermal preconditioning prevents fiber type transformation of the unloading induced-atrophied muscle in rats

Muscle atrophy is accompanied by a slow-to-fast transformation of the slow muscle, e.g., the soleus muscle, which is characterized by a decrease in the expression of the slow myosin heavy chain (MyHC) isoform. Heat stress before hindlimb unloading, i.e., thermal preconditioning, has been shown to reduce the rate of disuse-induced muscle atrophy. The present study examined whether thermal preconditioning could prevent a slow-to-fast transformation of the MyHC isoform through the induction of heat-shock protein (HSP) 72. Thermally preconditioned rats (Heat + HU) were individually placed in an environmentally controlled heat chamber for 1 h before hindlimb unloading for 2 weeks (HU). Although the mean fiber cross-sectional areas of the soleus muscle decreased in the HU and Heat + HU group, the loss of myofibrillar protein was attenuated in the Heat + HU group. Furthermore, a slow-to-fast transformation of MyHC isoform was inhibited in the Heat + HU group with the overexpression of HSP72. These results indicate that thermal preconditioning before hindlimb unloading attenuates the decrease of the slow MyHC isoform in the soleus muscle. Therefore, thermal preconditioning provides a new approach to prevent disuse-induced fiber type transformation of skeletal muscle.     

Functional regeneration in the hindlimb skeletal muscle of the mdx mouse

Summary The pattern of spontaneous skeletal muscle degeneration and clinical recovery in hindlimb muscles of the mdx mutant mouse was examined for functional and metabolic confirmation of apparent structural regeneration. The contractile properties, histochemical staining and myosin light chain and parvalbumin contents of extensor digitorum longus (EDL) and soleus (Sol) muscles of mdx and age-matched control mice were studied at 3–4 and 32 weeks. Histochemical staining (myofibrillar ATPase and NADH-tetrazolium reductase) revealed no significant change in slow-twitch-oxidative (SO) or fast-twitch-oxidative-glycolytic (FOG) fibre type proportions in mdx Sol apart from the normal age-related increase in SO fibres. At 32 weeks mdx EDL, however, showed significantly smaller fast-twitch-glycolytic (FG) and larger FOG proportions than those in control EDL. These fibre type distributions were confirmed by differential staining with antibodies to myosin slow-twitch and fast-twitch heavy chain isozymes. Frequency distribution of cross-sectional area for each fibre type showed a wider than normal range of areas especially in FOG fibres of mdx Sol, and FG fibres of mdx EDL, supporting previous observations using autoradiography of myofibre regeneration. Isometric twitch and tetanic tensions in Sol were significantly less than in controls at 4 weeks, but by 32 weeks, values were not different from age-matched controls. In mdx EDL at 3 weeks, twitch and tetanus tensions were significantly less, and time-to-peak twitch tensions were significantly faster than in control EDL. By 32 weeks, mdx EDL twitch and tetanus tensions expressed relative to muscle weight continued to be significantly lower than in age-matched controls, despite normal absolute tensions. The maximum velocity of shortening in 32-week mdx EDL was significantly lower than in control EDL. Myosin light chain distribution in mdx Sol exhibited significantly less light chain 2-slow (LC2s) and more light chain 1b-slow(LC1bs) at 32 weeks than age-matched control Sol. Gels of EDL from 32-week-old mdx mice showed significantly less light chain 2-fast-phosphorylated (LC2f-P) and light chain 3-fast (LC3f) and significantly more light chain 1-fast (LC1f) and light chain 2-fast (LC2f), but normal parvalbumin content compared to age-matched controls. These observations suggest that mdx hindlimb muscles are differentially affected by the disease process as it occurs in murine models of dystrophy. However, the uniqueness of mdx Sol and to a lesser extent EDL is that they also undergo an important degree of functional regeneration which is able to compensate spontaneously for degenerative influences of genetic origin. The mdx mutant may therefore be an important model for the study of regeneration by skeletal muscle, and of the nerve-muscle interactions which enable or restrict that regeneration.     

Fast and slow rat muscles degenerate and regenerate differently after whole crush injury

Summary The whole-crush injured rat skeletal muscle was used as a model to explore the regenerating potentialities of fast and slow muscles. Laminin was chosen to follow changes in basal lamina and desmin to visualize new muscular elements; they were revealed by immunofluorescence on cryostat sections of either fast (extensor digitorum longus) or slow (soleus) regenerating muscle. Soleus myolysis was rapid, extensive and heterogeneous. Basal laminae were nearly destroyed. In contrast, extensor digitorum longus maintained its basal lamina framework during myolysis. Soleus reconstruction began early, following the pattern of remaining basal laminae as closely as possible, but regeneration stagnated from day 16 and the regenerated muscle was fibrotic. In extensor digitorum longus, reconstruction progressed slower than in soleus, but regularly from the periphery toward the centre of the muscle. The regenerated extensor digitorum longus showed a quasi-normal structure from day 16. At the end of the process, the elimination of old basal lamina was completed in extensor digitorum longus, but was not achieved in soleus. We propose that the old basal lamina should help the initiation of reconstruction. This new model also underlines the importance of the turnover of basal laminae in muscular regeneration, and will be useful to understand the background of the different regenerative response of both muscles.