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.     

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.     

Upregulation of HSP72 in reloading rat soleus muscle after prolonged hindlimb unloading

To clarify the changes of heat shock protein (HSP) 72 in the rat soleus muscle after hind-limb unloading (HU) and during reloading, 7-week-old male Wistar rats were hind-limb-suspended for 9 weeks, thereafter ambulatory recovery was permitted for 8 weeks. The body and absolute soleus weights were significantly lower in the HU than in the age-matched control group after HU and during reloading. The soleus weight relative to body weight was also significantly lower in the HU than in the age-matched control group at the end of 9 weeks of suspension, but returned to the control level after 2 weeks of reloading. The HSP72 content decreased to 38% of the control level after HU and conversely increased to 165 and 175% of the control level after 2 and 4 weeks of reloading, respectively. The HSP72 content returned to the control level after 8 weeks of reloading. Thus our results showed that the expression of HSP72 was downregulated by HU and upregulated temporally over the level of the control during the reloading period, and they suggested that these down- and up-regulations of HSP72 may be related to many factors including mechanical stress or load applied to the muscle.     

Bupivacaine-induced regeneration of rat soleus muscle: ultrastructural and immunohistochemical aspects

The regeneration of soleus muscle injury induced by the bupivacaine model was studied ultrastructurally and immunohistochemically. Twenty-one young (age range 3-3.5 months) male Wistar rats were subjected to a single intramuscular injection of 1 mL of 0.5% Marcaine. The muscles were examined on biopsy days 1, 2, 3, 5, 7, 14, and 21. By day 1, mononuclear inflammatory cells had invaded the necrotic sarcoplasm. Degenerative morphological findings counted mainly for the hypercontracted fibers, dilation of sarcoplasmic reticulum, plasma membrane defects, mitochondrial alterations, and myofibril discontinuities. By day 2 proliferating myoblasts were seen with variety in shape, which fused on the day 3. Myotubes with multiple central nuclei and euchromatic nucleoli were formed by day 5. Asynchronous repair events were seen with bundles of myofilaments toward the core of the fibers, in contrast to the least mature distal growth cones, which had free myoblasts in proximity and formatted pseudopods. Chronologically asynchronous regeneration stages possibly suggested successive satellite cell activation profiles or heterogeneity in satellite cell population. In parallel with the electron microscopy, in light microscope immunocytochemistry, desmin- and vimentin-positive mononuclear cells were observed within the first 3 biopsy days, but as regeneration proceeded, desmin predominated over vimentin. Merosin immunoreactivity revealed preservation of the basal lamina, which is crucial for the stability and survival of myotubes. By day 21, fibers restored the overall control architecture.     

Embryonic myogenesis pathways in muscle regeneration.

Embryonic myogenesis involves the staged induction of myogenic regulatory factors and positional cues that dictate cell determination, proliferation, and differentiation into adult muscle. Muscle is able to regenerate after damage, and muscle regeneration is generally thought to recapitulate myogenesis during embryogenesis. There has been considerable progress in the delineation of myogenesis pathways during embryogenesis, but it is not known whether the same signaling pathways are relevant to muscle regeneration in adults. Here, we defined the subset of embryogenesis pathways induced in muscle regeneration using a 27 time-point in vivo muscle regeneration series. The embryonic Wnt (Wnt1, 3a, 7a, 11), Shh pathway, and the BMP (BMP2, 4, 7) pathway were not induced during muscle regeneration. Moreover, antagonists of Wnt signaling, sFRP1, sFRP2, and sFRP4 (secreted frizzled-related proteins) were significantly up-regulated, suggesting active inhibition of the Wnt pathway. The pro-differentiation FGFR4 pathway was transiently expressed at day 3, commensurate with expression of MyoD, Myogenin, Myf5, and Pax7. Protein verification studies showed fibroblast growth factor receptor 4 (FGFR4) protein to be strongly expressed in differentiating myoblasts and newly formed myotubes. We present evidence that FGF6 is likely the key ligand for FGFR4 during muscle regeneration, and further suggest that FGF6 is released from necrotic myofibers where it is then sequestered by basal laminae. We also confirmed activation of Notch1 in the regenerating muscle. Finally, known MyoD coactivators (MEF2A, p/CIP, TCF12) and repressors (Twist, Id2) were strongly induced at appropriate time points. Taken together, our results suggest that embryonic positional signals (Wnt, Shh, and BMP) are not induced in postnatal muscle regeneration, whereas cell-autonomous factors (Pax7, MRFs, FGFR4) involving muscle precursor proliferation and differentiation are recapitulated by muscle regeneration.     

Mechanics and fatigability of the rat soleus muscle during early reloading

In order to elucidate muscle functional changes by acute reloading, contractile and fatigue properties of the rat soleus muscle were investigated at three weeks of hindlimb suspension and the following 1 hr, 5 hr, 1 d, and 2 weeks of reloading. Compared to age-matched controls, three weeks of unloading caused significant changes in myofibrillar alignments, muscle mass relative to body mass (-43%), normalized tension (-35%), shortening velocity (+143%), and response times. Further significant changes were not observed during early reloading, because the transitional reverse process was gradual rather than abrupt. Although most of the muscle properties returned to the control level after two weeks of reloading, full recovery of the tissue would require more than the two-week period. Delayed recovery due to factors such as myofibrillar arrangement and fatigue resistance was apparent, which should be considered for rehabilitation after a long-term spaceflight or bed-rest.     

Reduced soleus muscle injury at long muscle length during contraction in the rat

Muscle injury was studied to test the hypotheses that maintaining the soleus muscle at a long muscle length during contraction prevents muscle injuries and that the prevention of initial muscle injuries reduces subsequent muscle damage. The rat sciatic nerve was stimulated for 30 min with plantar or dorsal flexion of the foot, and the time course of contraction-induced injuries was examined. The soleus muscle injuries were first classified into one of five types, and the percentages of aberrant sarcomere areas observed in the soleus muscle were then separately quantified by electron microscopy at 0, 1, 6, 12, and 24 h (n = 3) post-stimulation. At a short muscle length (plantar flexion) during contraction, the soleus muscle showed sarcomere hypercontraction (9.8 ± 2.5%, mean ± standard error) and Z-band disarrangement (31.0 ± 4.5%) at 0 h, sarcomere hypercontraction (6.7 ± 1.9%), Z-band disarrangement (28.0 ± 4.9%), and sarcomere hyperstretching (1.3 ± 1.3%) at 1 h, the absence of sarcomere hypercontraction, but Z-band disarrangement (6.7 ± 1.9%) and sarcomere hyperstretching (5.0 ± 1.8%) at 6 h, and myofilament disorganization at 12 and 24 h (5.2 ± 1.5 and 2.5 ± 1.0%, respectively). In contrast, the soleus muscles at a long muscle length (dorsal flexion) during contraction using a self-made brace showed alterations in 1.2–2.4% of sarcomeres at 0 h and afterwards. Desmin disappeared, and α-actinin immunostaining was weaker in areas of sarcomere hypercontraction, whereas dystrophin was always detected along the sarcoplasmic membrane, suggesting that the integrity of the sarcolemma was intact. These results indicate that initial and subsequent muscle injuries were significantly reduced at long muscle length during contraction, probably through the prevention of sarcomere hypercontraction, and that initial muscle injuries rapidly progress to other injuries or normal structure.     

Influence of chronic stretching upon rat soleus muscle during non-weight-bearing conditions

Morphological, contractile and histochemical properties as well as the myosin heavy chain (MHC) composition of rat soleus muscles were studied after 14 days of non-weight-bearing (NWB) and after immobilization of the foot in dorsiflexion of NWB rats. Significant reductions in soleus mass, fibre sizes and tetanic tension were found after 14 days of NWB. Furthermore, a transformation of the slow-twitch soleus muscle towards a faster type was characterized by a decrease in twitch time parameters, an increase in the fast-twitch type IIA fibre proportion and an increase in fast-twitch type MHC isoforms. Our results showed that the immobilization of the soleus muscle in a lengthened position during NWB not only prevented the loss of muscular mass and force output, but also counteracted the slow to faster shift in contractile and phenotypical parameters normally associated with NWB conditions.     

Decrease of MMP-9 activity improves soleus muscle regeneration

The regeneration of skeletal muscles relies on the function of satellite cells that are quiescent myogenic precursors associated with adult skeletal muscle fibers. Upon injury, the satellite cells are activated, divide extensively, and differentiate into new myofibers. These events are accompanied by the remodeling of the surrounding extracellular matrix, which is mediated by variety of factors, including matrix metalloproteinases (MMPs). Regeneration of certain type of muscles, such as Soleus slow twitch muscle, is often inefficient and hindered by the development of fibrosis. Here, we studied the effect of inhibition of MMP-9 and MMP-2 activity on the Soleus muscle regeneration in vivo and on the in vitro differentiation of myoblasts derived from this muscle. Using in situ and in-gel zymography, we tested the activity of these two MMPs in vivo, during regeneration of the muscle, and in vitro, during differentiation of the myoblasts. We also analyzed the histology of regenerating muscles and morphology of differentiating myoblasts. All these analyses showed that treatment with doxycycline and anti-MMP-9, but not MMP-2 antibody, significantly improved Soleus muscle regeneration and ameliorated development of excessive fibrosis, as well as delayed myoblast proliferation and differentiation in vitro.     

Effect of single and periodic contusion on the rat soleus muscle at different stages of regeneration

This work analyzed the rat soleus muscle after single and recurrent contusions at different stages of regeneration. A noninvasive contusion was produced by a type of drop‐mass equipment. The posterior region of the right hind limb received a trauma and both right and left soleus muscles were analyzed 1, 4, and 6 days after a single contusion (1×), and 6 and 30 days after periodic contusions (10×, one trauma per week for 10 weeks). Single contusion: there was no significant difference between right and left soleus muscle weight. All animals showed abundant signs of acute damage in the right soleus. AChE activity was identified in regeneration segments of the right soleus. Periodic contusions: there was an increase in the right soleus muscle weight (α = 5%) only in the animals evaluated 6 days after periodic contusions. The right soleus muscle showed a high incidence of chronic signs of damage, such as split fibers and a centralized nucleus, which predominated when compared with the acute signs. Right soleus muscles showed split fibers with AChE activity in both the proximal and middle regions. There was no difference in the incidence of muscle fiber types (I, II, and IIC) between right and left soleus muscles after periodic contusions. Skeletal muscle contusion is common in humans, especially in sport activities, where repetitive traumas are also frequent. The results of this work indicate that despite the regeneration process there is an important change in the morphological aspect of regenerated muscle fibers, which possibly affect muscle performance. Anat Rec 254:281–287, 1999. © 1999 Wiley‐Liss, Inc.     

The short-term influence of catecholamines on acid-base balance of rat soleus muscle in vitro

The short-term influence of catecholamines on surface pH (pHs) and intracellular pH (pHi) of superficial muscle fibres has been investigated in rat soleus in vitro using single-barrelled and double-barrelled glass micro-electrodes. pHs means the pH recorded at the surface of a muscle fibre. All measurements were performed in high-Ca2+ (10 mM) Ringer's solutions. Adrenaline caused an intracellular and surface acidification which increased with concentration in the range 6 X 10(-9)-6 X 10(-6) M. The effect was inhibited by propranolol (10(-5)M) but not by phentolamine (1.5 X 10(-7) M). Noradrenaline and isoproterenol (6 X 10(-6) M) also acidified the intracellular fluid. The relative effect of catecholamines on steady-state pHi was: adrenaline = isoproterenol greater than noradrenaline. Adrenaline (6 X 10(-9)-6 X 10(-6) M) did not accelerate pHi recovery following intracellular acid-loading by NH+4 or CO2. It is concluded that activation of beta-adrenoceptors by catecholamines causes an early intracellular acidosis presumably by enhancing synthesis of metabolic acids. The surface acidification seems at least partly due to non-ionic permeation of sarcolemma by metabolic acids, secondarily inducing accumulation of H+ ions at the cell surface.     

Force, relaxation and energy metabolism of rat soleus muscle during anaerobic contraction

Isolated soleus muscle of rat was stimulated electrically (2 Hz) for 7 min under anaerobic conditions. Isometric twitch tension decreased progressively and was 30% of the initial value at the end of stimulation. The decline in relative force was similar to that previously observed in fast twitch muscle and soleus can thus not be termed fatigue-resistant under anaerobic conditions. Phosphocreatine (PCr) decreased from (mean +/- SD) 61.1 +/- 4.4 at rest to 4.0 +/- 1.8 mmol kg-1 dry muscle (d.m.) after 7 min of stimulation, while lactate increased from 3.7 +/- 1.6 to 30 +/- 8 mmol kg-1 d.m. Energy was thus derived from complete utilization of PCr and a low rate of glycolysis resulting in an almost unchanged calculated intracellular pH. It is concluded that tension decline in soleus muscle is not due to decreased intracellular pH but is more related to the capacity to regenerate ATP at a sufficient rate. Contraction and relaxation time of the twitch remained practically constant during the stimulation period. In contrast prolonged activation of fast-twitch muscle results in a slowing of the relaxation of the twitch (Sahlin et al. 1981) and it has been suggested that this is caused by the decrease of intracellular pH. The constancy of both relaxation time and calculated pH in the fatigued soleus muscle is consistent with the hypothesis that there is a connection between these two parameters. In contrast to the twitch, relaxation of tension after a tetanus was prolonged in soleus. Hence, it appears that the rate limiting step for relaxation is different for a twitch than for a tetanus in soleus.     

Heat production and chemical change during isometric contraction of rat soleus muscle.

1. Methods are described whereby the soleus muscle of the rat may be used for the investigation of initial processes in the absence of oxidative recovery. 2. The anaerobic conditions employed had no effect on the concentration of phosphocreatine in resting muscle or the mechanical response during contraction. 3. Muscles were stimulated tetanically for 10 s at 17-18 degrees C. Measurements were made of the heat production and metabolic changes that occurred during a 13 s period following the first stimulus. 4. There was no detectable change in the concentration of ATP. Neither was there detectable activity of adenylate kinase or adenylate deaminase. The changes in the concentration of glycolytic intermediaries were undetectable or very small. 5. The change in the concentration of phosphocreatine was large and amounted to -127 +/- 11-4 mumol/mmol Ct (mean and S.E. of the mean, negative sign indicates break-down, Ct = free creatine + phosphocreatine) which is equivalent to about -2-13 mumol/g wet weight of muscle. The heat production was 6549 +/- 408 mJ/mmol Ct (mean and S.E. of mean) which is equivalent to about 110 mJ/g. 6. About 30% of the observed energy output is unaccounted for by measured metabolic changes. 7. The ratio of heat production (corrected for small amounts of glycolytic activity) to phosphocreatine hydrolysis was -49-7 +/- 5-6 kJ/mol (mean and S.E. of mean), in agreement with previous results using comparable contractions of frog muscle, but different from the enthalpy change associated with phosphocreatine hydrolysis under in vivo conditions (-34 kJ/mol). 8. The results support the notion that the discrepancy between energy output and metabolism is an indication of an unidentified process of substantial energetic significance that is common to a number of species.     

Non-uniform distribution of strain during stretch of relaxed skeletal muscle fibers from rat soleus muscle

Tension and regional average sarcomere length (L_s) behavior were examined during repeated stretches of single, permeabilized, relaxed muscle fibers isolated from the soleus muscles of rats. We tested the hypothesis that during stretches of single permeabilized fibers, the global fiber strain is distributed non-uniformly along the length of a relaxed fiber in a repeatable pattern. Each fiber was subjected to eight constant-velocity stretch and release cycles with a strain of 32% and strain rate of 54% s⁻¹. Stretch-release cycles were separated by a 4.5 min interval. Throughout each stretch-release cycle, sarcomere lengths were measured using a laser diffraction technique in which 20 contiguous sectors along the entire length of a fiber segment were scanned within 2 ms. The results revealed that: (1) the imposed length change was not distributed uniformly along the fiber, (2) the first stretch-release cycle differed from subsequent cycles in passive tension and in the distribution of global fiber strain, and (3) a characteristic “signature” for the L_s response emerged after cycle 3. The findings support the conclusions that longitudinal heterogeneity exists in the passive stiffness of individual muscle fibers and that preconditioning of fibers with stretch-release cycles produces a stable pattern of sarcomere strains.     

Reinnervation of partially denervated rat soleus muscle

The reinnervation of partially denervated rat soleus muscles by their interrupted, regenerating motor axons has been examined in adult white rats. If reinnervation occurred after the remaining, intact motor axons had sprouted to their full, maximal extent, then the regenerating axons formed synapses preferentially with denervated muscle fibers and not with fibers innervated by sprouts. The sprouted motor units retained their size as if no reinnervation had occurred. On the other hand, if reinnervation occurred early during the sprouting process, the sprouting motor units were never able to attain their maximal size. Further, some muscle fibers became innervated by both sprouted and regenerating axons. These "hyperinnervated" fibers lost their dual innervation within a few weeks. The sprouted axons seemed to be the nerve fibers preferentially eliminated from these hyperinnervated fibers, since during the loss of hyperinnervation the sprouted motor units decreased in size while the motor units formed by the regenerating axons did not change in size. It is proposed that the occurrence of hyperinnervation is influenced by the amount of time sprouting axons have to consolidate their synapses with muscle fibers. Further, it is proposed that on muscle fibers which can become hyperinnervated, the sprouted motor neurons are at a disadvantage in the competition for maintenance because of their larger unit sizes.     

Cadmium withdrawal contractures in rat soleus muscle fibres

Transient "Cd2+ withdrawal" contractures, with amplitudes of < or =60% peak tetanic tension, were seen when > or =3 mM Cd2+ was removed, after exposures lasting > or =5 min, from solutions bathing rat soleus fibres at 22 degrees C with Cl- as the major external anion. The minimum free [Cd2+] for withdrawal contractures was reduced twofold when the external anion was SO4(2-). Withdrawal contractures were not seen after removal of 3 mM Co2+, Zn2+ or La3+ and were not observed in rat extensor digitorum longus fibres. The contractures were not due to depolarization (membrane potential, Vm, did not change during Cd2+ removal) or to an influx of external Ca2+ (the transient tension increase was recorded when solutions either lacked Ca2+, or contained 2 mM Co2+). Cd2+ withdrawal contractures were abolished by inactivation of excitation-contraction coupling (ECC) following depolarization in 40 mM K+ for 20 min, and recovered from inactivation at the same time as twitch and tetanic contractions with repriming of ECC. Withdrawal contractures were depressed by agents that depress ECC, i.e. low [Ca2+], 2 mM Co2+, 30 mM Ca2+, 30 mM Mg2+ and 50 microM nifedipine. The results support a hypothesis in which withdrawing Cd2+ from the external solution induces a contracture by activating the voltage sensor for ECC.     

Heat-stress enhances proliferative potential in rat soleus muscle

The effects of heat-stress on proliferative potential in vivo were studied in rat skeletal muscle. Male Wistar rats (7-weeks-old) were divided into two groups: control (n=24) and heat-stressed (n=24). Rats in the experimental group were exposed to environmental heat-stress (41 degrees C for 60 min) in a heat chamber without anesthesia. The soleus muscles were dissected 1, 7, and 14 days after the heat exposure. The wet and dry weights of soleus muscle relative to body weight in the heat-stressed group were significantly higher than controls 7 days after the exposure (10.1% and 17.5%, respectively, p <0.05). The distribution of 5-bromo-2'-deoxyuridine and proliferating cell nuclear antigen-positive nuclei, that are the indicators for the cell proliferation, were increased by 2.2 and 5.1 times, respectively 1 day after heating (p <0.05). The expressions of heat shock protein 72 (58.0%) and phosphorylated p70S6 kinase (52.3%) were increased 1 day following heat exposure (p <0.05). These results suggest that heat-stress could promote the cell proliferation and induce muscular hypertrophy.     

Tenotomy-induced motor endplate alterations in rat soleus muscle

The effects of tenotomy on the ultrastructure of rat soleus muscle motor endplates were examined both qualitatively and quantitatively. Rat soleus muscle was studied 2 weeks following tenotomy and compared with normal littermates. The motor endplates from the tenotomized muscles were found to exhibit both degenerative and regenerative changes. Degeneration consisted of postjunctional fold breakdown, exposed junctional folds, myelin-like bodies within the sub-junctional sarcoplasm, and dense bodies within the Schwann cell cytoplasm. The regenerative changes consisted of several small nerve terminals occurring within the same primary synaptic cleft and several axons wrapped by the same Schwann cell. The results demonstrate that tenotomy induces denervation-like changes at endplates that lead to terminal sprouting within the neuromuscular junctional area and remodelling.