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 HARP during in vitro myogenesis and rat soleus muscle regeneration

Heparin affin regulatory peptide (HARP) is a heparin binding growth factor that belongs to a family of molecule whose biological function in myogenesis has been suspected without formal demonstration. In the present study, we investigated the expression and the distribution of HARP and its mRNA during soleus muscle regeneration using a crushed-induced regeneration model and also during differentiation of muscle satellite cells in primary cultures. We show that HARP mRNA and protein expression are increased during the regeneration process with a peak at day 5 after muscle crushing when new myotubes are formed. In situ hybridization and immunohistochemical studies showed that activated myoblasts expressed HARP at day two after crushing. Five days after muscle lesion, HARP is localised in newly formed myotubes as well as in prefused activated myoblasts. In regenerated myofibers, 15 days after crushing, expression of HARP was reduced. In vitro experiments using primary cultures of rat satellite cells indicated that HARP expression level increased during the differentiation process and peaked on fusion of myoblasts into myotubes. This is the first study demonstrating the presence of HARP in fusing myogenic cells suggests that this growth factor could play a function in myogenic differentiation.     

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.     

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.     

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.     

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.     

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.     

Bouts of passive stretching after immobilization of the rat soleus muscle increase collagen macromolecular organization and muscle fiber area

This study evaluated the effect of short bouts of stretching on the soleus muscle after immobilization, by measuring the birefringence of the intramuscular connective tissue (ICT) and the muscle fiber area. Thirty rats were divided into five groups: the left soleus was immobilized in the shortened position; after immobilization the animals remained free; after immobilization, the soleus was stretched daily (10 stretches of 60 sec followed by 30 sec rest); after immobilization, the soleus was stretched 3 times a week; control. Immobilization caused a loss of birefringence of the ICT and of muscle fiber area and only daily stretching increased both compared with the control (p< 0.01). In conclusion, short daily bouts of stretching after immobilization induced molecular reorganization of the collagen bundles and muscle fiber hypertrophy in the rat soleus.     

Development of tonic firing behavior in rat soleus muscle

Tonic firing behavior in soleus muscle of unrestrained rats aged 7 to 100 days was studied by chronic single-motor-unit and gross-electromyographic (EMG) recordings. Median motor-unit firing frequency at 10 days was 19–26 Hz and did not change appreciably after this time, whereas interval-to-interval firing variability was reduced with age. Two units with median frequencies 40 and 59 Hz were encountered in one 7-day-old rat. Integrated rectified gross EMG developed from being phasic only to predominantly tonic during the second and third postnatal week. From the end of the third week, rather short tonic periods with irregular amplitude were replaced by longer lasting constant-amplitude periods. Quantitatively, median duration of gross-EMG activity episodes more than doubled, while 90th-percentile values for episode duration increased 19-fold, from 7.4 s at 7 days to 140 s in adults. The main part of this increase took place after 22 days. Previous work in adult rats has indicated that descending monoaminergic innervation is essential for maintained tonic motoneuron activity, which probably is caused by depolarizing plateau potentials. Such innervation of the lumbar spinal cord matures gradually to an adult pattern and density 3–4 wk after birth. The present results, describing a concurrent considerable development of tonic firing behavior, support and extend these findings.     

Operant conditioning of reciprocal inhibition in rat soleus muscle

Operant conditioning of the H-reflex, the electrical analog of the spinal stretch reflex (SSR), induces activity-dependent plasticity in the spinal cord and might be used to improve locomotion after spinal cord injury. To further assess the potential clinical significance of spinal reflex conditioning, this study asks whether another well-defined spinal reflex pathway, the disynaptic pathway underlying reciprocal inhibition (RI), can also be operantly conditioned. Sprague-Dawley rats were implanted with electromyographic (EMG) electrodes in right soleus (SOL) and tibialis anterior (TA) muscles and a stimulating cuff on the common peroneal (CP) nerve. When background EMG in both muscles remained in defined ranges, CP stimulation elicited the TA H-reflex and SOL RI. After collection of control data for 20 days, each rat was exposed for 50 days to up-conditioning (RIup mode) or down-conditioning (RIdown mode) in which food reward occurred if SOL RI evoked by CP stimulation was more (RIup mode) or less (RIdown mode) than a criterion. TA and SOL background EMG and TA M response remained stable. In every rat, RI conditioning was successful (i.e., change > or =20% in the correct direction). In the RIup rats, final SOL RI averaged 171+/- 28% (mean +/- SE) of control, and final TA H-reflex averaged 114 +/- 14%. In the RIdown rats, final SOL RI averaged 37 +/- 13% of control, and final TA H-reflex averaged 60 +/- 18%. Final SOL RI and TA H-reflex sizes were significantly correlated. Thus like the SSR and the H-reflex, RI can be operantly conditioned; and conditioning one reflex can affect another reflex as well.     

Formation of unique vacuoles in tenotomized rat soleus muscle fibers

The formation of unique vacuoles in tenotomized rat soleus muscle fibers was examined by light and electron microscopy. After tenotomy at both proximal and distal tendons, virtually all muscle fibers underwent characteristic degenerative changes with a disorganization of myofibrils called the central core lesion, but eventually recovered. At 3 days after tenotomy, some muscle fibers showed small vacuoles in the sarcoplasm of the end segments, which were larger in diameter and paler in staining than those of the control fibers in light microscopy. At 5 days, more fibers formed larger vacuoles together with the extensive disorganization of myofibrils. Such vacuole formation was more conspicuous in the distal end than in the proximal end. At 1 week the myofibrillar disorganization was most extensive in the central areas, and vacuoles were considerably enlarged in some fibers to occupy most of the sarcoplasm near the fiber ends. Vacuoles decreased in number and size with time and could rarely be seen at 4 weeks postoperative. In thin-section electron microscopy, the early forms of vacuoles were often connected with the T-system tubules. The limiting membrane of such vacuoles possessed many caveolae, some of which appeared to be continuous with the T-system networks. The vacuole membrane was closely associated with the sarcoplasmic reticulum to form dyadic connections. In later stages, the vacuole membrane was lined in part with the basal lamina. From these findings, it can be concluded that the vacuoles are sarcolemmal in nature and derived from the T-system. The significances of the vacuole formation are discussed with special reference to the mechanism and fate of the vacuoles and their clinical implications.     

Lesions in the rat soleus muscle following eccentrically biased exercise

Morphological changes in skeletal muscle related to lenghening (eccentric) contractions have been noted by several laboratories. However, a systematic examination of skeletal muscle following repetitive ecentric ontractions is lacking. This study was undertaken to study lesions and determine their relative densities in rat soleus muscle 30 min following level or downhill treadmill exercise. Following fixation in situ by vascular perfusion, toluidine-blue-stained 1-μm sections of the muscle samples selected at 73-μm intervals were scanned with a light microscope. Three types of lesions were noted: focal disruptions in the A-band, localized dissolution of Z-lines, and clotting of muscle fibers. Soleus muscle from the caged controls and the tibialis anterior muscle from downhill-exercised rats were essentially free of lesions. Eighty-nine percent of the soleus m. lesions in the downhill runner group and 97% of those in the level runner group were A-band disruptions. A-band lesion density was significantly higher in the soleus muscle of the downhill runners compared to level runners with the highest incidence in the distal half. A-band lesion density was lower in soleus muscles from level runners; however, the highest intramuscular incidence was in the proximal rather than the distal end. The results indicate that a disruption of the A-band is a principal change within some skeletal muscle fibers 30 min following repetitive eccentric contractions.     

Ultrastructural and biochemical changes in rat soleus muscle following tenotomy

This study was designed to correlate changes in the rate-limiting enzymes of glycogen synthesis (glycogen synthase) and glycogen breakdown (glycogen phosphorylase) with the ultrastructural changes which occur in the soleus muscle following tenotomy. Soleus muscles were removed at 1, 2, 3, 7, 14, 21, and 63 days after tenotomy and were prepared for electron microscopy or frozen for enzyme analysis. In the first 7 days posttenotomy, soleus muscle fibers underwent a series of degenerative changes, while both synthase and phosphorylase activities decreased. Over the next 8 weeks the histological appearance of the soleus muscle eventually returned to normal while synthase and phosphorylase activities increased. We suggest that recovery from tenotomy involves an increase in the energy demands of the muscle, resulting in the increased activity of the key rate-limiting enzymes of muscle glycogen metabolism from the drastically reduced levels observed in the period before recovery begins.     

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.     

No change in skeletal muscle satellite cells in young and aging rat soleus muscle

Satellite cells are muscle stem cells capable of replenishing or increasing myonuclear number. It is postulated that a reduction in satellite cells may contribute to age-related sarcopenia. Studies investigating an age-related decline in satellite cells have produced equivocal results. This study compared the satellite cell content of young and aging soleus muscle in rat, using four different methods: dystrophin–laminin immunohistochemistry, MyoD immunohistochemistry, electron microscopy, and light microscopy of semi-thin sections. The absolute quantity of satellite cells increase with age, but satellite cell percentages were similar in young and aging soleus muscles. There were no differences in satellite cell quantity among MyoD immunohistochemistry, electron microscopy, and semi-thin sections. All three methods had significantly more satellite cells than with dystrophin–laminin immunohistochemistry. We conclude that satellite cell number does not decrease with age and postulate that satellite cell functionality may be responsible for age-related sarcopenia.