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Electrical stimulation (ES)-induced muscle contraction has multiple effects; however, mechano-responsiveness of bone tissue declines with age. Here, we investigated whether daily low-frequency ES-induced muscle contraction treatment reduces muscle and bone loss and ameliorates bone fragility in early-stage disuse musculoskeletal atrophy in aged rats. Twenty-seven-month-old male rats were assigned to age-matched groups comprising the control (CON), sciatic nerve denervation (DN), or DN with direct low-frequency ES (DN+ES) groups. The structural and mechanical properties of the trabecular and cortical bone of the tibiae, and the morphological and functional properties of the tibialis anterior (TA) muscles were assessed one week after DN. ES-induced muscle contraction force mitigated denervation-induced muscle and trabecular bone loss and deterioration of the mechanical properties of the tibia mid-diaphysis, such as the stiffness, but not the maximal load, in aged rats. The TA muscle in the DN+ES group showed significant improvement in the myofiber cross-sectional area and muscle force relative to the DN group. These results suggest that low-frequency ES-induced muscle contraction treatment retards trabecular bone and muscle loss in aged rats in early-stage disuse musculoskeletal atrophy, and has beneficial effects on the functional properties of denervated skeletal muscle.

For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells.

The effects of the intermittent incorporation of high-intensity downhill running sessions into long-term endurance training were assessed by examination of the plantaris muscle of rats. First, the intrinsic effects of a single session were evaluated in otherwise sedentary rats. The experimental group showed histological injuries in 2-3 days after the session. In addition, compared with the sedentary control, the experimental group showed a sevenfold increase in the fraction of type IIc fibers, and deceases of 74 and 88% in tetanic force evoked indirectly and directly, respectively, by electric stimulation. The injured muscle fibers showed regeneration within 21 days as evidenced by centrally located nuclei. Next, the effects of intermittently incorporated downhill-running sessions into a 9-week endurance training regimen were tested using two experimental groups: Training and Training + Downhill. On the first day of the 1st, 3rd, 5th, and 7th weeks of the training period, the rats in the Training + Downhill group experienced downhill-running sessions. After the endurance training period, the plantaris muscle of the two experimental groups demonstrated higher fatigue resistance with an increase in the type IIa fiber fraction, at an expense of the type IIb fiber fraction. Compared with the Training group, the Training + Downhill group had a higher type IIa fiber fraction, with clusters of 70-100 type IIa fibers. These results indicate that intermittent high-intensity sessions promote the fiber type transition induced by daily endurance training. However, the potentially adverse effect of fiber type cluster formation suggests that there is an optimum intensity and frequency of the high-intensity exercise sessions for better enhancing the effects of long-term endurance training.

We tested whether daily muscle electrical stimulation (ES) can ameliorate the decrease in cortical bone strength as well as muscle and bone geometric and material properties in the early stages of disuse musculoskeletal atrophy. 7-week-old male F344 rats were randomly divided into three groups: age-matched control group (Cont); a sciatic denervation group (DN); and a DN + direct electrical stimulation group (DN + ES). Denervated tibialis anterior (TA) muscle in the DN + ES group received ES with 16 mA at 10 Hz for 30 min/day, 6 days/week. Micro CT, the three-point bending test, and immunohistochemistry were used to characterize cortical bone mechanical, structural, and material properties of tibiae. TA muscle in the DN + ES group showed significant improvement in muscle mass and myofiber cross-sectional area relative to the DN group. Maximal load and stiffness of tibiae, bone mineral density estimated by micro CT, and immunoreactivity of DMP1 in the cortical bone tissue were also significantly greater in the DN + ES group than in the DN group. These results suggest that daily ES-induced muscle contraction treatment reduced the decrease in muscle mass and cortical bone strength in early-stage disuse musculoskeletal atrophy and is associated with a beneficial effect on material properties such as mineralization of cortical bone tissue.

The transplantation of myogenic cells is a potentially effective therapy for muscular dystrophy. However, this therapy has achieved little success because the diffusion of transplanted myogenic cells is limited. Hepatocyte growth factor (HGF) is one of the primary triggers to induce myogenic cell migration in vitro. However, to our knowledge, whether exogenous HGF can trigger the migration of myogenic cells (i.e. satellite cells) in intact skeletal muscles in vivo has not been reported. We previously reported a novel in vivo real-time imaging method in rat skeletal muscles. Therefore, the present study examined the relationship between exogenous HGF treatment and cell migration in rat intact soleus muscles using this imaging method. As a result, it was indicated that the cell migration velocity was enhanced in response to increasing exogenous HGF concentration in skeletal muscles. Furthermore, the expression of MyoD was induced in satellite cells in response to HGF treatment. We first demonstrated in vivo real-time imaging of cell migration triggered by exogenous HGF in intact soleus muscles. The experimental method used in the present study will be a useful tool to understand further the regulatory mechanism of HGF-induced satellite cell migration in skeletal muscles in vivo.

Jump training is a high-impact training regimen that increases bone volume in young bones. The aim of our study was to determine whether downregulation of adipogenesis that is associated with upregulation of osteogenesis is detected after jump training in growing rat tibiae. Four-week-old rats were jump trained for 1, 2, or 4 weeks for 5 days/week, and the height of jumping progressively increased to 35 cm. We performed morphometry to directly quantitate changes in bone volume and marrow adipocyte distribution in tibiae after the jump training. We also examined changes in the expression of osteogenic and adipogenic transcription factor proteins and mRNAs after the jump training. Four weeks of jump training induced an increase in trabecular bone volume, which was associated with the recruitment of runt-related transcription factor 2 expressing cells, as well as a decrease in marrow fat volume. However, peroxisome proliferator-activated receptor-gamma2 protein and mRNA expression levels did not change after high-impact jump training. The mRNA expression levels of the adipocyte differentiation genes CCAAT/enhancer-binding proteins (C/EBPs)alpha, C/EBPbeta, and C/EBPdelta also showed no change during the training period in jump-trained rats. We suggest that the levels of osteogenic factors that were upregulated by mechanical loading from high-impact jumping suppress adipogenesis in marrow rather than adipogenic transcription factors.

Aim: Activated satellite cells (SCs) have the ability to reacquire a quiescent, undifferentiated state. Pax7 plays a crucial role in allowing activated SCs to undergo self-renewal. Because the increase in the SC population is induced during overload-induced skeletal muscle hypertrophy, it is possible that Pax7-regulated SC self-renewal is involved in the modulation of the SC population during the functional overload of skeletal muscles. However, the characteristics of the expression patterns of Pax7 in SCs during the functional overload of adult skeletal muscles are poorly understood. Methods: Using immunohistochemical approaches, we examined the temporal and spatial expression patterns of Pax7 expressed in SCs during the functional overloading of rat skeletal muscles. Results: The time course of Pax7 expression in SCs was similar to that of the expression of the differentiation regulatory factor myogenin during the early stage of functional overload. However, the percentage of SCs that expressed Pax7 was markedly higher than that of the SCs that expressed myogenin. Coexpression of Pax7 and myogenin was not detected in SCs. In addition, the expression of cyclin-dependent kinase inhibitor p21, which regulates cell cycle arrest and differentiation, was not detected in Pax7-positive SCs. Conclusion: These results suggest that Pax7-regulated self-renewal of SCs may be induced during the early stage of functional overload and may contribute to modulating the SC population in hypertrophied muscles. Furthermore, it was suggested that the numbers of SCs which underwent self-renewal may be higher than that of SCs which were provided as the additional myonuclei for hypertrophying myofibres.

We evaluated the effects of brief, temporary denervation caused by ischiadic nerve-freezing on the processes of degeneration and regeneration of ultrastructural features in neuromuscular junction (NMJ) architecture in different types of rat skeletal muscle fibers. Nerve terminal (NT) area was decreased significantly 12 h after nerve freezing in both fast-twitch (FT) and slow-twitch (ST) fibers. One day after nerve freezing, some terminal axons were absent; decrease in NT area was remarkable in ST fibers, and there was retraction of Schwann cells and perineural epithelial cells. Fiber type-specific differences were observed in pattern of decrease in NT area between 24 h and 7 days after nerve freezing (there was significantly more decrease in FT fibers). The primary synaptic cleft became shallow, and the secondary junctional folds shorter and wider, but the basement lamina filling the subneural apparatus was unaltered. The number of secondary junctional folds decreased gradually between 6 h and 14 days after nerve freezing in both types of fiber. In control muscle fibers, synaptic vesicle density (SVD) per terminal area was significantly higher in FT fibers. The SVD densities decreased following nerve freezing-induced destruction of NMJs, and were minimal 3 days in FT fibers or 7 days ST fibers after nerve freezing. At 3 weeks, regeneration of both FT and ST fibers was well advanced, and all parameters had recovered to control values in FT fibers 28 days after nerve freezing. Severe degradation of the ultrastructural features in NMJs occurred due to temporary denervation during muscle fiber degeneration processes, and these structural changes were all reversible and fiber type-specific.

We examined the effect of running training on age-related changes in cardiac myosin isozyme composition in rats. Female Fischer 344 rats (6, 12, 20, and 27 months old) were divided into two groups: sedentary control and trained. The trained group rats were trained by treadmill running for up to 60 minutes per day, 5 days per week for 8 weeks at up to 30 m per minute. In sedentary control rats, the proportion of V1 myosin, that is, alpha-myosin heavy chain (MyHC) isoform, decreased progressively from 6 to 27 months of age. In the younger age groups (6 or 12 months old), there was a shift from V1 myosin to V3 myosin (beta-MyHC isoform) in trained hearts. However, the training program did not induce a cardiac myosin isozyme transition in older rats (20 or 27 months old). These results suggest that the mechanisms mediating the responses of cardiac muscle to running training alter during aging.

We compared the morphological features of the membrane systems involved in excitation-contraction (E-C) coupling during early postnatal development stages in rat skeletal muscles (tibialis anterior) denervated either at birth or 7 days after birth. Four obvious structural changes are observed in the arrangement of the transverse (T) tubule network and the disposition of triads following early postnatal denervation: (1) an increase in the longitudinal segments of the T tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolae clusters, (4) the appearance of pentads and heptads (i.e. a close apposition of two or three T tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum). The increased presence of longitudinal T tubules parallels the loss of cross striations, and this in turn is due to misalignment of the myofibrils. The clusters of caveolae appear almost exclusively in muscle fibres denervated at birth, and pentads and heptads are more frequently observed in muscles denervated at 7 days. The differential growth of muscle fibres in response to denervation leads to the formation of abnormal membrane systems involved in the E-C coupling with very unique morphological features, which differ from the case of denervation in adult muscle fibres.

Single fibres from tibialis anterior, extensor digitorum longus, gastrocnemius and soleus muscles in young (4–week–old) and adult (35–week–old) Wistar male rats were classified into three types on the basis of their enzyme–histochemical features: slow–twitch oxidative (SO), fast–twitch oxidative and glycolytic (FOG) and fast–twitch glycolytic (FG) fibres. Ultrastructural (volume density of mitochondria: Vmt and Z line width) and metabolic (phosphofructokinase: PFK and succinate dehydrogenase: SDH activities) profiles were measured. PFK activity in all types of fibres1 was higher in adult rats, and the difference between the two age–groups (adult/young) was largest between FG, FOG and SO fibres respectively. SDH activity and Vmt were lower in adult rats in a similar way in all fibres. A significant positive correlation was observed between the Vmt and SDH activity in both age–groups. This positive correlation was very specific in fast–twitch and slow–twitch fibres. Changes in the Vmt did not relate directly to the changes in fibre cross–sectional area. The overall pattern indicates that glycolytic capacity of fast–twitch fibres in flexor muscles (TA and EDL) is higher than in extensor muscles (GC and SOL), and that oxidative capacity of all types of fibre in extensor muscles is higher than in flexor muscles. These profiles were changed by growth, and may be related to the specific differences in pattern of activity of each skeletal muscle, and may reflect differences in the recruitment order of different muscles.

The recruitment of satellite cells, which are located between the basement membrane and the plasma membrane in myofibers, is required for myofiber repair after muscle injury or disease. In particular, satellite cell migration has been focused on as a satellite cell response to muscle injury because satellite cell motility has been revealed in cell culture. On the other hand, in situ, it is poorly understood how satellite cell migration is involved in muscle regeneration after injury because in situ it has been technically very difficult to visualize living satellite cells localized within skeletal muscle. In the present study, using quantum dots conjugated to anti-M-cadherin antibody, we attempted the visualization of satellite cells in both intact and injured skeletal muscle of rat in situ. As a result, the present study is the first to demonstrate in situ real-time imaging of satellite cells localized within the skeletal muscle. Moreover, it was indicated that satellite cell migration toward an injured site was induced in injured muscle while spatiotemporal change in satellite cells did not occur in intact muscle. Thus, it was suggested that the satellite cell migration may play important roles in the regulation of muscle regeneration after injury. Moreover, the new method used in the present study will be a useful tool to develop satellite cell-based therapies for muscle injury or disease.

We evaluated the degeneration and regeneration of neuromuscular junctions (NMJs) on the extensor digitorum longus muscle of Fischer 344 rats between 4 h and 3 weeks after bupivacaine hydrochloride (BPVC) injection, which induces muscle fiber necrosis, using histochemical staining by acetylcholine esterase (AchE)-silver and electron microscopy. Degeneration of muscle fibers and NMJs was observed 4 h after BPVC injection. One week after BPVC injection, some terminal axons were almost completely retracted, and the level of basal lamina-associated AchE in some NMJ regions had gradually disappeared. At that time, the depression contained a few, mostly pit-like or elongated oval invaginations: the incipient junctional folds and some NMJs did not have any secondary junctional fold. By 2 weeks after the BPVC injection, secondary junctional folds began to develop; however, the number of secondary junctional folds was clearly less than that in normal NMJs. At 3 weeks when regeneration of muscle fibers was well advanced, the staining for AchE at the end-plates became stronger and better-defined. The volume density of mitochondria in the terminal area of the terminal significantly decreased upon BPVC-induced destruction of the NMJ, and the density reached the lowest value 24 h after BPVC injection. Significant changes in the ultrastructural features of the architecture of NMJs occurred in skeletal muscle fibers damaged by BPVC during both the degeneration and regeneration processes. The changes in the ultrastructural and morphological features of the NMJ architecture during the regeneration of degenerated muscle fibers resembled those that occur during the differentiation of normal muscle fibers.

1. Physiological evidence suggests that excitation-contraction (E-C) coupling failure results from eccentric contraction-induced muscle injury because of structural and morphological damage to membrane systems directly associated with the E-C coupling processes within skeletal muscle fibres. In this study using rats, we observed the ultrastructural features of the membrane systems of fast-twitch (FT) and slow-twitch (ST) muscle fibres involved in E-C coupling following level and downhill running exercise. Our aim was to find out whether mechanically mediated events following eccentric exercise caused disorder in the membrane systems involved in E-C coupling, and how soon after exercise such disorder occurred. We also compared the morphological changes of the membrane systems between ST and FT muscle fibres within the same muscles. 2. Single muscle fibres were dissected from triceps brachii muscles of male Fischer 344 rats after level or downhill (16 deg decline) motor-driven treadmill running (18 m min(-1), 5 min running with 2 min rest interval, 18 bouts). All single muscle fibres were histochemically classified into ST or FT fibres. The membrane systems were visualized using Ca(2+)-K(3)Fe(CN)(6)-OsO(4) techniques, and observed by high voltage electron microscopy (120-200 kV). 3. There were four obvious ultrastructural changes in the arrangement of the transverse (t)-tubules and the disposition of triads after the downhill running exercise: (1) an increase in the number of longitudinal segments of the t-tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolar clusters, and (4) the appearance of pentads and heptads (close apposition of two or three t-tubule elements with three or four elements of terminal cisternae of the sarcoplasmic reticulum). The caveolar clusters appeared almost exclusively in the ST fibres immediately after downhill running exercise and again 16 h later. The pentads and heptads appeared almost exclusively in the FT fibres, and their numbers increased dramatically 2-3 days after the downhill running exercise. 4. The eccentric exercise led to the formation of abnormal membrane systems involved in E-C coupling processes. These systems have unique morphological features, which differ between ST and FT fibres, even within the same skeletal muscle, and the damage appears to be concentrated in the FT fibres. These observations also support the idea that eccentric exercise- induced E-C coupling failure is due to physical and chemical disruption of the membrane systems involved in the E-C coupling process in skeletal muscle.

We observed the morphological features of the membrane systems (sarcoplasmic reticulum, transverse tubules and triads) involved with the excitation-contraction coupling in rat soleus and extensor digitorum longus muscle following two disuse protocols: denervation and immobilization. The immobilized positions were: maximum dorsal flexor (soleus were stretched and extensor digitorum longus were shortened), maximum plantar flexor (soleus were shortened and extensor digitorum longus were stretched), and midway between the dorsal flexor and plantar flexor. The arrangement of the membrane systems was disordered following both disuse conditions. Increases in transverse tubule network were apparent; there were clearly more triads than in normal fibres, and pentadic and heptadic structures (i.e., a close approximation of two or three transverse tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum) were frequently appeared following both denervation and immobilization. The most notable difference between the influence of denervation and immobilization on the membrane systems is the time at which the pentads and heptads appeared. They appeared much earlier (1 week after denervation) in denervated than in immobilized (3 or 4 weeks after immobilization) muscle fibres. On the other hand, the frequency of pentads and heptads is clearly related to the fibre type (significantly higher in extensor digitorum longus) and to extent of atrophy. The different influences of immobilization in each leg position suggest that disuse, but with neurotrophic factor(s), influences on the membrane systems were affected by sarcomere length, and the neurotrophic factor(s) and muscle activity were not always necessary to form mew membrane systems in disuse skeletal muscle fibres.

The cell body size and succinate dehydrogenase (SDH) activity of spinal motoneurons innervating the superficial and deep regions of the tibialis anterior muscle were studied in rats ranging in postnatal age from 3 to 11 weeks, by retrograde neuronal labeling using fluorescent neuronal tracers. The motoneurons innervating the tibialis anterior muscle were located primarily at the L4 spinal cord segment and those innervating the superficial and deep regions of the muscle were distributed throughout the entire extent of the motoneuron pool. The distribution of the motoneurons during postnatal development was similar to that observed in the adult animal. The mean cell body size of the motoneurons innervating the superficial region of the muscle in rats from 5 to 11 weeks of age was greater than that innervating the deep region at corresponding ages. The mean SDH activity of the motoneurons innervating the deep region of the muscle increased during postnatal development, while there were no changes in the mean SDH activity of those innervating the superficial region during this period. At 11 weeks of age, the motoneurons innervating the deep region of the muscle had a higher mean SDH activity than those innervating the superficial region. An inverse relationship between cell body size and SDH activity of motoneurons innervating both the superficial and deep regions of the muscle was observed, independent of age. These results indicate that motoneurons innervating the superficial and deep regions of the rat tibialis anterior muscle have different developmental patterns with regard to cell body size and SDH activity.