Low-Frequency Electrical Stimulation of Denervated Skeletal Muscle Retards Muscle and Trabecular Bone Loss in Aged Rats.

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.

Electrical Stimulation of Denervated Rat Skeletal Muscle Ameliorates Bone Fragility and Muscle Loss in Early-Stage Disuse Musculoskeletal Atrophy.

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.

Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo.

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.

In Vivo Real-Time Imaging of Exogenous HGF-Triggered Cell Migration in Rat Intact Soleus Muscles.

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.

In situ real-time imaging of the satellite cells in rat intact and injured soleus muscles using quantum dots.

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.

Upregulation of osteogenic factors induced by high-impact jumping suppresses adipogenesis in marrow but not adipogenic transcription factors in rat tibiae.

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.

Changes in activities of daily living, physical fitness, and depressive symptoms after six-month periodic well-rounded exercise programs for older adults living in nursing homes or special nursing facilities.

A 6-month, twice weekly, well-rounded exercise program (47 sessions in total) comprised of a combination of aerobic, resistance and flexibility training was provided for institutionalized older adults aged 60 to 93. We analyzed the data of 18 older adults who could stand and had attended more than 10% of the classes (mean participation rate: 54%) to examine changes in activities of daily living (ADL), physical fitness tests and depressive moods. The mean (+/- standard deviation, range) age of the participants was 71.3 (+/- 15.6, 60-93) in men and 85.9 (+/- 5.8, 72-93) in women. Significant improvement in ADL of the hand manipulation domain and borderline significant improvement in ADL of the mobility domain were observed (McNemar test p = 0.011 and 0.072, respectively). A 6-minute walk distance increased significantly from 151.6 m to 236.6 m (p = 0.01, paired t-test), and the result of the Soda Pop test, which tests hand-eye coordination, also improved significantly from 35.2 sec to 25.3 sec (p = 0.01, paired t-test). These findings suggest that such a program could be effective in improving the ADL and physical fitness of the elderly.

Assembly of transverse tubule architecture in the middle and myotendinous junctional regions in developing rat skeletal muscle fibers.

The transverse (t)-tubule is responsible for the rapid inward spread of excitation from the sarcolemma to the inside of the muscle fiber, and the compartments of the t-tubule become highly and regularly organized during development. Although it is known that skeletal muscle fibers lengthen by adding sarcomeres at the myotendinous junction (MTJ) during development, no specific model exists for the assembly of new t-tubule architecture at the MTJ. We performed an electron-microscopic examination of the assembly of t-tubule architecture at the MTJ in developing rat skeletal muscle fibers. Although the longitudinally oriented t-tubule elements represent only a small fraction of the total t-tubule system in adult muscle fibers, they were observed at both A-band and I-band regions of middle and MTJ regions in early developmental stages, and gradually disappeared in the middle regions of muscle fibers during development; however, they remained in the MTJ even in adult muscle fibers. The frequency of pentads and heptads (two or three t-tubule elements with three or four elements of terminal cisternae, closely aligned with terminal cisternae of the sarcoplasmic reticulum) decreased during development, with sudden decrease between 7 and 10 weeks of age in the middle regions. Interestingly, although the frequency of decrease appeared to be higher in the middle region than in the MTJ regions in early (3- to 7-week) development, this pattern reversed, and the frequency of decrease was higher in the MTJ in later development (after 10 weeks of age). The MTJ maintained the features of immature membrane systems involved in e-c coupling much longer than the middle region of the fiber during development. The assembly of t-tubule architecture during postnatal development thus follows different processes in the middle and MTJ regions of skeletal muscle fibers.

Plasticity of neuromuscular junction architectures in rat slow and fast muscle fibers following temporary denervation and reinnervation processes.

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.

Plasticity of the transverse tubules following denervation and subsequent reinnervation in rat slow and fast muscle fibres.

We have studied the effects of short term denervation followed by reinnervation on the ultrastructure of the membrane systems and on the content of and distribution of key proteins involved in calcium regulation of fast-twitch (FT) extensor digitorum longus (EDL) and slow-twitch (ST) soleus (SOL) muscle fibres. Ischiadic nerve freezing resulted in total lack of neuromuscular transmission for 3 days followed by a slow recovery, but no decline in twitch force elicited by direct stimulation. The latter measurements indicate no significant atrophy within this time frame. The membrane systems of skeletal muscle fibres were visualized using Ca92+)-K3Fe(CN)6-OsO4 techniques and observed using a high voltage electron microscope. [3H]nitrendipine binding was used to detect levels of dihydropyridine receptor (DHPR) expression. The Ca2+ pumping free sarcoplasmic reticulum domains were not affected by the denervation, but the Ca2+ release domains were dramatically increased, particularly in the FT-EDL muscle fibres. The increase is evidenced by a doubling up of the areas of contacts between SR and transverse (t-) tubules, so that in place of the normal triadic arrangement, pentadic and heptadic junctions, formed by multiple interacting layers of ST and t-tubules are seen. Frequency of pentads and heptads increases and declines in parallel to the denervation and reinnervation but with a delay. Immunofluorecence and electron microscopy observations show presence of DHPR and ryanodine receptor clusters at pentads and heptads junctions. A significant (P < 0.01) positive correlation between the level of [3H]nitrendipine binding component and the frequency pentads and heptads was observed in both the FT-EDL and ST-SOL muscle fibres indicating that overexpression of DHPRs accompanies the build up extra junctional contacts. The results indicate that denervation reversibly affects the domains of the membrane systems involved in excitation-contraction coupling.

Degeneration and regeneration of neuromuscular junction architecture in rat skeletal muscle fibers damaged by bupivacaine hydrochloride.

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.

Age-related differences in the effect of running training on cardiac Myosin isozyme composition in rats.

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.