Essential role of satellite cells in the growth of rat soleus muscle fibers

Effects of gravitational loading or unloading on the growth-associated increase in the cross-sectional area and length of fibers, as well as the total fiber number, in soleus muscle were studied in rats. Furthermore, the roles of satellite cells and myonuclei in growth of these properties were also investigated. The hindlimb unloading by tail suspension was performed in newborn rats from postnatal day 4 to month 3 with or without 3-mo reloading. The morphological properties were measured in whole muscle and/or single fibers sampled from tendon to tendon. Growth-associated increases of soleus weight and fiber cross-sectional area in the unloaded group were approximately 68% and 69% less than the age-matched controls. However, the increases of number and length of fibers were not influenced by unloading. Growth-related increases of the number of quiescent satellite cells and myonuclei were inhibited by unloading. And the growth-related decrease of mitotically active satellite cells, seen even in controls (20%, P > 0.05), was also stimulated (80%). The increase of myonuclei during 3-mo unloading was only 40 times vs. 92 times in controls. Inhibited increase of myonuclear number was not related to apoptosis. The size of myonuclear domain in the unloaded group was less and that of single nuclei, which was decreased by growth, was larger than controls. However, all of these parameters, inhibited by unloading, were increased toward the control levels generally by reloading. It is suggested that the satellite cell-related stimulation in response to gravitational loading plays an essential role in the cross-sectional growth of soleus muscle fibers.     

Irreversible morphological changes in leg bone following chronic gravitational unloading of growing rats

Effects of gravitational unloading or loading on the growth and development of hindlimb bones were studied in rats. Male Wistar rats were hindlimb-unloaded or loaded at 2-G from the postnatal day 4 to month 3. The morphology and mineral content of tibia and fibula, as well as the mobility of ankle joints, were measured at the end of 3-month suspension or loading, and 1, 2, and 3 months after ambulation recovery. Growth-related increases of bone weight and mineral density were inhibited by unloading. But they were gradually recovered toward the control levels, even though they were still less than those in the age-matched controls after 3 months. None of the parameters were influenced by 2-G loading. However, here we report that chronic unloading causes abnormal morphological development in hindlimb bone of growing rats. Irreversible external bend of the shaft and rotation of the distal end of tibia, which limit the dorsiflexion of ankle joints, were induced following chronic gravitational unloading during developing period. It is also suggested that such phenomena are caused by the abnormal mechanical forces imposed by muscle utilization with altered patterns. The activity of ankle dorsiflexor was increased and that of plantarflexor was inhibited during unloading.     

Dependence of normal development of skeletal muscle in neonatal rats on load bearing

Antigravity function plays an important role in determining the morphological and physiological properties of the neuromuscular system. Inhibition of the normal development of the neuromuscular system is induced by hindlimb unloading during the neonatal period in rats. However, the role of gravitational loading on the development of skeletal muscle in rats is not well understood. It could be hypothesized that during the early postnatal period, i.e. when minimal weight-supporting activity occurs, the activity imposed by gravity would be of little consequence in directing the normal development of the skeletal musculature. We have addressed this issue by limiting the amount of postnatal weight-support activity of the hindlimbs of rats during the lactation period. We have focused on the development of three characteristics of the muscle fibers, i.e. size, myonuclear number and myosin heavy chain expression.     

Role of gravitational loading in the development of rat soleus muscle fibers

Effects of hindlimb unloading during the first 3 months after birth on the development of soleus muscle fibers were studied in rats. The mean absolute weigh and cross-sectional area of whole soleus muscle in the unloaded rats were -1/3 and 1/4 of those in the controls, respectively. But the unloading did not affect the lengths of muscle, at 90 degrees of ankle joint angle, and of muscle fibers sampled from tendon to tendon, and the total sarcomere number. Since the total number of fibers in soleus was not affected either, the inhibited increase of muscle mass following unloading was mainly due to the smaller CSA of individual fibers. Numbers of both myonuclei and satellite cells were significantly less in unloaded than control rats. The % distribution of fibers expressing pure type I myosin heavy chain was significantly less in unloaded than controls (-23 %). Further, muscle fibers with multiple innervation were noted in the unloaded rats. It is suggested that the development and/or differentiation of soleus muscle fibers are closely associated with gravitational loading and that the growth-associated increase in fiber number may be genetically programmed.     

Changes of bone morphology in response to hindlimb suspension of rats.

Effects of chronic hindlimb suspension or exposure to 2-G from postnatal day 4 to month 3 followed by ambulation recovery on the floor on the morphology of hindlimb bones were investigated in rats. The dorsi-flexion of the ankle was inhibited in the suspended group and such phenomena were not recovered at all. The mean weight and length of femur, tibia, and fibula were less than the cage controls at the end of suspension, but gradually increased during recovery. However, they were still less than those of the age-matched controls even after 3-month recovery. External bending of shaft and rotation of distal end of tibia were observed in the suspended group and these phenomena were not recovered at all. These morphological changes caused the inhibition of dorsi-flexion of ankle joints. The electromyogram activities of ankle plantar-flexors, soleus, plantaris, lateral gastrocnemius, were inhibited and those of dorsi-flexor, tibialis anterior, were increased during suspension. Typical changes in bone morphology were not induced by exposure to 2-G. It was suggested that gravitational unloading during developing period causes irreversible inhibition of normal bone growth. It was also indicated that the suspension-related changes in bone morphology may be caused by abnormal mechanical stress due to the altered mobilization of hindlimb muscles.     

Mechanical load-dependent regulation of satellite cell and fiber size in rat soleus muscle

The effects of mechanical unloading and reloading on the properties of rat soleus muscle fibers were investigated in male Wistar Hannover rats. Satellite cells in the fibers of control rats were distributed evenly throughout the fiber length. After 16 days of hindlimb unloading, the number of satellite cells in the central, but not the proximal or distal, region of the fiber was decreased. The number of satellite cells in the central region gradually increased during the 16-day period of reloading. The mean sarcomere length in the central region of the fibers was passively shortened during unloading due to the plantarflexed position at the ankle joint: sarcomere length was maintained at <2.1 µm, which is a critical length for tension development. Myonuclear number and domain size, fiber cross-sectional area, and the total number of mitotically active and quiescent satellite cells of whole muscle fibers were lower than control fibers after 16 days of unloading. These values then returned to control values after 16 days of reloading. These results suggest that satellite cells play an important role in the regulation of muscle fiber properties. The data also indicate that the satellite cell-related regulation of muscle fiber properties is dependent on the level of mechanical loading, which, in turn, is influenced by the mean sarcomere length. However, it is still unclear why the region-specific responses, which were obvious in satellite cells, were not induced in myonuclear number and fiber cross-sectional area. sarcomere     

Effects of gravitational loading on rat soleus muscle fibers following hindlimb suspension.

Effects of 16 days of hindlimb suspension and 16 days of ambulation recovery at 1-G or 2-G environment on the characteristics of soleus muscle fibers were studied in male Wistar Hannover rats. The mean cross-sectional area and myonuclear number in isolated single fibers at the termination of suspension were approximately 30% and 25% of the controls, respectively. Satellite cells were distributed evenly throughout the fiber length in the control. However, the number of satellite cells distributed at the middle of the fiber was less in the unloaded rats immediately after the termination of suspension. Both the numbers of quiescent and mitotic active satellite cell per fiber were approximately 57% less immediately after the termination of suspension than controls. The number of satellite cells at the end of fibers was increased first during the early phase of reloading. Subsequently, the number at the middle was gradually increased. The myonuclear number per fiber was also less (approximately 25%) in the unloaded than the age-matched control at the termination of suspension, but was increased following the recovery. Although the mean in vivo sarcomere length of the soleus muscle was shortened in response to plantarflexion of ankle joint, the length at the certain ankle joint angle was increased after 16 days of suspension due to sarcomere remodeling. The length at the proximal and distal, rather than the middle, portion of the fiber was stretched in both reloaded and control rats in response to dorsiflexion of the ankle joint. But it was noted that the magnitude of stretch was greater in the unloaded rats. It is suggested that the fiber end is more stimulated rapidly than the middle portion by the load applied to the muscle during the ambulation recovery.     

Developmental effects on myonuclear domain size of rat diaphragm fibers.

During early postnatal development in rat diaphragm muscle (Diam), significant fiber growth and transitions in myosin heavy chain (MHC) isoform expression occur. Similar to other skeletal muscles, Diam fibers are multinucleated, and each myonucleus regulates the gene products within a finite volume: the myonuclear domain (MND). We hypothesized that postnatal changes in fiber cross-sectional area (CSA) are associated with increased number of myonuclei so that the MND size is maintained. The Diam was removed at postnatal days 14 (P-14) and 28 (P-28). MHC isoform expression was determined by SDS-PAGE. Fiber CSA, myonuclear number, and MND size were measured using confocal microscopy. By P-14, significant coexpression of MHC isoforms was present with no fiber displaying singular expression of MHCNeo. By P-28, singular expression was predominant. MND size was not different across fiber types at P-14. Significant fiber growth was evident by P-28 at all fiber types (fiber CSA increased by 61, 93, and 147% at fibers expressing MHCSlow, MHC2A, and MHC2X, respectively). The number of myonuclei per unit of fiber length was similar across fibers at P-14, but it was greater at fibers expressing MHC2X at P-28. The total number of myonuclei per fiber also increased between P-14 and P-28 at all fiber types. Accordingly, MND size increased significantly by P-28 at all fiber types, and it became larger at fibers expressing MHC2X compared with fibers expressing MHCSlow or MHC2A. These results suggest that MND size is not maintained during the considerable fiber growth associated with postnatal development of the Diam.     

Load-dependent regulation of neuromuscular system

Roles of gravitational loading, sarcomere length, and/or tension development on the electromyogram (EMG) of soleus and afferent neurogram recorded at the L5 segmental level of spinal cord were investigated during parabolic flight of a jet airplane or hindlimb suspension in conscious rats. Both EMG and neurogram levels were increased when the gravity levels were elevated from 1-G to 2-G during the parabolic flight. They were decreased when the hindlimbs were unloaded by exposure to actual microgravity or by suspension. These phenomena were related to passive shortening of muscle fibers and/or sarcomeres. Unloading-related decrease in sarcomere length was greater at the central rather than the proximal and distal regions of fibers. These activities and tension development were not detected when the mean sarcomere length was less than 2.03 micrometers. It is suggested that load-dependent regulation of neuromuscular system is related to the tension development which is influenced by sarcomere length.     

Region-specific responses of adductor longus muscle to gravitational load-dependent activity in Wistar Hannover rats

Response of adductor longus (AL) muscle to gravitational unloading and reloading was studied. Male Wistar Hannover rats (5-wk old) were hindlimb-unloaded for 16 days with or without 16-day ambulation recovery. The electromyogram (EMG) activity in AL decreased after acute unloading, but that in the rostral region was even elevated during continuous unloading. The EMG levels in the caudal region gradually increased up to 6th day, but decreased again. Approximately 97% of fibers in the caudal region were pure type I at the beginning of experiment. Mean percentage of type I fibers in the rostral region was 61% and that of type I+II and II fiber was 14 and 25%, respectively. The percent type I fibers decreased and de novo appearance of type I+II was noted after unloading. But the fiber phenotype in caudal, not rostral and middle, region was normalized after 16-day ambulation. Pronounced atrophy after unloading and re-growth following ambulation was noted in type I fibers of the caudal region. Sarcomere length in the caudal region was passively shortened during unloading, but that in the rostral region was unchanged or even stretched slightly. Growth-associated increase of myonuclear number seen in the caudal region of control rats was inhibited by unloading. Number of mitotic active satellite cells decreased after unloading only in the caudal region. It was indicated that the responses of fiber properties in AL to unloading and reloading were closely related to the region-specific neural and mechanical activities, being the caudal region more responsive.     

β-Hydroxy-β-methylbutyrate reduces myonuclear apoptosis during recovery from hind limb suspension-induced muscle fiber atrophy in aged rats

β-Hydroxy-β-methylbutyrate (HMB) is a leucine metabolite shown to reduce protein catabolism in disease states and promote skeletal muscle hypertrophy in response to loading exercise. In this study, we evaluated the efficacy of HMB to reduce muscle wasting and promote muscle recovery following disuse in aged animals. Fisher 344×Brown Norway rats, 34 mo of age, were randomly assigned to receive either Ca-HMB (340 mg/kg body wt) or the water vehicle by gavage (n = 32/group). The animals received either 14 days of hindlimb suspension (HS, n = 8/diet group) or 14 days of unloading followed by 14 days of reloading (R; n = 8/diet group). Nonsuspended control animals were compared with suspended animals after 14 days of HS (n = 8) or after R (n = 8). HMB treatment prevented the decline in maximal in vivo isometric force output after 2 wk of recovery from hindlimb unloading. The HMB-treated animals had significantly greater plantaris and soleus fiber cross-sectional area compared with the vehicle-treated animals. HMB decreased the amount of TUNEL-positive nuclei in reloaded plantaris muscles (5.1% vs. 1.6%, P < 0.05) and soleus muscles (3.9% vs. 1.8%, P < 0.05). Although HMB did not significantly alter Bcl-2 protein abundance compared with vehicle treatment, HMB decreased Bax protein abundance following R, by 40% and 14% (P < 0.05) in plantaris and soleus muscles, respectively. Cleaved caspase-3 was reduced by 12% and 9% (P < 0.05) in HMB-treated reloaded plantaris and soleus muscles, compared with vehicle-treated animals. HMB reduced cleaved caspase-9 by 14% and 30% (P < 0.05) in reloaded plantaris and soleus muscles, respectively, compared with vehicle-treated animals. Although, HMB was unable to prevent unloading-induced atrophy, it attenuated the decrease in fiber area in fast and slow muscles after HS and R. HMB's ability to protect against muscle loss may be due in part to putative inhibition of myonuclear apoptosis via regulation of mitochondrial-associated caspase signaling.     

Role(s) of nucleoli and phosphorylation of ribosomal protein S6 and/or HSP27 in the regulation of muscle mass

Effects of 14 days of hindlimb unloading or synergist ablation-related overloading with or without deafferentation on the fiber cross-sectional area, myonuclear number, size, and domain, the number of nucleoli in a single myonucleus, and the levels in the phosphorylation of the ribosomal protein S6 (S6) and 27-kDa heat shock protein (HSP27) were studied in rat soleus. Hypertrophy of fibers (+24%), associated with increased nucleolar number (from 1–2 to 3–5) within a myonucleus and myonuclear domain (+27%) compared with the preexperimental level, was induced by synergist ablation. Such phenomena were associated with increased levels of phosphorylated S6 (+84%) and HSP27 (+28%). Fiber atrophy (–52%), associated with decreased number (–31%) and domain size (–28%) of myonuclei and phosphorylation of S6 (–98%) and HSP27 (–63%), and with increased myonuclear size (+19%) and ubiquitination of myosin heavy chain (+33%, P > 0.05), was observed after unloading, which inhibited the mechanical load. Responses to deafferentation, which inhibited electromyogram level (–47%), were basically similar to those caused by hindlimb unloading, although the magnitudes were minor. The deafferentation-related responses were prevented and nucleolar number was even increased (+18%) by addition of synergist ablation, even though the integrated electromyogram level was still 30% less than controls. It is suggested that the load-dependent maintenance or upregulation of the nucleolar number and/or phosphorylation of S6 and HSP27 plays the important role(s) in the regulation of muscle mass. It was also indicated that such regulation was not necessarily associated with the neural activity. rat soleus muscle; functional overload; deafferentation; 27-kDa heat shock protein; ubiquitination of myosin heavy chain     

Myonuclear domain and myosin phenotype in human soleus after bed rest with or without loading.

After 2 or 4 mo of bed rest (6 degrees head-down tilt) and 1 mo of ambulation, there was a tendency toward a higher percentage of fibers expressing fast myosin heavy chain (MHC) isoforms and a de novo appearance of fibers coexpressing type I+IIa+IIx and IIa+IIx MHC in human soleus fibers. After 2 and 4 mo of bed rest, the mean size of type I fibers decreased by 12 (P > 0.05) and 39%, respectively. Because myonuclear number/mm of fiber length was unchanged, myonuclear domain was smaller after bed rest than before. The mean size and myonuclear domain of type I fibers were largest after 1 mo of recovery. The effects of wearing an antigravity device (Penguin suit), which had a modest but continuous resistance at the knee and ankle (Penguin-1) or knee resistance without loading on the ankle (Penguin-2), for 10 consecutive h/day were determined during 2 mo of bed rest. Mean fiber sizes in Penguin-1, but not Penguin-2, group were maintained at or above pre-bed-rest levels, whereas neither group showed phenotype changes. Myonuclear domain in type I fibers was larger in Penguin-1 and smaller in Penguin-2 group post- compared with pre-bed rest, indicating that a single daily 10-h bout of modest muscle loading can prevent bed-rest-induced soleus fiber atrophy but has minimal effect on myosin phenotype. The specific adaptive cellular strategies involved may be a function of the duration and magnitude of the adaptive stimulus as well as the immediate activity history of the fiber before the newly changed functional demands.     

Running during recovery from hindlimb suspension induces transient muscle injury

The objectives were to study morphological adaptations of soleus muscle to decreased loading induced by hindlimb suspension and the effect of run training during the subsequent recovery period. Adult female Wistar rats were kept for 28 days with hindlimbs suspended. For the next 28 days, rats were assigned to a cage-sedentary or daily running group. Compared with control soleus muscles, 28 days of hindlimb suspension reduced the mass and fiber cross-sectional area to 58 and 53% of control values, respectively, and decreased type I fibers from 92 +/- 2 to 81 +/- 2%. During recovery, clusters of damaged fibers were observed in the soleus muscle, and this observation was more pronounced in trained animals. Type IIc fibers appeared transiently during recovery, and their presence was exacerbated with training, as IIc fibers increased to approximately 20% of the total by day 14 of recovery and were no longer evident at day 28. Although muscle wet mass does not differ as a result of mode of recovery at day 14, training transiently decreased the overall fiber area compared with sedentary recovery at this point. By day 28 of recovery the morphological characteristics of soleus muscle in the trained group did not differ from control muscle, whereas in the sedentary group muscle mass and overall fiber cross-sectional area were approximately 14% less than control values.     

Contractile properties, transversal stiffness and cytoskeletal protein content in Mongolian gerbils soleus fibers under long-term hindlimb suspension

Structural and functional changes in Mongolian gerbil soleus fibers were analyzed after a 31-day hindlimb suspension. Contractile properties of muscle fibers were studied by means of tensometry; the transversal stiffness of different parts of the contractile apparatus was measured by atomic force microscopy, resting calcium level was estimated by fluorescence microscopy by using Fluo-4-AM; cytoskeletal protein content was determined by western blotting. It was shown that after gravitational unloading the maximal force of contraction and specific tension of fiber were significantly reduced, as well as calcium sensitivity actually lowered. At the same time, the transversal stiffness of Z-disk in the relaxed and activated state was decreased significantly compared to the control group. Desmin content was at the control level, but alpha-actinin-2, main structural protein of Z-disk, became considerably less after a 31-day hindlimb suspension. Besides, resting calcium level remained at control values during the simulated gravitational unloading. The data suggest that Z-disk destruction, as a result of alpha-actinin-2 content reduction, leads to changes in the lattice spacing and decreases contractile properties.     

Sarcomeric cytoskeletal proteins and myosin phenotype in stretched soleus of hindlimb-suspended rats

Changes in sarcomeric cytoskeletal proteins of rat m. soleus fibers upon the chronic stretching against the background of gravitational unloading were analyzed and compared with changes in fiber size and myosin phenotype. For rats exposed to gravitational unloading in the usual microgravity-simulating experimental model (hindlimb suspension (HS) according to Morey-Holten), a considerable reduction in the mass of m. soleus (by 54%) and the area of its fibers of both slow-twitch (by 47%) and fast-twitch (37%) types compared with control animals was revealed. Moreover, the percent of fibers containing only slow isoforms of myosin heavy chains (MHC) for suspended animals was slightly smaller and the portion of fibers interacting only with antibodies against fast myosin isoforms was significantly higher than for control animals. For hindlimb-suspended rats, the titin/MHC and nebulin/MHC ratios appeared to be reduced almost by two times as compared with those for the contriol group of animals. Chronic immobilization of m. soleus in stretched state against the background of suspension leads to a partial or complete prevention of the reduction in muscle fiber sizes, the transformation of the myosin phenotype into fast one, and a decrease in relative content of sarcomeric cytoskeletal proteins.     

The contribution of muscle progenitor cells to maintaining morphological characteristics of unweighted rat soleus muscle during passive stretch

Skeletal muscle work hypertrophy is usually connected with muscle progenitor SC (satellite cells) activation with subsequent incorporation their nuclei into myofibers. Passive stretch of unloaded muscle was earlier established to prevent atrophic processes and be accompanied by enhanced protein synthesis. We hypothesized that elimination of SC proliferation capacity by gamma-irradiation would partly preavent stretched muscle fiber capability to maintain their size under condition of gravitational unloading. To assess the role of muscle progenitor (satellite) cells in development of passive stretch preventive effect SC proliferation was suppressed by local exposure to ionizing radiation (2500 Rad) and then subsequent hindlimb suspension or hindlimb suspension with concomitant passive stretch were carried out. Reduction of myofiber cross-sectional area and decrease in myo-nuclei number accompanying unloaded muscle atrophy were completely abolished by passive stretch both in irradiated and sham-treated animals. We concluded that satellite cells did not make essential contribution to passive stretch preventive action under condition of simulated weightlessness.     

Parameters of fibers cell respiration and desmin content in rat soleus muscle at early stages of gravitational unloading

The aim of the work was to study the parameters of fibers cell respiration and desmin content in Wistar rat soleus muscle after 1, 3, 7 and 14 days of gravitational unloading. Gravitational unloading was simulated by antiorthostatic hindlimb suspension. The parameters of cell respiration were determined using the polarography, and desmin content was assessed by means of Western blotting. The results showed that the intensity of cell respiration is reduced after three days of gravitational unloading, reaches a minimum level after seven days and slightly increases by the fourteenth day of hindlimb unloading, as well as the content of desmin, which, however, to the fourteenth day returns to the control level. Taking into account that mitochondrial function depends on the state of cytoskeleton the data allow us to assume that early reduction of the intensity of cell respiration under unloading could be caused by degradation of the protein desmin that determines intracellular localization of mitochondria.     

Size and myonuclear domains in Rhesus soleus muscle fibers: short-term spaceflight

The cross-sectional area (CSA), myonuclear number per mm of fiber length, and myonuclear domain (cytoplasmic volume/myonucleus) of mechanically isolated single fibers from biopsies of the soleus muscle of 5 vivarium control, 3 flight simulation and 2 flight (BION 11) Rhesus monkeys (Macaca [correction of Macacca] mulatta) were determined using confocal microscopy before and after a 14-day experimental period. Simulation monkeys were confined in chairs placed in capsules identical to those used during the flight. Fibers were classified as type I, type II or hybrid (containing both types I and II) based on myosin heavy chain (MHC) gel electrophoresis. A majority of the fibers sampled contained only type I MHC, i.e. 89, 62 and 68% for the control, simulation and flight groups, respectively. Most of the remaining fibers were hybrids, i.e. 8, 36 and 32% for the same groups. There were no significant pre-post differences in the fiber type composition for any of the experimental groups. There also were no significant pre-post differences in fiber CSA, myonuclear number or myonuclear domain. There was, however, a tendency for the fibers in the post-flight biopsies to have a smaller mean CSA and myonuclear domain (approximately 10%, p=0.07) than the fibers in the pre-flight biopsy. The combined mean cytoplasmic volume/myonucleus for all muscle fiber phenotypes in the Rhesus soleus muscle was approximately 25,000 micrometers3 and there were no differences in pre-post samples for the control and simulated groups. The cytoplasmic domains tended to be lower (p=0.08) after than before flight. No phenotype differences in cytoplasmic domains were observed. These data suggest that after a relatively short period of actual spaceflight, modest fiber atrophy occurs in the soleus muscle fibers without a concomitant change in myonuclear number.