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.     

Effects of hindlimb suspension on soleus muscle fibers and their spinal motoneurons in Wistar Hannover rats

Cross-sectional areas and succinate dehydrogenase (SDH) activities of soleus muscle fibers and their spinal motoneurons in male Wistar Hannover rats were determined after 16 days of hindlimb suspension. A decreased percentage of type I fibers and an increased percentage of type I+II fibers were observed after hindlimb suspension. Cross-sectional areas of all types of fibers were smaller in the hindlimb suspended than control rats. SDH activities of all types of fibers did not change after hindlimb suspension. Numbers, cross-sectional areas, or SDH activities of spinal motoneurons did not change after hindlimb suspension. It is suggested that spinal motoneurons innervating the rat soleus muscle are not affected by decreased neuromuscular activity on Earth and that gravity itself is important for maintaining of spinal motoneuron metabolic properties.     

Effects of body suspension on soleus muscle fibres and spinal motoneurones in the rat.

1. After 14 days of body suspension, fibre type composition and fibre cross-sectional area in the soleus muscle of 17-week-old male Sprague-Dawley rats were investigated. Oxidative enzyme activity of soleus motoneurones in the spinal cord was also examined. 2. After suspension, soleus muscle weight decreased by 44.2%, the cross-sectional area of SO and FOG fibres decreased by 60.4% and 58.6%, respectively. 3. The percentage of fibre types was not changed by suspension. However, ATPase activity after alkaline preincubation was markedly inhibited in FOG fibres. 4. Oxidative enzyme activity of soleus motoneurones was not changed by suspension. 5. This study demonstrates that using mature animals body suspension induces atrophy of fast- and slow-twitch fibres accompanied with the selective inhibition in ATPase activity of fast-twitch fibres, and without changes in histochemical profiles of the corresponding motoneurones.     

History-dependent mechanical properties of permeabilized rat soleus muscle fibers.

Permeabilized rat soleus muscle fibers were subjected to repeated triangular length changes (paired ramp stretches/releases, 0.03 l(0), +/- 0.1 l(0) s(-1) imposed under sarcomere length control) to investigate whether the rate of stiffness recovery after movement increased with the level of Ca(2+) activation. Actively contracting fibers exhibited a characteristic tension response to stretch: tension rose sharply during the initial phase of the movement before dropping slightly to a plateau, which was maintained during the remainder of the stretch. When the fibers were stretched twice, the initial phase of the response was reduced by an amount that depended on both the level of Ca(2+) activation and the elapsed time since the first movement. Detailed analysis revealed three new and important findings. 1) The rates of stiffness and tension recovery and 2) the relative height of the tension plateau each increased with the level of Ca(2+) activation. 3) The tension plateau developed more quickly during the second stretch at high free Ca(2+) concentrations than at low. These findings are consistent with a cross-bridge mechanism but suggest that the rate of the force-generating power-stroke increases with the intracellular Ca(2+) concentration and cross-bridge strain.     

Quantitative assessment of degenerative changes in soleus muscle after hindlimb suspension and recovery

The aim of this study was to quantify the degenerative and regenerative changes in rat soleus muscle resulting from 3-week hindlimb suspension at 45° tilt (HS group, n = 8) and 4-week normal cage recovery (HS-R group, n = 7). Degenerative changes were quantified by microscope examination of muscle cross sections, and the myosin heavy chain (MHC) composition of soleus muscles was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At the end of 3-week hindlimb suspension, histological signs of muscle degenerative changes were detected in soleus muscles. There was a significant variability in the percentage of fibres referred to as degenerating (%dg) in individual animals in the HS group [%dg = 8.41 (SEM 0.5)%, range 4.66%–14.08%]. Moreover, %dg varied significantly along the length of the soleus muscle. The percentage of fibres with internal nuclei was less than %dg in HS-soleus muscles [4.12 (SEM 0.3)%, range 1.24%–8.86%]. In 4-week recovery rats, the greater part of the fibres that were not referred to as normal, retained central nuclei [15.8 (SEM 2.2)%, range 6.2%–21.1%]. A significant increase in the slow isoform of MHC was recorded in the HS-R rats, compared to muscles from age-matched rats (P < 0.01). These results would suggest that a cycle of myofibre degeneration-regeneration occurred during HS and passive recovery, and that the increased accumulation of slow MHC observed in soleus muscles after recovery from HS could be related to the prevalence of newly formed fibres. Accepted: 14 October 1996     

Effect of hindlimb suspension on the functional properties of slow and fast soleus fibers from three strains of mice.

Cross-sectional area (CSA), peak Ca2+-activated force (Po), fiber specific force (Po/CSA), and unloaded shortening velocity (Vo) were measured in slow-twitch [containing type I myosin heavy chain (MHC)] and fast-twitch (containing type II MHC) chemically skinned soleus muscle fiber segments obtained from three strains of weight-bearing and 7-day hindlimb-suspended (HS) mice. HS reduced soleus slow MHC content (from approximately 50 to approximately 33%) in CBA/J and ICR strains without affecting slow MHC content in C57BL/6 mice ( approximately 20% of total MHC). Two-way ANOVA revealed HS-induced reductions in CSA, Po, and Po/CSA of slow and fast fibers from all strains. Fiber Vo was elevated post-HS, but not consistently across strains. No MHC x HS treatment interactions were observed for any variable for C57BL/6 and CBA/J mice, and the two significant interactions found for the ICR strain (CSA, Po) appeared related to inherent pre-HS differences in slow vs. fast fiber CSA. In the mouse HS models studied here, fiber atrophy and contractile dysfunction were partially dependent on animal strain and generally independent of fiber MHC isoform content.     

Expression of myosin heavy chain isoforms along intrafusal fibers of rat soleus muscle spindles after 14 days of hindlimb unloading.

Morphological, contractile, histochemical, and electrophoretical characteristics of slow postural muscles are altered after hindlimb unloading (HU). However, very few data on intrafusal fibers (IFs) are available. Our aim was to determine the effects of 14 days of hindlimb unloading on the morphological and immunohistochemical characteristics of IF in rat soleus muscle. Thirty-three control and 32 unloaded spindles were analyzed. The number and distribution of muscle spindles did not appear to be affected after unloading. There was no significant difference in number, cross-sectional area, and histochemical properties of IF between the two groups. However, after unloading, a significant decrease in slow type 1 MHC isoform and a slight increase in slow-tonic MHC expression were observed in the B and C regions of the bag1 fibers. The alpha-cardiac MHC expression was significantly decreased along the entire length of the bag2 fibers and in the B and C regions of the bag1 fibers. In 12 muscle spindles, the chain fibers expressed the slow type 1 and alpha-cardiac MHC isoforms over a short distance of the A region, although these isoforms are not normally expressed. The most striking finding of the study was the relative resistance of muscle spindles to perturbation induced by HU.     

Disorganization of honey-comb immunoreactive pattern of desmin and plectin in rat atrophic soleus muscle fiber induced by hindlimb suspension

Immunoreactivity for desmin, plectin and alpha-actinin was investigated in rat atrophic soleus muscle fibers induced by hindlimb suspension between 1 and 4 weeks (hindlimb suspension group, HSG), and compared with that of the control group (CG). Some of the HSG for 4 weeks were allowed unrestricted cage activity for 2 weeks as the recovery group (RG). In the cross-sectioned muscle fibers of the CG, desmin and plectin showed honey-comb immunoreactive patterns extending throughout the sarcoplasm. Superimposed images by double immunofluorescence labeling showed overlapping of both immunoreactivities. In the longitudinally sectioned profiles, superimposed images of alpha-actinin and desmin were overlapped at the level of Z-discs. The focal disorganization of the above honeycomb immunoreactive patterns, followed by the reduction of the cross-sectional area (CSA) of atrophic soleus muscle fibers and the appearance of Z-streaming, uniquely arose in the HSG from the first week and extended throughout the sarcoplasm in proportion to the suspension period. Such honey-comb patterns of both desmin and plectin were already restored in the RG at 2 weeks, followed by the disappearance of Z-streaming, prior to the recovery of the CSA. These findings indicate that the disorganization of topological and structural relationships of desmin and plectin with Z-discs surrounding individual myofibrils is primarily evoked, which leads to Z-streaming of atrophic soleus muscle fibers, and that the restoration of the muscle activity results in an early arrangement recovery of desmin and plectin around myofibrils.     

Effect of deafferentation on the size and myosin phenotype of muscle fibers during stretching of rat m. soleus and gravitational unloading

The study was purposed to evaluate the contribution of the reflectory and local components during the chronic stretch of the postural muscle to the attenuation of the unloading-induced fiber size reduction and changes in the myosin heavy chain (MHC) profile. The surgical unilateral deafferentation (dorsal rhizotomy) was used. It was shown that unilateral deafferentation didn't influence on the amelioration of unloading-induced fiber size reduction in chronically stretched soleus muscle. Thus, the results obtained in the present study don't confirm the hypothesis, supposing the predominant contribution of the muscle afferent activation to the attenuation of unloading-induced fiber atrophy. Deafferentation of unloaded as well as control rats leads to the increase of the percentage of fibers expressing slow MYC isoforms.     

Preventive effect of nucleoprotein on hindlimb unloading-induced capillary regression in rat soleus muscle

We investigated the preventive effects of nucleoprotein on capillary regression and mitochondrial dysfunction induced by unloading in the soleus muscle of rats. Nucleoprotein is a supplement made from soft roe of salmon, and its major components are nucleotides and protamine. Adult male Sprague-Dawley rats were divided randomly into control, hindlimb unloading (HU), and hindlimb unloading plus nucleoprotein administration (HU+ NP) groups. Hindlimb unloading was carried out for 2 weeks in the rats belonging to the HU and the HU+ NP groups. The rats of the HU+ NP group were administered nucleoprotein (500 mg/kg) using a feeding needle twice a day for 2 weeks. Hindlimb unloading resulted in capillary regression, decreased succinate dehydrogenase activity of the muscle fiber, and decreased PGC-1α expression in the soleus muscle. These effects were prevented by administration of nucleoprotein. Nucleoprotein appears to prevent capillary regression and mitochondrial dysfunction caused by unloading of the skeletal muscle. Therefore, nucleoprotein supplementation may be an effective therapy for maintaining capillary network and mitochondrial metabolism of the muscle fiber during an unloading period.     

Contractile characteristics of single skinned rat soleus muscle fibers under gravitational load: effects of a calcium-binding agent

Excessive intracellular calcium accumulation is believed to trigger the development of functional and structural changes in muscle fibers under microgravity conditions. The hypothesis was testified in the 14-day hindlimb suspension study with the application of a Ca(2+)-binding agent (10% EGTA). Twenty one rats were divided into 3 groups: cage controls (7), hindlimb-suspended rats that received intraperitoneal injections of saline (7), and hindlimb-suspended rats with EGTA treatment. Whereas the diameter of muscle fibers of unloaded rat soleus muscle was 20% less than in the control group (and there were no significant differences between rats with injections of EGTA and without them), the decrease of maximal tension was more pronounced (more than 50%). This discrepancy resulted in a decrease of maximal specific tension. The value of absolute tension in rats treated with placebo was by 52%, and in EGTA-treated rats by 41% less than in the control group. Thus, there were no significant differences in specific tension between this group and the control group. Obviously, the injections of EGTA prevented the effects of those mechanisms that induce a decline of tension in muscle fibers but are not linked with the reduction of fiber size. The Ca/tension curve in hindlimb-suspended saline-treated rats shifted to the right so that the pCa thresholds changed from 6.85 +/- 0.03 in cage controls to 6.70 +/- 0.04 (p < 0.05), which indicates that myofibrils of unloaded soleus are less sensitive to Ca2+. At the same time, the pCa threshold in EGTA-treated hindlimb-suspended rats was 6.93 +/- 0.02. It is concluded that chronic binding of excess calcium results in an increase in Ca sensitivity indices.     

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.     

Proteomic analysis of rat soleus muscle undergoing hindlimb suspension-induced atrophy and reweighting hypertrophy

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy.     

Effects of oxygen deprivation on incubated rat soleus muscle

Isolated soleus muscle deprived of oxygen produces more lactate and alanine than oxygen-supplied muscle. Oxygenated muscle synthesized glutamine, while anoxic muscle used this amino acid. Oxygen deprivation decreased adenine nucleotides leading to the efflux of nucleosides. Protein synthesis and degradation responded differently to anoxia. Synthesis almost completely ceased, while proteolysis increased. Therefore, protein degradation in soleus muscle is enhanced when energy supplies and oxygen tension are low.     

Contractile properties of the isolated rat soleus muscle and its single skinned soleus fibers at the early stage of gravitational unloading: Facts and hypotheses

The contractile properties of the postural soleus muscle were studied in rats at the early stage of gravitational unloading (three-day hindlimb suspension) with regard to different modes of muscle contraction (twitch and tetanic contraction of the isolated muscle and calcium-induced contraction of isolated skinned fibers). A significant (p < 0.01) enhancement of the peak twitch tension of the muscles of suspended rats without changes in time-dependent characteristics was observed, although the half-relaxation time tended to decrease. The fiber diameter did not change (42.37 ± 0.76 vs. 43.43 ± 1.15 μm in controls). The calcium-induced peak isometric tensions in control and unloaded soleus muscles were 37.6 ± 1.52 and 32.1 ± 1.05 mg, respectively (decrease significant at p < 0.05). No changes in threshold calcium concentration were recorded, but the pCa₅₀ value in unloaded muscles decreased from 6.05 ± 0.02 in controls to 5.97 ± 0.02 (p ≤ 0.05), indicating loss of myofibrillar calcium sensitivity. The cooperativity coefficient ηn in control animals was 3.46 ± 0.16, and in suspended ones it decreased to 3.08 ± 0.11 (p < 0.05). Analysis with the Fluo-4AM calcium probe demonstrated that the intracellular Ca²⁺ concentration increased significantly after hindlimb suspension, whereas the relative contents of titin or nebulin did not change.     

Adaptation in synergistic muscles to soleus and plantaris muscle removal in the rat hindlimb.

Although the soleus muscle comprises only 6% of the ankle plantar flexor mass in the rat, a major role in stance and walking has been ascribed to it. The purpose of this study was to determine if removal of the soleus muscle would result in adaptations in the remaining gastrocnemius and plantaris muscles due to the new demands for force production imposed on them during stance or walking. A second purpose was to determine whether the mass or the fiber type of the muscle(s) removed was a more important determinant of compensatory adaptations. Male Sprague-Dawley rats underwent bilateral removal of soleus muscle, plantaris muscle, or both muscles. For comparison, compensatory hypertrophy was induced in soleus and plantaris muscles by gastrocnemius muscle ablation. After forty days, synergist muscles remaining intact were removed. Mass, and oxidative, glycolytic, and contractile enzyme activities were determined. Despite its role in stance and slow walking, removal of the soleus muscle did not elicit a measurable alteration in muscle mass, or in citrate synthase, lactate dehydrogenase, or myofibrillar ATPase activity in gastrocnemius or plantaris muscles. Similarly, removal of the plantaris muscle, or soleus and plantaris muscles, had no effect on the gastrocnemius muscle, suggesting that this muscle was able to easily meet the new demands placed on it. These results suggest that amount of muscle mass removed, rather than fiber type, is the most important stimulus for compensatory hypertrophy. They also suggest that slow-twitch motor units in the gastrocnemius muscle play an important role during stance and locomotion in the intact animal.