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     

Time course of soleus muscle myosin expression during hindlimb suspension and recovery

This study examined the time course of adult rodent soleus muscle myofibril and myosin isoform protein expression after 4, 8, 16, 28, and 56 days of hindlimb unweighting by tail suspension (S). The time course of soleus muscle recovery (R) was also examined after 28 days of hindlimb unweighting with an additional 4, 8, 16, and 28 days of unrestricted cage activity. During suspension, soleus muscle myofibril protein rapidly decreased from 34.3 +/- 3.1 (1.96SE) mg/pair in the control (C) group to 6.9 +/- 1.4 (1.96SE) mg/pair in S (t = 56 days). The calculated first-order degradation rate constant for this loss was kd = 0.17 days-1 [half time (t1/2) = 4.1 days]. The estimated slow myosin (SM) isoform content decreased from 13.4 +/- 2.0 (1.96SE) mg/pair in C to 2.1 +/- 0.2 (1.96SE) mg/pair in S (kd = 0.19 days-1, t1/2 = 3.6 days). The relative proportion of other myosin isoforms was increased at 28 and 56 days of suspension, reflecting an apparent de novo synthesis and the loss of SM. Recovery of contractile protein after 28 days of suspension was slower for both the myofibril protein and the SM isoform (kd = 0.07 days-1, t1/2 = 10 days). These data suggest that loss of weight bearing specifically affected the mechanisms of contractile protein expression reflected in soleus muscle protein degradation processes. In addition, the expression of the myosin isoforms were apparently differentially affected by the loss of weight-bearing activity.     

Activity influences on soleus muscle myosin during rodent hindlimb suspension

This study examined the effect of stationary ground support (2 and 4 h/day) and uphill running (1.5 h/day, 20 m/min, 30% grade) activity patterns on soleus muscle atrophy and slow myosin loss during 4 wk of rodent hindlimb unweighting by tail suspension. We also examined the effect of uphill running during the last 4 wk of an 8-wk hindlimb unweighting program and during 4 wk of cage recovery after 4 wk of hindlimb unweighting. All forms of activity partially spared soleus muscle weight (mg), myofibril protein (mg/muscle pair and microgram/mg muscle), and relative and absolute slow myosin (SM) isoform content (% of total and mg/muscle pair, P less than 0.05). Relative to the normal control soleus muscle, the uphill running regimens resulted in 1) increased fast myosin isoform content and 2) diminished recovery of SM isoform content when coupled with cage activity recovery. Four weeks of cage recovery after 4 wk of hindlimb unweighting resulted in recovery of the relative SM isoform content to proportions exceeding normal control values, suggesting an apparent degradation of any normally existing fast myosin. These results indicate that, in the context of the hindlimb unweighting model, the mechanisms controlling the expression of soleus muscle SM and fast myosin genes can be affected differently by the diverse activities of stationary ground support, unrestricted cage activity, and programmed uphill running.     

Changes in fiber composition of soleus muscle during rat hindlimb suspension

Chronic reduction of gravitational load in the rear limbs of rats to simulate the influence of near-zero gravity in skeletal muscles has been shown previously to elicit atrophy in the soleus muscle. Use of this model by the present investigation indicates that soleus atrophy was characterized by a decline in the number of fibers in groups that contained the slow isoenzyme of myosin and which were classified as type I from intensity of staining to myofibrillar actomyosin adenosinetriphosphatase (ATPase) and to NADH tetrazolium reductase. Furthermore total fiber number was not changed, whereas fibers containing the intermediate isoenzyme and those classified as type IIa increased. There results could be explained by either a change in the composition within existing fibers or a simultaneous loss of slow fibers and de novo synthesis of intermediate and fast fibers. Evidence for transformation included an absence of embryonic or neonatal myosin in muscles from suspended rats and the constant fiber number that was unchanged by 4 wk of suspension. Furthermore although fiber areas of both groups of type I and IIa fibers declined during suspension, variability of the fiber areas within each group did not increase.     

Rat soleus muscle ultrastructure after hindlimb suspension

The aim of the present investigation was to determine, by quantitative electron microscopy, the effects of a 5-wk tail-suspension period on rat soleus muscle ultrastructure. A marked decline (-60%) in muscle mass occurred. The mean fiber cross-sectional area decreased to a greater extent (-75%) than the capillary-to-fiber ratio (-37%), leading to a higher capillary density (+148%) after hypokinesia. The total mitochondrial volume density remained unchanged, whereas the volume density of myofibrils was slightly but significantly reduced (-6%). A shift from subsarcolemmal to interfibrillar mitochondria occurred. Interfibrillar mitochondrial volume density was highest near the fiber border and decreased toward the fiber center. An increase in volume density of satellite cells suggested muscle regenerative events. Soleus atrophy with tail suspension greatly decreases the muscular volume but leaves the ultrastructural composition of muscle fibers relatively unaffected.     

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.     

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.     

Effect of hindlimb immobilization and recovery on compensatory hypertrophied rat plantaris muscle

Compensatory hypertrophy of the rat plantaris muscle (PLT) was induced by ipsilateral gastrocnemius muscle ablation. Following 8 weeks (wks) of hypertrophy, hindlimbs were cast immobilized (HI) for 4 weeks after which weight bearing was unrestricted for 8 wks (recovery). Compensatory hypertrophy increased PLT wet weight/body weight ratio (83%), muscle fiber cross-sectional areas (1.5 to 2 fold), and the percent of slow oxidative (% SO) fibers (2 fold) in the experimental compared to the contralateral sham control muscle. PLT protein content and maximal activities of phosphofructokinase (PFK), mitochondrial glycerol phosphate dehydrogenase, and succinate dehydrogenase were unaltered with muscle hypertrophy. HI produced significant decreases in PFK activity (50%) and muscle fiber cross-sectional areas (50%) but did not significantly change the histochemical myofibrillar ATPase profile. Following remobilization, muscle weight/body weight ratio and maximal enzyme activities recovered to that of aged matched controls. Muscle fiber areas returned to pre-immobilization sizes but were approximately 25% smaller than aged matched control hypertrophy muscles. The % SO fibers in the hypertrophied muscle remained higher than controls but did not return to pre-immobilization values. These results indicate that biochemical and histochemical characteristics of hypertrophied rat PLT recover from HI during 8 wks of normal weight bearing similar to that of normal control muscle. However, the recovery time period was insufficient to allow complete compensation of fiber size to that of the age-matched control animals.     

Periodic weight support effects on rat soleus fibers after hindlimb suspension

The morphological and histochemical properties of the rat soleus were studied after 1 wk of hindlimb suspension, one model that removes the weight-bearing function of the hindlimbs. To examine the effectiveness of weight support activity in maintaining soleus mass, fiber size, and succinate dehydrogenase (SDH) activity, the hindlimbs of adult male Sprague-Dawley rats were suspended (HS) and half of these rats were walked on a treadmill for 40 min/day (10 min every 6 h) at 5 m/min and a 19 degree grade (HS-WS). Significant reductions in soleus mass and fiber size were found after 1 wk of HS. Weight support activity decreased the atrophic response by approximately 50%. In the alkaline myofibrillar adenosine triphosphatase (ATPase) dark-staining fibers, SDH activity was higher in the HS than control rats, whereas it was similar to control in the HS-WS rats. Total SDH activity (SDH activity X cross-sectional area) in fibers staining lightly for ATPase in HS and HS-WS rats was lower than in control rats, whereas in the darkly stained ATPase fibers it was similar among the three groups. No changes were observed in fiber type percentages after 1 wk of HS or HS-WS. The results suggest that short-duration, daily weight support activity can ameliorate, but not prevent, soleus atrophy induced by HS. Furthermore, fiber cross-sectional area is more responsive to periodic weight support in dark than light ATPase fibers. These results also demonstrate that muscle fiber atrophy need not be associated with a loss in SDH activity.     

Size and metabolic properties of single muscle fibers in rat soleus after hindlimb suspension

The effects of 28 days of hindlimb suspension (HS) and HS plus 10 daily forceful lengthening contractions on rat soleus muscle fibers were studied. Compared with age-matched controls (CON), soleus wet weights of suspended rats were significantly decreased (approximately 49%). In HS rats, the light adenosinetriphosphatase (ATPase) fibers (staining lightly for myosin ATPase, pH = 8.8) atrophied more than the dark ATPase fibers (staining darkly for myosin ATPase, pH = 8.8). Single-fiber alpha-glycerophosphate dehydrogenase (GPD) and succinate dehydrogenase (SDH) activities and the proportion of dark ATPase fibers were higher in HS than CON rats. Daily forceful lengthening contractions did not prevent the suspension-induced changes. These results considered in conjunction with a collaborative study on the mechanical properties of HS rats (Roy et al., accompanying paper) suggest a shift in the contractile potential of the muscle following HS without a deficit in SDH, a metabolic property commonly associated with resistance to fatigue. The results support the view that soleus muscle fibers can change from a slow-twitch oxidative to a fast-twitch oxidative-glycolytic profile, but rarely to a fast-twitch glycolytic one, and that SDH and GPD activity per volume of tissue can be maintained or increased even when there are severe losses of contractile proteins.     

Contractile properties of rat soleus muscle after 15 days of hindlimb suspension

The properties of the contractile elements interacting to develop force in atrophied rat soleus muscle were studied by using single skinned fibers, which permitted direct access to the contractile apparatus. Muscle atrophy was induced by 15 days of hindlimb suspension. Suspension resulted in a decrease of maximal tension relative to an important decline in fiber diameter. Ca affinity of the contractile proteins was not changed insofar as the tension-pCa relationship was not shifted along the pCa axis. However, after hindlimb suspension 1) the value of the Hill coefficient from the tension-pCa curve was found to be higher, 2) a higher Ca threshold for activation was reported, and 3) a significant increase in contraction kinetics was described. All these results suggested that after suspension the mechanical properties of the slow-twitch soleus appeared to resemble more closely those of a fast-twitch muscle. Our results were in complete agreement with published histochemical data.     

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.     

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.