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.     

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.     

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 function of single muscle fibers after hindlimb suspension

The purpose of this investigation was to determine how muscle atrophy produced by the hindlimb suspension (HS) model alters the contractile function of slow- and fast-twitch single muscle fibers. After 2 wk of HS, small bundles of fibers were isolated from the soleus and the deep and superficial regions of the lateral and medial heads of the gastrocnemius, respectively. The bundles were placed in skinning solution and stored at -20 degrees C until studied. Single fibers were isolated and suspended between a motor arm and force transducer, the functional properties were studied, and subsequently the fiber type was established by myosin heavy chain (MHC) analysis on 1-D sodium dodecyl sulfate polyacrylamide gel electrophoresis. After HS, slow-twitch fibers of the soleus showed a significant reduction in fiber diameter (68 +/- 2 vs. 41 +/- 1 micron) and peak tension (1.37 +/- 0.01 vs. 0.99 +/- 0.06 kg/cm2), whereas the maximal shortening speed (Vmax) increased [1.49 +/- 0.11 vs. 1.92 +/- 0.14 fiber lengths (FL)/s]. A histogram showed two populations of fibers: one with Vmax values identical to control slow-twitch fibers and a second with significantly elevated Vmax values. This latter group frequently contained both slow and fast MHC protein isoforms. The pCa-force relation of the soleus slow-twitch fibers was shifted to the right; consequently, the free Ca2+ required for the onset of tension and for 50% of peak tension was significantly higher after HS. Slow-twitch fibers isolated from the gastrocnemius after HS showed a significant reduction in diameter (67 +/- 4 vs. 44 +/- 3 microns) and peak tension (1.2 +/- 0.06 vs. 0.96 +/- 0.07 kg/cm2), but Vmax was unaltered (1.70 +/- 0.13 vs. 1.65 +/- 0.18 FL/s). Fast-twitch fibers from the red gastrocnemius showed a significant reduction in diameter (59 +/- 2 vs. 49 +/- 3 microns) but no change in peak tension or Vmax. Fast-twitch fibers from the white superficial region of the medial head of the gastrocnemius were unaffected by HS. Collectively, these data suggest that the effects of HS on fiber function depend on the fiber type and location. Both slow-twitch type I and fast-twitch type IIa fibers atrophied; however, only slow-twitch fibers showed a decline in peak tension, and the increase in Vmax was restricted to a subpopulation of slow-twitch soleus fibers.     

Differential effects of mild therapeutic exercise during a period of inactivity on power generation in soleus type I single fibers with age

The purpose of this study was to investigate the effects of mild therapeutic exercise (treadmill) in preventing the inactivity-induced alterations in contractile properties (e.g., power, force, and velocity) of type I soleus single fibers in three different age groups. Young adult (5- to 12-mo-old), middle-aged (24- to 31-mo-old), and old (32- to 40-mo-old) F344BNF1 rats were randomly assigned to three experimental groups: weight-bearing control (CON), non-weight bearing (NWB), and NWB with exercise (NWBX). NWB rats were hindlimb suspended for 2 wk, representing inactivity. The NWBX rats were hindlimb suspended for 2 wk and received therapeutic exercise on a treadmill four times a day for 15 min each. Peak power and isometric maximal force were reduced following hindlimb suspension (HS) in all three age groups. HS decreased fiber diameter in young adult and old rats (-21 and -12%, respectively). Specific tension (isometric maximal force/cross-sectional area) was significantly reduced in both the middle-aged (-36%) and old (-23%) rats. The effects of the mild therapeutic exercise program on fiber diameter and contractile properties were age specific. Mild treadmill therapeutic exercise attenuated the HS-induced reduction in fiber diameter (+17%, 93% level of CON group) and peak power (μN·fiber length·s(-1)) (+46%, 63% level of CON group) in young adult rats. In the middle-aged animals, this exercise protocol improved peak power (+60%, 100% level of CON group) and normalized power (kN·m(-2)·fiber length·s(-1)) (+45%, 108% level of CON group). Interestingly, treadmill exercise resulted in a further reduction in shortening velocity (-42%, 67% level of CON group) and specific tension (-29%, 55% level of CON group) in the old animals. These results suggest that mild treadmill exercise is beneficial in attenuating and preventing inactivity-induced decline in peak power of type I soleus single fibers in young adult and middle-aged animals, respectively. However, this exercise program does not prevent the HS-induced decline in muscle function in the old animals.     

Intracellular Ca2+ transients in mouse soleus muscle after hindlimb unloading and reloading.

The objective of this study was to determine whether altered intracellular Ca(2+) handling contributes to the specific force loss in the soleus muscle after unloading and/or subsequent reloading of mouse hindlimbs. Three groups of female ICR mice were studied: 1) unloaded mice (n = 11) that were hindlimb suspended for 14 days, 2) reloaded mice (n = 10) that were returned to their cages for 1 day after 14 days of hindlimb suspension, and 3) control mice (n = 10) that had normal cage activity. Maximum isometric tetanic force (P(o)) was determined in the soleus muscle from the left hindlimb, and resting free cytosolic Ca(2+) concentration ([Ca(2+)](i)), tetanic [Ca(2+)](i), and 4-chloro-m-cresol-induced [Ca(2+)](i) were measured in the contralateral soleus muscle by confocal laser scanning microscopy. Unloading and reloading increased resting [Ca(2+)](i) above control by 36% and 24%, respectively. Although unloading reduced P(o) and specific force by 58% and 24%, respectively, compared with control mice, there was no difference in tetanic [Ca(2+)](i). P(o), specific force, and tetanic [Ca(2+)](i) were reduced by 58%, 23%, and 23%, respectively, in the reloaded animals compared with control mice; however, tetanic [Ca(2+)](i) was not different between unloaded and reloaded mice. These data indicate that although hindlimb suspension results in disturbed intracellular Ca(2+) homeostasis, changes in tetanic [Ca(2+)](i) do not contribute to force deficits. Compared with unloading, 24 h of physiological reloading in the mouse do not result in further changes in maximal strength or tetanic [Ca(2+)](i).     

Effects of mechanical over-loading on the properties of soleus muscle fibers, with or without damage, in wild type and mdx mice

Effects of mechanical over-loading on the characteristics of regenerating or normal soleus muscle fibers were studied in dystrophin-deficient (mdx) and wild type (WT) mice. Damage was also induced in WT mice by injection of cardiotoxin (CTX) into soleus muscle. Over-loading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mouse muscles by ablation of the distal tendons of plantaris and gastrocnemius muscles. All of the myonuclei in normal muscle of WT mice were distributed at the peripheral region. But, central myonuclei were noted in all fibers of WT mice regenerating from CTX-injection-related injury. Further, many fibers of mdx mice possessed central myonuclei and the distribution of such fibers was increased in response to over-loading, suggesting a shift of myonuclei from peripheral to central region. Approximately 1.4% branched fibers were seen in the intact muscle of mdx mice, although these fibers were not detected in WT mice. The percentage of these fibers in mdx, not in WT, mice was increased by over-loading (～51.2%). The fiber CSA in normal WT mice was increased by over-loading (p<0.05), but not in mdx and CTX-injected WT mice. It was suggested that compensatory hypertrophy is induced in normal muscle fibers of WT mice following functional over-loading. But, it was also indicated that muscle fibers in mdx mice are susceptible to mechanical over-loading and fiber splitting and shift of myonuclei from peripheral to central region are induced.     

Effect of inactivity and passive stretch on protein turnover in phasic and postural rat muscles

A state of hypokinesia and hypodynamia has been induced in the hindlimb muscles of the rat (100 g) using a suspension model. The ensuing muscle atrophy was assessed by reference to muscles in fully mobile control animals, which were either fed ad libitum or fed the same lower food intake of the suspended animals. Over a total of 7 days of suspension the slow-twitch postural soleus muscle underwent a much greater atrophy than the fast-twitch phasic extensor digitorum longus. Changes with respect to the position of the suspended foot, and hence muscle length, necessitate caution in comparing the extent of the atrophy between different muscle types. After 3 days of inactivity the atrophy of the soleus muscle was explained by a 21% decrease in the fractional rate of synthesis (measured in vivo) and a 100% increase in the rate of protein breakdown. The reduction in the synthetic rate was associated with a net loss (23%) of RNA and hence muscle ribosomes. In contrast when this inactive soleus muscle was permanently stretched the RNA content (44%) and protein synthetic rate increased (59%) markedly above control values. Although protein breakdown remained elevated in this stretched muscle, the extent of the atrophy in response to hypokinesia and hypodynamia was greatly reduced.     

Myosin subunits and contractile properties of single fibers from hypokinetic rat muscles

The effects of prolonged hypokinesia on the contractile properties and myosin isozymes of single fibers from the synergistic fast-twitch plantaris (PL) and slow-twitch soleus (SOL) skeletal muscles of adult rats were studied after 28 days of hindlimb suspension. There was a 31% increase in the mean maximal velocity of unloaded shortening (Vmax) among fibers from SOL with no change in the mean Vmax of fibers from PL after suspension. The myosin heavy and light chain (MHC and MLC) composition of bundles and the MHC composition of single fibers from control and suspended muscles were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase in the relative amount of fast-type MHC's in hypokinetic SOL and a smaller increase in the amount of fast-type MHC's in the PL. Relatively minor changes occurred in the MLC's during hypokinesia. As Vmax increased among individual fibers from control and suspended muscles, the relative amount of fast-type MHC's increased. The results demonstrate that the myosin isozyme composition of skeletal muscle, especially the heavy chains, is altered during hypokinesia, and this finding provides an explanation for changes in Vmax of rat single muscle fibers under the same conditions.     

Reloading of rat soleus after hindlimb unloading and serum insulin-like growth factor 1

The aim of this study was to elucidate the effects of 14- day hindlimb suspension (HS) and subsqquent reloading (3 or 7 days) on the m. soleus mass, muscle fiber cross-sectional area (CSA), soleus fiber properties and serum IGF-1 in rats. Rats were hindlimb suspended for 14 days or kept as controls (C, n = 7). Soleus muscles were isolated after HS (HS, n = 7) or after reambulation for either three (R3, n = 5) or seven days (R7, n = 6). Frozen serial sections of m. soleus were stained by primary monoclonal antibodies against MHCI. For measurement of concentration IGF-1 in the blood serum, test-system for IFA DSL-10-2800 Non-Extraction IGF-1 ELISA was used. Muscle mass was significantly reduced in HS (-35 %) but subsequently increased with reloading in R3 (-10 % to C) and was recovered to control values in R7 (+5 % to C). Fiber CSA was significantly reduced (-43 %) in HS and was greater in R7 than in HS and slightly greater than in R3. 14 days of HS resulted in a mean maximal tension reduced by 35 %. After 7 days of subsequent reloading the mean maximal specific tension was still low (-33 % to C) and didn't differ from HS level. The level in blood IGF-1 has obviously decreased during 14-day unloading by 48 %, remained at the same level in R3, and increased 10 fold in R7.     

Regulation of skeletal muscle dihydropyridine receptor gene expression by biomechanical unloading.

Biomechanical unloading of the rat soleus by hindlimb unweighting is known to induce atrophy and a slow- to fast-twitch transition of skeletal muscle contractile properties, particularly in slow-twitch muscles such as the soleus. The purpose of this study was to determine whether the expression of the dihydropyridine (DHP) receptor gene is upregulated in unloaded slow-twitch soleus muscles. A rat DHP receptor cDNA was isolated by screening a random-primed cDNA lambda gt10 library from denervated rat skeletal muscle with oligonucleotide probes complementary to the coding region of the rabbit DHP receptor cDNA. Muscle mass and DHP receptor mRNA expression were assessed 1, 4, 7, 14, and 28 days after hindlimb unweighting in rats by tail suspension. Isometric twitch contraction times of soleus muscles were measured at 28 days of unweighting. Northern blot analysis showed that tissue distribution of DHP receptor mRNA was specific for skeletal muscle and expression was 200% greater in control fast-twitch extensor digitorum longus (EDL) than in control soleus muscles. A significant stimulation (80%) in receptor message of the soleus was induced as early as 24 h of unloading without changes in muscle mass. Unloading for 28 days induced marked atrophy (control = 133 +/- 3 vs. unweighted = 62.4 +/- 1.8 mg), and expression of the DHP receptor mRNA in the soleus was indistinguishable from levels normally expressed in EDL muscles. These changes in mRNA expression are in the same direction as the 37% reduction in time to peak tension and 28% decrease in half-relaxation time 28 days after unweighting. Our results suggest that muscle loading necessary for weight support modulates the expression of the DHP receptor gene in the soleus muscle.     

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.     

The realization of a mechanical signal during gravitational unloading: The response of mTORC1 targets to eccentric contractions

The aim of this study was to assess the response of key mTORC1 substrates to a bout of contractile stimuli under different times of functional unloading. Functional unloading of hind-limb muscles was carried out by the method of antiorthostatic suspension. Twenty-eight Wistar rats were divided into four groups: control, and hindlimb suspension for 1, 3, and 7 days. After hindlimb suspension, isolated soleus muscles of rats were subjected to a bout of ex vivo eccentric contractions. The contents of phosphorylated forms of p70s6k and 4E-BP1 were then determined using western blotting. It was found that an eccentric load resulted in a significant increase in p70s6k phosphorylation and reduced 4E-BP1 phosphorylation both in control and suspended rats, but in the case of suspension the response was dramatically reduced. Thus, it can be concluded that a bout of eccentric contractions of isolated rat soleus muscle during functional unloading causes a weaker activation of the Akt-mTORC1-p70s6k signaling pathway compared with the control. This may indicate that it is important to maintain muscle tone for a more efficient muscle perception of an external mechanical signal and subsequent activation of anabolic signaling pathways.     

Stimulation of calcineurin Aalpha activity attenuates muscle pathophysiology in mdx dystrophic mice

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin Aalpha transgene (CnAalpha) was overexpressed in skeletal muscles of mdx (mdx CnAalpha*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnAalpha* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnAalpha* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnAalpha* than mdx mice. In the diaphragm, despite a slower phenotype and a approximately 35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnAalpha* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.     

Activity dependent characteristics of fast and slow muscle: biochemical and histochemical considerations

The effects of denervation and hindlimb suspension induced disuse on concentrations of ATP, phosphocreatine (PC), and fiber type profile were investigated in slow twitch soleus and fast twitch extensor digitorum longus (EDL) muscles. The results show that the soleus and EDL muscles differ in their dependency on loadbearing as a stimulus for maintaining normal energy metabolism and the biochemical and morphological characteristics of muscle fibers. As determined by R-P methodology, suspension reduced ATP and PC concentrations of the soleus to 26% and 56%, respectively, while, in EDL only, PC is reduced to 71% of control with no change in ATP. Both muscles, however, show identical losses in ATP and PC following denervation. The energy charge, an indicator of Pi availability in muscle was reduced significantly in both denervated muscles to 82% and 85% in soleus and EDL, respectively. No significant reduction of the energy charge was seen in the muscles from suspended rats. Thus, in parallel with the indirect regulation through muscle loadbearing, the nerve can effectively modulate the levels of high-energy phosphates more directly by some regulatory mechanisms independent of muscle type. Denervation and suspension disuse increased the proportion of type 2 fibers in the soleus with a concomitant decrease in type 1 fibers and a relative rise in the number of very small diameter fibers. The EDL showed only variation in fiber size.     

Development of rat muscle during short- and long-term hindlimb suspension

Histochemical and contractile properties of developing rat soleus (Sol) and plantaris (P) muscles were studied after hindlimb suspension to determine the effects of reduced activity levels on muscle development. Suspension (S) began at age 18 days and lasted for 14, 28, and 206 days, and results were compared with age-matched controls. Body weights were normal until 14 days and Sol growth was inhibited more than P, weighing 38 and 47% of controls at 46 and 224 days compared with 68 and 59% in P. The Sol did not develop into a slow-twitch (ST) muscle as evidenced by faster times to peak tension and half-relaxation times, faster times to develop 50% of maximum tetanic tension (Po) and a mean of 33% fewer ST fibers. Twitch tension and Po were lower in S-Sol and S-P, but force/cross-sectional area was unchanged. Fiber areas were smaller, but no structural changes characteristic of disuse atrophy were found. Fiber type populations were unchanged in P, and contractile properties were only minimally affected, demonstrating the greater importance of activity for ST muscles during development.     

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.     

Reloading of atrophied rat soleus muscle induces tenascin-C expression around damaged muscle fibers

The hypothesis was tested that mechanical loading, induced by hindlimb suspension and subsequent reloading, affects expression of the basement membrane components tenascin-C and fibronectin in the belly portion of rat soleus muscle. One day of reloading, but not the previous 14 days of hindlimb suspension, led to ectopic accumulation of tenascin-C and an increase of fibronectin in the endomysium of a proportion (8 and 15%) of muscle fibers. Large increases of tenascin-C (40-fold) and fibronectin (7-fold) mRNA within 1 day of reloading indicates the involvement of pretranslational mechanisms in tenascin-C and fibronectin accumulation. The endomysial accumulation of tenascin-C was maintained up to 14 days of reloading and was strongly associated with centrally nucleated fibers. The observations demonstrate that an unaccustomed increase of rat soleus muscle loading causes modification of the basement membrane of damaged muscle fibers through ectopic endomysial expression of tenascin-C.