Ciliary neurotrophic factor prevents unweighting-induced functional changes in rat soleus muscle.

The purpose of the present work was to see whether changes in rat soleus characteristics due to 3 wk of hindlimb suspension could be modified by ciliary neurotrophic factor (CNTF) treatment. Throughout the tail suspension period, the cytokine was delivered by means of an osmotic pump (flow rate 16 microg. kg(-1). h(-1)) implanted under the hindlimb skin. In contrast to extensor digitorum longus, CNTF treatment was able to reduce unweighting-induced atrophy in the soleus. Twitch and 146 mM potassium (K) tensions, measured in small bundles of unloaded soleus, decreased by 48 and 40%, respectively. Moreover, the time to peak tension and the time constant of relaxation of the twitch were 48 and 54% faster, respectively, in unloaded soleus than in normal muscle. On the contrary, twitch and 146 mM K contracture generated in CNTF-treated unloaded and normal soleus were not different. CNTF receptor-alpha mRNA expression increased in extensor digitorum longus and soleus unloaded nontreated muscles but was similar in CNTF-treated unloaded muscles. The present results demonstrate that exogenously provided CNTF could prevent functional changes occurring in soleus innervated muscle subject to unweighting.     

Profiles of connectin (titin) in atrophied soleus muscle induced by unloading of rats.

Responses of the properties of connectin molecules in the slow-twitch soleus (Sol) and fast-twitch extensor digitorum longus muscles of rats to 3 days of unloading with or without 3-day reloading were investigated. The wet weight (relative to body wt) of Sol, not of extensor digitorum longus, in the unloaded group was significantly less than in the age-matched control (P < 0.05). Immunoelectron microscopic analyses showed that a monoclonal antibody against connectin (SM1) bound to the I-band region close to the edge of the A band at resting length and moved reversibly away from the Z line as the muscle fibers were stretched. In Sol, the displacement of the SM1-bound dense spots in response to stretching decreased after hindlimb suspension. There were no changes in the molecular weights and the percent distributions of alpha- and beta-connectin in both muscles after hindlimb suspension. A significant increment of percent beta-connectin in Sol was observed after 3 days of reloading after hindlimb suspension (P < 0.05). It is suggested that the elasticity of connectin filaments in the I-band region of the atrophied Sol fibers was reduced relative to that of the control fibers. The lack of the elasticity in atrophied muscle fibers may cause a decrease in contractile function.     

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.     

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     

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.     

Increased response to insulin of glucose metabolism in the 6-day unloaded rat soleus muscle

Hind leg muscles of female rats (85-99 g) were unloaded by tail cast suspension for 6 days. In the fresh-frozen unloaded soleus, the significantly greater concentration of glycogen correlated with a lower activity ratio of glycogen phosphorylase (p less than 0.02). The activity ratio of glycogen synthase also was lower (p less than 0.001), possibly due to the higher concentration of glycogen. In isolated unloaded soleus, insulin (0.1 milliunit/ml) increased the oxidation of D-[U-14C]glucose, release of lactate and pyruvate, incorporation of D-[U-14C]glucose into glycogen, and the concentration of glucose 6-phosphate more (p less than 0.05) than in the weight-bearing soleus. At physiological doses of insulin, the percent of maximal uptake of 2-deoxy-D-[1,2-3H]glucose/muscle also was greater in the unloaded soleus. Unloading of the soleus increased by 50% the concentration of insulin receptors, due to no decrease in total receptor number during muscle atrophy. This increase may account for the greater response of glucose metabolism to insulin in this muscle. The extensor digitorum longus, which generally shows little response to unloading, displayed no differential response of glucose metabolism to insulin.     

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.     

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).     

Passive stretch reduces calpain activity through nitric oxide pathway in unloaded soleus muscles

Unloading in spaceflight or long-term bed rest induces to pronounced atrophy of anti-gravity skeletal muscles. Passive stretch partially resists unloading-induced atrophy of skeletal muscle, but the mechanism remains elusive. The aims of this study were to investigate the hypotheses that stretch tension might increase protein level of neuronal nitric oxide synthase (nNOS) in unloaded skeletal muscle, and then nNOS-derived NO alleviated atrophy of skeletal muscle by inhibiting calpain activity. The tail-suspended rats were used to unload rat hindlimbs for 2 weeks, at the same time, left soleus muscle was stretched by applying a plaster cast to fix the ankle at 35° dorsiflexion. Stretch partially resisted atrophy and inhibited the decreased protein level and activity of nNOS in unloaded soleus muscles. Unloading increased frequency of calcium sparks and elevated intracellular resting and caffeine-induced Ca(2+) concentration ([Ca(2+)]i) in unloaded soleus muscle fibers. Stretch reduced frequency of calcium sparks and restored intracellular resting and caffeine-induced Ca(2+) concentration to control levels in unloaded soleus muscle fibers. The increased protein level and activity of calpain as well as the higher degradation of desmin induced by unloading were inhibited by stretch in soleus muscles. In conclusion, these results suggest that stretch can preserve the stability of sarcoplasmic reticulum Ca(2+) release channels which prevents the elevated [Ca(2+)]i by means of keeping nNOS activity, and then the enhanced protein level and activity of calpain return to control levels in unloaded soleus muscles. Therefore, stretch can resist in part atrophy of unloaded soleus muscles.     

Respective effects of anabolic/androgenic steroids and physical exercise on isometric contractile properties of regenerating skeletal muscles in the rat

We examined the respective effects of anabolic-androgenic steroids and physical exercise on the contractile properties of regenerating fast and slow hindlimb skeletal muscles. Degeneration/regeneration of the left extensor digitorum longus muscles (EDL) and soleus of young Wistar male rats was induced by a snake venom (Notechis scutatus scutatus) injection. During muscle regeneration, experimental rats were either treated with nandrolone (NAN, nortestosterone, im, 2 mg X kg(-1) X week(-1), or endurance exercised on a treadmill (EXE, 60 min x day(-1), 10-40 m X min(-1). Twenty-one days after injury, isometric contractile properties of regenerating muscles were studied in situ. Neither the nandrolone treatment nor the physical exercise program was able to change significantly muscle contraction parameters both in twitch and tetanus in both regenerating EDL and soleus (p > 0.05). However, we observed a greater peak twitch tension in NAN versus grouped control and EXE EDL (p < 0.01). In conclusion, endurance exercise program or anabolic-androgenic steroid (nortestosterone) treatment did not significantly improve isometric contractile properties of regenerating slow and fast muscles in the male young rats.     

PGC-1α and FOXO1 mRNA levels and fiber characteristics of the soleus and plantaris muscles in rats after hindlimb unloading

Fifteen-week-old rats were subjected to unloading induced by hindlimb suspension for 3 weeks. The peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and forkhead box-containing protein O1 (FOXO1) mRNA levels and fiber profiles of the soleus and plantaris muscles in rats subjected to unloading (unloaded group) were determined and compared with those of age-matched control rats (control group). The body weight and both the soleus and plantaris muscle weights were lower in the unloaded group than in the control group. The PGC-1α mRNA was downregulated in the soleus, but not in the plantaris muscle of the unloaded group. The FOXO1 mRNA was upregulated in both the soleus and plantaris muscles of the unloaded group. The oxidative enzyme activity was reduced in the soleus, but not in the plantaris muscle of the unloaded group. The percentage of type I fibers was decreased and the percentages of type IIA and IIC fibers were increased in the soleus muscle of the unloaded group, whereas there was no change in fiber type distribution in the plantaris muscle of the unloaded group. Atrophy of all types of fibers was observed in both the soleus and plantaris muscles of the unloaded group. We conclude that decreased oxidative capacity and fiber atrophy in unloaded skeletal muscles are associated with decreased PGC-1α and increased FOXO1 mRNA levels.     

Increased phosphorylation of myosin light chain associated with slow-to-fast transition in rat soleus

In striated muscles myosin light chain (MLC)2 phosphorylation regulates calcium sensitivity and mediates sarcomere organization. Little is known about the changes in MLC2 phosphorylation in relation to skeletal muscle plasticity. We studied changes in MLC2 phosphorylation in rats receiving three treatment conditions causing slow-to-fast transitions: 1) atrophy induced by 14 days of hindlimb suspension (HS), 2) hypertrophy induced by 14 days of clenbuterol administration (CB), and 3) 14 days of combined treatment (CB-HS). Three variants of the slow (MLC2s) and two variants of the fast MLC2 (MLC2f) isoform were separated with two-dimensional electrophoresis and identified with monoclonal and polyclonal antibodies specific for MLC2; their relative proportions were densitometrically quantified. In control soleus muscle MLC2s predominated over MLC2f (91.4 ± 3.9% vs. 8.5 ± 3.9%) and was separated into two spots, the less acidic spot being 73.5 ± 4.3% of the total. All treatments caused a decrease of the less acidic unphosphorylated spot of MLC2s (CB: 64.1 ± 5.6%, HS: 62.4 ± 6.8%, CB-HS: 56.4 ± 4.4%), the appearance of a third more acidic variant of MLC2s (representing 3.9–5.9% of total MLC2s), an increase of MLC2f (CB: 30.9 ± 3.1%, HS: 23.9 ± 3.3%, CB-HS: 25.3 ± 3.9%), and the phosphorylation of a large fraction of MLC2f (CB: 30.4 ± 6.7%, HS: 28.7 ± 6.5%, CB-HS: 21.8 ± 2.1%). Treatment with alkaline phosphatase or with protein phosphatase 1 (PP1) removed the most acidic spots of both MLC2f and MLC2s. We conclude that in rat skeletal muscles an increase of MLC2 phosphorylation is associated with the slow-to-fast transition regardless of whether hypertrophy or atrophy develops. muscle atrophy; muscle hypertrophy; clenbuterol; hindlimb suspension     

Influence of brief daily tendon vibration on rat soleus muscle in non-weight-bearing situation.

The purpose of this study was to investigate whether tendon vibration could prevent soleus muscle atrophy during hindlimb unloading (HU). Mechanical vibrations with a constant low amplitude (0.3 mm) were applied (192 s/day) with constant frequency (120 Hz) to the Achilles tendon of the unloaded muscle during the 14-day HU period. Significant reductions in muscle mass (-41%), fiber size, maximal twitch (-54%), and tetanic tensions (-73%) as well as changes in fiber type and electrophoretic profiles and twitch-time parameters (-31% in the contraction time and -30% in the half relaxation time) were found after 14 days of HU when compared with the control soleus. Tendon vibration applied during HU significantly attenuated, but did not prevent, 1) the loss of muscle mass (17 vs. 41%); 2) the decrease in the fiber cross-sectional area of type IIA (-28 vs. -50%) and type IIC (-29 vs. -56%) fibers; and 3) the decrease in maximal twitch (-3 vs. -54%) and maximal tetanic tensions (-29 vs. -73%) and the half relaxation time (1 vs. -30%). Changes in the contraction time and in histological and electrophoretical parameters associated with HU were not counteracted. These findings suggest that tendon vibration can be used as a paradigm to counteract the atrophic process observed after HU.     

Hindlimb suspension reduces muscle regeneration

Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2′-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P<0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P<0.05) BrdU labeling in uninjured soleus muscles compared to weightbearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P>0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers. Accepted: 11 December 1997     

Unloading of juvenile muscle results in a reduced muscle size 9 wk after reloading.

The role of satellite cells and DNA unit size in determining muscle size was examined by inhibiting postnatal skeletal muscle development by using hindlimb suspension. Satellite cell mitotic activity and DNA unit size were determined in the soleus muscles from hindlimb-suspended and age-matched weight-bearing rats before the initiation of hindlimb suspension, at the conclusion of a 28-day hindlimb-suspension period, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-suspended rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb suspension, but they were the same (P > 0. 05) as those of weight-bearing rats 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, nuclei per millimeter, and DNA unit size for the hindlimb-suspended rats were significantly (P < 0.05) smaller than for the weight-bearing rats at all recovery times. Satellite cell mitotic activity was significantly (P < 0.05) higher in the soleus muscles from hindlimb-suspended rats 2 wk after reloading, but it was the same (P > 0.05) as in weight-bearing rats 9 wk after reloading. Juvenile soleus muscles failed to achieve normal muscle size 9 wk after reloading because there was incomplete compensation for the hindlimb-suspension-induced interruptions in myonuclear accretion and DNA unit size expansion.     

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 ovariectomy and hindlimb unloading on skeletal muscle

Female rats(7-8 mo old, n = 40) wererandomly placed into the intact control (Int) and ovariectomizedcontrol (Ovx) groups. Two weeks after ovariectomy, animals were furtherdivided into intact 2-wk hindlimb unloaded (Int-HU) and ovariectomizedhindlimb unloaded (Ovx-HU). We hypothesized that there would be greater hindlimb unloading-related atrophy in Ovx than in Int rats. In situcontractile tests were performed on soleus (Sol), plantaris (Plan),peroneus longus (Per), and extensor digitorum longus (EDL) muscles.Body weight and Sol mass were ~22% larger in Ovx than in Int groupand ~18% smaller in both HU groups than in Int rats (Ovx × HUinteraction, P < 0.05), and therewas a similar trend in Plan muscle (P < 0.07). There were main effects (P < 0.05) for both ovariectomy (growth) and hindlimb unloading(atrophy) on gastrocnemius mass. Mass of the Per and EDL muscles wasunaffected by either ovariectomy or hindlimb unloading. Time to peaktwitch tension for EDL and one-half relaxation times for Sol, Plan,Per, and EDL muscles were faster (P < 0.05) in Ovx than in Int animals. The results suggest that1) ovariectomy led to similarincreases of ~20% in body weight and plantar flexor mass;2) hindlimb unloading may haveprevented ovariectomy-related muscle growth;3) greater atrophy may have occurredin Sol and Plan of Ovx animals compared with controls; and4) removal of ovarian hormonalinfluence decreased skeletal muscle contraction times.