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).     

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that β2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21?days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4?mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9?days of treatment, while hypertrophy was observed only in EDL after 9?days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14?days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.     

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.     

Bowman-Birk inhibitor concentrate prevents atrophy, weakness, and oxidative stress in soleus muscle of hindlimb-unloaded mice.

Antigravity muscles atrophy and weaken during prolonged mechanical unloading caused by bed rest or spaceflight. Unloading also induces oxidative stress in muscle, a putative cause of weakness. We tested the hypothesis that dietary supplementation with Bowman-Birk inhibitor concentrate (BBIC), a soy protein extract, would oppose these changes. Adult mice were fed a diet supplemented with 1% BBIC during hindlimb unloading for up to 12 days. Soleus muscles of mice fed the BBIC-supplemented diet weighed less, developed less force per cross-sectional area, and developed less total force after unloading than controls. BBIC supplementation was protective, blunting decrements in soleus muscle weight and force. Cytosolic oxidant activity was assessed using 2',7'-dichlorofluorescin diacetate. Oxidant activity increased in unloaded muscle, peaking at 3 days and remaining elevated through 12 days of unloading. Increases in oxidant activity correlated directly with loss of muscle mass and were abolished by BBIC supplementation. In vitro assays established that BBIC directly buffers reactive oxygen species and also inhibits serine protease activity. We conclude that dietary supplementation with BBIC protects skeletal muscle during prolonged unloading, promoting redox homeostasis in muscle fibers and blunting atrophy-induced weakness.     

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.     

Differential localization of autolyzed calpains 1 and 2 in slow and fast skeletal muscles in the early phase of atrophy

Calpains have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. However, limited data are available about the specific involvement of each calpain in the early stages of muscle atrophy. The aims of this study were to determine whether calpains 1 and 2 are autolyzed after a short period of muscle disuse, and, if so, where in the myofibers the autolyzed products are localized. In the rat soleus muscle, 5 days of immobilization increased autolyzed calpain 1 in the particulate and not the soluble fraction. Conversely, autolyzed calpain 2 was not found in the particulate fraction, whereas it was increased in the soluble fraction after immobilization. In the less atrophied plantaris muscle, no difference was noted between the control and immobilized groups whatever the fraction or calpain. Other proteolytic pathways were also investigated. The ubiquitin-proteasome pathway was activated in both skeletal muscles, and caspase 3 was activated only in the soleus muscle. Taken together, our data suggest that calpains 1 and 2 are involved in atrophy development in slow type muscle exclusively and that they have different regulation and protein targets. Moreover, the activation of proteolytic pathways appears to differ in slow and fast muscles, and the proteolytic mechanisms involved in fast-type muscle atrophy remain unclear. Ca²⁺-dependent proteases; wasting; skeletal muscle; soluble and particulate fractions; immobilization     

Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy

In order to clarify the cellular mechanisms of denervation atrophy of skeletal muscle, we have studied protein turnover in denervated and control rat soleus muscles in vitro under different conditions. By 24 h after cutting the sciatic nerve, overall protein breakdown was greater in the denervated soleus than in the contralateral control muscle, and by 3 days, net proteolysis had increased about 3-fold. Since protein synthesis increased slightly following denervation, the rise in proteolysis must be responsible for the muscle atrophy and the differential loss of contractile proteins. Like overall proteolysis, the breakdown of actin (as shown by 3-methyl-histidine production by the muscles) increased each day after denervation and by 3 days was 2.5 times faster than in controls. Treatments that block the lysosomal and Ca2(+)-dependent proteolytic systems did not reduce the increase in overall protein degradation and actin breakdown in the denervated muscles (maintained in complete medium at resting length). However, the content of the lysosomal protease, cathepsin B, increased about 2-fold by 3 days after denervation. Furthermore, conditions that activate intralysosomal proteolysis (incubation without insulin or amino acids) stimulated proteolysis 2-3-fold more in the denervated muscles than in controls. Also, incubation conditions that activate the Ca2(+)-dependent pathway (incubation with Ca2+ ionophores or allowing muscles to shorten) were 2-3 times more effective in enhancing overall proteolysis in the denervated muscle. None of these treatments affected 3-methylhistidine production. Thus, multiple proteolytic systems increase in parallel in the denervated muscle, but a nonlysosomal process (independent of Ca2+) appears mainly responsible for the rapid loss of cell proteins, especially of myofibrillar components.     

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.     

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions.

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.     

Tetanic contractions impair sarcomeric Z-disk of atrophic soleus muscle via calpain pathway

The aim of this study was to determine whether or not over-activation of calpains during running exercise or tetanic contractions was a major factor to induce sarcomere lesions in atrophic soleus muscle. Relationship between the degrees of desmin degradation and sarcomere lesions was also elucidated. We observed ultrastructural changes in soleus muscle fibers after 4-week unloading with or without running exercise. Calpain activity and desmin degradation were measured in atrophic soleus muscles before or after repeated tetani in vitro. Calpain-1 activity was progressively increased and desmin degradation was correspondingly elevated in 1-, 2-, and 4-week of unloaded soleus muscles. Calpain-1 activity and desmin degradation had an additional increase in unloaded soleus muscles after repeated tetani in vitro. PD150606, an inhibitor of calpains, reduced calpain activity and desmin degradation during tetanic contractions in unloaded soleus muscles. The 4-week unloading decreased the width of myofibrils and Z-disk in soleus fibers. After running exercise in unloaded group, Z-disks of adjacent myofibrils were not well in register but instead were longitudinally displaced. Calpain inhibition compromised exercise-induced misalignment of the Z-disks in atrophic soleus muscle. These results suggest that tetanic contractions induce an over-activation of calpains which lead to higher degrees of desmin degradation in unloaded soleus muscle. Desmin degradation may loose connections between adjacent myofibrils, whereas running exercise results in sarcomere injury in unloaded soleus muscle.     

Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP

Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats 1, 2, or 4 days after adrenodemedullation. Adrenodemedullation reduced plasma epinephrine by 95% and norepinephrine by 35% but did not affect muscle norepinephrine content. In soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis increased by 15-20% by 2 days after surgery but returned to normal levels after 4 days. The rise in rates of protein degradation was accompanied by an increased activity of Ca(2+)-dependent proteolysis in both muscles, with no significant change in the activity of lysosomal and ATP-dependent proteolytic systems. In vitro rates of Ca(2+)-dependent proteolysis in soleus and EDL from normal rats decreased by ~35% in the presence of either 10(-5) M clenbuterol, a beta(2)-adrenergic agonist, or epinephrine or norepinephrine. In the presence of dibutyryl cAMP, proteolysis was reduced by 62% in soleus and 34% in EDL. The data suggest that catecholamines secreted by the adrenal medulla exert an inhibitory control of Ca(2+)-dependent proteolysis in rat skeletal muscle, mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.     

Mechanical loading regulates NOS expression and activity in developing and adult skeletal muscle

The hypothesis that changes in muscle activation and loadingregulate the expression and activity of neuronal nitric oxide (NO)synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNAconcentration in soleus muscles, which returned to controlconcentrations after return to weight bearing. Similarly, theconcentration of nNOS in cultured myotubes increased by application ofcyclic loading for 2 days. NO release from excised soleus muscles wasincreased significantly by a single passive stretch of 20% or bysubmaximal activation at 2 Hz, although the increases were not additivewhen both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on thepresence of extracellular calcium. Cyclic loading of cultured myotubesalso resulted in a significant increase in NO release. Together, thesefindings show that activity of muscle influences NO production in theshort term, by regulating NOS activity, and in the long term, byregulating nNOS expression.

     

Early biochemical consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation

1. One week after denervation several biochemical characteristics of the fast extensor digitorum longus and slow soleus muscles from adult rats were investigated and compared with the characteristics of the corresponding unoperated contralateral muscles. 2. After these short periods of denervation-induced atrophy, the isolated myosins showed unchanged ATPase (adenosine triphosphatase) activities, but there was the expected difference between fast and slow muscle. 3. The specific activities of several soluble enzymes and their characteristic patterns were found to be only slightly modified in both the extensor and soleus muscles after denervation, as were most of the activities measured in the isolated mitochondria. 4. The most significant modifications were in the isolated sarcoplasmic reticulum, and appeared to be specific to either slow or fast muscle. 5. Denervation of slow muscle led to a marked increase of Ca(2+)-transport rates, and of the specific activity of the Mg(2+)-activated K(+)-modulated Ca(2+)-stimulated ATPase, together with changes in the polyacrylamide-electrophoretic profiles of the microsomal membrane protein. Transformation of these several properties of slow muscle sarcoplasmic reticulum to those of fast muscle sarcoplasmic reticulum was further substantiated by electron-microscopic analysis after negative staining. Control experiments with tenotomized soleus muscle gave negative results. 6. The isolated sarcoplasmic reticulum from fast muscle showed a slight diminution of ATPase-linked Ca(2+)-transport activity and a selective increase of rotenone-insensitive NADH-cytochrome c reductase activity, in addition to a greater emphasis on slow-type electrophoretic components of the structural membrane protein. 7. The significance of these results in relation to specific differentiating influences from motor nerves is discussed.     

Expression and functional properties of four slow skeletal troponin T isoforms in rat muscles

We investigated the expression and functional properties of slow skeletal troponin T (sTnT) isoforms in rat skeletal muscles. Four sTnT cDNAs were cloned from the slow soleus muscle. Three isoforms were found to be similar to sTnT1, sTnT2, and sTnT3 isoforms described in mouse muscles. A new rat isoform, with a molecular weight slightly higher than that of sTnT3, was discovered. This fourth isoform had never been detected previously in any skeletal muscle and was therefore called sTnTx. From both expression pattern and functional measurements, it appears that sTnT isoforms can be separated into two classes, high-molecular-weight (sTnT1, sTnT2) and low-molecular-weight (sTnTx, sTnT3) isoforms. By comparison to the apparent migration pattern of the four recombinant sTnT isoforms, the newly described low-molecular-weight sTnTx isoform appeared predominantly and typically expressed in fast skeletal muscles, whereas the higher-molecular-weight isoforms were more abundant in slow soleus muscle. The relative proportion of the sTnT isoforms in the soleus was not modified after exposure to hindlimb unloading (HU), known to induce a functional atrophy and a slow-to-fast isoform transition of several myofibrillar proteins. Functional data gathered from replacement of endogenous troponin complexes in skinned muscle fibers showed that the sTnT isoforms modified the Ca(2+) activation characteristics of single skeletal muscle fibers, with sTnT2 and sTnT1 conferring a similar increase in Ca(2+) affinity higher than that caused by low-molecular-weight isoforms sTnTx and sTnT3. Thus we show for the first time the presence of sTnT in fast muscle fibers, and our data show that the changes in neuromuscular activity on HU are insufficient to alter the sTnT expression pattern.     

Caffeine-induced Ca(2+) release increases AMPK-dependent glucose uptake in rodent soleus muscle

Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because alpha-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-beta (ACCbeta) Ser221 and immunoprecipitated alpha(1)-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated alpha(2)-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCbeta and alpha(1)-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 microM), the CaM-competitive inhibitor KN-93 (10 microM), or the SR Ca(2+) release blocking agent dantrolene (10 microM) all inhibited ACCbeta phosphorylation and alpha(1)-AMPK phosphorylation, suggesting that SR Ca(2+) release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca(2+)-activated CaMKK may control alpha(1)-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.     

Muscle progenitor cell proliferation during passive stretch of unweighted soleus in dystrophin deficient mice

Dystrophin, subsarcolemmal protein communicating muscle fiber cytoskeleton to extracellular matrix, is believed to be involved in mechanical signal transduction. The experiment was carried out to assess the role of dystrophin in passive stretch-induced preventing unloaded muscle fiber atrophy and possible linkage between this protein and muscle progenitor (satellite cells) proliferation activity. The study was performed on two months old C57 black and mdx (dystrophin-deficient) mice. Passive stretch resulted in attenuating atrophy development in two fiber types of both C57 black and mdx mice. Altered dystrophin synthesis in mdx mice had virtually no effect on passive stretch preventive action. Thus the hypothesis about dystrophin key role in mediating stretch-induced hypertrophy effects didn't find its confirmation concerning gravitational unloading atrophy. Chronic hindlimb unloading downregulated SC proliferative activity in soleus muscle, passive stretch drastically increased proliferation both in C57 and mdx mice. Thus we observed no relationship between altered dystrophin synthesis and satellite cell proliferation activity in soleus muscle under conditions of simulated microgravity and concurrent passive stretch.     

Akt signaling in skeletal muscle: regulation by exercise and passive stretch

Akt/protein kinase B is a serine/threonine kinase that has emerged as a critical signaling component for mediating numerous cellular responses. Contractile activity has recently been demonstrated to stimulate Akt signaling in skeletal muscle. Whether physiological exercise in vivo activates Akt is controversial, and the initiating factors that result in the stimulation of Akt during contractile activity are unknown. In the current study, we demonstrate that treadmill running exercise of rats using two different protocols (intermediate high or high-intensity exhaustive exercise) significantly increases Akt activity and phosphorylation in skeletal muscle composed of various fiber types. To determine if Akt activation during contractile activity is triggered by mechanical forces applied to the skeletal muscle, isolated skeletal muscles were incubated and passively stretched. Passive stretch for 10 min significantly increased Akt activity (2-fold) in the fast-twitch extensor digitorum longus (EDL) muscle. However, stretch had no effect on Akt in the slow-twitch soleus muscle, although there was a robust phosphorylation of the stress-activated protein kinase p38. Similar to contraction, stretch-induced Akt activation in the EDL was fully inhibited in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin, whereas glycogen synthase kinase-3 (GSK3) phosphorylation was only partially inhibited. Stretch did not cause dephosphorylation of glycogen synthase on GSK3-targeted sites in the absence or presence of wortmannin. We conclude that physiological exercise in vivo activates Akt in multiple skeletal muscle fiber types and that mechanical tension may be a part of the mechanism by which contraction activates Akt in fast-twitch muscles.     

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.     

萎缩比目鱼肌间断强直收缩疲劳后恢复速率的影响因素

为研究模拟失重大鼠萎缩比目鱼肌强直收缩疲劳后恢复速率的影响因素,采用尾部悬吊模拟失重大鼠模型及离体骨骼肌条灌流技术,观测其在不同收缩模式下疲劳后的恢复过程。正常大鼠离体比目鱼肌条实验显示,10s短时程（S10P）与300s长时程（L10P）强直收缩轻度疲劳[强直收缩最大张力（P0）下降10％]后,在20min恢复期末,均可恢复至疲劳前P0,且恢复程度不受疲劳持续时间的影响;轻度疲劳后,在灌流液中加入10μmol／L钌红抑制肌浆网Ca^2＋释放功能,恢复速率减慢,恢复程度最大仅至94％P0,然后呈下降趋势,提示轻度疲劳可能仅抑制肌原纤维功能。60s短时程（S50P）与300s长时程（L50P）强直收缩中度疲劳（P0下降50％）后,在20min恢复期末,收缩张力分别恢复至95％P0和90％P0,表明中度疲劳持续时间影响恢复的速率;相同条件中度疲劳后,在灌流液中加入5mmol／L咖啡因促进肌浆网Ca62＋释放功能,恢复速率明显加快,无论疲劳持续时间长短,5min便可完全恢复,提示中度疲劳不仅抑制肌原纤维功能,还抑制肌浆网Ca^2＋释放功能。尾部悬吊1周的大鼠比目鱼肌明显萎缩,其重量／体重之比仅为对照大鼠的60％。采用短与长持续时间的轻与中度疲劳作用后,在20min恢复期末,收缩张力分别恢复至94％P0（S10P）、95％P0（L10P）、92％P0（S50P）、84％P0（L50P）,均与同步对照组有显著差异。以上结果提示：模拟失重1周大鼠萎缩的比目鱼肌,轻度与中度疲劳均可抑制肌原纤维功能与肌浆网Ca^2＋释放功能,使恢复速率减慢。