Role of insulin on exercise-induced GLUT-4 protein expression and glycogen supercompensation in rat skeletal muscle.

The purpose of this study was to investigate the role of insulin on skeletal muscle GLUT-4 protein expression and glycogen storage after postexercise carbohydrate supplementation. Male Sprague-Dawley rats were randomly assigned to one of six treatment groups: sedentary control (Con), Con with streptozocin (Stz/C), immediately postexercise (Ex0), Ex0 with Stz (Stz/Ex0), 5-h postexercise (Ex5), and Ex5 with Stz (Stz/Ex5). Rats were exercised by swimming (2 bouts of 3 h) and carbohydrate supplemented immediately after each exercise session by glucose intubation (1 ml of a 50% wt/vol). Stz was administered 72-h before exercise, which resulted in hyperglycemia and elimination of the insulin response to the carbohydrate supplement. GLUT-4 protein of Ex0 rats was 30% above Con in fast-twitch (FT) red and 21% above Con in FT white muscle. In Ex5, GLUT-4 protein was 52% above Con in FT red and 47% above Con in FT white muscle. Muscle glycogen in FT red and white muscle was also increased above Con in Ex5 rats. Neither GLUT-4 protein nor muscle glycogen was increased above Con in Stz/Ex0 or Stz/Ex5 rats. GLUT-4 mRNA in FT red muscle of Ex0 rats was 61% above Con but only 33% above Con in Ex5 rats. GLUT-4 mRNA in FT red muscle of Stz/C and Stz/Ex0 rats was similar but significantly elevated in Ex5/Stz rats. These results suggest that insulin is essential for the increase in GLUT-4 protein expression following postexercise carbohydrate supplementation.     

Calcineurin and heat shock protein 72 in functionally overloaded rat plantaris muscle

The involvement of calcineurin (CaN) and heat shock protein (Hsp) 72 in the regulation of fiber size and/or phenotype in response to functional overload (FO) was investigated. In one FO group, the plantaris muscle was overloaded by cutting the distal tendons (5-10 mm length) of the soleus and gastrocnemius of 3-week-old male Wistar rats. Cyclosporin A (CsA), a CaN inhibitor, was injected daily (5 mg/kg body weight, i.p.) in a second group of FO rats (FO+CsA group) for a 2-week period. Compared to age-matched controls (Con), the absolute and relative plantaris weights were increased in both FO groups: the hypertrophic response was attenuated in FO+CsA rats. The mean cross-sectional area of each fiber type was increased (approximately 2.0-fold) in the plantaris of FO rats: CsA treatment attenuated this effect, although the fibers were still larger than in Con rats. The percent composition of myosin heavy chain (MHC) IIb decreased from 54% in Con to 19% in FO rats, whereas types I, IIa, and IIx MHC increased in the FO rats. CsA treatment blunted the shifts in MHC isoforms: the FO+CsA group showed a smaller decrease in type IIb and a smaller increase in type IIx MHC than the FO group. The levels of CaN-A and -B proteins were higher (approximately 2.5-fold) in FO than Con rats, whereas these values were similar in Con and FO+CsA rats. Hsp72 protein levels were higher in FO (3.6-fold) and FO+CsA (5.2-fold) than Con rats, with the values being significantly higher in the FO+CsA than FO rats. CsA treatment in Con rats had no effects on muscle mass, fiber size, MHC composition, and Hsp72 or CaN levels. Combined, these results suggest that CaN levels are related to changes in both fiber size and phenotype, and that Hsp72 levels are more related to the levels of stress added to the muscle rather than to increases in the slow fiber phenotype in functionally overloaded rat plantaris muscles.     

Differential expression of caveolin-3 in mouse smooth muscle cells in vivo

Expression of caveolin-1 and -3 in mouse smooth muscle cells in vivo was examined by immunohistochemistry. Caveolin-1 was detected in almost all smooth muscles examined, except for the pupillary dilator muscle, whereas caveolin-3 was present only in smooth muscles of some specific tissues. In the eye, the pupillary sphincter muscle was intensely positive for caveolin-3, whereas the ciliary muscle and pupillary dilator muscle were negative. In the gastrointestinal tract, caveolin-3 was detected in the inner circular layer, but not in the outer longitudinal layer. Vascular smooth muscle cells of the resistance-sized artery in the uterus and corpus cavernosum were intensely positive for caveolin-3, whereas those of the aorta were only weakly positive and those of the vena cava were negative. Caveolin-3 was also detected in smooth muscle cells of the urinary bladder, ureter, prostatic vas deferens, and seminal vesicle. The different levels of caveolin-3 expression among various smooth muscle tissues were confirmed by Western blot analysis. Even within the same muscle, the relative expression levels of caveolin-1 and -3 were variable among neighboring cells, suggesting distinct fine regulation of expression of these two caveolins. Moreover, even in the same cell, caveolin-1 and -3 showed different distributions. These results indicate that the two caveolins form distinct caveolae in smooth muscles, and that caveolin-1 and -3 serve different functions. Their differential expression may therefore be related to the functional diversity of smooth muscles. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of the Japanese Government.     

Characterisation of caveolins from cartilage: expression of caveolin-1, -2 and -3 in chondrocytes and in alginate cell culture of the rat tibia

This study was performed to determine if rat articular chondrocytes express caveolin, the structural protein of caveolae, and to determine differences in the distribution of the caveolin subtypes 1, 2 and 3 in knee joints of newborn and adult rats. All three subtypes of caveolin were detected in adult cartilage by immunocytochemical staining. In newborn rats, only caveolin-1 was found in the hyaline cartilage. Caveolin-1, -2 and -3 messenger RNA and protein were also detected in chondrocyte cell cultures. Ultrastructural investigations of cell culture and cartilage tissue revealed the presence of caveolae at the plasma membrane of chondrocytes. These findings represent the first report on the different expression of caveolin isoforms, in particular the expression of the muscle cell-specific caveolin-3 in chondrocytes. There is evidence that caveolin-2 and -3 are upregulated during growth and development of articular cartilage, suggesting a role for caveolins in chondrocyte differentiation. Accepted: 4 May 1999     

Contractile properties and myosin expression in rats born and reared in hypergravity

The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

Detection of caveolin-3/caveolin-1/P2X7R complexes in mice atrial cardiomyocytes in vivo and in vitro

Caveolae and caveolins, structural components of caveolae, are associated with specific ion channels in cardiac myocytes. We have previously shown that P2X purinoceptor 7 (P2X7R), a ligand-gated ion channel, is increased in atrial cardiomyocytes of caveolin-1 knockout mice; however, the specific biochemical relationship of P2X7R with caveolins in the heart is not clear. The aim of this work was to study the presence of the P2X7R in atrial cardiomyocytes and its biochemical relationship to caveolin-1 and caveolin-3. Caveolin isoforms and P2X7R were predominantly localized in buoyant membrane fractions (lipid rafts/caveolae) prepared from hearts using detergent-free sucrose gradient centrifugation. Caveolin-1 knockout mice showed normal distribution of caveolin-3 and P2X7R to buoyant membranes indicating the importance of caveolin-3 to formation of caveolae. Using clear native-PAGE, we showed that caveolin-1, -3 and P2X7R contribute to the same protein complex in the membranes of murine cardiomyocytes and in the immortal cardiomyocyte cell line HL-1. Western blot analysis revealed increased caveolin-1 and -3 proteins in tissue homogenates of P2X7R knockout mice. Finally, tissue homogenates of atrial tissues from caveolin-3 knockout mice showed elevated mRNA for P2X7R in atria. The colocalization of caveolins with P2X7R in a biochemical complex and compensated upregulation of P2X7R or caveolins in the absence of any component of the complex suggests P2X7R and caveolins may serve an important regulatory control point for disease pathology in the heart.     

Immunolocalization of caveolin-1 and caveolin-3 in monkey skeletal,cardiac and uterine smooth muscles

Caveolin, a 20-24 kDa integral membrane protein, is a principal component of caveolar domains. Caveolin-1 is expressed predominantly in endothelial cells, fibroblasts, and adipocytes, while the expression of caveolin-3 is confined to muscle cells. However, their localization in various muscles has not been well documented. Using double-immunofluorescence labeling and confocal laser microscopy, we examined the localization of caveolins-1 and 3 in adult monkey skeletal, cardiac and uterine smooth muscles and the co-immunolocalization of these caveolins with dystrophin, which is a product of the Duchenne muscular dystrophy gene. In the skeletal muscle tissue, caveolin-3 was localized along the sarcolemma except for the transverse tubules, and co-immunolocalized with dystrophin, whereas caveolin-1 was absent except in the blood vessels of the muscle tissue. In cardiac muscle cells, caveolins-1 and -3 and dystrophin were co-immunolocalized on the sarcolemma and transverse tubules. In uterine smooth muscle cells, caveolin-1, but not caveolin-3, was co-immunolocalized with dystrophin on the sarcolemma.     

Altered caveolin-3 expression disrupts PI(3) kinase signaling leading to death of cultured muscle cells

Caveolae and their coat proteins, caveolins, co-ordinate multiple signaling pathways. Caveolin-3 is a muscle-specific caveolin isoform that is deficient in limb girdle muscular dystrophy type 1 C (LGMD1C). Paradoxically, overexpression of this protein also causes muscle degeneration in vivo. We hypothesize that altered membrane expression of caveolin-3 in muscle cells causes a degenerative phenotype by disrupting the co-ordination of signaling pathways that are critical to the maintenance of cell survival. Here, we show for the first time that, in normal muscle cells subjected to oxidative stress, the phosphatidylinositol (3) kinase (PI(3) kinase)-associated proteins PDK1 and Akt associate with caveolae where they bind to caveolin-3, and that normal activation of this pathway promotes cell survival. Either increased or decreased expression of caveolin-3 at the membrane caused an increased susceptibility to oxidative stress, and myotube survival was markedly improved by PI(3) kinase inhibition. This occurred concomitantly with altered phosphorylation of the pro-apoptotic proteins GSK3beta and Bad, despite normal levels of Akt activation. Taken together, our results demonstrate that altered caveolin-3 expression can change the outcome of PI(3) kinase activation from cell survival to cell death. These findings indicate that normal expression and localization of caveolin-3 are required to appropriately co-ordinate PI(3) kinase/Akt-mediated cell survival signaling, and suggest that this pathway may be an effective therapeutic target for the treatment of muscular dystrophies associated with caveolin-3 mutations.     

雌、雄草原田鼠外周骨骼肌肌球蛋白重链的表达

已往的研究对于实验室应用的各种啮齿类动物,如大鼠和小鼠骨骼肌蛋白表达的特性已有报道.然而,至今不清楚其它啮齿类动物如野生鼠骨骼肌蛋白的表达或性双态性的特征,而这些野生鼠的行为学、形态学及生理学特点均已有报道.已知骨骼肌的肌球蛋白重链(MHC)成分与其功能特性有关.我们研究了草原田鼠的肱三头肌、胫骨前肌、腓肠肌和比目鱼肌MHC蛋白表达的性别特性.应用SDS聚丙烯酰胺凝胶电泳法测定MHC Ⅰ型、Ⅱa型、Ⅱd/x和Ⅱb型的蛋白表达相对含量.结果表明:与雌鼠相比,雄鼠的比目鱼肌湿重较大,胫骨前肌的MHC Ⅱa蛋白量表达较高.未见骨骼肌重量及MHC蛋白表达含量在雌雄鼠间的性别差异.血中睾酮的浓度差异可能不影响外周骨骼肌蛋白的表达特性.然而,与过去在大鼠、兔和小鼠中的已报道的结果相比,草原田鼠骨骼肌MHC的表达显示了更多异质性.推测这可能与草原田鼠和其它小型哺乳类动物生存的自然环境和功能需要有关.     

LBNP treadmill exercise maintains spine function and muscle strength in identical twins during 28-day simulated microgravity.

The purpose of this study was to determine whether lower body negative pressure (LBNP) treadmill exercise maintains lumbar spinal compressive properties, curvature, and back muscle strength after 28 days of 6 degrees head-down tilt (HDT) bed rest (BR). We hypothesize that LBNP treadmill exercise will maintain lumbar spine compressibility, lumbar lordosis and back muscle strength after 28 days of 6 degrees HDT bed rest. Fifteen healthy identical twin pairs (14 women and 16 men) participated in this study. One identical twin was randomly assigned to the nonexercise control (Con) group, and their sibling was assigned to the exercise (Ex) group. The lumbar spine was significantly more compressible Post-BR compared with Pre-BR in the Con (P=0.01). Lumbar spine compressibility Post-BR was not significantly different compared with Pre-BR in the Ex group (P=0.89). In both the Con and Ex groups, there were no significant changes Post-BR in lumbar lordosis compared with Pre-BR. Back muscle strength significantly decreased in the Con group Post-BR (P=0.002), whereas in the Ex group back muscle strength was not significantly different from Pre-BR values. A significant increase in lumbar spine compressibility in the Con group suggests that spinal deconditioning to gravity occurs during 28-day bed rest. Changes in the mechanical properties of the lumbar spine may be an early indicator of lumbar intervertebral disk degeneration. Supine LBNP treadmill exercise provides axial loads to the lumbar spine and may prevent lumbar spine deconditioning associated with HDT bed rest.     

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).     

白藜芦醇通过调节SIRT1/NF-κB通路减轻力竭训练致大鼠肾脏炎症反应

白藜芦醇是天然存在的沉默信息调节因子2相关酶1(sirtuin1，SIRT1)小分子激动剂，其肾的保护作用已在多种肾疾病动物模型中得到了验证。然而，白藜芦醇是否能够改善力竭训练导致的大鼠肾损伤，以及是否通过SIRT1/NF κB信号通路调节运动性肾损伤大鼠肾炎症反应，尚缺乏系统研究。本研究将32只SD大鼠随机分为安静对照组(Con组)，白藜芦醇组（Rsv组），力竭运动组（Ex组），力竭运动+白藜芦醇组（Ex+Rsv组）。Rsv和Ex+Rsv组每天灌胃50 mg/kg体重剂量的白藜芦醇， Ex和Ex+Rsv组进行4周力竭训练，最后1次训练后24 h取材。本研究结果显示，与Con组相比，Ex组大鼠Scr（175.66 ± 16.08 vs.153.34 ± 8.67，P < 0.01）、BUN（6.67 ± 0.53 vs.5.37 ± 019，P < 0.01）和尿NGAL（9.01 ± 0.18 vs.7.48 ± 0.31，P < 0.01）水平均显著升高，Ex组大鼠肾组织NF κB P65在蛋白质水平表达显著升高（0.77 ± 010 vs. 0.27 ± 0.03，P < 0.01）；各组大鼠肾组织SIRT1在蛋白质水平表达上，Rsv组显著高于Con组（0.90 ± 0.14 vs. 0.43 ± 0.15，P < 0.05），Ex+Rsv组显著高于Ex组（1.0 ± 0.28 vs. 0.38 ± 0.12，P< 001）；与Ex组相比，Ex+Rsv组大鼠肾组织NF-κB P65（0.57 ± 0.13 vs. 0.77 ± 0.10，P < 0.05）和Ac-NF-κB P65（0.52 ± 0.13 vs. 0.78 ± 0.11，P < 0.05）在蛋白质水平表达显著降低。以上结果表明，4周大强度力竭运动导致大鼠出现运动性肾损伤，并激活大鼠肾NF-κB的表达。白藜芦醇可显著提高大鼠肾组织SIRT1在蛋白质水平的表达，并增加脱乙酰化作用，降低NF-κB P65蛋白质乙酰化修饰水平，进一步降低NF-κB的表达。白藜芦醇减轻力竭训练致大鼠肾的炎症反应的机制可能与SIRT1/NF-κB通路有关。     

Fiber type-specific immunostaining of the Na+,K+-ATPase subunit isoforms in skeletal muscle: age-associated differential changes

The expression of the Na(+),K(+)-ATPase alpha and beta subunit isoforms in rat skeletal muscle and its age-associated changes have been shown to be muscle-type dependent. The cellular basis underlying these findings is not completely understood. In this study, we examined the expression of Na(+),K(+)-ATPase isoforms in individual fiber types and tested the hypothesis that, with age, the changes in the expression of the isoforms differ among individual fibers. We utilized immunohistochemical techniques to examine the expression of the subunit isoforms at the individual fiber levels. Immunofluorescence staining of the subunit isoforms in both white gastrocnemius (GW) and red gastrocnemius (GR) revealed a predominance of staining on the sarcolemmal membrane. Compared to the skeletal muscle of 6-month-old rats, there were substantial increases in the levels of alpha1, beta1, and beta3 subunit isoforms, and decreases in the levels of alpha2 and beta2 in 30-month-old rats. In addition, we found distinct patterns of staining for the alpha1, alpha2, beta1, and beta2 isoforms in tissue sections from young and aged rats. Muscle fiber-typing was performed to correlate the pattern of staining with specific fiber types. Staining for alpha1 and alpha2 isoforms in the skeletal muscle of young rats was generally evenly distributed among the fibers of GW and GR, with the exception of higher alpha1 levels in slow-twitch oxidative Type I fibers of GR. By contrast, staining for the beta1 and beta2 isoforms in the mostly oxidative fibers and the mostly glycolytic fibers, respectively, was almost mutually exclusive. With age, there was a fiber-type selective qualitative decrease of alpha2 and beta2 in Type IIB fibers, and increase of beta1 in Type IIB fibers and beta2 in Type IID fibers of white gastrocnemius. These results provide, at the individual fiber level, a cellular basis for the differential expression of the Na(+),K(+)-ATPase subunit isoforms in the muscle groups. The data further indicate that the aged-associated changes in expression of the subunit isoforms occur in both a fiber-type specific as well as an across fiber-type manner. Because of the differing biochemical properties of the subunit isoforms, these changes add another layer of complexity in our understanding of the adaptation of the Na-pump in skeletal muscle with advancing age.     

Training-induced changes in skeletal muscle Na+-K+ pump number and isoform expression in rats with chronic heart failure.

The mechanisms responsible for the decrements in exercise performance in chronic heart failure (CHF) remain poorly understood, but it has been suggested that sarcolemmal alterations could contribute to the early onset of muscular fatigue. Previously, our laboratory demonstrated that the maximal number of ouabain binding sites (B(max)) is reduced in the skeletal muscle of rats with CHF (Musch TI, Wolfram S, Hageman KS, and Pickar JG. J Appl Physiol 92: 2326-2334, 2002). These reductions may coincide with changes in the Na(+)-K(+)-ATPase isoform (alpha and beta) expression. In the present study, we tested the hypothesis that reductions in B(max) would coincide with alterations in the alpha- and beta-subunit expression of the sarcolemmal Na(+)-K(+)-ATPase of rats with CHF. Moreover, we tested the hypothesis that exercise training would increase B(max) along with producing significant changes in alpha- and beta-subunit expression. Rats underwent a sham operation (sham; n = 10) or a surgically induced myocardial infarction followed by random assignment to either a control (MI; n = 16) or exercise training group (MI-T; n = 16). The MI-T rats performed exercise training (ET) for 6-8 wk. Hemodynamic indexes demonstrated that MI and MI-T rats suffered from severe left ventricular dysfunction and congestive CHF. Maximal oxygen uptake (Vo(2 max)) and endurance capacity (run time to fatigue) were reduced in MI rats compared with sham. B(max) in the soleus and plantaris muscles and the expression of the alpha(2)-isoform of the Na(+)-K(+)-ATPase in the red portion of the gastrocnemius (gastrocnemius(red)) muscle were reduced in MI rats. After ET, Vo(2 max) and run time to fatigue were increased in the MI-T group of rats. This coincided with increases in soleus and plantaris B(max) and the expression of the alpha(2)-isoform in the gastrocnemius(red) muscle. In addition, the expression of the beta(2)-isoform of the gastrocnemius(red) muscle was increased in the MI-T rats compared with their sedentary counterparts. This study demonstrates that CHF-induced alterations in skeletal muscle Na(+)-K(+)-ATPase, including B(max) and isoform expression, can be partially reversed by ET.     

Inhibition of PKCalpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide

Receptor-coupled contraction of smooth muscle involves recruitment to the plasma membrane of downstream effector molecules PKCalpha and rhoA but the mechanism of this signal integration is unclear. Caveolins, the principal structural proteins of caveolar plasma membrane invaginations, have been implicated in the organization and regulation of many signal transducing molecules. Thus, using laser scanning confocal immunofluorescent microscopy, we tested the hypothesis that caveolin is involved in smooth muscle signaling by investigating caveolin isoform expression and localization, together with the effect of a peptide inhibitor of caveolin function, in intact differentiated smooth muscle cells. All three main caveolin isoforms were identified in uterine, stomach, and ileal smooth muscles and assumed a predominantly plasma membranous localization in myometrial cells. Cytoplasmic introduction of a peptide corresponding to the caveolin-1 scaffolding domain-an essential region for caveolin interaction with signaling molecules--significantly inhibited agonist-induced translocation of both PKCalpha and rhoA. Translocation was unimpaired by a scrambled peptide and was unaltered in sham-treated cells. The membranous localization of caveolins, and direct inhibition of receptor-coupled PKCalpha and rhoA translocation by the caveolin-1 scaffolding domain, supports the concept that caveolins can regulate the integration of extracellular contractile stimuli and downstream intracellular effectors in smooth muscle.     

cav-p60 expression in rat muscle tissues

Caveolae are plasmalemmal invaginations of uncertain function. In view of the large number of hypotheses on caveolar functions, it is important to identify which components of caveolae are tissue specific and which are general. The only well-characterized major protein of caveolae is caveolin, which exists in three tissue-specific isoforms: caveolin-1, -2, and -3. Recently cav-p60 was characterized as a 60-kDa caveola-specific protein in adipocytes. The distributions of cav-p60 and caveolin isoforms in different rat muscle tissues were examined by immunofluorescence and immunoelectron microscopy. Cav-p60 was present in caveolae of skeletal and heart muscle, in vascular and intestinal smooth muscle, and in adipocyte caveolae. Furthermore cav-p60 was present in endothelial cells and cells of perineural sheaths. Caveolin-1 and -2 were present in adipocytes, endothelial cells, and cells of perineural sheaths. In all kinds of vascular and intestinal smooth muscle, caveolin-1 and -2 were present at high levels, whereas caveolin-3 expression was low or undetectable, depending on the specific smooth muscle subtype. High levels of caveolin-3 were found only in caveolae and T tubules of skeletal and heart muscle. We conclude that cav-p60 is a highly specific marker of caveolae in many if not all cell types having caveolae.     

Regulation of insulin response in skeletal muscle cell by caveolin status

Recent studies on the role of caveolin-1 in adipocytes showed that caveolin has emerged as an important regulatory element in insulin signaling but little is known on its role in skeletal muscle cells. In this study, we demonstrate for the first time that caveolin-1 plays a crucial role in insulin dependent glucose uptake in skeletal muscle cells. Differentiation of L6 skeletal muscle cells induce the expression of caveolin-1 and caveolin-3 with partial colocalization. However in contrast to adipocytes, phosphorylation of insulin receptor beta (IRbeta) and Akt/Erk was not affected by the respective downregulation of caveolin-1 or caveolin-3 in the muscle cells. Moreover, the phosphorylation of IRbeta was detected not only in the caveolae but also in the non-caveolae fractions of the muscle cells despite the interaction of IRbeta with caveolin-1 and caveolin-3. These data implicate the lack of relationship between caveolins and IRbeta pathway in the muscle cells, different from the adipocytes. However, glucose uptake was reduced specifically by downregulation of caveolin-1, but not that of caveolin-3. Taken together, these observations suggest that caveolin-1 plays a crucial role in glucose uptake in differentiated muscle cells and that the regulation of caveolin-1 expression may be an important mechanism for insulin sensitivity, implying the role of muscle cells for type 2 diabetes.     

Changes in myosin heavy chain isoform expression of overloaded rat skeletal muscles

• 1.1. The effect of functional overload produced by tenotomy of synergistic gastrocnemius muscle on the expression of myosin heavy chain (MHC) isoforms in the plantaris and soleus muscles of the rat was studied using gradient sodium dodecyl sulfate-acrylamide gel electrophoresis.
• 2.2. Five weeks tenotomy, the plantaris and soleus muscle weights induced by tenotomy of the gastrocnemius muscle were 44.3% (P < 0.005) and 37.4% (P < 0.005), respectively, heavier than the contralateral control muscles.
• 3.3. Although four types of MHC isoforms were observed in both control and experimental plantaris, the percentage of MHC isoforms in the control and experimental muscles differed; the hypertrophied plantaris muscle contained more HCI (P < 0.05), HCIIa and HCIId (P < 0.05) and less HCIIb (P < 0.05) than the control muscle.
• 4.4. The control soleus muscle contained two MHC isofonns, HCI and HCIIa. However, there was only a single HCI isoform in the hypertrophied soleus muscle.
• 5.5. These results indicate that overloading a skeletal muscle by removing its synergists produces not only the muscle hypertrophy but also the changes in the expression of MHC isofonns.