A proteomic assessment of muscle contractile alterations during unloading and reloading

Unloading of skeletal muscle causes atrophy and altered contractility. To identify major muscle proteins responding significantly to the altered loading and to elucidate how the contractile alterations reflect potential proteomic modifications, we examined protein expression in the rat soleus muscle during 3-week hindlimb suspension and 2-week reloading. Compared with unsuspended controls, experimental animals had a 0.5- to 0.6-fold decrease in tension during unloading and early reloading, comparable to 0.2- to 0.6-fold decreases in the protein levels of myosin light chain 1 (MLC1), alpha-actin, tropomyosin beta-chain, and troponins T1 and T2. The observed 1.4- to 1.6-fold increase in shortening velocity appears to reflect 1.2- to 9.0-fold increases in the protein levels of fast-type MLC2, glycolytic enzymes, and creatine kinase, and 0.2- to 0.3-fold decreases in slow-type troponins T1 and T2. The levels of three heat shock proteins (p20, alpha crystallin B chain, and HSP90) decreased during unloading but returned to control levels during reloading. These results imply that proteomic responses to unloading change overall myofibrillar integrity and metabolic regulation, resulting in altered contractility.     

Signal-transduction pathways that regulate visceral smooth muscle function. III. Coupling of muscarinic receptors to signaling kinases and effector proteins in gastrointestinal smooth muscles

Stimulation of muscarinic M3 and M2 receptors on gastrointestinal smooth muscle elicits contraction via activation of G proteins that are coupled to a diverse set of downstream signaling pathways and effector proteins. Many studies suggest a canonical excitation-contraction coupling pathway that includes activation of phospholipases, production of inositol 1,4,5-trisphosphate and diacylglycerol, release of calcium from the sarcoplasmic reticulum, activation of L-type calcium channels, and activation of nonselective cation channels. These events lead to elevated intracellular calcium concentration, which activates myosin light chain kinase to phosphorylate and activate myosin II thus causing contraction. In addition, muscarinic receptors are coupled to signaling pathways that modulate the effect of activator calcium. The Rho/Rho kinase pathway inhibits myosin light chain phosphatase, one of the key steps in sensitization of the contractile proteins to calcium. Phosphatidylinositol 3-kinases and Src family tyrosine kinases are also activated by muscarinic agonists. Src family tyrosine kinases regulate L-type calcium and nonselective cation channels. Src activation also leads to activation of ERK and p38 MAPKs. ERK MAPKs phosphorylate caldesmon, an actin filament binding protein. P38 MAPKs activate phospholipases and MAPKAP kinase 2/3, which phosphorylate HSP27. HSP27 may regulate cross-bridge function, actin filament formation, and actin filament attachment to the cell membrane. In addition to the well-known role of M3 muscarinic receptors to regulate myoplasmic calcium levels, the integrated effect of muscarinic activation probably also includes signaling pathways that modulate phospholipases, cyclic nucleotides, contractile protein function, and cytoskeletal protein function.     

Insight into skeletal muscle mechanotransduction: MAPK activation is quantitatively related to tension.

The mechanism by which mechanical forces acting through skeletal muscle cells generate intracellular signaling, known as mechanotransduction, and the details of how gene expression and cell size are regulated by this signaling are poorly understood. Mitogen-activated protein kinases (MAPKs) are known to be involved in mechanically induced signaling in various cell types, including skeletal muscle where MAPK activation has been reported in response to contraction and passive stretch. Therefore, the investigation of MAPK activation in response to mechanical stress in skeletal muscle may yield important information about the mechanotransduction process. With the use of a rat plantaris in situ preparation, a wide range of peak tensions was generated through passive stretch and concentric, isometric, and eccentric contractile protocols, and the resulting phosphorylation of c-Jun NH(2)-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAPKs was assessed. Isoforms of JNK and ERK MAPKs were found to be phosphorylated in a tension-dependent manner, such that eccentric > isometric > concentric > passive stretch. Peak tension was found to be a better predictor of MAPK phosphorylation than time-tension integral or rate of tension development. Differences in maximal response amplitude and sensitivity between JNK and ERK MAPKs suggest different roles for these two kinase families in mechanically induced signaling. A strong linear relationship between p54 JNK phosphorylation and peak tension over a 15-fold range in tension (r(2) = 0.89, n = 32) was observed, supporting the fact that contraction-type differences can be explained in terms of tension and demonstrating that MAPK activation is a quantitative reflection of the magnitude of mechanical stress applied to muscle. Thus the measurement of MAPK activation, as an assay of skeletal muscle mechanotransduction, may help elucidate mechanically induced hypertrophy.     

Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK

Our aim was to analyze the role of phosphatidylinositol 3-kinase (PI3K)-AKT and MAPK signaling pathways in the regulation of muscle mass and slow-to-fast phenotype transition during hindlimb unloading (HU). For that purpose, we studied, in rat slow soleus and fast extensor digitorum longus muscles, the time course of anabolic PI3K-AKT-mammalian target of rapamycin, catabolic PI3K-AKT-forkhead box O (FOXO), and MAPK signaling pathway activation after 7, 14, and 28 days of HU. Moreover, we performed chronic low-frequency soleus electrostimulation during HU to maintain exclusively contractile phenotype and so to determine more precisely the role of these signaling pathways in the modulation of muscle mass. HU induced a downregulation of the anabolic AKT, mammalian target of rapamycin, 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, and glycogen synthase kinase-3β targets, and an upregulation of the catabolic FOXO1 and muscle-specific RING finger protein-1 targets correlated with soleus muscle atrophy. Unexpectedly, soleus electrostimulation maintained 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, FOXO1, and muscle-specific RING finger protein-1 to control levels, but failed to reduce muscle atrophy. HU decreased ERK phosphorylation, while electrostimulation enabled the maintenance of ERK phosphorylation similar to control level. Moreover, slow-to-fast myosin heavy chain phenotype transition and upregulated glycolytic metabolism were prevented by soleus electrostimulation during HU. Taken together, our data demonstrated that the processes responsible for gradual disuse muscle plasticity in HU conditions involved both PI3-AKT and MAPK pathways. Moreover, electrostimulation during HU restored PI3K-AKT activation without counteracting soleus atrophy, suggesting the involvement of other signaling pathways. Finally, electrostimulation maintained initial contractile and metabolism properties in parallel to ERK activation, reinforcing the idea of a predominant role of ERK in the regulation of muscle slow phenotype.     

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate,but Not FAK

Background

Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency.

Principal Findings

Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts.

Conclusions

These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.     

Myosin filament assembly in an ever-changing myofilament lattice of smooth muscle

A major development in smooth muscle research in recent years is the recognition that the myofilament lattice of the muscle is malleable. The malleability appears to stem from plastic rearrangement of contractile and cytoskeletal filaments in response to stress and strain exerted on the muscle cell, and it allows the muscle to adapt to a wide range of cell lengths and maintain optimal contractility. Although much is still poorly understood, we have begun to comprehend some of the basic mechanisms underlying the assembly and disassembly of contractile and cytoskeletal filaments in smooth muscle during the process of adaptation to large changes in cell geometry. One factor that likely facilitates the plastic length adaptation is the ability of myosin filaments to form and dissolve at the right place and the right time within the myofilament lattice. It is proposed herein that formation of myosin filaments in vivo is aided by the various filament-stabilizing proteins, such as caldesmon, and that the thick filament length is determined by the dimension of the actin filament lattice. It is still an open question as to how the dimension of the dynamic filament lattice is regulated. In light of the new perspective of malleable myofilament lattice in smooth muscle, the roles of many smooth muscle proteins could be assigned or reassigned in the context of plastic reorganization of the contractile apparatus and cytoskeleton.     

Insulin-independent pathways mediating glucose uptake in hindlimb-suspended skeletal muscle.

Insulin resistance accompanies atrophy in slow-twitch skeletal muscles such as the soleus. Using a rat hindlimb suspension model of atrophy, we have previously shown that an upregulation of JNK occurs in atrophic muscles and correlates with the degradation of insulin receptor substrate-1 (IRS-1) (Hilder TL, Tou JC, Grindeland RF, Wade CE, and Graves LM. FEBS Lett 553: 63-67, 2003), suggesting that insulin-dependent glucose uptake may be impaired. However, during atrophy, these muscles preferentially use carbohydrates as a fuel source. To investigate this apparent dichotomy, we examined insulin-independent pathways involved in glucose uptake following a 2- to 13-wk hindlimb suspension regimen. JNK activity was elevated throughout the time course, and IRS-1 was degraded as early as 2 wk. AMP-activated protein kinase (AMPK) activity was significantly higher in atrophic soleus muscle, as were the activities of the ERK1/2 and p38 MAPKs. As a comparison, we examined the kinase activity in solei of rats exposed to hypergravity conditions (2 G). IRS-1 phosphorylation, protein, and AMPK activity were not affected by 2 G, demonstrating that these changes were only observed in soleus muscle from hindlimb-suspended animals. To further examine the effect of AMPK activation on glucose uptake, C2C12 myotubes were treated with the AMPK activator metformin and then challenged with the JNK activator anisomycin. While anisomycin reduced insulin-stimulated glucose uptake to control levels, metformin significantly increased glucose uptake in the presence of anisomycin and was independent of insulin. Taken together, these results suggest that AMPK may be an important mediator of insulin-independent glucose uptake in soleus during skeletal muscle atrophy.     

Muscle regeneration during hindlimb unloading results in a reduction in muscle size after reloading.

The hindlimb-unloading model was used to study the ability of muscle injured in a weightless environment to recover after reloading. Satellite cell mitotic activity and DNA unit size were determined in injured and intact soleus muscles from hindlimb-unloaded and age-matched weight-bearing rats at the conclusion of 28 days of hindlimb unloading, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-unloaded rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb unloading, but they were the same (P > 0.05) as those of weight-bearing rats 2 and 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, number of nuclei per millimeter, and DNA unit size were significantly (P < 0.05) smaller for the injured soleus muscles from hindlimb-unloaded rats than for the soleus muscles from weight-bearing rats at each recovery time. Satellite cell mitotic activity was significantly (P < 0.05) higher in the injured soleus muscles from hindlimb-unloaded rats than from weight-bearing rats 2 wk after reloading, but it was the same (P > 0.05) as in the injured soleus muscles from weight-bearing rats 9 wk after reloading. The injured soleus muscles from hindlimb-unloaded rats failed to achieve weight-bearing muscle size 9 wk after reloading, because incomplete compensation for the decrease in myonuclear accretion and DNA unit size expansion occurred during the unloading period.     

Localization of an actin binding domain in smooth muscle myosin light chain kinase

Phosphorylation of the regulatory light chain of myosin II by myosinlight chain kinase is important for regulating many contractile processes.Smooth muscle myosin light chain kinase has been shown to be associated withboth actin and myosin filaments in vitro and in vivo. In this report wedefine an actin binding region by using molecular deletions to generaterecombinant mutant proteins that were analyzed by co-sedimentation withF-actin. An actin binding region restricted to residues 2-42 in the animoterminus of the rabbit smooth muscle myosin light chain kinase wasidentified.     

The contractile apparatus of striated muscle in the course of atrophy and regeneration

Summary Changes in the contractile apparatus of denervated rat soleus muscles were investigated during the course of reinnervation.As observed earlier, in the course of denervation atrophy the ratio of myosin to actin filaments decreases because myosin filaments disappear faster than actin filaments (Jakubiec-Puka et al. 1981 a). After reinnervation the amount of myosin filaments and myosin heavy chains (myosin HC) in the muscle increased during the first few days; the increment of actin content was negligible. The proportion of myosin HC to actin remained lower than normal for about 30 days. The excess of actin filaments frequently observed in the newly-formed myofibrils reflects this disproportion.The results show a lability of myosin and suggest some cytoskeletal role for actin filaments.     

甲壳动物横纹肌的收缩蛋白质与调节机制

甲壳动物横纹肌肌原纤维的肌丝陈列,收缩蛋白质和收缩的Ca²⁺依赖性调节机制与脊椎动物横纹肌有不少差异.脊椎动物横纹肌、甲壳动物快肌与慢肌的粗丝与细丝的数量比依次为1:2,1:3和1:6,肌丝阵列各异.甲壳动物粗肌丝由肌球蛋白和副肌球蛋白组成,其分子装配与脊椎动物不同.细肌丝含有肌动蛋白、原肌球蛋白和肌钙蛋白,肌钙蛋白-T分子量较高,肌钙蛋白-C仅1个Ca²⁺结合位点.甲壳动物横纹肌兼有细肌丝调节与粗肌丝调节.     

The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle

Contractile stimulation induces actin polymerization in smooth muscle tissues and cells, and the inhibition of actin polymerization depresses smooth muscle force development. In the present study, the role of Cdc42 in the regulation of actin polymerization and tension development in smooth muscle was evaluated. Acetylcholine stimulation of tracheal smooth muscle tissues increased the activation of Cdc42. Plasmids encoding wild type Cdc42 or a dominant negative Cdc42 mutant, Asn-17 Cdc42, were introduced into tracheal smooth muscle strips by reversible permeabilization, and tissues were incubated for 2 days to allow for protein expression. Expression of recombinant proteins was confirmed by immunoblot analysis. The expression of the dominant negative Cdc42 mutant inhibited contractile force and the increase in actin polymerization in response to acetylcholine stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type Cdc42 had no significant effect on force, actin polymerization, or myosin light chain phosphorylation. Contractile stimulation increased the association of neuronal Wiskott-Aldrich syndrome protein with Cdc42 and the Arp2/3 (actin-related protein) complex in smooth muscle tissues expressing wild type Cdc42. The agonist-induced increase in these protein interactions was inhibited in tissues expressing the inactive Cdc42 mutant. We conclude that Cdc42 activation regulates active tension development and actin polymerization during contractile stimulation. Cdc42 may regulate the activation of neuronal Wiskott-Aldrich syndrome protein and the actin related protein complex, which in turn regulate actin filament polymerization initiated by the contractile stimulation of smooth muscle.     

Macrophages,not neutrophils,infiltrate skeletal muscle in mice deficient in P/E selectins after mechanical reloading

Our objective was to test the hypothesis that endothelial selectins, P and E selectins, are necessary for leukocyte migration after muscle injury from unloading/reloading. Mice hindlimbs were suspended for 10 days followed by reloading periods of 6 or 24 h after which the soleus muscle was dissected. Light microscopic observations showed that macrophages, but not neutrophils, were able to invade soleus muscles in mice deficient in P/E selectins (P/E-/-) during reloading periods. The recruitment efficiency of neutrophils after 6 and 24 h of reloading was minimal in P/E-/- mice relative to unloaded animals. The recruitment of macrophages in the soleus muscle was preserved in P/E-/- mice. The concentration of macrophages increased by 8.1-fold compared with unloaded muscles in double-mutant mice after 24 h of reloading. The accumulation of macrophages in reloaded muscles did not lead to fiber necrosis. Together, these findings indicate that macrophages can invade skeletal muscle through cellular mechanisms that do not involve P/E selectins during skeletal muscle reloading.     

Actin depolymerization factor/cofilin activation regulates actin polymerization and tension development in canine tracheal smooth muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.     

Role of mitogen-activated protein kinases in Thy-1-induced T-lymphocyte activation

Thy-1 (CD90) crosslinking by monoclonal antibodies (mAb) in the context of costimulation causes the activation of mouse T-lymphocytes; however, the associated signal transduction processes have not been studied in detail. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in Thy-1-mediated T-lymphocyte activation using mAb-coated polystyrene microspheres to crosslink Thy-1 and costimulatory CD28 on murine T-lymphocytes. Concurrent Thy-1 and CD28 crosslinking induced DNA synthesis by T-lymphocytes, as well as interleukin (IL)-2 and IL-2 receptor (IL-2R) α chain (CD25) expression. Increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal protein kinase (JNK) was also observed. Pharmacologic inhibition of ERK1/2 or JNK activation inhibited Thy-1-induced DNA synthesis and IL-2 production by T-lymphocytes. p38 MAPK inhibition also decreased DNA synthesis in Thy-1-stimulated T-lymphocytes; however, IL-2 production was increased in these cells. Inhibition of JNK, but not ERK1/2 or p38 MAPK, caused a marked reduction in Thy-1-induced CD25 expression. In addition, inhibition of p38 MAPK or JNK, but not ERK1/2, impaired the growth of IL-2-dependent CTLL-2 T-lymphocytes but did not substantially affect CD25 expression. Finally, exogenous IL-2 reversed the inhibitory effect of ERK1/2 or JNK inhibition on Thy-1-stimulated DNA synthesis by T-lymphocytes but did not substantially reverse JNK inhibition of CD25 expression. Collectively, these results suggest that during Thy-1-induced T-lymphocyte activation, ERK1/2 and JNK promoted IL-2 production whereas p38 MAPK negatively regulated IL-2 expression. JNK signalling was also required for CD25 expression. IL-2R signalling involved both p38 MAPK and JNK in CTLL-2 cells, whereas p38 MAPK was most important for IL-2R signalling in primary T-lymphocytes. MAPKs are therefore essential signalling intermediates for the Thy-1-driven proliferation of mouse T-lymphocytes.     

Neutrophil-induced skeletal muscle damage: a calculated and controlled response following hindlimb unloading and reloading

Neutrophils phagocyte necrotic debris and release cytokines, enzymes, and oxidative factors. In the present study, we investigated the contribution of neutrophils to muscle injury, dysfunction, and recovery using an unloading and reloading model. Mice were submitted to 10 days of hindlimb unloading and were transiently depleted in neutrophils with anti-Ly6G/Ly6C antibody prior to reloading. Leukocyte accumulation and muscle function were assessed immunohistologically and functionally in vitro. In addition, soleus muscles submitted to unloading and reloading were incubated in vitro with LPS (100 microg/ml) to determine whether exogenous stimulus would activate neutrophil response and produce extensive muscle damage. Contractile properties were recorded every hour for 6 h, and muscles were subsequently incubated in procion orange to assess muscle damage. Neutrophil depletion affected neither the loss in muscle force nor the time of recovery in atrophied and reloaded soleus muscles. However, atrophied and reloaded soleus muscles that contained high concentration of neutrophils experienced a 20% greater loss in force than atrophied and reloaded soleus muscles depleted in neutrophils following in vitro incubation with LPS. Procion orange dye also confirmed that neutrophils induced a 2.5-fold increase in muscle membrane damage in the presence of LPS. These results show that neutrophil infiltration during modified mechanical loading is highly regulated and efficiently eliminated, with no significant muscle fiber injury unless the activation state of neutrophils is modified by the presence of LPS.     

The ADP release step of the smooth muscle cross-bridge cycle is not directly associated with force generation.

When smooth muscle myosin subfragment 1 (S1) is bound to actin filaments in vitro, the light chain domain tilts upon release of MgADP, producing a approximately 3.5-nm axial motion of the head-rod junction (Whittaker et al., 1995. Nature. 378:748-751). If this motion contributes significantly to the power stroke, rigor tension of smooth muscle should decrease substantially in response to cross-bridge binding of MgADP. To test this prediction, we monitored mechanical properties of permeabilized strips of chicken gizzard muscle in rigor and in the presence of MgADP. For comparison, we also tested psoas and soleus muscle fibers. Any residual bound ADP was minimized by incubation in Mg2+-free rigor solution containing 15 mM EDTA. The addition of 2 mM MgADP, while keeping ionic strength and free Mg2+ concentration constant, resulted in a slight increase in rigor tension in both gizzard and soleus muscles, but a decrease in psoas muscle. In-phase stiffness monitored during small (<0.1%) 500-Hz sinusoidal length oscillations decreased in all three muscle types when MgADP was added. The changes in force and stiffness with the addition of MgADP were similar at ionic strengths from 50 to 200 mM and were reversible. The results with gizzard muscle were similar after thiophosphorylation of the regulatory light chain of myosin. These results suggest that the axial motion of smooth muscle S1 bound to actin, upon dissociation of MgADP, is not associated with force generation. The difference between the present mechanical data and previous structural studies of smooth S1 may be explained if geometrical constraints of the intact contractile filament array alter the motions of the myosin heads.     

Phosphorylation of rabbit skeletal muscle myosin in situ

Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.     

Improved insulin sensitivity with calorie restriction does not require reduced JNK1/2, p38, or ERK1/2 phosphorylation in skeletal muscle of 9-month-old rats

Calorie restriction [CR; ～40% below ad libitum (AL) intake] improves the health of many species, including rats, by mechanisms that may be partly related to enhanced insulin sensitivity for glucose disposal by skeletal muscle. Excessive activation of several mitogen-activated protein kinases (MAPKs), including JNK1/2, p38, and ERK1/2 has been linked to insulin resistance. Although insulin can activate ERK1/2, this effect is not required for insulin-mediated glucose uptake. We hypothesized that skeletal muscle from male 9-mo-old Fischer 344/Brown Norway rats CR (35-40% beginning at 3 mo old) versus AL rats would have 1) attenuated activation of JNK1/2, p38, and ERK1/2 under basal conditions; and 2) no difference for insulin-induced ERK1/2 activation. In contrast to our hypothesis, there were significant CR-related increases in the phosphorylation of p38 (epitrochlearis, soleus, and gastrocnemius), JNK1 (epitrochlearis and soleus), and JNK2 (gastrocnemius). Consistent with our hypothesis, CR did not alter insulin-mediated ERK1/2 activation. The greater JNK1/2 and p38 phosphorylation with CR was not attributable to diet effects on muscle oxidative stress (assessed by protein carbonyls and 4-hydroxynonenal protein conjugates). In muscles from the same rats used for the present study, we previously reported a CR-related increase in insulin-mediated glucose uptake by the epitrochlearis and the soleus (Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Am J Physiol Endocrinol Metab 300: E966-E978, 2011). The present results indicate that the improved insulin sensitivity with CR is not attributable to attenuated MAPK phosphorylation in skeletal muscle.