Control Soleus Research Articles

Metformin attenuates an increase of calcium-dependent and ubiquitin-proteasome markers in unloaded muscle.

Current study tested a hypothesis that during skeletal muscle unloading, calcium-dependent signaling pathways, markers of protein synthesis, and expression of E3 ubiquitin ligases can be regulated by metformin. Thirty-two male Wistar rats were randomly assigned into one of four groups: nontreated control (3C), control rats treated with metformin (3CM), 3 days of unloading/hindlimb suspension with placebo (3HS), and 3 days of unloading treated with metformin (3HSM). In soleus muscle of HS group level of phospho-AMP-activated protein kinase (p-AMPK) was decreased by 46% while ATP content was increased by 49% when compared with the control group. There was an increase of the level of phospho-CaMK II (483%) and an upregulation of Calcineurin (CaN), SERCA2a, and Calpain-1 mRNA expression (87%, 41%, and 62%, respectively, P < 0.05) in the HS group relative to the control. HS group also had increased mRNA expression of MuRF1, MAFbx, and ubiquitin (167%, 146%, and 191%, respectively, P < 0.05) when compared with the control soleus muscle. Metformin treatment impeded unloading-induced changes in soleus muscle. In conclusion, metformin treatment during 3 days of soleus muscle unloading: 1) prevented the decrease of p-AMPK and increase of ATP content; 2) affected regulation of calcium-dependent signaling pathways via level of CaMK II phosphorylation or CaMK II, CaN, SERCA2a, and Calpain-1 mRNA expression; 3) attenuated an increase in the expression of critical markers of ubiquitin-proteasome pathways MuRF1, MAFbx, and ubiquitin while not affecting the unloading-induced increase of ULK-1 marker of autophagic/lysosomal pathway.NEW & NOTEWORTHY Current study for the first time tested the hypothesis that during 3 days of soleus muscle unloading, calcium-dependent signaling pathways, markers of protein synthesis, and the expression of E3 ubiquitin ligases can be regulated by metformin. Treatment with metformin during unloading: prevented the decrease of p-AMPK and increase of ATP content, affected regulation of calcium-dependent signaling pathways, and attenuated an increase of critical markers of ubiquitin-proteasome pathways. Nevertheless, metformin treatment has not prevented soleus muscle atrophy.

μ-Crystallin in Mouse Skeletal Muscle Promotes a Shift from Glycolytic toward Oxidative Metabolism.

μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6–147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism.

Abstract 136: Denervation Induced Degradation of Motor Endplates can be Delayed Using Local Supplementation of Agrin at the Time of Injury.

PURPOSE: Robust muscle atrophy and fibrosis are cited as the prime causes for the permanence of motor deficits following surgical intervention for a denervation injury. Following a peripheral nerve transection, regenerating axons traverse long distances to reinnervate their target organs. However, due to the slow rate of axonal regrowth and a large segmental defect, muscle atrophy and destabilization of the neuromuscular junction (NMJ) usually proceeds before reinnervation occurs. Agrin is characterized as an essential component of NMJ formation and synaptogenesis. Here, we utilize an in vivo agrin deficient mouse nerve denervation model to both characterize the morphology of agrin deficient endplates as well as the response of the NMJ to local delivery of agrin following a denervation event. We found the endplates of agrin-deficient mice in a more degraded states than the endplates of wildtype mice following denervation. We also found that local delivery of agrin following denervation serves to transiently improve motor endplate morphology. We thus demonstrate the efficacy of using a biomolecular therapeutic approach to maintain and preserve the denervated NMJ. MATERIALS AND METHODS: A denervation model was created in 6-week-old mice from WT and agrin deficient C57BL/6 strains by excising 10 mm right sciatic nerve segment from the mid-thigh of the mice and suturing the proximal nerve stump to the gluteal muscle with 9-0 suture so as to prevent regeneration. Agrin deficient mice were either injected with supplemental agrin or PBS as a control at the site of injury. The downstream denervated and contralateral control soleus, plantaris and gastrocnemius muscles were harvested for immunohistochemistry, cryo-sections with H&E staining, and quantitative western blots at the 1,2,4,8 and 16-week timepoints. Quantification of motor endplate morphology was done using Volocity 3-D image software. Quantification of muscle fiber diameter was done using ImageJ software. RESULTS: Fluorescence confocal imaging of harvested soleus muscles revealed that agrin supplemented animals retained superior motor endplate morphology over control animals in all timepoints. The average surface area of agrin supplemented endplates were significantly greater than control endplates in all analyzed timepoints (p <0.05) CONCLUSION: Highlighting the importance of agrin, we have shown that even a single supplemental dose of agrin delivered locally at the site of injury is effective in preserving motor endplates in denervated mice hindlimbs even at the latest time points. This is consistent with our previous work that detailed MMP3 knock down maintained agrin at the NMJ and thereby preserved the motor endplates so as to improve function recovery. Taken together, these current experiments support the strategy to preserve motor endplates so as to prolong the window of opportunity for surgical intervention after a traumatic nerve injury.

Spinal and corticospinal pathways are differently modulated when standing at the bottom and the top of a three-step staircase in young and older adults.

This study investigated the modulation of spinal (group Ia afferents) and corticospinal pathways when young (22.7 ± 1.3years) and older adults (72.2 ± 7.9years) stood at the bottom and at the top of a three-step staircase equipped with force platforms. Changes in submaximal H-reflex amplitude (H 50) and slope of the H-reflex input-output relation (spinal pathway), and in amplitude of motor-evoked potentials (MEP) triggered by transcranial magnetic stimulation (corticospinal pathway) at two intensities (1.1× and 1.2× motor threshold) were recorded in soleus when subjects stood as steady as possible downstairs and upstairs. The centre of pressure (CoP) excursion was analyzed in the time and frequency domains in both conditions. Regardless of age, the mean CoP velocity was greater when standing upstairs (11.1 ± 3.5mms-1) than downstairs (9.0 ± 2.3mms-1; p = 0.002). The CoP power spectral density (PSD) in the 0-0.5Hz band was greater upstairs than downstairs (+18.4%; p = 0.03) whereas PSD in the 2-20Hz frequency band was lesser (-41%) upstairs than downstairs (p < 0.001), regardless of age. In both groups, the H 50 amplitude (-30.6%; p < 0.001) and slope of H-reflex input-output relation (-10.2%; p = 0.002) were lesser when standing upstairs than downstairs, whereas no significant difference was observed in MEP amplitude and silent period between balance conditions (p > 0.05). These results indicate a lower dependence on spinal pathway to control soleus motor neurones when standing upstairs than downstairs accompanied by a change in postural control. This suggests that healthy older adults preserved their ability to adjust postural control to environmental demands.

Intermittent stretch training of rabbit plantarflexor muscles increases soleus mass and serial sarcomere number.

In humans, enhanced joint range of motion is observed after static stretch training and results either from an increased stretch tolerance or from a change in the biomechanical properties of the muscle-tendon unit. We investigated the effects of an intermittent stretch training on muscle biomechanical and structural variables. The left plantarflexors muscles of seven anesthetized New Zealand (NZ) White rabbits were passively and statically stretched three times a week for 4 wk, while the corresponding right muscles were used as nonstretched contralateral controls. Before and after the stretching protocol, passive torque produced by the left plantarflexor muscles as a function of the ankle angle was measured. The left and right plantarflexor muscles were harvested from dead rabbits and used to quantify possible changes in muscle structure. Significant mass and serial sarcomere number increases were observed in the stretched soleus but not in the plantaris or medial gastrocnemius. This difference in adaptation between the plantarflexors is thought to be the result of their different fiber type composition and pennation angles. Neither titin isoform nor collagen amount was modified in the stretched compared with the control soleus muscle. Passive torque developed during ankle dorsiflexion was not modified after the stretch training on average, but was decreased in five of the seven experimental rabbits. Thus, an intermittent stretching program similar to those used in humans can produce a change in the muscle structure of NZ White rabbits, which was associated in some rabbits with a change in the biomechanical properties of the muscle-tendon unit.

Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

The expression of two cardiac myosin heavy chain (MyHC) isoforms in response to the thyroid status was studied in left ventricles (LVs) of Lewis rats. Major MyHC isoform in euthyroid and hyperthyroid LVs had a higher mobility on SDS-PAGE, whereas hypothyroid LVs predominantly contained a MyHC isoform with a lower mobility corresponding to that of the control soleus muscle. By comparing the MyHC profiles obtained under altered thyroid states together with the control soleus, we concluded that MyHCα was represented by the lower band with higher mobility and MyHCβ by the upper band. The identity of these two bands in SDS-PAGE gels was confirmed by western blot and mass spectrometry. Thus, in contrast to the literature data, we found that the MyHCα possessed a higher mobility rate than the MyHCβ isoform. Our data highlighted the importance of the careful identification of the MyHCα and MyHCβ isoforms analyzed by the SDS-PAGE.

Satellite cell proliferation is reduced in muscles of obese Zucker rats but restored with loading

The obese Zucker rat (OZR) is a model of metabolic syndrome, which has lower skeletal muscle size than the lean Zucker rat (LZR). Because satellite cells are essential for postnatal muscle growth, this study was designed to determine whether reduced satellite cell proliferation contributes to reduced skeletal mass in OZR vs. LZR. Satellite cell proliferation was determined by a constant-release 5-bromo-2-deoxyuridine (BrdU) pellet that was placed subcutaneously in each animal. Satellite cell proliferation, as determined by BrdU incorporation, was significantly attenuated in control soleus and plantaris muscles of the OZR compared with that shown in the LZR. To determine whether this attenuation of satellite cell activity could be rescued in OZR muscles, soleus and gastrocnemius muscles were denervated, placing a compensatory load on the plantaris muscle. In the LZR and the OZR after 21 days of loading, increases of approximately 25% and approximately 30%, respectively, were shown in plantaris muscle wet weight compared with that shown in the contralateral control muscle. The number of BrdU-positive nuclei increased similarly in loaded plantaris muscles from LZR and OZR. Myogenin, MyoD, and Akt protein expressions were lower in control muscles of OZR than in those of the LZR, but they were all elevated to similar levels in the loaded plantaris muscles of OZR and LZR. These data indicate that metabolic syndrome may reduce satellite cell proliferation, and this may be a factor that contributes to the reduced mass in control muscles of OZR; however, satellite cell proliferation can be restored with compensatory loading in OZR.

Influence of locomotor training on the structure and myosin heavy chains of the denervated rat soleus muscle

Objective: Mechanism of denervation atrophy remains poorly understood. In particular, the question about irreversibility of the late atrophy is still open. Therefore, in the present study, we investigated whether and how a passive movement can affect a progress of atrophy in rat soleus muscle. To address this issue, a locomotor training on a treadmill was applied to rats with their right hindlimb muscles denervated.Methods: The hindlimb muscles were denervated by cutting the sciatic nerve. Starting either 7 days or 1 month after the surgery, the animals were trained on a treadmill. Two months after denervation, the soleus muscle was investigated using light and electron microscopy and biochemical methods. Control soleus muscles were obtained from non-trained animals: the untreated and the 2-month denervated.Results: Locomotor training caused slight increase in denervated rat soleus muscle weight and significant increase in its fiber diameter. The training positively affected some of the factors that were believed to be the reasons of atrophy irreversibility, because of significant increase in the number of capillary blood vessels and muscle fiber nuclei with the concomitant decrease in the number of severely damaged muscle fibers and amount of collagen. Morphology of the contractile apparatus was also improved as more regular organization of sarcomeres and the hexagonal arrangement of myosin filaments was evident. Moreover, the amount of myosin heavy chains (MHC) significantly increased after training. The effects were more evident in the animals with longer training.Conclusion: Passive movement seems to attenuate some of the pathologic processes within the denervated muscle.

Coordinate expression of the 19S regulatory complex and evidence for ubiquitin-dependent telethonin degradation in the unloaded soleus muscle

Catabolic stimuli induce a coordinate expression of the 20S proteasome subunits in skeletal muscles. However, contradictory data have been obtained for the 19S regulatory complex (RC) subunits, which could reflect differential regulation at the transcriptional and/or translational level. To address this point we used a well-established model of muscle atrophy (hindlimb suspension) and determined the mRNA levels for 19S subunits belonging to both the base (non-ATPase S1, ATPases S7 and S8) and the lid (S14) of the 19S RC. Concomitant increased mRNA levels were observed for all studied subunits in rat soleus muscles after 9 days of unloading. In addition, analysis of polysome profiles showed a similar proportion of actively translated mRNA (50%) in unloaded and control soleus muscle. Furthermore, the repressed pool of messenger ribonucleoparticles (mRNPs) was low in both control (14%) and unloaded (15%) animals. Our data show that representative 19S subunits (S7 and S8) were efficiently translated, suggesting a coordinate production of 19S RC subunits. The 19S RC is responsible for the binding of polyubiquitin conjugates that are subsequently degraded inside the 20S proteasome core particle. We observed that soleus muscle atrophy was accompanied by an accumulation of ubiquitin conjugates. Purification of ubiquitin conjugates using the S5a 19S subunit followed by deubiquitination identified telethonin as a 26S proteasome substrate. In conclusion, muscle atrophy induces a concomitant expression of 26S proteasome subunits. Substrates to be degraded include a protein required for maintaining the structural integrity of sarcomeres.

O-GlcNAc level variations are associated with the development of skeletal muscle atrophy

O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory posttranslational modification of nucleocytoplasmic proteins, which consists of the attachment of N-acetylglucosamine to serine or threonine residues of a protein. This glycosylation is a ubiquitous posttranslational modification, which probably plays important roles in many aspects of protein function. Our laboratory has previously reported that, in skeletal muscle, proteins of the glycolytic pathway and energetic metabolism and contractile proteins were O-GlcNAc modified (Cieniewski-Bernard C, Bastide B, Lefebvre T, Lemoine J, Mounier Y, and Michalski JC. Mol Cell Proteomics 3: 577-585, 2004). O-GlcNAc has been recently demonstrated to play a role in modulating cellular function in response to nutrition and also in stress conditions. Therefore, we have investigated here the implication of the glycosylation/deglycosylation process in the development of atrophy in rat skeletal muscle after hindlimb unloading. The high O-GlcNAc level found in control soleus [compared with control extensor digitorum longus (EDL)] becomes lower in atrophied soleus. On the opposite side, the low rate of O-GlcNAc in control EDL reaches higher levels in EDL, not atrophied after hindlimb unloading. These variations in O-GlcNAc level are correlated with a variation of the O-GlcNAc process enzyme activities and could be associated with a differential expression of heat shock proteins. Our results suggest that O-GlcNAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy.

Effects of inactivity on fiber size and myonuclear number in rat soleus muscle

The effects of short-term (4 days) and long-term (60 days) neuromuscular inactivity on myonuclear number, size, and myosin heavy chain (MHC) composition of isolated rat soleus fibers were determined using confocal microscopy and gel electrophoresis. Inactivity was produced via spinal cord isolation (SI), i.e., complete spinal cord transections at a midthoracic and a high sacral level and bilateral deafferentation between the transection sites. Compared with control, there was an increase in the percentage of fibers containing the faster MHC isoforms after 60, but not 4, days of SI. The mean sizes of type I and type I+IIa fibers were 41 and 27% and 66 and 56% smaller after 4 and 60 days of SI, respectively. Thus atrophy occurred earlier than the shift in myosin heavy chain (MHC) profile. The number of myonuclei was approximately 30% higher in type I than type I+IIa fibers in control soleus, but after 60 days of SI these values were similar. The number of myonuclei per millimeter in type I fibers was significantly lower than control after 60 days of SI, whereas there was no change in type I+IIa fibers. Thus myonuclei were eliminated from fibers containing only type I MHC. Because the magnitude of the loss of myonuclei was less than the level of atrophy, the myonuclear domains of both type I and type I+IIa fibers were significantly lower than control. Thus chronic (60 days) inactivity results in smaller, faster fibers that contain a higher than normal amount of DNA per unit of cytoplasm. The absence of activation of muscle fibers that are normally the most active (pure type I fibers) resulted in most, but not all, fibers expressing some fast MHC isoforms. The results also indicate that a loss of myonuclei is not a prerequisite for sustained muscle fiber atrophy.

Skeletal muscle fibres synthesis in heart failure: Role of PGC-1α, calcineurin and GH

Background Patients with congestive heart failure (CHF) have decreased exercise capacity because of muscle fatigability. Symptoms are due to a specific myopathy with increased expression of fast type II fibres, fast MHCs and muscle atrophy. PGC-1α, a potent transcriptional coactivator for nuclear receptors, induces mitochondrial myogenesis and the preferential synthesis of slow fibres. IGF1-Calcineurin stimulation can lead to increased expression of PGC-1α. Methods We investigated the levels of PGC-1α during progression and regression of skeletal myopathy in the soleus muscle of rats with right heart failure secondary to monocrotaline-induced pulmonary hypertension. We used GH to stimulate the IGF1-calcineurin-PGC-1α axis. Results The slow MHC1 decreased from 90.6±0.5 to 71.7±2.2 in the CHF rats ( p<0.00001) and increased to 82.1±1.8 after GH ( p<0.00002). Western blot analysis showed that PGC-1α is significantly decreased in CHF, while it came back to control values after GH. Cytochrome c was decreased in CHF and returned to control values with GH. Troponin I was expressed solely as slow isoform in the control soleus, while the fast isoform appeared in CHF. Its expression returned to control values after GH. Conclusions We conclude that PGC-1α plays an important role in regulating slow fibres expression. PGC1-1α is in turn regulated by the IGF1-calcineurin axis. GH by increasing the circulating levels of IGF1, enhanced the expression of slow MHC1, TnI and the synthesis of mitochondria.

Effect of unloading on type I myosin heavy chain gene regulation in rat soleus muscle

Slow-twitch soleus, a weight-bearing hindlimb muscle, predominantly expresses the type I myosin heavy chain (MHC) isoform. However, under unloading conditions, a transition in MHC expression occurs from slow type I toward the fast-type isoforms. Transcriptional processes are believed to be involved in this adaptation. To test the hypothesis that the downregulation of MHC1 in soleus muscle following unloading is controlled through cis element(s) in the proximal region of the promoter, the MHC1 promoter was injected into soleus muscles of control rats and those subjected to 7 days of hindlimb suspension. Mutation analyses of six putative regulatory elements within the -408-bp region demonstrated that three elements, an A/T-rich, the proximal muscle-type CAT (betae3), and an E-box (-63 bp), play an important role in the basal level of MHC1 gene activity in the control soleus and function as unloading-responsive elements. Gel mobility shift assays revealed a diminished level of complex formation of the betae3 and E-box probes with nuclear extract from hindlimb suspension soleus compared with control soleus. Supershift assays indicated that transcriptional enhancer factor 1 and myogenin factors bind the betae3 and E-box elements, respectively, in the control soleus. Western blots showed that the relative concentrations of the transcriptional enhancer factor 1 and myogenin factors were significantly attenuated in the unloaded soleus compared with the control muscle. We conclude that the downregulation of MHC1 in response to unloading is due, in part, to a significant decrease in the concentration of these transcription factors available for binding the positive regulatory elements.

Myosin heavy chain isoform mRNA and protein levels after long-term paralysis

To assess the long-term influence of paralysis on muscle phenotypic mRNA and protein expression, the effects of spinal cord transection (ST) on myosin heavy chain (MyHC) isoform mRNA and protein levels in the soleus and medial gastrocnemius (MG) muscles of rats were analyzed. Control soleus contained predominantly MyHC-I with low amounts of MyHC-IIa and IIx mRNAs. After ST, MyHC-I mRNA decreased to ∼15%, MyHC-IIa was increased by 75–200%, and MyHC-IIx was elevated by 8–10×. Low level expression of MyHC-IIb was observed post-ST, suggesting that reduced activity is not a primary stimulus for MyHC-IIb expression. Adaptations in mRNA preceded protein adaptations in the soleus. Although MyHC-I protein in the MG was reduced post-ST, no other consistent changes occurred. The relative lack of adaptation to ST by the MG suggests that the reduced activity and load bearing encountered by the MG were insufficient to induce a change in muscle phenotype.

GMCSF and SDF treatment induces neovasculogenesis in a mouse ischemic hindlimb model

Abstract Introduction: Peripheral vascular disease is a prevalent condition associated with significant morbidity and mortality. Novel strategies to induce neovasculogenesis have revolved around ex-vivo expansion of endothelial progenitor cells (EPC’s) and their transplantation to ischemic tissue. The purpose of this study was to promote native in vivo expansion of EPC’s and targeting of progenitor cells to ischemic tissue utilizing GM-CSF, a bone marrow stimulant, and stromal derived factor (SDF), a known chemokine of EPC’s. Methods: A mouse ischemic hindlimb model was performed by ligation of the superficial femoral artery. 2.5ug of GM-CSF was administered subcutaneously into the experimental group (n = 5) on preoperative day one, on the operative day, and on post-operative day one. 1ug of SDF was injected into the hindlimb musculature after surgery in the experimental group. Saline was injected at these time points to the control group (n = 5). Two weeks later, the soleus muscle was harvested. Maximum force per contraction was obtained by isolated whole muscle mechanics of the experimental and control soleus muscle. Immunohistochemistry staining for von-willebrand’s factor, a marker of endothelial cells and CD133, a marker of EPC’s was performed. Results: The soleus muscle of the experimental groups had a higher maximum force per contraction (19 vs. 24 Newton/cm2, p Conclusions: Combined GMSCF and SDF therapy induces neovasculogenesis preserving muscle function in the murine ischemic hindlimb.

Motor Unit Properties in the Soleus Muscle after Its Distal Tendon Transfer to the Plantaris Muscle Tendon in the Rat

The aim of this study was to evaluate how a modification in the mechanical conditions under which a muscle is used could induce changes in the characteristics and the spinal drive of its motor units (MU). The distal tendon of the soleus muscle of Wistar rats was transferred to the distal stump of the plantaris muscle tendon. The EMG activity of the soleus was chronically recorded for 8 weeks, every other day, during a 1-min treadmill walk. After spinal ventral root splitting, individual MU contractile properties were measured in control soleus (102 MUs) or in transposed soleus muscles after 4 weeks (41 MUs) or 8 weeks (28 MUs). Muscle/body weight ratio did not vary after transposition, nor did MU tetanic forces. A decrease in MU twitch contraction times and in their half relaxation times was observed at weeks 4 and 8. MU tension-frequency curves varied significantly after tendon transfer, becoming closer to the curves of the fast MUs of the control group. During locomotion, we observed no change in the amplitude of rectified-filtered electromyographic activity, but a significant decrease in mean burst duration and an increase in the median frequency of the power density spectrum. Tendon transposition of the soleus muscle brought about adaptations in MU contractile properties and soleus spinal control.

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

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

Expression and neural control of follistatin versus myostatin genes during regeneration of mouse soleus.

Follistatin and myostatin are two secreted proteins involved in the control of muscle mass during development. These two proteins have opposite effects on muscle growth, as documented by genetic models. The aims of this work were to analyze in mouse, by using in situ hybridization, the spatial and temporal expression patterns of follistatin and myostatin mRNAs during soleus regeneration after cardiotoxin injury, and to investigate the influence of innervation on the accumulation of these two transcripts. Follistatin transcripts could be detected in activated satellite cells as early as the first stages of regeneration and were transiently expressed in forming myotubes. In contrast, myostatin mRNAs accumulated persistently throughout the regeneration process as well as in adult control soleus. Denervation significantly affected both follistatin and myostatin transcript accumulation, but in opposite ways. Muscle denervation persistently reduced the levels of myostatin transcripts as early as the young myotube stage, whereas the levels of follistatin mRNA were strongly increased in the small myotubes in the late stages of regeneration. These results are discussed with regard to the potential functions of both follistatin, as a positive regulator of muscle differentiation, and myostatin, as a negative regulator of skeletal muscle growth. We suggest that the belated up-regulation of the follistatin mRNA level in the small myotubes of the regenerating soleus as well as the down-regulation of the myostatin transcript level after denervation contribute to the differentiation process in denervated regenerating muscle.

Effect of thyroid hormones on acetylcholinesterase mRNA levels in the slow soleus and fast extensor digitorum longus muscles of the rat

In the rat, the level of acetylcholinesterase messenger RNA in the typical slow soleus muscles is only about 20-30% of that in the fast extensor digitorum longus muscles. The expression of contractile proteins in muscles is influenced by thyroid hormones and hyperthyroidism makes the slow soleus muscle faster. The influence of thyroid hormones on the levels of acetylcholinesterase messenger RNA level in the slow soleus and fast extensor digitorum longus muscle of the rat was studied in order to examine the effect of thyroid hormones on muscle acetylcholinesterase expression. Hyperthyroidism was induced in rats by daily thyroid hormone injection or thyroid hormone releasing tablet implantation. Hind-limb suspension was applied to produce muscle unloading. Muscle denervation or reinnervation was achieved by sciatic nerve transection or crush. Acetylcholinesterase messenger RNA levels were analyzed by Northern blots and evaluated densitometrically. Hyperthyroidism increased the levels of acetylcholinesterase messenger RNA in the slow soleus muscles close to the levels in the fast extensor digitorum longus. The effect was the same in the unloaded soleus muscles. Acetylcholinesterase expression increased also in the absence of innervation (denervation), in the presence of changed nerve activation pattern (reinnervation), and under enhanced tonic neural activation of the soleus muscle (electrical stimulation). However, the changes were substantially smaller than those observed in the control soleus muscles. Enhancement of acetylcholinesterase expression in the soleus muscles by the thyroid hormones is, therefore, at last in part due to hormonal effect on the muscle itself. On the contrary, increased level of the thyroid hormones had no influence on acetylcholinesterase expression in the normal fast extensor digitorum longus muscles. However, some enhancing influence was apparent whenever the total number of nerve-induced muscle activations per day in the extensor digitorum longus muscle was increased. Thyroid hormones seem to be an independent extrinsic factor of acetylcholinesterase regulation in the slow soleus muscle.

Glucose transporter content and glucose uptake in skeletal muscle constructs engineered in vitro.

Engineered muscle may eventually be used as a treatment option for patients suffering from loss of muscle function. The metabolic and contractile function of engineered muscle has not been well described; therefore, the purpose of this experiment was to study glucose transporter content and glucose uptake in engineered skeletal muscle constructs called myooids. Glucose uptake by way of 2-deoxyglucose and GLUT-1 and GLUT-4 transporter protein content was measured in basal and insulin-stimulated myooids that were engineered from soleus muscles of female Sprague-Dawley rats. There was a significant increase in the basal 2-deoxyglucose uptake of myooids compared with adult control (fivefold), contraction-stimulated (3.4-fold), and insulin-stimulated (threefold) soleus muscles (P = 0.0001, 0.0001, and 0.0001, respectively). In addition, there was a significant increase in the insulin-stimulated 2-deoxyglucose uptake of myooids compared with adult control soleus muscles in basal conditions (6.5-fold) and adult contraction-stimulated (4.5-fold) and insulin- stimulated (3.9-fold) soleus muscles (P = 0.0001, 0.0001, and 0.0001, respectively). There was a significant 30% increase in insulin-stimulated compared with basal 2-deoxyglucose uptake in the myooids. The myooid GLUT-1 protein content was 820% of the adult control soleus muscle, whereas the GLUT-4 protein content was 130% of the control soleus muscle. Myooid GLUT-1 protein content was 6.3-fold greater than GLUT-4 protein content, suggesting that the glucose transport of the engineered myooids is similar in several respects to that observed in both fetal and denervated skeletal muscle tissue.