Adult Mouse Skeletal Muscle Research Articles

Kinetics of nuclear-cytoplasmic translocation of Foxo1 and Foxo3A in adult skeletal muscle fibers

In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins that mediate muscle atrophy, making the nuclear concentration and nuclear-cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the insulin-like growth factor-1 (IGF-1)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway and to show that Akt mediates the efflux of nuclear Foxo1-GFP induced by IGF-1. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier chromosome region maintenance 1 by leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear-cytoplasmic shuttling of Foxo1 under control conditions before LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles ∼20-fold more slowly than Foxo1-GFP. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions.

Prevalence and elimination of sibling neurite convergence in motor units supplying neonatal and adult mouse skeletal muscle

During development, neurons form supernumerary synapses, most of which are selectively pruned leading to stereotyped patterns of innervation. During the development of skeletal muscle innervation, or its regeneration after nerve injury, each muscle fiber is transiently innervated by multiple motor axon branches but eventually by a single branch. The selective elimination of all but one branch is the result of competition between the converging arbors. It is thought that motor neurons initially innervate muscle fibers randomly, but that axon branches from the same neuron (sibling branches) do not converge to innervate the same muscle fiber. However, random innervation would result in many neonatal endplates that are co-innervated by sibling branches. To investigate whether this occurs we examined neonatal levator auris longus (LAL) and 4th deep lumbrical (4DL) muscles, as well as adult reinnervated deep lumbrical muscles (1-4) in transgenic mice expressing yellow fluorescent protein (YFP) as a reporter. We provide direct evidence of convergence of sibling neurites within single fluorescent motor units, both during development and during regeneration after nerve crush. The incidence of sibling neurite convergence was 40% lower in regeneration and at least 75% lower during development than expected by chance. Therefore, there must be a mechanism that decreases the probability of its occurrence. As sibling neurite convergence is not seen in normal adults, or at later timepoints in regeneration, synapse elimination must also remove convergent synaptic inputs derived from the same motor neuron. Mechanistic theories of synaptic competition should now accommodate this form of isoaxonal plasticity.

No evidence for inositol 1,4,5-trisphosphate–dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle

The presence and role of functional inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) in adult skeletal muscle are controversial. The current consensus is that, in adult striated muscle, the relative amount of IP3Rs is too low and the kinetics of Ca2+ release from IP3R is too slow compared with ryanodine receptors to contribute to the Ca2+ transient during excitation–contraction coupling. However, it has been suggested that IP3-dependent Ca2+ release may be involved in signaling cascades leading to regulation of muscle gene expression. We have reinvestigated IP3-dependent Ca2+ release in isolated flexor digitorum brevis (FDB) muscle fibers from adult mice. Although Ca2+ transients were readily induced in cultured C2C12 muscle cells by (a) UTP stimulation, (b) direct injection of IP3, or (c) photolysis of membrane-permeant caged IP3, no statistically significant change in calcium signal was detected in adult FDB fibers. We conclude that the IP3–IP3R system does not appear to affect global calcium levels in adult mouse skeletal muscle.

No role of muscle satellite cells in hypertrophy: further evidence of a mistaken identity?

No role of muscle satellite cells in hypertrophy: further evidence of a mistaken identity?

P38γ activity is required for maintenance of slow skeletal muscle size

p38γ kinase is highly enriched in skeletal muscle and is implicated in myotube formation. However, the activation status of p38γ in muscle is unclear. p38γ activity in slow and fast adult mouse skeletal muscle tissue was examined, as was the impact of p38γ deficiency on muscle development and gene expression. p38γ is preferentially activated in slow muscle, but it is inactive in fast muscle types. Furthermore, the loss of p38γ in mice led to decreased muscle mass associated with a smaller myofiber diameter in slow muscle, but there was no impact on fast muscle in either mass or myofiber diameter. Finally, p38γ-deficient muscle showed selective changes in genes related to muscle growth in slow muscle fibers. This study provides evidence that p38γ is selectively activated in slow skeletal muscle and is involved in the normal growth and development of a subset of skeletal muscle.

The critical role of VEGF in skeletal muscle angiogenesis and blood flow

VEGF (vascular endothelial growth factor) is well known as an important molecule in angiogenesis. Its inhibition is pursued as an anticancer therapy; its enhancement as therapy for tissue ischaemia. In the present paper, its role in skeletal muscle is explored, both at rest and after exercise. Muscle VEGF mRNA and protein are increased severalfold after heavy exercise. Whereas global VEGF knockout is embryonically lethal, muscle-specific knockout is not, providing models for studying its functional significance. Its deletion in adult mouse skeletal muscle: (i) reduces muscle capillarity by more than 50%, (ii) decreases exercise endurance time by approximately 80%, and (iii) abolishes the angiogenic response to exercise training. What causes VEGF to increase with exercise is not clear. Despite regulation by HIF (hypoxia-inducible factor), increased HIF on exercise, and PO2 falling to single digit values during exercise, muscle-specific HIF knockout does not impair performance or capillarity, leaving many unanswered questions.

Effect of Oxidative Stress on Homer Scaffolding Proteins

Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.

Characterization of the liver kinase B1-mouse protein-25 -Ste-20-related adaptor protein complex in adult mouse skeletal muscle

In liver, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of liver kinase B1 (LKB1), mouse protein 25 (MO25α/β), and Ste-20-related adaptor protein (STRADα/β); however, this complex has yet to be characterized in skeletal muscle. We demonstrate the expression of the LKB1-MO25-STRAD complex in skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these proteins in control and muscle-specific LKB1 knockout (LKB1(-/-)) mouse muscle. LKB1 detection in untreated control and LKB1(-/-) muscle lysates revealed two protein bands (50 and 60 kDa), although only the heavier band was diminished in LKB1(-/-) samples [55 ± 2.5 and 13 ± 1.5 arbitrary units (AU) in control and LKB1(-/-), respectively, P < 0.01], suggesting that LKB1 is not represented at 50 kDa, as previously cited. The 60-kDa LKB1 band was further confirmed following purification using polyethylene glycol (43 ± 5 and 8.4 ± 4 AU in control and LKB1(-/-), respectively, P < 0.01) and ion-exchange fast protein liquid chromatography. Mass spectrometry confirmed LKB1 protein detection in the 60-kDa protein band, while none was detected in the 50-kDa band. Coimmunoprecipitation assays demonstrated LKB1-MO25-STRAD complex formation. Quantitative PCR revealed significantly reduced LKB1, MO25α, and STRADβ mRNA in LKB1(-/-) muscle. These findings demonstrate that the LKB1-MO25-STRAD complex is the principal AMPKK in skeletal muscle.

Anti-LRP4 autoantibodies in AChR- and MuSK-antibody-negative myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disorder characterized by a defect in synaptic transmission at the neuromuscular junction causing fluctuating muscle weakness with a decremental response to repetitive nerve stimulation or altered jitter in single-fiber electromyography (EMG). Approximately 80% of all myasthenia gravis patients have autoantibodies against the nicotinic acetylcholine receptor in their serum. Autoantibodies against the tyrosine kinase muscle-specific kinase (MuSK) are responsible for 5-10% of all myasthenia gravis cases. The autoimmune target in the remaining cases is unknown. Recently, low-density lipoprotein receptor-related protein (LRP4) has been identified as the agrin receptor. LRP4 interacts with agrin, and the binding of agrin activates MuSK, which leads to the formation of most if not all postsynaptic specializations, including aggregates containing acetylcholine receptors (AChRs) in the junctional plasma membrane. In the present study we tested if autoantibodies against LRP4 are detectable in patients with myasthenia gravis. To this end we analyzed 13 sera from patients with generalized myasthenia gravis but without antibodies against AChR or MuSK. The results showed that 12 out of 13 antisera from double-seronegative MG patients bound to proteins concentrated at the neuromuscular junction of adult mouse skeletal muscle and that approximately 50% of the tested sera specifically bound to HEK293 cells transfected with human LRP4. Moreover, 4 out of these 13 sera inhibited agrin-induced aggregation of AChRs in cultured myotubes by more than 50%, suggesting a pathogenic role regarding the dysfunction of the neuromuscular endplate. These results indicate that LRP4 is a novel target for autoantibodies and is a diagnostic marker in seronegative MG patients.

Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells

Myogenesis is accompanied by an intensive metabolic remodeling. We investigated the mitochondrial reactive oxygen species (ROS) generation at different levels of skeletal muscle differentiation: in C2C12 myoblasts, in C2C12 myotubes and in adult mouse skeletal muscle. Differentiation was accompanied by an increase in mitochondrial content and respiratory chain activity. The detected ROS production levels correlated with mitochondrial content, being the lowest in the myoblasts. Unlike the adult skeletal muscle, myoblast ROS production was significantly stimulated by the complex I inhibitor rotenone. Our results show that mitochondria are an important ROS source in skeletal muscle cells. The substantial changes in mitochondrial ROS synthesis during skeletal muscle differentiation can be explained by intensive bioenergetic remodeling.

Teashirt-3, a Novel Regulator of Muscle Differentiation, Associates with BRG1-associated Factor 57 (BAF57) to Inhibit Myogenin Gene Expression

In adult muscles and under normal physiological conditions, satellite cells are found in a quiescent state but can be induced to enter the cell cycle by signals resulting from exercise, injury-induced muscle regeneration, or specific disease states. Once activated, satellite cells proliferate, self-renew, and differentiate to form myofibers. In the present study, we found that the zinc finger-containing factor Teashirt-3 (TSHZ3) was expressed in quiescent satellite cells of adult mouse skeletal muscles. We showed that following treatment with cardiotoxin TSHZ3 was strongly expressed in satellite cells of regenerating muscles. Moreover, immunohistochemical analysis indicated that TSHZ3 was expressed in both quiescent and activated satellite cells on intact myofibers in culture. TSHZ3 expression was maintained in myoblasts but disappeared with myotube formation. In C2C12 myoblasts, we showed that overexpression of Tshz3 impaired myogenic differentiation and promoted the down-regulation of myogenin (Myog) and up-regulation of paired-box factor 7 (Pax7). Moreover, knockdown experiments revealed a selective effect of Tshz3 on Myog regulation, and transcriptional reporter experiments indicated that TSHZ3 repressed Myog promoter. We identified the BRG1-associated factor 57 (BAF57), a subunit of the SWI/SNF complex, as a partner of TSHZ3. We showed that TSHZ3 cooperated with BAF57 to repress MYOD-dependent Myog expression. These results suggest a novel mechanism for transcriptional repression by TSHZ3 in which TSHZ3 and BAF57 cooperate to modulate MyoD activity on the Myog promoter to regulate skeletal muscle differentiation.

Increased Expression of Wild-Type or a Centronuclear Myopathy Mutant of Dynamin 2 in Skeletal Muscle of Adult Mice Leads to Structural Defects and Muscle Weakness

Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers.

G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation

After skeletal muscle injury, neutrophils, monocytes, and macrophages infiltrate the damaged area; this is followed by rapid proliferation of myoblasts derived from muscle stem cells (also called satellite cells). Although it is known that inflammation triggers skeletal muscle regeneration, the underlying molecular mechanisms remain incompletely understood. In this study, we show that granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is expressed in developing somites. G-CSFR and G-CSF were expressed in myoblasts of mouse embryos during the midgestational stage but not in mature myocytes. Furthermore, G-CSFR was specifically but transiently expressed in regenerating myocytes present in injured adult mouse skeletal muscle. Neutralization of endogenous G-CSF with a blocking antibody impaired the regeneration process, whereas exogenous G-CSF supported muscle regeneration by promoting the proliferation of regenerating myoblasts. Furthermore, muscle regeneration was markedly impaired in G-CSFR-knockout mice. These findings indicate that G-CSF is crucial for skeletal myocyte development and regeneration and demonstrate the importance of inflammation-mediated induction of muscle regeneration.

Ankyrin-B Interactions with Spectrin and Dynactin-4 Are Required for Dystrophin-based Protection of Skeletal Muscle from Exercise Injury

Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with β2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on β2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required β2 spectrin. Together, these results are consistent with a functional hierarchy beginning with β2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.

Response to the letter: “On the localization of ClC-1 in skeletal muscle fibers”

We are writing in response to the Letter to the Editor by [Lamb et al.][1] in this issue, which challenges the central conclusion of our recent study published in The Journal of General Physiology , namely that “functional ClC-1 channels in adult mouse skeletal muscle reside exclusively within the

Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle.

Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle. First, formamide-induced detubulation of single flexor digitorum brevis (FDB) muscle fibers from 15–16-day-old mice did not significantly alter macroscopic ClC-1 current magnitude (at −140 mV; −39.0 ± 4.5 and −42.3 ± 5.0 nA, respectively), deactivation kinetics, or voltage dependence of channel activation (V1/2 was −61.0 ± 1.7 and −64.5 ± 2.8 mV; k was 20.5 ± 0.8 and 22.8 ± 1.2 mV, respectively), despite a 33% reduction in cell capacitance (from 465 ± 36 to 312 ± 23 pF). In paired whole cell voltage clamp experiments, where ClC-1 activity was measured before and after detubulation in the same fiber, no reduction in ClC-1 activity was observed, despite an ∼40 and 60% reduction in membrane capacitance in FDB fibers from 15–16-day-old and adult mice, respectively. Second, using immunofluorescence and confocal microscopy, native ClC-1 channels in adult mouse FDB fibers were localized within the sarcolemma, 90° out of phase with double rows of dihydropyridine receptor immunostaining of the T-tubule system. Third, adenoviral-mediated expression of green fluorescent protein–tagged ClC-1 channels in adult skeletal muscle of a mouse model of myotonic dystrophy type 1 resulted in a significant reduction in myotonia and localization of channels to the sarcolemma. Collectively, these results demonstrate that the majority of functional ClC-1 channels localize to the sarcolemma and provide essential insight into the basis of myofiber excitability in normal and diseased skeletal muscle.

Increased Store-Operated Ca2+ Entry in Skeletal Muscle with Reduced Calsequestrin-1 Expression

Store-operated Ca2+ entry (SOCE) contributes to Ca2+ handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn2+ fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca2+ store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39°C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca2+ permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca2+ as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca2+ entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca2+ signaling in MH.

CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1

The neuromuscular disease myotonic dystrophy type I (DM1) affects multiple organ systems with the major symptoms being severe muscle weakness, progressive muscle wasting and myotonia. The causative mutation in DM1 is a CTG repeat expansion in the 3'-untranslated region of the DM protein kinase (DMPK) gene. RNA transcribed from the expanded allele contains the expanded CUG repeats and leads to the nuclear depletion of Muscleblind-like 1 (MBNL1) and to the increased steady-state levels of CUG-binding protein 1 (CUGBP1). The pathogenic effects of MBNL1 depletion have previously been tested by the generation of MBNL1 knockout mice, but the consequence of CUGBP1 overexpression in adult muscle is not known. In a DM1 mouse model expressing RNA containing 960 CUG repeats in skeletal muscle, CUGBP1 up-regulation is temporally correlated with severe muscle wasting. In this study, we generated transgenic mice with doxycycline-inducible and skeletal muscle-specific expression of CUGBP1. Adult mouse skeletal muscle overexpressing CUGBP1 reproduces molecular and physiological defects of DM1 tissue. The results from this study strongly suggest that CUGBP1 has a major role in DM1 skeletal muscle pathogenesis.

Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

Satellite cells/myoblasts account for the majority of muscle regenerative potential in response to injury and muscular adaptation to exercise. Although the ability to influence this process would provide valuable benefits for treating a variety of patients suffering from muscle loss, the regulatory mechanisms of myogenesis are not completely understood. We have tested the hypothesis that transforming growth factor-beta-activated kinase 1 (TAK1) is an important regulator of skeletal muscle formation. TAK1 is expressed in proliferating C2C12 myoblasts, and its levels are reduced upon differentiation of myoblasts into myotubes. In vivo, TAK1 is predominantly expressed in developing skeletal muscle of young mice. However, the expression of TAK1 was significantly up-regulated in regenerating skeletal muscle of adult mice. Overexpression of a dominant negative mutant of TAK1 or knockdown of TAK1 inhibited the proliferation and differentiation of C2C12 myoblasts. TAK1 was required for the expression of myogenic regulatory factors in differentiating myoblasts. Genetic ablation of TAK1 also inhibited the MyoD-driven transformation of mouse embryonic fibroblasts into myotubes. Inhibition of TAK1 suppressed the differentiation-associated activation of p38 mitogen-activated protein kinase (MAPK) and Akt kinase. Overexpression of a constitutively active mutant of MAPK kinase 6 (MKK6, an upstream activator of p38 MAPK) but not constitutive active Akt restored the myogenic differentiation in TAK1-deficient mouse embryonic fibroblasts. Insulin growth factor 1-induced myogenic differentiation was also found to involve TAK1. Collectively, our results suggest that TAK1 is an important upstream regulator of skeletal muscle cell differentiation.

Fatigue in Mouse Muscle Fibers: Role of Mitochondrial ATP-Sensitive Potassium Channels

Aim. The role of mitochondrial KATP (mitoKATP) channels on muscle fatigue was assessed in adult mouse skeletal muscle fibers.Methods. Muscle fatigue was produced by eliciting short repetitive tetani. Isometric tension and the rate of production of reactive oxygen species (ROS) were measured at room temperature (20-22°C) using a force transducer and the fluorescent indicator CM-H2DCFDA.Results. We found that opening mitoKATP channels with diazoxide (100 ∈μM) significantly reduced muscle fatigue. Fatigue tension was 34% higher in diazoxide-treated fibers relative to controls. This effect was blocked by the mitoKATP channel blocker 5-Hydroxydecanoate (5-HD), by the protein kinase C (PKC) inhibitor chelerythrine, and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester hydrochloride (L-NAME). We found that the rate of ROS production in muscle fibers incubated with diazoxide under non-fatigue and fatigue conditions was similar to control experiments, but that the increase in the rate of ROS production during recovery from fatigue was greatly reduced.Conclusions. A physiological role of mitoKATP channels on muscle fatigue is proposed.Diazoxide, an opener of mitoKATP channels, reduces muscle fatigue possibly through a preservation of mitochondrial volume and function.This work was supported in part by CONACYT grants 82667 (JS) and 60880 (MCG).