Mitochondrial Permeability Transition Causes Mitochondrial Reactive Oxygen Species- and Caspase 3-Dependent Atrophy of Single Adult Mouse Skeletal Muscle Fibers.

Elevated mitochondrial reactive oxygen species (mROS) and an increase in caspase-3 activity are established mechanisms that lead to skeletal muscle atrophy via the upregulation of protein degradation pathways. However, the mechanisms upstream of an increase in mROS and caspase-3 activity in conditions of muscle atrophy have not been identified. Based upon knowledge that an event known as mitochondrial permeability transition (MPT) causes an increase in mROS emission and the activation of caspase-3 via mitochondrial release of cytochrome c, as well as the circumstantial evidence for MPT in some muscle atrophy conditions, we tested MPT as a mechanism of atrophy. Briefly, treating cultured single mouse flexor digitorum brevis (FDB) fibers from adult mice with a chemical inducer of MPT (Bz423) for 24 h caused an increase in mROS and caspase-3 activity that was accompanied by a reduction in muscle fiber diameter that was able to be prevented by inhibitors of MPT, mROS, or caspase-3 (p < 0.05). Similarly, a four-day single fiber culture as a model of disuse caused atrophy that could be prevented by inhibitors of MPT, mROS, or activated caspase-3. As such, our results identify MPT as a novel mechanism of skeletal muscle atrophy that operates through mROS emission and caspase-3 activation.

α-Hydroxyisocaproic Acid Decreases Protein Synthesis but Attenuates TNFα/IFNγ Co-Exposure-Induced Protein Degradation and Myotube Atrophy via Suppression of iNOS and IL-6 in Murine C2C12 Myotube.

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.

Pulsed ultrasound prevents lipopolysaccharide-induced muscle atrophy through inhibiting p38 MAPK phosphorylation in C2C12 myotubes.

OBJECTIVE: Inflammation contributes to skeletal muscle atrophy via protein degradation induced by p38 mitogen-activated protein kinase (MAPK) phosphorylation. Meanwhile, pulsed ultrasound irradiation provides the mechanical stimulation to the target tissue, and has been reported to show anti-inflammatory effects. This study investigated the preventive effects of pulsed ultrasound irradiation on muscle atrophy induced by lipopolysaccharide (LPS) in C2C12 myotubes. METHODS: C2C12 myotubes were used in this research. The pulsed ultrasound (a frequency of 3 MHz, duty cycle of 20%, intensity of 0.5 W/cm2) was irradiated to myotube before LPS administration. RESULTS: The LPS increased phosphorylation of p38 MAPK and decreased the myofibril and myosin heavy chain protein (P < 0.05), followed by atrophy in C2C12 myotubes. The pulsed ultrasound irradiation attenuated p38 MAPK phosphorylation and myotube atrophy induced by LPS (P < 0.05). CONCLUSIONS: Pulsed ultrasound irradiation has the preventive effects on inflammation-induced muscle atrophy through inhibiting phosphorylation of p38 MAPK.

Propionate hampers differentiation and modifies histone propionylation and acetylation in skeletal muscle cells.

Protein acylation via metabolic acyl-CoA intermediates provides a link between cellular metabolism and protein functionality. A process in which acetyl-CoA and acetylation are fine-tuned is during myogenic differentiation. However, the roles of other protein acylations remain unknown. Protein propionylation could be functionally relevant because propionyl-CoA can be derived from the catabolism of amino acids and fatty acids and was shown to decrease during muscle differentiation. We aimed to explore the potential role of protein propionylation in muscle differentiation, by mimicking a pathophysiological situation with high extracellular propionate which increases propionyl-CoA and protein propionylation, rendering it a model to study increased protein propionylation. Exposure to extracellular propionate, but not acetate, impaired myogenic differentiation in C2C12 cells and propionate exposure impaired myogenic differentiation in primary human muscle cells. Impaired differentiation was accompanied by an increase in histone propionylation as well as histone acetylation. Furthermore, chromatin immunoprecipitation showed increased histone propionylation at specific regulatory myogenic differentiation sites of the Myod gene. Intramuscular propionylcarnitine levels are higher in old compared to young males and females, possibly indicating increased propionyl-CoA levels with age. The findings suggest a role for propionylation and propionyl-CoA in regulation of muscle cell differentiation and ageing, possibly via alterations in histone acylation.

Muscle fiber type specific alterations of mitochondrial respiratory function and morphology in aged female mice.

Mitochondrial dysfunction is considered to be a major cause of sarcopenia, defined as age-related muscle fiber atrophy and muscle weakness, as reduced mitochondrial respiration and morphological changes such as ragged red fibers (RRFs) are observed in aging muscles. However, the role of mitochondrial dysfunction in sarcopenia is not fully elucidated. Although previous studies have suggested that aging has a fiber type-specific effect on mitochondrial function, little is known about mitochondrial changes in individual fiber types. Here, we used C57BL/6NCr female mice to identify fiber type-specific pathological changes, examine the significance of pathological changes in sarcopenia, and identify possible mechanisms behind mitochondrial changes in slow-twitch soleus muscle (SOL) and fast-twitch extensor digitorum longus muscle (EDL). We observed reduced type I fiber-specific mitochondrial respiratory enzyme activity, impaired respiration, and subsarcolemmal mitochondrial accumulation in aged SOL, which was different from RRFs. These pathological alterations were not directly associated with fiber atrophy. Additionally, we found increased oxidative stress markers in aged SOL, suggesting that oxidative stress is involved in the pathological and functional changes in mitochondria. Meanwhile, obvious mitochondrial changes were not seen in aged EDL. Thus, age-related mitochondrial dysfunction is specific to the fiber type and may correlate with the muscle quality rather than the muscle mass.

Inhibition of xanthine oxidase in the acute phase of myocardial infarction prevents skeletal muscle abnormalities and exercise intolerance.

AIMS: Exercise intolerance in patients with heart failure (HF) is partly attributed to skeletal muscle abnormalities. We have shown that reactive oxygen species (ROS) play a crucial role in skeletal muscle abnormalities, but the pathogenic mechanism remains unclear. Xanthine oxidase (XO) is reported to be an important mediator of ROS overproduction in ischaemic tissue. Here, we tested the hypothesis that skeletal muscle abnormalities in HF are initially caused by XO-derived ROS and are prevented by the inhibition of their production. METHODS AND RESULTS: Myocardial infarction (MI) was induced in male C57BL/6J mice, which eventually led to HF, and a sham operation was performed in control mice. The time course of XO-derived ROS production in mouse skeletal muscle post-MI was first analysed. XO-derived ROS production was significantly increased in MI mice from Days 1 to 3 post-surgery (acute phase), whereas it did not differ between the MI and sham groups from 7 to 28 days (chronic phase). Second, mice were divided into three groups: sham + vehicle (Sham + Veh), MI + vehicle (MI + Veh), and MI + febuxostat (an XO inhibitor, 5 mg/kg body weight/day; MI + Feb). Febuxostat or vehicle was administered at 1 and 24 h before surgery, and once-daily on Days 1-7 post-surgery. On Day 28 post-surgery, exercise capacity and mitochondrial respiration in skeletal muscle fibres were significantly decreased in MI + Veh compared with Sham + Veh mice. An increase in damaged mitochondria in MI + Veh compared with Sham + Veh mice was also observed. The wet weight and cross-sectional area of slow muscle fibres (higher XO-derived ROS) was reduced via the down-regulation of protein synthesis-associated mTOR-p70S6K signalling in MI + Veh compared with Sham + Veh mice. These impairments were ameliorated in MI + Feb mice, in association with a reduction of XO-derived ROS production, without affecting cardiac function. CONCLUSION: XO inhibition during the acute phase post-MI can prevent skeletal muscle abnormalities and exercise intolerance in mice with HF.

Extraocular Muscle Reveals Selective Vulnerability of Type IIB Fibers to Respiratory Chain Defects Induced by Mitochondrial DNA Alterations.

Purpose: The purpose of this study was to gain insights on the pathogenesis of chronic progressive external ophthalmoplegia, thus we investigated the vulnerability of five extra ocular muscles (EOMs) fiber types to pathogenic mitochondrial DNA deletions in a mouse model expressing a mutated mitochondrial helicase TWINKLE. Methods: Consecutive pairs of EOM sections were analyzed by cytochrome C oxidase (COX)/succinate dehydrogenase (SDH) assay and fiber type specific immunohistochemistry (type I, IIA, IIB, embryonic, and EOM-specific staining). Results: The mean average of COX deficient fibers (COX-) in the recti muscles of mutant mice was 1.04 ± 0.52% at 12 months and increased with age (7.01 ± 1.53% at 24 months). A significant proportion of these COX- fibers were of the fast-twitch, glycolytic type IIB (> 50% and > 35% total COX- fibers at 12 and 24 months, respectively), whereas embryonic myosin heavy chain-expressing fibers were almost completely spared. Furthermore, the proportion of COX- fibers in the type IIB-rich retractor bulbi muscle was > 2-fold higher compared to the M. recti at both 12 (2.6 ± 0.78%) and 24 months (20.85 ± 2.69%). Collectively, these results demonstrate a selective vulnerability of type IIB fibers to mitochondrial DNA (mtDNA) deletions in EOMs and retractor bulbi muscle. We also show that EOMs of mutant mice display histopathological abnormalities, including altered fiber type composition, increased fibrosis, ragged red fibers, and infiltration of mononucleated nonmuscle cells. Conclusions: Our results point to the existence of fiber type IIB-intrinsic factors and/or molecular mechanisms that predispose them to increased generation, clonal expansion, and detrimental effects of mtDNA deletions.

Functional skeletal muscle model derived from SOD1-mutant ALS patient iPSCs recapitulates hallmarks of disease progression.

Recent findings suggest a pathologic role of skeletal muscle in amyotrophic lateral sclerosis (ALS) onset and progression. However, the exact mechanism by which this occurs remains elusive due to limited human-based studies. To this end, phenotypic ALS skeletal muscle models were developed from induced pluripotent stem cells (iPSCs) derived from healthy individuals (WT) and ALS patients harboring mutations in the superoxide dismutase 1 (SOD1) gene. Although proliferative, SOD1 myoblasts demonstrated delayed and reduced fusion efficiency compared to WT. Additionally, SOD1 myotubes exhibited significantly reduced length and cross-section. Also, SOD1 myotubes had loosely arranged myosin heavy chain and reduced acetylcholine receptor expression per immunocytochemical analysis. Functional analysis indicated considerably reduced contractile force and synchrony in SOD1 myotubes. Mitochondrial assessment indicated reduced inner mitochondrial membrane potential (ΔΨm) and metabolic plasticity in the SOD1-iPSC derived myotubes. This work presents the first well-characterized in vitro iPSC-derived muscle model that demonstrates SOD1 toxicity effects on human muscle regeneration, contractility and metabolic function in ALS. Current findings align with previous ALS patient biopsy studies and suggest an active contribution of skeletal muscle in NMJ dysfunction. Further, the results validate this model as a human-relevant platform for ALS research and drug discovery studies.

Damaged Myofiber-Derived Metabolic Enzymes Act as Activators of Muscle Satellite Cells.

Muscle satellite cells are normally quiescent but are rapidly activated following muscle damage. Here, we investigated whether damaged myofibers influence the activation of satellite cells. Our findings revealed that satellite cells are directly activated by damaged-myofiber-derived factors (DMDFs). DMDFs induced satellite cells to enter the cell cycle; however, the cells stayed at the G1 phase and did not undergo S phase, and these cells were reversible to the quiescent-like state. Proteome analysis identified metabolic enzymes, including GAPDH, as DMDFs, whose recombinant proteins stimulated the activation of satellite cells. Satellite cells pre-exposed to the DMDFs demonstrated accelerated proliferation ex vivo. Treatment with recombinant GAPDH prior to muscle injury promoted expansion of the satellite cell population in vivo. Thus, our results indicate that DMDFs are not only a set of biomarkers for muscle damage, but also act as moonlighting proteins involved in satellite cell activation at the initial step of muscle regeneration.

HDAC6 regulates microtubule stability and clustering of AChRs at neuromuscular junctions.

Microtubules (MTs) are known to be post-translationally modified at the neuromuscular junction (NMJ), hence increasing their stability. To date however, the function(s) of the dynamic MT network and its relative stability in the formation and maintenance of NMJs remain poorly described. Stabilization of the MT is dependent in part on its acetylation status, and HDAC6 is capable of reversing this post-translational modification. Here, we report that HDAC6 preferentially accumulates at NMJs and that it contributes to the organization and the stability of NMJs. Indeed, pharmacological inhibition of HDAC6 protects against MT disorganization and reduces the size of acetylcholine receptor (AChR) clusters. Moreover, the endogenous HDAC6 inhibitor paxillin interacts with HDAC6 in skeletal muscle cells, colocalizes with AChR aggregates, and regulates the formation of AChR. Our findings indicate that the focal insertion of AChRs into the postsynaptic membrane is regulated by stable MTs and highlight how an MT/HDAC6/paxillin axis participates in the regulation of AChR insertion and removal to control the structure of NMJs.

Comprehensive histological investigation of age-related changes in dermal extracellular matrix and muscle fibers in the upper lip vermilion.

OBJECTIVE: Few histological studies have directly examined age-related changes within the lips, although non-invasive investigations of such changes are increasing. Therefore, this study aimed to provide histological and molecular data on age-dependent alterations in the vermilion. METHODS: Upper vermilion specimens from 15 female Caucasian cadavers (age range, 27-78 years) were investigated histologically or immunohistochemically. RESULTS: Histologically, age-dependent decreases in areas occupied by hyaluronan and collagenous fibres in the dermis of upper vermilion were demonstrated. Elastic fibre content varied widely between individuals. The area occupied by muscle fibres in the orbicularis oris muscle region within the vermilion also correlated negatively with age. Immunohistochemically, signals of four proteins were attenuated in vermilion from older individuals compared with young individuals: procollagen type I, hyaluronan synthase (HAS)1, myosin heavy chain (MYH)2 (a component of fast-twitch oxidative muscle fibres) and MYH7 (a component of slow-twitch muscle fibres). In contrast, signals of cell migration inducing hyaluronidase 1 (CEMIP) were intensified in vermilion from older individuals. No marked differences between young and older individuals were seen in procollagen type III, HAS2, HAS3, hyaluronidase (HYAL)1, HYAL2, MYH1 or MYH4. CONCLUSION: Age-dependent decreases of hyaluronan in the dermis of vermilion were prominent, possibly due to both the decrease in synthesis (HAS1) and the increase in degradation (CEMIP). Furthermore, age-dependent decreases in collagenous fibres and two types of muscle fibre in the vermilion were also identified histologically. Type I collagen, MYH2 and MYH7 appear to represent the molecules responsible for these respective decrements.

OBJECTIF: Peu d'études histologiques ont examiné directement les changements liés à l'âge sur les lèvres, bien que les enquêtes non invasives de ces changements soient en augmentation. Par conséquent, cette étude visait à fournir des données histologiques et moléculaires sur les altérations liées à l'âge dans le vermillon. MÉTHODES: Des échantillons de vermillon supérieur provenant de 15 cadavres de femme Caucasiens (tranche d'âge, 27-78 ans) ont été étudiés histologiquement ou immuno-histochimiquement. RÉSULTATS: Histologiquement, des diminutions dépendant de l'âge dans les zones occupées par l'hyaluronane et les fibres de collagène dans le derme du vermillon supérieur ont été démontrées. La teneur en fibres élastiques variait considérablement entre les individus. La zone occupée par les fibres musculaires dans la région du muscle orbiculaire oris au sein du vermillon était également corrélée négativement avec l'âge. Immuno-histochimiquement, les signaux de quatre protéines ont été atténués dans vermillon des individus plus âgés que les jeunes: le procollagène type I, l'hyaluronane synthase (HAS) 1, la chaîne lourde de la myosine (MYH) 2 (un composant des fibres musculaires oxydatives à contraction rapide) et MYH7 (un composant des fibres musculaires à contraction lente). En revanche, les signaux du "cell migration inducing hyaluronidase 1 (CEMIP)" ont été intensifiés dans le vermillon des individus plus âgés. Aucune différence marquée entre les individus jeunes et âgés n'a été observée dans le procollagène type III, HAS2, HAS3, hyaluronidase (HYAL) 1, HYAL2, MYH1 et MYH4. CONCLUSION: Les diminutions dépendantes de l'âge du hyaluronane dans le derme du vermillon étaient importantes, probablement en raison à la fois de la diminution de la synthèse (HAS1) et de l'augmentation de la dégradation (CEMIP). En outre, les diminutions dépendantes de l'âge des fibres de collagène et de deux les types de fibres musculaires dans le vermillon ont également été identifiés histologiquement. Le collagène de type I, MYH2 et MYH7 semblent respectivement représenter les molécules responsables de ces diminutions.

Elevated MMP2 abundance and activity in mdx mice are alleviated by prenatal taurine supplementation.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disorder that leads to early death. The mdx mouse is a naturally occurring mutant model for DMD. It lacks dystrophin and displays peak muscle cell necrosis at ~28 days (D28), but in contrast to DMD, mdx mice experience muscle regeneration by D70. We hypothesized that matrix metalloproteinase-2 (MMP2) and/or MMP9 play key roles in the degeneration/regeneration phases in mdx mice. MMP2 abundance in muscle homogenates, measured by calibrated Western blotting, and activity, measured by zymogram, were lower at D70 compared with D28 in both mdx and wild-type (WT) mice. Importantly, MMP2 abundance was higher in both D28 and D70 mdx mice than in age-matched WT mice. The higher MMP2 abundance was not due to infiltrating macrophages, because MMP2 content was still higher in isolated muscle fibers where most macrophages had been removed. Prenatal supplementation with the amino acid taurine, which improved muscle strength in D28 mdx mice, produced approximately twofold lower MMP2 activity, indicating that increased MMP2 abundance is not required when muscle damage is attenuated. There was no difference in MMP9 abundance between age-matched WT and mdx mice (P > 0.05). WT mice displayed decreased MMP9 abundance as they aged. While MMP9 may have a role during age-related skeletal muscle growth, it does not appear essential for degeneration/regeneration cycles in the mdx mouse. Our findings indicate that MMP2 plays a more active role than MMP9 in the degenerative phases of muscle fibers in D28 mdx mice.

Alisol A-24-acetate promotes glucose uptake via activation of AMPK in C2C12 myotubes.

BACKGROUND: Alisol A-24-acetate (AA-24-a) is one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., which possesses multiple biological activities, including a hypoglycemic effect. Whether AA-24-a is a hypoglycemic-active compound of A. orientale (Sam.) Juz. is unclear. The present study aimed to clarify the effect and potential mechanism of action of AA-24-a on glucose uptake in C2C12 myotubes. METHOD: Effects of AA-24-a on glucose uptake and GLUT4 translocation to the plasma membrane were evaluated. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay. Cell membrane proteins were isolated and glucose transporter 4 (GLUT4) protein was detected by western blotting to examine the translocation of GLUT4 to the plasma membrane. To determine the underlying mechanism, the phosphorylation levels of proteins involved in the insulin and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways were examined using western blotting. Furthermore, specific inhibitors of key enzymes in AMPK signaling pathway were used to examine the role of these kinases in the AA-24-a-induced glucose uptake and GLUT4 translocation. RESULTS: We found that AA-24-a significantly promoted glucose uptake and GLUT4 translocation in C2C12 myotubes. AA-24-a increased the phosphorylation of AMPK, but had no effect on the insulin-dependent pathway involving insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/AKT). In addition, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the AKT substrate of 160 kDa (AS160), two proteins that act downstream of AMPK, was upregulated. Compound C, an AMPK inhibitor, blocked AA-24-a-induced AMPK pathway activation and reversed AA-24-a-induced glucose uptake and GLUT4 translocation to the plasma membrane, indicating that AA-24-a promotes glucose metabolism via the AMPK pathway in vitro. STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, also attenuated AA-24-a-induced glucose uptake and GLUT4 translocation. Moreover, STO-609 weakened AA-24-a-induced phosphorylation of AMPK, p38 MAPK and AS160. CONCLUSIONS: These results indicate that AA-24-a isolated from A. orientale (Sam.) Juz. significantly enhances glucose uptake via the CaMKKß-AMPK-p38 MAPK/AS160 pathway.

Aging reduces succinate dehydrogenase activity in rat type IIx/IIb diaphragm muscle fibers.

In aged rats, diaphragm muscle (DIAm) reduced specific force and fiber cross-sectional area, sarcopenia, is selective for vulnerable type IIx and/or IIb DIAm fibers, with type I and IIa fibers being resilient. In humans, the oxidative capacity [as measured by maximum succinate dehydrogenase (SDHmax) activity] of fast-type muscle is reduced with aging, with slow-type muscle being unaffected. We hypothesized that in aged Fischer rat DIAm exhibiting sarcopenia, reduced SDHmax activity would occur in type IIx and/or IIb fibers. Rats obtained from the NIA colony (6, 18, and 24 mo old) were euthanized, and ~2-mm-wide DIAm strips were obtained. For SDHmax and fiber type assessments, DIAm strips were stretched (approximately optimal length), fresh frozen in isopentane, and sectioned on a cryostat at 6 µm. SDHmax, quantified by intensity of nitroblue tetrazolium diformazan precipitation, was assessed in a fiber type-specific manner by comparing serial sections labeled with myosin heavy chain (MyHC) antibodies differentiating type I (MyHCSlow), IIa (MyHC2A), and IIx and/or IIb fibers. Isometric DIAm force and fatigue were assessed in DIAm strips by muscle stimulation with supramaximal pulses at a variety of frequencies (5-100 Hz) delivered in 1-s trains. By 24 mo, DIAm sarcopenia was apparent and SDHmax in type IIx and/or IIb fibers activity was reduced ~35% compared with 6-mo-old control DIAm. These results underscore the remarkable fiber type selectivity of type IIx and/or IIb fibers to age-associated perturbations and suggest that reduced mitochondrial oxidative capacity is associated with DIAm sarcopenia.NEW & NOTEWORTHY We examined the oxidative capacity as measured by maximum succinate dehydrogenase activity in older (18 or 24 mo old) Fischer 344 rat diaphragm muscle (DIAm) compared with young rats (6 mo old). In 24-mo-old rats, SDH activity was reduced in type IIx/b DIAm fibers. These SDH changes were concomitant with sarcopenia (reduced specific force and atrophy of type IIx/b DIAm fibers) at 24 mo old. At 18 mo old, there was no change in SDH activity and no evidence of sarcopenia.

Low Ouabain Doses and AMP-Activated Protein Kinase as Factors Supporting Electrogenesis in Skeletal Muscle.

Many motor disorders are associated with depolarization of the membrane of skeletal muscle fibers due to the impaired functioning of Na,K-ATPase. Here, we studied the role of ouabain (specific Na,K-ATPase ligand) and AMP-activated protein kinase (key regulator of muscle metabolism) in the maintenance of muscle electrogenesis; the levels of these endogenous factors are directly related to the motor activity. After 4-day intraperitoneal administration of ouabain (1 µg/kg daily), a hyperpolarization of sarcolemma was registered in isolated rat diaphragm muscles due to an increase in the electrogenic activity of Na,K-ATPase. In acute experiments, addition of nanomolar ouabain concentrations to the bathing solution resulted in the muscle membrane hyperpolarization within 15 min. The effect of ouabain reversed to membrane depolarization with the increase in the external potassium concentration. It is possible that Na,K-ATPase activation by ouabain may be regulated by such factors as specific subcellular location, interaction with molecular partners, and changes in the ionic balance. Preventive administration of the AMP-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; 400 mg/kg body weight daily for 7 days) in chronic experiments resulted in the stabilization of the endplate structure and abolishment of depolarization of the rat soleus muscle membrane caused by the motor activity cessation. The obtained data can be useful for creating approaches for correction of muscle dysfunction, especially at the early stages, prior to the development of muscle atrophy.

Transient induction of Cdk9 in the early stage of differentiation is critical for myogenesis.

Cdk9 is a serine-threonine protein kinase that has been recognized as a regulator of cardiac differentiation. Recently, we have reported that transient induction of Cdk9 using noncoding RNA targeting Cdk9 sequences results in efficient cardiac differentiation. Concerning Cdk9 regulatory roles, here, we proposed whether constant overexpression of Cdk9 might influence the differentiation of myoblast C2C12 cells into myotubes. We overexpressed Cdk9 in mouse myoblast C2C12 cells to investigate its regulatory roles on myogenic differentiation. Upon Cdk9 overexpression, the expression level of myogenic regulatory factors was determined. Moreover, the expression profile of three important myomiRs consist of miR 1, 133 and 206 was examined during the differentiation process. Although Cdk9 expression is necessary for inducing differentiation in the early stage of myogenesis, continuous Cdk9 expression inhibits differentiation by modulating myomiRs and myogenic gene expression. Our results indicate that the transient induction of Cdk9 in the early stage of differentiation is critical for myogenesis.

Distribution and activation of matrix metalloproteinase-2 in skeletal muscle fibers.

A substantial intracellular localization of matrix metalloproteinase 2 (MMP2) has been reported in cardiomyocytes, where it plays a role in the degradation of the contractile apparatus following ischemia-reperfusion injury. Whether MMP2 may have a similar function in skeletal muscle is unknown. This study determined that the absolute amount of MMP2 is similar in rat skeletal and cardiac muscle and human muscle (~10-18 nmol/kg muscle wet wt) but is ~50- to 100-fold less than the amount of calpain-1. We compared mechanically skinned muscle fibers, where the extracellular matrix (ECM) is completely removed, with intact fiber segments and found that ~30% of total MMP2 was associated with the ECM, whereas ~70% was inside the muscle fibers. Concordant with whole muscle fractionation, further separation of skinned fiber segments into cytosolic, membranous, and cytoskeletal and nuclear compartments indicated that ~57% of the intracellular MMP2 was freely diffusible, ~6% was associated with the membrane, and ~37% was bound within the fiber. Under native zymography conditions, only 10% of MMP2 became active upon prolonged (17 h) exposure to 20 µM Ca2+, a concentration that would fully activate calpain-1 in seconds to minutes; full activation of MMP2 would require ~1 mM Ca2+. Given the prevalence of intracellular MMP2 in skeletal muscle, it is necessary to investigate its function using physiological conditions, including isolation of any potential functional relevance of MMP2 from that of the abundant protease calpain-1.

Resveratrol regulates skeletal muscle fibers switching through the AdipoR1-AMPK-PGC-1α pathway.

This study was conducted to investigate the effect and underlying mechanism of Resveratrol (RES) in regulating skeletal muscle fiber-type switching. We found that RES had no effect on the body weight and food intake of Kunming mice (KM mice) that were orally administered with 400 mg kg-1 d-1 RES for 12 weeks. Notably, the RES administration significantly increased the expression of myosin heavy chain (MyHC) 1, MyHC2a, and MyHC2x in the extensor digitorum longus (EDL) and soleus (SOL) muscles. Furthermore, the muscle immunostaining of the results showed that the RES treatment led to the myofiber type transition from glycolytic to oxidative in muscles. The mRNA and protein levels of the adiponectin receptor (AdipoR), AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in EDL and SOL were drastically increased after RES treatment. Moreover, the plasma Adiponectin (AdipoQ) protein levels were higher in the RES-treated mice compared to the control mice. Moreover, the in vitro results further demonstrated that the 20 µM RES treatment increased the expression of AdipoR1, AdipoR2, AMPK, PGC-1α and MyHC1, but decreased the expression of MyHC2b in C2C12 myoblasts. Furthermore, mechanistic studies revealed that silencing the AdiopR1, not the AdiopR2, abolished the effect of RES on the expression of AMPK and PGC-1α in the C2C12 cells. These results indicated that RES could regulate skeletal fiber switching through the AdiopR1-AMPK-PGC-1α pathway. This work may provide a new strategy for enhancing endurance and relieving muscle diseases caused by oxidative muscle fiber deficiency.

Phenethyl isothiocyanate stimulates glucose uptake through the Akt pathway in C2C12 myotubes.

Phenethyl isothiocyanate (PEITC) is an aromatic isothiocyanate present in cruciferous vegetables. Several studies have shown that isothiocyanates regulate various intracellular signaling pathways, and thereby show anti-inflammatory and detoxifying activities. However, little is known about the effects of PEITC on glucose metabolism. In this study, we examined whether PEITC promotes glucose utilization in mouse skeletal muscle cells, C2C12 myotubes. PEITC induced glucose uptake, glucose transporter 4 (Glut4) translocation to the plasma membrane, and activation of Akt and ERK in C2C12 cells. Inhibition of Akt suppressed PEITC-induced Glut4 translocation and glucose uptake, whereas ERK inhibition did not. Furthermore, PEITC increased phosphorylation of ErbB2 and ErbB3. Treatment with a pan-ErbB inhibitor reduced Akt activation and the subsequent glucose uptake induced by PEITC. These results indicate that PEITC promotes glucose utilization through the ErbB/Akt pathway in C2C12 myotubes. PEITC may therefore serve as a dietary constituent with beneficial effects on the carbohydrate metabolism. Abbreviations: PEITC: phenethyl isothiocyanate; Glut4: glucose transporter 4; PI3K: phosphatidylinositide 3-kinase; Nrf2: erythroid-2-related factor; ARE: antioxidant response element; HO-1: heme oxygenase-1; NRG: neuregulin.

The distribution and time-dependent expression of HIPK2 during the repair of contused skeletal muscle in mice.

HIPK2 is an evolutionarily conserved serine/threonine kinase and is considered a co-regulator of an increasing number of transcription factors modulating a variety of cellular processes, including inflammation, proliferation and fibrosis. Skeletal muscle injuries repair is an overlapping event between inflammation and tissue repair. There are no reports about HIPK2 expression in skeletal muscles after trauma. A foundational study on distribution and time-dependent expression of HIPK2 was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR, which is expected to obtain a preliminary insight into the functions of HIPK2 during the repair of contused skeletal muscle in mice. An animal model of skeletal muscle contusion was established in 50 C57B6/L male mice. Samples were taken at 1, 3, 5, 7, 9, 14, 17, 21 and 28 days after contusion, respectively (5 mice at each posttraumatic interval). 5 mice were employed as control. No HIPK2-positive staining was detected in uninjured skeletal muscle. Intensive immunoreactivties of HIPK2 were observed in polymorphonuclear cells, round-shaped mononuclear cells, regenerated multinucleated myotubes and spindle-shaped fibroblastic cells in the contused tissue. The HIPK2-positive cells were identified as neutrophils, macrophages and myofibroblasts by double immunofluorescent procedure. HIPK2 protein and mRNA expression were remarkably up-regulated after contusion by Western blotting and qPCR analysis. The results demonstrated that the expression of HIPK2 is distributed in certain cell types and is time-dependently expressed in skeletal muscle after contusion, which suggested that HIPK2 may participate in the whole process of skeletal muscle wound healing, including inflammatory response, muscle regeneration and fibrogenesis.