The influence of skeletal muscle cell volume on the regulation of carbohydrate uptake and muscle metabolism

This study investigated the regulation of carbohydrate metabolism and glucose uptake through changes in skeletal muscle cell volume. Using an established invitro isolated whole muscle model, soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected from male rats and incubated in an organ bath containing Sigma medium-199 with 8 mM D-glucose altered to target osmolality (hypo-osmotic: HYPO, iso-osmotic: ISO, hyper-osmotic: HYPER; 190, 290, 400 mmol/kg). Muscles were divided into two groups; metabolite (MM) and uptake (MU). MM (N=48) were incubated for 60 minutes and were then immediately flash frozen. MU (N=24) were incubated for 30 minutes and then the extracellular fluid was exchanged for media containing ^H-glucose and ^'*C-mannitol and incubated for another 30 minutes. After the incubation, the muscles were freeze clamped. Results demonstrated a relative water decrease and increase in HYPER and HYPO, respectively. EDL and SOL glucose uptakes were found to be significantly greater in HYPER conditions. The HYPER condition resulted in significant alterations in muscle metabolite concentrations (lower glycogen, elevated lactate, and G-6-P) suggesting a catabolic cell state, and an increase in glycogen synthase transformation when compared to the HYPO group. In conclusion, skeletal muscle cell volume alters rates of glucose uptake with further alterations in muscle metabolites and glycogen synthase transformation.

The influence of skeletal muscle cell volume on carbohydrate metabolism in contracting skeletal muscle

This study investigated the regulation of carbohydrate metabolism through changes in skeletal muscle cell volume immediately post contraction and during recovery. Using an established in vitro isolated muscle strip model, soleus (SOL) and extensor digitorum longus (EDL) were dissected from male rats and incubated in an organ bath (perfused with 95% O2; 5% CO2, pH 7.4, temperature 25°C) containing medium- 199 altered to a target osmotic condition (iso-, hypo- or hyper-osmotic; 290, 1 80, 400 mmol/kg). Muscles were stimulated for 10 minutes (40 Hz SOL; 30 Hz EDL) and then either immediately flash frozen or allowed to recover for 20 minutes before subsequent metabolite and enzyme analysis. Results demonstrated a relative water decrease in HYPER vs. HYPOosmotic condition (n=8/group; p<0.05) regardless of muscle type. Specifically, the SOL HYPER condition had elevated metabolite concentrations after 10 minutes of stimulation in comparison to both HYPO and ISO (p<0.05), while EDL muscle did not show any significant difTerences between the HYPER or HYPO conditions. After 20 minutes of recovery, metabolic changes occurred in both SOL and EDL with the SOL HYPER condition showing greater relative changes in metabolite concentrations versus HYPO. The results of the current study have demonstrated that osmotic imbalance induces metabolic change within the skeletal muscle cell and muscle type may influence the mechanisms utilized for cell volume regulation.

The chicken ovalbumin upstream promoter-transcription factors modulate genes and pathways involved in skeletal muscle cell metabolism

The chicken ovalbumin upstream promoter-transcription factors ( COUP-TFs) are orphan members of the nuclear hormone receptor ( NR) superfamily. COUP-TFs are involved in organogenesis and neurogenesis. However, their role in skeletal muscle ( and other major mass tissues) and metabolism remains obscure. Skeletal muscle accounts for similar to 40% of total body mass and energy expenditure. Moreover, this peripheral tissue is a primary site of glucose and fatty acid utilization. We utilize small interfering RNA ( siRNA)-mediated attenuation of Coup-TfI and II ( mRNA and protein) in a skeletal muscle cell culture model to understand the regulatory role of Coup-Tfs in this energy demanding tissue. This targeted NR repression resulted in the significant attenuation of genes that regulate lipid mobilization and utilization ( including Ppar alpha, Fabp3, and Cpt-1). This was coupled to reduced fatty acid beta-oxidation. Additionally we observed significant attenuation of Ucp1, a gene involved in energy expenditure. Concordantly, we observed a 5-fold increase in ATP levels in cells with siRNA-mediated repression of Coup-TfI and II. Furthermore, the expression of classical liver X receptor ( LXR) target genes involved in reverse cholesterol transport ( Abca1 and Abcg1) were both significantly repressed. Moreover, we observed that repression of the Coup-Tfs ablated the activation of Abca1, and Abcg1 mRNA expression by the selective LXR agonist, T0901317. In concordance, Coup-Tf-siRNA-transfected cells were refractory to Lxr-mediated reduction of total intracellular cholesterol levels in contrast to the negative control cells. In agreement Lxr-mediated activation of the Abca1 promoter in Coup-Tf-siRNA cells was attenuated. Collectively, these data suggest a pivotal role for Coup-Tfs in the regulation of lipid utilization/cholesterol homeostasis in skeletal muscle cells and the modulation of Lxr-dependent gene regulation.

The effect of extracellular osmolality on cell volume and resting skeletal muscle metabolism

The purpose of the current investigation was to establish an in-l'itro skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated. whole muscle (SOL and EDL) was dissected from Long Evans rats and incubated for 60 min in Sigma Medium-199 (resting tension (lg). bubbled with 95:5% 02:C02, 30 ± 2°C, and pH 7.4). Media osmolality was altered to simulate hypo-osmotic (190 ± 10 Osm) (HYPO) or hyper-osmotic conditions (400 ± 10 Osm) (HYPER) while an iso-osmotic condition (290± 1 0 Osm) (CON) served as a control (n= 17.19.17). Following incubation, relative muscle water content decreased with HYPER and increased with HYPO in both muscle types (p<0.05). The cross-sectional area of HYPO SOL type I and type II fibres increased (p<0.05) while the EDL type 11 fibre area decreased in HYPER and increascd from HYPO exposure. Furthermore, HYPER exposure in both muscles lead to decreased ATP and phosphocreatine (p<0.05) and increased creatine and lactate (p<0.05) compared to CON. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acutc alterations in muscle water content and resting muscle metabolism.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3% of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

MicroRNA-499 expression distinctively correlates to target genes sox6 and rod1 profiles to resolve the skeletal muscle phenotype in nile tilapia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Statin therapy induces ultrastructural damage in skeletal muscle in patients without myalgia

Muscle pain and weakness are frequent complaints in patients receiving 3-hydroxymethylglutaryl coenzymeA (HMG CoA) reductase inhibitors (statins). Many patients with myalgia have creatine kinase levels that are either normal or only marginally elevated, and no obvious structural defects have been reported in patients with myalgia only. To investigate further the mechanism that mediates statin-induced skeletal muscle damage, skeletal muscle biopsies from statin-treated and non-statin-treated patients were examined using both electron microscopy and biochemical approaches. The present paper reports clear evidence of skeletal muscle damage in statin-treated patients, despite their being asymptomatic. Though the degree of overall damage is slight, it has a characteristic pattern that includes breakdown of the T-tubular system and subsarcolemmal rupture. These characteristic structural abnormalities observed in the statin-treated patients were reproduced by extraction of cholesterol from skeletal muscle fibres in vitro. These findings support the hypothesis that statin-induced cholesterol lowering per se contributes to myocyte damage and suggest further that it is the specific lipid/protein organization of the skeletal muscle cell itself that renders it particularly vulnerable.

The orphan nuclear receptor, NOR-1, is a target of beta-adrenergic signaling in skeletal muscle

beta-Adrenergic receptor (beta-AR) agonists induce Nur77 mRNA expression in the C2C12 skeletal muscle cell culture model and elicit skeletal muscle hypertrophy. We previously demonstrated that Nur77 (NR4A1) is involved in lipolysis and gene expression associated with the regulation of lipid homeostasis. Subsequently it was demonstrated by another group that beta-AR agonists and cold exposure-induced Nur77 expression in brown adipocytes and brown adipose tissue, respectively. Moreover, NOR-1 (NR4A3) was hyperinduced by cold exposure in the nur77(-/-) animal model. These studies underscored the importance of understanding the role of NOR-1 in skeletal muscle. In this context we observed 30-480 min of beta-AR agonist treatment significantly and transiently increased expression of the orphan nuclear receptor NOR-1 in both mouse skeletal muscle tissue (plantaris) and C2C12 skeletal muscle cells. Specific beta(2)-and beta(3)-AR agonists had similar effects as the pan-agonist and were blocked by the beta-AR antagonist propranolol. Moreover, in agreement with these observations, isoprenaline also significantly increased the activity of the NOR-1 promoter. Stable exogenous expression of a NOR-1 small interfering RNA (but not the negative control small interfering RNA) in skeletal muscle cells significantly repressed endogenous NOR-1 mRNA expression and led to changes in the expression of genes involved in the control of lipid use and muscle mass underscored by a dramatic increase in myostatin mRNA expression. Concordantly the myostatin promoter was repressed by NOR-1 expression. In conclusion, NOR-1 is highly responsive to beta-adrenergic signaling and regulates the expression of genes controlling fatty acid use and muscle mass.

Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox

Hypochlorous acid (HOCl) concentration-dependently decreased ATPase activity and SH groups of pure Ca-ATPase from sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle with IC(50) of 150 micromol/l and 6.6 micromol/l, respectively. This indicates that SH groups were not critical for impairment of Ca-ATPase activity. Pure Ca-ATPase activity was analysed individually with respect to both substrates, Ca(2+) and ATP. Concerning dependence of ATPase activity on HOCl (150 micromol/l) as a function of free Ca(2+) and ATP, V(max) of both dependences decreased significantly, while the affinities to individual substrates were not influenced, with the exception of the regulatory binding site of ATP. On increasing HOCl concentration, fluorescence of fluorescein-5-isothiocyanate (FITC) decreased, indicating binding of HOCl to nucleotide binding site of SERCA. A new fragment appeared at 75 kDa after HOCl oxidation of SR, indicating fragmentation of SERCA. Fragmentation may be associated with protein carbonyl formation. The density of protein carbonyl bands at 75 and 110 kDa increased concentration- and time-dependently. Trolox (250 micromol/l) recovered the Ca-ATPase activity decrease induced by HOCl, probably by changing conformational properties of the Ca-ATPase protein. Trolox inhibited FITC binding to SERCA.

Effects of Recession versus Tenotomy Surgery without Recession in Adult Rabbit Extraocular Muscle

PURPOSE. Surgical recession of an extraocular muscle (EOM) posterior to its original insertion is a common form of strabismus surgery, weakening the rotational force exerted by the muscle on the globe and improving eye alignment. The purpose of this study was to assess myosin heavy chain (MyHC) isoform expression and satellite cell activity as defined by Pax7 expression in recessed EOMs of adult rabbits compared with that in muscles tenotomized but not recessed and with that in normal control muscles. METHODS. The scleral insertion of the superior rectus muscle was detached and sutured either 7 mm posterior to its original insertion site (recession surgery) or at the same site (tenotomy). One day before euthanatization, the rabbits received bromodeoxyuridine (BrdU) injections. After 7 and 14 days, selected EOMs from both orbits were examined for changes in fast, slow, neonatal, and developmental MyHC isoform expression, Pax7 expression, and BrdU incorporation. RESULTS. Recession and tenotomy surgery resulted in similar changes in the surgical EOMs. These included a decreased proportion of fast MyHC myofibers, an increased proportion of slow MyHC myofibers, and increased BrdU-positive satellite cells. Similar changes were seen in the non-operated contralateral superior rectus muscles. The ipsilateral inferior rectus showed reciprocal changes to the surgical superior rectus muscles. CONCLUSIONS. The EOMs are extremely adaptive to changes induced by recession and tenotomy surgery, responding with modulations in fiber remodeling and myosin expression. These adaptive responses could be manipulated to improve surgical success rates. (Invest Ophthalmol Vis Sci. 2010;51:5646-5656) DOI:10.1167/iovs.10-5523

Cloning of a differentially expressed tropomyosin isoform from cultured rabbit aortic smooth muscle cells

The four known tropomyosin genes have highly conserved DNA and amino acid sequences, and at least 18 isoforms are generated by alternative RNA splicing in muscle and non-muscle cells. No rabbit tropomyosin nucleotide sequences are known, although protein sequences for alpha- and beta-tropomyosin expressed by rabbit skeletal muscle have been described. Subtractive hybridisation was used to select for genes differentially expressed in rabbit aortic smooth muscle cells (SMC), during the change in cell phenotype in primary culture that is characterised by a loss of cytoskeletal filaments and contractile proteins. This led to the cloning of a tropomyosin gene predominantly expressed in rabbit SMC during this change. The full-length cDNA clone, designated rabbit TM-beta, contains an open reading frame of 284 amino acids, 5' untranslated region (UTR) of I 17 base pairs and 3' UTR of 79 base pairs. It is closely related to the beta-gene isoforms in other species, with the highest homology in DNA and protein sequences to the human fibroblast isoform TM-1 (91.7% identity in 1035 bp and 93.3% identity in the entire 284 amino acid sequence of the protein), It differs from rabbit skeletal muscle P-tropomyosin (81.7% homology at the protein level) mainly in two regions at amino acids 189-213 and 258-283 suggesting alternative splicing of exons 6a for 6b and 9d for 9a. Since this TM-P gene was the only gene strongly enough expressed in SMC changing phenotype to be observed by the subtractive hybridisation screen, it likely plays a significant role in this process. (C) 2002 Published by Elsevier Science Ltd.

Evaluation of the rabbit as a model for Chagas disease - II: histopathologic studies of the heart, digestive tract and skeletal muscle

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, seventy one animals were inoculated with different Trypanosoma cruzi strains and routes. The rabbits were submitted to necropsy in acute (earlier than three months of infection), recent chronic (three to six months) and late chronic (later than six months) phases. Myocarditis, generally focal and endomysial, occurred in 94.1%, 66.7% and 70.8% of the infected rabbits respectively in the acute, recent chronic and late chronic phases. The myocardial inflammatory exudate was composed by mononuclear cells, and also polymorphonuclear cells in the acute phase. In most cases of the late chronic phase, the myocarditis was similar to that described in the indeterminate form of human chagasic patients. Initial fibrosis occurred in the three phases but was more severe and frequent in the early chronic. Advanced fibrosis occurred only in the late chronic phase. Tissue parasites occurred only in the acute phase. The digestive tract and skeletal muscles showed mild and occasional lesions. Our data indicate that experimentally infected chagasic rabbits repeat some lesions similar to that of humans chagasic patients, specially that of the indeterminate form. So, it may be a useful, however not an ideal, model.

Fish Glucose Transporter (GLUT)-4 Differs from Rat GLUT4 in Its Traffic Characteristics but Can Translocate to the Cell Surface in Response to Insulin in Skeletal Muscle Cells

In mammals, glucose transporter (GLUT)-4 plays an important role in glucose homeostasis mediating insulin action to increase glucose uptake in insulin-responsive tissues. In the basal state, GLUT4 is located in intracellular compartments and upon insulin stimulation is recruited to the plasma membrane, allowing glucose entry into the cell. Compared with mammals, fish are less efficient restoring plasma glucose after dietary or exogenous glucose administration. Recently our group cloned a GLUT4-homolog in skeletal muscle from brown trout (btGLUT4) that differs in protein motifs believed to be important for endocytosis and sorting of mammalian GLUT4. To study the traffic of btGLUT4, we generated a stable L6 muscle cell line overexpressing myc-tagged btGLUT4 (btGLUT4myc). Insulin stimulated btGLUT4myc recruitment to the cell surface, although to a lesser extent than rat-GLUT4myc, and enhanced glucose uptake. Interestingly, btGLUT4myc showed a higher steady-state level at the cell surface under basal conditions than rat-GLUT4myc due to a higher rate of recycling of btGLUT4myc and not to a slower endocytic rate, compared with rat-GLUT4myc. Furthermore, unlike rat-GLUT4myc, btGLUT4myc had a diffuse distribution throughout the cytoplasm of L6 myoblasts. In primary brown trout skeletal muscle cells, insulin also promoted the translocation of endogenous btGLUT4 to the plasma membrane and enhanced glucose transport. Moreover, btGLUT4 exhibited a diffuse intracellular localization in unstimulated trout myocytes. Our data suggest that btGLUT4 is subjected to a different intracellular traffic from rat-GLUT4 and may explain the relative glucose intolerance observed in fish.

Adult skeletal muscle stem cell migration is mediated by a blebbing/amoeboid mechanism

Adult skeletal muscle possesses a resident stem cell population called satellite cells which are responsible for tissue repair following damage. Satellite cell migration is crucial in promoting rapid tissue regeneration but is a poorly understood process. Furthermore, the mechanisms facilitating satellite cell movement have yet to be elucidated. Here the process of satellite cell migration has been investigated revealing that they undergo two distinct phases of movement; firstly under the basal lamina and then rapidly increasing their velocity when on the myofibre surface. Most significantly we show that satellite cells move using a highly dynamic blebbing based mechanism and not via lamellopodia mediated propulsion. We show that nitric oxide and non-canonical Wnt signalling pathways are necessary for regulating the formation of blebs and the migration of satellite cells. In summary, we propose that the formation of blebs and their necessity for satellite cell migration has significant implications in the future development of therapeutic regimes aimed at promoting skeletal muscle regeneration.

A hypoplastic model of skeletal muscle development displaying reduced foetal myoblast cell numbers, increased oxidative myofibres and improved specific tension capacity

The major component of skeletal muscle is the myofibre. Genetic intervention inducing over-enlargement of myofibres beyond a certain threshold through acellular growth causes a reduction in the specific tension generating capacity of the muscle. However the physiological parameters of a genetic model that harbours reduced skeletal muscle mass have yet to be analysed. Genetic deletion of Meox2 in mice leads to reduced limb muscle size and causes some patterning defects. The loss of Meox2 is not embryonically lethal and a small percentage of animals survive to adulthood making it an excellent model with which to investigate how skeletal muscle responds to reductions in mass. In this study we have performed a detailed analysis of both late foetal and adult muscle development in the absence of Meox2. In the adult, we show that the loss of Meox2 results in smaller limb muscles that harbour reduced numbers of myofibres. However, these fibres are enlarged. These myofibres display a molecular and metabolic fibre type switch towards a more oxidative phenotype that is induced through abnormalities in foetal fibre formation. In spite of these changes, the muscle from Meox2 mutant mice is able to generate increased levels of specific tension compared to that of the wild type.