Changes in phosphofructokinase isozymes during development of myoblasts to myotubes

The regulation of phosphofructokinase during development of C2C12 myoblasts to myotubes was investigated. Enzyme activity was markedly increased during myogenic development. The increase was observed when enzyme activity was measured under optimal conditions and was not due to changes in the allosteric kinetic properties of the enzyme. Immunoprecipitation of phosphofructokinase from [35S]methionine-labeled myogenic cells revealed that equal amounts of liver and muscle isozymes are present in myoblasts, while in myotubes there was a much higher level of the muscle isozyme. These results were confirmed using an immunoblotting technique. The increase in the level of muscle isozyme in myotubes is due to an increase in the rate of synthesis of the muscle isozyme and occurs in spite of a measurably small increase in its degradation rate. Northern blot analysis using a synthetic oligonucleotide probe showed a 25-fold increase in the level of muscle phosphofructokinase mRNA in myotubes. The conclusion is drawn that the increase in muscle isozyme in myotubes during myogenesis is due to an increase in its mRNA level.     

Up-Regulation of Adiponectin Expression in Antigravitational Soleus Muscle in Response to Unloading Followed by Reloading,and Functional Overloading in Mice

The purpose of this study was to investigate the expression level of adiponectin and its related molecules in hypertrophied and atrophied skeletal muscle in mice. The expression was also evaluated in C2C12 myoblasts and myotubes. Both mRNA and protein expression of adiponectin, mRNA expression of adiponectin receptor (AdipoR) 1 and AdipoR2, and protein expression of adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif 1 (APPL1) were observed in C2C12 myoblasts. The expression levels of these molecules in myotubes were higher than those in myoblasts. The expression of adiponectin-related molecules in soleus muscle was observed at mRNA (adiponectin, AdipoR1, AdipoR2) and protein (adiponectin, APPL1) levels. The protein expression levels of adiponectin and APPL1 were up-regulated by 3 weeks of functional overloading. Down-regulation of AdipoR1 mRNA, but not AdipoR2 mRNA, was observed in atrophied soleus muscle. The expression of adiponectin protein, AdipoR1 mRNA, and APPL1 protein was up-regulated during regrowth of unloading-associated atrophied soleus muscle. Mechanical loading, which could increase skeletal muscle mass, might be a useful stimulus for the up-regulations of adiponectin and its related molecules in skeletal muscle.     

Preferential accumulation of muscle type acylphosphatase in the nucleus during differentiation

In a previous paper we observed a direct involvement of acylphosphatase in differentiation, associated with enhanced levels of the enzyme in the cell. We have here investigated the subcellular localization of the two known acylphosphatase isoforms during this process. We show that in C2C12 myoblast cells, muscle type acylphosphatase accumulates in the nucleus during differentiation. The same pattern of accumulation is observed also in K562 erythroleukemia cells, although at a lower extent: this fact indicates that this phenomenon is not restricted to muscular cells but rather it could be of general importance in the differentiative process. The common type acylphosphatase, showing an 8-fold increase in the cytoplasm during differentiation, does not accumulate in the nucleus, suggesting distinct roles of the two isoenzymes in this process.     

Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.     

Changes in oleic acid oxidation and incorporation into lipids of differentiating L6 myoblasts cultured in normal or fatty acid-supplemented growth medium.

L6 myoblasts accumulate large stores of neutral lipid (predominantly triacylglycerol) when cultured in fatty acid-supplemented growth medium. No accumulation of neutral lipid was evident in myotubes (differentiated myoblasts) when treated similarly. Triacylglycerol accumulation was rapid and dependent on exogenous fatty acid concentration. Triacylglycerol content in myoblasts cultured in fatty acid-supplemented growth medium was approx. 3-fold higher than that in myotubes treated similarly and 2-3-fold higher than that in myoblasts cultured in normal growth medium. Incorporation studies using [I-14C]oleic acid showed that myoblasts and myotubes take up exogenous fatty acid at similar rates. However, cells cultured in fatty acid-supplemented growth medium remove more exogenous fatty acid than do cells cultured in normal growth medium. Over 90% of the incorporated label was found in phospholipid and triacylglycerol fractions in all situations studied. Myoblasts incorporated a more significant proportion (P less than 0.001) of label into triacylglycerol compared with that of myotubes. No differences in fatty acid oxidation rates were detected when differentiating L6 cells cultured in normal growth medium were compared with those cultured in fatty acid-supplemented growth medium. However, fatty acid oxidation rates were observed to increase 3-5-fold upon myoblast differentiation. We conclude that there is a marked change in the pattern of lipid metabolism when myoblasts (primarily triacylglycerol-synthesizing cells) differentiate into myotubes (primarily phospholipid-synthesizing cells). Understanding these changes, which coincide with normal muscle development, may be important, since a defect in this natural switch could explain the observed accumulation of lipid in muscle characteristic of some of the muscular dystrophies and other lipid-storage myopathies.     

Galactose enhances oxidative metabolism and reveals mitochondrial dysfunction in human primary muscle cells

Background

Human primary myotubes are highly glycolytic when cultured in high glucose medium rendering it difficult to study mitochondrial dysfunction. Galactose is known to enhance mitochondrial metabolism and could be an excellent model to study mitochondrial dysfunction in human primary myotubes. The aim of the present study was to 1) characterize the effect of differentiating healthy human myoblasts in galactose on oxidative metabolism and 2) determine whether galactose can pinpoint a mitochondrial malfunction in post-diabetic myotubes.

Methodology/Principal Findings

Oxygen consumption rate (OCR), lactate levels, mitochondrial content, citrate synthase and cytochrome C oxidase activities, and AMPK phosphorylation were determined in healthy myotubes differentiated in different sources/concentrations of carbohydrates: 25 mM glucose (high glucose (HG)), 5 mM glucose (low glucose (LG)) or 10 mM galactose (GAL). Effect of carbohydrates on OCR was also determined in myotubes derived from post-diabetic patients and matched obese non-diabetic subjects. OCR was significantly increased whereas anaerobic glycolysis was significantly decreased in GAL myotubes compared to LG or HG myotubes. This increased OCR in GAL myotubes occurred in conjunction with increased cytochrome C oxidase activity and expression, as well as increased AMPK phosphorylation. OCR of post-diabetic myotubes was not different than that of obese non-diabetic myotubes when differentiated in LG or HG. However, whereas GAL increased OCR in obese non-diabetic myotubes, it did not affect OCR in post-diabetic myotubes, leading to a significant difference in OCR between groups. The lack of an increase in OCR in post-diabetic myotubes differentiated in GAL was in relation with unaltered cytochrome C oxidase activity levels or AMPK phosphorylation.

Conclusions/Significance

Our results indicate that differentiating human primary myoblasts in GAL enhances aerobic metabolism. Because this cell culture model elicited an abnormal response in cells from post-diabetic patients, it may be useful in further studies of the molecular mechanisms of mitochondrial dysfunction.     

Ornithine decarboxylase is upregulated by the androgen receptor in skeletal muscle and regulates myoblast proliferation

The aim of this study is to determine if the Odc1 gene, which encodes ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is directly regulated by the androgen receptor (AR) in skeletal muscle myoblasts and if Odc1 regulates myoblast proliferation and differentiation. We previously showed that expression of Odc1 is decreased in muscle from AR knockout male mice. In this study, we show in vivo that Odc1 expression is also decreased >60% in muscle from male muscle-specific AR knockout mice. In normal muscle homeostasis, Odc1 expression is regulated by age and sex, reflecting testosterone levels, as muscle of adult male mice expresses high levels of Odc1 compared with age-matched females and younger males. In vitro, expression of Odc1 is 10- and 1.5-fold higher in proliferating mouse C(2)C(12) and human skeletal muscle myoblasts, respectively, than in differentiated myotubes. Dihydrotestosterone increases Odc1 levels 2.7- and 1.6-fold in skeletal muscle cell myoblasts after 12 and 24 h of treatment, respectively. Inhibition of ODC activity in C(2)C(12) myoblasts by α-difluoromethylornithine decreases myoblast number by 40% and 66% following 48 and 72 h of treatment, respectively. In contrast, overexpression of Odc1 in C(2)C(12) myoblasts results in a 27% increase in cell number vs. control when cells are grown under differentiation conditions for 96 h. This prolonged proliferation is associated with delayed differentiation, with reduced expression of the differentiation markers myogenin and Myf6 in Odc1-overexpressing cells. In conclusion, androgens act via the AR to upregulate Odc1 in skeletal muscle myoblasts, and Odc1 promotes myoblast proliferation and delays differentiation.     

Geranylgeranylaceton induces heat shock protein 72 in skeletal muscle cells

Effects of an antiulcer drug, geranylgeranylaceton (GGA), and/or heat-stress on 72 kDa heat shock protein (HSP72) expression and protein content in cultured skeletal muscle cells were studied. Mouse skeletal muscle cells (C(2)C(12)) were subjected to either 1) control (cultured at 37 degrees C without GGA), 2) GGA administration (10(-11) - 10(-8) M), 3) heat-stress at 41 degrees C for 60 min, or 4) GGA administration combined with heat-stress. Expression of HSP72 was up-regulated by GGA administration. Heat-stress further enhanced the GGA-related up-regulation of HSP72. Administration of GGA caused an increase of muscular protein content as a dose-dependent manner. Protein synthesis was also stimulated by heat-stress alone in myotubes. It was suggested that GGA stimulates the differentiation of myoblasts and protein synthesis. These observations may also suggest that the administration of GGA could be one of the useful tools to gain muscular mass not only in athletes, but also in patients during rehabilitation.     

Transforming Growth Factor-��-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-β-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.     

Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.     

Microinjection of calpastatin inhibits fusion in myoblasts

Rat satellite cells (RSC) were microinjected with purified calpastatin or m-calpain, and myoblasts from a C2C12 mouse line were microinjected with purified calpastatin. Microinjection with calpastatin completely prevented fusion of myoblasts from both sources, whereas microinjection with m-calpain significantly increased the rate of fusion of cultured RSC; 44% of the nuclei of RSC cultures were in multinucleated myotubes within 48 h after microinjection with m-calpain plus labeled dextran, whereas only 15% of the nuclei were in multinucleated myotubes after microinjection with dextran alone. Western analyses indicated that neither RSC nor C2C12 myoblasts contained detectable amounts of mu-calpain before fusion. The levels of calpastatin in C2C12 myoblasts increased as cells passed from the proliferative stage to the onset of fusion, and these levels increased substantially in both the C2C12 and the RSC cells as they progressed to the late or postfusion stage. Both RSC and C2C12 myoblasts contained an 80-kDa polypeptide that was labeled with an anti-m-calpain antibody in Western blots. The results are consistent with a role of the calpain system (m-calpain in these myoblast lines) in remodeling of the cytoskeletal/plasma membrane interactions during cell fusion.     

Prelamin A-mediated recruitment of SUN1 to the nuclear envelope directs nuclear positioning in human muscle

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.     

Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function

alpha-Dystroglycan (alpha-DG) is a laminin-binding protein and member of a glycoprotein complex associated with dystrophin that has been implicated in the etiology of several muscular dystrophies. To study the function of DG, C2 myoblasts were transfected stably with an antisense DG expression construct. Myotubes from two resulting clones (11F and 11E) had at least a 40-50% and 80-90% reduction, respectively, in alpha-DG but normal or near normal levels of alpha-sarcoglycan, integrin beta1 subunit, acetylcholine receptors (AChRs), and muscle-specific kinase (MuSK) when compared with parental C2 cells or three clones (11A, 9B, and 10C) which went through the same transfection and selection procedures but expressed normal levels of alpha-DG. Antisense DG-expressing myoblasts proliferate at the same rate as parental C2 cells and differentiate into myotubes, however, a gradual loss of cells was observed in these cultures. This loss correlates with increased apoptosis as indicated by greater numbers of nuclei with condensed chromatin and more nuclei labeled by the TUNEL method. Moreover, there was no sign of increased membrane permeability to Trypan blue as would be expected with necrosis. Unlike parental C2 myotubes, 11F and 11E myotubes had very little laminin (LN) on their surfaces; LN instead tended to accumulate on the substratum between myotubes. Exogenous LN bound to C2 myotubes and was redistributed into plaques along with alpha-DG on their surfaces but far fewer LN/alpha-DG plaques were seen after LN addition to 11F or 11E myotubes. These results suggest that alpha-DG is a functional LN receptor in situ which is required for deposition of LN on the cell and, further, implicate alpha-DG in the maintenance of myotube viability.     

Unusual laminin alpha2 processing in myoblasts from a patient with a novel variant of congenital muscular dystrophy

We recently described a novel congenital muscular dystrophy (CMD) syndrome characterized by mental retardation, microcephaly, and partial merosin deficiency on muscle biopsy. Linkage analysis excluded involvement of the known CMD loci. We now report on a study performed on the differentiation of cultured myoblasts from one patient affected by this condition to evaluate the potential to form myotubes and merosin processing in these cells. The differentiation rate was comparable to controls and myotubes were stable in culture. Biochemical analysis showed the expected 80-kDa merosin subunit in myoblasts. However, a shifted 60-kDa protein was detected in myotubes. Matrix-metalloproteinases (MMPs) zymography showed increased gelatinolytic activity, and immunoblotting identified an increased amount of membrane-type 1 matrix-metalloproteinase in pathological myotube preparations. Our results show that these CMD-derived myotubes contain a low molecular weight merosin. They further suggest that an altered regulation of MMPs can be involved in basal lamina damage.     

Myoblasts from affected and non-affected FSHD muscles exhibit morphological differentiation defects

Facioscapulohumeral dystrophy (FSHD) is a muscular hereditary disease with a prevalence of 1 in 20,000 caused by a partial deletion of a subtelomeric repeat array on chromosome 4q. However, very little is known about the pathogenesis as well as the molecular and biochemical changes linked to the progressive muscle degeneration observed in these patients. Several studies have investigated possible pathophysiological pathways in FSHD myoblasts and mature muscle cells but some of these reports were apparently in contradiction. The discrepancy between these studies may be explained by differences between the sources of myoblasts. Therefore, we decided to thoroughly analyze affected and unaffected muscles from patients with FSHD in terms of vulnerability to oxidative stress, differentiation capacity and morphological abnormalities. We have established a panel of primary myoblast cell cultures from patients affected with FSHD and matched healthy individuals. Our results show that primary myoblasts are more susceptible to an induced oxidative stress than control myoblasts. Moreover, we demonstrate that both types of FSHD primary myoblasts differentiate into multi-nucleated myotubes, which present morphological abnormalities. Whereas control myoblasts fuse to form branched myotubes with aligned nuclei, FSHD myoblasts fuse to form either thin and branched myotubes with aligned nuclei or large myotubes with random nuclei distribution. In conclusion, we postulate that these abnormalities could be responsible for muscle weakness in patients with FSHD and provide an important marker for FSHD myoblasts.