Simvastatin impairs ADP-stimulated respiration and increases mitochondrial oxidative stress in primary human skeletal myotubes

Statins, the widely prescribed cholesterol-lowering drugs for the treatment of cardiovascular disease, cause adverse skeletal muscle side effects ranging from fatigue to fatal rhabdomyolysis. The purpose of this study was to determine the effects of simvastatin on mitochondrial respiration, oxidative stress, and cell death in differentiated primary human skeletal muscle cells (i.e., myotubes). Simvastatin induced a dose-dependent decrease in viability of proliferating and differentiating primary human muscle precursor cells, and a similar dose-dependent effect was noted in differentiated myoblasts and myotubes. Additionally, there were decreases in myotube number and size following 48 h of simvastatin treatment (5 μM). In permeabilized myotubes, maximal ADP-stimulated oxygen consumption, supported by palmitoylcarnitine+malate (PCM, complex I and II substrates) and glutamate+malate (GM, complex I substrates), was 32-37% lower (P<0.05) in simvastatin-treated (5 μM) vs control myotubes, providing evidence of impaired respiration at complex I. Mitochondrial superoxide and hydrogen peroxide generation were significantly greater in the simvastatin-treated human skeletal myotube cultures compared to control. In addition, simvastatin markedly increased protein levels of Bax (proapoptotic, +53%) and Bcl-2 (antiapoptotic, +100%, P<0.05), mitochondrial PTP opening (+44%, P<0.05), and TUNEL-positive nuclei in human skeletal myotubes, demonstrating up-regulation of mitochondrial-mediated myonuclear apoptotic mechanisms. These data demonstrate that simvastatin induces myotube atrophy and cell loss associated with impaired ADP-stimulated maximal mitochondrial respiratory capacity, mitochondrial oxidative stress, and apoptosis in primary human skeletal myotubes, suggesting that mitochondrial dysfunction may underlie human statin-induced myopathy.     

Accumulation of mitochondrial DNA deletions in myotubes cultured from muscles of patients with mitochondrial myopathies

 Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percent- age of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA in the first patient’s cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation. Received: 16 April 1996/Accepted: 29 July 1996     

Activation of mitochondrial-driven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species production

While the acquisition of apoptosis resistance is part of the differentiation program of skeletal muscle cells, differentiated muscle cells can undergo apoptosis in response to physiological or pathological stimuli. The generation of reactive oxygen species by mitochondria plays a major role in the control of apoptosis in many cell types. Indeed their involvement in controlling apoptosis in differentiated muscle cells, or in generating resistance to apoptosis remains unknown. Moreover, differentiated muscle cells specifically express the uncoupling protein-3, a mitochondrial protein potentially involved in controlling reactive oxygen species production. To study the role of mitochondrial reactive oxygen species in the control of apoptosis in skeletal muscle cells, L6E9 myoblasts and myotubes were exposed to staurosporine, an inducer of apoptosis via mitochondrial pathways. Staurosporine activated apoptotic pathways (i.e. caspase-3 and caspase-9) increasing reactive oxygen species in myoblasts and, to a minor extent, in myotubes. However, the increase in reactive oxygen species was not needed to induce apoptosis nor was it involved in the differential sensitization of myoblasts and myotubes to apoptosis. Moreover, expression of uncoupling protein-3 in myotubes did not affect reactive oxygen species production, although it produced a slight sensitization for staurosporine-induced apoptosis. Results indicate that apoptotic activation in skeletal muscle cells mainly involves reactive oxygen species-independent mechanisms and that mitochondrial uncoupling protein-3 is not protective either for reactive oxygen species production or for apoptotic activation in muscle cells.     

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.     

PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle

The pathophysiology underlying mitochondrial dysfunction in insulin-resistant skeletal muscle is incompletely characterized. To further delineate this we investigated the interaction between insulin signaling, mitochondrial regulation, and function in C2C12 myotubes and in skeletal muscle. In myotubes elevated insulin and glucose disrupt insulin signaling, mitochondrial biogenesis, and mitochondrial bioenergetics. The insulin-sensitizing thiazolidinedione pioglitazone restores these perturbations in parallel with induction of the mitochondrial biogenesis regulator PGC-1alpha. Overexpression of PGC-1alpha rescues insulin signaling and mitochondrial bioenergetics, and its silencing concordantly disrupts insulin signaling and mitochondrial bioenergetics. In primary skeletal myoblasts pioglitazone also up-regulates PGC-1alpha expression and restores the insulin-resistant mitochondrial bioenergetic profile. In parallel, pioglitazone up-regulates PGC-1alpha in db/db mouse skeletal muscle. Interestingly, the small interfering RNA knockdown of the insulin receptor in C2C12 myotubes down-regulates PGC-1alpha and attenuates mitochondrial bioenergetics. Concordantly, mitochondrial bioenergetics are blunted in insulin receptor knock-out mouse-derived skeletal myoblasts. Taken together these data demonstrate that elevated glucose and insulin impairs and pioglitazone restores skeletal myotube insulin signaling, mitochondrial regulation, and bioenergetics. Pioglitazone functions in part via the induction of PGC-1alpha. Moreover, PGC-1alpha is identified as a bidirectional regulatory link integrating insulin-signaling and mitochondrial homeostasis in skeletal muscle.     

5-bromodeoxyuridine labeling of monomeric and catenated circular mitochondrial DNA in HeLa cells

Bromouracil labeling of the mitochondrial DNA in exponentially growing HeLa cells produces two hybrid mitochondrial DNA species, with density shifts of 41.9 and 54.0 mg/ml relative to unlabeled mitochondrial DNA, as well as heavy mitochondrial DNA, with a shift of 95.3 mg/ml. The two hybrid species result from the difference in thymine composition of the complementary strands of mitochondrial DNA. In addition, mitochondrial DNA with a density intermediate between the hybrid and unlabeled species was found. This quarter heavy mitochondrial DNA represents 25% (w/w) of the total DNA after eight hours of labeling, and forms two peaks with shifts of 20.6 and 27.0 mg/ml relative to unlabeled mitochondrial DNA. 70% (w/w) of the quarter heavy mitochondrial DNA is in catenated forms, while 30% (w/w) is monomeric. Degradation of the catenanes by shearing of purified quarter heavy mitochondrial DNA results in the appearance of hybrid and unlabeled mitochondrial DNA bands, demonstrating that the quarter heavy catenanes contain both hybrid and unlabeled submolecules. The implications of the structure of the quarter heavy catenanes on the mechanism of formation of catenanes are discussed.     

Inhibitory effect of p53 on mitochondrial content and function during adipogenesis

The p53 protein is known as a guardian of the genome and is involved in energy metabolism. Since the metabolic system is uniquely regulated in each tissue, we have anticipated that p53 also would play differential roles in each tissue. In this study, we focused on the functions of p53 in white adipose tissue (adipocytes) and skeletal muscle (myotubes), which are important peripheral tissues involved in energy metabolism. We found that in 3T3-L1 preadipocytes, but not in C2C12 myoblasts, p53 stabilization or overexpression downregulates the expression of Ppargc1a, a master regulator of mitochondrial biogenesis. Next, by using p53-knockdown C2C12 myotubes or 3T3-L1 preadipocytes, we further examined the relationship between p53 and mitochondrial regulation. In C2C12 myoblasts, p53 knockdown did not significantly affect Ppargc1a expression and mtDNA, but did suppress differentiation to myotubes, as previously reported. However, in 3T3-L1 preadipocytes and mouse embryonic fibroblasts, p53 downregulation enhanced both differentiation into adipocytes and mitochondrial DNA content. Furthermore, p53-depleted 3T3-L1 cells showed increase in mitochondrial proteins and enhancement of both Citrate Synthase and Complex IV activities during adipogenesis. These results show that p53 differentially regulates cell differentiation and mitochondrial biogenesis between adipocytes and myotubes, and provide evidence that p53 is an inhibitory factor of mitochondrial regulation in adipocyte lineage.     

Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.     

Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis.

Mammalian myogenesis is biphasic: primary myoblasts fuse to form primary myotubes, then secondary myoblasts align along the primary myotubes and form secondary myotubes, which comprise most of adult muscle. We provide evidence that an integrin (VLA-4) and its counter receptor (VCAM-1) have a role in secondary myogenesis. Both receptors are synthesized by cultured muscle cells: VLA-4 is induced as myotubes form, whereas VCAM-1 is present on myoblasts and myotubes. In vivo, both molecules are expressed at sites of secondary myogenesis, VLA-4 on primary and secondary myotubes, and VCAM-1 on secondary myoblasts and on regions of secondary myotubes apposed to primary myotubes. These patterns suggest that VLA-4-VCAM-1 interactions influence alignment of secondary myoblasts along primary myotubes and/or the fusion of secondary myoblasts. In support of the latter possibility, antibodies to VLA-4 or VCAM-1 inhibit myotube formation in culture.     

Statins interfere with the attachment of S. cerevisiae mtDNA to the inner mitochondrial membrane

Abstract

The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used Saccharomyces cerevisiae as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in S.cerevisiae by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins.     

Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress

In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.     

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.     

Versican Processing by a Disintegrin-like and Metalloproteinase Domain with Thrombospondin-1 Repeats Proteinases-5 and -15 Facilitates Myoblast Fusion

Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.     

Differentiation-induced radioresistance in muscle cells

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 microM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.     

Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Background

Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.

Methodology/Principal Findings

We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.

Conclusions/Significance

Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.     

Chemerin‐induced mitochondrial dysfunction in skeletal muscle

Chemerin is a novel adipocyte‐derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin‐mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real‐time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3‐I to LC3‐II and higher expression levels of Beclin1 and autophagy‐related protein‐5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine‐like receptor 1 or the use of mitochondria‐targeting antioxidant Mito‐TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin‐stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin‐induced mitochondrial autophagy likely through an AKT‐FoxO3α‐dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.     

Fabrication of skeletal muscle constructs by topographic activation of cell alignment

Skeletal muscle fiber construction for tissue-engineered grafts requires assembly of unidirectionally aligned juxtaposed myotubes. To construct such a tissue, a polymer microchip with linearly aligned microgrooves was fabricated that could direct myoblast adaptation under stringent conditions. The closely spaced microgrooves fabricated by a modified replica molding process guided linear cellular alignment. Examination of the myoblasts by immunofluorescence microscopy demonstrated that the microgrooves with subcellular widths and appropriate height-to-width ratios were required for practically complete linear alignment of myoblasts. The topology-dependent cell alignment encouraged differentiation of myoblasts into multinucleate, myosin heavy chain positive myotubes. The monolayer of myotubes formed on the microstructured chips allowed attachment, growth and differentiation of subsequent layers of linearly arranged myoblasts, parallel to the primary monolayer of myotubes. The consequent deposition of additional myoblasts on the previous layer of myotubes resulted in three-dimensional multi-layered structures of myotubes, typical of differentiated skeletal muscle tissue. The findings demonstrate that the on-chip device holds promise for providing an efficient means for guided muscle tissue construction.