1α, 25(OH)2D3 regulates agrin-induced acetylcholine receptor clustering through upregulation of rapsyn expression in C2C12 myotubes.

The active form of vitamin D (1α, 25-dihydroxyvitamin D3 [1α, 25(OH)2D3], referred to as 1,25D) has been suggested to play a pivotal role in skeletal muscle function and metabolism. However, the mechanisms through which 1,25D functions in this tissue remain to be elucidated. Recent studies have shown that vitamin D signaling regulates neuromuscular maintenance and improves locomotion in mice. In the present study, we examined the effects of 1,25D on neuromuscular synaptogenesis by measuring clustering of acetylcholine receptors (AChRs) in C2C12 myotubes. 1,25D treatment enhanced the agrin-induced AChR clustering in myotubes compared to treatment with agrin alone. Furthermore, siRNA-mediated knockdown of the vitamin D receptor (VDR) decreased the agrin-induced AChR clustering. 1,25D increased the expression of rapsyn, which is necessary for AChR clustering, while demonstrating no effect on other neuromuscular junction-related genes. In addition, rapsyn expression was dependent on 1,25D-VDR signaling. These results suggest that 1,25D-VDR signaling may regulate rapsin expression, resulting in the up-regulation of agrin-induced AChR clustering.

Cobalt chloride, a chemical hypoxia-mimicking agent, suppresses myoblast differentiation by downregulating myogenin expression.

Cobalt chloride can create hypoxia-like state in vitro (referred to as chemical hypoxia). Several studies have suggested that chemical hypoxia may cause deleterious effects on myogenesis. The intrinsic underlying mechanisms of myoblast differentiation, however, are not fully understood. Here, we show that cobalt chloride strongly suppresses myoblast differentiation in a dose-dependent manner. The impaired myoblast differentiation is accompanied by downregulation of myogenic regulatory factor myogenin. Under chemical hypoxia, myogenin stability is decreased at mRNA and protein levels. A muscle-specific E3 ubiquitin ligase MAFbx, which can target myogenin protein for proteasomal degradation, is upregulated along with changes in Akt/Foxo and AMPK/Foxo signaling pathways. A proteasome inhibitor completely prevents cobalt chloride-mediated decrease in myogenin protein. These results suggest that cobalt chloride might modulate myogenin expression at post-transcriptional and post-translational levels, resulting in the failure of the myoblasts to differentiate into myotubes.

Engineered topographical structure to control spatial cell density using cell migration.

Control of the spatial distribution of various cell types is required to construct functional tissues. Here, we report a simple topographical structure changed the spatial cell density. A concave curved boundary was designed, which allowed the spatial descent moving of cells and the change in spatial distributions of co-cultured cells. We utilized the difference in cell motility between myoblast cells (C2C12) and neuronal cells (PC12) to demonstrate the feasibility of spontaneous change in spatial cell density. Without the curved boundaries, high motility cells (C2C12) did not migrate to the adjacent area, which resulted in a slight temporal change (< 15%) in the spatial cell distribution. In contrast, with the curved boundaries, the cell density of the high motility cells in the groove to those cells on the ridge showed an increase exceeding 45%. On the other hand, the temporal change in the spatial cell distribution of low motility cells (PC12) was below 15% with or without the curved boundaries. In addition, as groove width increased, both cells displayed more initially gathering in groove. Importantly, these cell-type dependent results were also maintained under co-culture conditions. Our results suggest that designing topographical interfaces changes spatial cell density without any manipulation and is useful for multi-cellular constructs.

Pharmacological targeting of HSP90 with 17-AAG induces apoptosis of myogenic cells through activation of the intrinsic pathway.

We have shown that pharmacological inhibition of HSP90 ATPase activity induces apoptosis of myoblasts during their differentiation. However, the signaling pathways remain not fully characterized. We report that pharmacological targeting of HSP90 with 17-AAG activates the intrinsic pathway including caspase-dependent and caspase-independent pathways. 17-AAG induces the typical apoptotic phenotypes including PARP cleavage, chromatin condensation, and nuclear fragmentation with mitochondrial release of cytochrome c, Smac/DIABLO, procaspase-9 processing, and caspase-3 activation. AIF and EndoG redistribute from the mitochondria into the cytosol and are partially translocated to the nucleus in 17-AAG-treated cells. These results suggest that caspase-dependent and caspase-independent pathways should be considered in apoptosis of myogenic cells induced by inhibition of HSP90 ATPase activity.

Effects of ageing on expression of the muscle-specific E3 ubiquitin ligases and Akt-dependent regulation of Foxo transcription factors in skeletal muscle.

Controversy exists as to whether the muscle-specific E3 ubiquitin ligases MAFbx and MuRF1 are transcriptionally upregulated in the process of sarcopenia. In the present study, we investigated the effects of ageing on mRNA/protein expression of muscle-specific E3 ubiquitin ligases and Akt/Foxo signalling in gastrocnemius muscles of female mice. Old mice exhibited a typical sarcopenic phenotype, characterized by loss of muscle mass and strength, decreased amount of myofibrillar proteins, incidence of aberrant muscle fibres, and genetic signature to sarcopenia. Activation levels of Akt were lower in adult and old mice than in young mice. Consequently, Akt-mediated phosphorylation levels of Foxo1 and Foxo3 proteins were decreased. Nuclear levels of Foxo1 and Foxo3 proteins showed an overall increasing trend in old mice. MAFbx mRNA expression was decreased in old mice relative to adult mice, whereas MuRF1 mRNA expression was less affected by ageing. At the protein level, MAFbx was less affected by ageing, whereas MuRF1 was increased in old mice relative to adult mice, with ubiquitin-protein conjugates being increased with ageing. In conclusion, we provided evidence for no mRNA upregulation of muscle-specific E3 ubiquitin ligases and disconnection between their expression and Akt/Foxo signalling in sarcopenic mice. Their different responsiveness to ageing may reflect different roles in sarcopenia.

Mitochondria as a potential regulator of myogenesis.

Recent studies have shown that mitochondria play a role in the regulation of myogenesis. Indeed, the abundance, morphology, and functional properties of mitochondria become altered when the myoblasts differentiate into myotubes. For example, mitochondrial mass/volume, mtDNA copy number, and mitochondrial respiration are markedly increased after the onset of myogenic differentiation. Besides, mitochondrial enzyme activity is also increased, suggesting that the metabolic shift from glycolysis to oxidative phosphorylation as the major energy source occurs during myogenic differentiation. Several lines of evidence suggest that impairment of mitochondrial function and activity blocks myogenic differentiation. However, yet little is known about the molecular mechanisms underlying the regulation of myogenesis by mitochondria. Understanding how mitochondria are involved in myogenesis will provide a valuable insight into the underlying mechanisms that regulate the maintenance of cellular homeostasis. Here, we will summarize the current knowledge regarding the role of mitochondria as a potential regulator of myogenesis.

Spatial and temporal expression of hypoxia-inducible factor-1α during myogenesis in vivo and in vitro.

We investigated the spatial and temporal expression patterns of hypoxia-inducible factor-1α (HIF-1α) during muscle regeneration and myogenesis in a C2C12 cell culture system. The expression of HIF-1α synchronized with that of myogenic regulatory genes during muscle regeneration at both the mRNA and protein levels. The HIF-1α protein was localized in the nuclei of newly formed regenerating myofibers in three different muscle injury models, including freezing, bupivacaine injection, and muscular dystrophy. In myogenic cell culture, the HIF-1α protein was localized in the nucleus and cytoplasm of the majority of myoblasts and myotubes. HIF-1α protein expression decreased concomitant with the increased expression of MyoD and myogenin proteins after the induction of myogenic differentiation. We investigated the adaptive response of myoblasts to hypoxia-like conditions induced by treatment of cobalt chloride. This treatment allowed HIF-1α to accumulate and translocate to the nucleus to activate transcription of its target genes, suggesting that myoblasts adapted to acute hypoxia-like conditions through enhancing an HIF-1-dependent pathway. Our results provide insight into the possible involvement of HIF-1α in myogenesis in vivo and in vitro.

Muscle regeneration occurs to coincide with mitochondrial biogenesis.

We investigated the expression of the nuclear-encoded genes controlling the mitochondrial properties in the mouse gastrocnemius muscle to gain insight into the mitochondrial biogenesis that occurs during the muscle degeneration/regeneration induced by freezing. In addition, we tested whether the muscle regeneration is affected by pharmacologically blocking the mitochondrial protein synthesis to elucidate the possible involvement of mitochondrial biogenesis in muscle regeneration. The activity of citrate synthase dramatically increased soon after the initial injury when the myoblasts began to differentiate into myotubes, indicating that mitochondrial biogenesis occurs early during the muscle regeneration. At the same time, the expression of mitochondrial biogenesis-related genes including PGC-1ß, PRC, NRF-1, NRF-2, TFAM, mtSSB, fission 1, and Lon protease synchronized with that of the myogenic regulatory genes including MyoD and myogenin. The skeletal muscles forced to regenerate in the presence of chloramphenicol to block the mitochondrial protein synthesis were of poor repair with small myofibers and an increased amount of connective tissue. These results suggest that mitochondrial biogenesis activated early during the muscle regeneration and that mitochondrial biogenesis plays a role in muscle regeneration.

Mitochondrial adaptations in skeletal muscle to hindlimb unloading.

To gain insight into the regulation of mitochondrial adaptations to hindlimb unloading (HU), the activity of mitochondrial enzymes and the expression of nuclear-encoded genes which control mitochondrial properties in mouse gastrocnemius muscle were investigated. Biochemical and enzyme histochemical analysis showed that subsarcolemmal mitochondria were lost largely than intermyofibrillar mitochondria after HU. Gene expression analysis revealed disturbed or diminished gene expression patterns. The three main results of this analysis are as follows. First, in contrast to peroxisome proliferator-activated receptor γ coactivator 1 ß (PGC-1ß) and PGC-1-related coactivator, which were down-regulated by HU, PGC-1α was up-regulated concomitant with decreased expression of its DNA binding transcription factors, PPARα, and estrogen-related receptor α (ERRα). Moreover, there was no alteration in expression of nuclear respiratory factor 1, but its downstream target gene, mitochondrial transcription factor A, was down-regulated. Second, both mitofusin 2 and fission 1, which control mitochondrial morphology, were down-regulated. Third, ATP-dependent Lon protease, which participates in mitochondrial-protein degradation, was also down-regulated. These findings suggest that HU may induce uncoordinated expression of PGC-1 family coactivators and DNA binding transcription factors, resulting in reducing ability of mitochondrial biogenesis. Furthermore, down-regulation of mitochondrial morphology-related genes associated with HU may be also involved in alterations in intracellular mitochondrial distribution.

Pharmacological inhibition of HSP90 activity negatively modulates myogenic differentiation and cell survival in C2C12 cells.

Heat-shock protein90 (HSP90) plays an essential role in maintaining stability and activity of its clients. HSP90 is involved in cell differentiation and survival in a variety of cell types. To elucidate the possible role of HSP90 in myogenic differentiation and cell survival, we examined the time course of changes in the expression of myogenic regulatory factors, intracellular signaling molecules, and anti-/pro-apoptotic factors when C2C12 cells were cultured in differentiation condition in the presence of a HSP90-specific inhibitor, geldanamycin. Furthermore, we examined the effects of geldanamycin on muscle regeneration in vivo. Our results showed that geldanamycin inhibited myogenic differentiation with decreased expression of MyoD, myogenin and reduced phosphorylation levels of Akt1. Geldanamycin had little effect on the phosphorylation levels of p38MAPK and ERK1/2 but reduced the phosphorylation levels of JNK. Along with myogenic differentiation, geldanamycin increased apoptotic nuclei with decreased expression of Bcl-2. The skeletal muscles forced to regenerate in the presence of geldanamycin were of poor repair with small regenerating myofibers and increased connective tissues. Together, our findings suggest that HSP90 may modulate myogenic differentiation and may be involved in cell survival.

Pinpoint Delivery of Molecules by Using Electron Beam Addressing Virtual Cathode Display.

Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode. The kinetic energy of the incident primary electrons to the SiN membrane was completely blocked, whereas negative charges and leaking electric current appeared on the surface of the dielectric SiN membrane within a region of 100 nm. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). Increasing fluorescence of pre-diluted PI indicated local poration and transmembrane inflow at the virtual cathode position, as well as intracellular diffusion. The transmembrane inflows depended on beam duration time and acceleration voltage. At the post-molecular delivery, a slight decrease in intracellular PI fluorescence intensity indicates membrane recovery from the poration. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.

Directed cell migration in co-cultures by topographic curvature for heterogeneous tissue engineering.

Placing cells in the proper position is important for tissue engineering. Previous works addressed this subject in the way of controlling cell migration by micro- or nano-patterning the substrates. However, the problem of changing spatial cell density freely under co-culture conditions is remaining. To solve this problem, in this work, we report that C2C12 spatial cell density changes by the patterning geometric boundary of the topographical structures. In 48 h after seeding cells, at the linear boundary (ridge-groove) structures, C2C12 Groove/Ridge ratio was under 0.70 both under monoculture conditions and under co-culture conditions. In contrast, at the combining the linear boundary and the round boundary (ridge-groove + hole) structures, the ratio was over 0.89 under both culture conditions. This our finding will provide a new device which enables to manipulate spatial cell density under co-culture conditions for heterogeneous tissue engineering.

Habitual exercise induced resistance to oxidative stress.

We investigated whether habitual exercise (HE) modulates levels of oxidative DNA damage and responsiveness to oxidative stress induced by renal carcinogen Fe-nitrilotriacetic acid (Fe-NTA). During a ten week protocol, two groups of rats either remained sedentary or underwent swimming for 15--60 min per day, 5 days per week, with or without a weight equivalent to 5% of their body weight. Then we injected Fe-NTA and sacrificed the rats 1 h after the injection. We determined the activity of superoxide dismutase (SOD) in diaphragm and kidney, evaluated levels of 8-hydroxydeoxyguanosine (8OHdG), catalase, and glutathione peroxidase, and assayed OGG1 protein levels in kidney. SOD activity in the diaphragm and kidney was increased in HE rats. By itself, HE had no effect on the level of 8OHdG, but it did significantly suppress induction of 8OHdG by Fe-NTA, and the amount of suppression correlated with intensity of exercise. These results suggest that HE induces resistance to oxidative stress and, at least at the initiation stage, inhibits carcinogenesis.

Simple micropatterning method for enhancing fusion efficiency and responsiveness to electrical stimulation of C2C12 myotubes.

Cultured myotubes induced in vitro from myoblast cell lines have been widely used to investigate muscle functional properties and disease-related biological phenotypes. Until now, several cell patterning techniques have been applied to regulate in vitro myotube structures. However, these previous studies required specific geometry patterns or soft materials for inducing efficient myotube formation. Thus, more simple and easy handling method will be promising. In this study, we aimed to provide a method to form C2C12 myotubes with regulated sizes and orientations in simple line patterns. We used a poly(dimethylsiloxane) (PDMS) stamp and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to fabricate line patterns for myotube formation onto a culture dish. We confirmed that C2C12 myotubes of well-defined size and orientation were reproducibly formed. In particular, myotubes formed in the micropatterned lines showed the increased fusion efficiency. Then, functional dynamics in the micropatterned myotubes were detected and analyzed using a calcium imaging method. We confirmed micropatterning in line patterns enhanced the responsiveness of myotubes to external electrical stimulations. These results indicate that micropatterning myoblasts with the MPC polymer is a simple and effective method to form functional myotube networks.

Vitamin D signaling in myogenesis: potential for treatment of sarcopenia.

Muscle mass and strength progressively decrease with age, which results in a condition known as sarcopenia. Sarcopenia would lead to physical disability, poor quality of life, and death. Therefore, much is expected of an effective intervention for sarcopenia. Epidemiologic, clinical, and laboratory evidence suggest an effect of vitamin D on muscle function. However, the precise molecular and cellular mechanisms remain to be elucidated. Recent studies suggest that vitamin D receptor (VDR) might be expressed in muscle fibers and vitamin D signaling via VDR plays a role in the regulation of myoblast proliferation and differentiation. Understanding how vitamin D signaling contributes to myogenesis will provide a valuable insight into an effective nutritional strategy to moderate sarcopenia. Here we will summarize the current knowledge about the effect of vitamin D on skeletal muscle and myogenic cells and discuss the potential for treatment of sarcopenia.

Micropatterning C2C12 myotubes for orderly recording of intracellular calcium transients.

Reconstruction of skeletal muscle myotubes in vitro using myogenic cell lines have been widely carried out to study functional properties and disease-related biological changes of myotubes, such as intracellular calcium dynamics. However, the analysis of biological signals in isolated single myotubes or interactions among several myotubes is quite difficult problem because of the randomness in size, morphology and orientation of differentiated myotubes cultured on a conventional tissue culture dish. In the present study, we attempted to form uniform-size myotubes and detect intracellular calcium dynamics from the fabricated myotubes. We modified surfaces of culture dishes using a poly(-dimethylsiloxane) (PDMS) stamp and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to form line patterns for myotube formation. We could form uniform-size and -orientation C2C12 myotubes and detect intracellular calcium dynamics from it. This simple method would be a useful for studying properties in myotubes with specific sizes and morphologies.

Molecular mechanisms for age-associated mitochondrial deficiency in skeletal muscle.

The abundance, morphology, and functional properties of mitochondria decay in skeletal muscle during the process of ageing. Although the precise mechanisms remain to be elucidated, these mechanisms include decreased mitochondrial DNA (mtDNA) repair and mitochondrial biogenesis. Mitochondria possess their own protection system to repair mtDNA damage, which leads to defects of mtDNA-encoded gene expression and respiratory chain complex enzymes. However, mtDNA mutations have shown to be accumulated with age in skeletal muscle. When damaged mitochondria are eliminated by autophagy, mitochondrial biogenesis plays an important role in sustaining energy production and physiological homeostasis. The capacity for mitochondrial biogenesis has shown to decrease with age in skeletal muscle, contributing to progressive mitochondrial deficiency. Understanding how these endogenous systems adapt to altered physiological conditions during the process of ageing will provide a valuable insight into the underlying mechanisms that regulate cellular homeostasis. Here we will summarize the current knowledge about the molecular mechanisms responsible for age-associated mitochondrial deficiency in skeletal muscle. In particular, recent findings on the role of mtDNA repair and mitochondrial biogenesis in maintaining mitochondrial functionality in aged skeletal muscle will be highlighted.

Expression of nuclear-encoded genes involved in mitochondrial biogenesis and dynamics in experimentally denervated muscle.

The abundance, morphology, and functional properties of mitochondria become altered in response to denervation. To gain insight into the regulation of this process, mitochondrial enzyme activities and gene expression involved in mitochondrial biogenesis and dynamics in mouse gastrocnemius muscle was investigated. Sciatic nerve transactions were performed on mice, and then gastrocnemius muscles were isolated at days 5 and 30 after surgery. Muscle weight was decreased significantly by 15% and 62% at days 5 and 30 after surgery, respectively. The activity of citrate synthase, a marker of oxidative enzyme, was reduced significantly by 31% and 53% at days 5 and 30, respectively. Enzyme histochemical analysis revealed that subsarcolemmal mitochondria were largely lost than intermyofibrillar mitochondria at day 5, and this trend was further progressed at day 30 after surgery. Expression levels of peroxisome proliferator-activated receptor, γ coactivator 1 (PGC-1)α, estrogen-related receptor α (ERRα), and mitofusin 2 were down-regulated throughout the experimental period, whereas those of PGC-1ß, PRC, nuclear respiratory factor (NRF)-1, NRF-2, TFAM, and Lon protease were down-regulated at day 30 after surgery. These results suggest that PGC-1α, ERRα, and mitofusin 2 may be important factors in the process of denervation-induced mitochondrial adaptation. In addition, other PGC-1 family of transcriptional coactivators and DNA binding transcription factors may also contribute to mitochondrial adaptation after early response to denervation.

Transdermal delivery of a readthrough-inducing drug: a new approach of gentamicin administration for the treatment of nonsense mutation-mediated disorders.

To induce the readthrough of premature termination codons, aminoglycoside antibiotics such as gentamicin have attracted interest as potential therapeutic agents for diseases caused by nonsense mutations. The transdermal delivery of gentamicin is considered unfeasible because of its low permeability through the dermis. However, if the skin permeability of gentamicin could be improved, it would allow topical application without the need for systemic delivery. In this report, we demonstrated that the skin permeability of gentamicin increased with the use of a thioglycolate-based depilatory agent. After transdermal administration, the readthrough activity in skeletal muscle, as determined using a lacZ/luc reporter system, was found to be equivalent to systemic administration when measured in transgenic mice. Transdermally applied gentamicin was detected by liquid chromatography-tandem mass spectrometry in the muscles and sera of mice only after depilatory agent-treatment. In addition, expansion of the intercellular gaps in the basal and prickle-cell layers was observed by electron microscopy only in the depilatory agent-treated mice. Depilatory agent-treatment may be useful for the topical delivery of readthough-inducing drugs for the rescue of nonsense mutation-mediated genetic disorders. This finding may also be applicable for the transdermal delivery of other pharmacologically active molecules.

Endogenous expression of angiogenesis-related factors in response to muscle injury.

Gene therapy has developed a new strategy to treat a variety of ischemic diseases using angiogenic growth factors. However, the endogenous expression pattern of angiogenesis-related factors in response to muscle injury is not fully characterized. In the present study, we investigated the expression of angiogenesis-related factors, vascular endothelial growth factor, angiopoietin-1, -2, monocyte chemoattractant protein-1, and their receptors during muscle regeneration. Mice underwent freeze injury, and then the gastrocnemius muscles were isolated 1, 3, 5, 7, 10, 14, and 28 days after surgery. Generally, changes in gene expression were most dramatic during the early stage of muscle regeneration, and were attenuated as angiogenesis progressively developed and then returned to steady-state levels. VEGF mRNA began to increase from day 3 and peaked at day 5 after muscle injury. VEGF receptors, Flt-1, KDR/Flk-1, and neuropilin-1 mRNAs were increased from 3- to 9-fold at day 3 after muscle injury. At the same time, angiopoietin-1 and angiopoietin-2 mRNA were increased by 3- and 15-fold respectively, concomitantly with an increase in their receptors and Tie-2 mRNA. Finally, MCP-1 and CC-chemokine receptor 2 mRNAs were sharply up-regulated by 1600- and 100-fold, respectively, at day 3 after muscle injury. These results suggest that the molecular events implicated in angiogenesis occur at an early stage of muscle regeneration.