Negamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA.

Negamycin (NEG) is a ribosome-targeting antibiotic that exhibits clinically promising activity. Its binding site and mode of action have remained unknown. We solved the structure of the Thermus thermophilus ribosome bound to mRNA and three tRNAs, in complex with NEG. The drug binds to both small and large ribosomal subunits at nine independent sites. Resistance mutations in the 16S rRNA unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit as the primary site of antibiotic action in the bacterial and, possibly, eukaryotic ribosome. At this site, NEG contacts 16S rRNA as well as the anticodon loop of the A-site tRNA. Although the NEG site of action overlaps with that of tetracycline (TET), the two antibiotics exhibit different activities: while TET sterically hinders binding of aminoacyl-tRNA to the ribosome, NEG stabilizes its binding, thereby inhibiting translocation and stimulating miscoding.

A patient-derived iPSC model revealed oxidative stress increases facioscapulohumeral muscular dystrophy-causative DUX4.

Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient-derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2-derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS-induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS-induced DUX4 increase and identified ataxia-telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS-induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.

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.

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.

LIM homeobox transcription factor Lhx2 inhibits skeletal muscle differentiation in part via transcriptional activation of Msx1 and Msx2.

LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the -2097 to -1189 genomic region of Msx1 and two additional sites in the -536 to +73 genomic region of Msx2. These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors.

Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade.

Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade.

Dietary phosphorus overload aggravates the phenotype of the dystrophin-deficient mdx mouse.

Duchenne muscular dystrophy is a lethal X-linked disease with no effective treatment. Progressive muscle degeneration, increased macrophage infiltration, and ectopic calcification are characteristic features of the mdx mouse, a murine model of Duchenne muscular dystrophy. Because dietary phosphorus/phosphate consumption is increasing and adverse effects of phosphate overloading have been reported in several disease conditions, we examined the effects of dietary phosphorus intake in mdx mice phenotypes. On weaning, control and mdx mice were fed diets containing 0.7, 1.0, or 2.0 g phosphorus per 100 g until they were 90 days old. Dystrophic phenotypes were evaluated in cryosections of quadriceps and tibialis anterior muscles, and maximal forces and voluntary activity were measured. Ectopic calcification was analyzed by electron microscopy to determine the cells initially responsible for calcium deposition in skeletal muscle. Dietary phosphorus overload dramatically exacerbated the dystrophic phenotypes of mdx mice by increasing inflammation associated with infiltration of M1 macrophages. In contrast, minimal muscle necrosis and inflammation were observed in exercised mdx mice fed a low-phosphorus diet, suggesting potential beneficial therapeutic effects of lowering dietary phosphorus intake on disease progression. To our knowledge, this is the first report showing that dietary phosphorus intake directly affects muscle pathological characteristics of mdx mice. Dietary phosphorus overloading promoted dystrophic disease progression in mdx mice, whereas restricting dietary phosphorus intake improved muscle pathological characteristics and function.

Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation.

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor.

Low culture temperature inhibits myogenic differentiation through mitochondrial activity.

A previous study by our group reported that mouse and human myoblasts fail to express myogenin and to fuse into multi-nucleate myotubes when cultured at low temperature, such as 30°C, but that this activity is rescued by adding IGF-I and vitamin C to the culture medium. In the present study, we examined mitochondrial activity as a target of the inhibitory effects of the low culture temperature. It has been suggested that mitochondria regulate myogenesis. By using a mouse myoblast cell line C2C12, we demonstrate that the expression of cytochrome c oxidase subunit I (COX I), which is encoded in mitochondrial genome, increases during myogenic differentiation at the normal culture temperature (38°C), but that this up-regulation is inhibited at 30°C. The mitochondrial membrane potential also decreased at 30°C compared to the culture at 38°C. However, IGF-I and vitamin C rescued both COX I expression and mitochondrial membrane potential at 30°C as promoting muscle differentiation. We also find that the rescue of mitochondrial activity by IGF-I and vitamin C at 30°C occurred after the myogenin expression, which suggests that myogenin regulates mitochondrial function during myogenesis. We suggest that our low temperature-culture system may be suitable for use in studying the detailed mechanism of myogenin-related phenomena during myogenesis.

α1-Syntrophin-deficient mice exhibit impaired muscle force recovery after osmotic shock.

INTRODUCTION: α1-syntrophin, a member of the dystrophin complex, recruits membrane molecules, including aquaporin-4, at the sarcolemma. The physiological functions of α1-syntrophin are poorly understood. METHODS: We examined the physiological characteristics of α1-syntrophin-deficient muscles under osmotic stress conditions to test the possibility that mutant muscles are less tolerant of osmotic shock. RESULTS: Isolated muscle bundles from mutant mice showed markedly reduced force production after hypo-osmotic shock. In addition, the mutant muscle bundles showed delayed recovery of specific gravity after being exposed to hypo-osmotic conditions. Two consecutive exercise tests on the treadmill revealed their performance in the second test was significantly lower than for wild-type mice. Furthermore, mutant mice had higher serum lactate concentrations after treadmill exercise. CONCLUSIONS: Although the lack of α1-syntrophin from the sarcolemma does not lead to muscle degeneration, our results suggest that it may be partly involved in the pathophysiology of dystrophin-deficient Duchenne muscular dystrophy.

Bringing cell biology into classroom: tips to culture and observe skeletal muscle cells in high school and college.

Watching living cells through a microscope is much more exciting than seeing pictures of cells in high school and college textbooks. However, bringing cell cultures into the classroom is challenging for biology teachers since culturing cells requires sophisticated and expensive instruments such as a CO2 incubator and an inverted phase-contrast microscope. Here, we describe easy and affordable methods to culture and observe skeletal muscle cells using the L-15 culture medium, tissue culture flask, standard dry incubator, standard upright microscope, and modified Smartphone microscope. Watching natural living cells in a "Do-It-Yourself (DIY)" way may inspire more students' interest in cell biology.

IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures.

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30°C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30°C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30°C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.

Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling.

Adult skeletal muscle is able to repeatedly regenerate because of the presence of satellite cells, a population of stem cells resident beneath the basal lamina that surrounds each myofiber. Little is known, however, of the signaling pathways involved in the activation of satellite cells from quiescence to proliferation, a crucial step in muscle regeneration. We show that sphingosine-1-phosphate induces satellite cells to enter the cell cycle. Indeed, inhibiting the sphingolipid-signaling cascade that generates sphingosine-1-phosphate significantly reduces the number of satellite cells able to proliferate in response to mitogen stimulation in vitro and perturbs muscle regeneration in vivo. In addition, metabolism of sphingomyelin located in the inner leaflet of the plasma membrane is probably the main source of sphingosine-1-phosphate used to mediate the mitogenic signal. Together, our observations show that sphingolipid signaling is involved in the induction of proliferation in an adult stem cell and a key component of 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.

FGF2 induces ERK phosphorylation through Grb2 and PKC during quiescent myogenic cell activation.

Satellite cells are muscle-resident stem cells, which are located beneath the basement membrane of myofibers. Because the number of satellite cells is normally constant, there must be a tight regulation of satellite cell activation and self-renewal. However, the molecular mechanisms involved in satellite cell maintenance are largely unknown, and thus have become the subject of extensive study these days. Although RNA interference with a small interfering RNA has been widely used to investigate the role of specific gene products, inefficient knockdown of Grb2 expression occurred in quiescent reserve cells, a model for quiescent satellite cells, by ordinary transfection protocol. In this study we report that pretreatment with trypsin greatly enhanced siRNA delivery into quiescent reserve cells, resulting in efficient silencing of Grb2 expression. By applying a combination of Grb2-silencing and protein kinase C inhibitors, we demonstrated that extracellular signal-regulated kinase (ERK) phosphorylation induced with fibroblast growth factor 2 (FGF2) was dependent on both Grb2 and protein kinase C (PKC) with different kinetics. We concluded that the PKC-mediated pathway contributes to rapid initiation and termination of ERK phosphorylation, while the Grb2-mediated pathway contributes to delayed and sustained ERK phosphorylation.

Ectopic calcification is caused by elevated levels of serum inorganic phosphate in mdx mice.

Ectopic calcification occurs in the skeletal muscle of mdx mice, a dystrophin-deficient animal model of Duchenne muscular dystrophy. The purpose of this study was to clarify the mechanism of the calcification. The calcified deposits were identified as hydroxyapatite, a crystallized form of calcium phosphate, and the serum inorganic phosphate (Pi) level in the mdx mice was approximately 1.4 times higher than that in the normal B10 mice, suggesting that Pi plays a critical role in the ectopic calcification. When C2C12 mouse myoblasts were cultured under high-Pi conditions, myogenic differentiation was retarded while the expression of osteogenic markers such as osteocalcin and Runx2 were upregulated. This was followed by the generation of calcium deposition. Moreover, ectopic calcification reduced to an undetectable level in most of the mdx mice fed a Pi-reduced diet. We therefore conclude that the Pi-induced osteogenesis of muscle cells is responsible for ectopic calcification in the skeletal muscle of mdx mice.

Alpha1-syntrophin-deficient skeletal muscle exhibits hypertrophy and aberrant formation of neuromuscular junctions during regeneration.

Alpha1-syntrophin is a member of the family of dystrophin-associated proteins; it has been shown to recruit neuronal nitric oxide synthase and the water channel aquaporin-4 to the sarcolemma by its PSD-95/SAP-90, Discs-large, ZO-1 homologous domain. To examine the role of alpha1-syntrophin in muscle regeneration, we injected cardiotoxin into the tibialis anterior muscles of alpha1-syntrophin-null (alpha1syn-/-) mice. After the treatment, alpha1syn-/- muscles displayed remarkable hypertrophy and extensive fiber splitting compared with wild-type regenerating muscles, although the untreated muscles of the mutant mice showed no gross histological change. In the hypertrophied muscles of the mutant mice, the level of insulin-like growth factor-1 transcripts was highly elevated. Interestingly, in an early stage of the regeneration process, alpha1syn-/- mice showed remarkably deranged neuromuscular junctions (NMJs), accompanied by impaired ability to exercise. The contractile forces were reduced in alpha1syn-/- regenerating muscles. Our results suggest that the lack of alpha1-syntrophin might be responsible in part for the muscle hypertrophy, abnormal synapse formation at NMJs, and reduced force generation during regeneration of dystrophin-deficient muscle, all of which are typically observed in the early stages of Duchenne muscular dystrophy patients.

Renal involvement in the pathogenesis of mineral and bone disorder in dystrophin-deficient mdx mouse.

Duchenne muscular dystrophy is a severe muscular disorder, often complicated with osteoporosis, and impaired renal function has recently been featured. We aimed to clarify the involvement of renal function in the pathogenesis of mineral and bone disorder in mdx mice, a murine model of the disease. We clearly revealed renal dysfunction in adult mdx mice, in which dehydration and hypercalcemia were contributed. We also examined the effects of dietary phosphorus (P) overload on phosphate metabolism. Serum phosphate and parathyroid hormone (PTH) levels were significantly increased in mdx mice by dietary P in a dose-dependent manner; however, bone alkaline phosphatase levels were significantly lower in mdx mice. Additionally, bone mineral density in mdx mice were even worsened by increased dietary P in a dose-dependent manner. These results suggested that the uncoupling of bone formation and resorption was enhanced by skeletal resistance to PTH due to renal failure in mdx mice.