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.

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.

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.

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.

Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation.

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

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.

Structure-Activity Relationship Study of Leucyl-3-epi-deoxynegamycin for Potent Premature Termination Codon Readthrough.

(+)-Negamycin, isolated from Streptomyces purpeofuscus, shows antimicrobial activity against Gram-negative bacteria and readthrough activity against nonsense mutations. Previously, we reported that two natural negamycin analogues, 5-deoxy-3-epi-negamycin and its leucine adduct, have more potent readthrough activity in eukaryocytes (COS-7 cells) than negamycin but possess no antimicrobial activity and no in vitro readthrough activity in prokaryotic systems. In the present study, on leucyl-3-epi-deoxynegamycin, a structure-activity relationship study was performed to develop more potent readthrough agents. In a cell-based readthrough assay, the derivative 13b with an o-bromobenzyl ester functions as a prodrug and exhibits a higher readthrough activity against TGA-type PTC than the aminoglycoside G418. This ester (13b) shows an in vivo readthrough activity with low toxicity, suggesting that it has the potential for treatment of hereditary diseases caused by nonsense mutations.

Treatment with the anti-IL-6 receptor antibody attenuates muscular dystrophy via promoting skeletal muscle regeneration in dystrophin-/utrophin-deficient mice.

BACKGROUND: Chronic increases in the levels of the inflammatory cytokine interleukin-6 (IL-6) in serum and skeletal muscle are thought to contribute to the progression of muscular dystrophy. Dystrophin/utrophin double-knockout (dKO) mice develop a more severe and progressive muscular dystrophy than the mdx mice, the most common murine model of Duchenne muscular dystrophy (DMD). In particular, dKO mice have smaller body sizes and muscle diameters, and develop progressive kyphosis and fibrosis in skeletal and cardiac muscles. As mdx mice and DMD patients, we found that IL-6 levels in the skeletal muscle were significantly increased in dKO mice. Thus, in this study, we aimed to analyze the effects of IL-6 receptor (IL-6R) blockade on the muscle pathology of dKO mice. METHODS: Male dKO mice were administered an initial injection (200 mg/kg intraperitoneally (i.p.)) of either the anti-IL-6R antibody MR16-1 or an isotype-matched control rat IgG at the age of 14 days, and were then given weekly injections (25 mg/kg i.p.) until 90 days of age. RESULTS: Treatment of dKO mice with the MR16-1 antibody successfully inhibited the IL-6 pathway in the skeletal muscle and resulted in a significant reduction in the expression levels of phosphorylated signal transducer and activator of transcription 3 in the skeletal muscle. Pathologically, a significant increase in the area of embryonic myosin heavy chain-positive myofibers and muscle diameter, and reduced fibrosis in the quadriceps muscle were observed. These results demonstrated the therapeutic effects of IL-6R blockade on promoting muscle regeneration. Consistently, serum creatine kinase levels were decreased. Despite these improvements observed in the limb muscles, degeneration of the diaphragm and cardiac muscles was not ameliorated by the treatment of mice with the MR16-1 antibody. CONCLUSION: As no adverse effects of treatment with the MR16-1 antibody were observed, our results indicate that the anti-IL-6R antibody is a potential therapy for muscular dystrophy particularly for promoting skeletal muscle regeneration.

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.

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.

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.

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.

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.

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.

Discovery of natural products possessing selective eukaryotic readthrough activity: 3-epi-deoxynegamycin and its leucine adduct.

Herein we report the first discovery of natural readthrough products that do not display antimicrobial activity. Two natural negamycins, 3-epi-deoxynegamycin and its leucine adduct, isolated 37 years ago, were found to be potent readthrough agents against nonsense mutations of eukaryotes, but not prokaryotes, without displaying antimicrobial activity. These results suggest that the compounds are valuable leads for the development of readthrough drugs against nonsense-mediated genetic diseases without the potential for contributing to the emergence of drug-resistant bacteria.

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.

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.

Transdermal delivery of adriamycin to transplanted ehrlich ascites tumor in mice.

There is considerable interest in the skin as a site of anti-cancer drug application. Nevertheless, the skin poses a formidable barrier to drug penetration, thereby limiting topical and transdermal bioavailability. However, we previously showed that a thioglycolate-based depilatory agent increases the drug permeability of mouse skin. In the present report, we investigated the skin permeability and efficacy of the anti-cancer drug adriamycin increased when administered transdermally to mice in combination with a thioglycolate-based depilatory agent. Adriamycin in combination with depilatory treatment reduced Ehrlich tumor growth in hairless mice about the weight and size of harvested tumors. In addition, our delivery method for adriamycin increased the therapeutic effectiveness of this agent by decreasing toxicity. Moreover, measurement of adriamycin autofluorescence revealed that topically applied adriamycin penetrate the dermis after depilatory agent treatment. These results indicate that the transdermal delivery of anti-cancer drugs is feasible by handy pretreatment of the skin with a thioglycolate-based depilatory agent.

Hyperbaric hyperoxia accelerates fracture healing in mice.

Increased oxygen tension influences bone metabolism. This study comprised two main experiments: one aimed to determine the bone mineral apposition and bone formation rates in vivo under hyperbaric hyperoxia (HBO), and the other aimed to evaluate the effects of exposure to HBO on fracture healing. In experiment 1, male mice were exposed to HBO [90 min/day at 90% O2 at 2 atmospheres absolute (ATA) for 5 days]. In experiment 2, an open femur fracture model was created in mice, followed by exposure to HBO 5 times/week (90 min/day at 90% O2 at 2 ATA) for 6 weeks after surgery. In experiment 1, HBO treatment significantly increased the mineral apposition and bone formation rates in the lumbar vertebra and femur and type 1 collagen alpha 1 and alkaline phosphatase mRNA expression in the lumbar vertebra. In experiment 2, at 2 weeks after fracture, the fracture callus was significantly larger in the HBO group than in the non-HBO group. Furthermore, at 4 and 6 weeks after fracture, radiographic findings showed accelerated fracture healing in the HBO group. At 6 weeks after fracture, femur stiffness and maximum load were significantly higher in the HBO group than in the non-HBO group. Urinary 8-hydroxy-2'-deoxyguanosine and plasma calcium concentrations were not significantly different between groups. These results suggest that exposure to HBO enhances bone anabolism and accelerates fracture healing without causing oxidative DNA damage or disruption of plasma calcium homeostasis.

Ultrasound-enhanced delivery of morpholino with Bubble liposomes ameliorates the myotonia of myotonic dystrophy model mice.

Phosphorodiamidate morpholino oligonucleotide (PMO)-mediated control of the alternative splicing of the chloride channel 1 (CLCN1) gene is a promising treatment for myotonic dystrophy type 1 (DM1) because the abnormal splicing of this gene causes myotonia in patients with DM1. In this study, we optimised a PMO sequence to correct Clcn1 alternative splicing and successfully remedied the myotonic phenotype of a DM1 mouse model, the HSALR mouse. To enhance the efficiency of delivery of PMO into HSALR mouse muscles, Bubble liposomes, which have been used as a gene delivery tool, were applied with ultrasound exposure. Effective delivery of PMO led to increased expression of Clcn1 protein in skeletal muscle and the amelioration of myotonia. Thus, PMO-mediated control of the alternative splicing of the Clcn1 gene must be important target of antisense therapy of DM1.