Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are ...incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following
. Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level.
Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.
Peripheral muscle weakness and mass loss are characteristic features in severe COPD. We hypothesized that the phosphodiesterase-4 inhibitor roflumilast-induced cAMP may ameliorate proteolysis and ...metabolism in skeletal muscles of COPD patients with severe muscle wasting. In myogenic precursor cells (isolated from muscle biopsies and cultured up to obtain differentiated myotubes) from 10 severe COPD patients and 10 healthy controls, which were treated with 1 microM roflumilast N-oxide (RNO) for three time-cohorts (1h, 6h, and 24h), genes of antioxidant defense and oxidative stress marker, myogenesis and muscle metabolism, proteolysis (tyrosine release assay) and ubiquitin-proteasome system markers, autophagy, and myosin isoforms were analyzed using RT-PCR and immunoblotting. In COPD patients, at 6h-RNO treatment, myotube tyrosine release, total protein ubiquitination, and TRIM32 levels were significantly lower than healthy controls, whereas at 24h-RNO treatment, myotube MyHC-I and MyHC-IIx expression levels were upregulated in both patients and controls. In the 6h-RNO cohort, in patients and controls, myotube expression of NRF2 and its downstream antioxidants, Sirtuin-1, FN14, and IGF-I was upregulated, while that of MEF2C, MYOD, Myogenin, Myostatin, Atrogin-1, and MuRF-1 was downregulated. In myotubes of severe COPD patients with cachexia, roflumilast-induced cAMP signaling exerts beneficial effects by targeting muscle protein breakdown (tyrosine release) along with reduced expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In both patients and controls, roflumilast also favored antioxidant defense through upregulation of NRF2 pathway and that of the histone deacetylase Sirtuin-1, while it improved the expression of slow- and fast-twitch myosin isoforms. These findings show that muscle dysfunction and wasting may be targeted by roflumilast-induced cAMP signaling in COPD. These results have potential therapeutic implications as this PDE4 inhibitor is currently available for the treatment of systemic inflammation and exacerbations in patients with severe COPD.
Abstract As the largest organ in the human body, skeletal muscle is essential for breathing support, movement initiation, and maintenance homeostasis. It has been shown that programmed cell death ...(PCD), which includes autophagy, apoptosis, and necrosis, is essential for the development of skeletal muscle. A novel form of PCD called ferroptosis is still poorly understood in relation to skeletal muscle. In this study, we observed that the activation of ferroptosis significantly impeded the differentiation of C2C12 myoblasts into myotubes and concurrently suppressed the expression of OTUB1, a crucial deubiquitinating enzyme. OTUB1-silenced C2C12 mouse myoblasts were used to investigate the function of OTUB1 in ferroptosis. The results show that OTUB1 knockdown in vitro significantly increased C2C12 ferroptosis and inhibited myogenesis. Interestingly, the induction of ferroptosis resulting from OTUB1 knockdown was concomitant with the activation of autophagy. Furthermore, OTUB1 interacted with the P62 protein and stabilized its expression by deubiquitinating it, thereby inhibiting autophagy-dependent ferroptosis and promoting myogenesis. All of these findings demonstrate the critical role that OTUB1 plays in controlling ferroptosis, and we suggest that focusing on the OTUB1-P62 axis may be a useful tactic in the treatment and prevention of disorders involving the skeletal muscle.
Abstract Methionine is an essential amino critical to many cell functions including the synthesis of proteins. Supplementation of methionine in vivo is typically through L-methionine, DL-methionine, ...or a methionine hydroxy analog (MHA). The goal of this study was to compare the function of L-methionine, DL-methionine, and an MHA as a source of methionine to myoblasts in vitro. Avian myoblasts isolated from turkey embryos were plated in media containing varying concentrations of DL-methionine (DLM; 1.125 mg/mL or 0.56 mg/mL) or methionine hydroxy analog (MHA; 1.28 mg/mL or 0.64 mg/mL) as well as a methionine deficient negative control group and an L-methionine supplemented positive control group. The results of the proliferation assay exhibited cell division in the absence of methionine which was not significantly different than the positive control group. Results from the myoblast fusion assay revealed significantly greater myotube diameter between methionine supplemented groups compared to the methionine deficient negative control. The findings of this study show an ability for avian myoblasts to proliferate in the absence of methionine, the significance of which is discussed. Additionally, findings from the fusion assay suggest that DL-methionine and MHA are potential cost-effective substitutes for methionine supplementation during terminal differentiation of avian myoblasts.
Satellite cells are indispensable for skeletal muscle regeneration and hypertrophy by forming nascent myofibers (myotubes). They synthesize multi-potent modulator netrins (secreted subtypes: ...netrin-1, -3, and -4), originally found as classical neural axon guidance molecules. While netrin-1 and -3 have key roles in myogenic differentiation, the physiological significance of netrin-4 is still unclear. This study examined whether netrin-4 regulates myofiber type commitment and myotube formation. Initially, the expression profiles indicated that satellite cells isolated from the extensor digitorum longus muscle (EDL muscle: fast-twitch myofiber-abundant) expressed slightly more netrin-4 than the soleus muscle (slow-type abundant) cells. As netrin-4 knockdown inhibited both slow- and fast-type myotube formation, netrin-4 may not directly regulate myofiber type commitment. However, netrin-4 knockdown in satellite cell-derived myoblasts reduced the myotube fusion index, while exogenous netrin-4 promoted myotube formation, even though netrin-4 expression level was maximum during the initiation stage of myogenic differentiation. Furthermore, netrin-4 knockdown also inhibited MyoD (a master transcriptional factor of myogenesis) and Myomixer (a myoblast fusogenic molecule) expression. These data suggest that satellite cells synthesize netrin-4 during myogenic differentiation initiation to promote their own fusion, stimulating the MyoD–Myomixer signaling axis.
Display omitted
•Satellite cell-derived myoblasts abundantly synthesize netrin-4 during myogenic differentiation initiation.•Netrin-4 does not directly regulate specific myofiber types unlike semaphorin 3A and netrin-1.•Netrin-4–MyoD–Myomixer novel axis regulates myoblast–myoblast fusion.
Background
Plant-derived phytochemicals such as flavonoids have been explored to be powerful antioxidants that protect against oxidative stress-related diseases. In the present study, Morin, a ...flavonoid compound was studied for its antioxidant and antidiabetic properties in relation to oxidative stress in insulin resistant models conducted in rat skeletal muscle L6 cell line model.
Methods
Evaluation of antioxidant property of morin was assayed using in vitro methods such as cell viability by MTT assay, estimation of SOD and CAT activity and NO scavenging activity. The anti-oxidative nature of morin on L6 cell line was conducted by the DCF-DA fluorescent activity. Glucose uptake in morin treated L6 myotubes are accessed by 2-NBDG assay in the presence or absence of IRTK and PI3K inhibitors. Further glycogen content estimation due to the morin treatment in L6 myotubes was performed. Antioxidant and insulin signaling pathway gene expression was examined over RT-PCR analysis.
Results
Morin has a negligible cytotoxic effect at doses of 20, 40, 60, 80, and 100 µM concentration according to cell viability assay. Morin revealed that the levels of the antioxidant enzymes SOD and CAT in L6 myotubes had increased. When the cells were subjected to the nitro blue tetrazolium assay, morin lowered reactive oxygen species (ROS) formation at 60 µM concentration displaying 39% ROS generation in oxidative stress condition. Lesser NO activity and a drop in green fluorescence emission in the DCFDA assay, demonstrating its anti-oxidative nature by reducing ROS formation in vitro. Glucose uptake by the L6 myotube cells using 2-NBDG, and with IRTK and PI3K inhibitors (genistein and wortmannin) showed a significant increase in glucose uptake by the cells which shows the up regulated GLUT-4 movement from intracellular pool to the plasma membrane. Morin (60 µM) significantly enhanced the expression of antioxidant genes GPx, GST and GCS as well as insulin signalling genes IRTK, IRS-1, PI3K, GLUT-4, GSK-3β and GS in L6 myotubes treated cells.
Conclusion
Morin has the ability to act as an anti-oxidant by lowering ROS levels and demonstrating insulin mimetic activity by reversing insulin resistance associated with oxidative stress.
The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient ...overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force-frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.
Microfluidic‐based technologies enable the development of cell culture systems that provide tailored microenvironmental inputs to mammalian cells. Primary myoblasts can be induced to differentiate ...into multinucleated skeletal muscle cells, myotubes, which are a relevant model system for investigating skeletal muscle metabolism and physiology in vitro. However, it remains challenging to differentiate primary myoblasts into mature myotubes in microfluidics devices. Here we investigated the effects of integrating continuous (solid) and intermittent (dashed) walls in microfluidic channels as topological constraints in devices designed to promote the alignment and maturation of primary myoblast‐derived myotubes. The topological constraints caused alignment of the differentiated myotubes, mimicking the native anisotropic organization of skeletal muscle cells. Interestingly, dashed walls facilitated the maturation of skeletal muscle cells, as measured by quantifying myotube cell area and the number of nuclei per myotube. Together, our results suggest that integrating dashed walls as topographic constraints in microfluidic devices supports the alignment and maturation of primary myoblast‐derived myotubes.
A microfluidic device designed to promote the alignment and maturation of myotubes differentiated from primary myoblasts is described. Song and coworkers investigated the effects of integrating continuous and intermittent walls as topological constraints in microfluidics devices.
Fibro/Adipogenic Progenitors (FAPs) are muscle-interstitial progenitors mediating pro-myogenic signals that are critical for muscle homeostasis and regeneration. In myopathies, the ...autocrine/paracrine constraints controlling FAP adipogenesis are released causing fat infiltrates. Here, by combining pharmacological screening, high-dimensional mass cytometry and in silico network modeling with the integration of single-cell/bulk RNA sequencing data, we highlighted the canonical WNT/GSK/β-catenin signaling as a crucial pathway modulating FAP adipogenesis triggered by insulin signaling. Consistently, pharmacological blockade of GSK3, by the LY2090314 inhibitor, stabilizes β-catenin and represses PPARγ expression abrogating FAP adipogenesis ex vivo while limiting fatty degeneration in vivo. Furthermore, GSK3 inhibition improves the FAP pro-myogenic role by efficiently stimulating, via follistatin secretion, muscle satellite cell (MuSC) differentiation into mature myotubes. Combining, publicly available single-cell RNAseq datasets, we characterize FAPs as the main source of WNT ligands inferring their potential in mediating autocrine/paracrine responses in the muscle niche. Lastly, we identify WNT5a, whose expression is impaired in dystrophic FAPs, as a crucial WNT ligand able to restrain the detrimental adipogenic differentiation drift of these cells through the positive modulation of the β-catenin signaling.
PDF
