Rab10-CAV1 mediated intraluminal vesicle transport to migrasomes.

Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.

Fast and slow myofiber nuclei, satellite cells, and size distribution with lifelong endurance exercise in men and women.

We previously observed lifelong endurance exercise (LLE) influenced quadriceps whole-muscle and myofiber size in a fiber-type and sex-specific manner. The current follow-up exploratory investigation examined myofiber size regulators and myofiber size distribution in vastus lateralis biopsies from these same LLE men (n = 21, 74 ± 1 years) and women (n = 7, 72 ± 2 years) as well as old, healthy nonexercisers (OH; men: n = 10, 75 ± 1 years; women: n = 10, 75 ± 1 years) and young exercisers (YE; men: n = 10, 25 ± 1 years; women: n = 10, 25 ± 1 years). LLE exercised ~5 days/week, ~7 h/week for the previous 52 ± 1 years. Slow (myosin heavy chain (MHC) I) and fast (MHC IIa) myofiber nuclei/fiber, myonuclear domain, satellite cells/fiber, and satellite cell density were not influenced (p > 0.05) by LLE in men and women. The aging groups had ~50%-60% higher proportion of large (>7000 µm2) and small (<3000 µm2) myofibers (OH; men: 44%, women: 48%, LLE; men: 42%, women: 42%, YE; men: 27%, women: 29%). LLE men had triple the proportion of large slow fibers (LLE: 21%, YE: 7%, OH: 7%), while LLE women had more small slow fibers (LLE: 15%, YE: 8%, OH: 9%). LLE reduced by ~50% the proportion of small fast (MHC II containing) fibers in the aging men (OH: 14%, LLE: 7%) and women (OH: 35%, LLE: 18%). These data, coupled with previous findings, suggest that myonuclei and satellite cell content are uninfluenced by lifelong endurance exercise in men ~60-90 years, and this now also extends to septuagenarian lifelong endurance exercise women. Additionally, lifelong endurance exercise appears to influence the relative abundance of small and large myofibers (fast and slow) differently between men and women.

Inhibition of skeletal muscle differentiation by calciprotein particles in human primary myoblasts.

Sarcopenia is a common complication of chronic kidney disease (CKD) and has a detrimental effect on prognosis. Previous studies have explored the role of secondary calciprotein particles (CPP2) in determining the progression of complications and poor outcomes in patients with CKD. However, no study has demonstrated that CPP2 impairs skeletal myogenesis. Our study revealed that CPP2 exposure inhibits skeletal myogenesis by suppressing myotube formation and expression of skeletal muscle-specific myosin heavy chain and actin in human primary myoblasts. Moreover, CPP2 exposure altered the expression patterns of lineage-determinative transcription factors responsible for regulating myotube differentiation marker genes. This study first demonstrated that CPP2 interferes with myoblast differentiation and myotube formation in vitro.

The downregulation of genes encoding muscle proteins have a potential role in the development of scrotal hernia in pigs.

BACKGROUND: Testicular descent is a physiological process regulated by many factors. Eventually, disturbances in the embryological/fetal development path facilitate the occurrence of scrotal hernia, a congenital malformation characterized by the presence of intestinal portions within the scrotal sac due to the abnormal expansion of the inguinal ring. In pigs, some genes have been related to this anomaly, but the genetic mechanisms involved remain unclear. This study aimed to investigate the expression profile of a set of genes potentially involved with the manifestation of scrotal hernia in the inguinal ring tissue. METHODS AND RESULTS: Tissue samples from the inguinal ring/canal of normal and scrotal hernia-affected male pigs with approximately 30 days of age were used. Relative expression analysis was performed using qPCR to confirm the expression profile of 17 candidate genes previously identified in an RNA-Seq study. Among them, the Myosin heavy chain 1 (MYH1), Desmin (DES), and Troponin 1 (TNNI1) genes were differentially expressed between groups and had reduced levels of expression in the affected animals. These genes encode proteins involved in the formation of muscle tissue, which seems to be important for increasing the resistance of the inguinal ring to the abdominal pressure, which is essential to avoid the occurrence of scrotal hernia. CONCLUSIONS: The downregulation of muscular candidate genes in the inguinal tissue clarifies the genetic mechanisms involved with this anomaly in its primary site, providing useful information for developing strategies to control this malformation in pigs and other mammals.

A novel role for KIFC1-MYH9 interaction in triple-negative breast cancer aggressiveness and racial disparity.

African American (AA) women are twice as likely to develop triple-negative breast cancer (TNBC) as women of European descent. Additionally, AA women with TNBC present a much more aggressive disease course than their European American (EA) counterparts. Thus, there is an unmet clinical need to identify race-specific biomarkers and improve survival outcomes in AA patients with TNBC. The minus-end directed microtubule motor protein kinesin family member C1 (KIFC1) promotes centrosome clustering and chromosomal instability and is often overexpressed in TNBC. Previous findings suggest that KIFC1 plays a role in cell proliferation and migration in TNBC cells from AAs and that the levels of nuclear KIFC1 (nKIFC1) are particularly high in AA patients with TNBC. The nuclear localization of KIFC1 in interphase may underlie its previously unrecognized race-specific association. In this study, we found that in TNBC cells derived from AAs, nKIFC1 interacted with the tumor suppressor myosin heavy chain 9 (MYH9) over EA cells. Treatment of AA TNBC cells with commercial inhibitors of KIFC1 and MYH9 disrupted the interaction between KIFC1 and MYH9. To characterize the racial differences in the KIFC1-MYH9-MYC axis in TNBC, we established homozygous KIFC1 knockout (KO) TNBC cell lines. KIFC1 KO significantly inhibited proliferation, migration, and invasion in AA TNBC cells but not in EA TNBC cells. RNA sequencing analysis showed significant downregulation of genes involved in cell migration, invasion, and metastasis upon KIFC1 KO in TNBC cell lines from AAs compared to those from EAs. These data indicate that mechanistically, the role of nKIFC1 in driving TNBC progression and metastasis is stronger in AA patients than in EA patients, and that KIFC1 may be a critical therapeutic target for AA patients with TNBC.

Retinoic acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes.

BACKGROUND: Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocyte cells. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart. METHODS: CMs were derived from hiPSC (hi-PCS-CM) using different concentrations of RA (Control without RA, LRA with 0.05µM and HRA with 0.1 µM) between day 3-6 of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling hiPSC-CM at high cell density in a low collagen hydrogel. RESULTS: In the HRA group, hiPSC-CMs exhibited highest expression of contractile proteins MYH6, MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are marker genes for left ventricular CMs, was also the highest in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was functionally more similar to the left ventricle. RNAsequencing revealed that the heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. CONCLUSION: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHTs with a phenotype similar to that of the left ventricle or right ventricle.

OLFM4 promotes the progression of intestinal metaplasia through activation of the MYH9/GSK3ß/ß-catenin pathway.

BACKGROUND: Intestinal metaplasia (IM) is classified into complete intestinal metaplasia (CIM) and incomplete intestinal metaplasia (IIM). Patients diagnosed with IIM face an elevated susceptibility to the development of gastric cancer, underscoring the critical need for early screening measures. In addition to the complexities associated with diagnosis, the exact mechanisms driving the progression of gastric cancer in IIM patients remain poorly understood. OLFM4 is overexpressed in several types of tumors, including colorectal, gastric, pancreatic, and ovarian cancers, and its expression has been associated with tumor progression. METHODS: In this study, we used pathological sections from two clinical centers, biopsies of IM tissues, precancerous lesions of gastric cancer (PLGC) cell models, animal models, and organoids to explore the role of OLFM4 in IIM. RESULTS: Our results show that OLFM4 expression is highly increased in IIM, with superior diagnostic accuracy of IIM when compared to CDX2 and MUC2. OLFM4, along with MYH9, was overexpressed in IM organoids and PLGC animal models. Furthermore, OLFM4, in combination with Myosin heavy chain 9 (MYH9), accelerated the ubiquitination of GSK3ß and resulted in increased ß-catenin levels through the Wnt signaling pathway, promoting the proliferation and invasion abilities of PLGC cells. CONCLUSIONS: OLFM4 represents a novel biomarker for IIM and could be utilized as an important auxiliary means to delimit the key population for early gastric cancer screening. Finally, our study identifies cell signaling pathways involved in the progression of IM.

MYH7 R453C induced cardiac remodelling via activating TGF-ß/Smad2/3, ERK1/2 and Nox4/ROS/NF-κB signalling pathways.

Hypertrophic cardiomyopathy (HCM) is a monogenic cardiac disorder commonly induced by sarcomere gene mutations. However, the mechanism for HCM is not well defined. Here, we generated transgenic MYH7 R453C and MYH6 R453C piglets and found both developed typical cardiac hypertrophy. Unexpectedly, we found serious fibrosis and cardiomyocyte loss in the ventricular of MYH7 R453C, not MYH6 R453C piglets, similar to HCM patients. Then, RNA-seq analysis and western blotting identified the activation of ERK1/2 and PI3K-Akt pathways in MYH7 R453C. Moreover, we observed an increased expression of fetal genes and an excess of reactive oxygen species (ROS) in MYH7 R453C piglet models, which was produced by Nox4 and subsequently induced inflammatory response. Additionally, the phosphorylation levels of Smad2/3, ERK1/2 and NF-kB p65 proteins were elevated in cardiomyocytes with the MYH7 R453C mutation. Furthermore, epigallocatechin gallate, a natural bioactive compound, could be used as a drug to reduce cell death by adjusting significant downregulation of the protein expression of Bax and upregulated Bcl-2 levels in the H9C2 models with MYH7 R453C mutation. In conclusion, our study illustrated that TGF-ß/Smad2/3, ERK1/2 and Nox4/ROS pathways have synergistic effects on cardiac remodelling and inflammation in MYH7 R453C mutation.

Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent.

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac ß-myosin heavy chain (ß-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

Muscle-Protective Effect of Carnosine against Dexamethasone-Induced Muscle Atrophy in C2C12 Myotube.

This study investigated the protective effect of carnosine and its components (L-histidine and ß-alanine [HA]) against dexamethasone (Dex)-induced muscle atrophy in C2C12 myotubes. Myotubes were treated with Dex (10 µM) to induce muscle atrophy manifested by decreased myotube diameter, low myosin heavy chain content, and increased expression of muscle atrophy-associated ubiquitin ligases (Atrogin-1, MuRF-1, and Cbl-b). Carnosine (20 mM) treatment significantly improved the myotube diameter and MyHC protein expression level in Dex-treated C2C12 myotubes. It also downregulated the expression of Atrogin-1, MuRF-1, and Cbl-b and suppressed the expression of forkhead box O3 (FoxO3a) mediated by Dex. Furthermore, reactive oxygen species production was increased by Dex but was ameliorated by carnosine treatment. However, HA (20 mM), the component of carnosine, treatment was found ineffective in preventing Dex-induced protein damage. Therefore, based on above results it can be suggested that carnosine could be a potential therapeutic agent to prevent Dex-induced muscle atrophy compared to its components HA.

Identification of a third myosin-5a-melanophilin interaction that mediates the association of myosin-5a with melanosomes.

Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, we identify a third interaction between Myo5a and Mlph, that is, the interaction between the exon-G-encoded region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a transportation of melanosomes.

Basal ATP release signals through the P2Y2 receptor to maintain the differentiated phenotype of vascular smooth muscle cells.

BACKGROUND AND AIMS: Vascular smooth muscle cell (VSMC) dedifferentiation contributes substantively to vascular disease. VSMCs spontaneously release low levels of ATP that modulate vessel contractility, but it is unclear if autocrine ATP signaling in VSMCs is critical to the maintenance of the VSMC contractile phenotype. METHODS: We used pharmacological inhibitors to block ATP release in human aortic smooth muscle cells (HASMCs) for studying changes in VSMC differentiation marker gene expression. We employed RNA interference and generated mice with SMC-specific inducible deletion of the P2Y2 receptor (P2Y2R) gene to evaluate resulting phenotypic alterations. RESULTS: HASMCs constitutively release low levels of ATP that when blocked results in a significant decrease in VSMC differentiation marker gene expression, including smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), SM-22α and calponin. Basal release of ATP represses transcriptional activation of the Krüppel-Like Factor 4 (KFL4) thereby preventing platelet-derived growth factor-BB (PDGF-BB) from inhibiting expression of SMC contractile phenotype markers. SMC-restricted conditional deletion of P2Y2R evoked dedifferentiation characterized by decreases in aortic contractility and contractile phenotype markers expression. This loss was accompanied by a transition to the synthetic phenotype with the acquisition of extracellular matrix (ECM) proteins characteristic of dedifferentiation, such as osteopontin and vimentin. CONCLUSIONS: Our data establish the first direct evidence that an autocrine ATP release mechanism maintains SMC cytoskeletal protein expression by inhibiting VSMCs from transitioning to a synthetic phenotype, and further demonstrate that activation of the P2Y2R by basally released ATP is required for maintenance of the differentiated VSMC phenotype.

A Laing distal myopathy-associated proline substitution in the ß-myosin rod perturbs myosin cross-bridging activity.

Proline substitutions within the coiled-coil rod region of the ß-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Neuromuscular Junction Development Differs Between Extraocular and Skeletal Muscles and Between Different Extraocular Muscles.

Purpose: To determine whether development of neuromuscular junctions (NMJs) differs between extraocular muscles (EOMs) and other skeletal muscles. Methods: Mouse EOMs, diaphragm, and tibialis anterior (TA) were collected at postnatal day (P)0, P3, P7, P10, P14, and P21, and 12 weeks. Whole muscles were stained with α-bungarotoxin, anti-neurofilament antibody, and slow or fast myosin heavy chain antibody, and imaged with a confocal microscope. Images were quantified using Imaris software. Results: NMJs in the EOMs show a unique pattern of morphological development compared to diaphragm and TA. At P0, diaphragm and TA NMJs were oval plaques; EOM single NMJs were long, thin rods. NMJs in the three muscle types progress to mature morphology at different rates. At all ages, EOM single NMJs were larger, especially relative to myofiber size. The inferior oblique and inferior rectus muscles show delayed single NMJ development compared to other EOMs. NMJs on multiply-innervated fibers in the EOMs vary widely in size, and there were no consistent differences between muscles or over time. Incoming motor nerves formed complex branching patterns, dividing first into superficial and deep branches, each of which branched extensively over the full width of the muscle. Motor axons that innervate multiply-innervated fibers entered the muscle with the axons that innervate singly-innervated fibers, then extended both proximally and distally. EOM NMJs had more subsynaptic nuclei than skeletal muscle NMJs throughout development. Conclusions: EOMs show a unique pattern of NMJ development and have more subsynaptic nuclei than other muscles, which may contribute to the exquisite control of eye movements.

Sesamolin serves as an MYH14 inhibitor to sensitize endometrial cancer to chemotherapy and endocrine therapy via suppressing MYH9/GSK3ß/ß-catenin signaling.

BACKGROUND: Endometrial cancer (EC) is one of the most common gynecological cancers. Herein, we aimed to define the role of specific myosin family members in EC because this protein family is involved in the progression of various cancers. METHODS: Bioinformatics analyses were performed to reveal EC patients' prognosis-associated genes in patients with EC. Furthermore, colony formation, immunofluorescence, cell counting kit 8, wound healing, and transwell assays as well as coimmunoprecipitation, cycloheximide chase, luciferase reporter, and cellular thermal shift assays were performed to functionally and mechanistically analyze human EC samples, cell lines, and a mouse model, respectively. RESULTS: Machine learning techniques identified MYH14, a member of the myosin family, as the prognosis-associated gene in patients with EC. Furthermore, bioinformatics analyses based on public databases showed that MYH14 was associated with EC chemoresistance. Moreover, immunohistochemistry validated MYH14 upregulation in EC cases compared with that in normal controls and confirmed that MYH14 was an independent and unfavorable prognostic indicator of EC. MYH14 impaired cell sensitivity to carboplatin, paclitaxel, and progesterone, and increased cell proliferation and metastasis in EC. The mechanistic study showed that MYH14 interacted with MYH9 and impaired GSK3ß-mediated ß-catenin ubiquitination and degradation, thus facilitating the Wnt/ß-catenin signaling pathway and epithelial-mesenchymal transition. Sesamolin, a natural compound extracted from Sesamum indicum (L.), directly targeted MYH14 and attenuated EC progression. Additionally, the compound disrupted the interplay between MYH14 and MYH9 and repressed MYH9-regulated Wnt/ß-catenin signaling. The in vivo study further verified sesamolin as a therapeutic drug without side effects. CONCLUSIONS: Herein, we identified that EC prognosis-associated MYH14 was independently responsible for poor overall survival time of patients, and it augmented EC progression by activating Wnt/ß-catenin signaling. Targeting MYH14 by sesamolin, a cytotoxicity-based approach, can be applied synergistically with chemotherapy and endocrine therapy to eventually mitigate EC development. This study emphasizes MYH14 as a potential target and sesamolin as a valuable natural drug for EC therapy.

BMP-2 regulates the expression of myosin Va via smad in melan-a melanocyte.

Myosin Va (Myo Va) is one of three protein complexes involved in melanosome transport. In this study, we identified BMP-2 as an up-regulator of Myo Va expression using 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO). Our results showed that MNQO reduced the mRNA and protein expression of Myo Va and BMP-2 in melanocytes. Knockdown of BMP-2 by siRNA also affected Myo Va mRNA and protein expression, confirming that MNQO regulates Myo Va through BMP-2. Furthermore, phosphorylation of Smad1/5/8 by BMP2 treatment confirmed that the BMP-2/Smad signaling pathway regulates Myo Va expression in Melan-a melanocytes. Smad-binding elements were found in the Myo Va promoter and phosphorylated Smad1/5/8 bind directly to the Myo Va promoter to activate Myo Va transcription and BMP-2 enhances this binding. These findings provide insight into a new role for BMP-2 in Melan-a melanocytes and a mechanism of regulation of Myo Va expression that may be beneficial in the treatment of albinism or hyperpigmentation disorders.

Saururus chinensis (Lour.) Baill. extract promotes skeletal muscle cell differentiation by positively regulating mitochondrial biogenesis and AKT/mTOR signaling in vitro.

Promotion of myoblast differentiation by activating mitochondrial biogenesis and protein synthesis signaling pathways provides a potential alternative strategy to balance energy and overcome muscle loss and muscle disorders. Saururus chinensis (Lour.) Baill. extract (SCE) has been used extensively as a traditional herbal medicine and has several physiological activities, including anti­asthmatic, anti­oxidant, anti­inflammatory, anti­atopic, anticancer and hepatoprotective properties. However, the effects and mechanisms of action of SCE on muscle differentiation have not yet been clarified. In the present study, it was investigated whether SCE affects skeletal muscle cell differentiation through the regulation of mitochondrial biogenesis and protein synthesis in murine C2C12 myoblasts. The XTT colorimetric assay was used to determine cell viability, and myosin heavy chain (MyHC) levels were determined using immunocytochemistry. SCE was applied to C2C12 myotube at different concentrations (1, 5, or 10 ng/ml) and times (1,3, or 5 days). Reverse transcription­quantitative PCR and western blotting were used to analyze the mRNA and protein expression change of factors related to differentiation, mitochondrial biogenesis and protein synthesis. Treatment of C2C12 cells with SCE at 1,5, and 10 ng/ml did not affect cell viability. SCE promoted C2C12 myotube formation and significantly increased MyHC expression in a concentration­ and time­dependent manner. SCE significantly increased the mRNA and protein expression of muscle differentiation­specific markers, such as MyHC, myogenic differentiation 1, myogenin, Myogenic Factor 5, and ß­catenin, mitochondrial biosynthesis­related factors, such as peroxisome proliferator­activated receptor­gamma coactivator­1α, nuclear respirator factor­1, AMP­activated protein kinase phosphorylation, and histone deacetylase 5 and AKT/mTOR signaling factors related to protein synthesis. SCE may prevent skeletal muscle dysfunction by enhancing myoblast differentiation through the promotion of mitochondrial biogenesis and protein synthesis.

IGF1 promotes human myotube differentiation toward a mature metabolic and contractile phenotype.

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 day 6. 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.NEW & NOTEWORTHY 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.

A therapeutic leap: how myosin inhibitors moved from cardiac interventions to skeletal muscle myopathy solutions.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of ß myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity's role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

PHF2 regulates sarcomeric gene transcription in myogenesis.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.