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Investigative Ophthalmology & Visual... May 2024To determine whether development of neuromuscular junctions (NMJs) differs between extraocular muscles (EOMs) and other skeletal 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.
Topics: Animals; Neuromuscular Junction; Oculomotor Muscles; Mice; Muscle, Skeletal; Microscopy, Confocal; Mice, Inbred C57BL; Myosin Heavy Chains; Animals, Newborn; Female
PubMed: 38767908
DOI: 10.1167/iovs.65.5.28 -
BioRxiv : the Preprint Server For... May 2024Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and...
Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells.
Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.
PubMed: 38766013
DOI: 10.1101/2024.05.04.590649 -
ELife May 2024Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and...
Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, and and larger hibernators, and . We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in and during hibernation, whilst in and , changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in , which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.
Topics: Animals; Hibernation; Energy Metabolism; Skeletal Muscle Myosins; Ursidae; Adenosine Triphosphate; Muscle, Skeletal; Muscle Fibers, Skeletal; Proteomics
PubMed: 38752835
DOI: 10.7554/eLife.94616 -
Brain, Behavior, and Immunity Jul 2024Chronic pain is a heavily debilitating condition and a huge socio-economic burden, with no efficient treatment. Over the past decade, the gut microbiota has emerged as...
Chronic pain is a heavily debilitating condition and a huge socio-economic burden, with no efficient treatment. Over the past decade, the gut microbiota has emerged as an important regulator of nervous system's health and disease states. Yet, its contribution to the pathogenesis of chronic somatic pain remains poorly documented. Here, we report that male but not female mice lacking Myosin1a (KO) raised under single genotype housing conditions (KO-SGH) are predisposed to develop chronic pain in response to a peripheral tissue injury. We further underscore the potential of MYO1A loss-of-function to alter the composition of the gut microbiota and uncover a functional connection between the vulnerability to chronic pain and the dysbiotic gut microbiota of KO-SGH males. As such, parental antibiotic treatment modifies gut microbiota composition and completely rescues the injury-induced pain chronicity in male KO-SGH offspring. Furthermore, in KO-SGH males, this dysbiosis is accompanied by a transcriptomic activation signature in the dorsal root ganglia (DRG) macrophage compartment, in response to tissue injury. We identify CD206CD163 and CD206CD163 as the main subsets of DRG resident macrophages and show that both are long-lived and self-maintained and exhibit the capacity to monitor the vasculature. Consistently, in vivo depletion of DRG macrophages rescues KO-SGH males from injury-induced chronic pain underscoring a deleterious role for DRG macrophages in a Myo1a-loss-of function context. Together, our findings reveal gene-sex-microbiota interactions in determining the predisposition to injury-induced chronic pain and point-out DRG macrophages as potential effector cells.
Topics: Animals; Female; Male; Mice; Chronic Pain; Dysbiosis; Ganglia, Spinal; Gastrointestinal Microbiome; Macrophages; Mice, Inbred C57BL; Mice, Knockout; Myosin Type I
PubMed: 38710336
DOI: 10.1016/j.bbi.2024.05.010 -
Scientific Reports May 2024Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the...
Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature surface, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. It was previously documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration.
Topics: Humans; Actin Cytoskeleton; Actins; Cell Movement; Cofilin 1; Monocytes; Myosins; THP-1 Cells; Vascular Cell Adhesion Molecule-1
PubMed: 38702365
DOI: 10.1038/s41598-024-60971-1 -
PloS One 2024Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications....
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.
Topics: Animals; Mice; Cell Differentiation; Cell Line; Epigenesis, Genetic; Gene Knockout Techniques; Histone Demethylases; Histones; MEF2 Transcription Factors; Muscle Development; Muscle Fibers, Skeletal; Myoblasts; Myosin Heavy Chains; Sarcomeres; Transcription Factors; Transcription, Genetic
PubMed: 38701072
DOI: 10.1371/journal.pone.0301690 -
Cellular & Molecular Biology Letters May 2024Endometrial 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...
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 GSK3β-mediated β-catenin ubiquitination and degradation, thus facilitating the Wnt/β-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/β-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/β-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.
Topics: Humans; Female; Endometrial Neoplasms; Myosin Heavy Chains; Animals; Glycogen Synthase Kinase 3 beta; Cell Line, Tumor; beta Catenin; Mice; Cell Proliferation; Mice, Nude; Gene Expression Regulation, Neoplastic; Signal Transduction; Prognosis; Antineoplastic Agents; Middle Aged; Naphthoquinones
PubMed: 38698330
DOI: 10.1186/s11658-024-00583-9 -
American Journal of Physiology. Cell... May 2024Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal...
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.
Topics: Humans; Muscle Fibers, Skeletal; Insulin-Like Growth Factor I; Cell Differentiation; Muscle Contraction; Phenotype; Cells, Cultured; Glucose Transporter Type 4; Myosin Heavy Chains; Glucose; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Muscle, Skeletal
PubMed: 38690930
DOI: 10.1152/ajpcell.00654.2023 -
The Journal of Clinical Investigation May 2024The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac...
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 β 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.
Topics: Humans; Muscle, Skeletal; Cardiomyopathy, Hypertrophic; Myosin Heavy Chains; Distal Myopathies; Animals; Mutation; Myosins; Benzylamines; Uracil
PubMed: 38690729
DOI: 10.1172/JCI179958 -
The Journal of Clinical Investigation May 2024Proline substitutions within the coiled-coil rod region of the β-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal...
Proline substitutions within the coiled-coil rod region of the β-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.
Topics: Animals; Mice; Humans; Proline; Distal Myopathies; Amino Acid Substitution; Mutation, Missense; Cardiac Myosins; Myosin Heavy Chains; Female; Male; Mice, Transgenic; Muscle Contraction; Muscle, Skeletal
PubMed: 38690726
DOI: 10.1172/JCI172599