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The Journal of Physiology Jul 2019The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by...
KEY POINTS
The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied. Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform. Recovery from desensitization is determined by the AChR isoform-specific cytoplasmic M3-M4 domain. The co-expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering. These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions.
ABSTRACT
The neuromuscular junction (NMJ) is the site of a number of autoimmune and genetic disorders, many involving the muscle-type nicotinic acetylcholine receptor (AChR), although there are aspects of normal NMJ development and function that need to be better understood. In particular, there are still questions regarding the implications of the developmental switch from fetal to adult AChRs, as well as how their functions might be modified by rapsyn that clusters the AChRs. Desensitization of human muscle AChRs was investigated using the patch clamp technique to measure whole-cell currents in muscle-type (TE671/CN21) and non-muscle (HEK293) cell lines expressing either fetal or adult AChRs. Desensitization time constants were similar with both AChR isoforms but recovery time constants were shorter in cells expressing adult compared to fetal AChRs (P < 0.0001). Chimeric experiments showed that recovery from desensitization was determined by the M3-M4 cytoplasmic loops of the γ- and ε-subunits. Expression of rapsyn in TE671/CN21 cells induced AChR aggregation and also, surprisingly, shortened recovery time constants in both fetal and adult AChRs. However, this was not dependent on clustering because rapsyn also facilitated recovery from desensitization in HEK293 cells expressing a δ-R375H AChR mutant that did not form clusters in C2C12 myotubes. Thus, rapsyn interactions with AChRs lead not only to clustering, but also to a clustering independent faster recovery from desensitization. Both effects of rapsyn could be a necessary adjustment to the motor neuron firing rates that increase around the time of birth.
Topics: Cell Line; Cell Line, Tumor; HEK293 Cells; Humans; Motor Neurons; Muscle Cells; Muscle Fibers, Skeletal; Muscle Proteins; Neuromuscular Junction; Receptors, Cholinergic; Receptors, Nicotinic
PubMed: 31158924
DOI: 10.1113/JP277819 -
Development (Cambridge, England) Apr 2015The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and...
The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGFβ1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening.
Topics: Blotting, Western; Cell Differentiation; Cells, Cultured; Flow Cytometry; Humans; Immunohistochemistry; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pericardium; Pluripotent Stem Cells; Real-Time Polymerase Chain Reaction
PubMed: 25813541
DOI: 10.1242/dev.119271 -
Molecules (Basel, Switzerland) Jun 2020Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The...
Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The seeds of also known as morning glory, have been used as traditional medicine in East Asia. We focused on whether seeds have a suppressive effect on mutated KRAS-driven CRC as well as reserving muscle cell functions during CRC progression. Seeds of ( semen) were separated by chromatography and the active compound of semen (PN) was purified by HPLC. The compound PN efficiently suppressed the proliferation of mutated KRAS-driven CRC cells and their clonogenic potentials in a concentration-dependent manner. It also induced apoptosis of SW480 human colon cancer cells and cell cycle arrest at the G2/M phase. The CRC related pathways, including RAS/ERK and AKT/mTOR, were assessed and PN reduced the phosphorylation of AKT and mTOR. Furthermore, PN preserved muscle cell proliferation and myotube formation in cancer conditioned media. In summary, PN significantly suppressed mutated KRAS-driven cell growth and reserved muscle cell function. Based on the current study, PN could be considered as a promising starting point for the development of a nature-derived drug against KRAS-mutated CRC progression.
Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Humans; Ipomoea nil; Muscle Cells; Mutation; Proto-Oncogene Proteins p21(ras); Seeds
PubMed: 32580297
DOI: 10.3390/molecules25122864 -
Redox Biology Oct 2018Fibronectin (FN) occurs as both a soluble form, in plasma and at sites of tissue injury, and a cellular form in tissue extracellular matrices (ECM). FN is critical to...
Fibronectin (FN) occurs as both a soluble form, in plasma and at sites of tissue injury, and a cellular form in tissue extracellular matrices (ECM). FN is critical to wound repair, ECM structure and assembly, cell adhesion and proliferation. FN is reported to play a critical role in the development, progression and stability of cardiovascular atherosclerotic lesions, with high FN levels associated with a thick fibrotic cap, stable disease and a low risk of rupture. Evidence has been presented for FN modification by inflammatory oxidants, and particularly myeloperoxidase (MPO)-derived species including hypochlorous acid (HOCl). The targets and consequences of FN modification are poorly understood. Here we show, using a newly-developed MS protocol, that HOCl and an enzymatic MPO system, generate site-specific dose-dependent Tyr chlorination and dichlorination (up to 16 of 100 residues modified), and oxidation of Trp (7 of 39 residues), Met (3 of 26) and His (1 of 55) within selected FN domains, and particularly the heparin- and cell-binding regions. These alterations increase FN binding to heparin-containing columns. Studies using primary human coronary artery smooth muscle cells (HCASMC) show that exposure to HOCl-modified FN, results in decreased adherence, increased proliferation and altered expression of genes involved in ECM synthesis and remodelling. These findings indicate that the presence of modified fibronectin may play a major role in the formation, development and stabilisation of fibrous caps in atherosclerotic lesions and may play a key role in the switching of quiescent contractile smooth muscle cells to a migratory, synthetic and proliferative phenotype.
Topics: Amino Acid Sequence; Cell Adhesion; Cell Proliferation; Cells, Cultured; Fibronectins; Halogenation; Humans; Hypochlorous Acid; Myocytes, Smooth Muscle; Oxidation-Reduction; Peroxidase
PubMed: 30237127
DOI: 10.1016/j.redox.2018.09.005 -
Pflugers Archiv : European Journal of... Aug 2012Nicotinic acetylcholine receptors (nAChRs) are heteropentameric ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junction (NMJ)...
Nicotinic acetylcholine receptors (nAChRs) are heteropentameric ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junction (NMJ) and other peripheral and central synapses. At the NMJ, acetylcholine receptors (AChRs) are constantly exposed to mechanical stress resulting from muscle contraction. It is therefore of interest to understand if their function is influenced by mechanical stimuli. In this study, patch-clamp recordings showed that AChR channel activity was enhanced upon membrane stretching in both cultured Xenopus muscle cells and C2C12 myotubes. To examine how this property is physiologically regulated, effects of membrane-intrinsic and membrane-extrinsic factors on AChRs expressed in HEK293T cells were studied. As in muscle cells, AChR single channel currents recorded under cell-attached configuration were significantly increased-without change in current amplitude-when negative pressure was applied through the patch pipette. GsMTx-4, a peptide toxin that blocks mechanically activated cation channels, inhibited this effect on AChRs. The mechanosensitivity decreased when cells were treated with MβCD, latrunculin A or cytochalasin D, but increased when exposed to lysophosphatidylcholine, indicating contributions from both membrane lipids and the cytoskeleton. Rapsyn, which binds to AChRs and mediates their cytoskeletal interaction in muscle, suppressed AChR mechanosensitivity when co-expressed in HEK293T cells, but this influence of rapsyn was impaired following the deletion of rapsyn's AChR-binding domain or upon cytoskeletal disruption by cytochalasin D. These results suggest a mechanism for regulating AChR's mechanosensitivity through its cytoskeletal linkage via rapsyn, which may serve to protect the receptors and sarcolemmal integrity under high mechanical stress encountered by the NMJ.
Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Cytochalasin D; Cytoskeleton; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins; Ion Channel Gating; Lysophosphatidylcholines; Mechanotransduction, Cellular; Membrane Lipids; Muscle Cells; Muscle Fibers, Skeletal; Muscle Proteins; Neuromuscular Junction; Patch-Clamp Techniques; Peptides; Receptors, Nicotinic; Spider Venoms; Thiazolidines; Xenopus laevis
PubMed: 22733356
DOI: 10.1007/s00424-012-1132-9 -
Cells Sep 2023Olive flounder () muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached...
Olive flounder () muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.
Topics: Animals; Satellite Cells, Skeletal Muscle; Flounder; Myogenin; Desmin; Muscle Fibers, Skeletal; Muscle, Skeletal
PubMed: 37759547
DOI: 10.3390/cells12182325 -
International Journal of Molecular... Aug 2020Cellular stress has been considered a relevant pathogenetic factor in a variety of human diseases. Due to its primary functions by means of contractility, metabolism,... (Review)
Review
Cellular stress has been considered a relevant pathogenetic factor in a variety of human diseases. Due to its primary functions by means of contractility, metabolism, and protein synthesis, the muscle cell is faced with continuous changes of cellular homeostasis that require rapid and coordinated adaptive mechanisms. Hence, a prone susceptibility to cellular stress in muscle is immanent. However, studies focusing on the cellular stress response in muscular disorders are limited. While in recent years there have been emerging indications regarding a relevant role of cellular stress in the pathophysiology of several muscular disorders, the underlying mechanisms are to a great extent incompletely understood. This review aimed to summarize the available evidence regarding a deregulation of the cellular stress response in individual muscle diseases. Potential mechanisms, as well as involved pathways are critically discussed, and respective disease models are addressed. Furthermore, relevant therapeutic approaches that aim to abrogate defects of cellular stress response in muscular disorders are outlined.
Topics: Animals; Endoplasmic Reticulum Stress; Humans; Muscle Cells; Muscular Diseases; Oxidative Stress; Stress, Physiological; Unfolded Protein Response
PubMed: 32823799
DOI: 10.3390/ijms21165830 -
Scientific Reports Jul 2021Caenorhabditis elegans (C. elegans) can produce various motion patterns despite having only 69 motor neurons and 95 muscle cells. Previous studies successfully elucidate...
Caenorhabditis elegans (C. elegans) can produce various motion patterns despite having only 69 motor neurons and 95 muscle cells. Previous studies successfully elucidate the connectome and role of the respective motor neuron classes related to movement. However, these models have not analyzed the distribution of the synaptic and gap connection weights. In this study, we examined whether a motor neuron and muscle network can generate oscillations for both forward and backward movement and analyzed the distribution of the trained synaptic and gap connection weights through a machine learning approach. This paper presents a connectome-based neural network model consisting of motor neurons of classes A, B, D, AS, and muscle, considering both synaptic and gap connections. A supervised learning method called backpropagation through time was adapted to train the connection parameters by feeding teacher data composed of the command neuron input and muscle cell activation. Simulation results confirmed that the motor neuron circuit could generate oscillations with different phase patterns corresponding to forward and backward movement, and could be switched at arbitrary times according to the binary inputs simulating the output of command neurons. Subsequently, we confirmed that the trained synaptic and gap connection weights followed a Boltzmann-type distribution. It should be noted that the proposed model can be trained to reproduce the activity patterns measured for an animal (HRB4 strain). Therefore, the supervised learning approach adopted in this study may allow further analysis of complex activity patterns associated with movements.
Topics: Animals; Caenorhabditis elegans; Computer Simulation; Connectome; Locomotion; Models, Neurological; Motor Neurons; Muscle Cells; Nerve Net
PubMed: 34215774
DOI: 10.1038/s41598-021-92690-2 -
Zhong Nan Da Xue Xue Bao. Yi Xue Ban =... Aug 2021Vascular smooth muscle cell (VSMC) is one of the main cell types in blood vessel wall, and the VSMC apoptosis is involved in the development of atherosclerosis (AS). In...
Vascular smooth muscle cell (VSMC) is one of the main cell types in blood vessel wall, and the VSMC apoptosis is involved in the development of atherosclerosis (AS). In early AS, the apoptosis of VSMC is regarded as a compensatory mechanism for neointimal hyperplasia and lumen stenosis. However, this process will also accelerate the rupture of atherosclerotic plaques, resulting in serious cardiovascular complications. More studies on the related genes, RNA, and inducing factors of VSMCs apoptosis in occurrence and development of AS can provide scientific strategies for prevention and treatment of AS in different periods.
Topics: Apoptosis; Atherosclerosis; Cell Proliferation; Cells, Cultured; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Plaque, Atherosclerotic
PubMed: 34565732
DOI: 10.11817/j.issn.1672-7347.2021.200858 -
Mediators of Inflammation 2019Anti-inflammatory cytokine interleukin-4 (IL-4) promotes glucose tolerance and insulin sensitivity while reduces lipid deposits. However, the effects of IL-4 on energy...
Anti-inflammatory cytokine interleukin-4 (IL-4) promotes glucose tolerance and insulin sensitivity while reduces lipid deposits. However, the effects of IL-4 on energy metabolism in muscle, the largest insulin-targeting organ, remain obscure. The study aimed at addressing the roles of IL-4 in myocyte differentiation (myogenesis) and energy metabolism of muscle cells. Effects of IL-4 on myogenesis, and interaction between IL-4 and insulin on glucose metabolism of C2C12 myoblasts and the terminal differentiated myocytes were analyzed. IL-4 improved GLUT4 translocation and tended to elevate glucose uptake by boosting insulin signaling. In diabetic mice, transient and long-term IL-4 showed differential effects on insulin signaling and efficacy. The study provides evidence to address the roles of IL-4 in mediating whole-body muscle reservoir and glucose metabolism, as well as the interaction between immune responses and energy homeostasis. IL-4 has dual potential to act as an adjuvant therapeutic target for sarcopenia to preserve muscle mass and insulin resistance to improve insulin sensitivity, which implicates the regulation of immune system to the muscle differentiation and exercise performance.
Topics: Animals; Biological Transport; Blotting, Western; Cell Differentiation; Cell Line; Glucose; Glucose Transporter Type 4; Interleukin-4; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Muscle Cells; Muscle Development; Myoblasts; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 31814802
DOI: 10.1155/2019/4182015