Journal of Cellular Physiology

Complement C3 is a key factor in complement system. Our recently animal study found that C3 may regulate myocardial apoptosis through the intrinsic apoptosis pathway. The current work investigated if C3 regulation of apoptosis occurred in human cardiomyocytes. Our results showed that incubation of exogenous C3 reduced apoptosis in a cell culture system of human cardiomyocytes which did not inherently express C3. In addition, C3 inhibited intrinsic apoptosis pathway in a cell-free apoptosis system. Furthermore, pro-C3 was found to bind with an apoptotic factor, pro-caspase 3, in a cell-free system. Thus, we presented firsthand evidence that exogenous C3 is readily reduce apoptosis in human cardiomyocytes via interaction with the intrinsic apoptotic pathway.

Homeostatic calcium ion (Ca2+) fluxes between the endoplasmic reticulum, cytosol, and extracellular space occur not only in response to cell stimulation but also in unstimulated cells. Using murine astrocytes as a model, we asked whether there is a signaling function of these resting Ca2+-fluxes. The data showed that endoplasmic reticulum (ER) Ca{superscript 2} depletion, induced by sarcoplasmic/endoplasmic reticulum Ca{superscript 2}-ATPase (SERCA) inhibition, resulted to prolonged Ca{superscript 2} influx and mitochondrial fragmentation within 10 to 30 minutes. This mitochondrial fragmentation could be prevented in Ca2+- free medium or by inhibiting store-operated Ca2+ entry (SOCE). Similarly, attenuation of STIM proteins, which are vital ER Ca2+ sensors, protected mitochondrial morphology. On the molecular level, ER Ca2+ depletion, achieved either by removing extracellular Ca2+ or through acute SERCA inhibition, led to changes in gene expression of about 13% and 41% of the transcriptome within an hour, respectively. Transcriptome changes were associated with universal biological processes such as transcription, differentiation, or cell stress. Strong increase in expression was observed for the transcription factor ATF4, which is under control of the kinase PERK (EIF2AK3), a key protein involved in ER stress. Corroborating these findings, PERK was rapidly phosphorylated in Ca2+-free medium or after acute pharmacological inhibition of SOCE. In summary, resting, homeostatic Ca2+ fluxes prevent immediate- early cell stress and transcriptional reprogramming.

Contact sites between the mitochondria and endoplasmic reticulum (ER) regulate the exchange of lipids, Ca2+, and reactive oxygen species (ROS) across the two organelles. Mitofusin 2 (Mfn2) has been identified as one of the major components tethering these two organelles together. Several post-translational modifications (PTMs) of Mfn2 have been shown to modulate canonical (i.e., mitochondrial fusion) and non-canonical Mfn2 functions, such as mitophagy and activation of ER stress signaling. However, there is little information about whether any PTMs can regulate mitochondrial and ER tethering. Basal tyrosine phosphorylation of Mfn2 was detected by mass spectroscopy, but it is unknown whether Mfn2 is a substrate of mitochondria-localized tyrosine kinases. Here, we show that mitochondria-localized c-Src can phosphorylate the C-terminal tail of Mfn2, which decreases the distance between the mitochondria and ER and facilitates Ca2+ transfer from the ER to mitochondria, followed by changes in ROS generation and mitochondrial bioenergetics. Our findings suggest that tyrosine phosphorylation of Mfn2 may uniquely work to fine-tune ER-mitochondrial Ca2+ transport under physiological and pathological conditions.

The immune and metabolic responses of macrophages are closely linked, and mitochondria play a key role in polarizing them into pro-inflammatory (classical) and anti-inflammatory (alternative) states. Mitochondrial uncoupling protein 2 (UCP2) is involved in regulating macrophage inflammation and glucose metabolism; however, its regulatory mechanisms are unclear. We found that inflammatory stimuli reduce UCP2 expression and oxygen consumption rates (OCR), indicating mitochondrial suppression. Conversely, IL-4-activated macrophages displayed higher UCP2 levels and enhanced respiration. Under glucose deprivation, LPS-stimulated macrophages retained mitochondrial activity despite lower UCP2 levels. Pyruvate emerged as a key regulator of UCP2, blocking its mitochondrial entry reduced UCP2 expression. Additionally, hypoxia markedly decreased UCP2 levels in IL-4-activated macrophages, suggesting that hypoxia contributes to UCP2 suppression in pro-inflammatory macrophages. Notably, pro-inflammatory macrophages exhibit reduced reliance on UCP2 due to suppressed mitochondrial respiration. Pyruvate regulates UCP2 expression, highlighting the connection between glycolysis and mitochondrial metabolism. These findings may inform therapeutic strategies for diseases involving immune dysregulation.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

IntroductionOxidative stress is considered an essential mechanism in ICU-acquired weakness. The roles of oxidative stress in autophagy/mitophagy dysfunction remains elusive. Microtubule serves as an essential guide rail for auto/mitophagosome trafficking required for proper maturation of auto/mitophagosomes in normal circumstances, and microtubules network formation is regulated by signal transduction mechanisms involving Akt, GSK3{beta}, and the microtubule plus-end tracking molecule, EB1. We have investigated (1) whether oxidative stress affects this pathway, leading to the defective mitophagy response, and (2) whether trehalose, an auto/mitophagy modulator, can ameliorate these pathological conditions. MethodsBy stably transfecting markers for auto/mitophagy or MT synthesis, we have established a few new C2C12 myocyte cell lines, expressing, GFP-LC3, EB1-GFP, and/or tandem-fluorescence LC3 (tfLC3). To monitor microtubule network, the cells were stained by SiR-tubulin. The cells were cultured in the presence or absence of oxidative stress by hydrogen peroxide (H2O2) and treated with or without trehalose. The response of mitophagy parameters including vesicle motion and the maturation status was monitored by stimulating the cells with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an established mitophagy inducer, under a time-lapse confocal microscopy. Signal transduction mechanisms linking mitophagy to microtubule formation was analyzed by Western Blotting against Akt and GSK3{beta}. ResultsCells under the oxidative stress, showed abolished MT network formation, decreased microtubule synthesis by EB1, and a decrease in CCCP-invoked response of mitophagosome motion, perturbed mitophagosome maturation, and increased superoxide production. Signal resistance of Akt/GSK3{beta} pathway to mitophagic stimulation, was documented. Trehalose treatment reversed signal resistance, diminished MT synthesis, ameliorated the disturbed MT network, and improved maturation defects, suppressing the production of superoxide. ConclusionsOxidative stress decreases the response of mitophagy and abolishes microtubule network. Trehalose improves the synthetic ability of microtubule and normalized the disturbed microtubule network, resulting in the improvement of the perturbed mitophagosomes maturation under the oxidative stress.

Ischemic injury to the heart causes a loss of mitochondria function due to an increase in oxidative stress. MG53, also known as TRIM72, is highly expressed in striated muscle and is essential to repair damage to plasma membrane. We have shown that mg53-/- mice are more susceptible to ischemia-reperfusion injury, whereas treatment with exogenous recombinant human MG53 (rhMG53) reduces both infarct damage and restores cardiac function. This study assesses whether MG53 protects and repairs mitochondria injury after oxidative stress associated with myocardial infarction. We hypothesize that in addition to known cell membrane repair function, MG53 acts as a myokine to protect cardiomyocytes by maintaining mitochondrial function. A combination of in vivo and in vitro ischemia/reperfusion models were used to assess MG53s effect on mitochondria using biochemical assays and confocal microscopic imaging. Treatment with rhMG53 allowed cells to maintain a healthy mitochondrial membrane potential, reduced release of mitochondrial reactive oxygen species, and mitigated mitophagy. Mitochondrial localization of rhMG53 is mediated by exposure of and interaction with cardiolipin on the mitochondrial membrane. Our data demonstrates that rhMG53 protein preserves mitochondria integrity in cardiomyocytes during ischemia reperfusion-induced oxidative stress.

Insulin resistance results from several pathophysiologic mechanisms, including chronic tissue inflammation and defective insulin signaling. Pancreatic {beta}-cells hypersecretion (hyperinsulinemia), is a central hallmark of peripheral insulin resistance. However, the underlying mechanism by which hyperinsulinemia perpetuates towards the development of insulin resistance remains unclear and is still a bigger therapeutic challenge. Here, we found hyperinsulinemia triggers inflammation and insulin resistance by stimulating TLR4-driven inflammatory cascades. We show that hyperinsulinemia activates the TLR4 signaling through HMGB1, an endogenous TLR4 ligand emanating from hyperinsulinemia exposed immune cells and peripheral organs like adipose tissue and liver. Further, our observation suggests hyperinsulinemia ensuring hyperacetylation, nuclear-to-cytoplasmic shuttling and release of HMGB1 into the extracellular space. HMGB1 was also found to be elevated in serum of T2DM patients. We found that extracellular HMGB1 plays a crucial role to promote proinflammatory responses and provokes systemic insulin resistance. Importantly, in-vitro and in-vivo treatment with naltrexone, a TLR4 antagonist led to an anti-inflammatory phenotype with protection from hyperinsulinemia mediated insulin resistance. In-vitro treatment with naltrexone directly enhanced SIRT1 activity, blocked the release of HMGB1 into extracellular milieu, suppressed release of proinflammatory cytokines and ultimately led to insulin-sensitizing effects. These observations elucidate a regulatory network between pancreatic {beta}-cells, macrophage and hepatocytes and assign an unexpected role of TLR4 - HMGB1 signaling axis in hyperinsulinemia mediated systemic insulin resistance.\n\nGraphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Clinically, there is a positive correlation between BMI and the risk of tendinopathy. However, the underlying mechanisms are not understood. Dyslipidaemia and increased circulating free fatty acids (FFA) are associated with increased BMI. We hypothesised that increased FFA concentrations negatively affect rat tendon-derived cells (rTDCs) through mitochondrial-mediated mechanisms. rTDCs were isolated and treated with oleic acid (OA), stearic acid (SA), and palmitic acid (PA). Cell viability was assessed using AlamarBlue assay, and gene expression using real-time PCR. Cell respiration and reactive oxygen species (ROS) production were measured using high-resolution respirometry and MitoSox staining. PA transport into the mitochondria was blocked by pre-treatment with 50{micro}M etomoxir. Treatment with SA and PA at 10 {micro}g/ml decreased rTDC viability by 40% and 60%, respectively. PA decreased the gene expression of the tendon markers Scx and Tnmd, and increased the expression of Mmp3, Mmp13, and Ptgs2 (encoding Cox-2). FFA treatment increased the expression of Cpt1 and Pdk4, indicating an increase in mitochondrial FFA oxidation. PA, at 10 {micro}g/ml, increased cellular respiration and ROS production. Pre-treatment with etomoxir partially inhibited the effects of PA on cell viability, Mmp3 gene expression, ROS production, and cell respiration, but did not affect PA-induced inhibition of Scx or Tnmd expression. We found that increased saturated FFA concentrations in the microenvironment reduce cell viability and alter ROS production, respiration, and gene expression. Blocking PA transport into mitochondria partially reversed the negative effects of PA. Overall, an increase in saturated FFA concentrations may contribute to poor tendon health.

Estrogen deficiency increases bone resorption and is a major contributor to osteoporosis. However, the molecular mechanisms mediating the effects of estrogen on osteoclasts remain unclear. This study aimed at elucidating the early metabolic effects of RANKL - the essential cytokine for osteoclastogenesis - and 17-beta-estradiol (E2) on osteoclast progenitor cells, using RAW 264.7 macrophage cell line and bone marrow-derived macrophages as biological models. RANKL stimulated complex I activity, oxidative phosphorylation (OXPHOS), and mitochondria-derived ATP production, as early as 3 to 6 h. This up-regulation of mitochondrial bioenergetics was associated with an increased capacity to oxidize TCA cycle substrates, fatty acids, and amino-acids. E2 inhibited all effects of RANKL on mitochondria metabolism. In the presence of RANKL, E2 also decreased cell number and stimulated the mitochondrial-mediated apoptotic pathway, detected as early as 3h. Surprisingly, the pro-apoptotic effects of E2 were associated with an accumulation of p392S-p53 in mitochondria. These findings elucidate early effects of RANKL on osteoclast progenitor metabolism and suggest novel p53-mediated mechanisms that contribute to postmenopausal osteoporosis.

Saturated fatty acids impose lipotoxic stress on pancreatic {beta}-cells, leading to {beta}-cell failure and diabetes. In this study, we investigate the critical role of organellar Ca2+ disturbance on defective autophagy and {beta}-cell lipotoxicity. Palmitate, a saturated fatty acid, induced perilysosomal Ca2+ elevation, sustained mTORC1 activation on the lysosomal membrane, suppression of the lysosomal transient receptor potential mucolipin 1 (TRPML1) channel, and accumulation of undigested autophagosomes in {beta}-cells. These Ca2+ aberrations with autophagy defects by palmitate were prevented by a mTORC1 inhibitor or a mitochondrial superoxide scavenger. To alleviate perilysosomal Ca2+ overload, strategies such as lowering extracellular Ca2+, employing voltage-gated Ca2+ channel blocker or ATP-sensitive K+ channel opener effectively abrogated mTORC1 activation and preserved autophagy. Furthermore, redirecting perilysosomal Ca2+ into the endoplasmic reticulum (ER) with an ER Ca2+ ATPase activator, restores TRPML1 activity, promotes autophagic flux, and improves survival of {beta}-cells exposed to palmitate-induced lipotoxicity. Our findings suggest oxidative stress-Ca2+ overload-mTORC1 pathway involves in TRPML1 suppression and defective autophagy during {beta}-cell lipotoxicity. Restoring perilysosomal Ca2+ homeostasis emerges as a promising therapeutic strategy for metabolic diseases.

The ERG1A K+ channel modulates the protein degradation that contributes to skeletal muscle atrophy by increasing intracellular calcium concentration ([Ca2+]i) and enhancing calpain activity, but the mechanism by which the channel regulates the [Ca2+]i is not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca2+]i in C2C12 myotubes, using Fura-2 calcium assays, immunoblot, RT-qPCR, and electrophysiology. We hypothesized that HERG would modulate L-type calcium channel activity, specifically the Cav1.1 channel known to carry signal from the sarcoplasmic membrane of skeletal muscle to the sarcomeres of the myofibrils. However, we find that HERG has no effect on the amplitude of L-type channel current nor does it affect the mRNA levels nor protein abundance of the Cav1.1 channel. Instead we find that, although the rise in [Ca2+]i (induced by depolarization) is greater in myotubes over-expressing HERG relative to controls, the difference between the KCl-stimulated Ca2+ increase in control and HERG over-expressing cells cannot be accounted for by L-type channel mediated Ca2+ influx, which suggests that HERG could modulate excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca2+]i induced by depolarization is blocked by 2-APB, an inhibitor of ECCE (and SOCE). Further, we show data suggesting that HERG also modulates the activity of ryanodine receptors, a component of ECCE, as well as store operated calcium entry (SOCE). Therefore, we investigated the effect of HERG on calsequestrin1, a calcium buffering/binding protein known to modulate ryanodine receptor 1 and store operated Ca2+ entry activities. Indeed, we find that calsequestrin1 mRNA levels are decreased 0.83-fold (p<0.05) and the total protein abundance is lowered 77% (p<0.05) in myotubes over-expressing HERG relative to controls. In summary, the data show that ERG1A overexpression modulates [Ca2+]i in skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin1.

Skeletal muscle regulation is responsible for voluntary muscular movement in vertebrates. The genes of two essential proteins, teneurins and latrophilins (LPHN), evolving in ancestors of multicellular animals, form a ligand-receptor pair, and are now shown to be required for skeletal muscle function. Teneurins possess a bioactive peptide, termed the teneurin C-terminal associated peptide (TCAP) that interacts with the LPHNs to regulate skeletal muscle contractility strength and fatigue by an insulin-independent glucose importation mechanism. CRISPR-based knockouts and siRNA-associated knockdowns of LPHN-1 and-3 shows that TCAP stimulates an LPHN-mediated cytosolic Ca2+ signal transduction cascade to increase energy metabolism and enhance skeletal muscle function via increases in type-1 oxidative fiber formation and reduce the fatigue response. Thus, the teneurin/TCAP-LPHN system is presented as a novel mechanism likely to regulate the energy requirements and performance of skeletal muscle.

Mitochondria are an important organelle regulating energy homeostasis. Mitochondrial health and dynamics are crucial determinants of the outcome of several bacterial infections. SIRT3, a major mitochondrial sirtuin, along with SIRT1 regulates key mitochondrial functions. This led to considerable interest in understanding the role of SIRT1 and SIRT3 in governing mitochondrial functions during Salmonella infection. Here, we show that loss of SIRT1 and SIRT3 function either by shRNA-mediated knockdown or inhibitor treatment led to increased mitochondrial dysfunction with alteration in mitochondrial bioenergetics alongside increased mitochondrial superoxide generation in the Salmonella-infected macrophages. Consistent with dysfunctional mitochondria, mitophagy was induced along with altered mitochondrial fusion-fission dynamics in S. Typhimurium-infected macrophages. Additionally, the mitochondrial bioenergetic alteration promotes acidification of the infected macrophage cytosolic pH. This host cytosolic pH imbalance skewed the intra-phagosomal and intra- bacterial pH in the absence of SIRT1 and SIRT3, resulting in decreased SPI-2 gene expression. Our results suggest a novel role of SIRT1 and SIRT3 in maintaining the intracellular Salmonella niche by modulating the mitochondrial bioenergetics and dynamics in the infected macrophages. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/557159v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@486cbaorg.highwire.dtl.DTLVardef@da2fb0org.highwire.dtl.DTLVardef@70cd46org.highwire.dtl.DTLVardef@1b4d0bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mammalian hibernators experience profound cold stress and prolonged physical inactivity during torpor periods; however, it is unclear how skeletal muscle stem cells (satellite cells; SCs) respond to these challenges. In this study, we demonstrate that SCs from a mammalian hibernator, Syrian hamster exhibit remarkable resistance to cold-induced cell death, which is associated with intrinsically higher expression of the antioxidant enzyme GPX4, which likely contributes to the suppression of ferroptosis. RNA-seq analysis revealed widespread downregulation of myogenesis-related genes following cold exposure, which suggests suppression of the myogenic program. Consistently, SCs exposed to cold stress exhibited reduced activation and differentiation capacities upon subsequent rewarming, with an increased number of quiescent Pax7-positive/MyoD-negative cells. Muscle regeneration was markedly delayed during hibernation, accompanied by decreased SC activation and macrophage infiltration, suggesting that cold-induced suppression of SC function underlies limited regenerative capacity in hibernating hamsters. Our results provide insight into the unique physiology of mammalian hibernators: SC viability is preserved, whereas regenerative activity is selectively suppressed during hibernation.

ObjectiveThe objective of this study was to elucidate the role of Calcium calmodulin-dependent Kinase II (CaMKII) in articular chondrocytes and its involvement in osteoarthritis (OA) pathogenesis. By performing gain and loss of function experiments, the research aimed to determine how CaMKII modulates chondrocyte metabolism, anabolic and catabolic processes, hypertrophic differentiation, and autophagy within the articular cartilage. DesignArticular cartilage was harvested from patients undergoing joint replacement surgery for OA, and adult human articular chondrocytes (AHACs) were isolated and cultured. Recombinant adenoviruses were used to overexpress a constitutively active form of CaMKII{gamma} (AdCaMKII) or inhibit CaMKII activity (AdAIP). Various assays, including RT-PCR analysis, alcian blue staining of Micromass cultures, immunofluorescence, and Western blotting, were performed to assess the effects of CaMKII modulation on chondrocyte function. ResultsOverexpression of activated CaMKII{gamma} promoted anabolism, evidenced by increased expression of SOX9, COL2A1, and ACAN, and decreased MMP-13 levels. It also enhanced proteoglycan content in AHAC micromass cultures. Furthermore, CaMKII counteracted the catabolic effects of IL-1{beta} and preserved proteoglycan content. We also observed decreased chondrocyte proliferation and increased synthesis of hypertrophic marker Type X Collagen. CaMKII activation was found to induce autophagy, as indicated by increased phosphorylation of Beclin1 and decreased p62 expression. The anabolic effects of CaMKII were dependent on autophagy, as inhibition of autophagy with Bafilomycin prevented the CaMKII-induced increase in glycosaminoglycan content. ConclusionsCaMKII plays a significant role in modulating chondrocyte metabolism and maintaining cartilage homeostasis. It promotes anabolic processes, counteracts catabolic stimuli, and induces autophagy in articular chondrocytes. However, it also promotes hypertrophic differentiation, highlighting the complexity of CaMKII-mediated signalling in cartilage. Understanding these pathways could lead to new therapeutic strategies that leverage CaMKIIs anabolic potential while mitigating its pro-degenerative effects.

Mutations of EFHC1 gene have been identified in patients with epilepsies including juvenile myoclonic epilepsy (JME), and mice with Efhc1 deficiency exhibit epileptic phenotypes. Myoclonin1 protein encoded by EFHC1 is not expressed in neurons but in cells with motile cilia including those of choroid plexus and ependymal cells which form an epithelial layer lining brain ventricles. Detailed molecular basis of epilepsies caused by EFHC1 mutations, however, remain unclear. Here we report that myoclonin1 is well co-expressed with inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) at choroid plexus and ependymal cells and these two proteins bind each other. Endoplasmic reticulum (ER) of Efhc1-deficient mouse (Efhc1-/-) cells contains larger levels of calcium ions (Ca2+) than that of wild-type (WT) mice, and IP3-induced Ca2+ release (IICR) from ER is higher in Efhc1-/- cells than that of WT. Furthermore, myoclonin1 revealed to interact with PRKCSH, also known as a protein kinase C substrate 80K-H which interacts with IP3R1. Myoclonin1 further binds to IP3R2 and IP3R3. Thus, our results indicate that myoclonin1 modulates ER-Ca2+ homeostasis through interactions with IP3Rs and PRKCSH, and suggest that myoclonin1 dysfunctions cause impaired intracellular Ca2+ mobilization. Its relevance to the epileptic phenotypes of patients with EFHC1 mutations is now of interest.

Basal expression of the P2X7 receptor (P2X7R) improves mitochondrial metabolism, ATP synthesis and overall fitness of immune and non-immune cells. We investigated P2X7R contribution to energy metabolism and subcellular localization in fibroblasts (mouse embryo fibroblasts and HEK293 human fibroblasts), mouse microglia (primary brain microglia and the N13 microglia cell line), and heart tissue. The P2X7R localizes to mitochondria, and its lack a) decreases basal respiratory rate, ATP-coupled respiration, maximal uncoupled respiration, resting mitochondrial potential, mitochondrial matrix Ca2+ level, b) modifies expression pattern of oxidative phosphorylation (OxPhos) enzymes, and c) severely affects cardiac performance. Hearts from P2rx7-deleted versus WT mice are larger, heart mitochondria smaller, and stroke volume (SV), ejection fraction (EF), fractional shortening (FS) and cardiac output (CO), are significantly decreased. Accordingly, physical fitness of P2X7R-null mice is severely reduced. Thus, the P2X7R is a key modulator of mitochondrial energy metabolism and a determinant of physical fitness.

The differentiation of skeletal muscle myoblasts is a crucial step in muscle regeneration and hypertrophy. However, the phenotypic changes that occur in myoblasts just before myotube formation have remained poorly understood. In this study, we established a novel flow cytometric method to examine these changes. We identified two distinct populations in human desmin-positive myoblasts based on immunological NCAM (neural cell adhesion molecule) staining intensity after paraformaldehyde fixation. Our results indicate that these populations correspond to differentiated and proliferating myoblast phenotypes. Further analyses revealed that antigenicity for a polysialylated-NCAM (PSA-NCAM) antibody is strongly correlated with this fixation-resistant antigenicity of NCAM, suggesting that polysialylation is associated with myogenic differentiation. Using magnetic-activated cell sorting (MACS), we separated PSA-NCAM positive and negative myoblast fractions and demonstrated that PSA-NCAM positive cells show higher differentiation marker expression and faster myotube formation. These findings suggest that PSA-NCAM can serve as a valuable surface marker to distinguish between proliferating and differentiated myoblasts. Our method provides new insights into the phenotypic heterogeneity of myoblasts and offers an approach to studying the early stages of muscle cell differentiation before myotube formation.

PurposeMechanical stress on the ocular surface, such as from eye-rubbing, has been reported to lead to inflammation and various ocular conditions. We hypothesized that the mechanosensitive Piezo1 channel in the conjunctival epithelium contributes to the inflammatory response at the ocular surface after receiving mechanical stimuli. MethodsHuman conjunctival epithelial cells (HConjECs) were treated with Yoda1, a Piezo1-specific agonist, and various allergens to measure cytokine expression levels using qRT-PCR and Western blot. Piezo1 activation-induced intracellular signaling pathways were also investigated. Mechanical stretching experiments were conducted to simulate Piezo1 activation in HConjECs. In in vivo studies, using immunohistochemistry, rats were administered Yoda1 eye drops to examine the inflammatory response in the conjunctiva and Piezo1-induced signaling activation. ResultsHConjECs expressed functional Piezo1 channel, and its activation significantly increased IL-6 and IL-8 expression through the p38 MAPK-CREB pathway. Piezo1-induced [Ca2+]i elevation was crucial for the production of IL-6. Mechanical stretching mimicked these effects. In vivo, Yoda1 administration led to enhanced immunoreactivity of phospho-p38 MAPK and phospho-CREB and increased IL-6 in the rat conjunctival epithelium. Significant neutrophil infiltration was also observed after Piezo1 channel activation without affecting eosinophil numbers. ConclusionMechanical stress-induced Piezo1 channel activation in conjunctival epithelial cells can cause ocular inflammation by upregulating pro-inflammatory cytokines via the p38 MAPK-CREB pathway and promoting neutrophil infiltration. These findings suggest that mechanical stimuli on ocular surface tissues are significant risk factors for ocular inflammation. HighlightsO_LIPiezo1 channel activation increased IL-6 via p38 MAPK-CREB pathway in Human conjunctival epithelial cells (HConjECs). C_LIO_LIPiezo1 activation mimicked fungal extract but not pollen or dust mite. C_LIO_LIMechanical stretching mimicked Piezo1 effects, boosting IL-6 production in HConjECs. C_LIO_LIPiezo1 activation elevated p-p38 MAPK, p-CREB, and IL-6 in the rat conjunctiva. C_LIO_LIPiezo1 activation leads to neutrophil infiltration in the rat conjunctival epithelium. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/600298v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1c68f87org.highwire.dtl.DTLVardef@f9226corg.highwire.dtl.DTLVardef@42f264org.highwire.dtl.DTLVardef@1378c99_HPS_FORMAT_FIGEXP M_FIG C_FIG Mechanosensitive Piezo1 channel activation induces IL-6 expression in the conjunctival epithelial cells via elevation of [Ca2+]i and activation of p38 MAPK and transcription factor CREB. Mechanical stress stimulates Piezo1 channel and Ca2+ influx through it, which induces activation of p38 MAPK. A transcription factor CREB is phosphorylated after p38 MAPK activation, then promotes IL-6 transcription.