Excessive load promotes temporomandibular joint chondrocyte apoptosis via Piezo1/endoplasmic reticulum stress pathway.

Excessive load on the temporomandibular joint (TMJ) is a significant factor in the development of TMJ osteoarthritis, contributing to cartilage degeneration. The specific mechanism through which excessive load induces TMJ osteoarthritis is not fully understood; however, mechanically-activated (MA) ion channels play a crucial role. Among these channels, Piezo1 has been identified as a mediator of chondrocyte catabolic responses and is markedly increased in osteoarthritis. Our observations indicate that, under excessive load conditions, endoplasmic reticulum stress in chondrocytes results in apoptosis of the TMJ chondrocytes. Importantly, using the Piezo1 inhibitor GsMTx4 demonstrates its potential to alleviate this condition. Furthermore, Piezo1 mediates endoplasmic reticulum stress in chondrocytes by inducing calcium ion influx. Our research substantiates the role of Piezo1 as a pivotal ion channel in mediating chondrocyte overload. It elucidates the link between excessive load, cell apoptosis, and calcium ion influx through Piezo1. The findings underscore Piezo1 as a key player in the pathogenesis of TMJ osteoarthritis, shedding light on potential therapeutic interventions for this condition.

[Research progress on the role of endothelial mechanically sensitive ion channel protein Piezo1 in diseases].

Endothelial cells have important physiological functions and regulatory effects related to the occurrence and development of various diseases. Piezo1 is a mechanically sensitive ion channel protein, which is widely distributed in various tissues of the body and participates in the occurrence and development of various diseases. Piezo1 is highly expressed in endothelial cells and plays an important regulatory role in endothelial cell function. This article reviews the structure and function of Piezo1, the physiological function and pathological damage mechanism of endothelial cells, and the role of endothelial cell Piezo1 in various diseases, in order to understand the function and regulation mechanism of endothelial cell Piezo1, and provide new targets and strategies for the treatment of related diseases.

Stiffness sensing via Piezo1 enhances macrophage efferocytosis and promotes the resolution of liver fibrosis.

Tissue stiffening is a predominant feature of fibrotic disorders, but the response of macrophages to changes in tissue stiffness and cellular context in fibrotic diseases remains unclear. Here, we found that the mechanosensitive ion channel Piezo1 was up-regulated in hepatic fibrosis. Macrophages lacking Piezo1 showed sustained inflammation and impaired spontaneous resolution of early liver fibrosis. Further analysis revealed an impairment of clearance of apoptotic cells by macrophages in the fibrotic liver. Macrophages showed enhanced efferocytosis when cultured on rigid substrates but not soft ones, suggesting stiffness-dependent efferocytosis of macrophages required Piezo1 activation. Besides, Piezo1 was involved in the efficient acidification of the engulfed cargo in the phagolysosomes and affected the subsequent expression of anti-inflammation genes after efferocytosis. Pharmacological activation of Piezo1 increased the efferocytosis capacity of macrophages and accelerated the resolution of inflammation and fibrosis. Our study supports the antifibrotic role of Piezo1-mediated mechanical sensation in liver fibrosis, suggesting that targeting PIEZO1 to enhance macrophage efferocytosis could induce fibrosis regression.

The dysadherin/FAK axis promotes individual cell migration in colon cancer.

Dysregulation of cancer cell motility is a key driver of invasion and metastasis. High dysadherin expression in cancer cells is correlated with invasion and metastasis. Here, we found the molecular mechanism by which dysadherin regulates the migration and invasion of colon cancer (CC). Comprehensive analysis using single-cell RNA sequencing data from CC patients revealed that high dysadherin expression in cells is linked to cell migration-related gene signatures. We confirmed that the deletion of dysadherin in tumor cells hindered local invasion and distant migration using in vivo tumor models. In this context, by performing cell morphological analysis, we found that aberrant cell migration resulted from impaired actin dynamics, focal adhesion turnover and protrusive structure formation upon dysadherin expression. Mechanistically, the activation of focal adhesion kinase (FAK) was observed in dysadherin-enriched cells. The dysadherin/FAK axis enhanced cell migration and invasion by activating the FAK downstream cascade, which includes the Rho family of small GTPases. Overall, this study illuminates the role of dysadherin in modulating cancer cell migration by forcing actin dynamics and protrusive structure formation via FAK signaling, indicating that targeting dysadherin may be a potential therapeutic strategy for CC patients.

The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics.

Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity.

The activation thresholds and inactivation kinetics of poking-evoked PIEZO1 and PIEZO2 currents are sensitive to subtle variations in mechanical stimulation parameters.

PIEZO1 and PIEZO2 are mechanically activated ion channels that confer mechanosensitivity to various cell types. PIEZO channels are commonly examined using the so-called poking technique, where currents are recorded in the whole-cell configuration of the patch-clamp technique, while the cell surface is mechanically stimulated with a small fire-polished patch pipette. Currently, there is no gold standard for mechanical stimulation, and therefore, stimulation protocols differ significantly between laboratories with regard to stimulation velocity, angle, and size of the stimulation probe. Here, we systematically examined the impact of variations in these three stimulation parameters on the outcomes of patch-clamp recordings of PIEZO1 and PIEZO2. We show that the inactivation kinetics of PIEZO1 and, to a lesser extent, of PIEZO2 change with the angle at which the probe that is used for mechanical stimulation is positioned and, even more prominently, with the size of its tip. Moreover, we found that the mechanical activation threshold of PIEZO2, but not PIEZO1, decreased with increasing stimulation speeds. Thus, our data show that two key outcome parameters of PIEZO-related patch-clamp studies are significantly affected by common variations in the mechanical stimulation protocols, which calls for caution when comparing data from different laboratories and highlights the need to establish a gold standard for mechanical stimulation to improve comparability and reproducibility of data obtained with the poking technique.

Ion channel Piezo1 activation aggravates the endothelial dysfunction under a high glucose environment.

BACKGROUND: Vasculopathy is the most common complication of diabetes. Endothelial cells located in the innermost layer of blood vessels are constantly affected by blood flow or vascular components; thus, their mechanosensitivity plays an important role in mediating vascular regulation. Endothelial damage, one of the main causes of hyperglycemic vascular complications, has been extensively studied. However, the role of mechanosensitive signaling in hyperglycemic endothelial damage remains unclear. METHODS: Vascular endothelial-specific Piezo1 knockout mice were generated to investigate the effects of Piezo1 on Streptozotocin-induced hyperglycemia and vascular endothelial injury. In vitro activation or knockdown of Piezo1 was performed to evaluate the effects on the proliferation, migration, and tubular function of human umbilical vein endothelial cells in high glucose. Reactive oxygen species production, mitochondrial membrane potential alternations, and oxidative stress-related products were used to assess the extent of oxidative stress damage caused by Piezo1 activation. RESULTS: Our study found that in VECreERT2;Piezo1flox/flox mice with Piezo1 conditional knockout in vascular endothelial cells, Piezo1 deficiency alleviated streptozotocin-induced hyperglycemia with reduced apoptosis and abscission of thoracic aortic endothelial cells, and decreased the inflammatory response of aortic tissue caused by high glucose. Moreover, the knockout of Piezo1 showed a thinner thoracic aortic wall, reduced tunica media damage, and increased endothelial nitric oxide synthase expression in transgenic mice, indicating the relief of endothelial damage caused by hyperglycemia. We also showed that Piezo1 activation aggravated oxidative stress injury and resulted in severe dysfunction through the Ca2+-induced CaMKII-Nrf2 axis in human umbilical vein endothelial cells. In Piezo1 conditional knockout mice, Piezo1 deficiency partially restored superoxide dismutase activity and reduced malondialdehyde content in the thoracic aorta. Mechanistically, Piezo1 deficiency decreased CaMKII phosphorylation and restored the expression of Nrf2 and its downstream molecules HO-1 and NQO1. CONCLUSION: In summary, our study revealed that Piezo1 is involved in high glucose-induced oxidative stress injury and aggravated endothelial dysfunction, which have great significance for alleviating endothelial damage caused by hyperglycemia.

Relationship between ion currents and membrane capacitance in canine ventricular myocytes.

Current density, the membrane current value divided by membrane capacitance (Cm), is widely used in cellular electrophysiology. Comparing current densities obtained in different cell populations assume that Cm and ion current magnitudes are linearly related, however data is scarce about this in cardiomyocytes. Therefore, we statistically analyzed the distributions, and the relationship between parameters of canine cardiac ion currents and Cm, and tested if dividing original parameters with Cm had any effect. Under conventional voltage clamp conditions, correlations were high for IK1, moderate for IKr and ICa,L, while negligible for IKs. Correlation between Ito1 peak amplitude and Cm was negligible when analyzing all cells together, however, the analysis showed high correlations when cells of subepicardial, subendocardial or midmyocardial origin were analyzed separately. In action potential voltage clamp experiments IK1, IKr and ICa,L parameters showed high correlations with Cm. For INCX, INa,late and IKs there were low-to-moderate correlations between Cm and these current parameters. Dividing the original current parameters with Cm reduced both the coefficient of variation, and the deviation from normal distribution. The level of correlation between ion currents and Cm varies depending on the ion current studied. This must be considered when evaluating ion current densities in cardiac cells.

Central Channelopathies in Obesity.

As obesity has raised heightening awareness, researchers have attempted to identify potential targets that can be treated for therapeutic intervention. Focusing on the central nervous system (CNS), the key organ in maintaining energy balance, a plethora of ion channels that are expressed in the CNS have been inspected and determined through manipulation in different hypothalamic neural subpopulations for their roles in fine-tuning neuronal activity on energy state alterations, possibly acting as metabolic sensors. However, a remaining gap persists between human clinical investigations and mouse studies. Despite having delineated the pathways and mechanisms of how the mouse study-identified ion channels modulate energy homeostasis, only a few targets overlap with the obesity-related risk genes extracted from human genome-wide association studies. Here, we present the most recently discovered CNS-specific metabolism-correlated ion channels using reverse and forward genetics approaches in mice and humans, respectively, in the hope of illuminating the prospects for future therapeutic development.

Single-cell sequencing reveals the impact of endothelial cell PIEZO1 expression on thoracic aortic aneurysm.

INTRODUCTION: Thoracic aortic aneurysm (TAA) is a severe vascular disease that threatens human life, characterized by focal dilatation of the entire aortic wall, with a diameter 1.5 times larger than normal. PIEZO1, a mechanosensitive cationic channel, monitors mechanical stimulations in the environment, transduces mechanical signals into electrical signals, and converts them into biological signals to activate intracellular signaling pathways. However, the role of PIEZO1 in TAA is still unclear. METHODS: We analyzed a single-cell database to investigate the expression level of PIEZO1 in TAA. We constructed a conditional knockout mouse model of Piezo1 and used the PIEZO1 agonist Yoda1 to intervene in the TAA model mice established by co-administration of BAPN and ANG-II. Finally, we explored the effect of Yoda1 on TAA in vitro. RESULTS AND DISCUSSION: We observed decreased PIEZO1 expression in TAA at both RNA and protein levels. Single-cell sequencing identified a specific reduction in Piezo1 expression in endothelial cells. Administration of PIEZO1 agonist Yoda1 prevented the formation of TAA. In PIEZO1 endothelial cell conditional knockout mice, Yoda1 inhibited TAA formation by interfering with PIEZO1. In vivo and in vitro experiments demonstrated that the effect of Yoda1 on endothelial cells involved macrophage infiltration, extracellular matrix degradation, and neovascularization. This study highlights the role of PIEZO1 in TAA and its potential as a therapeutic target, providing opportunities for clinical translation.

Plp1-expresssing perineuronal DRG cells facilitate colonic and somatic chronic mechanical pain involving Piezo2 upregulation in DRG neurons.

Satellite glial cells (SGCs) of dorsal root ganglia (DRGs) are activated in a variety of chronic pain conditions; however, their mediation roles in pain remain elusive. Here, we take advantage of proteolipid protein (PLP)/creERT-driven recombination in the periphery mainly occurring in SGCs of DRGs to assess the role of SGCs in the regulation of chronic mechanical hypersensitivity and pain-like responses in two organs, the distal colon and hindpaw, to test generality. We show that PLP/creERT-driven hM3Dq activation increases, and PLP/creERT-driven TrkB.T1 deletion attenuates, colon and hindpaw chronic mechanical hypersensitivity, positively associating with calcitonin gene-related peptide (CGRP) expression in DRGs and phospho-cAMP response element-binding protein (CREB) expression in the dorsal horn of the spinal cord. Activation of Plp1+ DRG cells also increases the number of small DRG neurons expressing Piezo2 and acquiring mechanosensitivity and leads to peripheral organ neurogenic inflammation. These findings unravel a role and mechanism of Plp1+ cells, mainly SGCs, in the facilitation of chronic mechanical pain and suggest therapeutic targets for pain mitigation.

Neher and Sakmann: Single Ion Channels.

Most biological functions have their basis in a rapid change in cell membrane permeability. Hodgkin and Huxley showed regulation of the flow of molecules and ions between the nerve cell and its environment (Nobel Prize 1963), by recording macroscopic currents. Two Germans Neher and Sakmann showed that specific ion channels actually exist, and specific membrane proteins act as gates or agents for active transport regulating in physiological and pathological processes.

Exploring the Structural Attributes of Yoda1 for the Development of New-Generation Piezo1 Agonist Yaddle1 as a Vaccine Adjuvant Targeting Optimal T Cell Activation.

Piezo1, a mechano-activated ion channel, has wide-ranging physiological and therapeutic implications, with the ongoing development of specific agonists unveiling cellular responses to mechanical stimuli. In our study, we systematically analyzed the chemical subunits in Piezo1 protein agonist Yoda1 to comprehend the structure-activity relationship and push forward next-generation agonist development. Preliminary screening assays for Piezo1 agonism were performed using the Piezo1-mCherry-transfected HEK293A cell line, keeping Yoda1 as a positive control. We introduce a novel Piezo1 agonist Yaddle1 (34, 0.40 µM), featuring a trifluoromethyl group, with further exploration through in vitro studies and density functional theory calculations, emphasizing its tetrel interactions, to act as an ambidextrous wedge between the domains of Piezo1. In contrast to the poor solubility of the established agonist Yoda1, our results showed that the kinetic solubility of Yaddle1 (26.72 ± 1.8 µM at pH 7.4) is 10-fold better than that of Yoda1 (1.22 ± 0.11 µM at pH 7.4). Yaddle1 (34) induces Ca2+ influx in human CD4+ T cell, suggesting its potential as a vaccine adjuvant for enhanced T cell activation.

The Variety of Mechanosensitive Ion Channels in Retinal Neurons.

Alterations in intraocular and external pressure critically involve the pathogenesis of glaucoma, traumatic retinal injury (TRI), and other retinal disorders, and retinal neurons have been reported to express multiple mechanical-sensitive channels (MSCs) in recent decades. However, the role of MSCs in visual functions and pressure-related retinal conditions has been unclear. This review will focus on the variety and functional significance of the MSCs permeable to K+, Na+, and Ca2+, primarily including the big potassium channel (BK); the two-pore domain potassium channels TRAAK and TREK; Piezo; the epithelial sodium channel (ENaC); and the transient receptor potential channels vanilloid TRPV1, TRPV2, and TRPV4 in retinal photoreceptors, bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Most MSCs do not directly mediate visual signals in vertebrate retinas. On the other hand, some studies have shown that MSCs can open in physiological conditions and regulate the activities of retinal neurons. While these data reasonably predict the crossing of visual and mechanical signals, how retinal light pathways deal with endogenous and exogenous mechanical stimulation is uncertain.

Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore region.

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.

Mechanical gated ion channel Piezo1: Function, and role in macrophage inflammatory response.

Macrophages are present in many mechanically active tissues and are often subjected to varying degrees of mechanical stimulation. Macrophages play a crucial role in resisting pathogen invasion and maintaining tissue homeostasis. Piezo-type mechanosensitive channel component 1 (Piezo1) is the main cation channel involved in the rapid response to mechanical stimuli in mammals. This channel plays a crucial role in controlling blood pressure and motor performance and regulates urinary osmotic pressure and epithelial cell proliferation and division. In recent years, numerous studies have shown that in macrophages, Piezo1 not only plays a role in regulating the aforementioned physiological processes but also participates in multiple pathological processes such as inflammation and cancer. In this review, we summarize the research progress on Piezo1-mediated regulation of macrophage-mediated inflammatory responses through downstream signalling pathways and the aerobic glycolysis pathway.

Perivascular neurons instruct 3D vascular lattice formation via neurovascular contact.

The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.

The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development.

Lymphedema is a debilitating disease with no effective cure and affects an estimated 250 million individuals worldwide. Prior studies have identified mutations in piezo-type mechanosensitive ion channel component 1 (PIEZO1), angiopoietin 2 (ANGPT2), and tyrosine kinase with Ig-like and EGF-like domains 1 (TIE1) in patients with primary lymphedema. Here, we identified crosstalk between these molecules and showed that activation of the mechanosensory channel PIEZO1 in lymphatic endothelial cells (LECs) caused rapid exocytosis of the TIE ligand ANGPT2, ectodomain shedding of TIE1 by disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and increased TIE/PI3K/AKT signaling, followed by nuclear export of the transcription factor FOXO1. These data establish a functional network between lymphedema-associated genes and provide what we believe to be the first molecular mechanism bridging channel function with vascular signaling and intracellular events culminating in transcriptional regulation of genes expressed in LECs. Our study provides insights into the regulation of lymphatic function and molecular pathways involved in human disease.