Mechanically-activated Ion Channels Research Articles

PIEZO1 regulates leader cell formation and cellular coordination during collective keratinocyte migration.

The collective migration of keratinocytes during wound healing requires both the generation and transmission of mechanical forces for individual cellular locomotion and the coordination of movement across cells. Leader cells along the wound edge transmit mechanical and biochemical cues to ensuing follower cells, ensuring their coordinated direction of migration across multiple cells. Despite the observed importance of mechanical cues in leader cell formation and in controlling coordinated directionality of cell migration, the underlying biophysical mechanisms remain elusive. The mechanically-activated ion channel PIEZO1 was recently identified to play an inhibitory role during the reepithelialization of wounds. Here, through an integrative experimental and mathematical modeling approach, we elucidate PIEZO1's contributions to collective migration. Time-lapse microscopy reveals that PIEZO1 activity inhibits leader cell formation at the wound edge. To probe the relationship between PIEZO1 activity, leader cell formation and inhibition of reepithelialization, we developed an integrative 2D continuum model of wound closure that links observations at the single cell and collective cell migration scales. Through numerical simulations and subsequent experimental validation, we found that coordinated directionality plays a key role during wound closure and is inhibited by upregulated PIEZO1 activity. We propose that PIEZO1-mediated retraction suppresses leader cell formation which inhibits coordinated directionality between cells during collective migration.

Deciphering mechanically activated ion channels at the single-channel level in dorsal root ganglion neurons.

Mechanically activated (MA) ion channels confer somatosensory neurons with the ability to sense a wide range of mechanical stimuli. MA ion channel activity in somatosensory neurons is best described by the electrophysiological recordings of MA currents in cultured dorsal root ganglion (DRG) neurons. Biophysical and pharmacological characterization of DRG MA currents has guided the field in screening/confirming channel candidates that induce the currents and facilitate the mechanosensory response. But studies on DRG MA currents have relied mostly on whole-cell macroscopic current properties obtained by membrane indentation, and little is known about the underlying MA ion channels at the single-channel level. Here, by acquiring indentation-induced macroscopic currents as well as stretch-activated single-channel currents from the same cell, we associate macroscopic current properties with single-channel conductance. This analysis reveals the nature of the MA channel responsible for the ensemble response. We observe four different conductances in DRG neurons with no association with a specific type of macroscopic current. Applying this methodology to a Piezo2 expressing DRG neuronal subpopulation allows us to identify PIEZO2-dependent stretch-activated currents and conductance. Moreover, we demonstrate that upon Piezo2 deletion, the remaining macroscopic responses are predominantly mediated by three different single-channel conductances. Collectively, our data predict that at least two other MA ion channels exist in DRG neurons that remain to be discovered.

Optical control of PIEZO1 channels

PIEZO proteins are unusually large, mechanically-activated trimeric ion channels. The central pore features structural similarities with the pore of other trimeric ion channels, including purinergic P2X receptors, for which optical control of channel gating has been previously achieved with photoswitchable azobenzenes. Extension of these chemical optogenetics methods to mechanically-activated ion channels would provide tools for specific manipulation of pore activity alternative to non-specific mechanical stimulations. Here we report a light-gated mouse PIEZO1 channel, in which an azobenzene-based photoswitch covalently tethered to an engineered cysteine, Y2464C, localized at the extracellular apex of the transmembrane helix 38, rapidly triggers channel gating upon 365-nm-light irradiation. We provide evidence that this light-gated channel recapitulates mechanically-activated PIEZO1 functional properties, and show that light-induced molecular motions are similar to those evoked mechanically. These results push the limits of azobenzene-based methods to unusually large ion channels and provide a simple stimulation means to specifically interrogate PIEZO1 function.

Single-particle tracking and machine-learning classification reveals heterogeneous Piezo1 diffusion.

The mechanically-activated ion channel Piezo1 is involved in numerous physiological processes. Piezo1 is activated by diverse mechanical cues and is gated by membrane tension. The channel has been found to be mobile in the plasma membrane. We employed single particle tracking (SPT) of endogenously-expressed, tdTomato-tagged Piezo1 using Total Internal Reflection Fluorescence Microscopy in two cell types, mouse embryonic fibroblasts and liver sinusoidal endothelial cells. Application of SPT unveiled a surprising heterogeneity of Piezo1 mobility in the plasma membrane. Leveraging a machine learning technique, Piezo1 trajectories were sorted into distinct classes (“mobile,” “intermediate,” and “trapped”) by partitioning features that describe the geometric properties of a trajectory. To evaluate the effects of the plasma membrane properties on Piezo1 diffusion, we manipulated membrane composition by depleting or supplementing cholesterol or by adding margaric acid to stiffen the membrane. To address effects of channel activation on Piezo1 mobility, we treated cells with Yoda1, a Piezo1 agonist, and GsMTx-4, a channel inhibitor. We collected thousands of trajectories for each condition, and found that “mobile” Piezo1 in cells supplemented with cholesterol or margaric acid exhibited decreased mobility, whereas Piezo1 in cholesterol-depleted membranes demonstrated increased mobility, compared to their respective controls. Additionally, activation by Yoda1 increased Piezo1 mobility and inhibition by GsMTx-4 decreased Piezo1 mobility compared to their respective controls. The “mobile” trajectories were analyzed further by fitting the time-averaged mean-squared displacement as a function of lag time to a power-law model, revealing Piezo1 consistently exhibits anomalous subdiffusion. This suggests Piezo1 is not freely mobile, but that its mobility may be hindered by subcellular interactions. These studies illuminate the fundamental properties governing Piezo1 diffusion in the plasma membrane and set the stage to determine how specific cellular interactions may influence channel activity and mobility.

Piezo buffers mechanical stress via modulation of intracellular Ca2+ handling in the Drosophila heart.

Throughout its lifetime the heart is buffeted continuously by dynamic mechanical forces resulting from contraction of the heart muscle itself and fluctuations in haemodynamic load and pressure. These forces are in flux on a beat-by-beat basis, resulting from changes in posture, physical activity or emotional state, and over longer timescales due to altered physiology (e.g. pregnancy) or as a consequence of ageing or disease (e.g. hypertension). It has been known for over a century of the heart’s ability to sense differences in haemodynamic load and adjust contractile force accordingly (Frank, Z. biology, 1895, 32, 370–447; Anrep, J. Physiol., 1912, 45 (5), 307–317; Patterson and Starling, J. Physiol., 1914, 48 (5), 357–79; Starling, The law of the heart (Linacre Lecture, given at Cambridge, 1915), 1918). These adaptive behaviours are important for cardiovascular homeostasis, but the mechanism(s) underpinning them are incompletely understood. Here we present evidence that the mechanically-activated ion channel, Piezo, is an important component of the Drosophila heart’s ability to adapt to mechanical force. We find Piezo is a sarcoplasmic reticulum (SR)-resident channel and is part of a mechanism that regulates Ca2+ handling in cardiomyocytes in response to mechanical stress. Our data support a simple model in which Drosophila Piezo transduces mechanical force such as stretch into a Ca2+ signal, originating from the SR, that modulates cardiomyocyte contraction. We show that Piezo mutant hearts fail to buffer mechanical stress, have altered Ca2+ handling, become prone to arrhythmias and undergo pathological remodelling.

The Role of Mechanically-Activated Ion Channels Piezo1, Piezo2, and TRPV4 in Chondrocyte Mechanotransduction and Mechano-Therapeutics for Osteoarthritis.

Mechanical factors play critical roles in the pathogenesis of joint disorders like osteoarthritis (OA), a prevalent progressive degenerative joint disease that causes debilitating pain. Chondrocytes in the cartilage are responsible for extracellular matrix (ECM) turnover, and mechanical stimuli heavily influence cartilage maintenance, degeneration, and regeneration via mechanotransduction of chondrocytes. Thus, understanding the disease-associated mechanotransduction mechanisms can shed light on developing effective therapeutic strategies for OA through targeting mechanotransducers to halt progressive cartilage degeneration. Mechanosensitive Ca2+-permeating channels are robustly expressed in primary articular chondrocytes and trigger force-dependent cartilage remodeling and injury responses. This review discusses the current understanding of the roles of Piezo1, Piezo2, and TRPV4 mechanosensitive ion channels in cartilage health and disease with a highlight on the potential mechanotheraputic strategies to target these channels and prevent cartilage degeneration associated with OA.

Probing Piezo1 diffusion heterogeneity via single particle tracking and machine learning

The mechanically-activated ion channel Piezo1 plays diverse roles in various physiological processes, including neural stem/progenitor cell differentiation, vascular development, and keratinocyte wound healing. Piezo1 is activated by diverse mechanical cues and is thought to be gated by membrane tension. Interestingly, the channel was recently found to be mobile in the plasma membrane. However, Piezo1 subcellular diffusion regulation and how this behavior may contribute to channel function are poorly understood.

Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing

Keratinocytes, the predominant cell type of the epidermis, migrate to reinstate the epithelial barrier during wound healing. Mechanical cues are known to regulate keratinocyte re-epithelialization and wound healing however, the underlying molecular transducers and biophysical mechanisms remain elusive. Here, we show through molecular, cellular and organismal studies that the mechanically-activated ion channel PIEZO1 regulates keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls.

The science of “smell” and the noble for “hugs:” making “sense?”

David Julius and Ardem Patapoutian were jointly awarded the 2021 Nobel Prize in Physiology and Medicine for their discoveries of receptors for temperature and touch. It was a phenomenal moment for the scientific community, more for the discovery that forms the basic fabric of our everyday life—ever imagined how life would have been without feeling the aroma around? Or even the dangers of accidentally touching a heated object. Dr Julius and Dr Patapoutian, independently discovered key mechanisms of how living organisms sense heat, cold, and touch. The journey started when Dr Julius, at the University of California, San Francisco, used a key ingredient in hot chili peppers to identify a protein in nerve cells that respond to these stimuli. Using capsaicin, the pungent component of chili peppers, he provided fundamental insights into mechanisms of pain. Then using a meticulous cDNA library-based functional screening from sensory neurons to search for the gene(s) that could confer capsaicin sensitivity, Dr Julius identified for the first time a novel ion channel (now called transient receptor potential [TRP] vanilloid 1) belonging to the family of TRP ion channels associated with the pain sensitivity. A painful exercise indeed! While Prof Julius was exploring the oceans and skies to hunt for sensory pain pathways, quite independently, Dr Patapoutian of Scripps Research Institute La Jolla, California, was searching for a similar thing that seemed to bother him equally. How do we sense touches? After all, there are so many emotions packaged in this small five-letter word, touch. The mother’s touch is the first sensation that every single of us always cherishes. Dr Patapoutian research is centered around finding candidate genes in a mechanosensitive cell line that could respond to mechanical stimuli. After a thorough search, the team identified two mechanically-activated ion channels, PIEZO1 and PIEZO2, representing an entirely novel class of mechanical sensors-based ion channels. What is fascinating is the idea that discovery of the smell and touch receptor stretches far beyond just touch and temperature sensations only. Mutations in other TRP channels are involved in neurodegenerative disorders and skeletal dysplasia, while mutations in PIEZO channels help control critical functions such as respiration and blood pressure regulation. So now, the mysterious world surrounding us looks more transparent and clearer. The molecular landscape is defined with precision. We now have a chemical entity behind all these emotions and intuition. The warm hug that makes our day is now millions of ions crisscrossing the ion channels. There is a different side to this too. How will the world look if everything is defined as a chemical entity? Won’t we lose the charm? After all, so much within the subtleness remains charmful when wrapped within the veil of ignorance. Knowing too much about something steals the show.

Role of Piezo channels in group IV muscle afferent fiber mechanosensation in rats

During exercise, skeletal muscle afferentnerves are activated by both mechanical and chemical stimuli within contracting muscle, subsequently contributing to an increase in blood pressure (termed the exercise pressor reflex; EPR). Although metabolically-sensitive receptors mediating the EPR have been described, the mechano-gated channels responsible for mechanosensation in thin muscle afferents (i.e. group III and IV fibers) remain to be elucidated. Piezo channels are known to be mechanically-activated ion channels that play crucial roles in several mechanotransduction processes in various organs. Moreover, Piezo channels are expressed in sensory tissues including dorsal root ganglion associated with skeletal muscle group III and IV afferents. Evidence suggests that antagonism of Piezo channels reduces the pressor response to activation of the EPR in rats. However, whether antagonism of Piezo channels affects muscle afferent discharge during mechanical stimulation remains unknown. PURPOSE The purpose of this study was to determine the impact of GsMTx4, a selective Piezo channel inhibitor, on neuronal responses to mechanical stimulation in group IV muscle afferents. It was hypothesized that GsMTx4 reduces the neural response to mechanical stimulation in sensory afferent fibers innervating skeletal muscle. METHODS Sprague-Dawley rats were used in this experiment (8-12 wks old). Using the ex vivo extensor digitorum longus muscle-nerve preparation, the responsiveness of mechanically activated group IV fibers was assessed by obtaining single-fiber recordings. Action potentials elicited by a mechanical stimulus (0-196 mN ramped stimulus) were recorded before and 20-min after injection of either Krebs-buffer solution (control), gadolinium (a non-specific mechano-gated channel antagonist, 10 mM) or GsMTx4 (250 μM) near the receptive field. RESULTS Conduction velocities of the group IV fibers assessed ranged between 0.32 and 0.77 m/s (average 0.56±0.10 m/s, n=22). The percentage of group IV fibers unable to generate an action potential during mechanical stimulation after injection of Krebs-buffer, Gadolinium and GsMTx4 were 0% (0 out of 6 fibers), 29% (2 out of 7 fibers) and 22% (2 out of 9 fibers), respectively. Fibers whose action potentials were partially attenuated by Krebs-buffer, Gadolinium and GsMTx4 were 50% (3 out of 6 fibers), 29% (2 out of 7 fibers) and 56% (5 out of 9 fibers), respectively. Compared to control, the reduction in neural responsiveness to mechanical stimulation tended to be greater in GsMTx4 (-1±49 vs. -48±38 %, P=0.08) albeit similar to Gadolinium (-71±33%, P=0.32). CONCLUSIONS The data demonstrate, for the first time, that GsMTx4 reduces the neuronal response to mechanical stimulation in group IV muscle afferent fibers of rats. These findings suggest that Piezo channels may significantly contribute to mechanosensation in sensory afferent fibers innervating skeletal muscle.

Piezo1 activation gains traction

A major unanswered question in biology is how mechanical forces are generated, detected and transduced by cells to impact biochemical and genetic programs. Our work is aimed at uncovering the mechanical principles at play in cells and tissues using novel molecular, imaging and bioengineering tools. Here we present insights gleaned from non-invasive approaches to measure and manipulate mechanical signals in native cellular conditions. We find that the mechanically-activated ion channel Piezo1 transduces cell-generated traction forces: actomyosin-based traction forces evoke localized Piezo1 Ca2+ flickers in the absence of externally-applied mechanical forces. We examine the functional effects of cell-generated Piezo1 activity in neural stem/progenitor cells and in epithelial keratinocytes.

Disrupting ELKIN1-Dependent Mechanoelectrical Transduction Modulates Cell-Cell Interactions in Organotypic Tumour Spheroids

In the tumour microenvironment, cells within tumour masses can interact with neighbouring cells as well as the surrounding stroma. During melanoma progression, there are many changes to the mechanical properties of the cells and the surrounding tissue, and these mechanical perturbations can drive disease progression. We are studying how cells sense these mechanical changes with a focus on mechanoelectrical transduction: the channel-mediated conversion of mechanical inputs into an electrical signal. Mechanoelectrical transduction currents can be evoked in melanoma cells and in the secondary melanoma cell line, WM266-4, the ablation of TMEM87a/ELKIN1 led to a reduction in mechanically activated (MA) ion channel activity and the modulation of cell migration. To investigate how ELKIN1 impacts cell-cell interactions, we visualised cell patterning and dissociation using organotypic spheroids to mimic a tumour mass. in ELKIN1 deletion mutants, there is a significant increase in cell dissociation from the spheroids, suggesting that ELKIN1 plays a role in modulating cell-cell interactions. in chimeric spheroids formed from WT and ELKIN1-KO cells, we observed that the WT cells sorted to the core of the spheroid, whereas ELKIN1-KO cells sorted to the exterior of the spheroid. These functional changes were accompanied by changes to cell-cell and cell-substrate attachment properties. These data highlight that ELKIN1-dependent mechanoelectrical transduction influences cell-cell and cell-substrate interactions. in contrast, disruption of the expression of PIEZO1 (a well-characterised MA ion channel) did not exhibit similar effects.

Molecular Dynamics Simulations Studies of the Proton Channel Otopetrin and Other Mechanically-Activated Ion Channels

Molecular Dynamics Simulations Studies of the Proton Channel Otopetrin and Other Mechanically-Activated Ion Channels

Heterozygous Variants in the Mechanosensitive Ion Channel TMEM63A Result in Transient Hypomyelination during Infancy

Mechanically activated (MA) ion channels convert physical forces into electrical signals. Despite the importance of this function, the involvement of mechanosensitive ion channels in human disease is poorly understood. Here we report heterozygous missense mutations in the gene encoding the MA ion channel TMEM63A that result in an infantile disorder resembling a hypomyelinating leukodystrophy. Four unrelated individuals presented with congenital nystagmus, motor delay, and deficient myelination on serial scans in infancy, prompting the diagnosis of Pelizaeus-Merzbacher (like) disease. Genomic sequencing revealed that all four individuals carry heterozygous missense variants in the pore-forming domain of TMEM63A. These variants were confirmed to have arisen de novo in three of the four individuals. While the physiological role of TMEM63A is incompletely understood, it is highly expressed in oligodendrocytes and it has recently been shown to be a MA ion channel. Using patch clamp electrophysiology, we demonstrated that each of the modeled variants result in strongly attenuated stretch-activated currents when expressed in naive cells. Unexpectedly, the clinical evolution of all four individuals has been surprisingly favorable, with substantial improvements in neurological signs and developmental progression. In the three individuals with follow-up scans after 4 years of age, the myelin deficit had almost completely resolved. Our results suggest a previously unappreciated role for mechanosensitive ion channels in myelin development.

Mechanotransduction pathways and relevance to human OA

Purpose: We are investigating mechanoelectrical transduction (the conversion of a mechanical stimulus into an electrochemical signal) in chondrocytes. The force sensing proteins that mediate this conversion are mechanically-activated (MA) ion channels. The purpose of this study was to determine if we could directly measure MA ion channel activity in chondrocytes and to investigate how changing the mechanics of the cellular substrate impacts MA channel activity. Methods: In order to study MA channel activity a relevant mechanical stimulus must be applied to a cell whilst simultaneously monitoring the resulting flux of ions across the membrane. The gold standard for monitoring ion channel activity is to use electrophysiological techniques. We have isolated primary murine chondrocytes and used high speed pressure clamp to stretch a patch of the cell membrane. In addition, we have developed a specialized technique (using elastomeric pillar arrays) to apply deflections at contact points between the cell and the underlying substrate. Results: We found that the MA ion channels PIEZO1 and TRPV4 are both activated by substrate deflections, but only PIEZO1 is activated by membrane stretch. Given the changes in the mechanical properties of cartilage that accompany the development of osteoarthritis, we then investigated the effect of modulating substrate mechanics on TRPV4 and PIEZO1 activation. Conclusions: Our data suggest that MA channels may enable chondrocytes to integrate distinct mechanical inputs. In addition we predict that changes in tissue mechanics alone will disrupt signalling via PIEZO1 and TRPV4 in response to changing mechanical loads.

A hydrophobic gate in the inner pore helix is the major determinant of inactivation in mechanosensitive Piezo channels.

Piezo1 and Piezo2 belong to a family of mechanically-activated ion channels implicated in a wide range of physiological processes. Mechanical stimulation triggers Piezo channels to open, but their characteristic fast inactivation process results in rapid closure. Several disease-causing mutations in Piezo1 alter the rate of inactivation, highlighting the importance of inactivation to the normal function of this channel. However, despite the structural identification of two physical constrictions within the closed pore, the mechanism of inactivation remains unknown. Here we identify a functionally conserved inactivation gate in the pore-lining inner helix of mouse Piezo1 and Piezo2 that is distinct from the two constrictions. We show that this gate controls the majority of Piezo1 inactivation via a hydrophobic mechanism and that one of the physical constrictions acts as a secondary gate. Our results suggest that, unlike other rapidly inactivating ion channels, a hydrophobic barrier gives rise to fast inactivation in Piezo channels.

OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels.

Mechanically activated (MA) ion channels convert physical forces into electrical signals, and are essential for eukaryotic physiology. Despite their importance, few bona-fide MA channels have been described in plants and animals. Here, we show that various members of the OSCA and TMEM63 family of proteins from plants, flies, and mammals confer mechanosensitivity to naïve cells. We conclusively demonstrate that OSCA1.2, one of the Arabidopsis thaliana OSCA proteins, is an inherently mechanosensitive, pore-forming ion channel. Our results suggest that OSCA/TMEM63 proteins are the largest family of MA ion channels identified, and are conserved across eukaryotes. Our findings will enable studies to gain deep insight into molecular mechanisms of MA channel gating, and will facilitate a better understanding of mechanosensory processes in vivo across plants and animals.

The Flow Cytometric Osmotic Fragility Test Is an Effective Screening Method for Dehydrated Hereditary Stomatocytosis

Introduction: Dehydrated hereditary stomatocytosis (DHSt) or hereditary xerocytosis (HX) is a form of congenital hemolytic anemia characterized by red blood cell (RBC) dehydration. Heterozygous mutations in PIEZO1, a mechanically-activated ion channel, cause DHSt. Recently, KCNN4, which encodes the Gardos channel, has been found to be the second pathogenic gene for DHSt. DHSt is characterized by an alteration in the RBC morphology in target cells, stomatocytes, and/or echinocytes, and RBC deformability assessments by ektacytometry as well as RBC ion flux measurements are currently the standard laboratory tests for DHSt, but their use in laboratories is limited. The flow cytometric osmotic fragility (FCM-OF) test is a useful diagnostic test for hereditary spherocytosis (HS) and also for hereditary elliptocytosis (HE). In this study, we showed that the FCM-OF test could also successfully diagnose DHSt.Subjects: A total of 46 cases of RBC membrane disorders were examined, and tentative diagnoses were made based on the RBC morphology, acid glycerol lysis time, and eosin 5'-maleimide binding tests, resulting in HS (n=31), HE (n=6), and DHSt (n=9).Methods: The number of RBCs in isotonic and hypotonic buffers were measured by flow cytometry. The degree of osmotic fragility was expressed as the “percentage residual RBCs (%RRC)”. We confirmed the DHSt diagnosis by the massively paralleled sequencing using our custom panels targeting 68 hemolytic anemia-related genes with the next-generation sequencer.Results: Both HS and HE patients showed a decrease in %RRC; HS (18.0±8.9%, p<0.001) and HE (41.8±15.7%, p<0.001) compared to normal control (66.7±1.5%). DHSt patients showed a significant increase (112.6±34.5%, p<0.001) in FCM-OF. Additionally, next-generation sequencing revealed consistent causative gene mutations for DHSt; PIEZO1 (p.R2488Q and p.E2496ELE) or KCNN4 (p.P204R, p.A279T and p.R352H).Discussion: We examined 77 patients with congenital hemolytic anemia recently, and 59 cases were confirmed by diagnostic tests (76.6%). The results were as follows: 48 cases of RBC membrane abnormality (62.3%), 6 cases of RBC enzymopathy (7.8%), and 5 cases of hemoglobinopathy (6.5%). Of the cases of RBC membrane disorders, 31 cases of HS, 9 cases of DHSt, and 8 cases of HE were identified. These observations suggest that DHSt is the second-most common RBC membranopathy in Japan, and that the FCM-OF test and targeted sequencing efficiently discriminate DHSt from other RBC membrane disorders. DisclosuresNo relevant conflicts of interest to declare.

The Function of the Novel Mechanical Activated Ion Channel Piezo1 in the Human Osteosarcoma Cells

BackgroundThe Piezo1 protein ion channel is a novel mechanical activated ion channel which is related to mechanical signal transduction. However, the function of the mechanically activated ion channel Piezo1 had not been explored. In this study, we explored the function of the Piezo1 ion channel in human osteosarcoma (OS) cells related to apoptosis, invasion, and the cell proliferation.Material/MethodsReverse transcription polymerase chain reaction (RT-PCR) and western-blotting were used to detect the expression of the Piezo1 protein. CCK-8, Transwell experiments and AV-PI were used to detected cell proliferation, cell invasion and cell apoptosis.ResultsThe Piezo1 protein ion channel was highly expressed in human OS cells. The Piezo1-shRNA inhibited the expression of the Piezo1. We explored whether LV3-PIEZO1-homo-3201 could act as Piezo1-shRNA, which could then be an inhibitor of Piezo1. The expression of Piezo1 in the 2-hour stretch group were slightly higher than the 0-hour stretch group, and the difference was not statistically significant (n=3, p>0.05, one-way ANOVA). The apoptotic gene such as the Bax, BAD, caspase-3, and caspase-9 had the same characteristics as the Piezo1 expression under the stretch force. We also explored the invasion of Piezo1 in vivo using nude mice, and found that Piezo1-shRNA could inhibit the invasion of the OS cells.ConclusionsThe Piezo1 protein may be a novel, potential therapeutic target for OS.

Stretch‐activation of rat cardiac myocytes

Mechanically activated ion channels (MACs) in rat ventricular myocytes were activated by stretching membrane patches attached to microelectrodes. Charge injection from MACs, consisting mainly of inward K+, was sometimes of sufficient magnitude (0.2 pC) during brief (50 ms) periods, to trigger action currents. Action currents were shown to be extracellular records of action potentials. These results demonstrate the activation of myocytes by injection of approximately 4 pA from a few MACs in a membrane patch.