Piezo1 Activation Research Articles

Alteration of Piezo1 signaling in type 2 diabetic mice: Focus on endothelium and BKca channel

Aim: Piezo1, a mechanosensitive ion channel, plays a significant role in vascular physiology and disease. This study aimed to elucidate the altered signaling elicited by Piezo1 activation in the arteries of type 2 diabetes. Methods: Ten-to 12-week-old male C57BL/6 (control) and type 2 diabetic mice (db−/db−) were used. The second-order mesenteric arteries (~150 mm) were used for isometric tension experiments. Western blot analysis and immunofluorescence staining were performed to observe protein expression. Results: Piezo1 was significantly decreased in mesenteric arteries of type 2 diabetic mice compared to control mice, as analyzed by western blot and immunofluorescence staining. Piezo1 agonist, Yoda1, concentration-dependently induced relaxation of mesenteric arteries in both groups. Interestingly, the relaxation response was significantly greater in control mice than in db−/db− mice. The removal of endothelium reduced relaxation responses induced by Yoda1, which was greater in control mice than db−/db− mice. Furthermore, the relaxation response was reduced by pre-treatment with various types of K+ channel blockers in endothelium-intact arteries in control mice. In endothelium-denuded arteries, pre-incubation with charybdotoxin, an Ca2+-activated K+ channel (BKCa channel) blocker, significantly attenuated Yoda1-induced relaxation in db−/db− mice, while there was no effect in control mice. Co-immunofluorescence staining showed more structural coupling between Piezo1 and BKCa channel was observed in db−/db− mice than in control mice. Conclusions: The present study is the first report on the distinct aspects of vascular response induced by Piezo1 activation in the mesenteric resistance arteries in type 2 diabetic mice. National Research Foundation of Korea (RS-2023-00272628). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Piezo1 activity is regulated independently in principal and intercalated cells of the renal collecting duct

Water-electrolyte transport in the distal segments of the renal tubule, including the collecting duct (CD), is regulated by mechanical forces arising from changes in fluid flow and osmolarity. Such mechanical stress is known to directly activate Ca2+-permeable Piezo1 channel triggering various downstream effectors under different physiological conditions. However, the functional significance of Piezo1 in morphologically and physiologically distinct principal (PC) and intercalated (IC) cells of the CD is yet to be determined. Here, we implemented electrophysiological and imaging tools to investigate pharmacological Piezo1 activation in PC and IC of freshly isolated intact and split-opened CDs. For this purpose, we applied a variety of systemic treatments, including water restrictions, diuretic injections and dietary manipulations. We observed significantly greater intracellular Ca2+ ([Ca2+]i) signal in PC (AQP2-positive cells) than in IC (AQP2-negative) in freshly isolated split-opened CDs upon application of Piezo1 selective agonists, Yoda-1 and Jedi-2. Patch-clamp analysis in cell-attached configuration allowed us recording of a Yoda-1-activated non-selective channel with 18.6±0.7 pS conductance on both apical and basolateral membranes. These results are in a good agreement with immunofluorescent detection of Piezo1 at both apical and basolateral sides in renal sections and isolated split-opened collecting ducts. Acute stimulation of flow with furosemide injections (2 mg/kgBW, 18h and 2h prior to experimentation) stimulated the elevation of Yoda-1-induced Piezo1-dependent [Ca2+]i elevation in PCs, but not in ICs of split-opened collecting ducts. However, prolonged increased flow in the collecting duct by high K+ diet (for 1 week) decreased Yoda-1-dependent Ca2+ influx in the absence of apparent changes in Piezo1 levels pointing to its inactivation. This led to notable flattening of CD cells, suggesting cytoskeleton reorganization due to increased perfusion pressure existing during high K+ intake. At the same time, anti-diuresis with water restrictions led to reduced Piezo1-dependent [Ca2+]i elevation in PCs of split-opened CDs. Consistently, induction of metabolic acidosis with NH4Cl−supplemented water Piezo1 activity in IC, but not in PC. In summary, our results directly demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1 function in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-23POST1020372 (to K. Pyrshev). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

EGFR-mediated crosstalk between vascular endothelial cells and hepatocytes promotes Piezo1-dependent liver regeneration

Hepatocyte proliferation is essential for recovering liver function after injury. In liver surgery, the mechanical stimulation induced by hemodynamic changes triggers vascular endothelial cells (VECs) to secrete large amounts of cytokines that enhance hepatocyte proliferation and play a pivotal role in liver regeneration. Piezo1, a critical mechanosensory ion channel, can detect and convert mechanical forces into chemical signals, importing external stimuli into cells and triggering downstream biological effects. However, the precise role of Piezo1 in VECs, especially in terms of mediating liver regeneration, remains unclear. Here, we report on a potential mechanism by which early changes in hepatic portal hemodynamics activate Piezo1 in VECs to promote hepatocyte proliferation during the process of liver regeneration induced by portal vein ligation in rats. In this liver regeneration model, hepatocyte proliferation is mainly distributed in zone 1 and zone 2 of liver lobules at 24–48 h after surgery, while only a small number of Ki67-positive hepatocytes were observed in zone 3. Activation of Piezo1 promotes increased secretion of epiregulin and amphiregulin from VECs via the PKC/ERK1/2 axis, further activating epidermal growth factor receptor (EGFR) and ERK1/2 signals in hepatocytes and promoting proliferation. In addition, cytokines secreted by Piezo1-activated VECs can induce hepatocytes to undergo epithelial–mesenchymal transition. In the liver lobules, the expression of EGFR in hepatocytes of zone 1 and zone 2 is significantly higher than that in zone 3. The EGFR inhibitor gefitinib inhibits liver regeneration by suppressing the proliferation of hepatocytes in the middle zone. Thus, activation of Piezo1 in VECs promotes hepatocyte proliferation, suggesting mechanical stimulation regulates hepatocyte proliferation in zone 1 and zone 2 during portal vein ligation-induced liver regeneration. These data provide a theoretical basis for the regulation of liver regeneration through chemical signals mediated by mechanical stimulation.

AKAP150 and Caveolin 1 Facilitate the Formation of Distinct TRPV4 Signaling Microdomains in Vascular Smooth Muscle Cells

Ca2+ influx through transient receptor potential vanilloid 4 (TRPV4) channel is a well-established pathway in vascular smooth muscle cells (SMCs). We recently discovered two separate SMC TRPV4 (TRPV4SMC) channel subpopulations: constrictor TRPV4SMC channels that are activated by α1 adrenergic receptors (a1AR) through protein kinase C (PKC), and dilator TRPV4SMC channels that are activated by intraluminal pressure and couple with Ca2+-activated K+ (BK) channels. However, the underlying mechanism for the formation of these TRPV4SMC signaling microdomains is unclear. AKAP150 (A kinase anchoring protein 150), a PKC-anchoring protein, is essential for PKC activation of TRPV4SMC channels, and Caveolin-1 (Cav1) has been linked to enhanced activity of TRPV4 and BK channels. We hypothesized that distinct anchoring proteins facilitate the formation of spatially separated TRPV4SMC signaling microdomains. Therefore, we tested the possibility that AKAP150 mediates α1AR-induced activation of constrictor TRPV4SMC channels, and Cav1 provides a signaling scaffold for the intraluminal pressure-activated TRPV4SMC-BK channel signaling. Radiotelemetric recordings showed lower resting blood pressure in tamoxifen-inducible, SMC-specific AKAP150KO (AKAP150SMCKO) mice, suggesting that AKAP150SMC increases the resting blood pressure. On the contrary, Cav1SMCKO mice showed elevated resting blood pressure, suggesting that Cav1SMC decreases blood pressure. Furthermore, phenylephrine (PE, a1AR agonist)-induced TRPV4SMC currents and increase in blood pressure (1 mg/kg PE, intraperitoneal), and nerve stimulation-induced (electrical pulses; 50–150 V, 0.25-ms pulse, 15 Hz) TRPV4SMC channel activity were absent in AKAP150SMCKO mice, but not in Cav1SMCKO mice. TRPV4 channel-activated BK current was abrogated in SMCs from Cav1SMCKO mice, but not in SMCs from AKAP150SMCKO mice. Together, these data supported the concept that AKAP150 and Cav1 are required for the formation of constrictor and dilator TRPV4SMC signaling microdomains, respectively. Studies in other cell types have shown that mechanosensitive Piezo1 channels can increase the activity of TRPV4 channels. Therefore, we determined whether Piezo1 is required for intraluminal pressure- activation of the dilator TRPV4SMC channels. We postulated that intraluminal-pressure activates Cav1-scaffolded Piezo1SMC-TRPV4SMC-BK channel signaling. Piezo1SMCKO mice showed a loss of intraluminal pressure-induced TRPV4SMC channel activity and increased myogenic constriction in MAs. Importantly, Yoda1 (Piezo1 activator)-induced increase in BK current was absent in SMCs from Cav1SMCKO, TRPV4SMCKO, and Piezo1SMCKO mice, but not AKAP150SMCKO mice. Together, our results suggest that AKAP150SMC and Cav1SMC facilitate constrictor α1AR-PKC-TRPV4SMC and dilator Piezo1SMC-TRPV4SMC-BK channel signaling, respectively. Collectively, our data identify the anchoring proteins involved in the formation of distinct Ca2+ signaling microdomains with opposite effects on blood pressure. This work was supported by funding from the American Heart Association to YLC (POST833691), the National Institutes of Health to SKS (HL146914, HL142808, and HL147555). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1138: Impaired Flow-induced Vasodilation In Arterioles By Cholesterol-induced Suppression Of Endothelial Kir2.1

Dyslipidemia is a critical factor in endothelial dysfunction; however, the mechanisms are still not well understood. Our studies showed that cholesterol-induced suppression of endothelial flow-sensitive Kir2.1 channels contributes significantly to the impairment of flow-induced vasodilation (FIV) in hypercholesterolemic ApoE -/- mice. Notably, a CRISPR mouse generated to substitute Kir2.1 with its cholesterol-insensitive mutant via a single-point mutation results in a full rescue of FIV under hypercholesterolemia. To extend our observations to humans, 16 healthy subjects were recruited with LDL-cholesterol ranging from 51-153 mg/dl, and FIV was assessed in resistance arterioles isolated from gluteal adipose. Kir2.1-dependent FIV was assessed by transducing intact resistance arteries with the adenoviral construct expressing dominant-negative Kir2.1 (dn-Kir2.1) driven by an endothelial-specific ( Cdh5 ) promoter. Our results show that Kir2.1-dependent FIV of resistance arteries contributed ~50% of the vasodilatory response in participants with normal/low LDL levels (<100 mg/dL) and was sharply reduced in participants with LDL levels >100 mg/dL. A significant negative correlation was observed between FIV and LDL, which was accounted for by the loss of Kir2.1-dependent FIV component. Furthermore, impaired FIV in in arteries from participants with high-LDL was fully rescued by overexpressing WT-Kir2.1 in the endothelium. These data suggest that cholesterol-induced suppression of Kir2.1 is a major mechanism underlying endothelial dysfunction in hypercholesterolemia. Mechanistically, we showed previously that Kir2.1 is required for the flow-activation of Akt1/eNOS. Our new data show that Kir2.1 is also required for the upstream event of flow-induced activation of Piezo1, PECAM1, PI3K and that transducing the arteries with endothelial specific construct of Akt1 stably located at the membrane (myrAkt1) eliminates the dependence of FIV on Kir2.1. Based on our observation, we conclude that Kir2.1 is required for the translocation of Akt1 to the membrane, and the cholesterol suppression of Kir2.1 interferes Akt1-dependent flow-induced NO production.

Activation of Piezo-1 Prevents Human Microvascular Endothelial Dysfunction During Acute Inhibition of Ceramide Formation

Despite the well-known damaging effects of elevated intracellular ceramides on human microvascular function, our recent work has shown that inhibition of ceramide formation through neutral sphingomyelinase (NSmase) promotes endothelial dysfunction in arterioles from healthy adults, manifested as endothelial production of hydrogen peroxide (H2O2), rather than nitric oxide (NO), in response to flow (flow-induced dilation; FID). Recent evidence suggests that ceramide prevents inactivation of Piezo-1, a shear-sensitive Ca2+ channel and inhibition of Piezo-1 reduces endothelial nitric oxide synthase (eNOS) activation in response to flow. Together, we hypothesized that ceramide-induced Piezo-1 activation is required to maintain NO-mediated FID in human microvessels and thus agonism of Piezo-1 during NSmase inhibition will prevent endothelial dysfunction. Arterioles (100-250μm) from healthy adults were dissected from discarded surgical adipose tissue. Vessels were cannulated in organ chambers, pre-constricted with endothelin-1, and changes in internal diameter in response to increases in flow were measured with videomicroscopy. Peroxy yellow 1 (PY1) fluorescent probe was used to assess endothelial H2O2 production in human umbilical vein endothelial cells (HUVECs). Mean% maximal dilation or fluorescent intensity±SE with two-way repeated measures ANOVA is reported. FID was preserved with Piezo-1 agonism (Yoda-1, 1μM, 30min) during NSmase inhibition (GW4869 4μM, 30min; 73±16.8, n=4 vs control 73±8.2, n=9; p>0.05), however FID was diminished in the presence of the eNOS inhibitor L-NAME (100μM; -2.6±11.3, n=5; p<0.05). A similar switch to NO-mediated FID was seen with C2-ceramide (5μM)+GW4869 (70.1±9.7 vs +L-NAME -3±9.7, n=4 both; p<0.05). NSmase inhibition increased H2O2 production in HUVECs (5.2±7.4 vs 83.0±31.6, n=6; p<0.05), an effect that was diminished with Yoda-1 (5.1±7.5, n=4; p<0.05) and C2-ceramide (10.7±9.2, n=5; p<0.05). These data suggest a role for Piezo-1 in NSmase-induced NO-signaling and provide mechanistic insight into how ceramide may exert beneficial effects within the human microvasculature. This research is supported by the National Institute of Health (NHLBI) R01HL160752 (JKF) and the American Heart Association (AHA) Predoctoral Fellowship 909315 (GSK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Urothelial Piezo1 channels do not alter voiding or afferent nerve sensitivity in the mouse

The mechanosensitive piezo ion channels, Piezo1 and Piezo2, are expressed in tissues of the lower urinary tract, including the urothelium and sensory nerves. Interest in a role of piezo mechanosensitive ion channels in mediating sensory physiology in the lower urinary tract has been highlighted by the recent reports of putative voiding dysfunction in mice lacking Piezo2 channels in the lower extremities and sensory nerves (Marshall et al 2020) and Piezo1/2 in urothelium (Dalghi et al 2022). To investigate the potential role of urothelial Piezo1 channels in mediating normal function in the lower urinary tract, we generated a mouse line in which the Piezo1 gene can be conditionally knocked out in the urothelium (Piezo1UPKcKO mice). We examined spontaneous voiding behavior in male and female Piezo1UPKcKO and control mice during the light and dark phases of the day/night cycle. There were no gender differences in voiding parameters in our study (p=0.659, 3-way ANOVA), thus we did not isolate gender as a variable for further analysis. Piezo1UPKcKO mice (n=9) voided 6 ± 1 times per 24 hours, while the control mice (n=11) voided 7 ± 1 times per 24 hours (p = 0.7281). A clear nocturnal voiding pattern was observed with both Piezo1UPKcKO and control mice exhibiting more frequent voids during the dark cycle, but there were no differences in the number of voids, intermicturition intervals, or void volumes in any cycle between Piezo1UPKcKO and control mice. These results indicate that spontaneous voiding behavior remains fully intact in both male and female Piezo1UPKcKO mice during the light and dark phases of the day/night cycle. Furthermore, in an ex vivo bladder model where we measure afferent nerve output from the bladder during filling cycles, sensory nerve activity in the Piezo1UPKcKO mice was similar to that in bladders from control animals. The maximum frequency of afferent nerve activity during bladder filling reached 295 ± 28 Hz in control mice (n=6) and 336 ± 34 Hz in Piezo1UPKcKO mice (n=6; p = 0.368). Functional Piezo1 activity could be demonstrated in bladders from both Piezo1UPKcKO and control mice, as the pharmacological activator for Piezo1 channels, Yoda1 (5 μM) increased maximum afferent nerve activity during bladder filling by 60 ± 15 Hz in control mice (P = 0.0029) and 33 ± 15 Hz in Piezo1UPKcKO mice (P = 0.0567). In Ca2+ imaging experiments, we found that Yoda1 induced an increase in Ca2+ signaling events in urothelial tissue from control mice. Thus, it appears that urothelial cells express functional Piezo1 ion channels, but activity of these channels is not required for maintaining normal sensory nerve activity and voiding behavior. This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK125543). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of Piezo1 mechanotransduction in high-grade serous ovarian cancer: Insights from an in vitro model of collective detachment.

Slowing peritoneal spread in high-grade serous ovarian cancer (HGSOC) would improve patient prognosis and quality of life. HGSOC spreads when single cells and spheroids detach, float through the peritoneal fluid and take over new sites, with spheroids thought to be more aggressive than single cells. Using our in vitro model of spheroid collective detachment, we determine that increased substrate stiffness led to the detachment of more spheroids. We identified a mechanism where Piezo1 activity increased MMP-1/MMP-10, decreased collagen I and fibronectin, and increased spheroid detachment. Piezo1 expression was confirmed in omental masses from patients with stage III/IV HGSOC. Using OV90 and CRISPR-modified PIEZO1-/- OV90 in a mouse xenograft model, we determined that while both genotypes efficiently took over the omentum, loss of Piezo1 significantly decreased ascitic volume, tumor spheroids in the ascites, and the number of macroscopic tumors in the mesentery. These results support that slowing collective detachment may benefit patients and identify Piezo1 as a potential therapeutic target.

Mechano-sensor Piezo1 inhibits glucagon production in pancreatic α-cells

ObjectiveGlucagon is a critical hormone regulating glucose metabolism. It stimulates the liver to release glucose under low blood sugar conditions, thereby maintaining blood glucose stability. Excessive glucagon secretion and hyperglycemia is observed in individuals with diabetes. Precise modulation of glucagon is significant to maintain glucose homeostasis. Piezo1 is a mechanosensitive ion channel capable of converting extracellular mechanical forces into intracellular signals, thus regulating hormonal synthesis and secretion. This study aims to investigate the role of Piezo1 in regulating glucagon production in α cells. MethodsThe effects of Piezo1 on glucagon production were examined in normal- or high-fat diet fed α cell-specific Piezo1 knockout mice (Gcg-Piezo1−/−), and the murine pancreatic α cell line αTC1–6. Expression of Proglucagon was investigated by real-time PCR and western blotting. Plasma glucagon and insulin were detected by enzyme immunoassay. ResultsUnder both normal- and high-fat diet conditions, Gcg-Piezo1−/− mice exhibited increased pancreatic α cell proportion, hyperglucagonemia, impaired glucose tolerance, and activated pancreatic mTORC1 signaling. Activation of Piezo1 by its agonist Yoda1 or overexpression of Piezo1 led to decreased glucagon synthesis and suppressed mTOR signaling pathway in αTC1–6 cells. Additionally, the levels of glucagon in the medium were also reduced. Conversely, knockdown of Piezo1 produced opposite effects. ConclusionOur study uncovers the regulatory role of the Piezo1 ion channel in α cells. Piezo1 influences glucagon production by affecting mTOR signaling pathway.

Low-intensity pulsed ultrasound promotes the osteogenesis of mechanical force-treated periodontal ligament cells via Piezo1.

Low-intensity pulsed ultrasound (LIPUS) can accelerate tooth movement and preserve tooth and bone integrity during orthodontic treatment. However, the mechanisms by which LIPUS affects tissue remodeling during orthodontic tooth movement (OTM) remain unclear. Periodontal ligament cells (PDLCs) are pivotal in maintaining periodontal tissue equilibrium when subjected to mechanical stimuli. One notable mechano-sensitive ion channel, Piezo1, can modulate cellular function in response to mechanical cues. This study aimed to elucidate the involvement of Piezo1 in the osteogenic response of force-treated PDLCs when stimulated by LIPUS. After establishing rat OTM models, LIPUS was used to stimulate rats locally. OTM distance and alveolar bone density were assessed using micro-computed tomography, and histological analyses included hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining and immunohistochemical staining. GsMTx4 and Yoda1 were respectively utilized for Piezo1 functional inhibition and activation experiments in rats. We isolated human PDLCs (hPDLCs) in vitro and evaluated the effects of LIPUS on the osteogenic differentiation of force-treated hPDLCs using real-time quantitative PCR, Western blot, alkaline phosphatase and alizarin red staining. Small interfering RNA and Yoda1 were employed to validate the role of Piezo1 in this process. LIPUS promoted osteoclast differentiation and accelerated OTM in rats. Furthermore, LIPUS alleviated alveolar bone resorption under pressure and enhanced osteogenesis of force-treated PDLCs both in vivo and in vitro by downregulating Piezo1 expression. Subsequent administration of GsMTx4 in rats and siPIEZO1 transfection in hPDLCs attenuated the inhibitory effect on osteogenic differentiation under pressure, whereas LIPUS efficacy was partially mitigated. Yoda1 treatment inhibited osteogenic differentiation of hPDLCs, resulting in reduced expression of Collagen Ⅰα1 and osteocalcin in the periodontal ligament. However, LIPUS administration was able to counteract these effects. This research unveils that LIPUS promotes the osteogenesis of force-treated PDLCs via downregulating Piezo1.

The mechanisms of exercise improving cardiovascular function by stimulating Piezo1 and TRP ion channels: a systemic review.

Mechanosensitive ion channels are widely distributed in the heart, lung, bladder and other tissues, and plays an important role in exercise-induced cardiovascular function promotion. By reviewing the PubMed databases, the results were summarized using the terms "Exercise/Sport", "Piezo1", "Transient receptor potential (TRP)" and "Cardiovascular" as the keywords, 124-related papers screened were sorted and reviewed. The results showed that: (1) Piezo1 and TRP channels play an important role in regulating blood pressure and the development of cardiovascular diseases such as atherosclerosis, myocardial infarction, and cardiac fibrosis; (2) Exercise promotes cardiac health, inhibits the development of pathological heart to heart failure, regulating the changes in the characterization of Piezo1 and TRP channels; (3) Piezo1 activates downstream signaling pathways with very broad pathways, such as AKT/eNOS, NF-κB, p38MAPK and HIPPO-YAP signaling pathways. Piezo1 and Irisin regulate nuclear localization of YAP and are hypothesized to act synergistically to regulate tissue mechanical properties of the cardiovascular system and (4) The cardioprotective effects of exercise through the TRP family are mostly accomplished through Ca2+ and involve many signaling pathways. TRP channels exert their important cardioprotective effects by reducing the TRPC3-Nox2 complex and mediating Irisin-induced Ca2+ influx through TRPV4. It is proposed that exercise stimulates the mechanosensitive cation channel Piezo1 and TRP channels, which exerts cardioprotective effects. The activation of Piezo1 and TRP channels and their downstream targets to exert cardioprotective function by exercise may provide a theoretical basis for the prevention of cardiovascular diseases and the rehabilitation of clinical patients.

The role of the Piezo1 channel in osteoblasts under cyclic stretching: A study on osteogenic and osteoclast factors

ObjectivesOrthodontic tooth movement is a mechanobiological reaction induced by appropriate forces, including bone remodeling. The mechanosensitive Piezo channels have been shown to contribute to bone remodeling. However, information about the pathways through which Piezo channels affects osteoblasts remains limited. Thus, we aimed to investigate the influence of Piezo1 on the osteogenic and osteoclast factors in osteoblasts under mechanical load. Materials and methodsCyclic stretch (CS) experiments on MC3T3-E1 were conducted using a BioDynamic mechanical stretching device. The Piezo1 channel blocker GsMTx4 and the Piezo1 channel agonist Yoda1 were used 12 h before the application of CS. MC3T3-E1 cells were then subjected to 15% CS, and the expression of Piezo1, Piezo2, BMP-2, OCN, Runx2, RANKL, p-p65/p65, and ALP was measured using quantitative real-time polymerase chain reaction, western blot, alkaline phosphatase staining, and immunofluorescence staining. ResultsCS of 15% induced the highest expression of Piezo channel and osteoblast factors. Yoda1 significantly increased the CS-upregulated expression of Piezo1 and ALP activity but not Piezo2 and RANKL. GsMTx4 downregulated the CS-upregulated expression of Piezo1, Piezo2, Runx2, OCN, p-65/65, and ALP activity but could not completely reduce CS-upregulated BMP-2. ConclusionsThe appropriate force is more suitable for promoting osteogenic differentiation in MC3T3-E1. The Piezo1 channel participates in osteogenic differentiation of osteoblasts through its influence on the expression of osteogenic factors like BMP-2, Runx2, and OCN and is involved in regulating osteoclasts by influencing phosphorylated p65. These results provide a foundation for further exploration of osteoblast function in orthodontic tooth movement.

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.

Intestinal Piezo1 aggravates intestinal barrier dysfunction during sepsis by mediating Ca2+ influx

IntroductionIntestinal barrier dysfunction is a pivotal factor in sepsis progression. The mechanosensitive ion channel Piezo1 is associated with barrier function; however, its role in sepsis-induced intestinal barrier dysfunction remains poorly understood.MethodsThe application of cecal ligation and puncture (CLP) modeling was performed on both mice of the wild-type (WT) variety and those with Villin-Piezo1flox/flox genetic makeup to assess the barrier function using in vivo FITC-dextran permeability measurements and immunofluorescence microscopy analysis of tight junctions (TJs) and apoptosis levels. In vitro, Caco-2 monolayers were subjected to TNF-α incubation. Moreover, to modulate Piezo1 activation, GsMTx4 was applied to inhibit Piezo1 activation. The barrier function, intracellular calcium levels, and mitochondrial function were monitored using calcium imaging and immunofluorescence techniques.ResultsIn the intestinal tissues of CLP-induced septic mice, Piezo1 protein levels were notably elevated compared with those in normal mice. Piezo1 has been implicated in the sepsis-mediated disruption of TJs, apoptosis of intestinal epithelial cells, elevated intestinal mucosal permeability, and systemic inflammation in WT mice, whereas these effects were absent in Villin-Piezo1flox/flox CLP mice. In Caco-2 cells, TNF-α prompted calcium influx, an effect reversed by GsMTx4 treatment. Elevated calcium concentrations are correlated with increased accumulation of reactive oxygen species, diminished mitochondrial membrane potential, and TJ disruption.ConclusionsThus, Piezo1 is a potential contributor to sepsis-induced intestinal barrier dysfunction, influencing apoptosis and TJ modification through calcium influx-mediated mitochondrial dysfunction.

Pharmacological Activation of Piezo1 Channels Enhances Astrocyte-Neuron Communication via NMDA Receptors in the Murine Neocortex.

The Piezo1 mechanosensitive ion channel is abundant on several elements of the central nervous system including astrocytes. It has been already demonstrated that activation of these channels is able to elicit calcium waves on astrocytes, which contributes to the release of gliotransmitters. Astrocyte- and N-methyl-D-aspartate (NMDA) receptor-dependent slow inward currents (SICs) are hallmarks of astrocyte-neuron communication. These currents are triggered by glutamate released as gliotransmitter, which in turn activates neuronal NMDA receptors responsible for this inward current having slower kinetics than any synaptic events. In this project, we aimed to investigate whether Piezo1 activation and inhibition is able to alter spontaneous SIC activity of murine neocortical pyramidal neurons. When the Piezo1 opener Yoda1 was applied, the SIC frequency and the charge transfer by these events in a minute time was significantly increased. These changes were prevented by treating the preparations with the NMDA receptor inhibitor D-AP5. Furthermore, Yoda1 did not alter the spontaneous EPSC frequency and amplitude when SICs were absent. The Piezo1 inhibitor Dooku1 effectively reverted the actions of Yoda1 and decreased the rise time of SICs when applied alone. In conclusion, activation of Piezo1 channels is able to alter astrocyte-neuron communication. Via enhancement of SIC activity, astrocytic Piezo1 channels have the capacity to determine neuronal excitability.

Effect of Piezo1 Channel-Mediated Mechanotransduction on Osteogenic Differentiation and Interleukin-6 Secretion in Bone Mesenchymal Stem Cells Under Tensile Strain

Physical stimulation plays a crucial role in the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs). However, the mechanotransductive mechanisms remain uncleared. Recent studies have suggested that the Piezo1 channel is essential for transforming mechanical signals. Therefore, we investigate the Piezo1-mediated mechanisms in mechanical strain-regulated MSC osteogenic differentiation and release of proinflammatory cytokines. The tensile strain was applied to rat MSCs cultured in a monolayer to induce mechanical strain. The immuno-nanomagnetic bead enzyme-linked immunosorbent assay was employed to assess gene and protein expressions, as well as osteogenic biomarkers and interleukin-6 (IL-6) release, both in the presence or absence of a Piezo1 agonist/antagonist. Firstly, biophysical loading through mechanical strain was found to promote MSC osteogenic differentiation. Suppression of Piezo1 using GsMTx4 antagonist or transfection with Piezo1-siRNA effectively inhibited mechanical responses associated with osteogenic gene expressions and IL-6. Activation of Piezo1 by Yoda1 mimicked the effects induced by mechanical strain on osteogenic gene expressions and IL-6 release, which were associated with YAP activation, upregulation, and nuclear accumulation of β-catenin. In conclusion, these findings significantly enhance our understanding of MSC mechanotransduction and hold great promise for drug development to enhance skeletal mass.

Piezo1 channels restrain ILC2s and regulate the development of airway hyperreactivity.

Mechanosensitive ion channels sense force and pressure in immune cells to drive the inflammatory response in highly mechanical organs. Here, we report that Piezo1 channels repress group 2 innate lymphoid cell (ILC2)-driven type 2 inflammation in the lungs. Piezo1 is induced on lung ILC2s upon activation, as genetic ablation of Piezo1 in ILC2s increases their function and exacerbates the development of airway hyperreactivity (AHR). Conversely, Piezo1 agonist Yoda1 reduces ILC2-driven lung inflammation. Mechanistically, Yoda1 inhibits ILC2 cytokine secretion and proliferation in a KLF2-dependent manner, as we found that Piezo1 engagement reduces ILC2 oxidative metabolism. Consequently, in vivo Yoda1 treatment reduces the development of AHR in experimental models of ILC2-driven allergic asthma. Human-circulating ILC2s express and induce Piezo1 upon activation, as Yoda1 treatment of humanized mice reduces human ILC2-driven AHR. Our studies define Piezo1 as a critical regulator of ILC2s, and we propose the potential of Piezo1 activation as a novel therapeutic approach for the treatment of ILC2-driven allergic asthma.

Roles of mechanosensitive ion channel PIEZO1 in the pathogenesis of brain injury after experimental intracerebral hemorrhage

Secondary brain injury after intracerebral hemorrhage (ICH) is the main cause of poor prognosis in ICH patients, but the underlying mechanisms remain less known. The involvement of Piezo1 in brain injury after ICH was studied in a mouse model of ICH. ICH was established by injecting autologous arterial blood into the basal ganglia in mice. After vehicle, Piezo1 blocker, GsMTx4, Piezo1 activator, Yoda-1, or together with mannitol (tail vein injection) was injected into the left lateral ventricle of mouse brain, Piezo1 level and the roles of Piezo1 in neuronal injury, brain edema, and neurological dysfunctions after ICH were determined by the various indicated methods. Piezo1 protein level in neurons was significantly upregulated 24 h after ICH in vivo (human and mice). Piezo1 protein level was also dramatically upregulated in HT22 cells (a murine neuron cell line) cultured in vitro 24 h after hemin treatment as an in vitro ICH model. GsMTx4 treatment or together with mannitol significantly downregulated Piezo1 and AQP4 levels, markedly increased Bcl2 level, maintained more neurons alive, considerably restored brain blood flow, remarkably relieved brain edema, substantially decreased serum IL-6 level, and almost fully reversed the neurological dysfunctions at ICH 24 h group mice. In contrast, Yoda-1 treatment achieved the opposite effects. In conclusion, Piezo1 plays a crucial role in the pathogenesis of brain injury after ICH and may be a target for clinical treatment of ICH.

Gastric mechanosensitive channel Piezo1 regulates ghrelin production and food intake.

Ghrelin, produced mainly by gastric X/A-like cells, triggers a hunger signal to the central nervous system to stimulate appetite. It remains unclear whether X/A-like cells sense gastric distention and thus regulate ghrelin production. Here we show that PIEZO1 expression in X/A-like cells decreases in patients with obesity when compared to controls, whereas it increases after sleeve gastrectomy. Male and female mice with specific loss of Piezo1 in X/A-like cells exhibit hyperghrelinaemia and hyperphagia and are more susceptible to overweight. These phenotypes are associated with impairment of the gastric CaMKKII/CaMKIV-mTOR signalling pathway. Activation of PIEZO1 by Yoda1 or gastric bead implantation inhibits ghrelin production, decreases energy intake and induces weight loss in mice. Inhibition of ghrelin production by Piezo1 through the CaMKKII/CaMKIV-mTOR pathway can be recapitulated in a ghrelin-producing cell line mHypoE-42. Our study reveals a mechanical regulation of ghrelin production and appetite by PIEZO1 of X/A-like cells, which suggests a promising target for anti-obesity therapy.

Analysis of pressure-activated Piezo1 open and subconductance states at a single channel level

Mechanically activated Piezo1 channels undergo transitions from closed to open-state in response to pressure and other mechanical stimuli. However, the molecular details of these mechanosensitive gating transitions are unknown. Here, we used cell-attached pressure-clamp recordings to acquire single channel data at steady-state conditions (where inactivation has settled down), at various pressures and voltages. Importantly, we identify and analyze subconductance states of the channel which were not reported before. Pressure-dependent activation of Piezo1 increases the occupancy of open and subconductance state at the expense of decreased occupancy of shut-states. No significant change in the mean open time of subconductance states was observed with increasing negative pipette pressure or with varying voltages (ranging from -40 to -100 mV). Using Markov-chain modeling we identified a minimal four-states kinetic scheme, which recapitulates essential characteristics of the single channel data, including that of the subconductance level. This study advances our understanding of Piezo1 gating mechanism in response to discrete stimuli (such as pressure and voltage) and paves the path to develop cellular and tissue level models to predict Piezo1 function in various cell types.