Piezo1 Activation Research Articles

PIEZO1 activation may serve as an early tissue biomarker for the prediction of irradiation-induced salivary gland dysfunction

Irradiation (IR)-induced xerostomia is the most common side effect of radiation therapy in patients with head and neck cancer (HNC). Xerostomia diagnosis is mainly based on the patient's medical history and symptoms. Currently, no direct biomarkers are available for the early prediction of IR-induced xerostomia. Here, we identified PIEZO1 as a novel predictive tissue biomarker for xerostomia. Our data demonstrate that PIEZO1 is significantly upregulated at the gene and protein levels during IR-induced salivary gland (SG) hypofunction. Notably, PIEZO1 upregulation coincided with that of inflammatory (F4/80) and fibrotic markers (fibronectin and collagen fibers accumulation). These findings suggest that PIEZO1 upregulation in SG tissue may serve as a novel predictive marker for IR-induced xerostomia.

The activation of Piezo1 channel promotes invasion and migration via the release of extracellular ATP in cervical cancer

BackgroundThe mechanosensitive ion channel Piezo1 has emerged as a potential prognostic and therapeutic target in different types of cancers. The aim of this study was to determine the expression levels and underlying mechanisms of Piezo1 in the invasion and migration processes in cervical cancer. MethodsInitially, we employed qRT-PCR, western blot, and immunohistochemical staining techniques to assess the disparity in Piezo1 expression in cervical cancer tissues and cells. Subsequently, we conducted wound healing, transwell assays and phalloidin staining to observe the effects of stable Piezo1 silencing and Piezo1 selective agonist Yoda1 on the invasion and migration capabilities. The release of extracellular ATP was assessed using the enhanced ATP assay kit. Furthermore, we conducted rescue experiments to investigate whether the activation of Piezo1 facilitates cervical cancer invasion and migration through extracellular ATP. Finally, we constructed xenograft tumor models to determine weather the Piezo1 selective agonist Yoda1 influenced the tumor growth in vivo. ResultsIn our study, we found that Piezo1 expression was elevated in both cervical cancer tissues and cells, with the highest levels observed in patients with lymph node metastasis. Knocking down Piezo1 resulted in a significant reduction in the invasion and migration capabilities of cervical cancer cells, whereas the use of the Piezo1 selective agonist Yoda1 enhanced these capabilities. Moreover, the activation of Piezo1 channels was found to regulate the release of extracellular ATP. Mechanistically, the activation of Piezo1 might facilitate cervical cancer invasion, migration, and pseudopodium formation through the release of extracellular ATP. And Piezo1 was an important molecule for the tumor growth of cervical cancer in vivo. ConclusionOur findings revealed that Piezo1 facilitated the invasion and migration of cervical cancer by releasing extracellular ATP, which might hold potential as a valuable target for prognostic and therapeutic interventions in cervical cancer.

Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries.

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

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.

Mapping the unicellular transcriptome of the ascending thoracic aorta to changes in mechanosensing and mechanoadaptation during aging.

Aortic stiffening is an inevitable manifestation of chronological aging, yet the mechano-molecular programs that orchestrate region- and layer-specific adaptations along the length and through the wall of the aorta are incompletely defined. Here, we show that the decline in passive cyclic distensibility is more pronounced in the ascending thoracic aorta (ATA) compared to distal segments of the aorta and that collagen content increases in both the medial and adventitial compartments of the ATA during aging. The single-cell RNA sequencing of aged ATA tissues reveals altered cellular senescence, remodeling, and inflammatory responses accompanied by enrichment of T-lymphocytes and rarefaction of vascular smooth muscle cells, compared to young samples. T lymphocyte clusters accumulate in the adventitia, while the activation of mechanosensitive Piezo-1 enhances vasoconstriction and contributes to the overall functional decline of ATA tissues. These results portray the immuno-mechanical aging of the ATA as a process that culminates in a stiffer conduit permissive to the accrual of multi-gerogenic signals priming to disease development.

Multiplex, high-throughput method to study cancer and immune cell mechanotransduction

Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, whereby cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced Piezo1 activation. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm−2) increases activation of murine bone marrow-derived dendritic cells. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.

WED-058-YI High shear stress induced PIEZO1 activation promotes cytoskeleton remodelling and a nuclear deformation develops pro-thrombotic microenvironment and contributes to portal hypertension

WED-058-YI High shear stress induced PIEZO1 activation promotes cytoskeleton remodelling and a nuclear deformation develops pro-thrombotic microenvironment and contributes to portal hypertension

PIEZO1 as a new target for hyperglycemic stress-induced neuropathic injury: The potential therapeutic role of bezafibrate

Hyperglycemic stress can directly lead to neuronal damage. The mechanosensitive ion channel PIEZO1 can be activated in response to hyperglycemia, but its role in hyperglycemic neurotoxicity is unclear. The role of PIEZO1 in hyperglycemic neurotoxicity was explored by constructing a hyperglycemic mouse model and a high-glucose HT22 cell model. The results showed that PIEZO1 was significantly upregulated in response to high glucose stress. In vitro experiments have shown that high glucose stress induces changes in neuronal cell morphology and membrane tension, a key mechanism for PIEZO1 activation. In addition, high glucose stress upregulates serum/glucocorticoid-regulated kinase-1 (SGK1) and activates PIEZO1 through the Ca2+ pool and store-operated calcium entry (SOCE). PIEZO1-mediated Ca2+ influx further enhances SGK1 and SOCE, inducing intracellular Ca2+ peaks in neurons. PIEZO1 mediated intracellular Ca2+ elevation leads to calcium/calmodulin-dependent protein kinase 2α (CaMK2α) overactivation, which promotes oxidative stress and apoptosis signalling through p-CaMK2α/ERK/CREB and ox-CaMK2α/MAPK p38/NFκB p65 pathways, subsequently inducing synaptic damage and cognitive impairment in mice. The intron miR-107 of pantothenic kinase 1 (PANK1) is highly expressed in the brain and has been found to target PIEZO1 and SGK1. The PANK1 receptor is activated by peroxisome proliferator-activated receptor α (PPARα), an activator known to upregulate miR-107 levels in the brain. The clinically used lipid-lowering drug bezafibrate, a known PPARα activator, may upregulate miR-107 through the PPARɑ/PANK1 pathway, thereby inhibiting PIEZO1 and improving hyperglycemia-induced neuronal cell damage. This study provides a new idea for the pathogenesis and drug treatment of hyperglycemic neurotoxicity and diabetes-related cognitive dysfunction.

α-Catenin and Piezo1 Mediate Cell Mechanical Communication via Cell Adhesions.

Cell-to-cell distant mechanical communication has been demonstrated using in vitro and in vivo models. However, the molecular mechanisms underlying long-range cell mechanoresponsive interactions remain to be fully elucidated. This study further examined the roles of α-Catenin and Piezo1 in traction force-induced rapid branch assembly of airway smooth muscle (ASM) cells on a Matrigel hydrogel containing type I collagen. Our findings demonstrated that siRNA-mediated downregulation of α-Catenin or Piezo1 expression or chemical inhibition of Piezo1 activity significantly reduced both directional cell movement and branch assembly. Regarding the role of N-cadherin in regulating branch assembly but not directional migration, our results further confirmed that siRNA-mediated downregulation of α-Catenin expression caused a marked reduction in focal adhesion formation, as assessed by focal Paxillin and Integrin α5 localization. These observations imply that mechanosensitive α-Catenin is involved in both cell-cell and cell-matrix adhesions. Additionally, Piezo1 partially localized in focal adhesions, which was inhibited by siRNA-mediated downregulation of α-Catenin expression. This result provides insights into the Piezo1-mediated mechanosensing of traction force on a hydrogel. Collectively, our findings highlight the significance of α-Catenin in the regulation of cell-matrix interactions and provide a possible interpretation of Piezo1-mediated mechanosensing activity at focal adhesions during cell-cell mechanical communication.

Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca2+ dynamics

The pancreatic islet is surrounded by ECM that provides both biochemical and mechanical cues to the islet β-cell to regulate cell survival and insulin secretion. Changes in ECM composition and mechanical properties drive β-cell dysfunction in many pancreatic diseases. While several studies have characterized changes in islet insulin secretion with changes in substrate stiffness, little is known about the mechanotransduction signaling driving altered islet function in response to mechanical cues. We hypothesized that increasing matrix stiffness will lead to insulin secretion dysfunction by opening the mechanosensitive ion channel Piezo1 and disrupting intracellular Ca2+ dynamics in mouse and human islets. To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D. Our results indicate that increased scaffold stiffness causes insulin secretion dysfunction mediated by increases in Ca2+ influx and altered Ca2+ dynamics via opening of the mechanosensitive Piezo1 channel. Additionally, inhibition of Piezo1 rescued glucose-stimulated insulin secretion (GSIS) in islets in stiff scaffolds. Overall, our results emphasize the role mechanical properties of the islet microenvironment plays in regulating function. It also supports further investigation into the modulation of Piezo1 channel activity to restore islet function in diseases like type 2 diabetes (T2D) and pancreatic cancer where fibrosis of the peri-islet ECM leads to increased tissue stiffness and islet dysfunction.

Radiofrequency Treatment Attenuates Age-Related Changes in Dermal-Epidermal Junctions of Animal Skin.

The dermal-epidermal junction (DEJ) is essential for maintaining skin structural integrity and regulating cell survival and proliferation. Thus, DEJ rejuvenation is key for skin revitalization, particularly in age-related DEJ deterioration. Radiofrequency (RF) treatment, known for its ability to enhance collagen fiber production through thermal mechanisms and increase heat shock protein (HSP) expression, has emerged as a promising method for skin rejuvenation. Additionally, RF activates Piezo1, an ion channel implicated in macrophage polarization toward an M2 phenotype and enhanced TGF-β production. This study investigated the impact of RF treatment on HSP47 and HSP90 expression, known stimulators of DEJ protein expression. Furthermore, using in vitro and aged animal skin models, we assessed whether RF-induced Piezo1 activation and the subsequent M2 polarization could counter age-related DEJ changes. The RF treatment of H2O2-induced senescent keratinocytes upregulated the expression of HSP47, HSP90, TGF-β, and DEJ proteins, including collagen XVII. Similarly, the RF treatment of senescent macrophages increased Piezo1 and CD206 (M2 marker) expression. Conditioned media from RF-treated senescent macrophages enhanced the expression of TGF-β and DEJ proteins, such as nidogen and collagen IV, in senescent fibroblasts. In aged animal skin, RF treatment increased the expression of HSP47, HSP90, Piezo1, markers associated with M2 polarization, IL-10, and TGF-β. Additionally, RF treatment enhanced DEJ protein expression. Moreover, RF reduced lamina densa replication, disrupted lesions, promoted hemidesmosome formation, and increased epidermal thickness. Overall, RF treatment effectively enhanced DEJ protein expression and mitigated age-related DEJ structural changes by increasing HSP levels and activating Piezo1.

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.

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

BackgroundVasculopathy 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.MethodsVascular 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.ResultsOur 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.ConclusionIn 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.

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.