A mutation in F-actin polymerization factor suppresses the distal arthrogryposis type 5 PIEZO2 pathogenic variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.

Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Piezo1 mutant zebrafish as a model of idiopathic scoliosis.

Scoliosis is a condition where the spine curves sideways, unique to humans due to their upright posture. However, the cause of this disease is not well understood because it is challenging to find a model for experimentation. This study aimed to create a model for human idiopathic scoliosis by manipulating the function of mechanosensitive channels called Piezo channels in zebrafish. Zebrafish were chosen because they experience similar biomechanical forces to humans, particularly in relation to the role of mechanical force in scoliosis progression. Here we describe piezo1 and piezo2a are involved in bone formation, with a double knockout resulting in congenital systemic malformations. However, an in-frame mutation of piezo1 led to fully penetrant juvenile-onset scoliosis, bone asymmetry, reduced tissue mineral density, and abnormal intervertebral discs-resembling non-congenital scoliosis symptoms in humans. These findings suggest that functional Piezo channels responding to mechanical forces are crucial for bone formation and maintaining spine integrity, providing insights into skeletal disorders.

Involvement of mechano-sensitive Piezo1 channel in the differentiation of brown adipocytes.

Brown adipocytes expend energy via heat production and are a potential target for the prevention of obesity and related metabolic disorders. Piezo1 is a Ca2+-permeable non-selective cation channel activated by mechanical stimuli. Piezo1 is reported to be involved in mechano-sensation in non-sensory tissues. However, the expression and roles of Piezo1 in brown adipocytes have not been well clarified. Here, we generated a brown adipocyte line derived from UCP1-mRFP1 transgenic mice and showed that Piezo1 is expressed in pre-adipocytes. Application of Yoda-1, a Piezo1 agonist, suppressed brown adipocyte differentiation, and this suppression was significantly attenuated by treatment with a Piezo1 antagonist and by Piezo1 knockdown. Furthermore, the suppression of brown adipocyte differentiation by Yoda-1 was abolished by co-treatment with a calcineurin inhibitor. Thus, these results suggest that activation of Piezo1 suppresses brown adipocyte differentiation via the calcineurin pathway.

Mechanical Load and Piezo1 Channel Regulated Myosin II Activity in Mouse Lenses.

The cytoarchitecture and tensile characteristics of ocular lenses play a crucial role in maintaining their transparency and deformability, respectively, which are properties required for the light focusing function of ocular lens. Calcium-dependent myosin-II-regulated contractile characteristics and mechanosensitive ion channel activities are presumed to influence lens shape change and clarity. Here, we investigated the effects of load-induced force and the activity of Piezo channels on mouse lens myosin II activity. Expression of the Piezo1 channel was evident in the mouse lens based on immunoblot and immufluorescence analyses and with the use of a Piezo1-tdT transgenic mouse model. Under ex vivo conditions, change in lens shape induced by the load decreased myosin light chain (MLC) phosphorylation. While the activation of Piezo1 by Yoda1 for one hour led to an increase in the levels of phosphorylated MLC, Yoda1 treatment for an extended period led to opacification in association with increased calpain activity and degradation of membrane proteins in ex vivo mouse lenses. In contrast, inhibition of Piezo1 by GsMTx4 decreased MLC phosphorylation but did not affect the lens tensile properties. This exploratory study reveals a role for the mechanical load and Piezo1 channel activity in the regulation of myosin II activity in lens, which could be relevant to lens shape change during accommodation.

Mechanosensitivity in Pulmonary Circulation: Pathophysiological Relevance of Stretch-Activated Channels in Pulmonary Hypertension.

A variety of cell types in pulmonary arteries (endothelial cells, fibroblasts, and smooth muscle cells) are continuously exposed to mechanical stimulations such as shear stress and pulsatile blood pressure, which are altered under conditions of pulmonary hypertension (PH). Most functions of such vascular cells (e.g., contraction, migration, proliferation, production of extracellular matrix proteins, etc.) depend on a key event, i.e., the increase in intracellular calcium concentration ([Ca2+]i) which results from an influx of extracellular Ca2+ and/or a release of intracellular stored Ca2+. Calcium entry from the extracellular space is a major step in the elevation of [Ca2+]i, involving a variety of plasmalemmal Ca2+ channels including the superfamily of stretch-activated channels (SAC). A common characteristic of SAC is that their gating depends on membrane stretch. In general, SAC are non-selective Ca2+-permeable cation channels, including proteins of the TRP (Transient Receptor Potential) and Piezo channel superfamily. As membrane mechano-transducers, SAC convert physical forces into biological signals and hence into a cell response. Consequently, SAC play a major role in pulmonary arterial calcium homeostasis and, thus, appear as potential novel drug targets for a better management of PH.

Mechanical Stimulation-Induced Calcium Signaling by Piezo1 Channel Activation in Human Odontoblast Reduces Dentin Mineralization.

Odontoblasts play critical roles in dentin formation and sensory transduction following stimuli on the dentin surface. Exogenous stimuli to the dentin surface elicit dentinal sensitivity through the movement of fluids in dentinal tubules, resulting in cellular deformation. Recently, Piezo1 channels have been implicated in mechanosensitive processes, as well as Ca2+ signals in odontoblasts. However, in human odontoblasts, the cellular responses induced by mechanical stimulation, Piezo1 channel expression, and its pharmacological properties remain unclear. In the present study, we examined functional expression of the Piezo1 channel by recording direct mechanical stimulation-induced Ca2+ signaling in dentin matrix protein 1 (DMP-1)-, nestin-, and dentin sialophosphoprotein (DSPP)-immunopositive human odontoblasts. Mechanical stimulation of human odontoblasts transiently increased intracellular free calcium concentration ([Ca2+]i). Application of repeated mechanical stimulation to human odontoblasts resulted in repeated transient [Ca2+]i increases, but did not show any desensitizing effects on [Ca2+]i increases. We also observed a transient [Ca2+]i increase in the neighboring odontoblasts to the stimulated cells during mechanical stimulation, showing a decrease in [Ca2+]i with an increasing distance from the mechanically stimulated cells. Application of Yoda1 transiently increased [Ca2+]i. This increase was inhibited by application of Gd3+ and Dooku1, respectively. Mechanical stimulation-induced [Ca2+]i increase was also inhibited by application of Gd3+ or Dooku1. When Piezo1 channels in human odontoblasts were knocked down by gene silencing with short hairpin RNA (shRNA), mechanical stimulation-induced [Ca2+]i responses were almost completely abolished. Piezo1 channel knockdown attenuated the number of Piezo1-immunopositive cells in the immunofluorescence analysis, while no effects were observed in Piezo2-immunopositive cells. Alizarin red staining distinctly showed that pharmacological activation of Piezo1 channels by Yoda1 significantly suppressed mineralization, and shRNA-mediated knockdown of Piezo1 also significantly enhanced mineralization. These results suggest that mechanical stimulation predominantly activates intracellular Ca2+ signaling via Piezo1 channel opening, rather than Piezo2 channels, and the Ca2+ signal establishes intercellular odontoblast-odontoblast communication. In addition, Piezo1 channel activation participates in the reduction of dentinogenesis. Thus, the intracellular Ca2+ signaling pathway mediated by Piezo1 channels could contribute to cellular function in human odontoblasts in two ways: (1) generating dentinal sensitivity and (2) suppressing physiological/reactional dentinogenesis, following cellular deformation induced by hydrodynamic forces inside dentinal tubules.

Periphery signals generated by Piezo-mediated stomach stretch and Neuromedin-mediated glucose load regulate the Drosophila brain nutrient sensor.

Nutrient sensors allow animals to identify foods rich in specific nutrients. The Drosophila nutrient sensor, diuretic hormone 44 (DH44) neurons, helps the fly to detect nutritive sugar. This sensor becomes operational during starvation; however, the mechanisms by which DH44 neurons or other nutrient sensors are regulated remain unclear. Here, we identified two satiety signals that inhibit DH44 neurons: (1) Piezo-mediated stomach/crop stretch after food ingestion and (2) Neuromedin/Hugin neurosecretory neurons in the ventral nerve cord (VNC) activated by an increase in the internal glucose level. A subset of Piezo+ neurons that express DH44 neuropeptide project to the crop. We found that DH44 neuronal activity and food intake were stimulated following a knockdown of piezo in DH44 neurons or silencing of Hugin neurons in the VNC, even in fed flies. Together, we propose that these two qualitatively distinct peripheral signals work in concert to regulate the DH44 nutrient sensor during the fed state.

Piezo2 channel in nodose ganglia neurons is essential in controlling hypertension in a pathway regulated directly by Nedd4-2.

Baroreflex plays a crucial role in regulation of arterial blood pressure (BP). Recently, Piezo1 and Piezo2, the mechanically-activated (MA) ion channels, have been identified as baroreceptors. However, the underlying molecular mechanism for regulating these baroreceptors in hypertension remains unknown. In this study, we used spontaneously hypertensive rats (SHR) and NG-Nitro-l-Arginine (L-NNA)- and Angiotensin II (Ang II)-induced hypertensive model rats to determine the role and mechanism of Piezo1 and Piezo2 in hypertension. We found that Piezo2 was dominantly expressed in baroreceptor nodose ganglia (NG) neurons and aortic nerve endings in Wistar-Kyoto (WKY) rats. The expression of Piezo2 not Piezo1 was significantly downregulated in these regions in SHR and hypertensive model rats. Electrophysiological results showed that the rapidly adapting mechanically-activated (RA-MA) currents and the responsive neuron numbers were significantly reduced in baroreceptor NG neurons in SHR. In WKY rats, the arterial BP was elevated by knocking down the expression of Piezo2 or inhibiting MA channel activity by GsMTx4 in NG. Knockdown of Piezo2 in NG also attenuated the baroreflex and increased serum norepinephrine (NE) concentration in WKY rats. Co-immunoprecipitation experiment suggested that Piezo2 interacted with Neural precursor cell-expressed developmentally downregulated gene 4 type 2 (Nedd4-2, also known as Nedd4L); Electrophysiological results showed that Nedd4-2 inhibited Piezo2 MA currents in co-expressed HEK293T cells. Additionally, Nedd4-2 was upregulated in NG baroreceptor neurons in SHR. Collectively, our results demonstrate that Piezo2 not Piezo1 may act as baroreceptor to regulate arterial BP in rats. Nedd4-2 induced downregulation of Piezo2 in baroreceptor NG neurons leads to hypertension in rats. Our findings provide a novel insight into the molecular mechanism for the regulation of baroreceptor Piezo2 and its critical role in the pathogenesis of hypertension.

Functional Expression of Transient Receptor Potential and Piezo1 Channels in Cultured Interstitial Cells of Human-Bladder Lamina Propria.

The interstitial cells in bladder lamina propria (LP-ICs) are believed to be involved in sensing/afferent signaling in bladder mucosa. Transient receptor potential (TRP) cation channels act as mechano- or chemo-sensors and may underlie some of the sensing function of bladder LP-ICs. We aimed to investigate the molecular and functional expression of TRP channels implicated in bladder sensory function and Piezo1/Piezo2 channels in cultured LP-ICs of the human bladder. Bladder tissues were obtained from patients undergoing cystectomy. LP-ICs were isolated and cultured, and used for real-time reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and calcium-imaging experiments. At the mRNA level, TRPA1, TRPV2, and Piezo1 were expressed most abundantly. Immunocytochemical staining showed protein expression of TRPA1, TRPV1, TRPV2, TRPV4, TRPM8, as well as Piezo1 and Piezo2. Calcium imaging using channel agonists/antagonists provided evidence for functional expression of TRPA1, TRPV2, TRPV4, Piezo1, but not of TRPV1 or TRPM8. Activation of these channels with their agonist resulted in release of adenosine triphosphate (ATP) from LP-ICs. Inhibition of TRPV2, TRPV4 and Piezo1 blocked the stretch induced intracellular Ca2+ increase. Whereas inhibition of TRPA1 blocked H2O2 evoked response in LP-ICs. Our results suggest LP-ICs of the bladder can perceive stretch or chemical stimuli via activation of TRPV2, TRPV4, Piezo1 and TRPA1 channels. LP-ICs may work together with urothelial cells for perception and transduction of mechanical or chemical signals in human-bladder mucosa.

Expression and distribution of PIEZO1 in the mouse urinary tract.

The proper function of the organs that make up the urinary tract (kidneys, ureters, bladder, and urethra) depends on their ability to sense and respond to mechanical forces, including shear stress and wall tension. However, we have limited understanding of the mechanosensors that function in these organs and the tissue sites in which these molecules are expressed. Possible candidates include stretch-activated PIEZO channels (PIEZO1 and PIEZO2), which have been implicated in mechanically regulated body functions including touch sensation, proprioception, lung inflation, and blood pressure regulation. Using reporter mice expressing a COOH-terminal fusion of Piezo1 with the sequence for the tandem-dimer Tomato gene, we found that PIEZO1 is expressed in the kidneys, ureters, bladder, and urethra as well as organs in close proximity, including the prostate, seminal vesicles and ducts, ejaculatory ducts, and the vagina. We further found that PIEZO1 expression is not limited to one cell type; it is observed in the endothelial and parietal cells of the renal corpuscle, the basolateral surfaces of many of the epithelial cells that line the urinary tract, the interstitial cells of the bladder and ureters, and populations of smooth and striated muscle cells. We propose that in the urinary tract, PIEZO1 likely functions as a mechanosensor that triggers responses to wall tension.

The mechanosensitive Piezo1 channel: a three-bladed propeller-like structure and a lever-like mechanogating mechanism.

The evolutionarily conserved Piezo proteins, including Piezo1 and Piezo2, constitute a bona fide class of mechanosensitive (MS) cation channels, which play critical roles in various mammalian physiologies, including sensation of touch, proprioception and regulation of vascular development, and blood pressure. Furthermore, mutations in Piezos have been linked to various human genetic diseases, validating their potential as therapeutic targets. Thus, it is pivotal to understand how Piezo channels effectively convert mechanical force into selective cation permeation, and therefore precisely control the various mechanotransduction processes. On the basis of our recently determined cryoelectron microscopy structures of the full-length 2547-residue mouse Piezo1, structure-guided mutagenesis, and electrophysiological and pharmacological characterizations, here we focus on reviewing the key structural features and functional components that enable Piezo1 to employ a lever-like mechanogating mechanism to function as a sophisticated mechanotransduction channel.

Activation of Piezo1 but Not NaV1.2 Channels by Ultrasound at 43 MHz.

Ultrasound (US) can modulate the electrical activity of the excitable tissues, but the mechanisms underlying this effect are not understood at the molecular level or in terms of the physical modality through which US exerts its effects. Here, we report an experimental system that allows for stable patch-clamp recording in the presence of US at 43 MHz, a frequency known to stimulate neural activity. We describe the effects of US on two ion channels proposed to be involved in the response of excitable cells to US: the mechanosensitive Piezo1 channel and the voltage-gated sodium channel NaV1.2. Our patch-clamp recordings, together with finite-element simulations of acoustic field parameters indicate that Piezo1 channels are activated by continuous wave US at 43 MHz and 50 or 90 W/cm2 through cell membrane stress caused by acoustic streaming. NaV1.2 channels were not affected through this mechanism at these intensities, but their kinetics could be accelerated by US-induced heating.

Structure-based membrane dome mechanism for Piezo mechanosensitivity.

Mechanosensitive ion channels convert external mechanical stimuli into electrochemical signals for critical processes including touch sensation, balance, and cardiovascular regulation. The best understood mechanosensitive channel, MscL, opens a wide pore, which accounts for mechanosensitive gating due to in-plane area expansion. Eukaryotic Piezo channels have a narrow pore and therefore must capture mechanical forces to control gating in another way. We present a cryo-EM structure of mouse Piezo1 in a closed conformation at 3.7Å-resolution. The channel is a triskelion with arms consisting of repeated arrays of 4-TM structural units surrounding a pore. Its shape deforms the membrane locally into a dome. We present a hypothesis in which the membrane deformation changes upon channel opening. Quantitatively, membrane tension will alter gating energetics in proportion to the change in projected area under the dome. This mechanism can account for highly sensitive mechanical gating in the setting of a narrow, cation-selective pore.

Functional role of mechanosensitive ion channel Piezo1 in human periodontal ligament cells.

OBJECTIVE: To evaluate the function of Piezo1, an evolutionarily conserved mechanically activated channel, in periodontal ligament (PDL) tissue homeostasis under compressive loading. MATERIALS AND METHODS: Primary human PDL cells (hPDLCs) were isolated, cultured, and then subjected to 2.0 g/cm(2) static compressive loading for 0.5, 3, 6, and 12 hours, respectively. The expressions of Piezo1 and osteoclastogenesis marker gene were assessed by semiquantitative reverse transcription-polymerase chain reaction. In addition, Piezo1 inhibitor, GsMTx4, was used to block the function of Piezo1, and tumor necrosis factor-α was also used as a positive control. After 12 hours of compressive loading the PDLCs were co-cultured with murine monocytic cell line RAW264.7. Immunofluorescence, western blot, enzyme-linked immunosorbent assay, and tartrate-resistant acid phosphatase staining were also used to test the potency of PDLCs to induce osteoclastogenesis and the activation of nuclear factor (NF)-κB. RESULTS: Piezo1, cyclooxygenase-2, receptor activator of NF-κB ligand, and prostaglandin E2 were significantly upregulated under static compressive stimuli. GsMTx4 repressed osteoclastogenesis in the mechanical stress-pretreated PDLCs-RAW264.7 co-culture system. Furthermore, NF-κB signaling pathway was involved in the mechanical stress-induced osteoclastogenesis. CONCLUSIONS: Piezo1 exerts a transduction role in mechanical stress-induced osteoclastogenesis in hPDLCs.