Activation of Piezo1 Inhibits Kidney Cystogenesis.

The disruption of calcium signaling associated with polycystin deficiency has been proposed as the primary event underlying the increased abnormally patterned epithelial cell growth characteristic of Polycystic Kidney Disease. Calcium can be regulated through mechanotransduction, and the mechanosensitive cation channel Piezo1 has been implicated in sensing of intrarenal pressure and in urinary osmoregulation. However, a possible role for PIEZO1 in kidney cystogenesis remains undefined. We hypothesized that cystogenesis in ADPKD reflects altered mechanotransduction, suggesting activation of mechanosensitive cation channels as a therapeutic strategy for ADPKD. Here, we show that Yoda-1 activation of PIEZO1 increases intracellular Ca 2+ and reduces forskolin-induced cAMP levels in mIMCD3 cells. Yoda-1 reduced forskolin-induced IMCD cyst surface area in vitro and in mouse metanephros ex vivo in a dose-dependent manner. Knockout of polycystin-2 dampened the efficacy of PIEZO1 activation in reducing both cAMP levels and cyst surface area in IMCD3 cells. However, collecting duct-specific Piezo1 knockout neither induced cystogenesis in wild-type mice nor affected cystogenesis in the Pkd1 RC/RC model of ADPKD. Our study suggests that polycystin-2 and PIEZO1 play a role in mechanotransduction during cystogenesis in vitro , and ex vivo , but that in vivo cyst expansion may require inactivation or repression of additional suppressors of cystogenesis and/or growth. Our study provides a preliminary proof of concept for PIEZO1 activation as a possible component of combination chemotherapy to retard or halt cystogenesis and/or cyst growth.

Localized bioelectrical conduction block from radiofrequency gastric ablation persists after healing: safety and feasibility in a recovery model.

Gastric ablation has demonstrated potential to induce conduction blocks and correct abnormal electrical activity (i.e., ectopic slow-wave propagation) in acute, intraoperative in vivo studies. This study aimed to evaluate the safety and feasibility of gastric ablation to modulate slow-wave conduction after 2 wk of healing. Chronic in vivo experiments were performed in weaner pigs (n = 6). Animals were randomly divided into two groups: sham-ablation (n = 3, control group; no power delivery, room temperature, 5 s/point) and radiofrequency (RF) ablation (n = 3; temperature-control mode, 65°C, 5 s/point). In the initial surgery, high-resolution serosal electrical mapping (16 × 16 electrodes; 6 × 6 cm) was performed to define the baseline slow-wave activation profile. Ablation (sham/RF) was then performed in the mid-corpus, in a line around the circumferential axis of the stomach, followed by acute postablation mapping. All animals recovered from the procedure, with no sign of perforation or other complications. Two weeks later, intraoperative high-resolution mapping was repeated. High-resolution mapping showed that ablation successfully induced sustained conduction blocks in all cases in the RF-ablation group at both the acute and 2 wk time points, whereas all sham-controls had no conduction block. Histological and immunohistochemical evaluation showed that after 2 wk of healing, the lesions were in the inflammation and early proliferation phase, and interstitial cells of Cajal (ICC) were depleted and/or deformed within the ablation lesions. This safety and feasibility study demonstrates that gastric ablation can safely and effectively induce a sustained localized conduction block in the stomach without disrupting the surrounding slow-wave conduction capability.NEW & NOTEWORTHY Ablation has recently emerged as a tool for modulating gastric electrical activation and may hold interventional potential for disorders of gastric function. However, previous studies have been limited to the acute intraoperative setting. This study now presents the safety of gastric ablation after postsurgical recovery and healing. Localized electrical conduction blocks created by ablation remained after 2 wk of healing, and no perforation or other complications were observed over the postsurgical period.

Mechano-Sensing Channel PIEZO2 Enhances Invasive Phenotype in Triple-Negative Breast Cancer.

BACKGROUND: Mechanically gated PIEZO channels lead to an influx of cations, activation of additional Ca2+ channels, and cell depolarization. This study aimed to investigate PIEZO2's role in breast cancer. METHODS: The clinical relevance of PIEZO2 expression in breast cancer patient was analyzed in a publicly available dataset. Utilizing PIEZO2 overexpressed breast cancer cells, and in vitro and in vivo experiments were conducted. RESULTS: High expression of PIEZO2 was correlated with a worse survival in triple-negative breast cancer (TNBC) but not in other subtypes. Increased PEIZO2 channel function was confirmed in PIEZO2 overexpressed cells after mechanical stimulation. PIEZO2 overexpressed cells showed increased motility and invasive phenotypes as well as higher expression of SNAIL and Vimentin and lower expression of E-cadherin in TNBC cells. Correspondingly, high expression of PIEZO2 was correlated with the increased expression of epithelial-mesenchymal transition (EMT)-related genes in a TNBC patient. Activated Akt signaling was observed in PIEZO2 overexpressed TNBC cells. PIEZO2 overexpressed MDA-MB-231 cells formed a significantly higher number of lung metastases after orthotopic implantation. CONCLUSION: PIEZO2 activation led to enhanced SNAIL stabilization through Akt activation. It enhanced Vimentin and repressed E-cadherin transcription, resulting in increased metastatic potential and poor clinical outcomes in TNBC patients.

Specialized Mechanosensory Epithelial Cells in Mouse Gut Intrinsic Tactile Sensitivity.

BACKGROUND AND AIMS: The gastrointestinal (GI) tract extracts nutrients from ingested meals while protecting the organism from infectious agents frequently present in meals. Consequently, most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of the its external interface to optimize its function. Specialized sensory enteroendocrine cells (EECs) in GI epithelium interact intimately with luminal contents. A subpopulation of EECs express the mechanically gated ion channel Piezo2 and are developmentally and functionally like the skin's touch sensor- the Merkel cell. We hypothesized that Piezo2+ EECs endow the gut with intrinsic tactile sensitivity. METHODS: We generated transgenic mouse models with optogenetic activators in EECs and Piezo2 conditional knockouts. We used a range of reference standard and novel techniques from single cells to living animals, including single-cell RNA sequencing and opto-electrophysiology, opto-organ baths with luminal shear forces, and in vivo studies that assayed GI transit while manipulating the physical properties of luminal contents. RESULTS: Piezo2+ EECs have transcriptomic features of synaptically connected, mechanosensory epithelial cells. EEC activation by optogenetics and forces led to Piezo2-dependent alterations in colonic propagating contractions driven by intrinsic circuitry, with Piezo2+ EECs detecting the small luminal forces and physical properties of the luminal contents to regulate transit times in the small and large bowel. CONCLUSIONS: The GI tract has intrinsic tactile sensitivity that depends on Piezo2+ EECs and allows it to detect luminal forces and physical properties of luminal contents to modulate physiology.

Gastric ablation as a novel technique for modulating electrical conduction in the in vivo stomach.

Gastric motility is coordinated by underlying bioelectrical "slow wave" activity. Slow wave dysrhythmias are associated with motility disorders, including gastroparesis, offering an underexplored potential therapeutic target. Although ablation is widely used to treat cardiac arrhythmias, this approach has not yet been trialed for gastric electrical abnormalities. We hypothesized that ablation can create localized conduction blocks and modulate slow wave activation. Radiofrequency ablation was performed on the porcine serosa in vivo, encompassing a range of parameters (55-85°C, adjacent points forming a line, 5-10 s/point). High-resolution electrical mapping (16 × 16 electrodes; 6 × 6 cm) was applied to define baseline and acute postablation activation patterns. Tissue damage was evaluated by hematoxylin and eosin and c-Kit stains. Results demonstrated that RF ablation successfully induced complete conduction block and a full thickness lesion in the muscle layer at energy doses of 65-75°C for 5-10 s/point. Gastric ablation may hold therapeutic potential for gastric electrical abnormalities in the future.NEW & NOTEWORTHY This study presents gastric ablation as a new method for modulating slow wave activation and propagation in vivo, by creating localized electrical conduction blocks in the stomach, validated by high-resolution electrical mapping and histological tissue analysis. The results define the effective energy dose range for creating conduction blocks, while maintaining the mucosal and submucosal integrity, and demonstrate the electrophysiological effects of ablation. In future, gastric ablation can now be translated toward disrupting dysrhythmic slow wave activation.

microRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility.

OBJECTIVE: This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). DESIGN: All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. RESULTS: The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A, the gene that encodes the Na+ channel NaV1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na+ current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. CONCLUSIONS: A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na+ channel NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension.

BACKGROUND & AIMS: Mechanical forces contribute to portal hypertension (PHTN) and fibrogenesis. We investigated the mechanisms by which forces are transduced by liver sinusoidal endothelial cells (LSECs) into pressure and matrix changes. METHODS: We isolated primary LSECs from mice and induced mechanical stretch with a Flexcell device, to recapitulate the pulsatile forces induced by congestion, and performed microarray and RNA-sequencing analyses to identify gene expression patterns associated with stretch. We also performed studies with C57BL/6 mice (controls), mice with deletion of neutrophil elastase (NE-/-) or peptidyl arginine deiminase type IV (Pad4-/-) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice with LSEC-specific deletion of Notch1 (Notch1iΔEC). We performed partial ligation of the suprahepatic inferior vena cava (pIVCL) to simulate congestive hepatopathy-induced portal hypertension in mice; some mice were given subcutaneous injections of sivelestat or underwent bile-duct ligation. Portal pressure was measured using a digital blood pressure analyzer and we performed intravital imaging of livers of mice. RESULTS: Expression of the neutrophil chemoattractant CXCL1 was up-regulated in primary LSECs exposed to mechanical stretch, compared with unexposed cells. Intravital imaging of livers in control mice revealed sinusoidal complexes of neutrophils and platelets and formation of NETs after pIVCL. NE-/- and Pad4-/- mice had lower portal pressure and livers had less fibrin compared with control mice after pIVCL and bile-duct ligation; neutrophil recruitment into sinusoidal lumen of liver might increase portal pressure by promoting sinusoid microthrombi. RNA-sequencing of LSECs identified proteins in mechanosensitive signaling pathways that are altered in response to mechanical stretch, including integrins, Notch1, and calcium signaling pathways. Mechanical stretch of LSECs increased expression of CXCL1 via integrin-dependent activation of transcription factors regulated by Notch and its interaction with the mechanosensitive piezo calcium channel. CONCLUSIONS: In studies of LSECs and knockout mice, we identified mechanosensitive angiocrine signals released by LSECs which promote PHTN by recruiting sinusoidal neutrophils and promoting formation of NETs and microthrombi. Strategies to target these pathways might be developed for treatment of PHTN. RNA-sequencing accession number: GSE119547.

Neutrophil-induced genomic instability impedes resolution of inflammation and wound healing.

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of tissue injury associated with inflammatory bowel diseases (IBDs). The pathological effects of PMNs are largely attributed to the release of soluble mediators and reactive oxygen species (ROS). We identified what we believe is a new, ROS-independent mechanism whereby activated tissue-infiltrating PMNs release microparticles armed with proinflammatory microRNAs (miR-23a and miR-155). Using IBD clinical samples, and in vitro and in vivo injury models, we show that PMN-derived miR-23a and miR-155 promote accumulation of double-strand breaks (DSBs) by inducing lamin B1-dependent replication fork collapse and inhibition of homologous recombination (HR) by targeting HR-regulator RAD51. DSB accumulation in injured epithelium led to impaired colonic healing and genomic instability. Targeted inhibition of miR-23a and miR-155 in cultured intestinal epithelial cells and in acutely injured mucosa decreased the detrimental effects of PMNs and enhanced tissue healing responses, suggesting that this approach can be used in therapies aimed at resolution of inflammation, in wound healing, and potentially to prevent neoplasia.

EAVK segment "c" sequence confers Ca2+-dependent changes to the kinetics of full-length human Ano1.

Anoctamin1 (Ano1 and TMEM16A) is a calcium-activated chloride channel specifically expressed in the interstitial cells of Cajal (ICC) of the gastrointestinal tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full-length human Ano1 sequence includes an additional exon, exon "0," at the NH2 terminus. Ano1 with exon 0 [Ano1(0)] had a lower EC50 for intracellular calcium ([Ca2+]i) and faster chloride current (ICl) kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter ICl at low [Ca2+]i Exon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in the human stomach. Our aim was to determine the effect of EAVK deletion on Ano1(0)ICl parameters. By voltage-clamp electrophysiology, we examined ICl in HEK293 cells transiently expressing Ano1(0) with or without the EAVK sequence [Ano1(0)ΔEAVK]. The EC50 values of activating and deactivating ICl for [Ca2+]i were 438 ± 7 and 493 ± 9 nM for Ano1(0) but higher for Ano1(0)ΔEAVK at 746 ± 47 and 761 ± 26 nM, respectively. Meanwhile, the EC50 values for the ratio of instantaneous to steady-state ICl were not different between variants. Congruently, the time constant of activation was slower for Ano1(0)ΔEAVK than Ano1(0) currents at intermediate [Ca2+]i These results suggest that EAVK decreases the calcium sensitivity of Ano1(0) current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in the human stomach may function as a switch to increase sensitivity to [Ca2+]i via faster gating of Ano1.NEW & NOTEWORTHY Calcium-activated chloride channel anoctamin1 (Ano1) is necessary for normal slow waves in the gastrointestinal interstitial cells of Cajal. Exon 0 encodes the NH2 terminus of full-length human Ano1 [Ano1(0)], while exon 13 encodes residues EAVK on its first intracellular loop. Splice variants lack EAVK more often in the stomach than other tissues. Ano1(0) without EAVK [Ano1(0)ΔEAVK] has reduced sensitivity for intracellular calcium, attributable to slower kinetics. Differential expression of EAVK may function as a calcium-sensitive switch in the human stomach.

Mechanosensitive ion channel Piezo2 is inhibited by D-GsMTx4.

Enterochromaffin (EC) cells are the primary mechanosensors of the gastrointestinal (GI) epithelium. In response to mechanical stimuliEC cells release serotonin (5-hydroxytryptamine; 5-HT). The molecular details ofEC cell mechanosensitivity are poorly understood. Recently, our group found that human and mouseEC cells express the mechanosensitive ion channel Piezo2. The mechanosensitive currents in a humanEC cell model QGP-1 were blocked by the mechanosensitive channel blocker D-GsMTx4. In the present study we aimed to characterize the effects of the mechanosensitive ion channel inhibitor spider peptide D-GsMTx4 on the mechanically stimulated currents from both QGP-1 and human Piezo2 transfected HEK-293 cells. We found co-localization of 5-HT and Piezo2 in QGP-1 cells by immunohistochemistry. QGP-1 mechanosensitive currents had biophysical properties similar to dose-dependently Piezo2 and were inhibited by D-GsMTx4. In response to direct displacement of cell membranes, human Piezo2 transiently expressed in HEK-293 cells produced robust rapidly activating and inactivating inward currents. D-GsMTx4 reversibly and dose-dependently inhibited both the potency and efficacy of Piezo2 currents in response to mechanical force. Our data demonstrate an effective inhibition of Piezo2 mechanosensitive currents by the spider peptide D-GsMTx4.

Ion channelopathies in functional GI disorders.

In the gastrointestinal (GI) tract, abnormalities in secretion, absorption, motility, and sensation have been implicated in functional gastrointestinal disorders (FGIDs). Ion channels play important roles in all these GI functions. Disruptions of ion channels' ability to conduct ions can lead to diseases called ion channelopathies. Channelopathies can result from changes in ion channel biophysical function or expression due to mutations, posttranslational modification, and accessory protein malfunction. Channelopathies are strongly established in the fields of cardiology and neurology, but ion channelopathies are only beginning to be recognized in gastroenterology. In this review, we describe the state of the emerging field of GI ion channelopathies. Several recent discoveries show that channelopathies result in alterations in GI motility, secretion, and sensation. In the epithelium, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) or CFTR-associating proteins result in channelopathies with constipation or diarrhea as phenotypes. In the muscle, mutations in the SCN5A-encoded voltage-gated sodium channel NaV1.5 are associated with irritable bowel syndrome. In the sensory nerves, channelopathies of voltage-gated sodium channels NaV1.7 and NaV1.9 (encoded by SCN9A, SCN11A, respectively) manifest by either GI hyper- or hyposensation. Recent advances in structural biology and ion channel biophysics, coupled with personalized medicine, have fueled rapid discoveries of novel channelopathies and direct drug targeting of specific channelopathies. In summary, the emerging field of GI ion channelopathies has significant implications for functional GI disease stratification, diagnosis, and treatment.

A novel exon in the human Ca2+-activated Cl- channel Ano1 imparts greater sensitivity to intracellular Ca2.

Anoctamin 1 (Ano1; TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-"exon 0" upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(-0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl(-) currents between isoforms with varying concentrations of intracellular Ca(2+), extracellular anions, or Cl(-) channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(-0) in response to varying intracellular Ca(2+). The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(-0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(-0). In conclusion, human Ano1 containing exon 0 imparts its Cl(-) current with greater sensitivity to intracellular Ca(2+) and CACC inhibitors.

Hydrogen sulfide is a partially redox-independent activator of the human jejunum Na+ channel, Nav1.5.

Hydrogen sulfide (H(2)S) is produced endogenously by L-cysteine metabolism. H(2)S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through reduction-oxidation reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the H(2)S donor NaHS on Na(V)1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether H(2)S acts on Na(V)1.5 by redox reactions. Whole cell Na(+) currents were recorded in freshly dissociated human jejunum circular myocytes and Na(V)1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for H(2)S enzymes cystathionine ß-synthase and cystathionine γ-lyase from the human jejunum. NaHS increased native Na(+) peak currents and shifted the half-point (V(1/2)) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed Na(V)1.5 α subunit with EC(50)s in the 10(-4) to 10(-3) M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the Hg(2+)-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of Na(V)1.5 peak current. These studies show that H(2)S activates the gastrointestinal Na(+) channel, and the mechanism of action of H(2)S is partially redox independent.

Altered expression of Ano1 variants in human diabetic gastroparesis.

Diabetes affects many organs including the stomach. Altered number and function of interstitial cells of Cajal (ICC), the gastrointestinal pacemaker cells, underlie a number of gastrointestinal motility disorders, including diabetic gastroparesis. In the muscle layers, ICC selectively express Ano1, thought to underlie classical Ca(2+)-activated Cl(-) currents. Mice homozygous for Ano1 knock-out exhibit abnormal ICC function and motility. Several transcripts for Ano1 are generated by alternative splicing of four exons. Here, we report expression levels of transcripts encoded by alternative splicing of Ano1 gene in gastric muscles of patients with diabetic gastroparesis and nondiabetic control tissues. Expression of mRNA from two alternatively transcribed exons are significantly different between patients and controls. Furthermore, patients with diabetic gastroparesis express mRNA for a previously unknown variant of Ano1. The 5' end of this novel variant lacks exons 1 and 2 and part of exon 3. Expression of this variant in HEK cells produces a decreased density of Ca(2+)-activated Cl(-) currents that exhibit slower kinetics compared with the full-length Ano1. These results identify important changes in expression and splicing of Ano1 in patients with diabetic gastroparesis that alter the electrophysiological properties of the channel. Changes in Ano1 expression in ICC may directly contribute to diabetic gastroparesis.