Transient Receptor Potential Vanilloid 4 Antagonist Research Articles

Blockage of transient receptor potential vanilloid 4 prevents postoperative atrial fibrillation by inhibiting NLRP3-inflammasome in sterile pericarditis mice

The incidence of atrial fibrillation (AF) increases after surgery and is associated with the activation of NLRP3-inflammation. Our previous studies have found that transient receptor potential vanilloid 4 (TRPV4) blockade reduces the susceptibility to AF, but its molecular mechanisms remains unclear. Therefore, we hypothesized that blockage of TRPV4 reduces the incidence of AF by inhibiting NLRP3-inflammasome in sterile pericarditis (SP) mice. In this study, we established SP mice by dusting talcum powder on atrial surfaces. We first confirmed that genetic or pharmacological TRPV4 inhibition reduced the susceptibility to AF in SP mice. We also found that the expression level of NLRP3-inflammasome and inflammatory cytokines significantly increased in the atria of SP mice, which further increased in application the TRPV4 agonist GSK1016790A (GSK101) and decreased in application the TRPV4 antagonist GSK2193874. More importantly, ERK inhibitor (U0126) or NF-κB inhibitor (Bay11–7082) could partially reverse GSK101-induced NLRP3-inflammasome up-regulation. Interestingly, U0126 can reversed GSK101-induced NF-κB phosphorylation, but Bay11–7082 cannot change GSK101-induced ERK phosphorylation. Finally, we shown that the activation of NLRP3-inflammasome and ERK/NF-κB signaling pathway significantly reduced in TRPV4-knockout SP mice. Collectively, our studies indicate that blockage of TRPV4 prevents AF in SP mice by inhibiting NLRP3-inflammasome through the ERK/NF-κB signaling pathway.

Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist

Calcium-activated chloride channels (CaCCs) play important roles in many physiological processes and their malfunction is implicated in diverse pathologies such as cancer, asthma, and hypertension. TMEM16A and TMEM16B proteins are the structural components of the CaCCs. Recent studies in cell cultures and animal models have demonstrated that pharmacological inhibition of CaCCs could be helpful in the treatment of some diseases, however, there are few specific modulators of these channels. CaCCs and Transient Receptor Potential Vanilloid-4 (TRPV4) channels are co-expressed in some tissues where they functionally interact. TRPV4 is activated by different stimuli and forms a calcium permeable channel that is activated by GSK1016790A and antagonized by GSK2193874. Here we report that GSK2193874 enhances the chloride currents mediated by TMEM16B expressed in HEK cells at nanomolar concentrations and that GSK1016790A enhances native CaCCs of Xenopus oocytes. Thus, these compounds may be used as a tool for the study of CaCCs, TRPV4 and their interactions.

Activation of transient receptor potential vanilloid 4 exacerbates myocardial ischemia-reperfusion injury via JNK-CaMKII phosphorylation pathway in isolated mice hearts

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is involved in myocardial ischemia-reperfusion (IR) injury, yet its underlying molecular mechanism remains unclear. In this study, we isolated mice hearts for a Langendorff perfusion test and used HL-1 myocytes for in vitro assessments. We first confirmed that TRPV4 agonist (GSK101) enhanced myocardial IR injury, as demonstrated by the reduced recovery of cardiac function, larger myocardial infarct size, and more apoptotic cells. We also found that GSK101 could further increase the phosphorylation of JNK and CaMKII in isolated hearts during IR. Notably, GSK101 dose-dependently evoked the phosphorylation of JNK and CaMKII in isolated normal hearts. All above GSK101-induced effects could be significantly blocked by the pharmacological inhibition or genetic ablation of TRPV4. More importantly, JNK inhibition (with SP600125) or CaMKII inhibition (with KN93 or in transgenic AC3-I mice) could prevent GSK101-induced myocardial injury during IR. In HL-1 myocytes, GSK101 triggered Ca2+ influx and evoked the phosphorylation of JNK and CaMKII but these effects were abolished by removing extracellular Ca2+ or in the presence of a TRPV4 antagonist. Finally, we showed that in HL-1 myocytes and isolated hearts during IR, JNK inhibition significantly inhibited the phosphorylation of CaMKII induced by GSK101 but CaMKII inhibition had no effect on JNK activation induced by GSK101. Our data suggest that TRPV4 activation exacerbates myocardial IR injury via the JNK-CaMKII phosphorylation pathway.

Identification, Synthesis, and Characterization of a Major Circulating Human Metabolite of TRPV4 Antagonist GSK2798745.

GSK2798745, an antagonist of the transient receptor potential vanilloid 4 (TRPV4) ion channel, was recently investigated in clinical trials for the treatment of cardiac and respiratory diseases. Human plasma and urine samples collected from healthy volunteers following oral administration were analyzed to identify circulating and excreted metabolites of the parent drug. One major circulating metabolite (1) was found in pooled human plasma samples, accounting for approximately half of the observed drug-related material. Isolation of metabolite 1 from urine samples followed by MS and NMR studies led to a putative structural assignment of 1 where hydroxylation of GSK2798745 occurred on the central ring, producing a penta-substituted cyclohexane structure containing three stereocenters. Two unique chemical syntheses of the proposed structure were developed to confirm the identity of metabolite 1 and provide access to gram quantities for biological characterization.

Involvement of TRPV4 in changes in rapidly inactivating potassium channels in the early stage of pilocarpine-induced status epilepticus in mice.

The rapidly inactivating potassium current (IA ) is important in controlling neuronal action potentials. Altered IA function and K+ channel expression have been found in epilepsy, and activation of the transient receptor potential vanilloid 4 (TRPV4) channel is involved in epilepsy pathogenesis. This study examined whether TRPV4 affects Kv4.2 and K+ channel interacting protein (KCHIP) expression and IA changes following pilocarpine-induced status epilepticus (PISE) in mice. Herein, hippocampal protein levels of Kv4.2 and KCHIP2 increased 3 h-3 d and decreased 7-30 d; that of KCHIP1 increased 3-24 h and decreased 3-30 d post-PISE. The TRPV4 antagonist HC-067047 attenuated the increased protein levels of Kv4.2 and KCHIP2 but not that of KCHIP1 post-PISE. The TRPV4 agonist GSK1016790A increased hippocampal protein levels of Kv4.2 and KCHIP2 but had no effect on KCHIP1 expression. HC-067047 attenuated the increased IA in hippocampal pyramidal neurons 24 h and 3 d post-PISE. GSK1016790A increased IA in hippocampal pyramidal neurons, shifting the voltage-dependent inactivation curve toward depolarization. The GSK1016790A-induced increase of IA was blocked by protein kinase A and calcium/calmodulin-dependent kinase II antagonists but was unaffected by protein kinase C antagonists. We conclude that TRPV4 activation may be responsible for the increases of Kv4.2 and KCHIP2 expression in hippocampi and IA in hippocampal pyramidal neurons in PISE mice, which are likely compensatory measures for hyperexcitability at the early stage of epilepsy.

TRPV4 mediates cell damage induced by hyperphysiological compression and regulates COX2/PGE2 in intervertebral discs.

BackgroundAberrant mechanical loading of the spine causes intervertebral disc (IVD) degeneration and low back pain. Current therapies do not target the mediators of the underlying mechanosensing and mechanotransduction pathways, as these are poorly understood. This study investigated the role of the mechanosensitive transient receptor potential vanilloid 4 (TRPV4) ion channel in dynamic compression of bovine nucleus pulposus (NP) cells in vitro and mouse IVDs in vivo.MethodsDegenerative changes and the expression of the inflammatory mediator cyclooxygenase 2 (COX2) were examined histologically in the IVDs of mouse tails that were dynamically compressed at a short repetitive hyperphysiological regime (vs sham). Bovine NP cells embedded in an agarose‐collagen hydrogel were dynamically compressed at a hyperphysiological regime in the presence or absence of the selective TRPV4 antagonist GSK2193874. Lactate dehydrogenase (LDH) and prostaglandin E2 (PGE2) release, as well as phosphorylation of mitogen‐activated protein kinases (MAPKs), were analyzed. Degenerative changes and COX2 expression were further evaluated in the IVDs of trpv4‐deficient mice (vs wild‐type; WT).ResultsDynamic compression caused IVD degeneration in vivo as previously shown but did not affect COX2 expression. Dynamic compression significantly augmented LDH and PGE2 releases in vitro, which were significantly reduced by TRPV4 inhibition. Moreover, TRPV4 inhibition during dynamic compression increased the activation of the extracellular signal‐regulated kinases 1/2 (ERK) MAPK pathway by 3.13‐fold compared to non‐compressed samples. Trpv4‐deficient mice displayed mild IVD degeneration and decreased COX2 expression compared to WT mice.ConclusionsTRPV4 therefore regulates COX2/PGE2 and mediates cell damage induced by hyperphysiological dynamic compression, possibly via ERK. Targeted TRPV4 inhibition or knockdown might thus constitute promising therapeutic approaches to treat patients suffering from IVD pathologies caused by aberrant mechanical stress.

Modulation of Cerebrospinal Fluid Production by Transient Receptor Potential Vanilloid 4: Implications for Disorders of Brain Fluid Volume

Cerebrospinal fluid (CSF) secretion into the brain ventricular system is an active process via the choroid plexus (CP) - a specialized convoluted epithelial sheet surrounding a fenestrated capillary bed which is fed from the cerebral arterioles. The final CSF composition differs from a simple plasma filtrate implicating the CP as an important regulatory site controlling CSF composition and volume. Flux across this blood-CSF barrier is tightly controlled by ion and water transport proteins. Transient Receptor Potential Vanilloid 4 (TRPV4) is a polymodal, non-selective cation channel which functions as a regulatory hub protein in epithelia, including the CP. Our previous studies have shown that TRPV4-selective antagonists were effective at reducing ventriculomegaly in a genetic rat model of severe postnatal hydrocephalus. In CP, we found that activation of TRPV4 caused the influx of both Ca2+ and Na+ ions, and entry of both of these ions was selectively blocked by a TRPV4 antagonist. Systemic treatment with TRPV4 antagonists reduced TRPV4 mRNA expression in the CP in hydrocephalic rats, but not in normal rats, indicating a role for TRPV4 as part of a regulatory pathway in CSF production. To study the mechanism of CSF secretion by the CP, we utilized two continuous, high resistance CP cell lines: the porcine choroid plexus – Reims (PCP-R) and the human choroid plexus papilloma (HIBCPP) cells. The current experiments utilize Ussing chamber electrophysiological techniques, western blotting, qRT-PCR, and fluorescent immunolabeling. Immunolabeling and mRNA expression demonstrate a robust abundance for TRPV4 and other well-characterized CP membrane proteins in both cell lines. In both lines, TRPV4 activation stimulates a large change in short circuit current (ISC), a measure of net electrogenic ion flux. This change in ISCis coupled with a substantial change in transepithelial membrane conductance, a measure of cellular permeability. The transepithelial membrane transport changes due to TRPV4 activation in human and porcine cells are similar in overall effect, but differ in baseline, duration, and magnitude, indicating species-specific CP transport mechanisms. In both cases, these responses can be inhibited by either pre- or post-incubation with TRPV4 antagonists, substantiating the specificity to TRPV4 activation. The human cells, but not the porcine cells, demonstrate a substantial baseline transepithelial transport which is dependent on K+, Na+, and Cl- gradients. Furthermore, in both the porcine and human cells, the TRPV4 response is dependent upon K+ secretion, Cl- absorption, and Na+ secretion. These data confirm a role for TRPV4 in regulating ionic gradients, modulating both electrogenic and electroneutral transporters, and controlling CSF production. The promising results of these studies, coupled with published data from other investigators suggesting a role for TRPV4 in blood-brain-barrier permeability and astrocyte volume regulation, shows the importance of TRPV4 as a regulatory hub protein in brain fluid volume regulation.

Selective blockade of transient receptor potential vanilloid 4 reduces cyclophosphamide-induced bladder pain in mice

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel activated by various physical stimuli such as cell swelling and shear stress. TRPV4 is expressed in bladder sensory nerves and epithelium, and its activation produces urinary dysfunction in rodents. However, there have been few reports regarding its involvement in bladder pain. Therefore, we investigated whether TRPV4 is involved in bladder pain in mouse cystitis model. Intraperitoneal injection of cyclophosphamide (CYP; 300 mg/kg) produced mechanical hypersensitivity in the lower abdomen associated with a severe inflammatory bladder in mice. The mechanical threshold was reversed significantly in Trpv4-knockout (KO) mice. Repeated injections of CYP (150 mg/kg) daily for 4 days provoked mild bladder inflammation and persistent mechanical hypersensitivity in mice. Trpv4-KO mice prevented a reduction of the mechanical threshold without an alteration in bladder inflammation. A selective TRPV4 antagonist also reversed the mechanical threshold in chronic cystitis mice. Although expression of Trpv4 was unchanged in the bladders of chronic cystitis mice, the level of phosphorylated TRPV4 was increased significantly. These results suggest involvement of TRPV4 in bladder pain of cystitis mice. A TRPV4 antagonist might be useful for patients with irritable bladder pain such as those with interstitial cystitis/painful bladder syndrome.

Compressive mechanical stress enhances susceptibility to interleukin-1 by increasing interleukin-1 receptor expression in 3D-cultured ATDC5 cells

BackgroundMechanical overload applied on the articular cartilage may play an important role in the pathogenesis of osteoarthritis. However, the mechanism of chondrocyte mechanotransduction is not fully understood. The purpose of this study was to assess the effects of compressive mechanical stress on interleukin-1 receptor (IL-1R) and matrix-degrading enzyme expression by three-dimensional (3D) cultured ATDC5 cells. In addition, the implications of transient receptor potential vanilloid 4 (TRPV4) channel regulation in promoting effects of compressive mechanical loading were elucidated.MethodsATDC5 cells were cultured in alginate beads with the growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. The cultured cell pellet was seeded in collagen scaffolds to produce 3D-cultured constructs. Cyclic compressive loading was applied on the 3D-cultured constructs at 0.5 Hz for 3 h. The mRNA expressions of a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS4) and IL-1R were determined with or without compressive loading, and effects of TRPV4 agonist/antagonist on mRNA expressions were examined. Immunoreactivities of reactive oxygen species (ROS), TRPV4 and IL-1R were assessed in 3D-cultured ATDC5 cells.ResultsIn 3D-cultured ATDC5 cells, ROS was induced by cyclic compressive loading stress. The mRNA expression levels of ADAMTS4 and IL-1R were increased by cyclic compressive loading, which was mostly prevented by pyrollidine dithiocarbamate. Small amounts of IL-1β upregulated ADAMTS4 and IL-1R mRNA expressions only when combined with compressive loading. TRPV4 agonist suppressed ADAMTS4 and IL-1R mRNA levels induced by the compressive loading, whereas TRPV4 antagonist enhanced these levels. Immunoreactivities to TRPV4 and IL-1R significantly increased in constructs with cyclic compressive loading.ConclusionCyclic compressive loading induced mRNA expressions of ADAMTS4 and IL-1R through reactive oxygen species. TRPV4 regulated these mRNA expressions, but excessive compressive loading may impair TRPV4 regulation. These findings suggested that TRPV4 regulates the expression level of IL-1R and subsequent IL-1 signaling induced by cyclic compressive loading and participates in cartilage homeostasis.

September 2020 at a glance: focus on heart failure with preserved ejection fraction and medical therapy.

Type 2 diabetes mellitus and obesity increase heart failure (HF) risk.1-4 Dang et al.5 showed that delivery of longevity-associated variant of the human BPIFB4 gene in obese mice with diabetes improves cardiac function. Usefulness of biomarker measurements may be lower in patients with HF and preserved ejection fraction (HFpEF).6, 7 Henkens et al.8 performed a systematic review of studies investigating the diagnostic value of novel biomarkers for HFpEF. The risk of bias was high in all studies. The main limitations were use of case-control designs, poor diagnosis of HFpEF, absence of pre-specified cut-off points, lack of external validation, inadequate and variable reference standards. An analysis of TOPCAT Americas showed that spironolactone treatment was associated with a reduction in loop diuretic and thiazide doses. The reduction in the risk of HF hospitalizations with spironolactone (hazard ratio 0.77, 95% confidence interval 0.62–0.96; P = 0.021) was, however, independent of multiple variables, including loop diuretic dose and serum creatinine, thus showing it was not mediated by its diuretic effects.9 Elderly age, renal dysfunction and hyperkalaemia are major causes of spironolactone discontinuation.10 An analysis of TOPCAT Americas confirmed these data and also showed that these patients received lower spironolactone, but not placebo, doses. Spironolactone reduced the primary endpoint also in these subgroups while its withdrawal was associated with a two to four-fold higher risk of events. Thus, a low-dose strategy should be preferred instead of discontinuation.11 Larsen et al.12 showed that metformin can improve myocardial efficiency in insulin-resistant patients with HFrEF without diabetes. Sacubitril/valsartan improves outcome in HFrEF.13 An analysis of PARADIGM-HF showed that liver dysfunction is an independent predictor of outcomes in HFrEF patients and sacubitril/valsartan improved liver function, compared with enalapril.14 Oxidative stress and inflammation have a key role in HF.15 Vericiguat treatment was associated with reduction in inflammatory- and oxidative stress-related markers, such as high-sensitivity C-reactive protein and uric acid, and this may have a role for its beneficial effects.16 Inotropic drugs did not improve survival in HF.17, 18 Better results can be expected from drugs not acting through an increase in intracellular calcium. Danicamtiv, a novel cardiac myosin activator, improved stroke volume, global longitudinal and circumferential strain and left atrial function in experimental models and in a phase 2a trial in patients with HFrEF.19 In patients hospitalized for acute HF, istaroxime had beneficial effects on left ventricular function, without hypotension, tachycardia, or serious cardiac adverse events.20 Lung congestion is a leading cause of dyspnoea and exercise intolerance.21 Transient receptor potential vanilloid 4 (TRPV4) channel regulates fluid exchange in the lungs. A novel transient TRPV4 antagonist (GSK2798745) was tested in a pilot randomized, placebo-controlled trial aimed at the reduction of lung water content. The new drug was well tolerated and showed a trend towards improved gas transfer.22

TRPV4 Mediates Cardiac Fibrosis via the TGF-β1/Smad3 Signaling Pathway in Diabetic Rats.

Emerging evidence shows that the transient receptor potential vanilloid 4 (TRPV4) channel is involved in fibrosis in many organs. However, its role in diabetic cardiac fibrosis remains unclear. Our aim was to evaluate the expression level of TRPV4 in the diabetic heart and clarify its role in diabetes-induced cardiac fibrosis. A diabetic animal model was induced by a single intraperitoneal injection of streptozotocin into Sprague-Dawley rats. We also investigated cardiac fibroblasts isolated from neonatal Sprague-Dawley rats. TRPV4 expression was significantly upregulated in both diabetic myocardium and cardiac fibroblasts cultured in high-glucose medium. Masson's trichrome staining revealed that the TRPV4 antagonist HC067047 attenuated the diabetes-induced cardiac fibrosis. Furthermore, HC067047 reduced collagen Ι synthesis and suppressed the transforming growth factor beta 1 (TGF-β1) level as well as the phosphorylation of Smad3 in the diabetic heart. In addition, the TRPV4 antagonist inhibited the proliferation of cardiac fibroblasts, collagen Ι synthesis, and activation of the TGF-β1/Smad3 signaling pathway induced by high-glucose culture medium. Our findings demonstrate that the upregulation of TRPV4 expression mediates diabetic cardiac fibrosis via activation of the TGF-β1/Smad3 signaling pathway.

Targeting pulmonary capillary permeability to reduce lung congestion in heart failure: a randomized, controlled pilot trial.

Lung congestion in patients with heart failure (HF) has traditionally been treated using interventions that reduce pulmonary capillary hydrostatic pressure. The transient receptor potential vanilloid 4 (TRPV4) channel regulates fluid transit across the pulmonary capillary-interface, and represents a novel target to reduce lung water, independent of pulmonary capillary hypertension. This pilot study examined the safety and potential efficacy of TRPV4 blockade as a novel treatment for HF. In this randomized, double-blind, placebo-controlled crossover pilot trial, 11 subjects with chronic, compensated HF were treated with a novel TRPV4 antagonist (GSK2798745) or placebo. The primary endpoint was lung diffusing capacity for carbon monoxide (DLCO ) after 7 days of treatment with GSK2798745 as compared to placebo. Secondary endpoints included additional diffusion parameters, spirometry and safety assessments. Compared to placebo, treatment with GSK2798745 resulted in a trend to improvement in DLCO (placebo: -0.336 mL/mmHg/min; GSK2798745: +0.458 mL/mmHg/min; treatment difference: +0.793 mL/mmHg/min; 95% confidence interval: -0.925 to 2.512) that was not statistically significant. GSK2798745 was well-tolerated with no serious adverse events. In this pilot trial, GSK2798745 was found to be safe and well-tolerated, with a trend toward improved gas transfer. Further investigation is warranted in larger studies to determine whether treatment with TRPV4 antagonists or alternative treatments targeting capillary permeability might be effective to improve lung congestion, pulmonary gas transfer and clinical status in patients with acute or chronic HF.

Transient receptor potential vanilloid-4 contributes to stretch-induced hypercontractility and time-dependent dysfunction in the aged heart.

Cardiovascular disease remains the greatest cause of mortality in Americans over 65. The stretch-activated transient receptor potential vanilloid-4 (TRPV4) ion channel is expressed in cardiomyocytes of the aged heart. This investigation tests the hypothesis that TRPV4 alters Ca2+ handling and cardiac function in response to increased ventricular preload and cardiomyocyte stretch. Left ventricular maximal pressure (PMax) was monitored in isolated working hearts of Aged (24-27 months) mice following preload elevation from 5 to 20mmHg, with and without TRPV4 antagonist HC067047 (HC, 1 µmol/L). In preload responsive hearts, PMax prior to and immediately following preload elevation (i.e. Frank-Starling response) was similar between Aged and Aged+HC. Within 1 min following preload elevation, Aged hearts demonstrated secondary PMax augmentation (Aged>Aged+HC) suggesting a role for stretch-activated TRPV4 in cardiac hypercontractility. However, after 20 min at 20 mmHg Aged exhibited depressed PMax (Aged<Aged+HC) suggestive of TRPV4-dependent contractile dysfunction with sustained stretch. To examine stretch-induced Ca2+ homeostasis at the single-cell level, isolated cardiomyocytes were stretched 10-15% of slack length while measuring intracellular Ca2+ with fura-2. Uniaxial longitudinal stretch increased intracellular Ca2+ levels and triggered Ca2+ overload and terminal cellular contracture in Aged, but not Aged+HC. Preload elevation in hearts of young/middle-age (3-12 months) mice produced an initial PMax increase (Frank-Starling response) without secondary PMax augmentation, and cardiomyocyte stretch did not affect intracellular Ca2+ levels. Hearts of transgenic mice with cardiac-specific TRPV4 expression exhibited PMax similar to 3- to 12-month control mice prior to and immediately following preload elevation but displayed secondary PMax augmentation. Cardiomyocytes of mice with transgenic TRPV4 expression were highly sensitive to mechanical stimulation and exhibited elevated Ca2+ levels, Ca2+ overload, and terminal contracture upon cellular attachment and stretch. TRPV4 contributes to a stretch-induced increase in cardiomyocyte Ca2+ and cardiac hypercontractility, yet sustained stretch leads to cardiomyocyte Ca2+ overload and contractile dysfunction in the aged heart.

Pharmacological Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Alleviates Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

Transient receptor potential vanilloid 4 (TRPV4) is a member of the TRP channel family and is involved in diverse physiological and pathological processes. Accumulating evidence from in vitro studies indicates that TRPV4 has a potential role in liver fibrosis, but its precise role in the pathophysiological development of this condition is unclear. Exogenous interventions and endogenous reactions should be considered. This study used a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis to investigate the effects of intraperitoneal injection of the novel TRPV4 channel selective agonist GSK1016790A (GSK) and antagonist HC-067047 (HC). As compared with the CCl4 group, collagen fiber deposition and alpha-smooth muscle actin (α-SMA) levels were markedly higher and hepatic lobule disorganization was worse in the CCl4+GSK group, while collagen fiber deposition was significantly lower and hepatic lobule disorganization was less severe in the CCl4+HC group. The present findings suggest that activation of TRPV4 channels worsens liver fibrosis and that inhibition of TRPV4 channels may alleviate liver fibrosis in vivo.

Propofol Induces Cardioprotection Against Ischemia-Reperfusion Injury via Suppression of Transient Receptor Potential Vanilloid 4 Channel.

Ca2+ entry via the transient receptor potential vanilloid 4 (TRPV4) channel contributes to Ca2+ overload and triggers many pathophysiological conditions, including myocardial ischemia/reperfusion (I/R) injury. Propofol, a widely used intravenous anesthetic, attenuates myocardial I/R injury. However, the mechanism of propofol remains to be examined. The present study aims to test the hypothesis that propofol attenuates myocardial I/R injury through the suppression of TRPV4. We used a murine ex vivo model of myocardial I/R and in vitro cultured myocytes subjected to hypoxia/reoxygenation (H/R). Propofol or TRPV4 antagonist, HC-067047, attenuates myocardial I/R injury in isolated hearts. In addition, propofol, HC-067047, or TRPV4-siRNA attenuates H/R-induced intracellular Ca2+ concentration ([Ca2+]i) increase and cell viability reduction. On the contrary, TRPV4 agonist GSK1016790A exacerbates both ex vivo and in vitro myocardial injury. Pretreatment with propofol reverses the myocardial injury and intracellular Ca2+ overload induced by GSK1016790A at least in vitro. However, neither the combination of propofol and HC-067047 nor applying propofol to cells transfected with TRPV4-siRNA creates additional protective effects. In addition, propofol dose-dependently inhibits TRPV4-mediated Ca2+ entry induced by GSK1016790A and 4α-PDD. Propofol attenuates myocardial I/R injury partially through the suppression of TRPV4 channel and the subsequent inhibition of intracellular Ca2+ overload.

Significance of transient receptor potential vanilloid 4 and aquaporin 5 co-expression in the rat uterus at term

AimsAquaporins (AQPs) are channel proteins that facilitate the rapid passive movement of water. In our studies it was proved that the decreased AQP5 expression is followed by the increase of uterine contractility. The transient receptor potential vanilloid 4 (TRPV4) is a calcium channel, which is activated in response to osmotic changes. Our aim was to determine the possible role of AQP5 in this osmotic regulation of TRPV4, thus in pregnant uterine contraction. Main methodsWe used RT-PCR and Western blot techniques for the detection of the TRPV4 expression during pregnancy in rat uterus. The localization of AQP5 and TRPV4 was determined by immunohistochemical studies. The role of TRPV4 in uterus contraction was investigated in an isolated organ bath system. In vitro uterus contractions were stimulated with KCl and its effect was investigated with the selective TRPV4 agonist (RN1747) and antagonist (RN1734). Key findingsThe TRPV4 expression continuously increased from day 18 to the last day of pregnancy. The co-expression of TRPV4 and AQP5 in the myometrium and endometrium was determined in the late pregnant uterus. The TRPV4 antagonist and agonist significantly decreased and increased uterine contraction, respectively, especially on the last day of pregnancy. SignificanceWe presume the decreased AQP5 expression triggers hypertonic stress, which activates TRPV4 and increases uterus contraction on the day of labor. Based on these findings, we suppose the TRPV4 effect on uterus contraction is AQP5 control, which could be a new target in preterm birth therapy.

Transient receptor potential vanilloid 4 is involved in the upregulation of connexin expression following pilocarpine-induced status epilepticus in mice

Epilepsy is characterized by spontaneous seizures. Changes in the expression of the connexins (Cxs) have been reported to be involved in epileptogenesis. It has previously been shown that the transient receptor potential vanilloid 4 (TRPV4) plays an important role in the modulation of neuronal excitability, and that application of a TRPV4 antagonist blocks hyperthermia-induced seizures. Accordingly, in the present study, we sought to explore whether TRPV4 is involved in the regulation of Cx expression following pilocarpine-induced status epilepticus (PISE) in mice. We observed that TRPV4 protein levels in hippocampi increased 3 h to 30 d following PISE, peaking 1–3 d after induction, and that pre-application of the TRPV4 antagonist HC-067047 increased the latency to develop SE induced by pilocarpine and reduced the success rate of PISE preparation. We demonstrated that Cx43 protein levels followed a time profile similar to that of TRPV4, and further showed that the increase in Cx43 protein levels on 3 d post-PISE was markedly attenuated by HC-067047. In contrast, the corresponding increase in Cx32 protein levels lagged substantially behind, and these levels were unaffected by HC-067047. Similarly, the TRPV4 agonist GSK1016790A increased the mRNA and protein levels of Cx43, but not those of Cx32. We thus conclude that the upregulation of Cx43 expression by TRPV4 may be involved in the pathophysiology of epilepsy.

The Transient Receptor Potential Vanilloid 4 Agonist RN-1747 Inhibits the Calcium Response to Histamine

Background: Sensitization of transient receptor potential (TRP) cation channels probably contributes to intestinal hypersensitivity, a hallmark of gastrointestinal disorders. Histamine acting via histamine 1 receptor (H1R) to open TRP cation channels might also be involved. Method: The enterochromaffin cell line P-STS, responsive to histamine via H1R, was used as model to study possible synergism between histamine and TRP vanilloid 4 (TRPV4) pathways. Results: The TRPV4 antagonist RN-1734, but not HC-067047, inhibited the cytoplasmic calcium response to histamine in P-STS cells. However, also pre-incubation with the TRPV4 agonist RN-1747 strongly inhibited the calcium response to histamine in P-STS as well as HeLa cells. This inhibitory effect of RN-1747 was not due to its known TRP melastatin 8 (TRPM8) antagonism, as the TRPM8 antagonist RQ-00203078 showed no significant effect on the histamine-induced calcium response of P-STS or HeLa cells. Conclusion: The TRPV4 agonist RN-1747, and possibly also the structurally similar TRPV4 antagonist RN-1734, should be used with caution because of yet unidentified interference with histamine signaling via H1R.

Functional studies of TRPV4 in Choroid Plexus Epithelial Cells

Cerebrospinal fluid (CSF) is produced by the choroid plexus epithelial (CPe) cells, and is moved throughout the brain ventricular system in coordinated waves that are generated by ependymal cilia lining the ventricles and subsequently absorbed by the subarachnoid granulations. CSF is moved in a pulsatile motion which is thought to be influenced by multiple factors like blood pressure and circadian rhythms. Hydrocephalus is a blanket disease description which covers clinical manifestations from CSF over‐production to decreased absorption and can occur at any age. Patients with pediatric hydrocephalus have enlarged ventricles, failure to thrive, sleepiness, sunsetting of the eyes, and vomiting. The current treatment paradigm for hydrocephalus is exclusively surgical and includes CSF diversion by shunt or endoscopic third ventriculostomy. These surgeries have known morbidity and mortality rates, and variable patient outcomes. In order to study pediatric hydrocephalus, the lab has two mouse lines which develop hydrocephalus postnatally. These models have been backcrossed to different genetic backgrounds in order to characterize disease severity, and to select the models with the most viability to undergo further studies. Disease severity is characterized by ventricular area as a function of total brain area in Nissl stained brain slices at specific points from the bregma. Additional characterization is being performed by utilizing intracranial pressure (ICP) monitoring using a small probe inserted into the brain parenchyma. The models that are the most stable and have disease severity similar to pediatric hydrocephalus will be used to test a class of drugs known as Transient Receptor Potential Vanilloid 4 (TRPV4) antagonists. TRPV4 is a widely expressed, widely translated protein which functions in many epithelial tissues as well as in the vasculature, and several neuronal cell types. TRPV4 can function as an osmosensor, mechanosensor, or thermosensor, and can be activated by a number of different metabolites. Activation of TRPV4 results in cation influx into the epithelial cells which can secondarily affect other transporters. TRPV4 antagonists have been shown to ameliorate hydrocephalus in the rat model of pediatric hydrocephalus, the WPK rat. To further understand the in vivo mechanism of TRPV4, a CPe line (Porcine Choroid Plexus – Riems) was used to characterize the effects of pressure (both chronic and acute), and shear force on the TRPV4‐mediated ion flux and conductance. TRPV4‐mediated ion flux is potentiated by acute pressure on the apical side of the CPe cells. In addition, both chronic and acute pressure and shear stress increase TRPV4‐mediated conductance. Successfully completing drug studies in the mouse models of hydrocephalus, as well as characterizing the mechanism of action of the TRPV4 antagonist drugs in vivo will provide a solid basis for further preclinical pharmacology studies.Support or Funding InformationGrants from the Hydrocephalus Association and the Department of Defense Office of the Congressionally Directed Medical Research Programs (CDMRP).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preclinical Testing of TRPV4 Antagonists for the Treatment of Hydrocephalus

Cerebrospinal fluid (CSF) is a crucial component of the central nervous system that acts both mechanically and immunologically to support and protect the brain and spinal cord. Absorption and secretion of CSF must be properly maintained to achieve homeostasis. Some diseases, particularly hydrocephalus, experience an accumulation of CSF leading to an excess due to either overproduction or inadequate reabsorption. A small number of treatments are available to overcome this accumulation of CSF, all of which involve invasive surgical procedures that come with high risk and low success rates. The need for alternative treatments leads to a closer look at the choroid plexus, which consists of epithelial cells surrounding a fenestrated capillary network and produces a large portion of the CSF. This research looks specifically at the Transient Receptor Potential Vanilloid 4 (TRPV4), a non‐specific cation channel found in many secretory epithelia. When activated, TRPV4 allows an influx of Ca2+ which leads to downstream intracellular signaling and activation of Ca2+‐activated ion channels. The Wpk rat model, which contains a single point mutation in the TMEM67 gene coding for Mecklin, one of the proteins responsible for the Meckel‐Gruber Syndrome, was used to study possible treatments. Rats that are homozygous recessive for the TMEM67 gene develop a severe form of hydrocephalus that if, left untreated, will result in death by postnatal day 21 (P21). This rat model is analogous to pediatric hydrocephalus in humans. We have used the model to test a TRPV4 antagonist, RN1734, as a possible treatment of hydrocephalus. At P7, the animals received an MRI to measure the volume of the brain ventricles. Subsequently, the animals received daily intraperitoneal injections of the RN1734 through P14. At P15, the animals received a second MRI to observe changes over the course of the treatment. The animals were sacrificed on P15, and their brains, kidneys, and weights were collected. Upon sacrifice, homozygous animals are significantly smaller in body weight than their wild‐type counterparts. The brain weights of the vehicle treated homozygous and the wild‐type animals were not significantly different, however, the homozygous animals treated with the RN compound weighed statistically less than the wild‐type animals. Ventricular volume measurements by MRI revealed that the RN compound caused no effects on normal ventricular growth that occurred from day 7 through 14 in wild‐type and heterozygous animals, but it stopped the substantial ventricular enlargement that occurred over this time period in the homozygous animals. The successful results of the RN1734 treatment in the homozygous animals suggest TRPV4 antagonists for a potential pharmaceutical development for treating hydrocephalus.Support or Funding InformationHydrocephalus Association and Department of Defense Office of the Congressionally Directed Medical Research Programs (CDMRP).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.