Transient Receptor Potential Vanilloid 4 Inhibition Research Articles

TRPV4 blockade alleviates endoplasmic reticulum stress mediated apoptosis in hypoxia–induced cardiomyocyte injury

BackgroundHypoxia-induced myocardial injury remains to be a huge health issue worldwide. Transient receptor potential vanilloid 4 (TRPV4) is a high-flux Ca2+ channel that is involved in numerous cardiovascular diseases. However, the role of TRPV4 in myocardial hypoxic injury remains unclear. Accordingly, this study aimed to investigate the antiapoptotic activity of TRPV4 inhibition and elucidate the underlying mechanisms in myocardial hypoxic injury. MethodsThe ability of TRPV4 to modulate the endoplasmic reticulum stress (ERS) and apoptosis was assessed in vitro through the administration of the TRPV4 antagonist HC-067047 or the agonist GSK1016790A. Additionally, intracellular Ca2+ concentration was measured by Fluo-4 AM. ResultsTRPV4 expression was significantly upregulated in hypoxic H9c2 cells compared with that in normoxic cardiomyocytes, accompanied with increased intracellular Ca2+ levels. Conversely, TRPV4 inhibition alleviated ERS in hypoxic H9c2 cells and prevented apoptosis, whereas TRPV4 agonist exacerbated such events. Furthermore, H9c2 cell apoptosis was attenuated with the administration of 4-PBA, an ERS inhibitor. ConclusionTRPV4 inhibition alleviates hypoxia-induced H9c2 cell apoptosis by mitigating ERS.

TRPV4 differentially controls inflammatory cytokine networks during static and dynamic compression of the intervertebral disc.

The ion channel transient receptor potential vanilloid 4 (TRPV4) critically transduces mechanical forces in the IVD, and its inhibition can prevent IVD degeneration due to static overloading. However, it remains unknown whether different modes of loading signals through TRPV4 to regulate the expression of inflammatory cytokines. We hypothesized that TRPV4 signaling is essential duringstatic and dynamic loading to mediate homeostasis and mechanotransduction. Mouse functional spine units were isolated and either cyclically compressed for 5 days (1 Hz, 1 h, 10% strain) or statically compressed (24 h, 0.2 MPa). Conditioned media were monitored at 6 h, 24 h, 2 days, and 5 days, with and without TRPV4 inhibition. Effects of TRPV4 activation was also evaluated without loading. The media was analyzed for a panel of 44 cytokines using a microbead array and then a correlative network was constructed to explore the regulatory relationships during loading and TRPV4 inhibition. After the loading regimen, the IVDs were evaluated histologically for degeneration. Activation of TRPV4 led to an increase interleukin-6 (IL-6) family of cytokines (IL-6, IL-11, IL-16, and leukemia inhibitory factor [LIF]) and decreased the T-cell (CCL3, CCL4, CCL17, CCL20, CCL22, and CXCL10) and monocyte (CCL2 and CCL12) recruiting chemokines by the IVD. Dynamic and static loading each provoked unique chemokine correlation networks. The inhibition of TRPV4 during dynamic loading dysregulated the relationship between LIF and other cytokines, while the inhibition of TRPV4 during static loading disrupted the connectivity of IL-16 and VEGFA. We demonstrated that TRPV4 critically mediates the cytokine production following dynamic and static loading. The activation of TRPV4 upregulated a diverse set of cytokines that may suppress the chemotaxis of T-cells and monocytes, implicating the role of TRPV4 in maintaining the immune privilege of healthy IVD.

TRPV4-mediated mitochondrial dysfunction induces pyroptosis and cartilage degradation in osteoarthritis via the Drp1-HK2 axis

Osteoarthritis (OA) is an age-related chronic degenerative disease with complex pathophysiological mechanisms. Accumulating evidence indicates that nod-like receptor pyrin domain 3 (NLRP3) inflammasome-mediated pyroptosis of chondrocytes plays a crucial role in the OA progression. Transient Receptor Potential Vanilloid 4 (TRPV4), described as a calcium-permeable cation channel, isassociated with proinflammatory factors and pyroptosis. In this study, we studied the potential functions of TRPV4 in chondrocyte pyroptosis and cartilage degradation. We found that lipopolysaccharides(LPS)-induced mitochondrial reactive oxygen species (mtROS) accumulation aggravated chondrocyte pyroptosis and cartilage degeneration. TRPV4 induces dynamin-related protein 1 (Drp1) mitochondrial translocation through the Ca2+-calmodulin-dependent protein kinase II (CaMKII) signaling pathway, which subsequently caused the mitochondrial dysfunction (e.g., mPTP over opening; Δψm depolarization; ATP production decreased; mtROS accumulation), pyroptosis and extracellular matrix (ECM) degradation through hexokinase 2 (HK2) dissociation from mitochondrial membrane. Moreover, TRPV4 inhibition reversed Drp1-involved chondrocyte pyroptosis and cartilage degeneration in the anterior cruciate ligament transection (ACLT) mouse model. Our findings revealed the internal mechanisms underlying TRPV4 regulation in chondrocytes and its intrinsic therapeutic efficacy for OA.

Inhibition of TRPV4 remodels single cell polarity and suppresses the metastasis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a malignant tumor, frequently causing both intrahepatic and extrahepatic metastases. The overall prognosis of patients with metastatic HCC is poor. Recently, single-cell (sc) polarity is proved to be an innate feature of some tumor cells in liquid phase, and directly involved in the cell adhesion to blood vessel and tumor metastasis. Here, we characterize the maintained sc polarity of HCC cells in a suspension culture, and investigate its roles and regulatory mechanisms during metastasis. We demonstrate that transient receptor potential vanilloid 4 (TRPV4) is a promoting regulator of sc polarity via activating Ca2+-dependent AMPK/MLC/ERM pathway. This attenuates the adhesion of metastatic HCC cells to vascular endothelial cells. The reduction of cancer metastases can result from TRPV4 inhibition, which not only impacts the migration and invasion of tumor cells, but also prevents the adhesion to vascular endothelial cells. Additionally, we discover a brand-new TRPV4 inhibitor called GL-V9 that modifies the degree of sc polarization and significantly decreases the metastatic capacity of HCC cells. Taken together, our data shows that TRPV4 and calcium signal are significant sc polarity regulators in metastatic HCC, and that the pharmacological intervention that results in HCC cells becoming depolarized suggests a promising treatment for cancer metastasis.

Activation of TRPV4 channels promotes the loss of cellular ATP in organotypic slices of the mouse neocortex exposed to chemical ischemia.

The vertebrate brain has an exceptionally high energy need. During ischemia, intracellular ATP concentrations decline rapidly, resulting in the breakdown of ion gradients and cellular damage. Here, we employed the nanosensor ATeam1.03YEMK to analyse the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. We demonstrate that brief chemical ischemia, induced by combined inhibition of glycolysis and oxidative phosphorylation, results in a transient decrease in intracellular ATP. Neurons experienced a larger relative decline and showed less ability to recover from prolonged (>5min) metabolic inhibition than astrocytes. Blocking voltage-gated Na+ channels or NMDA receptors ameliorated the ATP decline in neurons and astrocytes, while blocking glutamate uptake aggravated the overall reduction in neuronal ATP, confirming the central role of excitatory neuronal activity in the cellular energy loss. Unexpectedly, pharmacological inhibition of transient receptor potential vanilloid 4 (TRPV4) channels significantly reduced the ischemia-induced decline in ATP in both cell types. Imaging with Na+ -sensitive indicator dye ING-2 furthermore showed that TRPV4 inhibition also reduced ischemia-induced increases in intracellular Na+ . Altogether, our results demonstrate that neurons exhibit a higher vulnerability to brief metabolic inhibition than astrocytes. Moreover, they reveal an unexpected strong contribution of TRPV4 channels to the loss of cellular ATP and suggest that the demonstrated TRPV4-related ATP consumption is most likely a direct consequence of Na+ influx. Activation of TRPV4 channels thus provides a hitherto unacknowledged contribution to the cellular energy loss during energy failure, generating a significant metabolic cost in ischemic conditions. KEY POINTS: In the ischemic brain, cellular ATP concentrations decline rapidly, which results in the collapse of ion gradients and promotes cellular damage and death. We analysed the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. Our results confirm the central role of excitatory neuronal activity in the cellular energy loss and demonstrate that neurons experience a larger decline in ATP and are more vulnerable to brief metabolic stress than astrocytes. Our study also reveals a new, previously unknown involvement of osmotically activated transient receptor potential vanilloid 4 (TRPV4) channels to the reduction in cellular ATP in both cell types and indicates that this is a consequence of TRPV4-mediated Na+ influx. We conclude that activation of TRPV4 channels provides a considerable contribution to the cellular energy loss, thereby generating a significant metabolic cost in ischemic conditions.

Mechanical overload induces TMJ disc degeneration via TRPV4 activation.

The temporomandibular joint (TMJ) disc cushions intraarticular stress during mandibular movements. While mechanical overloading is related to cartilage degeneration, the pathogenesis of TMJ disc degeneration is unclear. Here, we determined the regulatory role of mechanoinductive transient receptor potential vanilloid 4 (TRPV4) in mechanical overload-induced TMJ disc degeneration. We explored the effect of mechanical overload on the TMJ discs in a rat occlusal interference model in vivo, and by applying sustained compressive force in vitro. TRPV4 inhibition was delivered by small interfering RNA or GSK2193874; TRPV4 activation was delivered by GSK1016790A. The protective effect of TRPV4 inhibition was validated in the rat occlusal interference model. Occlusal interference induced TMJ disc degeneration with enhanced extracellular matrix degradation in vivo and mechanical overload promoted inflammatory responses in the TMJ disc cells via Ca2+ influx with significantly upregulated TRPV4. TRPV4 inhibition reversed mechanical overload-induced inflammatory responses; TRPV4 activation simulated mechanical overload-induced inflammatory responses. Moreover, TRPV4 inhibition alleviated TMJ disc degeneration in the rat occlusal interference model. Our findings suggest TRPV4 plays a pivotal role in the pathogenesis of mechanical overload-induced TMJ disc degeneration and may be a promising target for the treatment of degenerative changes of the TMJ disc.

Dental Pulp Stem Cell-Derived Exosomes Alleviate Mice Knee Osteoarthritis by Inhibiting TRPV4-Mediated Osteoclast Activation.

Osteoarthritis (OA) is a degenerative disease that causes chronic pain and joint swelling and even disables millions of patients. However, current non-surgical treatment for OA can only relieve pain without obvious cartilage and subchondral bone repair. Mesenchymal stem cell (MSC)-secreted exosomes have promising therapeutic effects on knee OA, but the efficacy of MSC-exosome therapy is not well determined, and the mechanisms involved are still unclear. In this study, we isolated dental pulp stem cell (DPSC)-derived exosomes by ultracentrifugation and determined the therapeutic effects of a single intra-articular injection of DPSC-derived exosomes in a mice knee OA model. The results showed that the DPSC-derived exosomes effectively improved abnormal subchondral bone remodeling, inhibited the occurrence of bone sclerosis and osteophytes, and alleviated cartilage degradation and synovial inflammation in vivo. Moreover, transient receptor potential vanilloid 4 (TRPV4) was activated during the progression of OA. Enhanced TRPV4 activation facilitated osteoclast differentiation, and TRPV4 inhibition blocked this process in vitro. DPSC-derived exosomes repressed osteoclast activation in vivo by inhibiting TRPV4 activation. Our findings demonstrated that a topical, single injection of DPSC-derived exosomes is a potential strategy for knee OA treatment, and that the exosomes regulated osteoclast activation by TRPV4 inhibition, which may act as a promising target for clinical OA treatment.

Modulation of TRPV4 protects against degeneration induced by sustained loading and promotes matrix synthesis in the intervertebral disc.

While it is well known that mechanical signals can either promote or disrupt intervertebral disc (IVD) homeostasis, the molecular mechanisms for transducing mechanical stimuli are not fully understood. The transient receptor potential vanilloid 4 (TRPV4) ion channel activated in isolated IVD cells initiates extracellular matrix (ECM) gene expression, while TRPV4 ablation reduces cytokine production in response to circumferential stretching. However, the role of TRPV4 on ECM maintenance during tissue-level mechanical loading remains unknown. Using an organ culture model, we modulated TRPV4 function over both short- (hours) and long-term (days) and evaluated the IVDs' response. Activating TRPV4 with the agonist GSK101 resulted in a Ca2+ flux propagating across the cells within the IVD. Nuclear factor (NF)-κB signaling in the IVD peaked at 6 h following TRPV4 activation that subsequently resulted in higher interleukin (IL)-6 production at 7 days. These cellular responses were concomitant with the accumulation of glycosaminoglycans and increased hydration in the nucleus pulposus that culminated in higher stiffness of the IVD. Sustained compressive loading of the IVD resulted in elevated NF-κB activity, IL-6 and vascular endothelial growth factor A (VEGFA) production, and degenerative changes to the ECM. TRPV4 inhibition using GSK205 during loading mitigated the changes in inflammatory cytokines, protected against IVD degeneration, but could not prevent ECM disorganization due to mechanical damage in the annulus fibrosus. These results indicate TRPV4 plays an important role in both short- and long-term adaptations of the IVD to mechanical loading. The modulation of TRPV4 may be a possible therapeutic for preventing load-induced IVD degeneration.

Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy.

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is expressed in hearts and implicated in cardiac remodeling and dysfunction. However, the effects of TRPV4 on pressure overload-induced cardiac hypertrophy remain unclear. In this study, we found that TRPV4 expression was significantly increased in mouse hypertrophic hearts, human failing hearts, and neurohormone-induced hypertrophic cardiomyocytes. Deletion of TRPV4 attenuated transverse aortic constriction (TAC)-induced cardiac hypertrophy, cardiac dysfunction, fibrosis, inflammation, and the activation of NFκB - NOD - like receptor pyrin domain-containing protein 3 (NLRP3) in mice. Furthermore, the TRPV4 antagonist GSK2193874 (GSK3874) inhibited cardiac remodeling and dysfunction induced by TAC. In vitro, pretreatment with GSK3874 reduced the neurohormone-induced cardiomyocyte hypertrophy and intracellular Ca2+ concentration elevation. The specific TRPV4 agonist GSK1016790A (GSK790A) triggered Ca2+ influx and evoked the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). But these effects were abolished by removing extracellular Ca2+ or GSK3874. More importantly, TAC or neurohormone stimulation-induced CaMKII phosphorylation was significantly blocked by TRPV4 inhibition. Finally, we show that CaMKII inhibition significantly prevented the phosphorylation of NFκB induced by GSK790A. Our results suggest that TRPV4 activation contributes to pressure overload-induced cardiac hypertrophy and dysfunction. This effect is associated with upregulated Ca2+/CaMKII mediated activation of NFκB-NLRP3. Thus, TRPV4 may represent a potential therapeutic drug target for cardiac hypertrophy and dysfunction after pressure overload.

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.

TRPV4: A trigger of pathological RhoA activation in neurological disease.

Transient receptor potential vanilloid 4 (TRPV4), a member of the TRP superfamily, is a broadly expressed, cell surface-localized cation channel that is activated by a variety of environmental stimuli. Importantly, TRPV4 has been increasingly implicated in the regulation of cellular morphology. Here we propose that TRPV4 and the cytoskeletal remodeling small GTPase RhoA together constitute an environmentally sensitive signaling complex that contributes to pathological cell cytoskeletal alterations during neurological injury and disease. Supporting this hypothesis is our recent work demonstrating direct physical and bidirectional functional interactions of TRPV4 with RhoA, which can lead to activation of RhoA and reorganization of the actin cytoskeleton. Furthermore, a confluence of evidence implicates TRPV4 and/or RhoA in pathological responses triggered by a range of acute neurological insults ranging from stroke to traumatic injury. While initiated by a variety of insults, TRPV4–RhoA signaling may represent a common pathway that disrupts axonal regeneration and blood–brain barrier integrity. These insights also suggest that TRPV4 inhibition may represent a safe, feasible, and precise therapeutic strategy for limiting pathological TRPV4–RhoA activation in a range of neurological diseases.

Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4) Mitigates Seizures.

Astrocytes are critical regulators of the immune/inflammatory response in several human central nervous system (CNS) diseases. Emerging evidence suggests that dysfunctional astrocytes are crucial players in seizures. The objective of this study was to investigate the role of transient receptor potential vanilloid 4 (TRPV4) in 4-aminopyridine (4-AP)-induced seizures and the underlying mechanism. We also provide evidence for the role of Yes-associated protein (YAP) in seizures. 4-AP was administered to mice or primary cultured astrocytes. YAP-specific small interfering RNA (siRNA) was administered to primary cultured astrocytes. Mouse brain tissue and surgical specimens from epileptic patient brains were examined, and the results showed that TRPV4 was upregulated, while astrocytes were activated and polarized to the A1 phenotype. The levels of glial fibrillary acidic protein (GFAP), cytokine production, YAP, signal transducer activator of transcription 3 (STAT3), intracellular Ca2+([Ca2+]i) and the third component of complement (C3) were increased in 4-AP-induced mice and astrocytes. Perturbations in the immune microenvironment in the brain were balanced by TRPV4 inhibition or the manipulation of [Ca2+]iin astrocytes. Knocking down YAP with siRNA significantly inhibited 4-AP-induced pathological changes in astrocytes. Our study demonstrated that astrocytic TRPV4 activation promoted neuroinflammation through the TRPV4/Ca2+/YAP/STAT3 signaling pathway in mice with seizures. Astrocyte TRPV4 inhibition attenuated neuroinflammation, reduced neuronal injury, and improved neurobehavioral function. Targeting astrocytic TRPV4 activation may provide a promising therapeutic approach for managing epilepsy.

Transient receptor potential vanilloid 4 promotes the growth of non-small cell lung cancer by regulating Foxp3.

Transient receptor potential vanilloid 4 (TRPV4) participates in malignant tumor. However, the role of TRPV4 in non-small cell lung cancer (NSCLC) remains unclear. In this study, we demonstrated TRPV4 was upregulated in NSCLC tissues and NSCLC cell lines. TRPV4 level in the NSCLC patients and cell lines were detected, and its function was studied both in vivo and vitro. The level of TRPV4 showed a positive correlation with tumor size of NSCLC patients. Activation TRPV4 by agonist GSK1016790A promoted cell proliferation and decreased apoptosis in A549 cells, and these effects were enhanced when the cells have overexpressed TRPV4. Moreover, GSK1016790A induced inhibitory effects on apoptosis of A549 cells was impaired when GSK1016790A used together with TRPV4 selective antagonist HC-067047, or impaired when the cells have already downregulated TRPV4 expression by TRPV4 siRNA. In vivo study, pharmacological inhibition of TRPV4 prevented A549 cells transplanted tumor growth. It was showed Foxp3 level was significantly increased in the NSCLC tissues, and showed a positive correlation with the level of TRPV4. Deactivation of TRPV4 using TRPV4 siRNA or HC-067047 significantly reduced expression of Foxp3 in GSK1016790A treated NSCLC cells. Moreover, downregulation Foxp3 by transfection of Foxp3 siRNA significantly impaired TRPV4 induced NSCLC cells proliferations in vitro. Antitumor effects caused by TRPV4 inhibition in NSCLC might be attributed to the suppression of Foxp3 which induced subsequent cell apoptosis. Thus, pharmacological inhibition of TRPV4 may be a promising option for NSCLC treatment.

TRPV4-induced M\xfcller cell gliosis and TNF-\u03b1 elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-\u03baB pathway

BackgroundGlaucoma, the leading cause of irreversible blindness worldwide, is a type of retinal disease characterized by the selective death of retinal ganglion cells (RGCs). However, the pathogenesis of glaucoma has not been fully elucidated. Transient receptor potential vanilloid 4 (TRPV4) is a pressure-sensitive and calcium-permeable cation channel. TRPV4 is widely distributed in the retina and its sustained activation leads to RGC death; indicating that TRPV4 may be a possible target for glaucoma treatment. Here, we investigated the effects of TRPV4 on RGC apoptosis in a rat model of chronic ocular hypertension (COH), then examined the mechanism underlying these effects.MethodsThe COH model was established by injection of micro-magnetic beads into the anterior chamber of adult male rats. The expression levels of TRPV4, glial fibrillary acidic protein, and inflammatory factors were assessed by immunohistochemistry and immunoblotting. RGC apoptosis and visual dysfunction were evaluated by TUNEL assay and photopic negative response. Functional expression of TRPV4 was examined by electrophysiology and calcium imaging. Real-time polymerase chain reaction and immunoblotting were employed to investigate the molecular mechanism underlying the effects of TRPV4 on tumor necrosis factor-α (TNF-α) release.ResultsWe found that TRPV4 played an essential role in glaucoma, such that high levels of TRPV4 expression were associated with elevated intraocular pressure. Furthermore, TRPV4 activation was involved in glaucoma-induced RGC apoptosis and RGC-related reductions in visual function. Mechanistic investigation demonstrated that TRPV4 activation led to enhanced Müller cell gliosis and TNF-α release via the JAK2/STAT3/NF-kB pathway, while TRPV4 inhibition could reverse these effects. Finally, TRPV4 activation could lead to elevated expression of TNF receptor 1 in RGCs, while inhibition of TNF-α could reduce TRPV4-mediated RGC apoptosis.ConclusionsTRPV4 activation induces Müller cell gliosis and TNF-α elevation via the JAK2/STAT3/NF-κB pathway, which may exacerbate RGC apoptosis in glaucoma; these results suggest that TRPV4 can serve as a therapeutic target in glaucoma treatment.

Abstract 13709: TRPV4 Enhances Ca 2+ Transients and Promotes Arrhythmic Ca 2+ Signaling Following Hypoosmotic Stress and Ischemia-Reperfusion

Introduction: Transient Receptor Potential Vanilloid 4 (TRPV4) is an osmotically activated Ca 2+ channel which enhances cardiomyocyte Ca 2+ cycling and may contribute to arrhythmic Ca 2+ signals in hypoosmotic stress and Ischemia-Reperfusion (I/R). Hypothesis: TRPV4 overexpression enhances Ca 2+ transient (CaT) amplitude and arrhythmic Ca 2+ events in left ventricular (LV) cardiomyocytes exposed to hypoosmotic stress and Langendorff hearts following I/R. Methods: Mouse LV cardiomyocytes from TRPV4 Overexpressors (OEX) and non-transgenic controls (CTL) were exposed to hypoosmotic stress (250 mOsm, 30min). CaT amplitude (paced 0.5 Hz) and arrhythmic Ca 2+ events (prolonged kinetics, spontaneous CaT, Ca 2+ waves) were monitored via confocal imaging (fluo-4, 100Hz). I/R (45min global ischemia, 30min reperfusion) experiments were conducted in OEX and CTL Langendorff hearts expressing the GCaMP6f Ca 2+ sensor (OEX-GCaMP, CTL-GCaMP). CaT and arrhythmic Ca 2+ signals (Ca 2+ waves, alternans, Ca 2+ overload) were monitored in the LV myocardium. Experiments were conducted in the absence and presence of TRPV4 inhibition by HC067047 (HC, 1uM). Results: Incidence of arrhythmic Ca 2+ signals during minutes 0-15 of hypoosmotic stress was increased in OEX cardiomyocytes vs CTL (78 vs 20%, P<0.05, N=9, 5) and prevented in OEX by HC (11%, P<0.05, N=9). Arrhythmia score was increased in OEX vs CTL (1.3±0.7 vs. 0.1±0.1, P<0.05, N=9, 5) and decreased in OEX by HC (0.2±0.2, P<0.05, N=9). Following hypoosmotic stress, CaT amplitude was elevated in OEX vs CTL (F/F 0 = 3.6±0.4 vs 2.1±0.4, P<0.05, N=7, 4) and this effect in OEX was prevented by HC (F/F 0 =2.4±0.4, P<0.05, N=8). Langendorff OEX-GCaMP hearts subjected to I/R demonstrated increased systolic Ca 2+ (F/F 0 =1.8±0.09 vs 1.3±0.1, P<0.05, N=5, 6) during minutes 10-30 of reperfusion, which was attenuated by HC (F/F 0 =1.4±0.08, P<0.05, N=4). OEX-GCaMP hearts were more likely to demonstrate arrhythmic Ca 2+ signals during minutes 15-30 of reperfusion than CTL-GCaMP (27/487 vs 0/415 cells, p<0.05 N=5, 6) and this effect in OEX-GCaMP was prevented by HC (0/633 cells, p<0.05, N=4). Conclusions: TRPV4 activation enhances Ca 2+ transients and promotes arrhythmic Ca 2+ signals, and may provide a novel anti-arrhythmic target in I/R.

Adaptive study design to assess effect of TRPV4 inhibition in patients with chronic cough.

ObjectiveAirway sensory nerves involved in the cough reflex are activated by adenosine triphosphate (ATP) agonism of P2X purinoceptor 3 (P2X3) receptors. Transient receptor potential vanilloid 4 (TRPV4) channel activation causes ATP release from airway cells, and it is hypothesised that a TRPV4-ATP-P2X3 axis contributes to chronic cough. An adaptive study was run to determine if TRPV4 inhibition, using the selective TRPV4 channel blocker GSK2798745, was effective in reducing cough.MethodsA two-period randomised, double blinded, placebo-controlled crossover study was designed with interim analyses for futility and sample size adjustment. Refractory chronic cough patients received either GSK2798745 or placebo once daily for 7 days with a washout between treatments. Pharmacokinetic samples were collected for analysis of GSK2798745 at end of study. The primary end-point was total cough counts assessed objectively during day-time hours (10 h) following 7 days of dosing.ResultsInterim analysis was performed after 12 participants completed both treatment periods. This showed a 32% increase in cough counts on Day 7 for GSK2798745 compared to placebo; the pre-defined negative criteria for the study were met and the study was stopped. At this point 17 participants had been enrolled (mean 61 years; 88% female), and 15 had completed the study. Final study results for posterior median cough counts showed a 34% (90% credible interval: −3%, +85%) numerical increase for GSK2798745 compared to placebo.ConclusionThere was no evidence of an anti-tussive effect of GSK2798745. The study design allowed the decision on lack of efficacy to be made with minimal participant exposure to the investigational drug.

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.

Metabolic regulation of mitochondrial reactive oxygen species (mtROS) production and transient receptor potential vanilloid‐4 (TRPV4) activation in microvascular endothelial cells in PAH

Rationale In pulmonary arterial hypertension (PAH), highly proliferative microvascular endothelial cells (MVECs) are thought to contribute to vaso-occlusive lesion formation and increased pulmonary artery pressures. Using MVECs isolated from the Sugen/Hypoxia (SuHx) model of PAH, we recently showed that increased mtROS production activates the TRPV4 calcium channel and contributes to increased MVEC proliferation in vitro. However, the mechanistic basis for sustained mtROS production in MVECs isolated from SuHx rats (SuHx-MVECs) was unclear. Along with increased mtROS production, we observed evidence of significant mitochondrial dysfunction, including increased fragmentation and glycolytic shift. Since mtROS production can be increased by changes in the type of fuel (e.g. fatty acids)used to generate TCA metabolites, we hypothesized that use of non-glucose sources (specifically, increased fatty acid oxidation - FAO) could be the source of mitochondrial dysfunction and mtROS production in SuHx-MVECs. Methods Intracellular Ca2+ concentration ([Ca2+]i) was measured using Fura-2AM loaded MVEC in a flow chamber perfused with modified Krebs solution. Mitochondrial morphology was measured computationally using validated algorithms applied to confocal images of mitochondria in N- and SuHx-MVEC stained with MitoTracker. Lipid droplets were measured using BODIPY staining. Targeted metabolomics were performed on MVEC cell lysates using a standardized LC/MS approach as described previously (Suresh et al., AJP-Lung 2019). Human PAH metabolomics and genomics data were obtained from NIH Metabolomics Workbench or GEO Omnibus, respectively, and analyzed using R. Results Despite evidence of glycolytic shift (decreased oxygen consumption rate, increased extracellular acidification rate, increased extracellular lactate levels), levels of TCA metabolites, measured using targeted metabolomics, were not significantly lower in SuHx-MVECs compared to normoxic (N-MVEC) controls, suggesting presence of anaplerosis. Serum free fatty acids (FFA) were increased in SuHx rats at 5 weeks, while intracellular lipid droplet number was increased in SuHx-MVECs. Analysis of human PAH metabolomics and genomics data revealed increased serum FFA levels and increased expression of enzymes involved in breaking down long chain fatty acids for use in the TCA cycle (i.e. FAO). In SuHx-MVECs, untargeted metabolomics and metabolite-specific assays revealed increased levels of beta-hydroxybutyrate (BOHB), a ketone body produced by FAO. In N-MVECs, BOHB supplementation was sufficient to increase TRPV4 sensitivity to pharmacologic agonists. In contrast, inhibition of FAO (with Etoximir) in SuHx-MVECs attenuated basal Ca2+ and mitochondrial fragmentation to a similar extent as TRPV4 inhibition or mtROS quenching. Conclusion We conclude that intermediary signaling molecules such as BOHB and mtROS, produced as a consequence of increased FAO, act in concert to sensitize and activate the TRPV4 channel in lung MVECs. These data suggest an exciting role for metabolism as a regulator of TRPV4 channel activity in PAH.

Arrhythmogenesis in the aged heart following ischaemia-reperfusion: role of transient receptor potential vanilloid 4.

Cardiomyocyte Ca2+ homoeostasis is altered with ageing and predisposes the heart to Ca2+ intolerance and arrhythmia. Transient receptor potential vanilloid 4 (TRPV4) is an osmotically activated cation channel with expression in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in Ca2+ handling and arrhythmogenesis following ischaemia-reperfusion (I/R), a pathological scenario associated with osmotic stress. Cardiomyocyte membrane potential was monitored prior to and following I/R in Langendorff-perfused hearts of Aged (19-28 months) male and female C57BL/6 mice ± TRPV4 inhibition (1 μM HC067047, HC). Diastolic resting membrane potential was similar between Aged and Aged HC at baseline, but following I/R Aged exhibited depolarized diastolic membrane potential vs. Aged HC. The effects of TRPV4 on cardiomyocyte Ca2+ signalling following I/R were examined in isolated hearts of Aged cardiac-specific GCaMP6f mice (±HC) using high-speed confocal fluorescence microscopy, with cardiomyocytes of Aged exhibiting an increased incidence of pro-arrhythmic Ca2+ signalling vs. Aged HC. In the isolated cell environment, cardiomyocytes of Aged responded to sustained hypoosmotic stress (250mOsm) with an increase in Ca2+ transient amplitude (fluo-4) and higher incidence of pro-arrhythmic diastolic Ca2+ signals vs. Aged HC. Intracardiac electrocardiogram measurements in isolated hearts following I/R revealed an increased arrhythmia incidence, an accelerated time to ventricular arrhythmia, and increased arrhythmia score in Aged vs. Aged HC. Aged exhibited depolarized resting membrane potential, increased pro-arrhythmic diastolic Ca2+ signalling, and greater incidence of arrhythmia when compared with Young (3-5 months). TRPV4 contributes to pro-arrhythmic cardiomyocyte Ca2+ signalling, electrophysiological abnormalities, and ventricular arrhythmia in the aged mouse heart.