The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-, which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2ϩ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC 50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4␣-phorbol 12,13-didecanoate (4␣-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4 ϩ/ϩ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4 Ϫ/Ϫ bladders. TRPV4 activation with GSK1016790A contracted TRPV4 ϩ/ϩ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4 Ϫ/Ϫ bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4␣-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4 ϩ/ϩ and TRPV4 Ϫ/Ϫ mice revealed an enhanced bladder capacity in the TRPV4 Ϫ/Ϫ mice. Infusion of GSK1016790A into the bladders of TRPV4 ϩ/ϩ mice induced bladder overactivity with no effect in TRPV4 Ϫ/Ϫ mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.Transient receptor potential (TRP) vanilloid 4 (TRPV4), a member of the TRP superfamily of cation channels, has been implicated in a number of physiological processes, including osmoregulation (Liedtke and Friedman, 2003;Mizuno et al., 2003), hearing (Tabuchi et al., 2005), thermal and mechaniThis work was supported by GlaxoSmithKline Pharmaceuticals. Article, publication date, and citation information can be found at

The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca 2ϩ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4 Ϫ/Ϫ null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-L-arginine methyl ester; L-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (L-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.Evidence suggests that the transient receptor potential (TRP) vanilloid subtype 4 (V4), a member of the TRP family, is a thermo/osmo/mechanosensitive cationic channel that regulates intracellular Ca 2ϩ -homeostasis and cell volume (for review, see Plant and Strotmann, 2007). The TRPV4 message is expressed in cardiovascular tissues (heart and blood vessels), and evidence of functional expression has been demonstrated in vascular smooth muscle and endothelial cells (Earley, 2006;Inoue et al., 2006;Yang et al., 2006). In the endothelium, activation of TRPV4 by ligands or shearstress triggers nitric oxide (NO)-dependent vasorelaxation (Kohler et al., 2006). These studies suggest that TRPV4 activation is linked mechanistically to NO generation during the process of endothelial mechanotransduction.TRPV4 also seems to play a role in fluid distribution and integrity of endothelial/epithelial barriers. It is important to note that TRPV4 activation in the lung microvasculature Article, publication date, and citation information can be found at

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Significance Cardiac hypertrophy and dysfunction in response to sustained hormonal and mechanical stress are sentinel features of most forms of heart disease. Activation of non–voltage-gated transient receptor potential canonical channels TRPC3 and TRPC6 may contribute to this pathophysiology and provide a therapeutic target. Effects from combined selective inhibition have not been tested previously. Here we report the capability of highly selective TRPC3/6 inhibitors to block pathological hypertrophic signaling in several cell types, including adult cardiac myocytes. We show in vivo redundancy of each channel; individual gene deletion was not protective against sustained pressure overload, whereas combined deletion ameliorated the response. These data strongly support a role for both channels in cardiac disease and the utility of selective combined inhibition.

The effects of left ventricular hypertrophy (LVH) on the generation of phase 2 early afterdepolarization (EAD) and transmural dispersion of repolarization (TDR) were assessed using arterially perfused rabbit ventricular wedge preparations. Transmembrane action potentials from epicardium, subendocardium, and endocardium were simultaneously recorded together with a transmural ECG. Transmural action potential duration (APD) was also mapped. LVH (renovascular hypertension model) produced significant prolongation in ventricular APD and QT interval. Preferential APD prolongation in subendocardium and endocardium was associated with a marked increase in TDR. Phase 2 EADs were generated from subendocardium or endocardium in all LVH rabbits (15 of 15) in the absence of APD prolonging agents at basic cycle lengths of 2,000-4,000 ms. Phase 2 EAD could produce "R on T" extrasystoles, initiating polymorphic ventricular tachycardia (VT). This study provides the first direct evidence from intracellular recordings that phase 2 EAD could be generated from rabbit intact hypertrophied LV wall in the absence of APD prolonging agents, resulting in R on T extrasystoles capable of initiating polymorphic VT under enhanced TDR.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.

Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the β-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process.

Transient Receptor Potential Vanilloid 4 (TRPV4) is a member of the Transient Receptor Potential (TRP) superfamily of cation channels. TRPV4 is expressed in the vascular endothelium in the lung and regulates the integrity of the alveolar septal barrier. Increased pulmonary vascular pressure evokes TRPV4-dependent pulmonary edema, and therefore, inhibition of TRPV4 represents a novel approach for the treatment of pulmonary edema associated with conditions such as congestive heart failure. Herein we report the discovery of an orally active, potent, and selective TRPV4 blocker, 3-(1,4'-bipiperidin-1'-ylmethyl)-7-bromo--(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-quinolinecarboxamide (GSK2193874, ) after addressing an unexpected off-target cardiovascular liability observed from studies. GSK2193874 is a selective tool for elucidating TRPV4 biology both and .