Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy

Chronic neurohormonal and mechanical stresses are central fea-tures of heart disease. Increasing evidence supports a role forthe transient receptor potential canonical channels TRPC3 andTRPC6 in this pathophysiology. Channel expression for both is nor-mally very low but is increased by cardiac disease, and geneticgain- or loss-of-function studies support contributions to hypertro-phy and dysfunction. Selective small-molecule inhibitors remainscarce, and none target both channels, which may be useful giventhe high homology among them and evidence of redundant sig-naling. Here we tested selective TRPC3/6 antagonists (GSK2332255Band GSK2833503A; IC50,3–21 nM against TRPC3 and TRPC6) andfound dose-dependent blockade of cell hypertrophy signaling trig-gered by angiotensin II or endothelin-1 in HEK293T cells as well as inneonatal and adult cardiac myocytes. In vivo efficacy in mice andrats was greatly limited by rapid metabolism and high protein bind-ing, although antifibrotic effects with pressure overload were ob-served. Intriguingly, although gene deletion of TRPC3 or TRPC6alone did not protect against hypertrophy or dysfunction frompressure overload, combined deletion was protective, support-ing the value of dual inhibition. Further development of thispharmaceutical class may yield a useful therapeutic agent forheart disease management.Fil: Seo, Kinya. Johns Hopkins Medical Institutions. Department of Medicine; Estados UnidosFil: Rainer, Peter P.. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unidos. Medical University of Graz. Department of Medicine; AustriaFil: Shalkey Hahn, Virginia. Johns Hopkins Medical Institutions. Department of Medicine; Estados UnidosFil: Lee, Dong-ik. Johns Hopkins Medical Institutions. Department of Medicine; Estados UnidosFil: Jo, Su-Hyun. Kangwon National University School of Medicine; Corea del Sur. Johns Hopkins Medical Institutions. Department of Medicine; Estados UnidosFil: Andersen, Asger. Aarhus University Hospital. Department of Cardiology; DinamarcaFil: Liu, Ting. Johns Hopkins Medical Institutions. Department of Medicine; Estados UnidosFil: Xu, Xiaoping. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados UnidosFil: Willette, Robert N.. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados UnidosFil: Lepore, John J.. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados UnidosFil: Marino, Joseph P.. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados UnidosFil: Birnbaumer, Lutz. ational Institute of Environmental Health Sciences; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Schnackenberg, Christine G.. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados UnidosFil: Kass, David A.. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unido

The elevated blood pressure of human GRK4γ A142V transgenic mice is not associated with increased ROS production

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the sensitivity of GPCRs, including dopamine receptors. The GRK4 locus is linked to, and some of its polymorphisms are associated with, human essential hypertension. Transgenic mice overexpressing human (h) GRK4γ A142V on a mixed genetic background (C57BL/6J and SJL/J) have impaired renal D1-dopamine receptor (D 1R) function and increased blood pressure. We now report that hGRK4γ A142V transgenic mice, in C57BL/6J background, are hypertensive and have higher blood pressures than hGRK4γ wild-type transgenic and nontransgenic mice. The hypertensive phenotype is stable because blood pressures in transgenic founders and F6 offspring are similarly increased. To determine whether the hypertension is associated with increased production of reactive oxygen species (ROS), we measured renal NADPH oxidase (Nox2 and Nox4) and heme oxygenase (HO-1 and HO-2) protein expressions and urinary excretion of 8-isoprostane and compared the effect of Tempol on blood pressure in hGRK4γ A142V transgenic mice and D5R knockout (D 5-/-) mice in which hypertension is mediated by increased ROS. The expressions of Nox isoforms and HO-2 and the urinary excretion of 8-isoprostane were similar in hGRK4γ A142V transgenic mice and their controls. HO-1 expression was increased in hGRK4γ A142V relative to hGRK4γ wild-type transgenic mice. In contrast with the hypotensive effect of Tempol in D5-/- mice, it had no effect in hGRK4γ A142V transgenic mice. We conclude that the elevated blood pressure of hGRK4γ A142V transgenic mice is due mainly to the effect of hGRK4γ A142V transgene acting via D1R and increased ROS production is not a contributor

Dataset for: Orai and TRPC channel characterisation in FcεRI- mediated calcium signaling and mediator secretion in human mast cells

Inappropriate activation of mast cells via the FcεRI receptor leads to the release of inflammatory mediators and therefore symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signalling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcεRI initiated mediator release. However, until now the role of TRPC calcium channels in FcεRI- mediated human mast cell signalling has not been published. Here we show evidence for the expression of Orai 1,2 and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line; but, we only find evidence of functional contribution of Orai and not TRPC channels to FcεRImediated calcium entry. Calcium imaging experiments, utilising an Orai selective antagonist (Synta66) showed the contribution of Orai to FcεRI-mediated signalling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcεRI- mediated calcium signalling in human mast cells. Similarly, inactivation of STIM1- regulated TRPC1 in human mast cells (as tested by transfecting LAD2 cells with STIM1-KK684-685EE - TRPC1 gating mutant) failed to alter FcεRI- mediated calcium signalling in LAD2 human mast cells. Mediator release assays confirm that FcεRI- mediated calcium influx through Orai is necessary for histamine and TNFα release but is differentially involved in the generation of cytokines and eicosanoids