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

Abstract

Chronic neurohormonal and mechanical stresses are central features of heart disease. Increasing evidence supports a role for the transient receptor potential canonical channels TRPC3 and TRPC6 in this pathophysiology. Channel expression for both is normally very low but is increased by cardiac disease, and genetic gain- or loss-of-function studies support contributions to hypertrophy and dysfunction. Selective small-molecule inhibitors remain scarce, and none target both channels, which may be useful given the high homology among them and evidence of redundant signaling. Here we tested selective TRPC3/6 antagonists (GSK2332255B and GSK2833503A; IC50, 3-21 nM against TRPC3 and TRPC6) and found dose-dependent blockade of cell hypertrophy signaling triggered by angiotensin II or endothelin-1 in HEK293T cells as well as in neonatal and adult cardiac myocytes. In vivo efficacy in mice and rats was greatly limited by rapid metabolism and high protein binding, although antifibrotic effects with pressure overload were observed. Intriguingly, although gene deletion of TRPC3 or TRPC6 alone did not protect against hypertrophy or dysfunction from pressure overload, combined deletion was protective, supporting the value of dual inhibition. Further development of this pharmaceutical class may yield a useful therapeutic agent for heart disease management.

Keywords: Gq-coupled protein receptors; calcium; ion channels; myocardial; nuclear factor of activated T cells.

Fig. 1.

Efficacy of TRPC3/6 inhibitor GSK255B on hormone-induced hypertrophic signaling in cells. NFAT activation by Ang II is blocked in a dose dependent manner by GSK255B (0.01, 0.1, and 1 μM) in HEK293T cells overexpressing TRPC3 (A) or TRPC6 (B). Cells without channel up-regulation show less Ang II-stimulated NFAT, and no response to the inhibitors, supporting selectivity. An inactive control compound (GSK383A) is also tested at 0.1 μM concentration, and shows no impact. P values shown are for one-way ANOVA or KW test; symbols denote results of unpaired or post hoc multiple-comparison tests: ^#P < 0.05 vs. inactive control; *P < 0.05 and ^†P < 0.005 vs. vehicle control and Ang II + pcDNA; ^§P < 0.005 vs. Ang II + TRPC3 (or TRPC6) and P < 0.005 vs. vehicle + pcDNA. (C) GSK255B (10 μM) blocks calcium entry stimulated by PE (20 μM) in rat neonatal cardiac myocytes. TG, thapsigargin (1 μM). (D) GSK255B blocks NFAT activation by Ang II in HEK293T cells expressing a mutant TRPC6 channel with T70 and S322 mutated to glutamic acid (SETE). Data were analyzed by one-way ANOVA or the Kruskal–Wallis test in the Ang II treatment group for all three TRPC6 channel types. Post hoc testing: *P < 0.01 vs. non–Ang II-stimulated control for pcDNA, TRPC6-WT, or TRPC6-SETE transfected cells; ^#P < 0.005 vs. corresponding response (with or without Ang II) for pcDNA or TRPC6-SETE transfected cells; ^†P < 0.05 vs. other groups in one-way ANOVA (horizontal line identifies groups). ^§n = 3 for this group.

Fig. 2.

Effects of TRPC3/6 inhibitor GSK503A on isolated cardiomyocytes. (A) GSK503A (0.1, 1, 5, and 10 μM) dose-dependently inhibits ET-1–stimulated Rcan-1 luciferase activity in rat neonatal myocytes. ^#P < 0.05 vs. inactive control (GSK678A); *P < 0.05 vs. vehicle control. (B) Current-voltage (I-V) relationship for L-type Ca²⁺ current (I-V_Ca-L) in adult mouse cardiomyocytes treated with GSK503A (10 µM). (C) Gene expression of hypertrophy-related genes and TRPC channels in adult myocytes exposed to 24 h of ET-1 with or without coinhibition of TRPC3/6 (GSK503A, 10 μM). n = 6–9 for each condition. Rcan1: *P < 0.03 vs. control, ^#P = 0.011 vs. ET-1; Nppa: *P < 0.01 vs. control, ^#P < 0.03 vs. ET-1; Nppb: *P < 0.001 vs. control, ^#P < 0.03 vs. ET-1; Trpc1: *P = 0.06 vs. control; Trpc6: *P = 0.01 vs. control, ^#P < 0.05 vs. ET-1.

Fig. 3.

Echocardiographic analyses of LV mass and fractional shortening in mice selectively lacking (A) Trpc3 (C3-/-), (B) Trpc6 (C6^−/−), or (C) both genes and their respective littermate controls (WT) each subjected to TAC. P values denote (group) × (time) interaction based on ANCOVA; symbols identify interaction terms for pairwise covariance analysis versus sham control (*P < 0.001; ^†P < 0.02; ^§P < 0.01) or WT-TAC (^#P < 0.02). Sham control data combine both littermates and KOs for each group, because there was no significant difference between them.

Fig. 4.

Analyses of morphometric and molecular parameters of hypertrophy in mice selectively lacking Trpc3, Trpc6, or both genes and then subjected to TAC. (A) Heart weight/tibia length (at 6–7 wk for dKO and controls and 3 wk for other models). (B) Myocyte cross-sectional area assessed by wheat germ agglutinin staining at the terminal study for each model. P values for one-way ANOVA for each genotype, post hoc test results: *P < 0.05 vs. sham; ^#P < 0.05 vs. WT-TAC. (C) Response of hypertrophy-related fetal genes (Nppa and Nppb) for each model. *P < 0.05 vs. sham; ^#P < 0.05 vs. WT-TAC; ^§P = 0.07 vs. WT-TAC.