In vivo selective inhibition of TRPC6 by antagonist BI 749327 ameliorates fibrosis and dysfunction in cardiac and renal disease

Abstract

Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC 50 13 nM against mouse TRPC6, t 1/2 8.5-13.5 hours) with 85-and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis. TRPC6 | ion channels | calcium | nuclear factor of activated T cells | fibrosis

Ca2+ Signaling in Cardiac Fibroblasts and Fibrosis-Associated Heart Diseases

“…Most of the TRPC channels, TRPC1, TRPC3, TRPC6, TRPC7, and the heterotetrameric channel complexes TRPC3/6/7, TRPC1/4/5, and TRPC1/TRPC4 [202], have been demonstrated to be important mediators of pathological hypertrophy, and may serve as therapeutic targets [201,203]. Indeed, the TRPC3 blocker Pyrazole-3 (Pyr3) [166], the combined TRPC3 and TRPC6 channel blockers GSK2332255B and GSK2833503A [204], and the TRPC6 channel blocker BI-749327 [38], are effective at attenuating pathological remodeling and improving heart function. Although these studies provide strong evidence that TRPC channels in cardiac myocytes play an essential role in mediating hypertrophic remodeling, it remains unclear whether TRPC channels in cardiac fibroblasts contribute to pathological remodeling.…”

“…Increasing the differentiation of fibroblasts is, therefore, essential for initiating and perpetuating the fibrogenesis cascade [28][29][30][31][32][33][34] and the formation of fibrosis, which is involved in various pathological cardiac remodeling processes [9,35] (Figure 1). Numerous signaling pathways are involved in the activation of cardiac fibrogenesis [36], among which intracellular Ca 2+ has been found to play a particularly critical role [37][38][39]. The Ca 2+ signaling mechanisms in cardiac fibroblasts, however, are not fully understood.…”

TRPC Channels in Proteinuric Kidney Diseases

“…In a separate study, Wu et al found that TRPC6 knockout provided partial protection from renal fibrosis in mice subjected to UUO, and a comparable beneficial effect was observed in obstructed kidneys following treatment with the non-selective TRPC inhibitor BTP2 [76]. Moreover, a similar beneficial effect was observed by another group in a mouse UUO model using the selective TRPC6 inhibitor BI-749327 [166]. In the Wu et al study, the authors also examined the effects of soluble klotho in the UUO model on the basis of the following observations: (1) the investigators had previously demonstrated that soluble klotho inhibits TRPC6 exocytosis and TRPC6 currents in the heart [167], and (2) treatment with soluble klotho inhibits renal fibrosis in obstructed kidneys [168].…”

Analysis of Mrgprb2 Receptor-Evoked Ca2+ Signaling in Bone Marrow Derived (BMMC) and Peritoneal (PMC) Mast Cells of TRPC-Deficient Mice

“…The contribution of TRPC6 to the compound 48/80-evoked current was elaborated by genetic deletion of TRPC6 expression. The deduced concept of TRPC6 as an indispensable constituent of cation channels activated by compound 48/80 in PMCs could be corroborated in future studies by experimental evidence showing that acute blockage of these currents using TRPC6-specific antagonists such as Pyrazolo [1,5-a] pyrimidines antagonist (62) or BI 749327 (63).…”