Cyclic GMP/PKG-dependent inhibition of TRPC6 channel activity and expression negatively regulates cardiomyocyte NFAT activation

Abstract

Increased cyclic GMP from enhanced synthesis or suppressed catabolism (e.g. PDE5 inhibition by sildenafil, SIL) activates protein kinase G (PKG) and blunts cardiac pathological hypertrophy. Suppressed calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling appears to be involved, though it remains unclear how this is achieved. One potential mechanism involves activation of Cn/NFAT by calcium entering via transient receptor potential canonical (TRPC) channels (notably TRPC6). Here, we tested the hypothesis that PKG blocks Cn/NFAT activation by modifying and thus inhibiting TRPC6 current to break the positive feedback loop involving NFAT and NFAT-dependent TRPC6 upregulation. TRPC6 expression rose with pressure-overload in vivo, and angiotensin (ATII) or endothelin (ET1) stimulation in neonatal and adult cardiomyocytes in vitro. 8Br-cGMP and SIL reduced ET1-stimulated TRPC6 expression and NFAT dephosphorylation (activity). TRPC6 upregulation was absent if its promoter was mutated with non-functional NFAT binding sites, whereas constitutively active NFAT triggered TRPC6 expression that was not inhibited by SIL. PKG phosphorylated TRPC6, and both T70 and S322 were targeted. Both sites were functionally relevant, as 8Br-cGMP strongly suppressed current in wild-type TRPC6 channels, but not in those with phospho-silencing mutations (T70A, S322A or S322Q). NFAT activation and increased protein synthesis stimulated by ATII or ET1 was blocked by 8Br-cGMP or SIL. However, transfection with T70A or S322Q TRPC6 mutants blocked this inhibitory effect, whereas phosphomimetic mutants (T70E, S322E, and both combined) suppressed NFAT activation. Thus PDE5-inhibition blocks TRPC6 channel activation and associated Cn/NFAT activation signaling by PKGdependent channel phosphorylation.

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

“…TRPC6 contains two residues in the N terminus, T70 and S322, that when phosphorylated by protein kinase G (PKG) potently suppress channel conductance and corresponding NFAT activation (9,21). Thus, we tested whether the TRPC3/6 antagonists were redundant or additive to this potent posttranslational regulation.…”

“…For evaluation of the effect of GSK503A in adult myocytes, hearts from C57BL/6J mice were first subjected to pressure overload (i.e., aortic constriction) to augment basal channel expression (9). Myocytes were then isolated and were subjected to 24-h stimulation with 0.1 μM ET-1 with or without 10 μM GSK503A.…”

“…TRPC3 and TRPC6 are highly homologous nonvoltage-gated and nonselective cation channels (6,7). They are normally expressed at very low levels in myocytes, but levels are increased in cardiac disease, such as pressure overload (3,4,8,9). TRPC6 is essential for the transformation of fibroblasts to myofibroblasts and corresponding scar formation (10).…”

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis

“…Furthermore, activation of TRPC channels followed by elevated [Ca 2+ ] i may contribute to the induction of the cardiac hypertrophy gene response (8)(9)(10)(11). Again, TRPC3 and TRPC6 channels are negatively regulated by cGKI (12)(13)(14).…”

“…This effect is postulated to be caused by an increased activity of RGS2 (19), and in part by the direct regulation of TRPC3/6 conductance by cGKI (11,14) mentioned above. However, two clinical studies did not report positive therapeutic results after prolonged treatment of diastolic dysfunction with sildenafil (20,21).…”