Abstract 3087: Cardiomyocyte Protein Kinase G Activation by Phosphodiesterase-5 Inhibition Suppresses Canonical Transient Receptor Potential (TRPC) Channel 6 Expression/Activity and Cardiac Hypertrophy Signaling
Inhibition of cGMP selective phosphodiesterase type 5 (PDE5) blocks cardiac pathological hypertrophy by suppressing Gαq and calcineurin (Cn)-nuclear factor of activated T-cells (NFAT) signaling. Canonical transient receptor potential (TRPC) calcium channel expression and activity (notably TRPC6) have been recently shown to couple to Cn-NFAT activation and cardiac hypertrophy. Here, we show that the PDE5A antagonist, sildenafil (SIL) blocks TRPC6 gene up-regulation in pressure-overload hypertrophy in mice, and inhibits angiotensin II (AII) and endothelin-1 (ET1) stimulated Trpc6 expression in cultured neonatal and adult cardiomyocytes. This regulation is mimicked by cGMP stimulation, and dependent on protein kinase G (PKG) activation. Deletion of Trpc6 promotor NFAT binding sites depressed ET-1-mediated Trpc6 expression, but was not sufficient to prevent SIL-mediated inhibition. TRPC6 current measured by patch clamp is strongly suppressed by cGMP- PKG activation in HEK cells, and phospho-silencing mutations at potential PKG targets (T70A, S322Q) largely blocked this suppression. AII or ET1 increased NFAT activation (luciferase-reporter) and protein synthesis (leucine incorporation) in neonatal rat cardiomyocytes, and SIL (or cGMP) inhibited this activation. Transfection with TRPC6T70A or TRPC6S322Q blocked inhibitory effects of cGMP or SIL, whereas phospho-mimetic mutants (T70E, S322E) constitutively reduced NFAT activation induced by either agonist. Given that TRPC channel activity regulates TRPC6 gene regulation and has a critical role in sustained NFAT activation of pathological hypertrophy, these results identify a central mechanism underlying cGMP/PKG (and SIL) inhibition of NFAT activation (and thus myocyte hypertrophy) mediated by the direct phosphorylation of TRPC6 at one of two residues, and subsequent inhibition of channel conductance of calcium.