Phosphorylation of TRPC6 channels at Thr69 is required for anti-hypertrophic effects of phosphodiesterase 5 inhibition

Abstract

Activation of Ca(2+) signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca(2+) signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca(2+) influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca(2+) spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca(2+) influx itself or molecule(s) upstream of Ca(2+) influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr(69) and prevented TRPC6-mediated Ca(2+) influx. Substitution of Ala for Thr(69) in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G alpha(q)-mediated signaling, did not affect the suppression of receptor-activated Ca(2+) influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.

FIGURE 1.

Inhibition of PDE5 suppresses agonist-induced cardiomyocyte hypertrophic responses through inhibition of DAG-mediated Ca²⁺ signaling. A–C, effects of PDE5-I on agonist-induced hypertrophic responses (actin reorganization (A), BNP expression (B), and protein synthesis (C)). Cardiomyocytes were treated with PDE5-I (10 μm) for 20 min before the addition of Ang II (1 μm) or ET-1 (100 nm). Scale bar, 50 μm. D, effects of PDE5-I on NFAT activation induced by ET-1 and OAG (30 μm). E–H, average time courses of Ca²⁺ responses induced by ET-1 (E), OAG (F), and KCl (H) in the absence or presence of PDE5-I. G, effects of PDE5-I on OAG-induced increase in membrane potential (MP). Cardiomyocytes were treated with OAG for 20 min, and maximal increase in MP was calculated from peak changes in DiBAC₄(3) fluorescence intensity (21). H, voltage-dependent Ca²⁺ influx was evoked by KCl (8 mm) for 8 min, and nitrendipine (10 μm) was added to inhibit the activities of voltage-dependent Ca²⁺ channels.

FIGURE 2.

Inhibition of PDE5 does not suppress the agonist-independent cardiomyocyte hypertrophic responses. A and C, effects of PDE5-I on hypertrophic responses (actin reorganization, protein synthesis, and increases in area of cardiomyocytes) induced by KCl and phorbol 12-myristrate 13-acetate (PMA). Cardiomyocytes were stimulated with Ang II (1 μm), ET-1 (100 nm), KCl (5 mm), or phorbol 12-myristrate 13-acetate (1 μm) for 48 h. B and C, effects of PDE5-I on hypertrophic growth (B) and protein synthesis (C) in green fluorescent protein- and CA-NFAT-expressing cardiomyocytes. Scale bar, 50 μm. **, p < 0.01; n.s., no significance.

FIGURE 3.

TRPC6 mediates ET-1-induced cardiomyocyte hypertrophic responses. A, Ca²⁺ responses induced by ET-1 (100 nm) in LacZ- and DN-TRPC6-overexpressing cardiomyocytes. B, results of the frequency of Ca²⁺ oscillations induced by ET-1 or OAG (30 μm). Cardiomyocytes were pretreated with PDE5-I (10 μm) 35 min before agonist stimulation. C and D, effects of DN-TRPC6 on ET-1-induced actin reorganization and increase in cell size (C) and protein synthesis (D). Scale bar, 50 μm.

FIGURE 4.

PKG-dependent suppression of TRPC6-mediated Ca²⁺ influx by PDE5 inhibition. A, average time courses of Ca²⁺ responses induced by OAG (30 μm) in vector and TRPC6-expressing HEK293 cells with or without PDE5-I. B, peak increases in [Ca²⁺]_i induced by OAG in vector- and TRPC6-expressing cells. HEK293 cells were treated with S-nitroso-N-acetyl-dl-penicillamine (SNAP) (100 μm), 8-Br-cGMP (100 μm), or PDE5-I (10 μm) for 30 min before the addition of OAG (30 μm). C and D, concentration-dependent (C) and time-dependent (D) suppression of OAG-induced [Ca²⁺]_i increases by PDE5-I. E and F, effects of PDE5-I on OAG-induced Ca²⁺ influx-mediated [Ca²⁺]_i increases in TRPC6 (WT)- and TRPC6 (T69A)-expressing cells. HEK293 cells were treated with KT5823 (1 μm) for 30 min before the addition of OAG. *, p < 0.05 versus vector (white bar); #, p < 0.05 versus TRPC6 (−) control (black bar).

FIGURE 5.

Specific recognition of TRPC6 phosphorylation at Thr⁶⁹ by a phospho-specific antibody. A, phosphorylation of TRPC6 at Thr⁶⁹ induced by PKG activation in vector-, TRPC3-, TRPC6-, and TRPC7-expressing HEK293 cells. HEK293 cells were treated with 8-Br-cGMP (100 μm) for 2 h. B, optimization of dilution of anti-phospho-TRPC6 antibody. Antibody (0.56 mg/ml) was diluted with Tris-buffered saline plus 0.1% Tween 20 (TBS-T) and incubated with blots for 1 h at room temperature. C, effect of treatment with a TRPC6-blocking peptide on the recognition of TRPC6 phosphorylation by this antibody. Blots were incubated with phospho-TRPC6 antibody diluted 1:1000 in TBS-T with phospho-TRPC6-blocking peptide or non-phosphorylated TRPC6-blocking peptide (10 μg/ml) for 1 h at room temperature.

FIGURE 6.

PKG-dependent phosphorylation of Thr⁶⁹ in TRPC6 by PDE5 inhibition. A, PKG-dependent phosphorylation of TRPC6 proteins at Thr⁶⁹ in TRPC6 (WT)- and TRPC6 (T69A)-expressing HEK293 cells. HEK293 cells were treated with KT5823 (1 μm) for 20 min before the addition of 8-Br-cGMP (100 μm) and PDE5-I (10 μm) for 30 min. B, PKG-dependent TRPC6 phosphorylation by PDE5-I. Cardiomyocytes were treated with KT5823 for 20 min before the addition of PDE5-I (10 μm) for 1 h. C, effects of sildenafil on the phosphorylation of TRPC6 in mice. One week after oral administration with sildenafil (100 mg/kg/day), hearts were lysed with radioimmune precipitation buffer, and 100 μg of proteins were applied to SDS-PAGE.

FIGURE 7.

Phosphorylation of Thr⁶⁹ is essential for the anti-hypertrophic effects of PDE5 inhibition. A and B, effects of PDE5-I on ET-1-induced NFAT activation (A) and BNP gene expression (B) in TRPC6 (T69A)-expressing cardiomyocytes. C and D, effects of PDE5-I on the ET-1-induced protein synthesis (C) and increase in the size of TRPC6 (T69A)-overexpressing cardiomyocytes (D). Cardiomyocytes were treated with PDE5-I (10 μm) for 20 min before the addition of ET-1 (100 nm). Scale bar, 50 μm. *, p < 0.05; **, p < 0.01 versus without (−) ET-1 within vector. #, p < 0.05 versus vector with (+) ET-1. †, p < 0.01 versus TRPC6-WT with ET-1.

FIGURE 8.

Inhibition of PDE5 suppresses Ca²⁺ responses and cardiomyocyte hypertrophic responses induced by mechanical stretch. A, typical traces of Ca²⁺ responses induced by mechanical stretch (MS) in the absence or presence of PDE5-I, KT5823, or BTP2. Cardiomyocytes were treated with PDE5-I (10 μm) or BTP2 (5 μm) for 35 min before MS. DN-TRPC6 proteins were expressed using adenoviral infection. B–D, effects of PDE5-I, BTP2, and DN-TRPC6 on the MS-induced NFAT activation (B) and hypertrophic responses (BNP gene expression (C) and protein synthesis (D)).

FIGURE 9.

PDE5-I suppresses ET-1-induced Ca²⁺ responses in RGS2/4-down-regulated cardiomyocytes. A and B, average time courses of Ca²⁺ responses induced by ET-1 in the absence or presence of PDE5-I in control siRNA-treated cardiomyocytes (A) and RGS2/4 siRNAs-treated cardiomyocytes (B). C, peak Ca²⁺ releases (0 mm Ca²⁺) and Ca²⁺ influx-mediated increases in [Ca²⁺]_i (2 mm Ca²⁺) induced by ET-1 (100 nm). D, effects of knockdown of RGS2/4 proteins on PKG-dependent inhibition of ET-1-induced NFAT activation by PDE5-I. *, p < 0.05 versus control siRNA-treated cardiomyocytes without PDE5-I; #, p < 0.05 versus RGS2/4 siRNA-treated cardiomyocytes without PDE5-I.