Phosphorylation of TRPC6 channels at Thr69 is required for anti-hypertrophic effects of phosphodiesterase 5 inhibition.
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.
Nishida
Motohiro
M
Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.
Watanabe
Kenta
K
Sato
Yoji
Y
Nakaya
Michio
M
Kitajima
Naoyuki
N
Ide
Tomomi
T
Inoue
Ryuji
R
Kurose
Hitoshi
H
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D006332:Cardiomegaly / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control; D002460:Cell Line; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000235:genetics / Q000378:metabolism*; D004075:Diglycerides / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D008297:Male; D051379:Mice; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000378:metabolism*; D058986:Phosphodiesterase 5 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000009:adverse effects / Q000494:pharmacology; D010879:Piperazines / Q000009:adverse effects / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D011687:Purines / Q000009:adverse effects / Q000494:pharmacology; D051381:Rats; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000009:adverse effects / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel
D004075:Diglycerides; D058986:Phosphodiesterase 5 Inhibitors; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C492146:Trpc6 protein, mouse; C492145:Trpc6 protein, rat; D011189:Potassium Chloride; D000068677:Sildenafil Citrate; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517382:Pde5a protein, mouse; C517384:Pde5a protein, rat; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium