Nitric Oxide-cGMP-PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell-Derived Cardiomyocytes

Cardiac hypertrophy is an abnormal enlargement of heart muscle. It frequently results in congestive heart failure, which is a leading cause of human death. Previous studies demonstrated that the nitric oxide (NO), cyclic GMP (cGMP), and protein kinase G (PKG) signaling pathway can inhibit cardiac hypertrophy and thus improve cardiac function. However, the underlying mechanisms are not fully understood. Here, based on the human embryonic stem cell-derived cardiomyocyte (hESC-CM) model system, we showed that Orai1, the pore-forming subunit of store-operated Ca²⁺ entry (SOCE), is the downstream effector of PKG. Treatment of hESC-CMs with an α-adrenoceptor agonist phenylephrine (PE) caused a marked hypertrophy, which was accompanied by an upregulation of Orai1. Moreover, suppression of Orai1 expression/activity using Orai1-siRNAs or a dominant-negative construct Orai1^G98A inhibited the hypertrophy, suggesting that Orai1-mediated SOCE is indispensable for the PE-induced hypertrophy of hESC-CMs. In addition, the hypertrophy was inhibited by NO and cGMP via activating PKG. Importantly, substitution of Ala for Ser³⁴ in Orai1 abolished the antihypertrophic effects of NO, cGMP, and PKG. Furthermore, PKG could directly phosphorylate Orai1 at Ser³⁴ and thus prevent Orai1-mediated SOCE. Together, we conclude that NO, cGMP, and PKG inhibit the hypertrophy of hESC-CMs via PKG-mediated phosphorylation on Orai1-Ser-34. These results provide novel mechanistic insights into the action of cGMP-PKG-related antihypertrophic agents, such as NO donors and sildenafil.