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

Abstract

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 Ca 21 entry (SOCE), is the downstream effector of PKG. Treatment of hESC-CMs with an a-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 Ser 34 in Orai1 abolished the antihypertrophic effects of NO, cGMP, and PKG. Furthermore, PKG could directly phosphorylate Orai1 at Ser 34 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. STEM CELLS 2015;33:2973-2984 SIGNIFICANCE STATEMENTCardiac hypertrophy is an abnormal enlargement of cardiomyocytes. It contributes to congestive heart failure. Nitric oxide donors and cGMP-phosphodiesterase inhibitors have been used in clinical trials to treat cardiac hypertrophy. However, the underlying mechanisms of these agents are not fully understood. Here, with the use of human embryonic stem cell-derived cardiomyocytes as the model, we uncovered a novel mechanism underlying the inhibitory effect of these agents on cardiac hypertrophy. We found that nitric oxide and cGMP, via protein kinase G-mediated phosphorylation on Orai1 proteins, inhibit the cardiomyocyte hypertrophy. This study provides novel mechanistic insights into the action of nitric oxide-related anti-hypertrophic agents.

Gastrodin Inhibits Store-Operated Ca2+ Entry and Alleviates Cardiac Hypertrophy

“…The molecular determinants of SOCE include STIM1 and Orai1, in which STIM1 serves as a Ca 2+ sensor in sarco/endoplasmic reticulum whereas Orai1 functions as the pore-forming subunit for Ca 2+ permeation. Previous studies from us and others presented the data consistent with the hypothesis that SOCE and its molecular determinants STIM1 and Orai1 can promote hypertrophy in rat neonatal cardiomyocytes, adult cardiomyocytes and embryonic stem-cell derived cardiomyocytes (Hunton et al, 2002; Voelkers et al, 2010; Hulot et al, 2011; Luo et al, 2012; Wang et al, 2015). …”

“…Previous reports suggest that SOCE is an important immediate signal that contribute to agonist-induced cardiac hypertrophy (Hunton et al, 2002; Voelkers et al, 2010; Hulot et al, 2011; Luo et al, 2012; Wang et al, 2015). This was confirmed in the present study.…”

“…Thus, a more recent trend in anti-hypertrophic therapy focuses on small molecules that act inside the cardiomyocytes targeting crucial signaling cascades that alter cardiomyocyte remodeling, including the inhibitors for histone deacetylase (Schiattarella and Hill, 2015) and G protein-coupled receptor kinase (Traynham et al, 2016). SOCE is a major Ca 2+ signaling pathway that plays a key role in promoting cardiac hypertrophy (Hunton et al, 2002; Voelkers et al, 2010; Hulot et al, 2011; Luo et al, 2012; Wang et al, 2015). Pro-hypertrophic role of SOCE has been demonstrated both in the pressure overload-induced hypertrophic models (Hulot et al, 2011; Luo et al, 2012) and the neurohormonal agent-induced hypertrophic models (Hunton et al, 2002; Ohba et al, 2009; Voelkers et al, 2010; Wang et al, 2015).…”

“…SOCE is a major Ca 2+ signaling pathway that plays a key role in promoting cardiac hypertrophy (Hunton et al, 2002; Voelkers et al, 2010; Hulot et al, 2011; Luo et al, 2012; Wang et al, 2015). Pro-hypertrophic role of SOCE has been demonstrated both in the pressure overload-induced hypertrophic models (Hulot et al, 2011; Luo et al, 2012) and the neurohormonal agent-induced hypertrophic models (Hunton et al, 2002; Ohba et al, 2009; Voelkers et al, 2010; Wang et al, 2015). Therefore, SOCE and its molecular determinants STIM1 and Orai1 offer attractive targets for anti-hypertrophic therapy.…”

Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells

“…The Orai1 N-terminal intracellular region contains several phosphorylation sites. Ser-27 and -30 are the main phosphorylation sites targeted by PKCβ1 [20], while Ser-34 was found to be a target of protein kinase G (PKG) [36] and, more recently, Zhang and coworkers revealed that Ser-34 is also a PKA phosphorylation site [21]. Phosphorylation of Orai1 at any of these serine residues was demonstrated to result in Orai1 channel inactivation and reduction of Ca 2+ influx.…”