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Nitric Oxide-cGMP-PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell-Derived
Y Wang1,2,3, Z C Li1,2, P Zhang1,2
1Li Ka Shing Institute of Health Sciences and School of Biomedical Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Hong Kong, People's Republic of China.
Insights
Nitric oxide (NO), cyclic GMP (cGMP), and protein kinase G (PKG) inhibit cardiac hypertrophy by phosphorylating Orai1. This PKG-mediated phosphorylation prevents Orai1-mediated store-operated calcium entry (SOCE), offering new therapeutic insights.
Area of Science:
- Cardiovascular Biology
- Cell Signaling
- Stem Cell Research
Background:
- Cardiac hypertrophy, an enlargement of heart muscle, leads to heart failure and death.
- The nitric oxide (NO)-cyclic GMP (cGMP)-protein kinase G (PKG) pathway is known to inhibit cardiac hypertrophy.
- The precise molecular mechanisms by which this pathway exerts its antihypertrophic effects remain incompletely understood.
Purpose of the Study:
- To elucidate the downstream molecular targets of the NO-cGMP-PKG pathway in cardiac hypertrophy.
- To investigate the role of Orai1, a component of store-operated calcium entry (SOCE), in cardiac hypertrophy.
- To determine if PKG directly interacts with and regulates Orai1 function.
Main Methods:
- Utilized human embryonic stem cell-derived cardiomyocytes (hESC-CMs) as a model system.
- Induced cardiac hypertrophy using phenylephrine (PE) and assessed Orai1 expression and activity.
- Employed Orai1-siRNAs and dominant-negative constructs to inhibit Orai1 function.
- Investigated the effects of NO, cGMP, and PKG activators on hypertrophy and Orai1 phosphorylation.
- Performed site-directed mutagenesis to examine the role of Orai1 Serine 34 (Ser34) phosphorylation.
Main Results:
- Phenylephrine-induced cardiac hypertrophy in hESC-CMs was associated with increased Orai1 expression.
- Inhibition of Orai1 suppressed PE-induced hypertrophy, indicating Orai1-mediated SOCE is crucial for hypertrophy development.
- NO, cGMP, and PKG activation inhibited hypertrophy, and this effect was abolished when Orai1 Ser34 was mutated to Alanine (Ala).
- PKG was found to directly phosphorylate Orai1 at Ser34, thereby inhibiting Orai1-mediated SOCE.
Conclusions:
- The NO-cGMP-PKG signaling pathway inhibits cardiac hypertrophy in hESC-CMs by directly phosphorylating Orai1 at Ser34.
- PKG-mediated phosphorylation of Orai1 at Ser34 prevents Orai1-mediated SOCE, a key contributor to hypertrophy.
- These findings reveal a novel mechanism for the antihypertrophic effects of NO and cGMP-related agents, such as sildenafil.
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(2+) 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 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.
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