Osteopontin & β2-adrenergic receptor signaling in cardiac fibrosis

Abstract

Objective: Test the role of osteopontin in anti-fibrotic β2AR signaling in cardiac myocytes. Background: Cardiac β2-adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. β2AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates generation of cyclic 3`, 5`-adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac). Epac1 inhibits cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory and pro-fibrotic cytokine, including in the heart. Methods: We used the H9c2 cardiac myocyte cell line & the real-time qPCR, co-immunoprecipitation/western blotting, and ELISA techniques. Results: The cardiotoxic hormone aldosterone upregulates OPN via the mineralocorticoid receptor (MR) in H9c2 cardiomyocytes. This is prevented by β2AR-activated GPCR-kinase (GRK)-5. GRK5 directly phosphorylates and inhibits the MR in cardiomyocytes. Additionally, CRISPR-mediated OPN deletion enhances β AR-dependent cAMP generation in H9c2 cardiomyocytes and upregulates Epac1. OPN deletion also enables the β AR to completely abrogate transforming growth factor (TGF)-β-dependent fibrosis in H9c2 cardiomyocytes. Mechanistically, OPN interacts with Gsa subunits to facilitate recruitment of GRK2, the major GRK phosphorylating and desensitizing the cardiac β2AR. This, in turn, augments the GRK2-dependent functional desensitization of the β2AR, thereby opposing this receptor`s anti-fibrotic cAMP/Epac1 signaling. Conclusion: We have uncovered a direct inhibitory effect of OPN in cardiac β2AR`s anti-fibrotic signaling via facilitation of GRK2-mediated receptor desensitization. Thus, OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis. Grants: PFRDG FY2018-19 #33546