Deletion of osteopontin enhances β₂-adrenergic receptor-dependent anti-fibrotic signaling in cardiomyocytes

Cardiac β2-adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. The β2 AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates the 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 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory cytokine, which also mediates fibrosis in several tissues, including the heart. OPN underlies several cardiovascular pathologies, including atherosclerosis and cardiac adverse remodeling. We found that the cardiotoxic hormone aldosterone transcriptionally upregulates OPN in H9c2 rat cardiac myoblasts—an effect prevented by endogenous β2 AR activation. Additionally, CRISPR-mediated OPN deletion enhanced cAMP generation in response to both β1 AR and β2 AR activation in H9c2 cardiomyocytes, leading to the upregulation of Epac1 protein levels. These effects rendered β2 AR stimulation capable of completely abrogating transforming growth factor (TGF)-β-dependent fibrosis in OPN-lacking H9c2 cardiomyocytes. Finally, OPN interacted constitutively with Gα s subunits in H9c2 cardiac cells. Thus, we uncovered a direct inhibitory role of OPN in cardiac β2 AR anti-fibrotic signaling via cAMP/Epac1. OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.