Deletion of Osteopontin Enhances β2-Adrenergic Receptor-Dependent Anti-Fibrotic Signaling in Cardiomyocytes

Abstract

Cardiac β2-adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. The β2AR 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 β2AR activation. Additionally, CRISPR-mediated OPN deletion enhanced cAMP generation in response to both β1AR and β2AR activation in H9c2 cardiomyocytes, leading to the upregulation of Epac1 protein levels. These effects rendered β2AR stimulation capable of completely abrogating transforming growth factor (TGF)-β-dependent fibrosis in OPN-lacking H9c2 cardiomyocytes. Finally, OPN interacted constitutively with Gαs subunits in H9c2 cardiac cells. Thus, we uncovered a direct inhibitory role of OPN in cardiac β2AR anti-fibrotic signaling via cAMP/Epac1. OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.

Osteopontin: A Promising Therapeutic Target in Cardiac Fibrosis

“…Many studies have demonstrated the efficacy of partial or complete OPN inhibition in a variety of preclinical models of diseases, including CH [15,37,39], coronary artery disease [24,28,58,59], DCM [46], diabetic cardiomyopathy [48], heart failure [52], and cardiac fibrosis [13,15,38,39,60]. The earliest study by Matsui et al demonstrated that the Ang-II-induced elevation in blood pressure, collagen deposition, and development of cardiac fibrosis was significantly reduced in OPN −/− mice compared to wild type (WT) mice [38].…”

“…β-adrenergic receptor (AR) blockers have demonstrated significant survival benefits and have become standard therapy for adults battling ischemic heart disease. Current appreciation of β-receptor signaling involves activating pathways by which these receptors crosstalk with other signaling pathways; making the β1-AR the "cardiotoxic subtype" whereas the β2-AR appears to be the "cardioprotective" one [60]. While the more well-known β1-AR mediates the positive inotropic and chronotropic actions of the sympathetic nervous system, β2-AR has been shown to exert several cardioprotective effects in the heart post-MI, such as inhibition of fibrosis as well as inflammation and apoptosis [5].…”

“…While the more well-known β1-AR mediates the positive inotropic and chronotropic actions of the sympathetic nervous system, β2-AR has been shown to exert several cardioprotective effects in the heart post-MI, such as inhibition of fibrosis as well as inflammation and apoptosis [5]. As a Gs protein-coupled receptor (GPCR), β2-AR antifibrotic effects have been attributed to the stimulation and generation of cyclic 3 ,5 -adenosine monophosphate (cAMP), which activates both protein kinase A (PKA) and Epac, an exchange protein directly activated by cAMP known to inhibit cardiac fibroblast activation and fibrosis [60]. Pollard et al sought to delineate the role of OPN on β-AR-regulated cardiomyoblast fibrosis, using the H9c2 rat cardiac myoblast cell line [60].…”

“…As a Gs protein-coupled receptor (GPCR), β2-AR antifibrotic effects have been attributed to the stimulation and generation of cyclic 3 ,5 -adenosine monophosphate (cAMP), which activates both protein kinase A (PKA) and Epac, an exchange protein directly activated by cAMP known to inhibit cardiac fibroblast activation and fibrosis [60]. Pollard et al sought to delineate the role of OPN on β-AR-regulated cardiomyoblast fibrosis, using the H9c2 rat cardiac myoblast cell line [60]. Interestingly, the study demonstrated that CRISPR-mediated OPN deletion enriched the release of cAMP, leading to the upregulation of Epac protein levels, and abrogating TGF-β-induced fibrosis, in response to β2-AR activation in H9c2 cells [60].…”

GRK2-Mediated Crosstalk Between β-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo

“…previously [15,33,35]. Densitometry was performed with the AlphaView software (Protein Simple) in the linear range of signal detection (on non-saturated bands).…”