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.
Highlights
Cardiac fibrosis is a fundamental process mediating the adverse remodeling of the heart post-myocardial infarction (MI) or other ischemic injury, as well as in heart failure (HF) [1]
All adrenergic receptors (ARs) belong to the G protein-coupled receptor (GPCR) superfamily and both β1AR and β2AR activate the Gs protein signaling pathway in cardiac cells, leading to the activation of adenylyl cyclase (AC) and subsequent cyclic 3,5 -adenosine monophosphate synthesis [3]. cAMP is a major second messenger inside cells, and in cardiac myocytes it is responsible for the stimulation of cardiac contractility thanks mainly to activation of its effector kinase protein kinase A (PKA), which phosphorylates a variety of substrates to facilitate contraction in the cardiac myocyte [4,10,11]
Using the H9c2 rat cardiac myoblast cell line as our cell model, which endogenously expresses both β1AR and β2AR, as well as OPN [25,26], we found that OPN substantially impedes β2AR-induced, cAMP/Epac1-dependent anti-fibrotic signaling in cardiac cells
Summary
Cardiac fibrosis is a fundamental process mediating the adverse remodeling of the heart post-myocardial infarction (MI) or other ischemic injury, as well as in heart failure (HF) [1]. The main cell type driving the fibrotic process is activated myofibroblasts, with significant contributions by monocytes/macrophages, lymphocytes, mast cells, vascular endothelial cells, and cardiomyocytes All of these produce and secrete key pro-fibrotic factors such as reactive oxygen species, proteases, and metalloproteinases; and fibrosis-promoting growth factors, predominantly transforming growth factor (TGF)-β [2]. Epac is expressed ubiquitously and is quite abundant in the heart, including in cardiac fibroblasts [14] This Epac isoform has been documented to decrease collagen expression in response to βAR activation in rat cardiac fibroblasts and its expression is downregulated in the heart post-MI and in response to pro-fibrotic stimuli [12,14,15,16]. Epac blocks collagen and DNA synthesis in rat cardiac fibroblasts, and was very recently reported to block atrial fibroblast activation, migration, and secretion of fibrotic mediators in post-MI mice and in HF dogs [9,14]
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