Aldosterone And Osteopontin Promote Cardiac Fibrosis Via GRK2‐dependent β 2 ‐Adrenergic Receptor Dysfunction

Background Cardiac β 2 ‐adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. β 2 AR is a G s 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 (Epac1/2). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro‐inflammatory and pro‐fibrotic cytokine, including in the heart. OPN expression is known to be induced by the cardiotoxic hormone aldosterone in various tissues and OPN has been reported to dysregulate β 2 AR signaling and function in bone cells. Thus, we hypothesized here that aldosterone upregulates OPN in the heart in order to perturb β 2 AR anti‐fibrotic function. Methods We used the rat cardiomyoblast cell line H9c2 and human cardiac fibroblasts (HCFs). We measured OPN levels and fibrosis markers via real time PCR and β 2 AR function via cAMP accumulation studies and co‐immunoprecipitation/western blotting for several downstream signaling mediators. We also deleted OPN via CRISPR/Cas9 to examine its role in cardiac β 2 AR function and fibrosis. Results Aldosterone upregulates OPN via the mineralocorticoid receptor (MR) in H9c2 cardiomyocytes. This is prevented by β 2 AR activation with salbutamol (albuterol), which stimulates GPCR‐kinase (GRK)‐5 to phosphorylate the cardiac MR and inhibit its transcriptional activity. Importantly, CRISPR/Cas9‐mediated OPN gene deletion enhances β 2 AR‐dependent cAMP generation in H9c2 cardiomyocytes and upregulates Epac1 protein levels. The functional result of this is that β 2 AR’s inhibition of transforming growth factor (TGF)‐β‐dependent fibrosis in H9c2 cardiomyocytes is robustly augmented. In addition, pharmacological inhibition of GRK2, the major GRK that phosphorylates and desensitizes the cardiac β 2 AR, with Cmpd101 or OPN CRIPSR/Cas9‐mediated knockout completely abrogates isoproterenol (agonist)‐induced β 2 AR functional desensitization in HCFs (Isoproterenol’s ΔEC 50 : 94±16 μM in control HCFs, p<0.05, n=4; 6±7.2 μM in Cmpd101‐treated HCFs, Not significant at p=0.05, n=4; 5.3+6.1 μM in OPN knockout HCFs, Not significant at p=0.05, n=4). Mechanistically, OPN dysregulates the β 2 AR via enhancement of GRK2‐mediated functional desensitization, since OPN forms a multi‐protein complex with GRK2 and GRK2‐interacting protein (GIT)‐1 in HCFs, thereby facilitating GRK2 plasma membrane recruitment. This reduces β 2 AR’s anti‐fibrotic cAMP signaling in the heart. Conclusions We have uncovered a direct inhibitory effect of OPN on cardiac β 2 AR’s anti‐fibrotic signaling: facilitation of GRK2‐mediated receptor desensitization. Since aldosterone/MR upregulates OPN, a combination of an MR blocker with OPN or GRK2 blockade could be of value in cardiac fibrosis treatment. Support or Funding Information 1) American Heart Association (A.L.)2) NSU President’s Faculty Research & Development Grant (A.L.)