Antagonistic Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Reveal GRK5-Mediated Cardioprotection via Mineralocorticoid Receptor Inhibition

Abstract

Aldosterone (Aldo), when overproduced, is a cardiotoxic hormone underlying heart failure and hypertension. Aldo exerts damaging effects via the mineralocorticoid receptor (MR) but also activates the antiapoptotic G protein-coupled estrogen receptor (GPER) in the heart. G protein-coupled receptor (GPCR)-kinase (GRK)-2 and -5 are the most abundant cardiac GRKs and phosphorylate GPCRs as well as non-GPCR substrates. Herein, we investigated whether they phosphorylate and regulate cardiac MR and GPER. To this end, we used the cardiomyocyte cell line H9c2 and adult rat ventricular myocytes (ARVMs), in which we manipulated GRK5 protein levels via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and GRK2 activity via pharmacological inhibition. We report that GRK5 phosphorylates and inhibits the cardiac MR whereas GRK2 phosphorylates and desensitizes GPER. In H9c2 cardiomyocytes, GRK5 interacts with and phosphorylates the MR upon β2-adrenergic receptor (AR) activation. In contrast, GRK2 opposes agonist-activated GPER signaling. Importantly, GRK5-dependent MR phosphorylation of the MR inhibits transcriptional activity, since aldosterone-induced gene transcription is markedly suppressed in GRK5-overexpressing cardiomyocytes. Conversely, GRK5 gene deletion augments cardiac MR transcriptional activity. β2AR-stimulated GRK5 phosphorylates and inhibits the MR also in ARVMs. Additionally, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis and oxidative stress in ARVMs. In conclusion, GRK5 blocks the cardiotoxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder beneficial effects of Aldo through GPER. Thus, cardiac GRK5 stimulation (e.g., via β2AR activation) might be of therapeutic value for heart disease treatment via boosting the efficacy of MR antagonists against Aldo-mediated cardiac injury.

GRK5 is an Essential Co-repressor for Cardiac Mineralocorticoid Receptor Antagonism induced by Finerenone but not Eplerenone

“…The H9c2 rat cardiomyoblast cell line was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured as previously described [11,16,17]. Neonatal rat ventricular myocytes (NRVMs) were isolated and cultured, as previously described [18].…”

“…Neonatal rat ventricular myocytes (NRVMs) were isolated and cultured, as previously described [18]. Recombinant lentiviruses encoding for wild-type full-length GRK5 or for empty vector (control) (OriGene Technologies, Rockville, MD, USA) were propagated and purified via CsCl density gradient ultracentrifugation, as described previously [11,19]. For CRISPR/Cas9-mediated GRK5 gene deletion, a gRNA sequence was custom-synthesized by Sigma-Aldrich (target ID: RN0000391809, target sequence: 5'-GTGGTTTGAATTTATGCGG-3') and incorporated into a lentiviral vector (Sigma-Aldrich).…”

“…Cell extracts were prepared, as described previously [11,20], in a 20-mM Tris pH 7.4 buffer containing 137 mM NaCl, 1% Nonidet P-40, 20% glycerol, 10 mM phenylmethylsulfonylfluoride Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 September 2020 doi:10.20944/preprints202009.0374.v1…”

“…Luciferase reporter activity assay was performed, as described previously, by transfecting the cells with the LightSwitch™ luciferase reporter gene vector under the influence of the MR promoter (Active Motif, Inc., Carlsbad, CA, USA) [11]. The measurements were done the next day with the manufacturer's LightSwitch™ assay kit and according to the manufacturer`s instructions.…”

“…Both phosphorylate GPCRs but also non-GPCR substrates [6][7][8][9][10]. We recently showed that GRK5 blocks the cardio-toxic MR-dependent effects of aldosterone in the heart by directly phosphorylating the cardiac MR and inhibiting its transcriptional activity [11].…”