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.
Highlights
Aldosterone (Aldo) is among the hormones for which levels are elevated and contributes significantly to the pathogenesis and morbidity/mortality of heart disease, including hypertension and chronic heart failure (CHF) [1]
We have uncovered a new unconventional substrate for cardiac GRK5, the mineralocorticoid receptor (MR), which results in cardioprotective effects of this kinase against Aldo
The major limitation of our study is that its findings await in vivo validation. We have confirmed this novel role for cardiac GRK5 in a bona fide cardiomyocyte cell system (ARVMs) and we have delineated the underlying signaling mechanism
Summary
Aldosterone (Aldo) is among the hormones for which levels are elevated and contributes significantly to the pathogenesis and morbidity/mortality of heart disease, including hypertension and chronic heart failure (CHF) [1]. Most of the pathophysiological effects of Aldo in the heart and vessels, which can cause essential hypertension and trigger cardiac hypertrophy, fibrosis, and adverse remodeling, are thought to result from MR activation, whereas several studies suggest that GPER activation may be beneficial in the myocardium, partly due to activation of antiapoptotic mediators, such as Epidermal Growth Factor Receptor (EGFR) and extracellular signal-regulated kinase (ERK)1/2 [2,3,4,5,6,7]). The MR is known to be phosphorylated at various Ser/Thr residues, including Ser-843 of the ligand-binding domain and Ser-601 of the N-terminal domain [11] These phosphorylating events occur in the cytoplasm, and some of them result in MR inhibition either via direct repression of transcriptional activity (e.g., the Ser-843 phosphorylation) or via cytosolic retention, i.e., blockade of nuclear translocation (e.g., the Ser-601 phosphorylation) [11]. We sought to investigate the potential involvement of GRK2 and -5 in the regulation of cardiac Aldo signaling via the MR and/or GPER
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