Differential roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling
Authors: Maning Jennifer , McCrink Katie Anne , Brill Ava , Desimine Victoria , Aukszi Beatrix , Lymperopoulos Anastasios 
Published: November 2023

Background

Heart failure (HF) is the leading cause of death in the western world and new and innovative treatments are urgently needed. Aldosterone (Aldo) is one of several cardio‐toxic hormones, whose levels are increased in post‐myocardial infarction (MI) HF, and contributes significantly to the morbidity and mortality of the disease. Aldo binds to its cognate receptor, the mineralocorticoid receptor (MR), a nuclear receptor expressed in various tissues including the heart. However, Aldo also exerts MR‐independent (its so‐called “non‐genomic”) actions, which are very rapid, take place at the plasma membrane and do not involve gene transcription. Several of these non‐genomic actions of Aldo are mediated by the G protein‐coupled receptor 30 (GPR30) or G protein‐coupled estrogen receptor (GPER), a plasma membrane‐residing G protein‐coupled receptor (GPCR). Aldo causes a plethora of detrimental effects in the myocardium, such as abnormal activation of L‐type Ca2+ currents and activation of the ryanodine receptor in ventricular myocytes, while elevated levels of Aldo are associated with essential hypertension and can trigger cardiac hypertrophy, fibrosis, and adverse remodeling. Whether these pathophysiological effects of Aldo in the heart result from its non‐genomic or genomic (MR‐dependent) signaling pathways (or both) is still a matter of debate. Recent studies suggest that cardiac GPER activation by Aldo may actually be beneficial in the myocardium, as it activates the anti‐apoptotic extracellular signal‐regulated kinases (ERKs). Most GPCRs undergo functional desensitization due to phosphorylation by GPCR‐kinases (GRKs), followed by binding of βarrestins which uncouple the receptor from its cognate G proteins. GRKs are a family of seven serine/threonine protein kinases, whose most abundant members in the heart are GRK2 and GRK5. GRK2 upregulation in the failing myocardium is one of the molecular hallmarks of HF and cardiac GRK2 inhibition has already been established as a therapeutic strategy for this disease. In contrast, the role of cardiac GRK5 in the pathophysiology of HF is not as clearly defined. Both GRK2 and −5 are also known to phosphorylate a variety of non‐GPCR substrates. Recently, both GRK2 and −5 were shown to get activated by the MR in the heart to induce apoptosis and hypertrophy, respectively. On the other hand, the MR was recently shown to get phosphorylated by unidentified kinase(s) at Ser‐843, inside its ligand binding domain, an event that diminishes the MR's capacity to activate transcription, i.e. essentially inhibits its function.

Hypothesis

In the heart, GRK5 phosphorylates the MR at Ser‐843 and inhibits its function, whereas GRK2 phosphorylates and desensitizes GPER.

Methods

We utilized the rat cardiomyocyte cell line H9c2, which endogenously expresses MR, GPER, GRK2 and GRK5. We performed co‐immunoprecipitation experiments to detect GRK‐GPER and GRK‐MR interactions. We measured protein phosphorylation via western blotting with anti‐phosphoserine antibodies. MR transcriptional activity was measured via a luciferase reporter assay.

Results

GRK5, but not GRK2, interacts with, and phosphorylates the MR in H9c2 cardiomyocytes, constitutively and in response to beta‐adrenoceptor stimulation. In contrast, GRK2, but not GRK5, phosphorylates and desensitizes GPER upon beta‐adrenoceptor stimulation in H9c2 cells. The GRK5‐phosphorylated MR appears incapable of activating gene transcription, since MR luciferase reporter activity is markedly depressed upon GRK5 overexpression. In contrast, it is markedly enhanced upon GRK5 siRNA‐mediated knockdown in H9c2 cells.

Conclusions

The pathological MR‐dependent Aldo signaling in the heart may be blocked by GRK5, while its beneficial signaling through GPER may be inhibited by GRK2. Thus, a strategy combining GRK5 potentiation with simultaneous GRK2 inhibition may beneficially modulate cardiac Aldo signaling for HF therapy.

Support or Funding Information

1) American Heart Association's Scientist Development Grant2) NSU President's Faculty Research & Development Grant