Gαq-mediated Activation of GRK2 by Mechanical Stretch in Cardiac Myocytes: THE ROLE OF PROTEIN KINASE C

Ricky Malhotra,Karen M D'Souza,Michelle L Staron,Konstantin G Birukov,Ilona Bodi,Shahab A Akhter

doi:10.1074/jbc.m110.109272

Abstract

G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of beta-adrenergic receptor (beta-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and beta-AR signaling. To eliminate neurohormonal influences, neonatal rat ventricular myocytes were subjected to cyclical equi-biaxial stretch. A hypertrophic response was confirmed by "fetal" gene up-regulation. GRK2 activity in cardiac myocytes was increased 4.2-fold at 48 h of stretch versus unstretched controls. Adenylyl cyclase activity was blunted in sarcolemmal membranes after stretch, demonstrating beta-AR desensitization. The hypertrophic response to mechanical stretch is mediated primarily through the G alpha(q)-coupled angiotensin II AT(1) receptor leading to activation of protein kinase C (PKC). PKC is known to phosphorylate GRK2 at the N-terminal serine 29 residue, leading to kinase activation. Overexpression of a mini-gene that inhibits receptor-G alpha(q) coupling blunted stretch-induced hypertrophy and GRK2 activation. Short hairpin RNA-mediated knockdown of PKC alpha also significantly attenuated stretch-induced GRK2 activation. Overexpression of a GRK2 mutant (S29A) in cardiac myocytes inhibited phosphorylation of GRK2 by PKC, abolished stretch-induced GRK2 activation, and restored adenylyl cyclase activity. Cardiac-specific activation of PKC alpha in transgenic mice led to impaired beta-agonist-stimulated ventricular function, blunted cyclase activity, and increased GRK2 phosphorylation and activity. Phosphorylation of GRK2 by PKC appears to be the primary mechanism of increased GRK2 activity and impaired beta-AR signaling after mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and beta-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.

Highlights

Utilizing an in vitro model of cyclical biaxial mechanical stretch to exclude the effects of the complex neurohormonal milieu, we studied the effects of mechanical stretch-induced hypertrophy in cardiac myocytes on ␤-AR signaling, which plays an important role in the regulation of cardiac function
Chronic Mechanical Stretch Leads to ␤-AR Desensitization— A hypertrophic phenotype in response to mechanical stretch was confirmed in primary cultures of neonatal rat ventricular myocytes subjected to cyclical equi-biaxial stretch on the Flexcell apparatus through up-regulation of the fetal genes atrial natriuretic factor (ANF) and ␤-MHC
In this study we have demonstrated that activation of hypertrophic signaling in cardiac myocytes by mechanical stretch, an in vitro model of pressure overload hypertrophy [34], leads to impaired ␤-AR signaling, which is critical in the regulation of cardiac function

Summary

THE ROLE OF PROTEIN KINASE C*

PKC is known to dent [5] and independent manner [6] Consistent with these phosphorylate GRK2 at the N-terminal serine 29 residue, lead- findings, we have previously shown that signaling through ing to kinase activation. Previous work has shown that PKC␣ functions as GRK2 activity and impaired ␤-AR signaling after mechanical a novel regulator of cardiac contractility through effects on stretch. Pharmacoof PKC and ␤-AR signaling regulated by GRK2 may be an impor- logical and gene therapy-based inhibition of conventional PKC tant mechanism in the transition from compensatory ventricu- isoforms in mice have been shown to enhance cardiac contraclar hypertrophy to heart failure. Activation of GRK2 by Mechanical Stretch in Cardiac Myocytes receptor kinase-2 (GRK2) in vitro [18, 19], and GRK2 is known to be a critical regulator of ventricular function [20, 21]. This study demonstrates cross-talk between hypertrophic signaling and ␤-AR signaling, which may represent an important mechanism in the transition from compensatory myocardial hypertrophy to ventricular dysfunction and heart failure

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS