G Protein-coupled Receptor Kinase-2 Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

Karen M D'Souza,Ricky Malhotra,Shahab A Akhter,Jennifer L Philip,Michelle L Staron,Tiju Theccanat,Valluvan Jeevanandam

doi:10.1074/jbc.m111.218263

Karen M D'Souza, Ricky Malhotra + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m111.218263

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Abstract

Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

Highlights

Alloproteinase activity dominates the early, adaptive wound healing response after myocardial infarction (MI), enhanced collagen synthesis is a feature of the later stages of healing and results in increased extracellular matrix (ECM) deposition (3)
Previous work suggests that increased cAMP production inhibits collagen synthesis via an inhibition of rat fibroblast to myofibroblast transformation, no studies have examined the effects of increased cAMP on myofibroblast formation in the human heart, and, importantly, there have been no studies investigating how Cardiac fibroblasts (CF) ␤-AR signaling and adenylyl cyclase activity may be regulated, in response to profibrotic and hypertrophic stimuli such as myocardial infarction, which can transition to a heart failure (HF) phenotype
Fibroblasts Isolated from Failing Human Left Ventricles Display a Myofibroblast-predominant Phenotype—CF isolated from failing human left ventricles had a 2.2-fold increase in ␣-SMA expression by protein immunoblotting, indicative of transformation to a myofibroblast phenotype compared with the normal control CF (Fig. 1A)

Summary

Introduction

Alloproteinase activity dominates the early, adaptive wound healing response after MI, enhanced collagen synthesis is a feature of the later stages of healing and results in increased ECM deposition (3). Increased collagen types I, III, and VI expression in failing CF is clearly illustrated in the confocal imaging studies relative to normal control ventricular fibroblasts (Fig. 1E).

Results

Conclusion