Interaction of G-protein coupled receptor kinase 2 (GRK2) and phosphoinositide 3-kinase γ (PI3Kγ) mediates cardiac hypertrophy and NFAT activation.

<p>(A-C) GRK2 and PI3Kγ interaction promotes cardiac hypertrophy. (A) Representative immunofluorescence images and (B) quantification of GRK2-PI3Kγ-interaction by proximity ligation assay and quantification of cardiac myocyte size (C) in cells with and without pharmacological PI3Kγ inhibition by Wortmannin; for modulation of GRK2 levels neonatal rat ventricular myocytes (NRVMS) were transduced with an adenovirus harboring GRK2 (AdGRK2) or β-galactosidase/LacZ (AdLacZ) or were not treated with an adenovirus (control); GRK2-PI3Kγ-interaction is visualized by proximity ligation assay were each green dot represents GRK2 and PI3Kγ alignment (green), α-Actinin (red), DAPI (blue), 18–34 individual cells from 2 independent cell preparations were analyzed, * P < 0.05, ** P < 0.01. Scale bar (A): 20 μm. (D) GRK2-PI3Kγ-interaction promotes proteinkinase B (Akt) phosphorylation. Representative Western Blots of Akt phosphorylation at residues Ser 473 (p_SAKT) and Thr 308 (p_TAKT), total Akt expression, GRK2 expression and GAPDH under respective conditions, Akt protein expression normalized to untreated (-) cells, n = 3–8, * P < 0.05, ** P < 0.01, *** P < 0.001. (E, F) GRK2-PI3Kγ-interaction promotes phosphorylation of glycogen synthase kinase 3β (GSK3β). Representative Western Blot (E) and quantification (F) of pGSK3β to total GSK3β (n = 4–8) and GSK3β to GAPDH (n = 2–3), values normalized to untreated control (-), ** P < 0.01, *** P < 0.001. (G) PI3Kγ inhibition (by Wortmannin) and Akt inhibition (by MK2206) abrogate NFAT activation induced by adenoviral GRK2 overexpression. (B) Transcriptional NFAT activity was evaluated using the NFAT-luciferase-reporter assay system. NRVM were transduced with an adenovirus harboring luciferase (AdLuc, MOI 25) plus either an adenovirus harboring LacZ (AdLacZ, MOI 25) or GRK2 (AdGRK2, MOI 25) for 48h. Data are normalized to AdLacZ without inhibition, n = 12–18 from 3 different cell preparations, *** P <0.001.