G protein-coupled receptor kinase 2 promotes cardiac hypertrophy

<div><p>The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity.</p></div

G-protein coupled receptor kinase 2 (GRK2) regulates proteinkinase B (Akt) phosphorylation.

<p>(A-E) G-protein coupled receptor (GPCR) promoted Akt phosphorylation depends on GRK2. (A) 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. Samples shown on each lane are blotted, antibody stained and developed on the same western blot membrane. Vertical dotted line indicates 2 excluded samples with non ANG II/PE GPCR stimulation. Quantification of p_SAKT under PE (B; n = 4) and ANG II (C; n = 3) and p_TAKT under PE (D; n = 3) and ANG II (E; n = 4) stimulation and total Akt under respective treatment conditions, values normalized to control scrambled siRNA (Scr), * P < 0.05. (F) GRK2 overexpression promotes full Akt phosphorylation. Representative Western Blots and quantification of p_SAKT (n = 7), p_TAKT (n = 7) and total Akt (n = 4) expression after transfection with an adenovirus harboring GRK2 (AdGRK2) or β-galactosidase/LacZ as control (AdLacZ), GAPDH served as loading control. Values normalized to untreated control (-). * P < 0.05, *** P < 0.001.</p

Genetic knockout of GRK2 abolishes GRK2-dependent NFAT activation.

<p>(A, B) GRK2 knockout attenuates regulator of calcineurin 1 (RCAN) transcription and thus NFAT activation following transverse aortic constriction. (B) Experimental protocol. Transverse aortic constriction (TAC), organ harvest at study end (Harvest). (C) mRNA expression of NFAT dependent RCAN in GRK2KO mice and respective WT control mice at 1 day and 4 days following TAC operation. n = 4 for sham WT (d1+d4), n = 4 for sham KO (d1+d4), n = 8–10 for TAC WT (d1+d4) and n = 9–10 for TAC GRK2 group (d1+d4). *** P < 0.001. (C) Illustration of proposed signaling pathway for G-protein coupled receptor kinase 2 (GRK2) mediated cardiac hypertrophy. Angiotensin II (ANG II), phenylephrine (PE), phosphoinositide 3-kinase γ (PI3Kγ), protein kinase B (Akt), glycogen synthase kinase 3 β (GSK3β), nuclear factor of activated T-cells (NFAT).</p

G-protein coupled receptor kinase 2 (GRK2) expression is regulated by G-protein coupled receptor (GPCR) stimulation <i>in vitro</i>.

<p>(A) representative Western Blots of GRK2 expression in neonatal rat ventricular myocytes (NRVMs) after stimulation with Phenylephrine (PE), or Angiotensin II (ANG II), GAPDH as loading control. (B) Quantification of GRK2 protein expression after PE stimulation normalized to GAPDH and unstimulated (-) NRVM as control. Samples shown on each lane are blotted, antibody stained and developed on the same western blot membrane. Vertical dotted line indicates 2 excluded samples with non ANG II/PE GPCR stimulation. n = 10, * P < 0.05. (C) Quantification of GRK2 protein expression after ANG II stimulation normalized to GAPDH, n = 11, *** P < 0.001. (D) GRK2 knockdown by siRNA. Treatment of NRVM with siRNA against GRK2 (siGRK2, n = 6) and scrambled siRNA as control (Scr, n = 4), ** P < 0.01. (E) Adenoviral GRK2 overexpression. Representative Western Blots of GRK2 protein expression in untreated NRVMS, NRVMs treated with a control adenovirus harboring β-galactosidase (AdLacZ) and NRVMs after treatment with an adenovirus expressing GRK2 (AdGRK2), MOI = multiplicity of infection. (F) Quantification of GRK2 protein expression following AdLacZ or AdGRK2 transfection normalized to GAPDH as loading control and normalized to untreated cells (-), n = 2.</p

Anti-hypertrophic effects of GRK2 knock down can be abolished by GSK3β inhibition.

<p>(A) Quantification of cardiac myocyte cell size and (B) representative immunofluorescence stains of neonatal rat ventricular myocytes (NRVMs) with or without siRNA mediated GRK2 knockdown, GPCR stimulation by ANGII and/or GSK3β inhibition by lithium chloride (LiCl). Quantification of (C) phosphorylated GSK3β to total-GSK3β protein expression (n = 6–12) and (D) GSK3β to GAPDH (n = 5) including (E) representative Western Blots under respective treatment conditions, all values normalized to scrambled control (Scr). >200 NRVM per condition from 3 independent cell preparations were analyzed for cell size determination, * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bar (B): 50 μm.</p

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.</p

G-protein coupled receptor kinase 2 (GRK2) promotes cardiac myocyte hypertrophy.

<p>(A-C) GRK2 knockdown by siGRK2 attenuates G-protein coupled receptor (GPCR) induced cardiac hypertrophy. (A) Quantification of cardiac myocyte cell size from immunofluorescence stains of neonatal rat ventricular myocytes (NRVMs) following siRNA mediated GRK2 knockdown and GPCR stimulation by phenylephrine (PE), >200 NRVM per condition from 3 independent cell preparations were analyzed, *** P < 0.001. (B) Quantification of cardiac myocyte cell size following siRNA mediated GRK2 knockdown and Angiotensin II (ANG II) treatment, >100 NRVM per condition from 3 independent cell preparations were analyzed, **, P < 0.01. (C) Representative immunofluorescence images of NRVMs under respective conditions. α-Actinin is stained red for demarcation of cell dimensions, blue DAPI staining marks nuclei. (D, E) GRK2 overexpression promotes hypertrophy. (D) Quantification of cardiac myocyte cell size from immunfluorescence stains of NRVMs following treatment with an adenovirus harboring GRK2 (AdGRK2) or β-galactosidase/LacZ as control (AdLacZ), >100 NRVM per condition from 3 independent cell preparations were analyzed, *, P < 0.05. (E) Representative images of immunofluorescence of NRVMs (DAPI and α-Actinin) following AdGRK2 or AdLacZ transduction compared or untreated (-) cells. Scale bar (C and E): 50 μm.</p

G-protein coupled receptor kinase 2 (GRK2) regulates nuclear factor of activated T-cells (NFAT) nuclear activity via glycogen synthase kinase 3β (GSK3β).

<p>(A, B) G-protein coupled receptor (GPCR) induced nuclear NFAT activity is decreased by GRK2 knockdown. (A) Representative immunofluorescence images of neonatal rat ventricular cardiac myocytes (NRVMs) under respective treatment conditions; NFAT subunits are marked using a proximity ligation assay (respective antibodies against each NFAT c1 and c4) resulting in green fluorescence, α-Actinin (red), DAPI (blue). (B) Transcriptional NFAT activity was evaluated using the NFAT-luciferase-reporter assay system, n = 3–4, * P< 0.05; ** P < 0.01. (C-G) GPCR stimulation results in increased GSK3β phosphorylation mediated by GRK2. (C) Representative Western Blots of GSK3β and phosphorylated GSK3β (pGSK3β) under respective treatment conditions, GAPDH served as loading control. Samples shown on each lane are blotted, antibody stained and developed on the same western blot membrane. Vertical dotted line indicates 2 excluded samples with non ANG II/PE GPCR stimulation. Quantification of GSK3β phosphorylation and total GSK3β protein under PE (D, n = 4) and ANG II (E; n = 6) stimulation, all values normalized to scrambled control (Scr), * P< 0.05, ** P < 0.01. (F, G) GRK2 overexpression promotes GSK3β phosphorylation. (F) Representative Western Blots of pGSK3β and GSK3β after transfection with an adenovirus harboring GRK2 (AdGRK2) or β-galactosidase/LacZ as control (AdLacZ) or untreated cells (-), GAPDH served as loading control. (G) Quantification of pGSK3β and total GSK3β with and without GRK2 overexpression, n = 3, * P < 0.05.</p