Gβγ-independent recruitment of G-protein coupled receptor kinase 2 drives tumor necrosis factor α-induced cardiac β-adrenergic receptor dysfunction

Abstract

Background: Proinflammatory cytokine tumor necrosis factor-α (TNFα) induces β-adrenergic receptor (βAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known.

Methods and results: Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate βAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling βAR desensitization independent of sympathetic overdrive. TNFα-mediated βAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in β2AR-overexpressing human embryonic kidney 293 cells showed significant βAR desensitization, GRK2 upregulation, and recruitment to the βAR complex following TNFα. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated βAR phosphorylation and GRK2 recruitment on TNFα. Furthermore, TNFα-mediated βAR phosphorylation was not blocked with βAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gβγ-sequestering peptide βARK-ct could not prevent βAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the βAR is Gβγ independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNFα-mediated βAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility, showing that TNFα-induced βAR desensitization is GRK2 dependent.

Conclusions: TNFα-induced βAR desensitization is mediated by GRK2 and is independent of Gβγ, uncovering a hitherto unknown cross-talk between TNFα and βAR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

Keywords: G-protein coupled receptor kinase 2; heart failure; inflammation; phosphoinositide 3-kinase; tumor necrosis factor-α; tumor necrosis factor-α receptor 2; β-adrenergic receptor.

Figure 1

Cardiac dysfunction in Myo-Tg mice is associated with βAR desensitization and is independent of sympathetic overdrive. (A) In vitro isoproterenol (I) (closed bars) stimulated cardiac adenylyl cyclase activity compared to vehicle (V) (open bars) in the Wt and Myo-Tg mice of 4, 8, 12, 16 and 36 weeks of age (n=6–8), *p< 0.001 versus respective I Wt, ^#p<0.01 versus I Wt (all ages) & I Myo (4 & 8 weeks). (B) A plot of correlation between adenylyl cyclase activity and % fractional shortening (% FS) in Wt and Myo-Tg mice. *p< 0.01 versus Wt, ^#p<0.05 versus Myo 4 & 8 weeks. (C) Plasma epinephrine levels of Wt and Myo-Tg mice at 8 and 12 weeks (n=5–7). (D) Plasma norepinephrine levels of Wt and Myo-Tg mice at 8 and 12 weeks (n=5–7). (E) Immunoblotting for GRK 2, 3, 5, 6 and β-actin from cardiac lysates of Wt and Myo-Tg mice at 4, 12, 16, and 36 weeks of age. (F) Summary data of densitometric analysis of GRK2 (n=6–8), *p< 0.001 versus Myo-Tg at 4 weeks.

Figure 2

βAR desensitization is caused by TNFα mediated upregulation of GRK2. (A) cAMP generation following ISO challenge in HEK-FLAG-β₂AR cells (ISO) compared to untreated control cells (C), ISO re-challenge following ISO pre-treatment (ISO+ISO) or ISO challenge following pre-treatment with combination of cytokines (TGFβ+TNFα+IL-6+IL-13=Cyto), (n=5–6), *p< 0.001 versus ISO, **p< 0.001 versus ISO. (B) cAMP generation following ISO challenge in HEK-FLAG-β₂AR cells (ISO) compared to untreated control cells (C), ISO re-challenge following ISO pre-treatment (ISO/ISO), ISO challenge following TNFα pre-treatment (ISO/TNFα), ISO challenge following TGFβ pre-treatment (ISO/TGFβ), ISO challenge following IL-6 pre-treatment (ISO/IL-6) or ISO challenge following IL-13 pre-treatment (ISO/IL-13), (n=4–5), *p< 0.001 versus ISO, **p< 0.005 versus ISO, TGFβ, IL-6 & IL-13. (C) Immunoblots of GRK 2, 3, 5, and 6 following treatment of HEK-FLAG-β₂AR cells with TGFβ, TNFα, IL-6 or IL-13 for 60 minutes. (D) Densitometric analysis of the same. (n=5–6), *p< 0.001 versus C. (E) FLAG-β₂AR phosphorylation was visualized by confocal microscopy using anti-phospho-β₂AR antibody (green) following ISO stimulation or TNFα treatment for 5 or 60 minutes. Nucleus was visualized by DAPI (blue) staining. Scale bar: 10 μm.

Figure 3

βAR desensitization by TNFα is GRK2 dependent. (A) Immunoblots to assess phospho-β₂AR, GRK2 and FLAG-β₂AR on the plasma membranes of TNFα and ISO treated HEK-FLAG-β₂AR cells, (n=4). (B) Levels of β₂AR phosphorylation and GRK2 co-immunoprecipitating with FLAG-β₂AR from plasma membrane fractions of HEK-FLAG-β₂AR cells following TNFα or ISO, (n=4). (C) Representative autoradiograph showing β₂AR phosphorylation upon TNFα or ISO treatment following metabolic ^[32]Pi labeling of HEK-FLAG-β₂AR cells (n=4). (D) HEK 293 cells were transfected with FLAG-β₂AR Wt or Serine 355/356 mutant cDNA constructs, metabolically labeled with ^[32]Pi, treated with TNFα. Representative autoradiograph showing β₂AR phosphorylation following immunoprecipitation with anti-FLAG antibody (n=4). (E) Effect of GRK2 knock down by siRNA on phosphorylation of β₂ARs following the stimulation of HEK-FLAG-β₂AR cells with ISO or TNFα. (F) Cumulative data showing significant loss of β₂AR phosphorylation due to knock down of GRK2 by siRNA (right panel), (n=3–4), *p< 0.001 versus Vehicle (Veh), ^# p<0.005 versus ctrl siRNA -TNFα or ISO.

Figure 4

TNFα mediated βAR desensitization is agonist independent. (A) FLAG-β₂AR phosphorylation was visualized by confocal microscopy using anti-phospho-β₂AR antibody (green) following ISO stimulation or TNFα treatment (60 minutes) in the presence and absence of β-blocker propranolol. Nucleus was visualized by DAPI (blue) staining. Scale bar: 10 μm. (B) Immunoblots of phospho-β₂AR and FLAG-β₂AR following TNFα treatment of HEK-FLAG-β₂AR cells in the presence or absence of propranolol. Densitometric analysis of the same is shown on the lower panel. (n=4), *p< 0.001 versus Veh, ^#p< 0.001 versus Veh. (C) β-Arrestin (green) recruitment to the plasma membrane was visualized by confocal microscopy using double stable cells expressing GFP-β-Arrestin and HA-β₂AR following ISO or TNFα in the presence or absence of β-blocker propranolol. Scale bar: 10 μm

Figure 5

PI3K regulates TNFα-mediated GRK2 recruitment to the receptor complex. (A) Effect of PI3K inhibition by LY294002 on β₂AR phosphorylation and GRK2 recruitment to the plasma membrane following TNFα (n=5). (B) FLAG-β₂AR phosphorylation was visualized by confocal microscopy using anti-phospho-β₂AR antibody (green) following TNFα in the presence or absence of PI3K inhibitor LY294002. (C) Lysates from HEK-FLAG-β₂AR cells following TNFα or vehicle treatment were immunoblotted for PI3Kγ, (n=4).

Figure 6

Cardiac dysfunction in TNFα-Tg mice is associated with βAR desensitization (A) Cardiac lysates from Wt and TNFα-Tg mice 6 or 20 weeks of age were immunoblotted for GRK 2, 3, 5, 6, phospho-β₂AR and adenylyl cyclase V/VI (n=6). (B) In vitro ISO (I) (closed bars) stimulated cardiac adenylyl cyclase activity compared to vehicle (V) (open bars) in the Wt and TNFα-Tg mice of 6 or 20 weeks (n=6), *p< 0.005 versus respective I Wt samples (6 or 20 weeks). (C) βAR density on the plasma membranes isolated from the hearts of Wt and TNFα-Tg mice (n=6) of 6 or 20 weeks, *p< 0.001 versus 20 weeks Wt. (D) Plasma epinephrine (left panel) and norepinephrine (right panel) levels in Wt and TNFα-Tg mice at 6 weeks (n=6).

Figure 7

βAR desensitization by TNFα is G_βγ independent. (A) Representative echocardiography images from Wt or βARK-ct-Tg pre- and post-TNFα treatment for 2 weeks. (B) % fractional shortening (% FS) from Wt or βARK-ct-Tg mice with or without TNFα treatment. (n=6), *p< 0.005 versus Vehicle (both Wt and βARK-ct-Tg). (C) In vitro ISO (I) (closed bars) stimulated cardiac adenylyl cyclase activity compared to vehicle (V) (open bars) in the hearts of Wt and βARK-ct-Tg mice following 2 weeks of ISO or TNFα3 treatment. (n=6), *p< 0.05 versus in vitro ISO (I) stimulated 2 weeks vehicle treated Wt or βARK-ct-Tg or 2 weeks ISO treated βARK-ct-Tg cardiac membranes. (D) Upper panel: Cardiac lysates from Wt or βARK-ct-Tg mice given a bolus of Vehicle (AA-Ascorbic Acid or Sal-saline), TNFα or ISO were immunoblotted for phospho-β₂AR. Lower panel: The blot was stripped and re-probed for β-actin.

Figure 8

βAR desensitization by TNFα is GRK2 dependent (A) Representative tracings of isolated myocytes from GRK2 floxed mice (GRK2 f/f) or GRK2 knockout mice (GRK2 del) following pre-treatment with ISO or TNFα. (B & C) Cell-contractility measurements upon ISO in myocytes from GRK2 f/f or GRK2 del mice pre-treated with Veh, ISO or TNFα. *p < 0.01 versus ISO + ISO GRK2 f/f; #p < 0.01 versus TNFα + ISO GRK2 f/f (n =5, ~30 cells/experiment). (D) Plasma membrane from mouse aortic endothelial cells of Wt, TNFR1 or TNFR2 knock out mice (TNFR1^−/− or TNFR2^−/−) treated with Veh or TNFα were immunoblotted for phospho-β₂AR. The blots were stripped and re-probed for GRK2 and β-actin. (E) TNFR1 was immunoprecipitated from cardiac lysates of Wt or TNFR2^−/− mice and TNFR2 was immunoprecipitated from Wt or TNFR1^−/− mice and immunoblotted for co-immunoprecipitating GRK2 (n=6). (F) Illustration depicting mechanism of TNFα-mediated desensitization of βAR through TNFR2 and GRK2.