Internalized β2-Adrenergic Receptors Oppose PLC-Dependent Hypertrophic Signaling

Abstract

Background: Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to β-adrenergic receptor (β-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. β1-AR (β1-adrenergic receptor) and β2-ARs (β2-adrenergic receptor) are the 2 major subtypes of β-ARs present in the human heart; however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of β1-ARs drives detrimental cardiac remodeling while β2-AR signaling is protective. The underlying molecular mechanisms for cardiac protection through β2-ARs remain unclear.

Methods: β2-AR signaling mechanisms were studied in isolated neonatal rat ventricular myocytes and adult mouse ventricular myocytes using live cell imaging and Western blotting methods. Isolated myocytes and mice were used to examine the roles of β2-AR signaling mechanisms in the regulation of cardiac hypertrophy.

Results: Here, we show that β2-AR activation protects against hypertrophy through inhibition of phospholipaseCε signaling at the Golgi apparatus. The mechanism for β2-AR-mediated phospholipase C inhibition requires internalization of β2-AR, activation of Gi and Gβγ subunit signaling at endosome and ERK (extracellular regulated kinase) activation. This pathway inhibits both angiotensin II and Golgi-β1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD (protein kinase D) and histone deacetylase 5 phosphorylation and protection against cardiac hypertrophy.

Conclusions: This reveals a mechanism for β2-AR antagonism of the phospholipase Cε pathway that may contribute to the known protective effects of β2-AR signaling on the development of heart failure.

Keywords: GTP-binding proteins; heart failure; hypertrophy; receptors, adrenergic; type C phospholipases.

Figure 1.. A pathway downstream of β2-ARs suppresses β1-AR stimulation of PLCε at the Golgi.

A) AVMs were transduced with adenoviruses expressing GFP-FAPP-PH for 18 hours prior to fixation and staining for GM130, a cis-Golgi marker. Scale bars = 20μm B) NRVMs were transduced with FAPP-PH-GFP and imaged by a live cell confocal microscopy. NRVMs were stimulated with dobutamine at either 10 μM or 100 nM at the arrow and remaining fluorescence intensity at the Golgi apparatus was monitored over time. C) AVMs transduced with GFP-FAPP-PH were stimulated with dobutamine at either 10 μM or 100 nM at the arrow indicated and Golgi associated fluorescence was monitored over time. NRVMs (D and F) and AVMs (E) were pretreated with a selective β2-AR antagonist ICI-118,551 (50 nM) for 30 min before stimulation with dobutamine at the indicated concentrations and Golgi PI4P hydrolysis was measured. G) AVMs were pretreated with either Sal (100 nM) or vehicle control 30 min before imaging and dobutamine was added at the arrow. Data for B, D, and F were from at least n=8 cells from 3 separate preparations of NRVMs and analyzed with a two-way repeated measures ANOVA (sphericity corrected) with Tukey’s post-hoc test. Data for C, G and E were collected from n=3–5 cells from at least 3 independent preparations of AVMs. C and G were analyzed with a Wilcoxon test, and E was analyzed with a Friedman test and Dunn’s post-hoc test. (See Table S1 for exact cell numbers).

Figure 2.. PLCε is the likely target for β2-AR-dependent inhibition of Golgi PI4P hydrolysis.

A) Diagram of β2-AR-dependent blockade of cpTOME-AM mediated activation of EPAC/PLCε. B) AVMs were pretreated with or without Sal (100 nM) before stimulation with cpTOME-AM (10 μM) and Golgi PI4P associated fluorescence was monitored with time. C) Diagram of β2-AR-dependent blockade of AngII mediated activation of PLCε at the Golgi apparatus. D) AVMs were pretreated with or without Sal (100 nM) before stimulation with Angiotensin II (1 μM) and Golgi PI4P associated fluorescence was monitored. Data are from n=3–11 cells from at least 3 independent preparations of AVMs (See Table S1 for exact cell numbers). Data in A were analyzed with a Friedman test and Dunn’s post hoc analysis; Data in B were analyzed with a repeated measures mixed effects model with Tukey’s post-hoc analysis.

Figure 3.. β2-AR-Gi-Gβγ signaling axis counters activation of PLCε.

A) Diagram of possible signaling pathways downstream of β2-ARs. B) AVMs were pretreated with or without the PKA inhibitor, myrPKI (1 μM) 30 min before stimulation with dobutamine at the indicated concentrations and Golgi PI4P hydrolysis was assessed. C) AVMs were pretreated with either PTX (100 ng/ml) or vehicle control overnight and followed by pretreatment with Sal 30 min before imaging and dobutamine was added at arrow. D) Representative images of NRVMs cotransfected with siGLO red/Gα_i2-siRNA mix (1:1 20 nmol each) and co-transduced with FAPP adenovirus. Scale bar = 40 μm E) NRVMs were transfected with Gα_i2 targeting siRNA or control scrambled siRNA. 48 hours after transfection cells were treated with the indicated agonists. F) AVMs were pretreated with the Gβγ inhibitor, gallein (10 μM) or vehicle control for 30 min prior to the pretreatment with Sal for 30 min and dobutamine was added at the arrow. Data for B, C and F are n=3–6 from 3 independent preparations of AVMs and (See Table S1 for exact cell numbers) and were analyzed Friedman’s test with Dunn’s post-hoc analysis. E; data are n=6–7 from 3 independent preparations of NRVMs and analyzed with a repeated measures mixed effects model (sphericity corrected) with Tukey’s post-hoc test.

Figure 4.. β2-AR-ERK signaling axis counters activation of PLCε.

A) AVMs were pretreated with the MEK inhibitor, PD0325901 (100 nM) for 30 min prior to the pretreatment with Sal for 30 min and dobutamine was added at the arrow. B) NRVMs were transfected with ERK2 targeting siRNA or control scrambled siRNA. 48 hours after transfection cells were treated with the indicated agonists. C) AVMs were pretreated with or without the EGF receptor inhibitor, gefitinib (10 μM) for 2 hours before the pretreatment with Sal for 30 min and dobutamine was added at the arrow. D) Top panel: Acutely isolated adult ventricular myocytes were pretreated or without gefitinib for 2 hours before the treatment with Sal for indicated times followed by western blotting for p-ERK and total ERK. Shown are representative western blots from three independent preparations of AVMs. Bottom panel, NRVMs were treated with or without gefitinib for 2 h followed by 10 min treatment with EGF followed by western blotting. E) Quantitation of data in D from three AVM preparations (FOB=fold over basal) with the 30 min time analyzed with an unpaired Mann-Whitney U test. Data in A and C were analyzed with a Friedman test and Dunn’s post-hoc test. Data in B was analyzed with a two-way repeated measures ANOVA (sphericity corrected) with Tukey’s post-hoc test. E. See table S1 for exact cell numbers.

Figure 5.. β2-AR-dependent blockade of PLCε activation relies on endosomal Gβγ.

A) AVMs were pretreated with the dynamin inhibitor Dyngo-4a (40 μM) for 30 min prior to pretreatment with Sal for 30 min and dobutamine was added at the arrow. Data was analyzed with a Friedman test with Dunn’s post-hoc test. 4–5 cells from N=3 cell preparations. B) Representative images showing Sal or Iso mediated β2-AR internalization. NRVMs were transfected with Flag-β2-ARs for 24 hours. Cells were then labeled with M1-Flag-488 for 10min at 37°C and then treated with negative control, Sal (100 nM), or Iso (10 μM) at 37°C. Images were acquired at the indicated times by confocal microscopy. Scale bars = 10 μm. C-D) Acutely isolated adult ventricular myocytes were pretreated in the presence or absence of Dyngo-4A before the treatment with Sal for indicated times followed by western blotting for p-ERK and total ERK. Shown is a representative western blot from four independent preparations of AVMs with quantitation shown in D. Data at the 30 min time was analyzed with a Mann Whitney U test N=4 independent cell preparations. E) Diagram of blockade of Gβγ signaling at endosomal membranes. F) Representative image of AVMs expressing FYVE-GRK2ct. Scale bar = 10 μm. G) AVMs were transduced with adenoviruses expressing FYVE-mApple-GRK2ct and FAPP-PH-GFP for 18 hours before imaging. AVMs were stimulated with Dobutamine alone or in the presence of Sal. Golgi associated PI4P fluorescence intensities at 0 and 30 min were measured. Data was analyzed with a Kruskal-Wallis test. N=4. See table S1 for exact cell numbers.

Figure 6.. Nuclear PKD activation downstream of PLCε is suppressed by β2-AR activation.

A-B) NRVMs were pretreated with either Sal or vehicle before addition of AngII (1 μM) for 30 min and followed by western blotting for p-PKD and total PKD. Shown is a representative western blot from four separate preparations of NRVMs. Quantitation is shown in B. C) Representative images of AVMs expressing nuclear-DKAR. D) The nuclear region of AVMs expressing nDKAR was selected and the CFP/YFP ratio was measured before and after addition of AngII (1 μM) for 30 min addition in the presence with or without pretreatment with Sal for 30 min. Scale bars = 20 μm. E) Heart lysates from WT mice infused with either saline (Veh) or salmeterol (Sal) (25 μg/kg/day) together with or without AngII (1.5 mg/kg/day) for 14 days. Western blotting was performed to determine the level of p-PKD, p-HDAC, total HDAC and GAPDH. Shown is a representative western blot from 4 mice each group. F) Quantitation of PKD phosphorylation from E. G) Quantitation of HDAC phosphorylation from E. H) Heart weight/body weight (HW/BW) ratios from AngII and AngII+Sal treated mice. N=4 animals each condition. All data were analyzed with a Kruskal-Wallis test.

Figure 7.. Animals were implanted with osmotic pumps containing Saline (NC), Salmeterol (Sal), AngII or AngII+Sal for 14 days followed by echocardiography, sacrificed and tissue harvesting.

A) Scale bar on hearts is 2 mm and 1 mm on HE stained sections. B) Cells were stained with WGA and myocyte area was quantitated using Image J. Scale bar is 60 μm. Data were analyzed from at least 300 cells from 3 stained sections each condition (See Table S1 for exact cell numbers) using a nested hierarchical unpaired One-Way ANOVA with Tukey’s post-hoc test. C) Echocardiography analysis of fractional shortening (FS), systolic left ventricular inside dimension (LVIDs), and diastolic left ventricular inside dimension (LVIDd). Data in A and C were analyzed using an ordinary one-way unpaired ANOVA with Tukey’s post-hoc test. See Table S1 for animal numbers.

Figure 8.. Activation of β2-ARs inhibits dobutamine induced cardiomyocyte hypertrophic growth and requires endosomal Gβγ signaling.

A and B, Neonatal rat ventricular myocytes (NRVMs) were transduced with β-galactosidase expressing adenovirus for 6 hours then stimulated with dobutamine for 42 hours in the presence of Sal or vehicle control followed by fixation. A, Cells were stained for α-actinin to identify cardiomyocytes and quantitated for cell area by image J. B, Cells were stained for ANF (atrial natriuretic factor) expression and with DAPI (4’,6-diamidino- 2-phenylindole) to identify nuclei. The fluorescence intensity of ANF rings was quantified by image J. C and D, Same as A except NRVMs were transduced with FYVE (Fab 1, YOTB, Vac 1, and EEA1 domain)-mApple-GRK2ct (G proteincoupled receptor kinase 2 c-terminus) expressing adenovirus before stimulation with dobutamine in the presence of Sal or vehicle control (NC), followed by fixation. C, Cell area. D, ANF expression. FOB is fold over basal. Cell size was quantified from n=1283–1383 cells from 6 separate wells each condition, repeated with 2 separate preparations of NRVMs. ANF was quantified from n=890–1348 cells from 6 wells each condition, repeated with 2 separate preparations of NRVMs. Scale bars is 40 μm. All data were analyzed with an ordinary one-way ANOVA with Tukey’s post-hoc test. NC indicates negative control.