Internalized β2-Adrenergic Receptors Inhibit Subcellular Phospholipase C-Dependent Cardiac Hypertrophic Signaling

Figures

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  Figure 1.. A pathway downstream of β2ARs suppresses β1AR stimulation of PLCε at the Golgi. A) AVMs were transduced with adenoviruses expressing FAPP-PH-GFP for 18 hours prior to fixation and staining for GM130, a cis-Golgi marker. 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 FAPP-PH-GFP 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 β2AR 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. Images taken for PI4P hydrolysis were collected from n=3–5 cells from at least 3 independent preparations of AVMs and at least n=8 cells from 3 separate preparations of NRVMs. Data was analyzed with a…

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  Figure 2.. PLCε is the likely target for β2AR-dependent inhibition of Golgi PI4P hydrolysis. A) Diagram of β2AR-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 β2AR-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. Images for PI4P hydrolysis were taken from n=3–8 cells from 3 independent preparations of AVMs. Data was analyzed with a two-way unpaired ANOVA with Sidak’s post-hoc test. *p<0.05; ****p<0.00001 using GraphPad Prism 9.

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  Figure 3.. β2AR-Gi-Gβγ-ERK signaling axis counters activation of PLCε. A) Diagram of possible signaling pathways downstream of β2ARs. 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) 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. E) AVMs were pretreated with the ERK inhibitor, PD0325901 (100 nM) for 30 min prior to the pretreatment with Sal for 30 min and dobutamine was added at the arrow. F) 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. G-H) 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…

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  Figure 4.. β2AR-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 two-way unpaired ANOVA with Sidak’s post-hoc test **p<.001, ns=not significant. B) Representative images showing Sal or Iso mediated β2-AR internalization. NRVMs were transfected with Flag-β2ARs 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. 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 was analyzed using an unpaired two-way ANOVA with Sidak’s post-hoc test. *p<0.05; ****p<0.00001. E) Diagram of blockade of Gβγ signaling at endosomal membranes. F) Representative image of AVMs expressing…

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  Figure 5.. Nuclear PKD activation downstream of PLCε is suppressed by β2AR 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 to 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. 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. Data was analyzed with a one-way ANOVA with Sidak’s…

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  Figure 6.. Activation of by β2ARs inhibits dobutamine induced cardiomyocyte hypertrophic growth. (A-B) NRVMs were stimulated with dobutamine for 48 hours in the presence of Sal or vehicle control and followed by fixation and staining for α-actinin to identify cardiomyocytes and quantitated for cell area by image J. (C-D) NRVMs were stimulated with dobutamine for 48 hours in the presence of Sal or vehicle control and followed by fixation, cells were stained for ANF expression and with DAPI to identify nuclei. The fluorescence intensity of ANF rings was quantified by image J. Data was quantified from at least n=350 cells from 3 separate preparations of NRVMs. Data was analyzed with a one-way ANOVA with Tukey’s post-hoc test. *p<0.05; *p<0.001 using GraphPad Prism 9.