Golgi localized β1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy
- University of Michigan School of Medicine, United States
- University of California, San Francisco, United States
Figures
Figure 1 with 5 supplements
Dobutamine induces PI4P hydrolysis through the activation of internal βARs.
(A) NRVMs were transduced with FAPP-PH-GFP and stimulated as indicated. Time lapse live cell microscopy was used to quantitate Golgi associated FAPP-PH-GFP fluorescence was quantitated as previously …
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Figure 1—source data 1
PI4P hydrolysis is stimulated by dobutamine and inhibited by a cell permeable antagonist.
- https://doi.org/10.7554/eLife.48167.005
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Figure 1—source data 2
Dobutamine but not Iso stimulates Mini Gs translocation.
- https://doi.org/10.7554/eLife.48167.006
Figure 1—figure supplement 1
Staining of endogenous β1AR in cardiac myocytes.
Mouse neonatal cardiac myocytes were stained with anti-β1AR (abcam ab3442-1:100) and anti-TGN38 (Biorad AHP499G-1:1000) using the saponin permeabilization protocol described in the Materials and meth…
Figure 1—figure supplement 2
Disruption of the Golgi apparatus reverses mini-Gs protein recruitment to the perinuclear region by dobutamine.
NRVMs were transfected with β1-AR and NES-Venus-mini-Gs, followed by viral transduction with CFP-Giantin. Representative images of dobutamine-mediated NES-Venus-mini-Gs recruitment (100 nM, Upper …
Figure 1—video 1
Dobutamine activates β1ARs in the Golgi apparatus.
Confocal images of β1-AR-overexpressing cardiomyocytes with NES-Venus-mini-Gs, treated with 100 nM dobutamine. Total time represented by the movie is 10 min and pictures were taken every 15 s. …
Figure 1—video 2
Isoproterenol does not activate β1ARs in the Golgi apparatus.
Confocal images of β1-ARs-overexpressing cardiomyocytes with NES-Venus-mini-Gs, treated with 100 nM isoproterenol. Total time represented by the movie is 10 min and pictures were taken every 15 …
Figure 1—video 3
Disruption of the Golgi apparatus inhibits activation of β1ARs in the Golgi apparatus.
Reversal of mini Gs recruitment to Golgi membrane after addition of Brefeldin A. Total time represented by the movie is 30 min and pictures were taken every 30 s. Dobutamine was added at 2 min …
Figure 2
Dobutamine-mediated PI4P hydrolysis requires Epac and mAKAPβ bound PLCε.
NRVMs were transduced with FAPP-PH-GFP, stimulated with treatments as indicated and Golgi associate fluorescence was monitored with time. (A) PLCε knockdown prevents dobutamine-mediated PI4P …
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Figure 2—source data 1
Effects of inhibition of PLCε, Epac and Gβγ on dobutamine stimulated PI4P hydrolysis.
- https://doi.org/10.7554/eLife.48167.011
Figure 3
β1ARs in the Golgi are required for PI4P hydrolysis stimulated by dobutamine.
NRVMs were transduced with FRB-CFP-Nb80 and FKBP-GalT-mApple containing adenovirus, along with adenovirus containing FAPP-PH-GFP for 24 hr. (A) Confocal fluorescence images of NRVMs expressing …
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Figure 3—source data 1
Effects of Golgi targeted NB80 and β1AR-specific antagonism on dobutamine stimulated PI4P hydrolysis.
- https://doi.org/10.7554/eLife.48167.013
Figure 4 with 2 supplements
The physiological neurotransmitter, norepinephrine, can induce PI4P hydrolysis through internal receptors and can activate βARs at the Golgi.
(A) NRVMs were transduced with FAPP-PH-GFP and stimulated with norepinephrine (10 μM, left) in the presence of metoprolol (100 μM, center) or sotalol (5 mM, right) and analyzed as in Figure 1A. Data …
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Figure 4—source data 1
NE stimulates PI4P hydrolysis.
- https://doi.org/10.7554/eLife.48167.015
Figure 4—video 1
Norepinephrine activates β1ARs in the Golgi apparatus.
Representative confocal images of β1-ARs-overexpressing cardiomyocytes with NES-Venus-mini-Gs, treated with 10 µM norepinephrine. This confocal plane was chosen to emphasize both PM and Golgi …
Figure 4—video 2
Norepinephrine activates β1ARs in the Golgi apparatus.
Representative confocal images of β1-ARs-overexpressing cardiomyocytes with NES-Venus-mini-Gs, treated with 10 µM norepinephrine. This confocal plane was chosen to emphasize Golgi Venus-mini-Gs …
Figure 5 with 2 supplements
Norepinephrine requires OCT transporters but not receptor internalization to stimulated PI4P hydrolysis.
NRVMs were transduced with FAPP-PH-GFP and stimulated with norepinephrine (10 μM) in the presence of corticosterone (100 μM, (A), abacavir (10 μM, (B), lamotrigine (10 μM, (C) or Dyngo (40 μM, (D). …
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Figure 5—source data 1
NE requires membrane transport by OCT3 and Golgi resident βARs to stimulate PI4P hydrolysis.
- https://doi.org/10.7554/eLife.48167.020
Figure 5—figure supplement 1
Receptor internalization does not contribute to Golgi β1AR localization.
(A) Dyngo blocks Isoproterenol induced β1-AR internalization. NRVMs were transfected with FLAG-β1-AR receptors and allowed to express for 48 hr. Cells were then stimulated with either vehicle …
Figure 5—video 1
Inhibition of receptor internalization does not alter activation of β1ARs in the Golgi apparatus.
Confocal images of β1-ARs-overexpressing cardiomyocytes with NES-Venus-mini-Gs, pretreated with 40 µM Dyngo. Total time represented by the movie is 30 min and pictures were taken every 30 s. Dyngo …
Figure 6
Dobutamine and NE stimulate PI4P hydrolysis in Adult Ventricular Myocytes (AVMs).
Freshly isolated mouse AVMs were infected with FAPP-PH-GFP (100 MOI) for 24 hr. (A) representative image of an AVM expressing FAPP-PH-GFP showing strong labeling surrounding the nucleus. (B) …
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Figure 6—source data 1
NE-Stimulated PI4P hydrolysis in adult cardiac myocytes requires OCT3.
- https://doi.org/10.7554/eLife.48167.023
Figure 7
Dobutamine induced cardiomyocyte hypertrophy requires intracellular βARs.
(A) Dobutamine induces internal-receptor dependent increases in cell area. NRVMs were transduced with YFP virus prior to stimulation for 48 hr with dobutamine in the presence of the indicated …
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Figure 7—source data 1
Dobutamine-stimulated cardiomyocyte hypertrophy is blocked by a cell permeable βAR antagonist.
- https://doi.org/10.7554/eLife.48167.025
Figure 8
Dobutamine and norepinephrine induced cardiomyocyte hypertrophy requires Golgi-localized βARs.
Norepinephrine induced hypertrophy also requires agonist internalization. Norepinephrine induces internal-receptor dependent increases in cell area (A) and ANF expression (B). NRVMs were stimulated …
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Figure 8—source data 1
NE-stimulated hypertrophy requires OCT3 and Golgi resident βARs.
- https://doi.org/10.7554/eLife.48167.027
Figure 9
Signal transduction by cell surface and Golgi β1ARs.
β1ARs are located on both the plasma membrane and the Golgi apparatus in cardiac myocytes. Stimulation of cell surface β1ARs leads to production of cytosolic cAMP but this cAMP cannot access the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Rat) | NRVM | Freshly isolated myocytes from neonatal rats | ||
| Recombinant DNA reagent | NES-Venus-mini-Gs | Nevin Lambert, Augusta University, GA. Wan et al. (2018) | ||
| Recombinant DNA reagent (human) | FLAG-β1 adrenergic receptor | Addgene | RRID:Addgene_14698 | |
| Recombinant DNA reagent | CFP Giantin | Irannejad et al. (2017) | Golgi Marker Adenovirus expressing CFP Giantin MOI 50 | |
| Transfected construct (Rat) | PLCε-RA1 | Zhang et al. (2013) | Adenovirus expressing the RA1 domain fromPLCE1 MOI 50 | |
| Transfected construct (Rat) | PLCE1-shRNA | Zhang et al. (2013) | Adenovirus expressing PLCE1 shRNA MOI 50 | |
| Transfected construct | FAPP-PH-GFP | Zhang et al. (2013) | Adenovirus expressing FAPP-PH-GFP for PI4P detection MOI 50 | |
| Transfected construct | CFP-Nb80-FRB | This paper and Irannejad et al. (2017) | In this paper adenovirus was created for expressing CFP-Nb80-FRB previously created in Irannejad et al. (2017) MOI 50 | |
| Transfected construct | FKBP-mApple-GalT | This paper and Irannejad et al. (2017) | In this paper adenovirus was created for expressing FKBP-mApple-GalT previously created in Irannejad et al. (2017) MOI 50 | |
| Antibody | Anti-β1 adrenergic receptor (rabbit polyclonal) | Abcam | #ab3442 RRID: AB_10890808 | 1:100 dilution |
| Antibody | Anti-TGN38 (sheep anti Rat polyclonal) | Biorad | #AHP499G RRID:AB_2203272 | 1:1000 dilution |
| Antibody | M2-FLAG-Cy3 (mouse monoclonal) | Sigma | A9594 RRID:AB_439700 | 5 ug/mL |
| Chemical compound, drug | Butanedione-monoxime (BDM) | Sigma | 112135 | Myosin blocker |
| Chemical compound, drug | Isoproterenol (ISO) | Sigma | 1351005 | βAR agonist |
| Chemical compound, drug | Dyngo | Abcam | Ab120689 | Dynamin inhibitor |
| Chemical compound, drug | Sotalol | Sigma | S0278 | βAR antagonist |
| Chemical compound, drug | HJC0726 | Xiaodong Cheng, UT Houston Health Science Center. Zhu et al. (2015) | Epac inhibitor | |
| Chemical compound, drug | Brefeldin A | Biolegend | 420601 | Golgi disruptor |
| Chemical compound, drug | Gallein | Sigma | 371708 | G protein βγ subunit inhibitor |
| Chemical compound, drug | Corticosterone | Tocris | 3685 | Oct3 inhibitor |
| Chemical compound, drug | Metoprolol | Sigma | M5391 | βAR antagonist |
| Chemical compound, drug | Dobutamine | Tocris | 0515 | βAR agonist |
| Chemical compound, drug | Lamotrigine | Tocris | 2289 | OCT3 blocker |
| Chemical compound, drug | Abacavir | Tocris | 4148 | OCT3 blocker |
| Chemical compound, drug | Norepinephrine (NE) | Sigma | A0937 | AR agonist |
| Chemical compound, drug | Rapamycin (Rapa) | Tocris | 1292 | |
| Peptide, recombinant protein | Collagenase Type II | Worthington | CLS-2 | |
| Commercial assay or kit | cAMP Elisa | ENZO | ADI-900–066 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.48167.029
