Distinct functions of cardiac β-adrenergic receptors in the T-tubule vs. outer surface membrane
- Université Paris-Saclay, Inserm, UMR-S 1180, France
- Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, France
- Collège de France, CIRB, INSERM U1050/CNRS UMR 7241, France
- Université Paris Saclay, CEA, Département Médicaments et Technologies pour la Santé, SIMoS, France
- Université Paris-Saclay, CNRS, BioCIS, France
Figures
Figure 1 with 1 supplement
Localization of PEG-FITC in freshly isolated adult rat ventricular cardiomyocytes.
Typical confocal images (A, C, E) and plot profiles representing the fluorescence intensity measured across the cell (B, D, F) of adult rat ventricular myocytes (ARVMs) incubated during 15 min with either 100 µM PEG5000-FITC (A, B), 100 µM free fluorescein (C, D), or 100 µM PEG5000-FITC after a 1-hr treatment with 0.25 U/ml neuraminidase (+nmd) (E, F). Summary fluorescence intensity for cells stained with FITC and PEG-FITC either in control (−, blue) conditions or after treatment with neuraminidase (+, green) (G). Bars represent the mean ± SEM. n/N = 8–18/2–4. Kruskal–Wallis test with multiple comparisons was used: ****p < 0.0001; ns, non-significant.
Figure 1—figure supplement 1
PEG-Isoprenaline synthesis.
Top, concept of chemical grafting between PEG5000 and Iso; bottom, chemical coupling between PEG and Iso using carbodiimide reaction.
Figure 2
Binding properties of Iso and PEG-Iso on β-ARs.
β1- or β-AR Chinese Hamster Ovary (CHO) membrane preparations (2.5 μg/triplicate) were incubated for 2 hr with 0.25 nM [125Iodo]cyanopindolol and increasing concentrations of unlabeled Iso or PEG-Iso. (A) Example of a typical competition binding of Iso and PEG-Iso on β1-ARs. (B) Example of a typical competition binding of Iso and PEG-Iso on β2-ARs. Ki values were, respectively, 2.0 ± 0.7 µM and 1.4 ± 0.7 mM for Iso and PEG-Iso on β1-ARs; 2.1 ± 0.4 µM and 0.4 ± 1.0 mM for Iso and PEG-Iso on β2-ARs (N = 5). (C) Time evolution of solvent-accessible surface area (SASA) of Iso (black trace) and PEG-Iso (blue trace) using molecular dynamics simulations. Two representative conformations of PEG-Iso with large or small SASA are shown.
Figure 3
Effects of Iso and PEG-Iso on cytosolic cAMP in adult rat ventricular cardiomyocytes.
Freshly isolated adult rat ventricular myocytes (ARVMs) were infected with an adenovirus encoding the Epac-SH187 FRET-based cytosolic cAMP sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment showing the time course of CFP/YFP ratio during successive applications of four increasing Iso concentrations: 0.3, 1, 10, and 100 nM. (B) Similar experiment showing the time course of CFP/YFP ratio during successive applications of four increasing PEG-Iso concentrations: 10 and 100 nM, 1 and 10 µM. Pseudo-color images shown above the main graphs were taken at times indicated by the corresponding letters in the graphs. (C, D) Summary data from several similar experiments as in (A) and (B), respectively. The bars show the mean ± SEM of the data shown by symbols. Sixteen cells from 3 rats were used in (C); 32 cells from 4 rats in (D). One-way ANOVA and Tukey’s multiple comparisons post hoc test were used: **p < 0.01; ***p < 0.001; ns, non-significant. (E) Comparison of the concentration–response curves in (C) and (D). A fit of the data to the Michaelis–Menten equation allowed us to estimate Emax and EC50 values for the effects of Iso and PEG-Iso, as well as 95% confidence intervals (CI 95%) for each parameter: Emax = 168.9% (CI 95% 141.1, 196.8) and EC50 = 4.6 nM (CI 95% 1.3, 7.9) for Iso; Emax = 107.3% (CI 95% 98.1, 116.5) and EC50 = 297.5 nM (CI 95% 174.1, 420.9) for PEG-Iso. The two curves are statistically different at p < 0.05 (*) since there was no overlap of CI 95% for each parameter.
Figure 4
Comparison of the effects of PEG-Iso and Iso on ICa,L.
The whole-cell patch-clamp technique was applied to adult rat ventricular myocytes (ARVMs) after 24 hr culture. (A) Typical experiment showing the time course of peak ICa,L current density upon application of 10 nM Iso and during washout. (B) Similar experiment showing the response of peak ICa,L current density to 1 µM PEG-Iso. Individual current traces shown above the graphs were taken at times indicated by the corresponding letters on the graphs. (C) Mean ± SEM. increase in peak ICa,L in response to 10 nM Iso or 1 µM PEG-Iso stimulation. Student’s unpaired t-test: ns, non-significant, n = 11–12.
Figure 5
Comparison of the effects of PEG-Iso and Iso on sarcomere shortening.
Representative traces of sarcomere shortening recorded in adult rat ventricular myocytes (ARVMs) paced at 0.5 Hz and loaded with Fura-2 AM 1 µM showing the effects of Iso 1 and 3 nM (A) and PEG-Iso 100 and 300 nM(B). The bars in (A) and (B) show the mean ± SEM of the data shown by symbols. (C, D) Average time-to-50% relaxation of sarcomere shortening from experiments shown in (A) and (B), respectively. Thirty cells from 4 rats were used in (A) and (C); 34 cells from 4 rats in (B) and (D). One-way ANOVA and Tukey’s post hoc test: *p < 0.05; ***p < 0.001; ns, non-significant.
Figure 6
Comparison of the effects of PEG-Iso and Iso on Ca2+ transients.
Representative traces of Ca2+ transients recorded in adult rat ventricular myocytes (ARVMs) paced at 0.5 Hz and loaded with Fura-2 AM (1 µM) showing the effect of Iso 1 and 3 nM (A) and PEG-Iso 100 and 300 nM (B). The bars in (A) and (B) show the mean ± SEM with individual data points denoted by symbols. (C, D) Exponential time constant (Tau) of relaxation of Ca2+ transients from experiments shown in (A) and (B), respectively. Thirty cells from 4 rats were used in (A) and (C); 34 cells from 4 rats in (B) and (D). One-way ANOVA and Tukey’s post hoc test: *p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant.
Figure 7
Comparison of the effects of PEG-Iso and Iso on cytosolic PKA activity.
Freshly isolated adult rat ventricular myocytes (ARVMs) were infected with an adenovirus encoding the AKAR3-NES FRET-based PKA sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment showing the time course of YFP/CFP ratio during successive applications of three increasing Iso concentrations: 0.3, 1, and 3 nM. (B) Similar experiment showing the time course of YFP/CFP ratio during successive applications of three increasing PEG-Iso concentrations: 10, 30, and 100 nM. Pseudo-color images shown above the main graphs were taken at times indicated by the corresponding letters in the graphs. (C, D) Summary data from several similar experiments as in (A) and (B), respectively. The bars show the mean ± SEM of the data shown by symbols. Three rats and 8–10 cells were used in (C); 3–5 rats and 13–18 cells in (D). One-way ANOVA and Tukey’s multiple comparisons post hoc test: **p < 0.01; ***p < 0.001.
Figure 8
Comparison of the effects of PEG-Iso and Iso on nuclear cAMP activity.
Freshly isolated adult rat ventricular myocytes (ARVMs) were infected with an adenovirus encoding the nuclear Epac-SH187-3NLS FRET-based nuclear cAMP sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment showing the time course of CFP/YFP ratio during successive applications of increasing Iso concentrations and (B) a dose–response of increasing PEG-Iso concentrations. Pseudo-color images shown above the main graphs were taken at times indicated by the corresponding letters in the graphs. (C, D) Summary data from several similar experiments as in (A) and (B), respectively. The bars show the mean ± SEM of the data shown by symbols. Two rats and 6 cells were used in (C) and 6 cells from 3 rats in (D). One-way ANOVA and Tukey’s multiple comparisons post hoc test were used: *p < 0.05; **p < 0.01.
Figure 9 with 6 supplements
Comparison of the effects of PEG-Iso and Iso on nuclear PKA activity.
Freshly isolated adult rat ventricular myocytes (ARVMs) were infected with an adenovirus encoding the AKAR3-NLS FRET-based PKA sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment showing the time course of YFP/CFP ratio during successive applications of three increasing Iso concentrations: 0.3, 1, and 3 nM. (B) Similar experiment showing the time course of YFP/CFP ratio during successive applications of three increasing PEG-Iso concentrations: 10, 30, and 100 nM. Pseudo-color images shown above the main graphs were taken at times indicated by the corresponding letters in the graphs. (C, D) Summary data from several similar experiments as in (A) and (B), respectively. The bars show the mean ± SEM of the data shown by symbols. Three rats and 10–12 cells were used in (C) 3–5 rats and 10–17 cells in (D). One-way ANOVA and Tukey’s multiple comparisons post hoc test: *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 9—figure supplement 1
Comparison of the effects of PEG-Iso and Iso on nuclear proteins phosphorylation levels by PKA.
Freshly isolated adult rat ventricular myocytes (ARVMs) were treated with or without Iso 10 nM or PEG-Iso 1 µM during 15 min. Nuclear proteins were extracted, and proteins phosphorylated by PKA were revealed using a phospho-PKA substrate antibody. (A) Total proteins labeling as a loading control. (B) Immunoblot for phosphorylation of PKA substrates using a specific phospho-PKA antibody. (C) Quantification of phospho-PKA levels normalized on total protein, N = 3. One-way ANOVA and Tukey’s post hoc test: *p < 0.05; **p < 0.01. (see also Figure 9—figure supplement 1—source data 1). The bars show the mean ± SEM of the data.
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Figure 9—figure supplement 1—source data 1
TIF files containing original western blots for Figure 9—figure supplement 1A, B.
- https://cdn.elifesciences.org/articles/84243/elife-84243-fig9-figsupp1-data1-v2.zip
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Figure 9—figure supplement 1—source data 2
PRISM, PDF, and Excel files for the western blot analysis in Figure 9—figure supplement 1A–C.
- https://cdn.elifesciences.org/articles/84243/elife-84243-fig9-figsupp1-data2-v2.zip
Figure 9—figure supplement 2
Plot of cytosolic and nuclear PKA activity as a function of cytosolic cAMP in response to Iso and PEG-Iso.
For three concentrations of Iso (0.3, 1, and 10 nM) and PEG-Iso (10 nM, 100 nM, and 1 µM), the average percent increase (± SEM) in YFP/CFP ratio for cytosolic PKA activity (A, taken from Figure 7) and nuclear PKA activity (B, taken from Figure 8) are plotted as a function of the average percentage increase (± SEM) in CFP/YFP ratio for cytosolic cAMP (taken from Figure 3).
Figure 9—figure supplement 3
PEG-Iso cytosolic cAMP and nuclear cAMP/PKA responses after treatment of cells with neuraminidase.
Freshly isolated adult rat ventricular myocytes (ARVMs) were infected with an adenovirus encoding the (A, B) Epac-SH187 FRET-based cytosolic cAMP sensor, (C, D) Epac-SH187-3NLS FRET-based nuclear cAMP, or (E, F) AKAR-NLS FRET-based nuclear PKA sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. On the day of experimentation, a subset of cells was pretreated with 0.25 U/ml neuraminidase (+nmd, green) for 1 hr at 37°C. All cells were washed in physiological solution before use and responses to 10 μM PEG-Iso (blue) with and without neuraminidase were recorded. An equivalent Iso concentration of 100 nM Iso (black) was used as a negative control for the effects of neuraminidase. Symbols are from individual cells together with the mean ± SEM. Unpaired t-test was performed in each condition: *p < 0.05; **p < 0.01. n = 4–13. (G) A representative increase in nuclear PKA response to PEG-Iso ± neuraminidase.
Figure 9—figure supplement 4
Detubulation normalizes the effect of Iso and PEG-Iso.
T-tubules were chemically removed by incubating the cells with 300 µM imipramine for 15 min. Loading the cells with 2 µM Di4Anepps on a 10x spinning disk confocal (20 µm scale bar) showed that control cells had a dense (a) intracellular T-tubule network, while this had been completely detubulated (b) in imipramine-treated cells. FRET experiments showed that without T-tubules and only outer surface membrane (OSM) β-ARs, the response to stimulation with Iso (black) and PEG-Iso (blue) elicited the same cytosolic cAMP (c), cytosolic PKA (d), and nuclear PKA (e) responses. Data points are of the mean ± SEM. ns, not significant. Tested by two-way ANOVA. Confocal images were checked for detubulation in N = 3 isolations. FRET n = 2–12.
Figure 9—figure supplement 5
Effect of permeant (propranolol) and impermeant (sotalol) β-blockers on nuclear PKA response to β-AR stimulation by Iso.
Freshly isolated adult rat ventricular myocytes (ARVMs) were infected with an adenovirus encoding the AKAR-NLS FRET-based nuclear PKA sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. On the day of experimentation, cells were pretreated with either vehicle, propranolol 1 µM (red) or sotalol 25 µM (orange) for 15 min prior to stimulation with Iso 10 nM (black). (a) Representative traces of typical responses to Iso stimulation alone or with β-blockers being preincubated for 15 min and added with Iso. (b, c) Baseline and percentage of YFP/CFP increase after Iso with or without β-blockers, respectively. The bars present mean ± SEM. with N = 3 rats, n = 8–13 cells with 15–26 nuclei. One-way ANOVA and Tukey’s multiple comparisons post hoc test were performed: ***p < 0.001.
Figure 9—figure supplement 6
T-tubule network is still robust up to 48 hr post-isolation.
The density of T-tubules was quantified in adult rat ventricular myocytes after 0 (a), 24 (b), and 48 hr (c) post-isolation using 2 µM Di4Anepps on a 10x spinning disk confocal. Summary quantification supports a decrease in the T-tubule network over 48 hr but still a robust network at the end of the experimental time frame (d). One-way ANOVA with Tukey’s post hoc test performed; n = 13–31. ***p < 0.001; ****p < 0.0001. The bars show the mean ± SEM of the data.
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Distinct functions of cardiac β-adrenergic receptors in the T-tubule vs. outer surface membrane
eLife 14:e84243.
https://doi.org/10.7554/eLife.84243
