The β1 adrenoceptor (AR) blocker metoprolol normalises βAR cascade and caveolar protein expression and restores β1AR responsiveness in failing right ventricular myocytes

Figures

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  Fig. 1. β blocker (BB) treatment in monocrotaline (MCT) animals partially restores the amplitude of RV cardiomyocyte shortening and relaxation kinetics in response to β1AR stimulation. All recordings were made in the presence of the β2AR antagonist ICI 118,551 (ICI). A. Representative traces of cell shortening (as % resting cell length) at baseline (solid line) and following stimulation with 100 nM isoprenaline (ISO; broken line) in control (CON), MCT animals treated with metoprolol (MCT + BB) and the MCT group. B. Log10 concentration-response relationship for fractional shortening (as % resting length) with ISO. C. Log10 concentration-response relationship for time to half (t0.5) relaxation with ISO. D. Fractional shortening with 100 nM ISO. E. t0.5 relaxation with 100 nM ISO. Symbols in B + C represent mean ± SEM of n = 4–66 cells from 5 to 6 rats per group. Open symbols in D + E show individual cell values, bars show mean ± SEM of cell values and cell numbers, and crosses show mean values for each animal. Hierarchical statistics with Bonferroni post hoc correction were performed on D + E to account for multiple cells being isolated from each heart [26]. Number of cells shown on bars,…

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  Fig. 2. β blocker (BB) treatment in monocrotaline (MCT) animals partially restores the amplitude and kinetics of the Ca2+ transient in response to β1AR stimulation. All recordings were made in the presence of the β2AR antagonist ICI 118,551 (ICI). A. Representative traces of individual Ca2+ transients (expressed as % change from baseline in fura-2 ratio units, RU) in absence (solid line) and presence (broken line) of 100 nM isoprenaline (ISO) in control (CON), MCT animals treated with metoprolol (MCT + BB) and the MCT group. B. Log10 concentration-response relationship for Ca2+ transient amplitude response to ISO. The response is expressed as % change from ICI baseline C. Log10 concentration-response relationship for change in t0.5 Ca2+ transient decay with ISO. D. Ca2+ transient amplitude with 100 nM ISO (expressed as % change from ICI). E. t0.5 transient decay with 100 nM ISO (expressed as % change from mean ICI). Symbols in B + C represent mean ± SEM of n = 6–27 cells from 5 to 6 rats per group. Open symbols in D + E show individual cell values, bars show mean ± SEM of cell values and number of cells, and crosses show mean values for each animal. Hierarchical statistics with…

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  Fig. 3. β blocker (BB) treatment does not affect the inotropic and lusitropic response to β2AR stimulation in monocrotaline (MCT) animals. All recordings were made in the presence of the β1AR antagonist CGP 20712A. A. The proportion of cells showing a positive inotropic response to zinterol (ZIN; 1–10 μM) was not different in RV myocytes from the 3 groups (P > 0.05; Lancaster's mid-P test with Bonferroni post hoc correction; number of cells shown on bars; N = 5–7 animals). B. Fractional shortening (expressed as % resting length) and C. time to half (t0.5) relaxation with 10 μM ZIN in the presence of CGP. Data are from cells that showed a positive inotropic response to ZIN. Open symbols in B + C show individual cell values, bars show mean ± SEM of cell values and cell numbers, and crosses show mean values for each animal. Hierarchical statistics with Bonferroni post hoc correction were performed on B + C to account for multiple cells being isolated from each heart [26]. Cells were isolated from 2 to 7 rats per group. 2 CON and 1 MCT + BB cell in panel B, and 3 CON, 1 MCT + BB and 1 MCT cell in panel C were automatically excluded as outliers > 2SD from the mean and were not included in…

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  Fig. 4. β blocker (BB) treatment normalises total expression of proteins of the β1AR cascade and the caveolar domain in monocrotaline (MCT) animals. A. Western blot from the same gel of β1AR, adenyl cyclase (AC) 5/6 and G protein receptor kinase (GRK) 2 in RV myocardial homogenate. B. Protein expression normalised to GAPDH and expressed as % mean control (CON) value. C. Western blot from the same gel of caveolin (Cav) and cavin in RV myocardial homogenate. D. Expression of caveolar proteins normalised to GAPDH and expressed as % mean control (CON) value. Data in B. and D. are mean + SEM, N = 6 animals in each group. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA); # P < 0.05 (Kruskal Wallis).

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  Fig. 5. The impact of monocrotaline (MCT) and β blocker (BB) treatment on the membrane and cellular distribution of key elements of the βAR cascade and its regulators. Equal volumes of each pooled (4–5, 9–12) or individual (8) fractions were loaded. Fractions 4–5 contain caveolae; fractions 9–12 contain non-caveolar membranes and cytosolic proteins. The same amount of total protein was loaded for each heart. Distribution is shown as % of the sum of fractions 4–12 for A. β1AR B. AC5/6C. GRK2 and D. Cav3. Data are mean + SEM (N = 6 in each group, apart from A where N = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA).

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  Fig. 6. Computer simulation of mean/compartmentalised cAMP and Ca2+ transients in response to β1AR stimulation in a cardiac myocyte. Experimental data from control (CON), MCT rats treated with metoprolol (MCT + BB) and the MCT group were used to modify the model of Heijman et al. [30], as outlined in Supplementary Table 1. The response to 100 nM isoprenaline (ISO) was modelled in the presence of β2AR blockade. A. Mean cAMP levels (left panel) under basal conditions and with ISO stimulation. Mean cAMP was predicted based on the relative volume of 3 functional compartments: caveolar (CAV), extra-caveolar (ECAV) and cytosolic (CYT) which are 2, 4 and 67.8% respectively. For comparison, experimental data from Leineweber et al. [31] in rat RV myocyte homogenates is shown (right panel). B. Cyclic AMP levels under basal conditions and with ISO stimulation in individual compartments from the model. C. Time course of Ca2+ transient amplitude to ISO. D. Individual Ca2+ transients in absence (solid lines) and presence (broken lines) of ISO. E. Ca2+ transient amplitude in response to ISO (expressed as % of baseline). F. Ca2+ transient decay in response to ISO (expressed as % of baseline).

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  Fig. 7. Computational modelling of target protein phosphorylation in response to β1AR stimulation. A. Fraction of protein kinase A (PKA) phosphorylated L-type Ca2+ channel (LTCC), ryanodine receptor 2 (RyR2), phospholamban (PLB) and troponin I (TnI) with (+) and without (−) ISO stimulation. B. Fraction of Ca2+/calmodulin-dependent kinase II (CaMKII) phosphorylated LTCC, RyR2 and PLB.

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  Fig. 8. Simulation of the contribution of changes in individual β1AR cascade proteins and Cav 3 to the response to β1AR stimulation. For this simulation, mean cAMP was used as the output measure. Baseline and ISO bars show cAMP in each group with all experimental changes enabled (i.e. the same as Fig. 6A left panel). Additional simulations were performed with each change in protein expression or distribution disabled in turn (indicated by red line) to assess their individual impact on the cAMP response in MCT and MCT + BB groups. For each panel, the dotted line indicates the ISO response with all protein changes enabled. In MCT, cAMP increased most when the disease-associated decrease in expression of AC5/6, and to a lesser extent β1AR, was restored. With BB treatment in the MCT group, AC5/6 and β1AR expression contributed approximately equally to the change in cAMP. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)