Loss of caveolin-3-dependent regulation ofICain rat ventricular myocytes in heart failure

Figures

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  Fig. 1.. β2-Adrenergic potentiation of L-type Ca2+ current (ICa) in sham and coronary artery ligated (CAL) myocytes. A: representative ICa traces (elicited by step depolarization to 0 mV) recorded from intact and detubulated (DT) myocytes isolated from sham hearts. Overlapping traces were from the same cell and were recorded under control conditions and after application of 1 and 3 µmol/l zinterol (in the presence of 10 µmol/l atenolol). Vertical scale bar = 1 nA; horizontal scale bar = 50 ms. B: time course of changes in mean normalized peak ICa (±SE) of intact (n = 5) and DT (n = 6) sham myocytes during superfusion with control solution (containing 10 μmol/l atenolol) and 1 and 3 µmol/l zinterol. ICa, elicited by step depolarization to 0 mV at 0.1 Hz, was expressed as a percentage of control measured just before application of the first concentration of zinterol. C: mean changes in ICa elicited by application of 1 and 3 µmol/l zinterol to intact (1 μmol/l: n = 7 and 3 μmol/l: n = 9) and DT (1 μmol/l: n = 7 and 3 μmol/l: n = 9] sham myocytes. Data were subjected to two-way ANOVA: β2-agonism P < 0.001; DT P < 0.001; interaction P < 0.001. *P < 0.05 and ***P < 0.001, Bonferroni post hoc…

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  Fig. 2.. Increase in L-type Ca2+ current (ICa) through direction activation of adenylyl cyclase in sham and coronary artery ligated (CAL) myocytes. A: representative ICa traces (elicited by step depolarization to 0 mV) recorded in the absence and presence of forskolin (FSK; 10 μmol/l and 0.5 mmol/l CaCl2) from intact and detubulated (DT) myocytes isolated from sham hearts. Overlapping traces were taken from the same myocytes under control conditions and after 3-min perfusion with FSK. Vertical scale bar = 2 pA/pF; horizontal scale bar = 100 ms. B: mean ICa density-voltage relationships from intact (n = 12) and DT (n = 14) sham myocytes in the absence and presence of FSK. Not significant (ns), P > 0.05, *P < 0.05; two-way ANOVA with Bonferroni post hoc test, intact vs. DT cells. C: effect of FSK on peak ICa density (elicited at −10 mV) recorded in intact and DT sham myocytes. Data were subjected to two-way ANOVA: FSK P < 0.001, DT P < 0.01, interaction ns. ***P < 0.001, Bonferroni post hoc test. D: representative ICa traces recorded in the absence and presence of FSK (10 μmol/l and 0.5 mmol/l CaCl2) from intact and DT myocytes isolated from CAL hearts. Conditions and scale were as in A. E:…

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  Fig. 3.. Constitutive regulation of basal L-type Ca2+ current (ICa) by caveolin-3 (Cav-3). A: representative ICa traces recorded from intact and detubulated (DT) myocytes isolated from sham hearts. Overlapping traces were taken from different myocytes that had either undergone incubation with C3SD peptide (1 µmol/l) or were untreated. Vertical scale bar = 2 pA/pF; horizontal scale bar = 100 ms. B: mean ICa density-voltage relations from untreated intact sham cells (n = 16) and intact sham cells treated with C3SD peptide (n = 16). **P < 0.01, two-way ANOVA with Bonferroni post hoc test, untreated vs. C3SD-treated cells. C: mean ICa density-voltage relations from untreated sham DT cells (n = 20) and sham DT cells treated with C3SD peptide (n = 10). Not significant (ns), P > 0.05; two-way ANOVA with Bonferroni post hoc test, untreated vs. C3SD-treated cells. D: effect of C3SD on peak ICa density (elicited at 0 mV) recorded from intact and DT sham myocytes. Data were subject to two-way ANOVA: C3SD ns, DT P < 0.001, interaction P < 0.01. **P < 0.01 and ***P < 0.001, Bonferroni post hoc test. E: representative ICa traces recorded from intact and DT myocytes isolated from coronary artery ligated…

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  Fig. 4.. Role of PKA in caveolin-3 (Cav-3)-dependent regulation of basal L-type Ca2+ current (ICa) in sham and coronary artery ligated (CAL) myocytes. A: representative ICa traces recorded from intact untreated (control) and C3SD-treated (C3SD) myocytes isolated from sham hearts. Overlapping traces were taken from the same myocytes before and after application of the PKA inhibitor H-89 (20 µmol/l). Vertical scale bar = 2 pA/pF; horizontal scale bar = 100 ms. B: mean ICa density-voltage relationship curves recorded from intact myocytes isolated from sham hearts that were either untreated (n = 16) or treated with C3SD (n = 16) before and after application of H-89. Control data are the same as those shown in Fig. 3B. ***P < 0.001, two-way ANOVA with Bonferroni post hoc test, absence vs. presence of H-89. C: effect of PKA inhibition on mean peak ICa density (elicited at 0 mV) in sham myocytes that were untreated or treated with C3SD. Data were subjected to two-way ANOVA: C3SD not significant (ns), H-89 P < 0.001, interaction ns. *P < 0.05, **P < 0.01, and ***P < 0.001, Bonferroni post hoc test. D: representative ICa traces recorded from untreated and C3SD-treated myocytes isolated from CAL…

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  Fig. 5.. Localization of PKA-dependent regulation of basal L-type Ca2+ current (ICa) in sham and coronary artery ligated (CAL) myocytes. A: representative ICa traces recorded from intact and detubulated (DT) myocytes isolated from sham hearts. Overlapping traces were taken from the same myocytes before and after application of the PKA inhibitor H-89 (20 µmol/l). Vertical scale bar = 2 pA/pF; horizontal scale bar = 100 ms. B: mean ICa density-voltage relationship curves recorded from myocytes isolated from sham hearts that were either intact (n = 17) or DT (n = 8) before and after application of H-89. ***P < 0.001, two-way ANOVA with Bonferroni post hoc test, control vs. H-89. C: effect of PKA inhibition on mean peak ICa density (elicited at 0 mV) in intact or DT sham myocytes under control conditions or after PKA inhibition (H-89). Data were subjected to two-way ANOVA: H-89 P < 0.001, DT P < 0.001, interaction P < 0.05. ***P < 0.001, Bonferroni post hoc test. D: representative ICa traces recorded from intact and DT myocytes isolated from CAL hearts; overlapping traces were taken from the same myocytes before and after application of the PKA inhibitor H-89. Conditions and scale were as in…

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  Fig. 6.. A: mean L-type Ca2+ current (ICa) density at 0 mV measured in intact (total cell membrane) and detubulated (DT) cells (surface membrane) and calculated at the t-tubules (t-tubule membrane) for sham myocytes. Correction for incomplete detubulation was applied (see methods). Control conditions and treatment with H-89 are shown. B: mean ICa density at 0 mV measured in intact (total cell membrane) and DT cells (surface membrane) and calculated at the t-tubules (t-tubule membrane) for coronary artery ligated (CAL) myocytes. Correction for incomplete detubulation was applied (see methods). Control conditions and treatment with H-89 are shown. *P < 0.05 and **P < 0.01, Student’s t-test.

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  Fig. 7.. Schema summarizing the role of caveolin-3 (Cav-3) in the regulation of L-type Ca2+ current (ICa) in normal ventricular myocytes and in heart failure. A: regulation of ICa in normal cardiac myocytes. L-type Ca2+ channel (LTCC) density is greatest in the t-tubules, where Cav-3 coordinates a signaling domain involving β2-adrenoceptors (β2ARs), adenylyl cyclase (Ad Cyc), PKA, and the LTCC α1c-subunit Cav1.2. β2-Adrenoceptors coupled with LTCCs are located exclusively in the t-tubules. Adenylyl cyclase, PKA, and Cav1.2 are also located outside of Cav-3 signaling domains, both within and without t-tubules. Activation of adenylyl cyclase, either via β2-adrenoceptors or directly, augments LTCC activity through the production of cAMP. B: remodeling of ICa regulation in heart failure. The Cav-3 signaling complex is disrupted. β2-Adrenoceptors are located both within the t-tubules and on the surface sarcolemma. LTCC density is more evenly distributed between t-tubules and surface sarcolemma. The role of Cav-3 in the regulation of ICa is lost in heart failure. Schema represents the simplest explanation of the data. Other mechanisms are possible; for example, β2-adrenoceptors may be located…