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

Abstract

β₂-Adrenoceptors and L-type Ca²⁺ current ( I_Ca) redistribute from the t-tubules to the surface membrane of ventricular myocytes from failing hearts. The present study investigated the role of changes in caveolin-3 and PKA signaling, both of which have previously been implicated in this redistribution. I_Ca was recorded using the whole cell patch-clamp technique from ventricular myocytes isolated from the hearts of rats that had undergone either coronary artery ligation (CAL) or equivalent sham operation 18 wk earlier. I_Ca distribution between the surface and t-tubule membranes was determined using formamide-induced detubulation (DT). In sham myocytes, β₂-adrenoceptor stimulation increased I_Ca in intact but not DT myocytes; however, forskolin (to increase cAMP directly) and H-89 (to inhibit PKA) increased and decreased, respectively, I_Ca at both the surface and t-tubule membranes. C3SD peptide (which decreases binding to caveolin-3) inhibited I_Ca in intact but not DT myocytes but had no effect in the presence of H-89. In contrast, in CAL myocytes, β₂-adrenoceptor stimulation increased I_Ca in both intact and DT myocytes, but C3SD had no effect on I_Ca; forskolin and H-89 had similar effects as in sham myocytes. These data show the redistribution of β₂ -adrenoceptor activity and I_Ca in CAL myocytes and suggest constitutive stimulation of I_Ca by PKA in sham myocytes via concurrent caveolin-3-dependent (at the t-tubules) and caveolin-3-independent mechanisms, with the former being lost in CAL myocytes. NEW & NOTEWORTHY In ventricular myocytes from normal hearts, regulation of the L-type Ca²⁺ current by β₂-adrenoceptors and the constitutive regulation by caveolin-3 is localized to the t-tubules. In heart failure, the regulation of L-type Ca²⁺ current by β₂-adrenoceptors is redistributed to the surface membrane, and the constitutive regulation by caveolin-3 is lost.

Keywords: L-type Ca2+ current; cAMP; caveolin-3; heart failure; myocardial infarction.

Fig. 1.

β₂-Adrenergic potentiation of L-type Ca²⁺ current (I_Ca) in sham and coronary artery ligated (CAL) myocytes. A: representative I_Ca 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 I_Ca (±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. I_Ca, 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 I_Ca 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 test. D: representative I_Ca traces (elicited by step depolarization to 0 mV) recorded from intact and DT myocytes isolated from CAL hearts. Conditions and scale were as in A. E: time course of changes in mean normalized peak I_Ca of intact (n = 5) and DT (n = 4) CAL myocytes during superfusion with control solution (containing 10 μmol/l atenolol) and 1 and 3 μmol/l zinterol. F: mean changes in I_Ca elicited by application of 1 and 3 µmol/l zinterol to intact (1 μmol/l: n = 5 and 3 μmol/l: n = 19) and DT (1 μmol/l: n = 4 and 3 μmol/l: n = 8) CAL myocytes. Data were subjected to two-way ANOVA: β₂-agonism P < 0.001, DT not significant, interaction not significant. **P < 0.01 and ***P < 0.001, Bonferroni post hoc test.

Fig. 2.

Increase in L-type Ca²⁺ current (I_Ca) through direction activation of adenylyl cyclase in sham and coronary artery ligated (CAL) myocytes. A: representative I_Ca 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 I_Ca 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 I_Ca 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 I_Ca 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: mean I_Ca density-voltage relationships from intact (n = 18) and DT (n = 12) CAL myocytes in the absence and presence of FSK. ns, P > 0.05, *two-way ANOVA with Bonferroni post hoc test, intact vs. DT cells. F: effect of FSK on peak I_Ca density (elicited at −10 mV) recorded in intact and DT CAL 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.

Fig. 3.

Constitutive regulation of basal L-type Ca²⁺ current (I_Ca) by caveolin-3 (Cav-3). A: representative I_Ca 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 I_Ca 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 I_Ca 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 I_Ca 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 I_Ca traces recorded from intact and DT myocytes isolated from coronary artery ligated (CAL) hearts. Conditions and scale were as in A; overlapping traces were taken from different myocytes that had either undergone incubation with C3SD peptide (1 µmol/l) or were untreated. F: mean I_Ca density-voltage relations from untreated intact CAL cells (n = 14) and intact CAL cells treated with C3SD peptide (n = 15). ns, P > 0.05, two-way ANOVA with Bonferroni post hoc test, untreated vs. C3SD-treated cells. G: mean I_Ca density-voltage relations from untreated DT CAL cells (n = 22) and DT CAL cells treated with C3SD peptide (n = 7). ns, P > 0.05, two-way ANOVA with Bonferroni post hoc test, untreated vs. C3SD-treated cells. H: effect of C3SD on peak I_Ca density (elicited at 0 mV) recorded from intact and DT CAL myocytes. Data were subjected to two-way ANOVA: C3SD ns, DT ns, interaction ns.

Fig. 4.

Role of PKA in caveolin-3 (Cav-3)-dependent regulation of basal L-type Ca²⁺ current (I_Ca) in sham and coronary artery ligated (CAL) myocytes. A: representative I_Ca 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 I_Ca 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 I_Ca 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 I_Ca traces recorded from untreated and C3SD-treated myocytes isolated from CAL hearts before and after application of the PKA inhibitor H-89 (20 µmol/l). Conditions and scale were as in A. E: mean I_Ca density-voltage relationship curves recorded from intact myocytes isolated from CAL hearts that were either untreated (n = 14) or treated with C3SD (n = 15) before and after application of H-89. Control data are the same as those shown in Fig. 3F. ***P < 0.001, two-way ANOVA with Bonferroni post hoc test, absence vs. presence of H-89. F: effect of PKA inhibition on mean peak I_Ca density (elicited at 0 mV) in CAL myocytes that were treated or untreated with C3SD. Data were subjected to two-way ANOVA: C3SD ns, H-89 P < 0.001, interaction ns. ***P < 0.001, Bonferroni post hoc test.

Fig. 5.

Localization of PKA-dependent regulation of basal L-type Ca²⁺ current (I_Ca) in sham and coronary artery ligated (CAL) myocytes. A: representative I_Ca 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 I_Ca 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 I_Ca 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 I_Ca 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 A. E: mean I_Ca density-voltage relationship curves recorded from myocytes isolated from CAL hearts that were either intact (n = 14) or DT (n = 9) before and after application of H-89. ***P < 0.001, two-way ANOVA with Bonferroni post hoc test, control vs. H-89. F: effect of PKA inhibition on mean peak I_Ca 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 not significant, interaction not significant. ***P < 0.001, Bonferroni post hoc test.

Fig. 6.

A: mean L-type Ca²⁺ current (I_Ca) 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 I_Ca 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.

Fig. 7.

Schema summarizing the role of caveolin-3 (Cav-3) in the regulation of L-type Ca²⁺ current (I_Ca) in normal ventricular myocytes and in heart failure. A: regulation of I_Ca 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 α_1c-subunit Ca_v1.2. β₂-Adrenoceptors coupled with LTCCs are located exclusively in the t-tubules. Adenylyl cyclase, PKA, and Ca_v1.2 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 I_Ca 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 I_Ca is lost in heart failure. Schema represents the simplest explanation of the data. Other mechanisms are possible; for example, β₂-adrenoceptors may be located in both the cell surface and t-tubule membranes in normal cardiac myocytes, but the coupling of β₂-adrenoceptors with LTCCs is confined to the t-tubules.