Loss of caveolin-3-dependent regulation of I_Ca 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.

INTRODUCTION

L-type Ca²⁺ current (I_Ca) plays a key role in excitation-contraction (EC) coupling in cardiac ventricular myocytes: activation of L-type Ca²⁺ channels (LTCCs) during the action potential causes influx of Ca²⁺ that triggers Ca²⁺ release via ryanodine receptors (RyRs) in the adjacent sarcoplasmic reticulum (SR) membrane (2, 8). Previous work has shown that the function of many of the key proteins involved in EC coupling, including LTCCs and RyRs, occurs predominantly at the t-tubules: invaginations of the surface membrane that enable near-synchronous SR Ca²⁺ release, and thus contraction, throughout the cell (18, 21, 28). The mechanism for the localization of I_Ca at the t-tubules is less clear, although it has been suggested that the caveolar protein caveolin-3 (Cav-3) plays a role in the localization of I_Ca, possibly via a mechanism involving cAMP/PKA signaling pathways (1, 5, 9, 24).

Cav-3 is also involved in the localization of cAMP signaling via β₂-adrenoceptors to the t-tubules, and it has been proposed that LTCCs and β₂-adrenoceptors are colocalized in a Cav-3 signaling microdomain (1, 5, 7, 23, 30). It has been shown that Cav-3 plays a critical role in the constitutive maintenance of I_Ca at the t-tubule (5). In heart failure, there is redistribution of β₂-adrenoceptors from the t-tubular to the surface membrane so that they become more uniformly distributed across the cell membrane (22, 27). This redistribution is associated with a change from localized to more diffuse signaling in response to β₂-adrenergic stimulation (27). We (6) have recently shown in a coronary artery ligation (CAL) model in that rat that ventricular I_Ca is also redistributed from the t-tubules to the surface sarcolemma in heart failure.

We hypothesized that the redistribution of I_Ca after CAL is due to loss of Cav-3-dependent localization at the t-tubules, which may be secondary to the decreased expression of Cav-3 observed in heart failure. Thus, changes in the localization of the β₂-signaling pathway in heart failure may be associated with a loss of constitutive regulation of I_Ca by PKA at the t-tubules. Therefore, we further investigated the relationship between the distribution of I_Ca and changes in Cav-3/β₂-adrenergic signaling observed after CAL in rats (6).

METHODS

Animals and surgical procedures.

All procedures were performed in accordance with United Kingdom legislation and approved by the University of Bristol Ethics Committee. This study was conducted in parallel with other investigations using cells from the same animals to investigate ventricular and atrial cellular remodeling in heart failure and thereby conformed with the reduction component of the 3Rs (“replace, reduce, refine”) (3, 6, 16). Adult male Wistar rats (~250 g) were subjected to either ligation of the left anterior descending coronary artery (CAL; 10 animals) or equivalent surgery without ligation (sham; 12 animals). Operations were conducted under general anesthesia [ketamine (75 mg/kg) and medetomidine (0.5 mg/kg ip)] with appropriate analgesia [buprenorphine (0.05 mg/kg sc)], as previously described (6). Data regarding changes in cardiac morphology and function as well as in cell morphology in these groups of animals have been previously published (3, 6).

Myocyte isolation.

Left ventricular myocytes were isolated from the hearts ~18 wk after surgery as previously described (5). Animals were euthanized under pentobarbitone anesthesia, and the heart was quickly excised and Langendorff perfused at 8 ml/min (37°C), initially with Tyrode solution (see Solutions below) plus 0.75 mmol/l CaCl₂ for 4 min and then in nominally Ca²⁺-free solution for 4 min and finally plus 1 mg/ml collagenase (Worthington) for 10 min. The left ventricle was then excised and shaken in collagenase-containing solution at 37°C for 5–7 min, filtered, and centrifuged. The supernatant was discarded, and the pellet was resuspended in Kraftbrühe solution and stored at 4°C for 2–10 h before use on the day of isolation (20). Detubulation (DT) of myocytes (physical and functional uncoupling of the t-tubules from the surface membrane) was achieved using formamide-induced osmotic shock, as previously described (21).

Solutions.

Tyrode solution for cell isolation contained (in mmol/l) 130 NaCl, 5.4 KCl, 0.4 NaH₂PO₄, 4.2 HEPES, 10 glucose, 1.4 MgCl₂, 20 taurine, and 10 creatinine; pH 7.4 (NaOH). Kraftbrühe solution for cell storage contained (in mmol/l) 90 l-glutamic acid, 30 KCl, 10 HEPES, 1 EGTA, 5 Na pyruvate, 20 taurine, 20 glucose, 5 MgCl₂, 5 succinic acid, 5 creatine, 2 Na₂ATP, and 5 β-OH butyric acid; pH 7.4 with KOH. For patch-clamp experiments, cells were superfused with solution that contained (in mmol/l) 133 NaCl, 1 MgSO₄, 1 CaCl₂, 1 Na₂HPO₄, 10 glucose, 10 HEPES [pH 7.4 (NaOH)]; 5 mmol/l CsCl was added to inhibit K⁺ currents. The pipette solution contained (in mmol/l) 110 CsCl, 20 TEA-Cl, 0.5 MgCl₂, 5 Mg-ATP, 5 BAPTA, 10 HEPES, and 0.4 GTP-Tris; pH 7.2 (CsOH). BAPTA was used to inhibit Ca²⁺-dependent inactivation of I_Ca (33).

Selective β₂-adrenoceptor stimulation was achieved as previously described (5) using the β₂-adrenoceptor agonist zinterol (1 and 3 μmol/l) in the presence of the β₁-adrenoceptor-selective antagonist atenolol (10 μmol/l); cells were superfused with atenolol alone for at least 4 min before superfusion with zinterol in the presence of atenolol. Under these conditions, the effects of 1 and 3 μmol/l zinterol could be completely abolished by 100 nM ICI-118,551, a β₂-adrenoceptor-selective antagonist (5). The plant alkaloid forskolin (10 μmol/l) was used to activate adenylyl cyclase directly (31). C3SD, a short peptide encompassing the Cav-3 scaffolding domain, was used to disrupt binding of Cav-3 to its protein partners as previously described (5, 13, 15, 23); myocytes were incubated in 1 µmol/l TAT-C3SD for at least 45 min before use. PKA was inhibited using H-89 (20 μmol/l) (11, 17).

Recording and analysis of I_Ca.

Myocytes were placed in a chamber mounted on a Nikon Diaphot inverted microscope. Membrane currents and cell capacitance were recorded using the whole cell patch-clamp technique using an Axopatch 200B, Digidata 1322A analog-to-digital converter, and pClamp 10 (Axon Instruments). Pipette resistance was typically 2–4 MΩ when filled with pipette solution, and pipette capacitance and series resistance were compensated by ~70%. Currents were activated from a holding potential of −80 mV by a 100-ms step depolarization to –40 mV (to inactivate Na⁺ current) followed by steps to potentials between −50 and +80 mV for 500 ms before repolarization to the holding potential at a frequency of 0.2 Hz. I_Ca amplitude (in pA) was measured as the difference between peak inward current and current at the end of the depolarizing pulse and was normalized to cell capacitance [in pF; a function of membrane area (25)] to calculate I_Ca density (in pA/pF). Surface membrane current density was obtained from currents measured in DT myocytes, whereas t-tubular membrane current density was calculated by subtraction of surface from whole cell currents and corrected for incomplete DT, as previously described (5, 6, 19, 21). DT efficiency, measured from images of intact and DT cells stained with di-8-ANEPPS, was ~84% and was not different between wild-type and CAL myocytes (6). To correct for incomplete DT, the distribution of membrane capacitance and I_Ca between the t-tubule and surface membrane was calculated as previously described (6). As we have previously reported, there was no statistically significant difference between sham and CAL myocytes in the degree of osmotic shock-induced DT, nor was there any relationship between the whole cell capacitance and time of recording (6).

Statistics.

Data are expressed as means ± SE of n myocytes. Statistical analysis was performed using GraphPad Prism (GraphPad Software). I_Ca density-voltage relationship curves were analyzed using repeated-measures ANOVA with voltage and the corresponding intervention (i.e., DT, H-89, or C3SD) as factors. I_Ca properties elicited by a step depolarization to a single voltage were analyzed by two-way ANOVA. Post hoc tests used the Bonferroni correction. The limit of statistical confidence was taken as P < 0.05. Errors in derived variables (specifically I_Ca density at the t-tubule membrane) and the subsequent statistical analysis (unpaired Student’s t-test) were calculated using propagation of errors from the source measurements (6, 14).

RESULTS

Effect of CAL on the response to β₂-adrenoceptor stimulation.

In intact ventricular myocytes from sham hearts, selective activation of β₂-adrenoceptors (1 and 3 μmol/l zinterol in the presence of 10 μmol/l atenolol) caused a significant, concentration-dependent increase of I_Ca, which reached ~140% of control in the steady state in the presence of 3 μmol/l of the β₂-agonist (Fig. 1A, left, B, and C). In contrast, in DT cells, 3 μmol/l zinterol did not increase I_Ca (Fig. 1A, right, B, and C). In CAL myocytes, 3 μmol/l zinterol caused an increase of ~40% in intact myocytes and ~29% in DT myocytes (Fig. 1, D–F). Thus, because I_Ca recorded in DT cells represents the current at the surface membrane, it appears that in sham myocytes, the response of I_Ca to β₂-adrenoceptor stimulation occurs predominantly at the t-tubule membrane. However, after CAL, the β₂-adrenergic response redistributes and occurs at both the cell surface and t-tubule membranes. These data also show that the DT procedure per se is not responsible for the lack of response to zinterol observed in sham myocytes.

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.

Effect of CAL on the response to forskolin.

To investigate whether the distribution of the β₂-adrenergic response was due to localization of a downstream component of the signaling pathway, we used forskolin (10 μmol/l) to activate adenylyl cyclase directly, to increase cAMP in the absence of adrenoceptor stimulation. Superfusion with forskolin (10 μmol/l) increased I_Ca in both intact and DT myocytes from sham hearts (Fig. 2A). The corresponding mean I_Ca density-voltage relationships for intact and DT myocytes are shown in Fig. 2B. Figure 2C shows the effect of forskolin on I_Ca density at a test potential of −10 mV in sham intact and DT myocytes. Forskolin also caused an increase in I_Ca in both intact and DT myocytes from CAL hearts (Fig. 2, D–F). These data show that the increase in I_Ca in response to forskolin was similar in intact sham and CAL myocytes. More importantly, these data also show that forskolin caused a significant increase in the amplitude of I_Ca in DT sham myocytes, which was similar to that observed in DT CAL myocytes. Thus, it appears that adenylyl cyclase and PKA are present at both the surface and t-tubule membranes in both sham and CAL myocytes and can stimulate I_Ca to a similar extent at either site. It is unlikely, therefore, that the lack of effect of zinterol in DT sham myocytes was due to absence of the components of the cAMP signaling pathway (i.e., adenylyl cyclase and PKA) at the cell surface but may be due to the absence of β₂-adrenoreceptors.

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.

Effect of CAL on the response to C3SD.

Since Cav-3 has been implicated in the localization of β₂-adrenoceptor/cAMP signaling at the t-tubules, we investigated the effect of acutely inhibiting Cav-3 binding to its partner proteins by pretreatment of cells with C3SD peptide (5, 15). I_Ca density was reduced in intact sham myocytes treated with the C3SD peptide (Fig. 3A, left, and B). However, treatment with C3SD had no effect on I_Ca density in DT sham myocytes (Fig. 3A, right, and C). The effects of treatment with C3SD on I_Ca density at 0 mV in intact and DT myocytes from sham hearts are shown in Fig. 3D. In contrast to its effect in sham myocytes, C3SD had no effect on I_Ca density in intact CAL myocytes (Fig. 3E, left, F, and H), nor did C3SD have any effect on I_Ca in DT CAL myocytes (Fig. 3E, right, G, and H). Thus, there appears to be no Cav-3-dependent regulation of I_Ca at the surface membrane in either sham or CAL myocytes. However, there does appear to be Cav-3-dependent stimulation of I_Ca at the t-tubules of sham myocytes, which is absent in CAL cells.

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.

Effect of CAL on the response to H-89 in the absence and presence of C3SD.

Since it has been suggested that Cav-3-dependent stimulation of t-tubular I_Ca is via a PKA-dependent mechanism (5), we investigated the effect of the PKA inhibitor H-89 on I_Ca density after CAL and the effect of C3SD on the response to H-89. Inhibition of PKA (20 μmol/l H-89) decreased I_Ca in untreated and C3SD-treated sham myocytes, indicating that constitutive stimulation of I_Ca by PKA that did not require Cav-3 (Fig. 4, A–C). Moreover, there was no difference in the I_Ca density-voltage relations of untreated and C3SD-treated cells in the presence of H-89 (Fig. 4B), demonstrating that the effects of H-89 and C3SD treatment were not summative. Thus, in the presence of PKA inhibition, treatment with C3SD peptide was without effect on I_Ca density, indicating that PKA activity was required for the constitutive regulation of I_Ca by Cav-3 in sham myocytes. Similarly, H-89 decreased I_Ca density to the same level in both untreated and C3SD-treated CAL myocytes, indicating constitutive regulation of I_Ca by PKA (Fig. 4, D–F). C3SD was without effect on I_Ca density in either the absence or presence of H-89 (Fig. 4, E and F). These data show that in sham myocytes, there is constitutive stimulation of I_Ca by PKA that is mediated both via Cav-3-dependent (localized to the t-tubule membrane) and Cav-3-independent mechanisms. Although the constitutive regulation by Cav-3 was lost in CAL myocytes, constitutive regulation of I_Ca via PKA remained.

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.

Effect of DT on the constitutive regulation of I_Ca by PKA.

To further investigate the site of constitutive PKA-dependent regulation, the response to H-89 was determined in DT myocytes. I_Ca density was reduced by H-89 in both intact and DT sham myocytes (Fig. 5, A–C). I_Ca density was also reduced by DT in both the presence or absence of PKA inhibition (Fig. 5C). H-89 also reduced I_Ca density in intact and DT myocytes from CAL hearts (Fig. 5, D–F). However, in contrast to sham myocytes, in CAL, I_Ca density was similar in intact and DT myocytes in either the presence or absence of PKA inhibition (Fig. 5F).

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.

The calculated current densities at the cell surface and in the t-tubule membrane before and after inhibition of PKA are shown in Fig. 6. These data show that in sham myocytes without inhibition of PKA, I_Ca density was significantly greater in the t-tubule membrane than at the cell surface, consistent with previous reports (5, 6, 16). In contrast, in CAL myocytes, there was no difference in I_Ca density between t-tubule and surface membranes (Fig. 6B). Inhibition of PKA caused a broadly similar fractional decrease in I_Ca at the surface membrane in both sham and CAL myocytes, so that surface membrane I_Ca remained larger in CAL than sham myocytes. Thus, constitutive stimulation of basal I_Ca by PKA at the cell surface was similar in the two cell types. H-89 also decreased t-tubular I_Ca density in both cell types so that it was smaller in CAL than sham myocytes. However, after inhibition of PKA, I_Ca density remained higher at the t-tubules than in the surface membrane in sham myocytes, whereas in CAL myocytes, I_Ca density was smaller at the t-tubules than at the surface membrane. Thus, it appears not only that I_Ca redistributes from the t-tubules to the cell surface in heart failure but also that constitutive regulation of t-tubular I_Ca by PKA is increased in these cells (cf. Fig. 6, A and B).

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.

DISCUSSION

This study presents two novel findings regarding the regulation of I_Ca in heart failure. First, stimulation of I_Ca by β₂-adrenoceptors, but not by adenylyl cyclase/PKA, is localized to the t-tubules in sham myocytes and redistributes to the cell surface after CAL. Second, it demonstrates constitutive stimulation of I_Ca by PKA in sham myocytes that is mediated both via Cav-3-dependent (at the t-tubules) and Cav-3-independent mechanisms, whereas in CAL myocytes, constitutive regulation by Cav-3 is lost, although that via PKA remains at both sites. Thus, the present study advances previous findings from our laboratory that Cav-3 plays a role in the regulation of I_Ca at the t-tubule by PKA and β₂-adrenoceptors in normal myocytes (5, 9) and that I_Ca is redistributed from the t-tubules to the surface sarcolemma in CAL-induced heart failure (6). Interestingly, although constitutive PKA-dependent stimulation of I_Ca at the cell surface appeared to be the same in both sham and CAL myocytes, constitutive stimulation of t-tubular I_Ca appeared to increase in CAL myocytes, helping to maintain t-tubular I_Ca. Figure 7 shows schematic diagrams illustrating the regulation of I_Ca by β₂-adrenoceptors, Cav-3, and PKA in normal cells (Fig. 7A) and in heart failure (Fig. 7B).

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.

Localization of I_Ca regulation by PKA in sham myocytes.

The Ca_v1.2 pore-forming α-subunit of ventricular LTCCs has been shown to be colocalized with Cav-3, adenylyl cyclase, PKA, and the β₂-adrenoceptor (1). Stimulation of β₂-adrenoceptors in cardiac myocytes activates adenylyl cyclase, causing a local increase of cAMP, activation of PKA, and thereby phosphorylation and stimulation of colocalized LTCCs (1). The present data show that β₂-adrenoceptor stimulation of I_Ca in sham myocytes occurs predominantly at the t-tubules (Fig. 1), although direct activation of adenylyl cyclase using forskolin increased I_Ca at both the t-tubular and surface membranes (Fig. 2). Thus, in normal myocytes, adenylyl cyclase and the downstream pathway is present at both the t-tubular and surface membranes, but the β₂-adrenoceptor is present only at the t-tubules, consistent with previous work showing t-tubular localization of β₂-adrenoceptor signaling in normal ventricular myocytes (27). Pretreatment with C3SD peptide decreased basal I_Ca at the t-tubules but not at the surface membrane in sham myocytes (Fig. 3), showing that Cav-3 plays a role in the constitutive stimulation of I_Ca at the t-tubules but not at the surface membrane in normal cells (Fig. 7A). These data are entirely consistent with our previous report (5) in which we showed that pretreatment with C3SD abolished both the constitutive regulation of I_Ca at the t-tubule and the response to β₂-adrenoceptors in myocytes from unoperated animals. In contrast, inhibition of PKA using H-89 in the present study decreased I_Ca at both the surface and t-tubule membranes, presumably reflecting the loss of tonic activity of the adenylyl cyclase/cAMP/PKA pathway at both the surface and t-tubular membranes (Figs. 5 and 7A). Although it has been suggested that H-89 may have nonspecific effects independent of PKA inhibition (26), we (9) have previously shown that basal I_Ca was decreased by a peptide inhibitor of PKA, PKI. Moreover, we have recently shown that H-89 was without effect on basal I_Ca in rat atrial myocytes from the same hearts as used in the present study, demonstrating both regional differences in the role of PKA in the regulation of I_Ca and that H-89 was without direct effect on I_Ca per se (3). The regulation of basal I_Ca by constitutive PKA activity has also been previously demonstrated in rat ventricular myocytes (4, 5, 9).

While the inhibitory effect of H-89 in sham myocytes was not abolished by pretreatment of the cells with C3SD, H-89 reduced basal I_Ca to the same mean amplitude in C3SD-treated and untreated cells (Fig. 4), indicating that the effects of C3SD and H-89 were not summative. Thus, PKA is required for the constitutive regulation of I_Ca by Cav-3 at the t-tubules in sham myocytes, but there is an additional Cav-3-independent constitutive regulation of I_Ca by PKA. As basal I_Ca density in DT sham myocytes was reduced by H-89 but not by C3SD, it can be concluded that PKA is also involved in the constitutive regulation of I_Ca at the surface sarcolemma through a mechanism independent of Cav-3. Taken together, these data suggest a role for Cav-3 in coordinating a complex of signaling proteins including LTCC, PKA, and the β₂-adrenoceptor at the t-tubule membrane in normal ventricular myocytes (1, 5, 27). Although Cav-3 is important to the constitutive maintenance of I_Ca by PKA at the t-tubule in normal ventricular myocytes, it does not appear to be required for localizing I_Ca density at the t-tubule membrane, because the difference in I_Ca density between t-tubule and surface sarcolemma was maintained after inhibition of PKA (Fig. 6A).

Regulation of I_Ca by PKA in CAL myocytes.

In contrast to sham myocytes, I_Ca increased in response to β₂-adrenergic stimulation in both intact and DT CAL myocytes (Fig. 1). Moreover, C3SD had no effect on I_Ca in CAL myocytes (Fig. 3). However, as in sham myocytes, forskolin increased (Fig. 2), and H-89 decreased (Figs. 4, 5, and 6), I_Ca at both the surface and t-tubular membranes. The simplest explanation of these data is that the normal Cav-3-dependent localization of β₂-adrenoceptor signaling at the t-tubules is disrupted in CAL myocytes, so that the β₂-adrenoceptor is distributed across both the surface and t-tubular membranes and can stimulate adenylyl cyclase/PKA and thus LTCCs at both sites, even without Cav-3 regulation; this is consistent with the redistribution of β₂-adrenoceptor cAMP signaling in heart failure (27) and demonstrates that Cav-3 is not required for β₂-adrenoceptor stimulation of adenylyl cyclase/PKA, which are already present at both sites (Fig. 7B). Interestingly, in the presence of H-89, I_Ca density was similar in the t-tubular and surface membranes of CAL myocytes, suggesting that LTCCs are also redistributed in heart failure (6). The mechanisms underlying the redistribution of β₂-adrenoceptors and LTCCs away from the t-tubules, resulting in a more uniform distribution across the cell membrane, are unclear; presumably, the redistribution of Cav-3 to noncholesterol-rich membranes in heart failure leads to a loss of Cav-3 from the t-tubules and the consequent disruption of Cav-3-dependent complexes containing LTCC/adenylyl cyclase/PKA/β₂-adrenoceptors (29). Cav-3 likely plays a role in the localization of the β₂-adrenoceptor to the t-tubule so that the loss of Cav-3 regulation from the t-tubule membrane in heart failure contributes directly to the redistribution of the receptor to the surface sarcolemma and the loss of localization of β₂-adrenoceptor signaling to the t-tubule in failing myocytes (1, 5, 27, 32). Alternatively, in principle, it is possible that β₂-adrenoceptors are more uniformly distributed between the t-tubule and surface membranes and that Cav-3 may be responsible for the localization of adenylyl cyclase/PKA signaling to the β₂-adrenoceptors in the t-tubules. Consistent with either of these proposals, treatment of normal ventricular myocytes with C3SD peptide has been shown to antagonize β₂-adrenoceptor-mediated increases in I_Ca (5). Moreover, overexpression of Cav-3 restored the localization of β₂-adrenoceptor signaling to the t-tubules in failing cells, implying a direct role for Cav-3 in the localization of the receptors and/or receptor signaling to the t-tubules, presumably via binding with the scaffolding domain (32). However, the observation that zinterol stimulates I_Ca at the surface membrane of CAL myocytes, in which C3SD has no effect on I_Ca, suggests that β₂-adrenoceptor stimulation can stimulate adenylyl cyclase/PKA even without Cav-3 binding. The role of Cav-3 in the loss of t-tubular localization of β₂-adrenoceptor signaling in heart failure might be tested in future studies by investigating the effect of C3SD peptide on the response of CAL myocytes to β₂ stimulation. On the other hand, Cav-3 does not seem to play a direct role in the localization of LTCCs to the t-tubule because 1) interference of Cav-3 binding to its partners in intact sham myocytes by treatment with C3SD peptide had no effect on I_Ca in the presence of PKA inhibition (Fig. 4), indicating that PKA activity was required for the Cav-3-dependent regulation of LTCCs, and 2) PKA was not required for the concentration of I_Ca at the t-tubule in sham myocytes (Fig. 4). Although Cav-3-dependent regulation of t-tubular I_Ca by PKA was lost in CAL myocytes, they showed an increased ratio of basal t-tubular I_Ca density to t-tubule I_Ca density in the presence of H-89 compared with sham myocytes (CAL: basal −5.9 ± 1.6 pA/pF and H-89 −1.7 ± 0.8 pA/pF; sham: basal −12.9 ± 3.0 pA/pF and H-89 −7.4 ± 1.6 pA/pF), indicating that the contribution of PKA to the maintenance of t-tubular I_Ca was augmented in heart failure. This is consistent with increased PKA-dependent regulation of basal whole cell I_Ca in failing human ventricular myocytes (10). Nevertheless, the mechanism for the increased constitutive regulation of t-tubular I_Ca by PKA in heart failure remains unclear.

Functional implications of regulation of I_Ca by PKA.

Previous work has shown that I_Ca occurs predominantly in the t-tubules, in close proximity to RyRs in the SR membrane, allowing efficient coupling between Ca²⁺ entry via I_Ca and Ca²⁺ release from the SR (21, 28). The present work shows that even in the presence of PKA inhibition, I_Ca still occurs predominantly in the t-tubules of sham myocytes, suggesting a higher concentration of LTCCs in the t-tubules. The observation that β₂-adrenoceptor stimulation of I_Ca is normally localized to the t-tubules is consistent with the importance of this site for the normal regulation of EC coupling and the potential detrimental effects of a whole cell increase of cAMP.

In CAL myocytes, although there was little change in whole cell I_Ca density, there was redistribution of I_Ca so that it was more uniformly distributed across the surface and t-tubular membranes. Unless accompanied by parallel redistribution of RyRs, which, to the best of our knowledge, does not occur, the reduced Ca²⁺ entry at the t-tubules will result in less effective coupling of Ca²⁺ entry and release and increased numbers of “orphaned” RyRs, resulting in a smaller, slower Ca²⁺ transient and thus contraction. However, the present work shows that increased local constitutive stimulation of I_Ca by PKA helps to maintain I_Ca at the t-tubules, which will help ameliorate these deleterious effects.

It has been proposed that a subpopulation of LTCCs in surface membrane caveolae play a role in cardiac hypertrophy (12, 24). The observation that C3SD has little effect on I_Ca in DT sham or CAL myocytes suggests that Cav-3 binding has little effect on LTCC function at the cell surface, although it remains possible that downstream effects of I_Ca are altered.

Summary.

The present study shows that Cav-3 plays a vital role in the coordination of PKA-dependent regulation of both basal and β₂-adrenoceptor stimulation of I_Ca in myocytes from healthy hearts. The colocalization by Cav-3 is lost in heart failure, and both β₂-adrenoceptors and LTCCs are redistributed from the t-tubular to surface sarcolemma membranes. The role of Cav-3 in the redistribution in heart failure remains unclear, but the data are consistent with a shift in Cav-3 from cholesterol-rich to noncholesterol-rich membranes (29).

GRANTS

This work was funded by British Heart Foundation Grants PG/10/91/28644, PG/14/65/31055, and RG/12/10/29802.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.M.B. performed experiments; S.M.B. analyzed data; S.M.B., C.H.K., M.B.C., C.H.O., and A.F.J. interpreted results of experiments; S.M.B. prepared figures; S.M.B., C.H.K., C.H.O., and A.F.J. drafted manuscript; S.M.B., C.H.K., M.B.C., C.H.O., and A.F.J. edited and revised manuscript; S.M.B., C.H.K., M.B.C., C.H.O., and A.F.J. approved final version of manuscript; C.H.O. and A.F.J. conceived and designed research.