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Am J Physiol Heart Circ Physiol 286: H186-H194, 2004. First published September 11, 2003; doi:10.1152/ajpheart.00272.2003
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Intracellular Ca2+ regulates responsiveness of cardiac L-type Ca2+ current to protein kinase A: role of calmodulin

Kenneth B. Walsh and Qi Cheng

Department of Pharmacology, Physiology, and Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina 29208

Submitted 26 March 2003 ; accepted in final form 8 September 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca2+ current (ICa) is affected during transient increases in intracellular Ca2+ concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca2+ external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 µM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca2+ concentration was reduced to 0.5 mM or replaced with Ba2+. This Ca2+-dependent inhibition was also observed when the cells were treated with 1 µM isoproterenol, 100 µM 3-isobutyl-1-methylxanthine, or 500 µM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca2+/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca2+-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba2+. Thus Ca2+ entering the myocyte through the voltage-gated Ca2+ channel regulates the PKA responsiveness of ICa.

heart; calcium channels; {beta}-adrenergic; cAMP

THE ENZYME cAMP-dependent protein kinase A (PKA) regulates the activity of a large and diverse group of cellular proteins, including ion channel proteins in excitable and non-excitable tissues (for review see Refs. 25 and 26). In cardiac ventricular cells, stimulation of PKA by the hormone norepinephrine and the {beta}-adrenergic receptor agonist isoproterenol augments the plateau phase of the action potential by stimulating the L-type Ca2+ current (ICa) (5, 7, 18). Numerous studies have suggested that modulation of the L-type or CaV1.2 channel by PKA occurs subsequent to a direct phosphorylation of the {alpha}1-subunit or associated {beta}-subunits of the protein (6, 9, 28). In addition to PKA, other kinases, such as protein kinase C, cGMP-dependent protein kinase, and Ca2+/calmodulin (CaM)-dependent protein kinase, regulate the activity of the CaV1.2 channel (20).

Ca2+ entering the cardiac myocyte through the CaV1.2 channel causes a strong and rapid inactivation of ICa (11, 24). Recent studies have suggested that this Ca2+-dependent inactivation (CDI) results from binding of Ca2+ and calmodulin (CaM) to an IQ motif located in the COOH-terminal tail of the {alpha}1-subunit. Mutations within the IQ motif that prevent the binding of CaM to the {alpha}1-subunit disrupt CDI (34, 47). In addition, CaM mutants lacking Ca2+-binding sites in their EF-hand domains act in a dominant-negative manner to prevent CDI (2, 33). CaM may also mediate CDI and facilitation of P- and Q-type channels (23). Thus Ca2+ and CaM are functionally linked to voltage-gated Ca2+ channels.

In a previous study, we measured ICa from cultured neonatal rat ventricular myocytes displaying two different morphologies (41). Myocytes plated on flat or nonaligned collagen membranes spread out and display a stellar-shaped morphology. In contrast, myocytes plated on aligned collagen organize along a common axis and exhibit an elongated, rod-like shape. Compared with the flat, stellar-shaped myocytes, the aligned cells express a high level of the CaV1.2 {alpha}1-subunit and possess a large ICa density. However, the ability of PKA to augment ICa is diminished in these myocytes. The goal of the present study was to determine the mechanism of this insensitivity and test the hypothesis that CaM plays a role in this regulation. It is reported that the PKA responsiveness of ICa can be restored in the aligned myocytes by lowering the external Ca2+ concentration, replacing external Ca2+ with Ba2+, dialyzing a CaM inhibitory peptide into the cells, or expressing a mutant CaM protein. Ca2+ channels in adult mouse ventricular myocytes also displayed a stronger response to PKA when external Ca2+ was substituted with Ba2+. Thus Ca2+ entering the myocyte through the voltage-gated Ca2+ channel may act in a feedback manner to regulate the PKA responsiveness of ICa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and culture of cardiac ventricular myocytes. Neonatal rat ventricular myocytes were isolated and cultured as described previously (4, 37). Briefly, heart ventricles were removed from neonatal pups (3–4 days of age), minced into 1-mm3 pieces, and subjected to collagenase (type B, Boehringer Mannheim Biochemicals) dissociation (4). For preparation of the aligned myocytes, collagen (type I, Celtrix) was applied to the peripheral edges of the culture dish containing Silastic membranes (Specialty Manufacturing). While the dish was tilted, a small sterile scraper was used to draw the collagen solution across the Silastic membrane. Excess collagen was then aspirated, and the culture dish was transferred to a 37°C incubator. When plated on this oriented or aligned collagen substrate, the cells exhibit an in vivo-like phenotype with a rod-shaped appearance (37, 41). Myocytes were cultured in DMEM (GIBCO) supplemented with 10% horse serum (Flow Laboratories) and maintained in a humidified atmosphere of 5% CO2 at 37°C. After the cells were allowed 2–3 days for attachment, Silastic membranes were transferred to a recording chamber for patch-clamp measurements. In some experiments, cardiac myocytes were isolated from adult mouse hearts using retrograde aortic dialysis (40). These cells were stored in 132 mM NaCl external solution (see below) and used within 1–10 h of isolation.

Recording procedure and measurement of voltage-gated Ca2+ currents. The patch-clamp method (15) was used to record the whole cell ICa using L/M EPC-7 (Adams and List) and Axopatch 200 (Axon Instruments) amplifiers. Our procedure for measurement and analysis of this current has been described previously (40, 41). Pipettes with resistance of 1–2 M{Omega} when filled with internal solution were made from Prism glass capillaries (Dagan). All experiments were conducted on isolated, noncoupled myocytes at room temperature (22–24°C). For the measurement of ICa, cells were placed in an external solution consisting of (in mM) 132 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 5 dextrose, and 5 HEPES, with pH adjusted to 7.4 with NaOH (280 mosM). Na+ current was blocked with 10 µM tetrodotoxin, and the Na+ channels were inactivated by maintaining the myocytes at a holding potential of –40 mV. The normal internal solution consisted of (in mM) 60 CsCl, 50 Cs-aspartate, 2 MgCl2, 1 CaCl2, 11 EGTA, 3 ATP, and 10 HEPES, with pH adjusted to 7.3 with CsOH (280 mosM). The ratio of EGTA to CaCl2 sets the free intracellular Ca2+ concentration to ~10 nM (12). Ca2+ concentrations were also set to 1 and 100 nM by adjusting the amount of added CaCl2 (12). For the experiments shown in Fig. 4, internal solutions containing 10 mM BAPTA with 1 mM CaCl2 and 10 mM BAPTA without CaCl2 were compared with the normal internal solution.



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  		Fig. 4. Ca2+-dependent inhibition of Ca2+ current regulation measured under various conditions. A: currents recorded during a 400-ms voltage step applied to 0 mV. Current traces were fit with a biexponential function (in 1 mM Ca2+: {tau}fast = 22 ms, {tau}slow = 146 ms, where {tau} is time constant) or a monoexponential function (in 1 mM Ba2+: {tau} = 138 ms). B: percent increase in peak ICa or IBa measured in the presence of forskolin. Values are means ± SE of 4–8 myocytes. Results obtained with internal solutions containing 10 mM BAPTA with and without 1 mM CaCl2 are plotted in the first 2 columns. *Significantly different (P < 0.05) from 1 mM Ca2+ external solution (ANOVA).

 

In some experiments, peptide inhibitors of protein kinase C, Ca2+/CaM protein kinase, and CaM were added to the Cs+ internal solution and dialyzed into the myocytes. After disruption of the cell membrane, chemicals in the internal solution move into the myocytes by diffusion. Previous studies have shown that peptides of this size (~1,000–2,000 Da) reach 90% of the pipette concentration within 3–5 min (18). To allow for diffusion of the peptides into the cells, experiments were performed 5–10 min after establishment of the whole cell configuration.

Membrane currents were recorded with 12-bit analog-to-digital converters (Axon Instruments). In most experiments, data were sampled at 10 kHz and filtered at 2 kzH with a low-pass Bessel filter (Frequency Devices). Current-voltage relations were obtained by applying 40-ms voltage steps, from a holding potential of –40 mV, to potentials ranging from –60 to +50 mV. Voltage pulses (in 10-mV increments) were applied at 4- to 5-s intervals to allow adequate time for Ca2+ channel recovery from inactivation. Series resistance was compensated to give the fastest possible capacity transient without producing oscillations. With this procedure, >70% of the series resistance could be compensated. Averaged current values are means ± SE. Data were adjusted for liquid junction potentials that occurred between the pipette solution and bath solution and between the reference electrode and the bath (40).

Adenovirus infection. On day 1 of cell culture, myocytes were infected with adenovirus expressing {beta}-galactosidase (LacZ) or a CaM molecule with aspartate-to-alanine mutations in all four EF-hand Ca2+-binding sites (CaM1234). The cDNA encoding CaM1234 was kindly provided by Dr. John Adelman (Vollum Institute, Portland, OR). Viruses were constructed using the pAdEasy system (Stratagene), and viral titer was determined on all stocks using the Adeno-X kit (BD Biosciences). Myocytes were infected at matched multiplicities of infection of 20. Ca2+ current recordings and Western blot analysis were performed 2–3 days after infection.

Western blot analysis. Our procedure for preparing cell lysates and performing Western blot analysis has been described in detail previously (41). For Western blots, proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels using a mini-PROTEAN cell (Bio-Rad). Proteins were transferred to polyvinylidene difluoride membranes using a mini-Trans-Blot apparatus (Bio-Rad). For immunodetection, membranes were first blocked in PBS containing 0.1% Tween 20, 5% Carnation nonfat dry milk, and 0.025% sodium azide. An antibody to CaM (1:500; Upstate Biotechnology) was incubated with the membranes overnight. After primary antibody treatment, the membranes were washed with PBS-0.1% Tween 20 and incubated with a secondary antibody (horseradish peroxidase-conjugated goat IgG; Jackson ImmunoResearch). Immunoreactive protein bands were visualized on X-ray film (Kodak) using the enhanced chemiluminescence method (Pierce).

Drugs and chemicals. Forskolin, IBMX, 8-bromo-cAMP, and isoproterenol were purchased from Sigma Chemical (St. Louis, MO). Tetrodotoxin, protein kinase C-{beta} C2–4 inhibitor, bisindolylmaleimide I, autocamtide-2-related inhibitory peptide, KN-93, cyclosporin A, and CaM inhibitory peptide (myosin light chain kinase peptide) were purchased from Calbiochem (San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PKA regulation of ICa in cardiac ventricular myocytes. Figure 1A shows ICa recorded from a neonatal rat ventricular myocyte. As reported previously (41), cells cultured on aligned collagen membranes express a large ICa when measured in external solution containing 1 mM Ca2+. In 10 cells, the ICa density measured during a voltage step to 0 mV was 12 ± 1 pA/pF. Addition of 2 µM forskolin to stimulate PKA caused a relatively small change in the amplitude of ICa, with increases of 64, 23, and 20% at –20, –10, and 0 mV, respectively (Fig. 1B). The small response of ICa was not due to "rundown" of the current in the presence of external Ca2+, because the basal ICa does not decrease during the time course of these experiments (41).



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  		Fig. 1. Regulation of Ca2+ currents in aligned neonatal ventricular myoyctes. A: currents recorded in 1 mM Ca2+ external solution during voltage steps, in 10-mV increments, to potentials ranging –30 to +10 mV. B: average current-voltage relation for L-type Ca2+ current (ICa) measured under control conditions and in the presence of 2 µM forskolin (n = 10 cells). Currents were normalized to cell membrane capacity.

 

One possible explanation for the small regulatory effect of forskolin on ICa could be that Ca2+ entering the myocytes through the L-type channels decreases the responsiveness of the current to PKA. To test this hypothesis, Ca2+ in the external solution was replaced with the permeant divalent cation Ba2+. As shown in Figs. 2 and 3, forskolin caused a 100% increase in the peak Ba2+ current (IBa). Consistent with previous studies of cardiac ICa regulation (26), this increase was associated with a leftward shift in the current-voltage relation (Fig. 2). The ability of ICa to respond to PKA stimulation was also examined in the presence of various concentrations of external Ca2+ and free internal Ca2+ (Fig. 3). Compared with 1 mM external Ca2+, increasing external Ca2+ to 2 and 4 mM caused no further depression in the response of ICa to forskolin. In contrast, forskolin produced a strong increase in ICa when external Ca2+ was lowered to 0.5 mM. In addition to the standard 10 nM Ca2+ internal solution, we prepared and tested internal solutions containing 100 and 1 nM internal Ca2+. Although increasing internal Ca2+ from 10 to 100 nM produced no change in the PKA responsiveness of ICa, decreasing internal Ca2+ to 1 nM resulted in a significant enhancement in current regulation (P < 0.05 compared with 10 nM Ca2+ internal solution).



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  		Fig. 2. Regulation of Ba2+ currents (IBa) in aligned neonatal ventricular myoyctes. A: currents recorded in 1 mM Ba2+external solution during voltage steps, in 10-mV increments, to potentials from –30 to +10 mV. B: average current-voltage relation for IBa measured under control conditions and in the presence of 2 µM forskolin (n = 10 cells). Currents were normalized to cell membrane capacity. Peak IBa under control conditions was 9 ± 1 pA/pF, which is not statistically different from basal ICa (12 ± 1 pA/pF, P > 0.05; Fig. 1).

 


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  		Fig. 3. Effect of external Ca2+ and Ba2+ concentrations on Ca2+ current regulation. A: currents recorded during a voltage step applied to 0 mV in the presence and absence of 2 µM forskolin (For). B: percent increase in peak ICa or IBa measured in the presence of forskolin. Values are means ± SE of 5–10 myocytes. Results obtained with 1 and 100 nM free Ca2+internal (Int) solutions were recorded with 1 mM Ca2+ external solution. Con, control. *Significantly different (P < 0.05) from 1 mM Ca2+ external solution (ANOVA).

 

Ca2+ entering excitable cells through voltage-gated Ca2+ channels causes a strong inactivation of ICa (11, 24). Figure 4 shows ICa and IBa recorded during a 400-ms depolarization to 0 mV using the normal 11 mM EGTA-containing internal solution. In the presence of 1 mM external Ca2+, the aligned myocytes displayed two components of current inactivation: a fast component with a time constant of 16 ± 1 ms and a slow component with a time constant of 106 ± 6 ms (n = 6 cells). As expected, replacement of Ca2+ with Ba2+ eliminated the fast CDI (Fig. 4). This large CDI suggests that the Ca2+ concentration in the vicinity of the channel reaches significant levels (38). Because the Ca2+ chelator BAPTA has faster binding kinetics than EGTA, we hypothesized that the regulation of ICa might be enhanced in internal solution containing 11 mM BAPTA. However, when measured in 1 mM external Ca2+ solution, there was no significant difference in the regulation of ICa by forskolin in aligned cells dialyzed with BAPTA or EGTA (cf. Figs. 3B and 4B).

Cardiac ventricular myocytes express type V and VI isoforms of adenylyl cyclase (30). These isoforms are inhibited by 100 nM–10 µM intracellular Ca2+ (30). To determine whether the Ca2+-dependent inhibition of ICa regulation was related to suppression of adenylyl cyclase activity, the {beta}-adrenergic-PKA signaling pathway was stimulated "upstream" and "downstream" of the adenylyl cyclase enzyme. As was the case with forskolin, augmentation of ICa by the {beta}-adrenergic agonist isoproterenol was much greater in 1 mM Ba2+ external solution than in 1 mM Ca2+ external solution (Fig. 4B). A combination of the phosphodiesterase inhibitor IBMX (100 µM) and the membrane-permeable cAMP analog 8-bromo-cAMP (500 µM) was used to elevate intracellular cAMP levels independently of adenylyl cyclase. Consistent with the results with isoproterenol and forskolin, cAMP regulation of ICa was significantly reduced in the presence of Ca2+ external solution (Fig. 4B).

The results displayed in Figs. 1,2,3,4 suggest that Ca2+ entering through the voltage-gated Ca2+ channel regulates the PKA responsiveness of ICa in neonatal myocytes. However, this inhibitory action could be a consequence of culturing neonatal cells on aligned collagen membranes and maintaining the myocytes in culture for 3–4 days. Thus it was important to determine whether this inhibitory effect of Ca2+ occurs under more appropriate physiological conditions. Because transgenic mice are now commonly used in studies of myocardial regulation, the effect of forskolin was also determined on ventricular myocytes isolated from adult mice. As shown in Fig. 5, the ability of forskolin to augment ICa was significantly greater (P < 0.01) in Ba2+ external solution. Elevation of cAMP using IBMX and 8-bromo-cAMP also produced a greater response in Ba2+ external solution. Thus, for the neonatal rat and adult mouse myocytes, replacement of extracellular Ca2+ with Ba2+ increased the response of ICa to PKA.



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  		Fig. 5. Regulation of Ca2+ currents in adult mouse ventricular myocytes. A: currents recorded from adult mouse myocytes during a voltage step applied to 0 mV in the presence and absence of 100 µM IBMX and 500 µM 8-bromo-cAMP. B: percent increase in peak ICa or IBa measured in the presence of forskolin or IBMX/8-bromo cAMP (cAMP). Values are means ± SE in 6–9 myocytes. *Significantly different (P < 0.05) from 1 mM Ca2+ external solution.

 

Mechanism of Ca2+-dependent inhibition. Even in the presence of the Ca2+ chelators EGTA and BAPTA, a transient rise in intracellular Ca2+ might reduce the PKA regulation of ICa in the neonatal cells by activating a Ca2+-dependent effector pathway. Given the high concentration of CaM within the myocardium and the high Ca2+-binding affinity of this protein, we postulated that CaM might play a role in ICa regulation. Using various chemical inhibitors, we tested four Ca2+-dependent signaling molecules, including protein kinase C, Ca2+/CaM-dependent protein kinase, protein phosphatase 2a (calcineurin), and CaM, as possible mediators of this inhibition. Protein kinase C isoforms were inhibited by dialyzing cells with the protein kinase C-{beta} C2–4 inhibitor (200 nM) (17) or through addition of 100 nM bisindolylmaleimide I (16, 17) to the external solution. Ca2+/CaM-dependent protein kinase was inhibited by addition of 50 µM autocamtide-2-related inhibitory peptide to the internal solution or by application of external solution containing 1 µM KN-93 (39). To inhibit calcineurin, myocytes were incubated overnight in the presence of cyclosporin A (500 ng/ml) (29). Compared with the results obtained in the absence of these agents (Fig. 3), none of the inhibitors effectively enhanced the response of ICa in the aligned myocytes to forskolin (Fig. 6B). In contrast, cellular dialysis with a CaM inhibitory peptide (10 µM) (21) resulted in a significant increase in the ability of ICa to respond to forskolin in 1 mM Ca2+ external solution (Fig. 6). This regulatory effect was absent when the myocytes were dialyzed with an inactive CaM inhibitory peptide. The presence of the CaM inhibitory peptide did not significantly change the response of IBa to forskolin (88 ± 9% increase, n = 3 cells; results not shown). As expected, dialysis of the myocytes with a PKA inhibitory peptide completely blocked the response of ICa to forskolin (Fig. 6).



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  		Fig. 6. Ca2+ current regulation during inhibition of Ca2+-dependent signaling molecules. A: currents recorded during a voltage step applied to 0 mV in the presence and absence of 2 µM forskolin. B: percent increase in peak ICa measured in the presence of forskolin. Values are means ± SE in 4–8 myocytes. *Significantly different (P < 0.05) from 1 mM Ca2+ external solution. Myocytes were treated with 1 µM KN-93 (KN), 100 nM bisindolylmaleimide I (BIS-I), 50 µM autocamtide-2-related inhibitory peptide (AC-2), 500 ng/ml cyclosporin A (CsA), 200 nM protein kinase C-{beta} C2–4 inhibitor (C{beta}C), 10 µM calmodulin (CaM) inhibitory peptide (CaM Inhib), 10 µM inactive CaM inhibitory peptide (CaM Con), or 20 µM protein kinase A inhibitory peptide (PKI).

 

To further investigate the importance of CaM in regulating ICa, myocytes were infected with adenovirus expressing a CaM molecule with mutations in all four EF-hand, Ca2+-binding sites (CaM1234). Previous studies have shown that CaM1234 functions in a dominant-negative manner to inhibit endogenous CaM (2, 33). Adenovirus infection resulted in a strong expression of CaM1234 as revealed by Western blot analysis (Fig. 7A). To confirm that CaM1234 functions in the neonatal cells to inhibit CaM, we measured the effect of this mutant on CDI in 1 mM Ca2+ external solution. As reported for adult rat myocytes (2), expression of CaM1234 was associated with a complete elimination of CDI (Fig. 7B). We next examined the effect of CaM1234 on the regulatory properties of the Ca2+ channel. Compared with myocytes infected with control LacZ virus, expression of CaM1234 enhanced the responsiveness of ICa to forskolin (Fig. 7C). Overall, forskolin increased the peak ICa by 104 ± 15% (n = 8 cells) vs. 18 ± 5% (n = 6 cells) in myocytes infected with CaM1234 and LacZ adenovirus, respectively.



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  		Fig. 7. Effect of CaM1234 expression on Ca2+ current regulation. A: Western blots for CaM were obtained from myocyte cultures infected with LacZ (lane 1) or CaM1234 (lane 2) adenovirus. As expected (1, 33), CaM1234 had a slightly lower molecular weight than the endogenous CaM. B: currents recorded during a 400-ms voltage step applied to 0 mV. Current traces were fit with a biexponential function (LacZ: {tau}fast = 26 ms, {tau}slow = 138 ms) or a monoexponential function (CaM1234: {tau} = 171 ms). For purpose of display, CaM1234 current was scaled to superimpose on the LacZ record. In 6 cells expressing CaM1234, {tau} = 165 ± 9 ms. C: currents recorded during a voltage step applied to 0 mV in the presence and absence of 2 µM forskolin.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Regulation of ICa during {beta}-adrenergic stimulation. The major finding of the present study is that Ca2+ entering neonatal rat and adult mouse ventricular myocytes can regulate the responsiveness of ICa to agents that act by stimulating PKA. This inhibitory effect occurred at a site beyond the synthesis of cAMP by adenylyl cyclase, because addition of the phosphodiesterase inhibitor IBMX, along with the cell membrane-permeable cAMP analog 8-bromo-cAMP, did not restore ICa responsiveness in the presence of 1 mM external Ca2+. As discussed below, increases in intracellular Ca2+ may limit PKA-mediated increases in ICa through a mechanism involving the Ca2+-binding protein CaM.

Cardiac Ca2+ channels are strongly regulated by {beta}-adrenergic receptor agonists and other agents that stimulate PKA (5, 7, 18). When measured in 1.8–2 mM external Ca2+, {beta}-adrenergic receptor/adenylyl cyclase stimulation produces a 80–120% increase in ICa in adult rat ventricular myocytes (14, 19, 26). These findings are consistent with previous results demonstrating that forskolin increases ICa by 80–100% in myocytes isolated from 4-wk-old animals (41). Increases of similar magnitude in ICa have been reported in freshly isolated (19) and cultured neonatal rat myocytes (13). In neonatal myocytes cultured on flat, nonaligned collagen, ICa is increased >100% in the presence of forskolin and IBMX (41). However, significantly smaller PKA-mediated increases in ICa (50–70%) have been reported by some investigators using adult mouse ventricular myocytes (3, 8, 35). Sako et al. (35) found that regulation of the L-type channel by isoproterenol and forskolin in the mouse heart was enhanced when Ba2+ was used as a charge carrier instead of Ca2+. On the basis of these results, the authors suggested that Ca2+ entering the myocytes acts to inhibit adenylyl cyclase. In our study, the response of the Ca2+ channels to isoproterenol, forskolin, and IBMX/cAMP was augmented in the aligned neonatal and mouse myocytes when Ba2+ was substituted for Ca2+. Thus our findings extend beyond the previous studies and suggest that Ca2+ acts at a site beyond cAMP synthesis to decrease PKA regulation of ICa (see below).

Numerous studies have demonstrated that the {beta}-adrenergic responsiveness of cardiac myocytes is depressed under conditions that result in dysfunctional intracellular Ca2+ handling. For example, {beta}-adrenergic-induced increases in ICa are diminished in adult rat myocytes isolated from hypertrophied and infarcted hearts (36, 46). Transgenic mice overexpressing the G protein Gq (27) or the Ca2+-binding protein calsequestrin (22) display cardiac hypertrophy with a ventricular ICa that responds poorly to isoproterenol. Cardiac-specific overexpression of the CaV1.2 {alpha}1-subunit in transgenic mice results in marked hypertrophy with accompanying heart failure (31). Interestingly, inotropic responses and increases in ICa during {beta}-adrenergic stimulation are diminished in the {alpha}1-transgenic hearts (32). This diminution in {beta}-adrenergic responsiveness occurs before overt pathological symptoms are displayed, indicating that elevations in intracellular Ca2+, caused by overexpression of the {alpha}1-subunit, regulate PKA signaling independent of cardiomyopathy-related changes in gene transcription (31, 32).

Role of intracellular Ca2+ and CaM in regulation of ICa. It has long been appreciated that serum hypercalcemia attenuates the positive inotropic actions of {beta}-adrenergic agonists in animal and human heart (1, 45). Isoproterenol-mediated increases in cardiac myocyte cAMP accumulation are greatly enhanced on removal of extracellular Ca2+ or in the presence of the Ca2+ channel blockers D-600 and nifedipine (44). This Ca2+-dependent inhibition of {beta}-adrenergic signaling has previously been attributed to the cardiac expression of Ca2+-sensitive isoforms of adenylyl cyclase (30). Type V and VI isoforms of adenylyl cyclase, which are present in ventricular myocytes, are inhibited by submicmolar concentrations of Ca2+ (30). Although inhibitory effects on adenylyl cyclase may have occurred in the adult mouse and aligned neonatal myocytes, our results indicate that Ca2+ acts at a site downstream from cAMP production to attenuate the response of ICa to PKA stimulation. This conclusion is supported by the finding that Ca2+ channel responsiveness was suppressed in the presence of Ca2+, even when cAMP levels were increased by addition of IBMX and 8-bromo-cAMP. In addition, we previously showed that ICa in the aligned cells does not respond to direct intracellular application of cAMP (41). Finally, dialysis of the myocytes with a CaM inhibitory peptide, but not a control CaM peptide, restored the ability of forskolin to fully augment ICa.

CaM is a ubiquitously expressed, Ca2+-binding protein that modulates the activity of a large number of proteins. Recent data provide convincing support for the involvement of CaM in CDI and facilitation of cardiac Ca2+ channels. Binding of CaM to an IQ motif in the COOH terminus of the CaV1.2 {alpha}1-subunit appears to be involved in CDI and facilitation. Overexpression of a mutant CaM (CaM1234), defective in Ca2+ binding, was found to function in a dominant-negative manner to eliminate CDI (2, 33). Furthermore, dialysis of cardiac myocytes with an IQ-binding peptide and other peptides that mimic CaM enhances the facilitation of ICa (43). These findings not only demonstrate that CaM serves as a mediator of CDI and facilitation but suggest that CaM may be tethered to the {alpha}1-subunit. A close physical association between CaM and the {alpha}1-subunit might also explain how elevations in intracellular Ca2+ in the vicinity of the Ca2+ channel pore regulate the responsiveness of ICa to PKA. Although the inhibitor experiments presented in Fig. 6 would initially rule out an involvement of Ca2+/CaM-dependent protein kinase, binding of Ca2+/CaM may activate other signaling molecules associated with the channel. Recently, Ca2+-dependent binding of CaM to the {alpha}1-subunit was shown to be linked to the activation of the small G protein Ras and mitogen-activated protein kinases (10). Alternatively, Ca2+ regulation of ICa might exist at another intracellular or, possibly, extracellular site. However, the inability of intracellular BAPTA dialysis to restore Ca2+ channel responsiveness and the enhancement in PKA regulation obtained with CaM1234 suggest that this site is closely associated with the channel. Future experiments manipulating the expression of CaM and other Ca2+-dependent proteins will provide more information on the relation between intracellular Ca2+, the L-type Ca2+ channel, and the {beta}-adrenergic regulatory pathway.


   ACKNOWLEDGMENTS
 
The authors thank Kathryn J. Long for preparing the neonatal myocytes used in the study and Dr. Steven P. Wilson and Annette Smith for constructing the LacZ and CaM1234 adenoviruses.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-45789, the American Heart Association, and the Research Development Fund of the University of South Carolina School of Medicine.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. B. Walsh, Dept. of Pharmacology, Physiology, and Neuroscience, School of Medicine, Univ. of South Carolina, Columbia, SC 29208 (E-mail: walsh{at}med.sc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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