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Am J Physiol Heart Circ Physiol 293: H2826-H2835, 2007. First published August 24, 2007; doi:10.1152/ajpheart.00101.2007
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beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes

Leon P. Collis,1 Shekhar Srivastava,1 William A. Coetzee,1 and Michael Artman2

1Department of Pediatrics, Program in Pediatric Cardiology, New York University School of Medicine, New York, New York; and 2Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, Iowa

Submitted 24 January 2007 ; accepted in final form 20 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Selective stimulation of beta2-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta2-AR stimulation on L-type Ca2+ current (ICa,L) during postnatal development. ICa,L was measured (37°C; either Ca2+ or Ba2+ as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca2+ transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta2-ARs (with either 100 nM zinterol or 1 µM isoproterenol in the presence of the beta1-AR antagonist, CGP20712A) stimulated ICa,L twofold in newborns but not in adults. The beta2-AR-mediated increase in Ca2+ transient amplitude in newborns was due exclusively to the augmentation of ICa,L. Zinterol increased the rate of inactivation of ICa,L and increased the Ca2+ flux integral. The beta2-AR inverse agonist, ICI-118551 (500 nM), but not the beta1-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 µM), PKI 6-22 amide (10 µM), and Rp-cAMP (100 µM), all failed to prevent the response to zinterol but completely blocked responses to selective beta1-AR stimulation of ICa,L in newborns. Our results demonstrate that in addition to the conventional beta1-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta2-AR-mediated, PKA-insensitive pathway that stimulates ICa,L. This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta2-AR agonists.

development; protein kinase A; adenosine 3',5'-cyclic monophosphate

AFTER BIRTH, THERE IS A developmental increase in the sensitivity of the myocardium to beta1-AR agonists and a decline in beta2-AR responsiveness (26, 34, 36, 38). Consequently, selective stimulation of beta2-ARs has been shown to contribute to positive inotropy (26, 34), lusitropy (26, 34), and chronotropy (8) in newborn rodent cardiac myocytes. However, the underlying mechanism behind these ontogenic changes remains unconfirmed. In the adult, the heterogeneity of responses to beta2-AR agonists has been attributed to the differences in activation and/or expression of G{alpha}i, which together with G{alpha}s, is downstream of the beta2-AR. G{alpha}i counteracts the phosphorylation events promoted by activation of G{alpha}s that normally elicit positive inotropy and lusitropy (25, 45). However, age-dependent differences in the compartmentalization of the beta2-AR signal cascade and coupling between beta2-ARs and Gi do not appear to account for enhanced beta2-AR-mediated inotropic responses in the newborn (34).

Stimulation of L-type Ca2+ channels by beta-AR agonists in adult cardiac myocytes is primarily through the cAMP/PKA cascade (23). Other signaling pathways have also been proposed, particularly in regard to the beta2-AR. For example, in newborn myocytes, activation of beta2-ARs increases chronotropy independent of PKA (8) and elevates cAMP without activating PKA (10). This suggests that beta2-AR signaling may invoke alternative pathways to those observed in adults (23).

The present study used an electrophysiological approach combined with Ca2+ fluorescence measurements to examine the effects of beta2-AR stimulation on L-type Ca2+ current (ICa,L) in newborn ventricular cardiac myocytes. Although ICa,L has a minimal role in newborn excitation-contraction (EC) coupling under basal conditions (3, 13, 16, 18), enhancement of ICa,L during beta-AR stimulation may serve as a significant source for increasing intracellular Ca2+ concentration ([Ca2+]i) in the newborn. We activated beta2-ARs using a low concentration of zinterol (100 nM), which has previously been shown to be selective for beta2-ARs in newborn cardiac myocytes (27, 34). Prior studies of beta2-AR-mediated responses in newborn cardiac preparations have used primary cultures of newborn myocytes (8, 26, 34). Although these preparations exhibit spontaneous contractions and beta-AR responsiveness, there are modifications in underlying beta-AR signaling pathways and EC coupling that occur during the cell culture process (28). In light of this, we studied freshly isolated newborn and adult rabbit ventricular cardiac myocytes under voltage-clamp conditions to record ICa,L and Ca2+ transients during beta2-AR stimulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation. Ventricular myocytes were isolated from the hearts of New Zealand White rabbits at two different age groups: 1 to 5 days old (both sexes) and adult males (>150-day old; left ventricle only). Rabbits were heparinized (500 IU/kg) and anesthetized using pentobarbital sodium (60 mg/kg body wt). Isolation involved retrograde Langendorff perfusion with a collagenase-based digestion described previously (3). Myocytes were stored in Krebs bicarbonate solution (3) at room temperature until used. All experiments were performed within 8 h of cell isolation and were performed in accordance with the recommendations from the National Institutes of Health "Guiding Principles in the Care and Use of Animals" and approved by the Institutional Laboratory Animal Care and Use Committee at New York University School of Medicine. A total of 30 newborn and 6 adult rabbits were used for myocyte studies. Individual numbers of preparations (preps) are presented in RESULTS.

Electrophysiology. Adult and newborn ventricular myocytes were patch clamped in whole cell mode at 37°C. Micropipettes (1–2 M{Omega} for adult, and 4–5 M{Omega} for newborn) were used. ICa,L was measured by using the methods previously described (49) with slight modifications. Seal and cell rupture was achieved in standard Tyrode solution, and myocytes were voltage clamped to –80 mV and perfused with the modified ICa,L bath solution. Currents were measured with a patch-clamp amplifier (Axopatch 200B, Axon Instruments) and low-pass filtered (–3 dB at 1 kHz). The offset potential was corrected by zeroing the potential before touching the surface of the cell with the pipette tip. Cell capacitance, access resistance, and membrane seal resistance were measured using a +5-mV step pulse (pClamp v.8.1, Axon Instruments) at a holding potential of –80 mV. Access resistances between 2 and 10 M{Omega} were deemed adequate. Cell capacitance and series resistance were compensated, the latter to at least 70%. Clamp potentials were offset to correct for a shift in calculated junctional potential of –0.7 mV (Clampex v.8.1; Axon Instruments). ICa,L was measured with a prepulse from –80 to –50 mV for 200 ms to inactivate T-type Ca2+ current (ICa,T) and Na+ current (INa). A step-pulse series from –50 to +50 mV ({Delta}10 mV; 0.1 Hz) was used to assess the shift in voltage dependence of peak ICa,L, whereas a single pulse from –50 mV to –10 mV (0.05 Hz) was used to examine the effect of the drug on peak ICa,L with time. Steady-state activation parameters in newborn myocytes were extracted from conditioning steps using a double-voltage protocol ({Delta}10 mV; 0.01 Hz; GoGoFig. 3A, top). Inactivation parameters were extracted from a test pulse to –10 mV that followed the conditioning pulses after a 5-ms step to –80 mV (Fig. 3A, top). Time dependence of inactivation was assessed in newborn myocytes, using the voltage protocol shown (0.1 Hz; Fig. 3B, top). At the close of the experiment, 10 µM nifedipine was applied to block ICa,L. This component was numerically subtracted from the ICa,L traces.


Figure 1
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  		Fig. 1. beta2-Adrenergic receptor (AR) stimulation of Ca2+ transients in newborn (Nb) myocytes is mediated by L-type Ca2+ current (ICa,L). A, left: representative ICa,L traces during stimulation with 100 nM zinterol (Zint) in Nb and adult (Ad) cardiac myocytes, recorded at –10 and 0 mV, respectively. Current was nifedipine (Nif) sensitive. A, right: current-voltage (I-V) plots of peak ICa,L density in Nb (n = 6) and Ad (n = 6) cardiac myocytes. In Nb myocytes, the increase in current density with Zint was voltage dependent (2-factor ANOVA with replication; P < 0.05; n = 6). *P < 0.05, (control vs. Zint; n = 6). Errors bars are means ± SE. B: peak ICa,L and Ca2+ transients recorded concomitantly from Nb myocytes dialyzed with indo-1. C: representative Ca2+ transients at peak ICa,L (–10 mV; 200 ms) in Nb myocytes exposed to Nif and Zint plus Nif. D: representative Ca2+ transients measured with voltage clamp to +50 mV (200 ms) before and after Zint application. Arbitrary units are fluorescence {Delta}F1/F0 units. Voltage-clamp protocol for all transients was a 200-ms prepotential step to –50 mV, followed by a 200-ms step to either –10 or +50 mV. Vm, test potential.

 

Figure 2
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  		Fig. 2. The effect of Zint is mediated by beta2-ARs. A: representative peak ICa,L traces in CGP20712A (CGP)-treated Nb and Ad cardiac myocytes during stimulation with isoproterenol (Iso). B: representative peak ICa,L in a Nb myocyte during application with Zint following pretreatment with CGP or ICI-118551 (ICI). C: effect of various beta-adrenergic interventions on ICa,L in Nb myocytes, expressed as percent increase in ICa,L relative to basal ICa,L. Each intervention had a significant effect on ICa,L (P < 0.05) compared to basal values (represented by the horizontal dashed line). **P < 0.05, beta-AR agonist vs. beta-AR agonist plus beta-AR blocker. Error bars are means ± SE. ICa, Ca2+ current. n, Number of experiments.

 

Figure 3
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  		Fig. 3. Effect of Zint on activation, availability, and recovery from inactivation of ICa,L. A: steady-state activation and inactivation curves for ICa,L in newborn myocytes. Data were fit using a Boltzmann function: [Go – Gmax]/[1 + exp(V – V0.5)/k] + Gmax. Activation was expressed as percent maximal conductance (Gca) at each voltage step, using: GCa = ICa,L/(Vm – Vrev ), where Vm is test potential and Vrev is the apparent reversal potential for Ca2+, estimated from the current-voltage (I-V) relationship. For Vm > 20 mV, GCa was assumed the value of the slope for the linear regression line plotted through the I-V relationship at Vm > 20 mV (OriginPro 7.0, OriginLab). Error bars are means ± SE (n = 5 experiments). B: time-dependence recovery curves for ICa,L in Nb myocytes during application with Zint. Peak current during the test pulse was expressed as a percentage of peak current during the inactivation pulse. Errors bars are means ± SE (n = 5 experiments). Curves were generated using the voltage protocols shown (A and B, top).

 
Concomitant measurements of Ca2+ transients and ICa,L. The bath solution consisted of Tyrode buffer with 1.8 mM Ca2+ and equimolar CsCl replacing KCl. Newborn (1–5 day old) ventricular myocytes were patch clamped in whole cell mode with 3–5-M{Omega} pipettes. Background fluorescence was assessed after gigaseal formation but before rupture. Fluorescence measurements were obtained using a Delta Ram fluorescence system [Photon Technology International (PTI)]. After 10 min of dialysis, 10 cycles (0.2 Hz) of step clamps (–50 mV for 200 ms, and then –10 mV for 200 ms) were used. Fluorescence was recorded as the ratio, F = F405/F485 (Felix; v.1.42, PTI). The relative change in fluorescence ({Delta}F1/F0; F1 = peak, F0 = resting) was assessed before and after application of zinterol and nifedipine (10 µM). Peak Ca2+ transient was averaged over the last five cycles at steady state.

Solutions. Tyrode saline contained (in mM) 137 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, 0.33 NaH2PO4, and 1.8 CaCl adjusted with NaOH to pH 7.4. ICa,L bath solution contained (in mM) 140 tetraethylammonium (TEA), 6 CsCl, 5 HEPES, 2 CaCl2, 1 MgCl2, 10 glucose, 0.5 niflumic acid, pH adjusted to 7.4 with TEA-OH. Pipette solution for recording ICa,L contained (in mM) 125 CsCl, 20 HEPES, 10 Mg-ATP, 5 BAPTA (tetracesium salt), 0.3 GTP (Tris salt), buffered to 7.2 with CsOH. Mg-ATP and GTP were included to minimize rundown during whole cell patch clamp. Rundown was negligible, where 95 ± 6%, 87 ± 4%, and 80 ± 3% (n = 4) of initial peak ICa,L remained after 10, 30, and 40 min, respectively. Most experiments were completed after 20 min of postrupture. To block the influence of sarcoplasmic reticulum Ca2+ release on beta2-AR stimulation, 10 µM ryanodine was included in the pipette. The pipette solution for concomitant measurement of ICa,L and Ca2+ transients contained (in mM) 130 K+-glutamate, 9 KCl, 10 NaCl, 1 MgCl2, 5 MgATP, 10 HEPES, and 0.01 indo-1 K+ pentapotassium salt (Molecular Probes), pH adjusted to 7.2 with KOH.

Drug application. The beta-AR agonists, zinterol (gift of Bristol-Myers Squibb) and isoproterenol (Iso, 1 µM; Sigma-Aldrich), were added for 3 min. The beta-blockers, CGP20712A (CGP; 500 nM; Sigma-Aldrich) and ICI-118551 (ICI; 500 nM; Sigma-Aldrich) were applied for 10 min before beta-AR stimulation. The membrane-permeable PKA blocker N-{2-[(p-bromocinnamyl)amino]ethyl}-5- isoquinoline-sulfonamide (H89; 10 µM; Sigma-Aldrich) and the Ca2+/calmodulin kinase II (CaMKII) blocker, KN93 (5 µM; Calbiochem), were added for 20 min before stimulation with zinterol. The PKA blockers, adenosine 3',5'-cyclic phosphorothioate-Rp (Rp-cAMP; 100 µM; Calbiochem) and PKI 6-22 amide (PKI; 10 µM; Calbiochem), were dialyzed intracellularly via the patch pipette for at least 5 min before beta-AR stimulation. IBMX (100 µM; 5 min; Sigma-Aldrich) was used to inhibit phosphodiesterases (PDEs), and 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP; 100 µM; Calbiochem) was used to increase intracellular cAMP concentration. ADP ribosylation of Gi was catalyzed with pertussis toxin (PTX; 200 ng/ml at 37°C for 3 h). In some experiments, equimolar BaCl2 replaced CaCl2 in the bath solution.

Statistics. Results are presented as means ± SE (unless stated otherwise). Statistical significance was determined by Student's t-test for paired data (unless stated otherwise) and a one-way ANOVA for current-voltage (I-V) relationships. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Stimulation of beta2-ARs augments ICa,L in newborns but not adults. To investigate the amplitude and kinetics of ICa,L, all ancillary currents were blocked and 2 mM Ca2+ was used as a charge carrier with heavily buffered [Ca2+]i. Under these conditions, zinterol increased nifedipine-sensitive ICa,L almost twofold in newborn myocytes (Fig. 1A and Table 1; P < 0.05; 10 preps). The effect of zinterol was reversed with washout (Fig. 1A, top). In contrast to newborns, zinterol had no effect on peak ICa,L in adult rabbit myocytes (Fig. 1A; control: 17.3 ± 0.7; zinterol: 16.3 ± 0.8 pA/pF; n = 5; 3 preps). The presence of minor residual inward current with 10 µM nifedipine in the adult suggests newborn myocytes are more sensitive to nifedipine and that 10 µM nifedipine cannot completely block all ICa,L in adult myocytes. Mean cell capacitance increased from 22.7 ± 1.3 (n = 30) in newborns to 131.0 ± 8.6 pF (n = 7) in adult myocytes. There was also a twofold increase in ICa,L density postnatally (Fig. 1A). In addition, the adult I-V plot for ICa,L showed a +10-mV shift in voltage dependence of peak ICa,L relative to the newborn (Fig. 1A; I-V curves). In newborn myocytes, zinterol induced a slight hyperpolarizing shift in the voltage dependence of peak ICa,L (Fig. 1A; I-V curve) with no change in the reversal potential for ICa,L ({approx}+50 mV). In newborn myocytes, the time constants of inactivation of ICa,L were significantly reduced by zinterol, using both biexponential or monoexponential fits (Table 1; P < 0.05). In addition, zinterol increased the integral of peak ICa,L and corresponding [Ca2+]i flux (in amol/pF) by twofold (Table 1). Thus, despite faster inactivation, the total Ca2+ influx through L-type channels was more than doubled in the presence of zinterol.


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  		Table 1. Effects of zinterol on ICa,L in newborn ventricular myocytes

 
Zinterol induces a concomitant increase in Ca2+ transient and ICa,L in newborns. ICa,L and Ca2+ transients were recorded simultaneously in newborn myocytes to verify that the zinterol-induced increase in ICa,L leads to a functional increase in [Ca2+]i. Zinterol augmented peak ICa,L from 3.3 ± 0.3 to 5.2 ± 0.7 pA/pF (P < 0.05; n = 5; 4 preps) and increased the amplitude of the Ca2+ transient by 25.1 ± 4.8% (Fig. 1B; {Delta}F ratio units: 1.0 ± 0.1 to 1.25 ± 0.1; P < 0.05; n = 5). Basal ICa,L was significantly lower compared with the control data in Fig. 1A due to the reduced Ca2+ buffering that was necessary for recording Ca2+ transients. Under these nominal Ca2+ buffering conditions, a monoexponential curve was the best fit for the inactivation profile for ICa,L. As with the data in Fig. 1A, {tau}1 was significantly decreased with zinterol (basal: 25.5 ± 1.7; zinterol: 22.0 ± 1.2 ms; P < 0.05; n = 5). To examine whether the zinterol-mediated increase in ICa,L accounted for this observed increase in Ca2+ transient, ICa,L was blocked with nifedipine or by voltage clamping to the apparent reversal potential for ICa,L (+50 mV) in newborn myocytes. After a 3-min perfusion with 10 µM nifedipine, ICa,L was completely blocked (Fig. 1C). This current shows a negligible amount of residual inward and outward current, possibly from incomplete inactivation of INa, ICa,T, and K+ current. This was partly due to an abbreviated prepulse of only 200 ms at –50 mV that was necessary to prevent hypercontraction of newborn myocytes. These residual inward currents were also observed at +50 mV (Fig. 1D), suggesting this is not ICa,L. At 0 mV and in the presence of nifedipine, the amplitude of the Ca2+ transient was reduced by 23.6% [Fig. 1C; {Delta}F (ratio units): 1.2 ± 0.4 to 0.9 ± 0.3; n = 4; P < 0.05]. This was coupled with a 6% decrease in the level of diastolic fluorescence (Fig. 1C; 1.05 ± 0.3 to 0.97 ± 0.3 {Delta}F units; n = 5; P < 0.05). Subsequent application of zinterol in nifedipine-treated myocytes failed to increase the amplitude of the Ca2+ transient [Fig. 1C; {Delta}F (ratio units): 0.9 ± 0.3 to 1.1 ± 0.3; n = 4; P > 0.05]. At +50 mV, ICa,L was absent (Fig. 1D), yet the Ca2+ transient amplitude was increased by 51.6% (–10 mV: 1.1 ± 0.1 {Delta}F; +50 mV: 1.7 ± 0.1 {Delta}F; n = 8; P < 0.05). Nifedipine had no effect on the Ca2+ transient amplitude at +50 mV (control: 1.7 ± 0.2; nifedipine: 1.7 ± 0.2; n = 4), suggesting that residual ICa,L does not contribute to Ca2+ influx at +50 mV. At +50 mV, zinterol failed to increase the Ca2+ transient (Fig. 1D; {Delta}F units; control: 1.6 ± 0.2; zinterol: 1.7 ± 0.2; n = 4). Taken together, these results suggest that zinterol increases the amplitude of Ca2+ transients exclusively by stimulation of ICa,L.

Zinterol increases ICa,L in newborn myocytes by beta2-AR stimulation. In newborns, the nonselective beta-AR agonist, Iso (1 µM), increased ICa,L to a similar magnitude as zinterol (peak ICa,L; control: 8.0 ± 0.8; Iso: 13.4 ± 1.9 pA/pF; n = 6; P < 0.05; 4 preps), although this increase was less than that observed in adult myocytes (peak ICa,L; control: 16.1 ± 0.9; Iso: 41.3 ± 4.5 pA/pF; P < 0.05; n = 4). In newborns, beta2-AR stimulation using Iso in combination with the beta1-AR blocker, CGP (500 nM) increased ICa,L to a similar extent with zinterol alone (Fig. 2, A and C; n = 4; 2 preps) but failed to stimulate ICa,L in adult myocytes (Fig. 2A; n = 4; 3 preps). This provides additional evidence that activation of beta2-ARs fails to stimulate Ca2+ channels in the adult and confirms that CGP is an effective blocker of beta1-ARs. CGP alone did not alter basal current significantly (peak ICa,L; control: 15.9 ± 1.5; CGP: 14.1 ± 2.0 pA/pF; n = 4; P < 0.05; 2 preps).

Zinterol mediated its effect on ICa,L via activation of beta2-ARs. Evidence for this assertion is as follows: the increase in ICa,L was blocked by the beta2-AR inverse agonist ICI (Fig. 2, B and C; peak ICa,L; control: 11.2 ± 2.2; zinterol: 9.6 ± 1.5 pA/pF; n = 4; P < 0.05; 3 preps). The beta1-AR blocker, CGP, failed to block the effect of zinterol in newborn myocytes (Fig. 2, B and C; peak ICa,L; control: 8.1 ± 2.1; zinterol: 12.7 ± 4.2 pA/pF; n = 4; P < 0.05; 2 preps). Interestingly, ICI by itself did not significantly alter basal ICa,L (peak ICa,L; control: 10.3 ± 1.7; ICI: 9.7 ± 1.5 pA/pF; P > 0.05; n = 5; 3 preps), suggesting there is little constitutive beta2-AR stimulation of ICa,L in newborn rabbit myocytes. This result also implies that ICI exhibits negligible inverse agonist activity as previously suggested (14).

Zinterol affects the voltage-dependence of ICa,L in newborn myocytes. The negative shift of peak ICa,L with zinterol was further studied by analyzing the voltage dependence of steady-state activation and inactivation in newborn myocytes. Zinterol shifted the steady-state activation and inactivation curves by –5.0 and –5.6 mV, respectively (Fig. 3A and Table 1; n = 5; P < 0.05; 3 preps), whereas the slope factor (k) was not significantly affected (Table 1). The window current (overlap of inactivation and activation curves) was similar for both control and zinterol, indicating that the fraction of steady-state current relative to maximum conductance was not affected.

The time course of recovery from inactivation was significantly, though only mildly, prolonged by zinterol (Fig. 3B and Table 1; 3 preps). This was possibly a secondary effect of increased Ca2+ influx with zinterol stimulation.

beta1-AR but not beta2-AR stimulation is mediated by PKA in the newborn. Selective stimulation of beta1-ARs in newborn myocytes was achieved using Iso (1 µM) in ICI-pretreated cells (Fig. 4A). The effect of Iso on peak ICa,L was 18% less in the presence of ICI than with ISO alone, suggesting ISO is an agonist for both beta1-ARs and beta2-ARs in the newborn. As with selective beta2-AR stimulation, a slight negative shift in voltage dependence of the I-V relationship was observed. beta1-AR stimulation of ICa,L in newborns was completely blocked at all potentials by intracellular dialysis of the PKA-specific inhibitor, Rp-cAMP (100 µM; Fig. 4B). The I-V curve shows that this block was not due to a shift in the voltage dependence of peak ICa,L (Fig. 4B, right), and Rp-cAMP did not affect basal ICa,L (Fig. 4, A and B). However, Rp-cAMP failed to prevent the increase in peak ICa,L by zinterol (ICa,L increased by 66 ± 16%; Fig. 4, C and D; n = 7; 5 preps). The I-V relation shows that this increase was not mediated by a shift in voltage dependence of peak ICa,L. To eliminate the possibility of limited blockade of PKA by Rp-cAMP, additional PKA inhibitors were tested. Intracellular dialysis with the peptide, PKI (10 µM), and bath application of the synthetic PKA inhibitor, H89 (10 µM; 20 min exposure), did not prevent the zinterol-mediated increase in ICa,L (Fig. 4D). However, H89 and PKI did reduce mean basal ICa,L by 26% and 23%, respectively (Fig. 4C; P < 0.05; unpaired t-test; 4 and 3 preps, respectively). Prolonged exposures (>30 min) of H89 decreased basal ICa,L by 42.1% to 4.4 ± 0.7 pA/pF (n = 4; P < 0.05; 2 preps), suggesting either a role for constitutive PKA at rest or possible a secondary effect of the H89 and PKI compounds. These data indicate that in newborn myocytes, there is a substantial PKA-independent component to stimulation of ICa,L by zinterol.


Figure 4
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  		Fig. 4. beta2-AR-mediated stimulation of ICa,L does not depend on activation of PKA in Nb myocytes. A: representative traces of peak ICa,L (left) and I-V curves (right) in Nb myocytes pretreated with ICI and Iso to stimulate beta1-ARs exclusively. B: as above, but myocytes were dialyzed with 100 µM Rp-cAMP to inhibit PKA. C: representative traces of peak ICa,L (left) and I-V curves (right) showing effect of Zint after dialysis with Rp-cAMP. Error bars are means ± SE. *P < 0.05; n = 4 experiments for all plots. D: summary data showing the effect of the PKA blockers, H89 (10 µM; bath applied), PKI (10 µM; dialyzed), and Rp-cAMP (100 µM; dialyzed) on beta2-AR stimulation of peak ICa,L in Nb myocytes (n, number of experiments). *P < 0.05 (PKA blocker only vs. PKA blocker + Zint). Errors bars are means ± SE. **P < 0.05 (basal ICa,L vs. PKA blocker only).

 
The effect of zinterol is not Ca2+ or CaMKII dependent. To investigate whether changes in [Ca2+]i during voltage clamp regulate the beta2-AR response, we used Ba2+ (2 mM) as a charge carrier. In the newborn, switching to Ba2+ exhibited a mild slowing of inactivation for both time constants (Fig. 5A; {tau}fast: 9.9 ± 0.8 to 23.7 ± 1.3; {tau}slow: 79 ± 3.2 to 89.5 ± 2.1 ms; n = 4; 2 preps). In the adult, whereas {tau}fast decreased by a similar magnitude with Ba2+, {tau}slow was markedly prolonged ({tau}fast: 19.4 ± 1.8 to 35.6 ± 2.4 ms; {tau}slow: 69.5 ± 5.7 to 114.2 ± 6.8 ms; n = 3; P < 0.05; Fig. 5A, inset). In the newborn, zinterol stimulated Ba2+ current (IBa) similar to ICa,L (92 ± 24%; Fig. 5, B and D; n = 4; 2 preps) and decreased the inactivation time constants ({tau}fast: 23.7 ± 1.3 to 21.3 ± 1.0; {tau}slow: 81.2 ± 2.1 to 61.6 ± 5.0 ms; n = 4; P < 0.05). This suggests that the beta2-AR response is not mediated by Ca2+ influx through Ca2+ channels.


Figure 5
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  		Fig. 5. A: representative peak ICa,L in a Nb myocyte with 2 mM Ca2+ as the initial charge carrier and then substituted with 2 mM Ba2+. Inset: comparison of peak ICa,L and Ba2+ current (IBa) in the Ad cardiac myocyte. B: representative recording of peak IBa (2 mM Ba2+) in a Nb myocyte before and after treatment with Zint. C: representative peak ICa,L with and without Zint after pretreatment with shown interventions. Incubation with pertussis toxin (PTX; 200 ng/ml; 3 h) did not alter control ICa,L but enhanced the response to Zint. A representative trace of Zint stimulation in a cell without PTX is shown for reference. In addition, cells were either incubated for IBMX (100 µM) or dialyzed with 100 µM 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) before treatment with Zint. D: summary of the influence of various pharmacological interventions on the response of peak ICa,L to Zint in Nb myocytes. Error bars are means ± SE. *P < 0.05, control vs. Zint. **P < 0.05, basal ICa,L for treatment vs. basal ICa,L for control. {ddagger}P < 0.05, unpaired t-test; Zint (n = 12 experiments) vs. treatment + Zint (see text for sample sizes).

 
Ca2+/CaMKII has been implicated as an alternative pathway to PKA in beta-AR signaling in cardiac myocytes (40). To examine the possible role of CaMKII in the newborn, we used the CaMKII inhibitor KN93 (5 µM). Interestingly, KN93 reduced basal ICa,L in the newborn myocyte (Fig. 5D; P < 0.05; n = 6; 3 preps). The inactive analog of KN93 (KN92; 5 µM) also reduced basal ICa,L (Fig. 5D; P < 0.05; n = 4; 2 preps), suggesting a nonspecific interaction between the Ca2+ channel and these compounds. Nevertheless, both KN93 and KN92 failed to prevent or augment the effect of zinterol on ICa,L, suggesting that CaMKII is not involved in mediating the beta2-AR effects on ICa,L in newborn myocytes.

Downstream mediators of beta2-AR stimulation. To assess whether Gi restricts beta2-AR signaling in the newborn myocyte, myocytes were incubated with PTX, which catalyzes the ADP-ribosylation of the {alpha}-subunits of Gi. PTX treatment augmented the zinterol-mediated increase in ICa,L by 68 ± 48% (Fig. 5, C and D; P < 0.05; unpaired t-test; n = 7; 5 preps) but did not affect the level of basal ICa,L (Fig. 5D).

Myocytes were exposed to a saturating concentration of the PDE blocker, IBMX (100 µM), to maximally increase cAMP (1). IBMX increased basal peak ICa,L significantly (Fig. 5, C and D; P < 0.05; n = 4; 2 preps). With this concentration of IBMX, zinterol did not further increase the amplitude of peak ICa,L (Fig. 5, C and D; n = 4; 2 preps), suggesting saturation of the cAMP pathway. This effect was replicated by dialyzing the cells with 100 µM 8-CPT-cAMP (Fig. 5, C and D; P < 0.05; n = 4).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Developmental change in the effect of selective beta2-AR stimulation on ICa,L. This study was motivated by the observation that cardiac myocytes from newborn, but not adult, rats exhibit a robust positive inotropic response to beta2-AR agonists (26). To characterize the potential mechanisms for this developmental change, we measured the effects of beta2-AR stimulation on ICa,L using the selective beta2-AR agonist, zinterol, and the nonselective beta-AR agonist, Iso, in the presence of the beta1-AR blocker, CGP. Stimulation of beta2-ARs using either protocol significantly increased ICa,L in freshly isolated newborn rabbit myocytes but had no effect on ICa,L in adult myocytes. This also demonstrated the efficacy of CGP as a beta1-AR blocker, by preventing any stimulatory action of Iso in the adult. In the newborn, CGP failed to block the effect of zinterol, which supports the claim that 100 nM zinterol acts exclusively on beta2-ARs. A well-documented beta2-AR inverse agonist, ICI (14), also blocked any stimulatory action of zinterol. The relative response to Iso was greater in adults than in newborn myocytes. This is consistent with a relative lower density of beta-ARs at birth in the rabbit (36) and an increased efficacy of Iso to stimulate ICa,L with age (32).

In addition to our present findings in rabbits, beta2-AR stimulation has been previously shown to increase contractility in the newborn rat (26, 34) and newborn mouse (8). However, there is a lack of information on the specific Ca2+ sources and transport pathways involved in this process. We have shown that increased ICa,L plays a major role in the augmented response to beta2-AR agonists in newborn rabbits. The diminished responsiveness to beta2-AR-mediated stimulation of Ca2+ current (ICa) in the adult has been observed in a number of species, including mouse (42), rat (6, 27), and dog (30). Thus the age-related changes in beta2-AR responsiveness may be a general developmental phenomenon that is not necessarily exclusive to the species that we chose for these studies (rabbit).

Stimulation of Ca2+ transient amplitude by beta2-AR agonists is attributable to increased ICa,L. Zinterol increased the Ca2+ transient amplitude in newborn ventricular myocytes. This increase was not secondary to beta2-AR-mediated changes in action potential duration (APD), since experiments were performed under voltage-clamp conditions. We have previously shown that newborn cardiac myocytes are particularly sensitive to APD (13, 16). In the newborn, Ca2+ transients had a larger amplitude at +50 versus –10 mV, suggesting a significant role for reverse-mode Na+-Ca2+ exchanger (NCX)-dependent Ca2+ influx (18). To eliminate the possibility of beta2-AR regulation of NCX in the newborn, we used nifedipine and/or voltage clamp to +50 mV to block L-type Ca2+ channels. In both cases, the response to zinterol was inhibited, indicating that the effect on Ca2+ transient was attributable solely to the increase in ICa,L and not from Ca2+ influx via NCX. Therefore, at a constant duration of depolarization, beta2-AR agonists enhance Ca2+ transient amplitude primarily through increased ICa,L.

We have previously shown that under basal conditions, the Ca2+ transient in the newborn rabbit is relatively less reliant on Ca2+ influx through the L-type Ca2+ channel as either a direct source of "activator" Ca2+ or as the "trigger" for Ca2+ release from intracellular stores (13, 17). In addition, there is a significantly diminished fractional release from the sarcoplasmic reticulum in the newborn on a beat-to-beat basis (3). In fact, reverse-mode NCX current can provide sufficient Ca2+ influx to support contraction in newborn myocytes, and there is evidence that contraction and relaxation at birth are predominately controlled by NCX (16, 17). However, beta-AR-mediated regulation of NCX remains controversial, and nonselective beta-AR stimulation in the rabbit heart does not regulate NCX activity (12). Thus, if the newborn heart is more dependent on NCX and NCX is not regulated by adrenergic stimulation, how does the newborn increase cardiac contractility during stress? Our present results demonstrate that in newborn myocytes, beta2-AR stimulation increases peak [Ca2+]i during the Ca2+ transient and that this response is dependent predominately on increased ICa. Based on these data, we propose that although there may be a limited contribution of ICa to Ca2+ transient amplitude in newborn myocytes under basal conditions (17, 41), in times of stress and/or adrenergic stimulation, recruitment of additional Ca2+ influx via increased ICa may be an important mechanism for increasing contractile function at birth.

The effects of beta2-AR stimulation on the kinetics of ICa,L. Contrary to previous reports (29, 44, 48), beta2-AR stimulation decreased both {tau}fast (Ca2+ dependent) and {tau}slow (voltage dependent) of ICa,L. The former could have resulted from greater influx of Ca2+ ions, as a consequence of an increase in ICa,L amplitude with zinterol. This correlates with the shift in steady-state inactivation toward hyperpolarized levels as a result of the negative shift in steady-state activation. This leftward shift in activation curves has been attributed to the phosphorylation of residues within the voltage sensor of the Ca2+ channel (22), the result of which would facilitate the opening of Ca2+ channels earlier in depolarization. These data, together with the lack of effect of zinterol on the reversal potential and threshold of activation, indicate that zinterol augments ICa,L by increasing the single-channel open probability, similar to the effect seen with phosphorylation of Ca2+ channels by PKA (23, 48).

In addition, the rate of inactivation for peak IBa was also increased by zinterol for both time constants. The fast component here was not due to Ca2+-dependent inactivation since Ba2+ was the charge carrier. The implies that beta2-AR stimulation can accelerate the voltage-dependent inactivation of ICa,L, which differs from the effect seen during PKA phosphorylation of ICa,L (48). The basis of this effect remains unclear, although a decrease in {tau}slow was reported in newborn rabbit myocytes during nonselective beta-AR stimulation (24). Interestingly, {tau}slow was increased markedly by Ba2+ in the adult but only mildly augmented in the newborn. Differences in the inactivation of Ba2+ currents have been attributed to the alternative splicing of exons in the {alpha}1C-subunit of the Ca2+ channel (37). A developmental shift (postnatal to adult) from the IVS3A to the IVS3B mRNA isoforms of the {alpha}1C-subunit has recently been identified in the rabbit (18). Although this switch in isoforms has been attributed to a downregulation of ICa,L-dependent EC coupling (5), as seen in the newborn, it could also confer properties such as localization and interaction with downstream regulators of adrenergic signaling.

Although zinterol accelerated the inactivation rate of ICa,L, more Ca2+ entered the newborn myocytes in the presence of zinterol. In fact, the estimated Ca2+ flux value in the presence of zinterol was similar to that for adult rabbit myocytes under basal conditions using the same voltage step and extracellular Ca2+ concentration (49). This helps to explain the increase in Ca2+ transient amplitude observed with zinterol since the zinterol-mediated increase in [Ca2+]i is ICa,L dependent.

Modulation of beta2-AR responses by intracellular Ca2+. An increase in peak ICa density during postnatal maturation has been previously reported in newborn rabbit cardiac myocytes (20, 41). However, we found that peak ICa,L in newborn cells was twofold greater than that reported in previous studies, which also used freshly isolated myocytes (20, 41). BAPTA enhances peak ICa,L and reduces ICa,L inactivation compared with EGTA as an intracellular Ca2+ buffer (35), which likely explains the observed differences in ICa,L amplitude between our data and those of previous investigators. This is further exemplified by the Ca2+-transient experiments, where nominal Ca2+ buffering significantly reduced basal peak ICa,L. The degree of Ca2+ buffering and therefore the level of [Ca2+]i can regulate cell signaling by modulating Ca2+-sensitive adenylyl cyclases (35). Interestingly, intracellular dialysis of BAPTA prevented beta2-AR-stimulated phosphorylation of the S1928 residue of the {alpha}1c-subunit of adult cardiac Ca2+ channels (19). However, this phosphorylation event was not coupled to functional changes in ICa,L magnitude. We show that in both high and nominal Ca2+ buffering, zinterol augmented ICa,L by a similar magnitude. This suggests that modifications in free [Ca2+]i near Ca2+ channels influence the kinetics of the Ca2+ current but does not affect signaling by beta2-ARs. Indeed, Ba2+ currents were augmented by a similar magnitude by zinterol, suggesting that Ca2+ entering through Ca2+ channels does not regulate beta2-AR-mediated events. This contrasts with studies where Ba2+ enhances PKA-mediated increases in ICa,L, presumably through relieving inhibition on Ca2+-sensitive adenylyl cyclases (35, 47) and/or inhibition of calmodulin (39). In regard to beta2-AR regulation, newborn myocytes appear to lack this Ca2+ feedback mechanism, particularly since PKA is not involved.

Downstream mediators of beta2-AR signaling. Few prior studies have attempted to identify the underlying mechanisms for the enhanced response to beta2-AR agonists in the newborn heart, and the results to date are inconclusive. In adult myocytes, beta2-AR stimulation of ICa,L is unmasked by PTX treatment, suggesting that under control conditions, Gi plays a role in restricting beta2-AR downstream signaling (42). Differences in beta2-AR responsiveness among species and developmental stages have been attributed to the degree of beta2-AR-Gi coupling (43). Here we have shown that PTX exposure augments beta2-AR stimulation of ICa,L in newborn myocytes. Therefore, Gi is still an active component of beta2-AR signaling in the newborn and does not account for the enhanced responsiveness to beta2-AR agonists under basal conditions. Indeed, G{alpha}i expression has been shown to be greater in the newborn heart relative to the adult heart (34).

We have also demonstrated that in the newborn, ICa,L is maximally activated by inhibition of PDEs and with the dialysis of a cAMP analog. A saturating dose (1) of the nonselective PDE inhibitor, IBMX, increased ICa,L by threefold in the newborn and blocked further stimulation by zinterol. We obtained a similar result by dialyzing myocytes with 8-CPT-cAMP, a cAMP analog. These results suggest that either cAMP upregulation interferes with beta2-AR signaling in cardiac myocytes or that beta2-AR agonists are ineffective after maximal cAMP stimulation. Indeed, beta2-AR stimulation has been shown to robustly increase intracellular cAMP in newborn cardiac myocytes (8, 26, 34). Although the possibility of beta2-AR-cAMP coupling remains disputed (26, 27, 34), pharmacological data presented here suggest that beta2-ARs stimulate ICa,L in a cAMP-dependent, but PKA-independent, manner.

In adult rabbit ventricular myocytes, the CaMKII complex has been implicated as a mediator of L-type Ca2+ channel function (2). The effects of chronic stimulation of beta-ARs on EC coupling have also been shown to be mediated by CaMKII, wherein the cAMP/PKA pathway is desensitized and the CaMKII pathway becomes sensitized (40). However, blockade of CaMKII did not modulate the effect of beta2-AR activation on ICa,L in newborn myocytes. The effects of chronic beta2-AR stimulation in newborn myocytes remain to be investigated.

beta2-ARs but not beta1-ARs stimulate ICa,L independently of PKA in newborn cardiac myocytes. beta1-AR-mediated stimulation of ICa,L depends on activation of PKA (23). This is consistent with the previous observation that PKA blockade inhibited beta1-AR-mediated chronotropy in newborn cardiac myocytes (8). Our present results indicate a PKA-independent component to the coupling between beta2-ARs and L-type Ca2+ channels in newborns. In particular, Rp-cAMP, although able to prevent beta1-AR stimulation, failed to suppress the beta2-AR-mediated increase in ICa,L, even after 10 min of intracellular dialysis. The PKA blockers H89 and PKI significantly reduced basal ICa,L, suggesting that either PKA is constitutively active in the newborn or that they had a nonspecific action on ICa,L, as described previously for H89 (9, 33). Constitutively active PKA may be the result the higher [Ca2+]i buffering used in this study (47). However, Rp-cAMP did not reduce basal ICa,L, and the discrepancy remains. It is possible that PKI and H89 were more effective at blocking PKA when constitutively active. For example, PKI is a competitive peptide blocker of the catalytic domain of PKA, whereas Rp-cAMP is a competitive antagonist of cAMP for PKA. Constitutively active PKA may be a result of the catalytic domain outnumbering the regulatory domain or becoming activated independently of cAMP (21). In this case, Rp-cAMP would be less effective.

With the use of a nonselective beta-AR agonists, a PKA-independent component to beta-AR signaling has been previously identified in newborns but was absent in adult rabbit ventricular myocytes (24). Our data suggest that this component is coupled to the beta2-AR pathway, whereas beta1-AR-mediated stimulation of ICa,L requires activation of PKA. This is further verified by the fact that stimulation of newborn cardiac beta1-ARs, but not beta2-ARs, is able to phosphorylate PKA-dependent substrates (10). It remains to be determined whether the PTX-augmented component to newborn beta2-AR signaling is dependent on PKA. PKA-independent pathways in beta2-AR signaling have been previously demonstrated in cardiac myocytes (8) and in other cell types (4, 7). CaMKII (40) has been implicated in this PKA-independent signaling. However, we have shown that blockade of this pathway does not suppress the effect of beta2-AR-mediated stimulation of ICa,L in newborn rabbit myocytes. Stimulation of ICa,L may involve a direct interaction between Gs, the beta2-AR and the L-type Ca2+ channel, although this proposal is controversial (7, 46). More recently, the cAMP-dependent Rap-GTP exchange factor (Epac) has been implicated in beta-AR signaling in mouse myocytes (31), which might serve as a possible pathway that is PKA independent. Other possible molecular mechanisms have been proposed for the robust stimulation of ICa,L via beta-ARs, although they remain inconclusive. For instance, the main consensus site for PKA-dependent phosphorylation, S1928, is not required for beta1-AR- (11) or beta2-AR-mediated increases in Ca2+ currents (19). One novel alternative is via ahnak, a ICa,L repressor protein that binds to the beta-subunit of the Ca2+ channel. PKA phosphorylates and relieves the inhibition of ahnak, similar to the mechanism of phospholamban (15). If PKA is not involved in beta2-AR signaling during sympathetic stimulation in the newborn, the question remains as to what are the sources and sites of phosphorylation, if any, for downstream targets of beta2-ARs.

In summary, our results demonstrate that the developmental difference in the inotropic response to beta2-AR receptor agonists is attributable to an increase ICa,L that occurs in newborns but not in adults. Although the precise cellular mechanisms remain to be determined, it appears that this response in newborns does not require activation of PKA. This pathway may play an important role in providing additional inotropic support to the newborn heart under conditions of increased sympathetic stimulation.


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 ABSTRACT
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This work was supported by the National Institute of Child Health and Human Development Grant HD-39988 (to M. Artman).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Artman, Dept. of Pediatrics, 2636 JCP, Univ. of Iowa Carver College of Medicine, 200 Hawkins Dr., Iowa City, IA 52242 (e-mail: michael-artman{at}uiowa.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.


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 MATERIALS AND METHODS
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Dopamine increases L-type calcium current more in newborn than adult rabbit cardiomyocytes via D1 and {beta}2 receptors
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2327 - H2335.
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