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Am J Physiol Heart Circ Physiol 292: H295-H303, 2007. First published August 25, 2006; doi:10.1152/ajpheart.00719.2006
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Developmental changes in time course of recovery from inactivation in L-type calcium currents of rabbit ventricular myocytes

Takao Namiki, Ronald W. Joyner, and Mary B. Wagner

Department of Pediatrics, Emory University School of Medicine, and Children’s Healthcare of Atlanta, Atlanta, Georgia

Submitted 6 July 2006 ; accepted in final form 24 August 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The mechanisms of recovery from inactivation of the L-type calcium current (ICa) are not well established, and recovery is affected by many experimental conditions. Little is known about developmental changes of recovery from inactivation of ICa. We studied developmental changes of recovery from inactivation in ICa using isolated adult and newborn (1–4 days) rabbit ventricular myocytes. We used broken-patch and perforated-patch techniques with physiological extracellular ionic concentrations of calcium and sodium and interpulse conditioning potentials of –80 or –50 mV. We also maximized ICa with forskolin. We found that recovery from inactivation did not differ between adult and newborn cells when either EGTA or BAPTA was used to buffer intracellular calcium. Maximizing ICa with forskolin slowed recovery from inactivation in newborn but not in adult cells. In contrast, when the intracellular buffering of the cell was left nearly intact (perforated patch), recovery from inactivation (half-time of recovery) in the newborn cells was significantly slower than for the adult cells when either a conditioning potential of –80 mV (140 ± 9 vs. 58 ± 4 ms, newborn vs. adult; P < 0.05) or –50 mV (641 ± 106 vs. 168 ± 15 ms, newborn vs. adult; P < 0.05) was used. Forskolin significantly increased half-time of recovery for both adult and newborn cells. Dialysis with no calcium buffer showed a slower recovery from inactivation in newborn cells. Intracellular dialysis with a calcium buffer masked differences in recovery from inactivation of ICa between newborn and adult rabbit ventricular cells.

voltage clamp; calcium buffering; perforated patch; forskolin

IT IS WELL KNOWN THAT THE cardiac L-type Ca2+ current (ICa) plays a crucial role in the regulation of cardiac activity. The decay of ICa during depolarization is called inactivation, which is a characteristic feature of Ca2+ channels in myocytes. The inactivation of ICa is well studied and is regulated by both voltage and intracellular Ca2+ concentration ([Ca2+]). The Ca2+-dependent process of inactivation may serve as a negative feedback mechanism for regulating Ca2+ entry into heart cells (19). In general, ICa inactivation is fastest when the intracellular [Ca2+] is not buffered by cell dialysis (by using the perforated-patch voltage clamp), slower when the broken-patch technique is used with EGTA dialysis, even slower with the use of a faster Ca2+ buffer such as BAPTA, and slowest of all when Ba2+ replaces Ca2+ as the charge carrier [see review by Bers (7)]. All of these results are consistent with inactivation arising from a combination of voltage dependence and dependence on the transient rise of Ca2+ near the channel either from Ca2+ entry through the channel or from release of Ca2+ from the sarcoplasmic reticulum, with Ca2+ binding to calmodulin as an intermediate step (25, 26, 35). Our group (22) previously reported that the time constant of inactivation (using EGTA with the dialysis-patch technique) was greater in newborn than in adult rabbit ventricular cells, and the same developmental difference has recently been shown to be present with the use of the perforated-patch technique (14).

On the other hand, the mechanisms of recovery from inactivation of ICa are still not well established. The recovery from inactivation is affected by the holding potential, the voltage and duration of the conditioning pulse, the frequency of the coupled pulses, and the extracellular ionic milieu. When ICa was increased by increasing extracellular [Ca2+], by applying beta-adrenoreceptor agonists or by dialyzing cAMP analogs, the time course of recovery from inactivation was estimated to be faster (28, 29, 31) or slower (3, 11). The reason for this diversity is not well known. These different findings and interpretations concerning recovery from inactivation may result from the use of different voltage-clamp protocols, different solutions, and different species, but the influences of ICa density, beta-adrenoreceptor agonist, and extracellular [Ca2+] suggest that intracellular Ca2+ may be an important variable at the magnitude of ICa.

In steady-state conditions, a persistent elevation of intracellular [Ca2+] decreases ICa (16). During repetitive stimulation, intracellular [Ca2+] changes affect the magnitude of ICa in a complex manner, producing decreases in ICa because of inactivation, although in some situations producing potentiation of ICa (31). Repetitive activation of ICa in guinea pig ventricular cells can cause potentiation of the peak amplitude of ICa and slow its inactivation. Such Ca2+-dependent potentiation may facilitate the positive force frequency relationship in many cardiac cells and has been reported to involve Ca2+-dependent activation of calmodulin-dependent protein kinase II (34). Recent mathematical models of ICa have reproduced the time course of recovery from inactivation by incorporation of a state model of four closed states, one open state, and three inactivated states (9). These closed states represent the inactivation either from voltage alone or from interactions between the channel and a "tethered" calmodulin-binding ancillary subunit bound to calcium or a combination of slower voltage inactivation and the fast Ca2+-dependent inactivation. Mitochondria may also play a role in recovery from inactivation by removing Ca2+ from the subcellular space (27) . Slowing of the rate of recovery from inactivation has been shown to be important in producing the negative force-frequency relationship for cells from failing human ventricles (2, 30). In studies on ventricular cells surviving in the epicardial border zone of the canine-healed infracted heart, Dun et al. (10) showed slowing of the rate of recovery of inactivation of ICa compared with cells from the normal zone.

Little is known about the developmental changes of rates of recovery from inactivation in the L-type Ca2+ channel of mammalian cells. Wetzel et al. (33) showed that recovery from inactivation had voltage dependency and that the mean time constant of recovery was greater for neonatal than for adult myocytes at each membrane potential from –80 to –40 mV, although more prominently different at more depolarized potentials. However, these data were obtained with a high concentration (10 mM) of Ca2+ in the bath solution and zero Na+ in the bath solution. The time course of recovery from inactivation at physiological extracellular [Ca2+] and Na+ concentration ([Na+]) has not yet been evaluated. In the present work, we have evaluated developmental changes in recovery from inactivation at a more physiological extracellular [Ca2+] (1.8 mM) and at a physiological [Na+] (130 mM) by using tetrodotoxin to block Na+ currents. We have also compared the effects of using either EGTA or BAPTA as an intracellular Ca2+ buffer, as well as the effects of a physiological buffering, utilizing the perforated-patch condition on the kinetics of recovery from inactivation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of ventricular cells. New Zealand White adult (1.5–2 kg) and newborn (1–4 days old) rabbits were used. All animal protocols were approved by the Institutional Animal Care and Use Committee of Emory University. The enzymatic procedure for single cell isolation was previously described (22). Briefly, after rabbits were heparinized and anesthetized with pentobarbital sodium (50 mg/kg), hearts were rapidly removed and the aorta was cannulated. A dissected heart was perfused, at 36°C, for 4–5 min with oxygenated normal Tyrode solution and then with nominally Ca2+-free solution for 5–6 min. The perfusate was then switched to the same solution containing 0.08–0.15 mg/ml collagenase (Yakult Japan) and 0.05 mg/ml protease (type XIV; Sigma) for 5–7 min, followed by perfusion with storage solution for 4 min. The ventricles were placed in storage solution, chopped finely, and triturated to disperse isolated cells and then filtered through a 100-µm mesh. Cells were used within 8 h of dissociation.

Solutions and drugs. Normal Tyrode solution contained (in mmol/l) 148.8 NaCl, 4.0 KCl, 1.8 CaCl2, 0.53 MgCl2, 0.33 NaH2PO4, 5.0 HEPES, and 5.0 glucose, pH adjusted to 7.4 with NaOH. The composition of Ca2+-free solution was the same as that of normal Tyrode solution except that CaCl2 was omitted. Storage solution contained (in mmol/l) 140 potassium glutamate, 25 KCl, 10 KH2PO4, 0.5 EGTA, 1 MgSO4, 20 taurine, 5 HEPES, and 10 dextrose. ICa test solution contained (in mmol/l) 130 NaCl, 1.8 CaCl2, 20 CsCl, 0.53 MgCl2, and 10 HEPES, pH 7.4 with KOH. Tetrodotoxin (0.01–0.03 mmol/l) was added in all test solution to block Na+ current. EGTA pipette solution was (in mmol/l) 110 CsOH, 90 aspartic acid, 20 CsCl, 10 tetraethylammonium Cl, 5 HEPES, 5 MgATP, 5 Na2 creatine phosphate, 0.4 GTP(Tris), 0.1 leupeptin, and 10 EGTA, pH 7.2 with KOH. For the BAPTA pipette solution, we substituted 10 mM BAPTA for the EGTA. For perforated-patch recording, the pipette solution contained (in mM) 110 cesium glutamate, 20 CsCl, 1.1 MgCl2, 1.8 CaCl2, and 5 HEPES (pH 7.2). Just before use, the perforated-patch pipette tip was filled with antibiotic-free pipette solution by dipping the tip for a few seconds. The rest of the pipette was backfilled with pipette solution to which had been added 240 µg/mg amphotericin B. Forskolin stock solution (10 mM) was dissolved in DMSO and then diluted to 10 µM in test solution at time of use.

Electrophysiology. Voltage-clamp experiments were performed in the whole cell clamp configuration of the broken-patch or perforated-patch recording by use of an Axopatch 200 patch-clamp amplifier [Axon Instruments (Molecular Devices), Sunnyvale, CA]. Pipettes had resistances of 1–2 M{Omega} for adult cells and 2–4 M{Omega} for newborn cells. In broken-patch recordings, after high resistance was achieved, the cell membrane was disrupted by suction. Series resistance and cell capacitance compensation were done. At least 10 min were allowed for the intercellular dialysis to reach equilibrium. In perforated-patch recordings, the pipette was used in the cell attached mode, resulting in a relatively low resistance access to the cell interior, whereas the intracellular constituents remained largely undisturbed. The newborn cells were significantly smaller than the adult cells, as measured by cell capacitance [16 ± 1 pF (newborn, n = 34) vs. 78 ± 3 pF (adult, n = 40); means ± SE]. Series resistance, before compensation, was 9.7 ± 0.6 (n = 61) for cells in the broken-patch and 10.7 ± 1.5 (n = 13) for cells in the perforated-patch groups.

We define the time course of recovery from inactivation using a two-pulse protocol. Cells were depolarized to +15 mV from a holding potential of –80 mV for a 400- or 800-ms conditioning pulse. The test pulse was an identical depolarization to +15 mV for the same length of time after a variable recovery interval at a conditioning voltage (Vcond) level of –50 or –80 mV. The experiments were performed at room temperature (21–23°C). The elicited ICa was filtered at a corner frequency of 2 kHz, digitized at 200-µs intervals, and stored and analyzed on a computer with pCLAMP software (Axon Instruments). The peak amplitude in ICa was measured as the difference between the inward peak and the end of the current trace. The degree of recovery from inactivation was calculated individually as the half-maximum activation time (t1/2) by fitting the data to a single rising exponential equation. Data are presented as means ± SE. Statistical significance was determined by ANOVA using SysStat, with pair-wise comparisons using Tukey’s post hoc test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The time course of recovery from inactivation with EGTA internal solution. Figure 1 shows data from an adult ventricular cell (Fig. 1A) and from a newborn ventricular cell (Fig. 1B). For both cells, we superimposed data from seven current traces. Each trace includes an initial current response to a voltage step from –80 to +15 mV and then the response to a second depolarization from Vcond (where Vcond is either –80 or –50 mV) to +15 mV, which is delayed from the first voltage step by 50, 100, 200, 500, 1,000, or 2,000 ms. As this time delay is increased, the membrane current (ICa) progressively increases back to the level of the initial current response. For both cells, we included a horizontal dotted line, which indicates the current level for a return to 50% of the initial current response. In Fig. 1A, top, for the adult cell with a Vcond of –80 mV, the recovery to 50% of the initial current response occurs after ~100 ms. When the cell is held at Vcond = –50 mV (Fig. 1A, bottom), the time required to reach 50% recovery is increased to ~500 ms.


Figure 1
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  		Fig. 1. Membrane Ca2+ current (ICa) recorded using the dialysis-patch-clamp technique with 10 mM EGTA in the pipette from an adult (A) and a newborn (B) ventricular cell. For both A and B, we superimposed data from 7 current traces. Each trace includes an initial current response to a voltage step from –80 to +15 mV and then the response to a second depolarization from –80 to +15 mV, which is delayed from the first voltage step by 50, 100, 500, 1,000, or 2,000 ms. We included a horizontal dotted line that indicates the current level for a return to 50% of the initial current response. A and B, top: conditioning voltage (Vcond) level of –80 mV. A and B, bottom: Vcond of –50 mV. Recovery from inactivation is slowed for both newborn and adult cells when Vcond = –50 mV.

 
Figure 1B shows data in the same format as in Fig. 1A but for an isolated newborn ventricular cell. For Vcond = –80 mV, the recovery of ICa to 50% of the control level occurs at 100 ms, which is very similar to that obtained in Fig. 1A for the adult cell. Increasing Vcond to –50 mV again results in an increase in the time to reach 50% recovery, similar to the results obtained for the adult cell. For six adult and seven newborn cells, respectively, the t1/2 of ICa was 105 ± 9 vs. 97 ± 6 ms (means ± SE) for Vcond of –80 mV and 425 ± 49 vs. 482 ± 34 ms for a Vcond of –50 mV. There was no significant difference between adult and newborn t1/2 of recovery from inactivation for either Vcond; however, for both adult and newborn, the t1/2 for recovery was significantly prolonged with Vcond = –50 mV vs. Vcond = –80 mV.

To examine the effect of maximizing the ICa on recovery from inactivation, we added 10 µM forskolin to the external solution. With forskolin, ICa density was significantly increased for both adult and newborn cells. Figure 2 shows results of the same adult and newborn cells with the EGTA internal solution as in Fig. 1 but with forskolin in the bath. For the adult cell (Fig. 2A), although the peak ICa is much larger than in control, the time for recovery from inactivation of ICa is almost unchanged, occurring at ~100 ms for Vcond = –80 mV (top) and at ~500 ms for Vcond = –50 mV (bottom). In contrast, for the newborn cell (Fig. 2B) with 10 µM forskolin, t1/2 for recovery increased to ~200 ms for Vcond = –80 mV (top) and dramatically increased to nearly 2,000 ms for Vcond = –50 mV (bottom). For four adult cells and four newborn cells with 10 µM forskolin, t1/2 for recovery of ICa was 88 ± 6 vs. 193 ± 9 ms (adult vs. newborn) for Vcond = –80 mV and 356 ± 27 vs. 1,699 ± 131 ms for Vcond = –50 mV.


Figure 2
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  		Fig. 2. Data under the same conditions as Fig. 1 (dialysis patch, 10 mM EGTA) but with 10 µM forskolin in the external solution to maximize the density of ICa. A: recovery of ICa for an adult cell for Vcond = –80 mV (top) or –50 mV (bottom). B: recovery of ICa for a newborn cell for Vcond = –80 mV (top) or –50 mV (bottom). Recovery is significantly slower for the newborn cell (when compared with control) but unchanged for the adult cell.

 
As summarized in Table 1, t1/2 for recovery from inactivation was not different for adult vs. newborn cells in the control external solution, determined with Vcond of either –80 or –50 mV, although results with –50 mV were longer than those for –80 mV. The addition of forskolin to the external solution, to maximize ICa, did not significantly change t1/2 for recovery from inactivation at either Vcond in the adult cells. However, the addition of forskolin to the external solution for the newborn cells significantly prolonged t1/2 for recovery from inactivation with Vcond of –80 mV (~2-fold) and with Vcond of –50 mV (~4-fold). Additionally, the addition of forskolin resulted in a significant difference between newborns and adults.


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  		Table 1. Analysis of recovery kinetics for ICa with 10 mM EGTA in the pipette

 
These effects are more easily seen in Fig. 3, in which we plot the data in each part as adult vs. newborn cells. For each time interval at the Vcond ({Delta}t), we plot the percent recovery of ICa. Figure 3, top left, shows the recovery from inactivation time course for Vcond = –80 mV in control solution. Figure 3, top right, shows the recovery from inactivation time course for Vcond = –50 mV in control solution. As shown, in control solution, there is no difference between the adult and the newborn cells, although recovery is slowed when Vcond = –50 mV. Figure 3, bottom, shows the results when 10 µM forskolin was used to maximize ICa, with Vcond = –80 mV (bottom left) and Vcond = –50 mV (bottom right). As shown, the time course of recovery from inactivation is significantly slower for newborn than for adult cells.


Figure 3
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  		Fig. 3. Summary of results for adult (AD, solid symbols) and newborn (NB, open symbols) cells for time course of percent recovery from inactivation in control (squares) vs. forskolin (triangles) solutions with the EGTA internal perfusion. Top left: recovery from inactivation time course for Vcond of –80 mV, control solution. Top right: recovery from inactivation time course for Vcond of –50 mV, control solution. Bottom left: recovery from inactivation time course for Vcond of –80 mV, 10 µM forskolin solution. Bottom right: recovery from inactivation time course for Vcond of –50 mV, 10 µM forskolin solution. Right panels (Vcond = –50 mV) are plotted at an extended time course compared with left. There is no difference between adult and newborn recovery from inactivation in control solution, but the newborn is slower than the adult in forskolin solution.

 
Time course of recovery from inactivation with BAPTA internal solution. Because Ca2+ buffering with EGTA may have been incomplete, we performed similar experiments with BAPTA in the pipette to achieve more complete Ca2+ buffering. Figure 4 shows results for an adult cell (Fig. 4A) and a newborn cell (Fig. 4B) for which we did used the same double-pulse protocol as used in Fig. 1 (with control external solution), except that the EGTA in the internal solution was replaced with BAPTA. Because inactivation of ICa is slowed when BAPTA is used as a buffer, we increased the pulse duration to 800 ms for both the first and second pulses. For the adult cell, t1/2 for recovery from inactivation with Vcond = –80 mV (Fig. 4A, top) is somewhat less than 100 ms; for Vcond = –50 mV (Fig. 4A, bottom), t1/2 of recovery is between 200 and 500 ms. For 10 adult cells, t1/2 for recovery from inactivation was 86 ± 3 and 328 ± 21 ms (Vcond of –80 vs. –50 mV, respectively). Similar results were seen for the newborn cell (Fig. 4B). For five newborn cells, t1/2 for recovery from inactivation was 61 ± 8 and 227 ± 39 ms (Vcond of –80 vs. –50 mV, respectively).


Figure 4
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  		Fig. 4. Experiments were repeated with the dialysis-patch technique with 10 mM BAPTA to strongly buffer intracellular Ca2+ in the pipette. The dual-pulse protocol was used to examine recovery from inactivation. A: membrane currents from an adult ventricular cell with Vcond = –80 mV (top) and Vcond = –50 mV (bottom). B: membrane currents from a newborn ventricular cell with Vcond = –80 mV (top) and Vcond = –50 mV (bottom). There is no difference in the half-time (t1/2) of recovery from inactivation between the adult and newborn cells.

 
We again maximized ICa with 10 µM forskolin and BAPTA in the pipette, and the results are seen in Fig. 5. Figure 5A shows the results for an adult cell. The t1/2 of recovery at either Vcond is similar in the presence of forskolin to that in control solution (compare to Fig. 4A). For 10 adult cells, t1/2 for recovery from inactivation was 77 ± 3 and 291 ± 26 ms (Vcond of –80 vs. –50 mV, respectively), not significantly different from the control values. For a newborn cell (Fig. 5B), t1/2 for recovery from inactivation is between 100 and 200 ms (top; Vcond = –80 mV) and ~500 ms (bottom; Vcond = –50 mV). For five newborn cells, t1/2 for recovery from inactivation was 135 ± 3 and 599 ± 33 ms (Vcond of –80 vs. –50 mV, respectively), which is significantly longer than in control solution for both Vcond values.


Figure 5
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  		Fig. 5. Data under the same conditions as in Fig. 4 (dialysis patch, 10 mM BAPTA) but with 10 µM forskolin in the external solution to maximize the density of ICa. A: recovery of ICa for an adult cell with Vcond = –80 mV (top) or –50 mV (bottom). B: recovery of ICa for a newborn cell with Vcond = –80 mV (top) or –50 mV (bottom). Recovery is significantly slower for the newborn cell (when compared with control) but unchanged for the adult cell.

 
The data for which BAPTA was used as a Ca2+ buffer in the internal solution are summarized in Table 2. Note that the amplitude of ICa in control tends to be larger with BAPTA buffering than with EGTA for both adult and newborn cells, but t1/2 for recovery from inactivation is similar. The observations seen with EGTA buffering (Table 1) that recovery is slower with Vcond of –50 mV compared with –80 mV and that forskolin increases t1/2 of recovery for newborns but not adults was also seen when BAPTA was used in the internal solution.


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  		Table 2. Analysis of recovery kinetics for ICa: effect of cell dialysis

 
Time course of recovery from inactivation with perforated-patch technique. Because BAPTA provides a fast and strong buffer of Ca2+, we wanted to compare these results with those obtained when the cell’s normal cytoplasmic Ca2+ buffering was not modified by the dialysis of an internal solution. This would provide a more physiological comparison of recovery from inactivation between the newborn and adult cells. We therefore repeated the recovery from inactivation experiments on adult and newborn cells using the perforated-patch technique. For the adult cells, an example is shown in Fig. 6A in which we show the results obtained with the control external solution (black lines) and the forskolin external solution (gray lines) with a conditioning time of 500 ms (top: Vcond of –80 mV; bottom: Vcond of –50 mV). For both values of Vcond, the conditioning interval is set to 500 ms. Note that, as expected, forskolin increases the amplitude of the ICa. With Vcond = –80 mV, in both control and forskolin, the recovery from inactivation is nearly complete by 500 ms of conditioning time. In contrast, for Vcond of –50 mV, the recovery from inactivation is nearly complete by 500 ms for the control solution but is ~50% for the forskolin solution. During the conditioning time, there is a small inward current apparent for both values of Vcond.


Figure 6
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  		Fig. 6. ICa recovery from inactivation recordings using the perforated-patch technique to investigate conditions with undisturbed intracellular Ca2+ buffering. Black traces, control conditions; gray traces, 10 µM forskolin. Data are shown for a conditioning time interval of 500 ms. A: adult cell with Vcond = –80 mV (top) or –50 mV (bottom). Recovery is complete in control solution; however, at Vcond = –50 mV, forskolin delays recovery. B: newborn cell with Vcond = –80 mV (top) or –50 mV (bottom). Recovery is slowed by forskolin at both Vcond.

 
When we applied the same perforated-patch technique to a newborn cell (Fig. 6B), we also observed a large increase in the ICa with the forskolin solution compared with the control solution. For Vcond of –80 mV, there is less recovery from inactivation at 500 ms for both the control and forskolin solutions compared with the adult cell (Fig. 6A, top). For Vcond of –50 mV, there is very little recovery from inactivation at 500 ms for either the control or the forskolin solution. Another difference when the newborn and adult cells are compared is that there is much more prominent inward current; this slowly decays during the conditioning period, is greater in magnitude at Vcond of –80 mV than at –50 mV, and is not affected by the addition of forskolin.

Figure 7 shows a summary of the time course of recovery from inactivation, with the perforated-patch technique, for adult and newborn cells in the control external solution (top left and top right: Vcond of –80 and –50 mV, respectively) and also in the forskolin solution (bottom left and bottom right: Vcond of –80 and –50 mV, respectively). Note that the time course of recovery from inactivation is faster under all conditions of external solution and that values of Vcond for adult cells compared with those for newborn cells with the perforated-patch technique. These data are summarized in Table 2 and are compared with the results obtained when BAPTA was dialyzed into the cells to buffer Ca2+. In control solution with the perforated-patch technique, recovery from inactivation is significantly slower in newborn than in adult cells, which was not true when BAPTA was used in the pipette. Additionally, recovery in the newborn cells with the perforated patch is significantly slower than with BAPTA buffering (Fig. 8). With the addition of forskolin, the recovery from inactivation was again slower in the newborn cell than when BAPTA was used as internal solution. In the adult, recovery tends to be faster in control but slower in forskolin with the perforated-patch technique than with buffering BAPTA, but these results did not reach statistical significance. Additionally, with forskolin, t1/2 of recovery was increased in the newborn cell compared with control, similar to the results seen with BAPTA buffering. In contrast, in the adult with perforated patch, forskolin (compared to control) increased t1/2 for recovery from inactivation, which did not occur when BAPTA was used to buffer intracellular Ca2+.


Figure 7
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  		Fig. 7. Summary of results for adult (solid symbols) and newborn (open symbols) cells for time course of percent recovery from inactivation in control (squares) vs. forskolin (triangles) solutions using the perforated patch. Top left: recovery from inactivation time course for Vcond of –80 mV, control solution. Top right: recovery from inactivation time course for Vcond of –50 mV, control solution. Bottom left: recovery from inactivation time course for Vcond of –80 mV, 10 µM forskolin solution. Bottom right: recovery from inactivation time course for Vcond of –50 mV, 10 µM forskolin solution. Right panels (Vcond = –50 mV) are plotted at an extended time course compared with left. Under all conditions, there is a significant difference between adult and newborn recovery from inactivation.

 

Figure 8
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  		Fig. 8. Bar graphs summarizing the comparisons of the time course of recovery of ICa measured with the use of either BAPTA dialysis to buffer Ca2+ or perforated patch to leave the intracellular buffering undisturbed. The t1/2 of recovery is plotted for Vcond of –80 mV (left) or –50 mV (right). *Values that are significantly different (P < 0.05). Using perforated patch unmasks differences in recovery from inactivation between adult and newborn ventricular cells.

 
In the experiments described above, we compared the time course of recovery from inactivation for the ICa under different Ca2+ buffering conditions: adding either EGTA or BAPTA with the dialysis patch or using the cell’s intrinsic buffering with the perforated patch. Under control conditions, there is a difference between adult and newborn recovery only when the perforated patch was used. When the ICa was dramatically increased with forskolin, if Ca2+ was buffered with either EGTA or BAPTA, t1/2 of recovery from inactivation was unchanged in the adult cells but slowed in the newborn cells. In contrast, when the perforated patch was used, forskolin slowed recovery from inactivation in both the adult and newborn cells. We suggest that the difference between recovery from inactivation for newborn vs. adult cells is due to decreased intrinsic Ca2+ buffering in the newborn cells, which becomes unmasked when perforated patch is used. Additionally, increasing Ca2+ flux using forskolin in newborn cells, under all buffering conditions, slows recovery from inactivation; however, in adult cells, it slows recovery only when perforated patch is used. Using forskolin to increase the Ca2+ flux can be problematic because forskolin increases phosphorylation and thus could be having effects on recovery from inactivation distinct from the effects produced by increasing the size of the ICa. Thus we performed additional experiments in which we used the dialysis patch (as for the EGTA and BAPTA) data but simply omitted both EGTA and BAPTA from the internal solution, thus not imposing any extrinsic Ca2+ buffering on the cytoplasm. The recovery from inactivation curves for these experiments are shown in Fig. 9 (left: Vcond of –80 mV; right: Vcond of –50 mV). As can be seen in Fig. 9, under conditions of dialysis patch with no pipette Ca2+ buffering, recovery from inactivation is slower in the newborn than in the adult, similar to the results seen with perforated patch (Fig. 7, top left and top right) but in contrast to dialysis with Ca2+ buffering by either EGTA (Table 1) or BAPTA (Table 2). The t1/2 for recovery from inactivation for Vcond of –80 mV was significantly slower in the newborn cells (102 ± 6 ms, n = 5) than in the adult cells (63.8 ± 6 ms, n = 5; P < 0.002). For Vcond of –50 mV, recovery from inactivation in the newborn cells tended to be slower than in the adult cells, but this difference did not reach statistical significance [316 ± 41 ms (newborn: n = 5) vs. 212 ± 24 ms (adult: n = 5); P = 0.059]. These results show that the differences in the time course of recovery from inactivation of the ICa between newborn and adult cells becomes apparent with dialysis with no extrinsic Ca2+ buffering, even without altering the size of ICa. This demonstrates that neither the perforated-patch technique nor enhanced Ca2+ channel phosphorylation is necessary to show developmental differences in recovery from inactivation; elimination of extrinsically supplied Ca2+ buffering is sufficient.


Figure 9
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  		Fig. 9. Summary of results for adult ({blacksquare}) and newborn ({square}) cells for time course of percent recovery from inactivation of the ICa in control solution with dialysis patch and no Ca2+ buffering. Left: recovery from inactivation time course for Vcond of –80 mV. Right: recovery from inactivation time course for Vcond of –50 mV. Newborn cells have a slowed recovery from inactivation when there is no extrinsic Ca2+ buffering using the broken-patch technique.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There have been a large number of comparisons of ICa properties between newborn and adult rabbit ventricular myocytes (1, 15, 17, 18, 21–24, 32, 33), most of them done with the EGTA-containing dialysis-patch technique, with one recent study using the perforated-patch technique (14). There is general agreement that the amplitude (pA) of ICa increases with age, with most of this difference being accounted for by increases in cell capacitance, although the current density (pA/pF) remains somewhat smaller in newborn than in adult cells (14, 22). There is also a significant body of literature that the Na+/Ca2+ exchange current (as well as the mRNA and protein levels) is larger in newborn than in adult cells (4, 5, 8, 13) and that the Na+/Ca2+ exchange current may play a more prominent role in excitation-contraction coupling in newborn than in adult cells (20). Furthermore, intracellular Ca2+ buffering power has been shown to be two to three times lower in newborn than in adult myocytes (6).

Our results indicate that, when the dialysis-patch technique is used, with either EGTA or BAPTA buffering intracellular Ca2+, the time course of recovery from inactivation at –80 mV is the same for newborn and adult myocytes and remains unchanged when the density of ICa is dramatically increased by application of forskolin for adult cells but is slowed by application of forskolin to the newborn cells. We suggest that the large increase in ICa for the newborn cells may overcome the added buffer capacity of the EGTA or BAPTA with the forskolin application, particularly because the newborn cells have a significantly larger surface-to-volume ratio than adult cells and thus may experience a larger increase in intracellular [Ca2+] for a similar increase in ICa density. For Vcond of –50 mV (vs. –80 mV) for the EGTA or BAPTA conditions, the recovery from inactivation is slower but remains the same for the newborn and adult cells except when forskolin is applied to newborn cells, where the same phenomenon of slowed recovery for newborn cells is seen as for Vcond of –80 mV but now occurs with even slower recovery. In the earlier report from Wetzel et al. (32), they showed a slower recovery from inactivation for newborn than for adult cells; this study used an elevated [Ca2+] of 10 mM, which may account for this difference.

The use of the perforated-patch technique removes the effect of the added Ca2+ buffering of the EGTA or BAPTA with the dialysis-patch technique, forcing the cell to rely on its intrinsic Ca2+ buffering capacity. This uncovers an intrinsic difference between adult and newborn cells, with now newborn cells having significantly slower recovery from inactivation under control conditions. In addition, the use of forskolin now slows the recovery from inactivation in both the adult and newborn cells. Furthermore, using the broken patch with no added Ca2+ buffering and without augmenting ICa also shows a developmental difference in the time course of recovery from inactivation, with newborns being slower than adults. These results suggest that the newborn may have an intrinsically lower Ca2+ buffering capacity than the adults. The developmental differences in recovery from inactivation are shown at Vcond of either –80 or –50 mV, with more exaggerated effects at the –50-mV level. The slowing of the recovery from inactivation with Vcond of –50 mV compared with that found at Vcond of –80 mV occurs with the perforated patch and with the dialysis patch with either EGTA or BAPTA. Although this may be partly due to a voltage-dependent change in the time constant for recovery of ICa, there may also be a significant contribution of slower clearing of intracellular Ca2+ by the Na+/Ca2+ exchange pump due to the depolarization. The large inward current present during the conditioning pulse to either –80 or –50 mV for the newborn cells (and a much smaller inward current for the adult cells) under perforated-patch conditions either in control solution or in the forskolin solution may be attributed to Na+/Ca2+ exchange current, although we have not further studied this current component. The larger amplitude of this current in newborn than in adult cells is consistent with the larger expression of Na+/Ca2+ exchange in the newborn cells (4, 5, 8, 13) and the decreased Ca2+ buffering capacity (without added intracellular Ca2+ buffering by EGTA or BAPTA) of newborn cells (6). Note that this current appears to saturate with respect to the amplitude of ICa, being at the same level and time course in the control and forskolin solutions but being decreased at –50 mV compared with –80 mV and appearing to be turned off very quickly with the depolarization to +10 mV. This is consistent with recent reports from Ginsburg and Bers (12), suggesting that increases in cAMP do not increase Na+/Ca2+ exchange pump activity in adult rabbit myocytes.

One limitation of our study is that the experiments were performed at room temperature to ensure stability of the cell recordings; thus, to extrapolate these results to a physiological temperature, would be uncertain. This study emphasizes the observation that intracellular dialysis with a Ca2+ buffer (whether it is EGTA or BAPTA) can alter the kinetics of ICa and, in this study, masked differences in recovery from inactivation of ICa between newborn and adult rabbit ventricular cells. The difference in recovery from inactivation with development was unmasked by using either no extrinsic Ca2+ buffering and the broken patch or using the perforated patch and the cell’s intrinsic Ca2+ buffering.


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 ABSTRACT
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-077485 (R. W. Joyner).


   ACKNOWLEDGMENTS
 
Present address of T. Namiki: Chiba Prefectural Tohgane Hospital, Tohgane City, Chiba 283-8588, Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. B. Wagner, Dept. of Pediatrics, Emory Univ. School of Medicine, 2015 Uppergate Drive, Atlanta, GA 30322 (e-mail: mwagner{at}cellbio.emory.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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 DISCUSSION
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