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(Circulation Research. 2002;90:165.)
© 2002 American Heart Association, Inc.

Cellular Biology

Modulation of Ca²⁺ Release in Cardiac Myocytes by Changes in Repolarization Rate

Role of Phase-1 Action Potential Repolarization in Excitation-Contraction Coupling

Rajan Sah, Rafael J. Ramirez, Peter H. Backx

From the Departments of Physiology and Medicine, Heart and Stroke/Richard Lewar Centre, Division of Cardiology at the University Health Network, University of Toronto, Toronto, Canada.

Correspondence to Dr Peter H. Backx, Toronto General Hospital, CCRW 3-802, 101 College St, Toronto, Ontario M5G 2C4, Canada. E-mail p.backx{at}utoronto.ca

	Abstract

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The early rate of action potential (AP) repolarization varies in the mammalian heart regionally, during development, and in disease. We used confocal microscopy to assess the effects of changes in repolarization rate on spatially resolved sarcoplasmic reticulum (SR) Ca²⁺ release. The kinetics and peak amplitude of Ca²⁺ transients were reduced, and the amplitude, frequency, and temporal synchronization of Ca²⁺ spikes decreased as the rate of repolarization was slowed. The first latencies and temporal dispersion of Ca²⁺ spikes tracked closely with the time to peak and the width of the L-type Ca²⁺ current (I_Ca,L), suggesting that the effects of repolarization on excitation-contraction coupling occur primarily via changes in I_Ca,L. Next, we examined the effect of changes in the rapid early repolarization rate (phase 1) of a model human AP on SR Ca²⁺ release by varying the amount of transient outward K⁺ current. Slowing of phase-1 repolarization also caused a loss of temporal synchrony and recruitment of Ca²⁺-release events, associated with a reduced amplitude and lengthened time to peak of I_Ca,L. Isoproterenol application enhanced and largely resynchronized SR Ca²⁺ release, while it increased the magnitude and shortened the time to peak of I_Ca,L. Our data demonstrate that membrane repolarization modulates the recruitment and synchronization of SR Ca²⁺ release via I_Ca,L and illustrate a physiological role for the phase-1 notch of the AP in optimizing temporal summation and recruitment of Ca²⁺-release events. The effects of slowing phase-1 repolarization can be overcome by ß-adrenergic stimulation.

Key Words: confocal microscopy • electrophysiology • calcium sparks • heart disease

	Introduction

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In cardiac myocytes, the intracellular Ca²⁺ transient is mediated by Ca²⁺-induced Ca²⁺ release via the close interaction of voltage-gated L-type Ca²⁺ channels (VLCCs) and SR Ca²⁺-release channels (RyRs), followed by Ca²⁺ removal from the cytosol, predominantly by sarcoplasmic reticulum (SR) Ca²⁺-ATPase and the Na⁺-Ca²⁺ exchanger.¹ Altered expression, function, and interaction of these Ca²⁺-handling proteins have been associated with the characteristically slowed kinetics and blunted amplitude of the Ca²⁺ transient observed in failing cardiac myocytes from animals^2–4 and humans.⁵ Despite extensive investigation, the precise mechanisms responsible for such alterations in the intracellular Ca²⁺ transient remain incompletely understood, although changes in SR Ca²⁺ content,⁶ coupling between RyRs and VLCCs,⁴ and VLCC density³ are likely to be contributing factors.

Several studies have also shown that heart disease is associated with reductions in repolarizing K⁺ currents, which cause alterations in the action potential (AP) profile.^7–9 Although the typical electrophysiological consequences of increased AP duration in heart disease include QT prolongation¹⁰ and an enhanced propensity for cardiac arrhythmias,¹¹ alterations in AP profile, particularly when caused by reductions in transient outward K⁺ current (I_to), are also known to influence L-type Ca²⁺ current (I_Ca,L),¹² intracellular Ca²⁺ transients,^9,13 and excitation-contraction (E-C) coupling.¹⁴ Therefore, reductions in I_to and the associated loss of the early repolarization notch might contribute significantly to changes in the kinetics and amplitude of SR Ca²⁺ release in heart disease.

Consistent with a link between altered AP repolarization and SR Ca²⁺ release, recent studies have shown that slowed AP repolarization¹⁵ and impaired SR Ca²⁺ release occur in rabbit myocytes after myocardial infarction.³ Furthermore, the depressed kinetics and amplitudes of Ca²⁺ transients from failing rabbit myocytes were linked to asynchronous Ca²⁺ release and poorly coordinated summation of Ca²⁺ sparks.³ Other studies have also suggested that temporal synchronization of Ca²⁺ release is an important determinant of peak systolic [Ca²⁺]_i and cardiac inotropy in rat ventricular myocytes, independent of SR Ca²⁺ load, under resting conditions¹⁶ and after ß-adrenergic stimulation.¹⁷ Therefore, modifying the time course of SR Ca²⁺ release in response to changes in the AP profile may indeed provide an additional mechanism whereby the Ca²⁺ transient can be altered in normal and diseased myocardium.

In the present study, confocal Ca²⁺ imaging was combined with patch-clamp recordings of myocytes to investigate the connection between the rate of membrane repolarization and time course of SR Ca²⁺ release. We find that the rate of repolarization influences the time course and magnitude of the whole-cell Ca²⁺ transient, independent of SR Ca²⁺ load, by modulating the recruitment and synchronization of fundamental Ca²⁺ release events via changes in I_Ca,L. Extending these results to physiological waveforms, we find early phase-1 repolarization of the AP to be critical for maintaining optimal firing and synchronization of release events. Asynchronous Ca²⁺ release after the slowing of phase-1 repolarization could be largely overcome by ß-adrenergic stimulation. Thus, we propose a novel paradigm in which altered AP morphology contributes to reduced as well as asynchronous SR Ca²⁺ release, which can be reversed by ß-adrenergic stimulation.

	Materials and Methods

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Cardiomyocyte Isolation
Cardiac ventricular myocytes were isolated and electrophysiological recordings were performed as previously described.^14,18

Confocal Ca²⁺ Imaging
Myocytes were imaged with a Fluoview confocal microscope (Olympus) and scanned by using the 488-nm spectrum line.

Measurement of Whole-Cell Ca²⁺ Transients
Ca²⁺ transients were measured in myocytes dialyzed with (mmol/L) KCl 140, HEPES 10, MgCl₂ 1, NaCl 7, MgATP 7, and fluo 3 pentapotassium salt 0.060, adjusted to pH 7.2 with KOH, and superfused at room temperature (20°C to 23°C at 1 mL · min^-1) with extracellular solution of the following composition (mmol/L): NaCl 140, KCl 4, HEPES 10, MgCl₂ 1, CaCl₂ 2, and D-glucose 10, adjusted to 7.4 with NaOH (solution A).

Measurement of Ca²⁺ Spikes
Ca²⁺ spikes were measured in myocytes superfused with solution A (20°C to 23°C) and dialyzed with the following solution (mmol/L): KCl 140, HEPES 10, MgCl₂ 1, NaCl 10, MgATP 7, fluo 3 0.75, EGTA 3, and CaCl₂ 1.55, adjusted to pH 7.2 with KOH. The stimulus waveforms were applied every 15 seconds. In some experiments, Ca²⁺ spikes were measured after 4 minutes of exposure to solution A plus isoproterenol (ISO, 200 nmol/L, Sigma Chemical Co).

Measurement of I_Ca,L
I_Ca,L was measured in a separate group of experiments by using a pipette solution containing (mmol/L) aspartic acid 120, CsOH 120, HEPES 10, MgCl₂ 1, MgATP 7, tetraethylammonium chloride 10, EGTA 4, and CaCl₂ 2, adjusted to pH 7.2 with CsOH, and sodium-free extracellular solution containing (mmol/L) choline chloride 135, HEPES 10, MgCl₂ 1, CaCl₂ 2, and D-glucose 10, adjusted to 7.4 with CsOH (solution B). Stimulus waveforms were applied every 15 seconds, and the effects of ß-adrenergic stimulation were tested after 4 minutes of exposure to solution B plus 200 nmol/L ISO.

Statistical Analysis
Data are presented as mean±SEM; n refers to the sample size. A value of P<0.05 was considered to be statistically significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

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Changes in [Ca²⁺]_i With Ramp Repolarization
After a loading train of eight 100-ms voltage steps to 10 mV, myocytes were stimulated by use of ramp depolarizations (Figure 1B, broken line; see Materials and Methods). Figure 1 shows 1.6-second confocal line scans (Figure 1A) and corresponding fluo 3 transients (Figure 1B) derived from these line scans in response to the voltage-ramp stimulus. As summarized in Figure 1C, peak fluorescence and Ca²⁺ transient amplitudes progressively decreased (P<0.005) as the repolarization period was lengthened from 50 to 500 ms. The time course of Ca²⁺ release also depended on the repolarization rate. As repolarization rates were decreased, the leading edge of the line scans became increasingly diffuse, indicating a more asynchronous pattern of Ca²⁺ release and more slowly rising Ca²⁺ transients (Figures 1A and 1B). A plot of the time to peak of the Ca²⁺ transient versus repolarization time (Figure 1D) revealed a strong and significant (P<0.001) positive correlation between these parameters. Prolonged repolarization times were also associated with slowed rates of decay of the Ca²⁺ transients. Figure 1E shows that the time from peak systolic [Ca²⁺]_i to 50% decline of the transient increased progressively and significantly (P<0.005) with longer ramps. Collectively, these results show that slowed repolarization influences the amplitude and temporal profile of Ca²⁺ transients.

View larger version (47K): [in this window] [in a new window]   Figure 1. Effect of repolarization rate on Ca2+ release. A, Line scans (1.6 seconds) were taken during 50-, 200-, and 500-ms voltage ramps after a train of eight 100-ms steps to 10 mV. In line scans taken during 200- and 500-ms ramps, Ca2+ release was sufficiently asynchronous to resolve individual Ca2+ sparks (arrows). B and C, Intracellular Ca2+ transients (solid line, B) triggered by the superimposed voltage ramps (dotted line, B) were derived from the line scans above and show a significant (P<0.005) decreasing relationship (C) between [Ca2+]i and ramp duration. D and E, Time to peak (D) and time to 50% decay (E) of Ca2+ transients were correlated strongly (R=0.999) and increased significantly (P<0.005) with decreasing repolarization rate. *P<0.05 vs 50-ms ramp data; P<0.05 vs both 50- and 200-ms ramp data.

Repolarization Rate Modulates Ca²⁺ Release via Changes in I_Ca,L
The basis for the effect of the repolarization rate on the intracellular Ca²⁺ transient was further investigated by measuring the frequency, amplitude, and synchronization of Ca²⁺ spikes, because the magnitude and shape of the whole-cell Ca²⁺ transient is determined by the number and timing of such fundamental Ca²⁺-release events.^3,16,17 For these experiments, myocytes were dialyzed with a K⁺-based solution with high concentrations of fluo 3 (0.75 mmol/L) and EGTA (3 mmol/L), similar to those in previous studies,^16,19 whereas I_Ca,L was isolated in a separate set of experiments by dialysis with a Cs⁺-based solution (see Materials and Methods). Figure 2 shows that slower repolarization rates increase the temporal dispersion of Ca²⁺ spikes in line scans (Figure 2)C and surface plots (Figure 2D) while they also decrease the peak amplitude and slow the time course of I_Ca,L (Figure 2B, Table 1). Similar results were also observed with the use of voltage ramps peaking at 40 mV (data not shown), establishing that these effects are also observed at more physiological membrane potentials. As summarized in Figures 3A and 3B, both the amplitude and total number of triggered Ca²⁺ spikes decreased significantly (P<0.05) and progressively as the repolarization time was lengthened, with a pattern similar to that observed with the Ca²⁺ transient amplitudes (Figure 1C). Because the amplitude of Ca²⁺-release events under these experimental conditions reflects the number of activated Ca²⁺-release units within the active SR junction,¹⁷ these data demonstrate that slower repolarization rates reduce the numbers of SR-release units that contribute to Ca²⁺ spikes as well as the total number of Ca²⁺ spikes.

View larger version (62K): [in this window] [in a new window]   Figure 2. Repolarization rate modulates the synchrony and amplitude of Ca2+ spikes. A, Stimulus ramps of 50-, 200-, and 500-ms duration were applied to rat ventricular myocytes. B, Representative ICa,L traces were triggered by the voltage ramps shown in panel A above (see Table 1). C and D, Line scans (C) and surface plots (D) show a gradual reduction in number, intensity, and a loss of temporal synchronization of Ca2+ spikes with diminished repolarization rate. Note that ICa,L was measured in a set of experiments separate from the Ca2+ spike measurements (see Materials and Methods).

View this table: [in this window] [in a new window]   Table 1. L-Type Ca2+ Current Properties During Voltage Ramps

View larger version (23K): [in this window] [in a new window]   Figure 3. Repolarization rate alters microscopic properties of Ca2+ release. A and B, Both Ca2+ spike amplitude (A) and frequency (B) decreased significantly (P<0.05) with slower repolarization rates. C, The first latency of Ca2+ spike firing was correlated strongly with time to peak of trigger ICa,L (R=0.99); each increased significantly (P<0.001) with diminished repolarization rate. D, Standard deviation of Ca2+ spike latency and full widths at half maxima of ICa,L were also correlated (R=0.99) and increased significantly (P<0.001) with reductions in repolarization rate. *P<0.05 vs 50-ms ramp data; P<0.05 vs both 50- and 200-ms ramps.

Efficient temporal summation of individual Ca²⁺-release events can be just as important as the frequency of triggered events in determining the peak Ca²⁺ transient amplitude¹⁷; therefore, we also analyzed the temporal distribution and coherence of Ca²⁺ spikes triggered by different voltage ramps by using the first latency of Ca²⁺ spikes and standard deviation of latency as described previously.¹⁷ To assess the time profile of I_Ca,L during voltage ramps, the time to peak and full width at half-maximum I_Ca,L were also measured. Both the first latency of Ca²⁺ spikes and time to peak of I_Ca,L increased linearly and significantly (P<0.001) with the repolarization rate (data not shown), and as shown in Figure 3C, they were strongly correlated (R=0.99). These results are entirely expected, inasmuch as the peak of the whole-cell I_Ca,L reflects the product of the open probability of Ca²⁺ channels and the unitary Ca²⁺ current (i), both of which vary complexly with voltage during ramps and are tightly linked to SR Ca²⁺ release.²⁰ This tight connection is further established in Figure 3D, which shows a strong linear correlation (R=0.99, Figure 3D) between the standard deviation of Ca²⁺ spike latency and the width of I_Ca,L, which were also shown to increase significantly (P<0.001) with the repolarization rate (data not shown). Nevertheless, the Ca²⁺ transient amplitude and profile depends in a complex manner on Ca²⁺-release processes (ie, Ca²⁺ spikes) combined with Ca²⁺ reuptake. Taken together, our data indicate that the whole-cell Ca²⁺ transient profile can be altered through changes in repolarization rate by influencing the recruitment and temporal coherence of fundamental Ca²⁺-release events via corresponding changes in the I_Ca,L profile.

Effect of Altered Phase-1 AP Repolarization on SR Ca²⁺ Release
I_to is a critical determinant of early phase-1 AP repolarization. To examine the consequences of I_to changes and phase-1 repolarization on SR Ca²⁺ release, APs were generated by using a computer model in which I_to density was varied to alter early AP repolarization, as seen in human myocardium.^21,22 Two AP waveforms with different rates of phase-1 repolarization were then used as command waveforms on rat (Figure 4) and rabbit (Figure 6) ventricular myocytes. The early repolarization rates of fast (high I_to) and slow (low I_to) phase-1 APs (AP duration at 30% repolarization) were comparable to those of the 50- and 200-ms ramps, respectively (see online Table 1 in the data supplement available at http://www.circresaha.org). As expected from the ramp data, with slowed phase-1 AP repolarization, peak I_Ca,L was reduced (P<0.001), whereas the time to peak and duration of I_Ca,L were each increased (P<0.01) (Table 2 and Figure 4B). As shown in Figures 4 and 5, there was also a reduction (P<0.005) in amplitude (A_fast=2.24±0.19, A_slow=1.80±0.15; n=10) and total number (69.4±4.2%, n=9) of Ca²⁺ spikes triggered by slow versus fast phase-1 repolarization. In addition, the mean first latency of firing of Ca²⁺ spikes (Lat) was lengthened (P<0.005) 2.7-fold by slowed phase-1 repolarization (Lat_fast=8.41±0.72 ms, Lat_slow=22.4±3.7 ms; n=10), along with a 3.8-fold increase (P<0.05) in the standard deviation of latency (L_SD,fast=5.0±0.7 ms, L_SD,slow=19.1±5.0 ms; n=10; Figures 5C and 5D). The similarities in the trend of I_Ca,L and SR Ca²⁺ release with prolonged voltage ramps and the slowing of phase-1 AP repolarization suggest that the rate of early repolarization is critical for the recruitment and synchronization of Ca²⁺ release.

View larger version (76K): [in this window] [in a new window]   Figure 4. Effect of fast and slow phase-1 AP repolarization and ß-adrenergic stimulation on Ca2+ spikes. A, AP waveforms applied to rat ventricular myocytes in the absence (left and center) and presence of 200 nmol/L ISO (right). B, Representative ICa,L traces in myocytes under the conditions shown directly above (see Table 2). C and D, Line scans (600 ms, C) and surface plots (D) showing loss of recruitment and temporal synchronization of Ca2+-release events in myocytes stimulated with slow phase-1 AP (center), which was effectively reversed by ß-adrenergic stimulation (right). Note that ICa,L was measured in a separate set of experiments from Ca2+ spike measurements (see Materials and Methods).

View larger version (66K): [in this window] [in a new window]   Figure 6. Effect of slowed phase-1 repolarization and ISO on Ca2+ release in rabbit ventricular myocytes. A, AP waveforms applied to rabbit ventricular myocytes in the absence (left and center) and presence of 200 nmol/L ISO (right). B, Representative ICa,L traces in rabbit myocytes under conditions shown directly above (see Table 2). C, Line scans (600 ms) also showing loss of recruitment and temporal synchronization of Ca2+-release events in rabbit myocytes stimulated with slow phase-1 AP, which was reversed by ß-adrenergic stimulation. D and E, Ca2+ spike intensity (D) and frequency (E) showing a significant reduction by slowing of phase-1 AP repolarization and an increase with ISO application. F, Latency of Ca2+ spike firing and time to peak of trigger ICa,L. G, Standard deviation of Ca2+ spike latency and full width at half-maximum ICa,L with slow phase-1 AP repolarization and after ISO application. Note that ICa,L was measured in a separate set of experiments from Ca2+ spike measurements (see Materials and Methods).

View this table: [in this window] [in a new window]   Table 2. L-Type Ca2+ Current Properties in Rat and Rabbit Myocytes During AP Clamps With and Without ISO

View larger version (33K): [in this window] [in a new window]   Figure 5. Slowing of phase-1 repolarization and ISO alters microscopic properties of Ca2+ release. A and B, Ca2+ spike amplitude (A) and frequency (B) were significantly (P<0.05) reduced by slowing of phase-1 AP repolarization and were restored by ISO application. C, First latency of Ca2+ spike firing and time to peak of trigger ICa,L were significantly (P<0.05) lengthened when stimulated with slow phase-1 AP and largely restored with ß-adrenergic activation. D, Standard deviation of Ca2+ spike latency and full width at half maximum of ICa,L were both increased significantly (P<0.05) on stimulation with slow phase-1 AP. ISO application significantly (P<0.05) reduced the standard deviation of Ca2+ spike firing, whereas the full width at half maximum of ICa,L actually increased. *P<0.05 for slow vs fast phase-1 AP data; P<0.05 for slow phase-1 AP in the absence vs presence of ISO application.

Although freshly isolated rat cardiac myocytes are commonly used to study E-C coupling, it is possible that fundamental species differences in E-C coupling^23,24 may exist between rodents, larger mammals, and humans. To assess whether our observations in rodents are applicable to larger mammals, experiments were repeated on freshly isolated rabbit ventricular myocytes. Slowing phase-1 repolarization in rabbit myocytes also resulted in reductions (P<0.05) of the amplitude (A_fast=2.50±0.11, A_slow=2.16±0.10; n=5) and the number of active Ca²⁺-release sites (72±7%, n=5). The temporal distribution of release events (Figure 6F) revealed a 2.5-fold increase (P<0.001) in first latencies of firing with slow (Lat_slow=17.4±1.6 ms) versus rapid (Lat_fast=7.1±0.7 ms) early repolarization, as in the rat. The temporal dispersion of Ca²⁺ release (Figure 6G) was also increased (P<0.005) by slow repolarization in rabbit myocytes (L_SD,fast=3.3±0.3 ms, L_SD,slow=8.4±1.1 ms). Thus, SR Ca²⁺ release in both rat and rabbit myocytes responds in a similar fashion to changes in phase-1 AP repolarization.

Effects of ß-Adrenergic Stimulation
ß-Adrenergic receptor stimulation is known to influence SR Ca²⁺ release^25–27 and has been recently shown to be involved in the synchronization of Ca²⁺ release in rat cardiac myocytes¹⁷; therefore, we determined whether activation of the ß-adrenergic signaling cascade (200 nmol/L ISO) could resynchronize the Ca²⁺ release in myocytes stimulated with the slow phase-1 AP. As shown in Figures 4 and 6B (right), ISO significantly (P<0.05) increased peak I_Ca,L and shortened the time to peak of the current (Table 2) in both rat and rabbit myocytes. Associated with these changes in I_Ca,L after ISO application was a significant increase in amplitude (A_slow+ISO=2.25±0.19 in rats, A_slow+ISO=2.94±0.16 in rabbits; P<0.01) and percentage of total Ca²⁺-release events (111±7.2% in rats, 125.2±8.45% in rabbits; P<0.05) to levels comparable to those observed with the rapid phase-1 AP (Figures 5 and 6). Similarly, the temporal distribution and dispersion of release events was resynchronized (Figures 4C and 4D; Figure 6C, right), as illustrated by the reduced mean latency (Lat_slow+ISO=11.53±1.6 ms in rats and 10.0±1.1 ms in rabbits) and standard deviation of latency (L_SD,slow+ISO= 6.4±0.8 ms in rats and 4.5±0.6 ms in rabbits; Figures 5C and 5D, Figures 6F and 6G). These results demonstrate that ß-adrenergic stimulation is capable of enhancing E-C coupling sufficiently to reverse the asynchronous Ca²⁺ release that occurs when early repolarization is slowed.

	Discussion

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Membrane Repolarization Modulates Ca²⁺ Release via Alterations in I_Ca,L
AP repolarization varies regionally in normal myocardium²¹ and is impaired in diseased myocardium.^7–9 Although normal AP repolarization is believed to be important primarily for maintaining coordinated electrical activity and reducing the propensity for cardiac arrhythmias,¹¹ alterations in AP profile are also known to influence I_Ca,L,¹² intracellular Ca²⁺ transients,^9,13 and E-C coupling.¹⁴ Our studies show that altering the rate of repolarization profoundly affects both the magnitude and time course of SR Ca²⁺ release. A profound and monotonic decrease in the amplitude (Figure 1C) and an increase in the time to peak of Ca²⁺ transients (Figure 1D) were observed in response to reductions in the repolarization rate. Visualization of the fundamental Ca²⁺-release events that constitute the Ca²⁺ transient revealed that the delayed time to peak of the Ca²⁺ transient with longer ramps (Figures 1B and 1D) resulted from increases in first latency of Ca²⁺-release events (Figure 3C), whereas the reduced Ca²⁺ transient amplitudes were due to reductions in the recruitment (Figures 3A and 3B) and in the temporal summation of Ca²⁺-release events (Figure 3D). Underlying these changes in the synchronization of SR Ca²⁺ release was a close correlation between the time to peak of I_Ca,L and the first latency of Ca²⁺ spikes. This correlation is expected because the probability of spark occurrence has been shown to be proportional to I_Ca,L · i (ie, open probability multiplied by i²).²⁰ Thus, alterations in the time course and magnitude of I_Ca,L via changes in the repolarization rate will produce complementary changes in the recruitment and temporal summation of elementary Ca²⁺-release events, which ultimately influence the amplitude and kinetics of the whole-cell Ca²⁺ transient, albeit in a complex manner.

Longer ramps were also associated with mild slowing of the decay rate of Ca²⁺ transients in rat myocytes (Figure 1E). This observation was initially surprising because relaxation of the transient is traditionally considered to be dependent primarily on reuptake via the SR Ca²⁺-ATPase and extrusion by the Na⁺-Ca²⁺ exchanger.¹ When Ca²⁺ release is rapid and synchronous, these Ca²⁺-removal processes likely dominate the decay of the intracellular Ca²⁺ transient, as observed with the 50-ms ramp. On the other hand, with longer ramps (500 ms), the time periods of SR Ca²⁺ release and reuptake partially overlap (data not shown), such that very-late-release events effectively slow the early decay of the Ca²⁺ transient (Figures 2C and 2D). However, slowed repolarization will also decrease forward-mode activity of the voltage-dependent Na⁺-Ca²⁺ exchanger, thereby also contributing to slowed relaxation. The connection between AP prolongation and intracellular Ca²⁺ handling has been suggested previously to account for impaired intracellular Ca²⁺ relaxation in human heart failure⁵ and clearly warrants further study.

Our results are consistent with a recent report showing a connection between trigger I_Ca,L and the recruitment and synchronization of Ca²⁺ release in rat ventricular myocytes by use of voltage steps in the presence and absence of either ß-adrenergic stimulation or I_Ca,L agonist (FPL).¹⁷ In failing rabbit myocytes, Litwin et al³ also demonstrated that a reduction of I_Ca,L was associated with asynchronous late-firing Ca²⁺ sparks, resulting in blunted Ca²⁺ transients with slowed kinetics similar to our results in rat ventricular myocytes stimulated with slowly repolarizing ramps (Figures 1A and 1B, 200- and 500-ms ramps). Because changes in AP repolarization have also been shown previously to alter trigger I_Ca,L,^9,12–14 we speculated that the rate of phase-1 repolarization of the AP may also influence SR Ca²⁺ release in a manner similar to that observed with ramps.

Phase-1 AP Repolarization Is Critical for Optimizing SR Ca²⁺ Release
We extended the modulatory role of membrane repolarization rate by examining the effects of different rates of phase-1 repolarization of the AP on Ca²⁺ release. Using computer-generated APs like those recorded in human epicardial and endocardial myocytes²¹ or in failing versus nonfailing human ventricular myocytes,²² we observed a significant loss in the amplitude, number, and temporal synchronization of Ca²⁺ spikes after stimulation with the slow phase-1 AP compared with the rapid phase-1 AP, which is associated with a reduction in peak I_Ca,L and a shift in the time to peak of I_Ca,L. Although changes in overall AP duration after I_to reduction in human heart disease is somewhat controversial, it is generally agreed that there is loss of the phase-1 notch in failing myocytes (see review by Nabauer and Kaab²⁸). Furthermore, the role of I_to in setting the rate of early repolarization has been well established in model studies,^12,22 in studies in vitro,²⁹ and in studies in vivo.³⁰ Our findings suggest an important physiological role for I_to and the phase-1 notch of the human AP in optimizing the synchronization and recruitment of Ca²⁺-release events by increasing the driving force for Ca²⁺ influx and enhancing trigger I_Ca,L. This relationship between I_to density, phase-1 AP repolarization, and I_Ca,L has also been shown theoretically in a recent canine AP computer model,¹² in which selective reductions in K_v4.3-based I_to result in elevation of the AP notch and a decrease in peak I_Ca,L. Thus, reductions in I_to and in the rate of early repolarization might contribute to the slowed, blunted Ca²⁺ transient observed in failing human myocardium⁵ by decreasing trigger I_Ca,L and impairing SR Ca²⁺ release. Indeed, this effect will further exacerbate the impairment in SR Ca²⁺ handling observed in heart failure that has been linked to reductions in SR Ca²⁺ content,⁶ ß-adrenergic receptor desensitization,³¹ RyR and VLCC coupling,⁴ and VLCC density.³

Interestingly, the early repolarization rates of the fast and slow phase-1 APs were comparable to those of the 50- and 200-ms ramps, respectively, as indicated by the AP duration at 30% repolarization (see online Table 1), and resulted in a similar trend toward desynchronization of Ca²⁺ release with slowed repolarization. On the other hand, late repolarization of both APs, as indicated by AP duration at 50% and 90% repolarization, differed markedly from either the 50- or 200-ms ramp and also showed no correlation with the magnitude or kinetics of SR Ca²⁺ release. Collectively, this suggests that early AP repolarization mediated by I_to is more important with respect to E-C coupling than is late repolarization of the AP.

ß-Adrenergic Stimulation Promotes Synchronous Firing of Ca²⁺-Release Events
Recent studies have demonstrated that ß-stimulation enhances E-C coupling²⁷ and promotes synchronization of Ca²⁺ release in normal¹⁷ and failing³ myocardium. We also found that ß-adrenergic stimulation effectively restores temporal synchrony and enhances the recruitment of Ca²⁺-release events triggered by the slow phase-1 AP. Similarly, the magnitude and kinetics of I_Ca,L triggered by the slow phase-1 AP was partially restored on ß-adrenergic stimulation, with the exception of the width at half-maximum I_Ca,L and the total integrated I_Ca,L, which actually increase. ß-Adrenergic stimulation may enhance SR Ca²⁺ release via increases in the I_Ca,L trigger,¹⁷ increases in SR Ca²⁺ load,²⁶ or enhancements in cross signaling between L-type Ca²⁺ channels and RyRs.²⁷ Although direct effects of ISO on I_Ca,L may contribute in part to the enhanced recruitment and synchronization of Ca²⁺ release observed in the present study, the modest increase in I_Ca,L amplitude and the discrepancy between the increase in width of I_Ca,L and Ca²⁺ release after ß-adrenergic stimulation suggest that other mechanisms may also contribute to the observed effect. Regardless, the ability of ß-adrenergic stimulation to restore normal SR Ca²⁺ release to a failing myocyte may represent an ideal compensatory mechanism by which the desynchronizing effect of slow phase-1 AP repolarization can be countered. However, in the setting of heart failure, the ultimate onset of ß-receptor desensitization³¹ may minimize this compensation, effectively uncovering the latent deficiency in E-C coupling resulting from impaired early repolarization.

In summary, we have demonstrated that the rate of membrane repolarization modulates the recruitment and synchronization of SR Ca²⁺-release events primarily via alterations in I_Ca,L and that this influences the amplitude as well as the rise and decay of the intracellular Ca²⁺ transient. Furthermore, our results suggest a putative physiological role for the phase-1 notch of the AP in optimizing the temporal summation and recruitment of Ca²⁺-release events. Our results also suggest that asynchronous Ca²⁺ release resulting from slowed phase-1 repolarization after I_to reduction in failing myocytes can be acutely overcome by ß-adrenergic stimulation but may ultimately lead to impaired systolic and diastolic function due to ß-receptor desensitization. According to this paradigm, therapeutic agents designed to accelerate phase-1 repolarization and reestablish the AP notch may prove to be beneficial by possibly improving both systolic and diastolic dysfunction.

	Acknowledgments

 
This study was supported by a Canadian Institutes of Health Research (CIHR) grant to Dr Backx. R. Sah is the recipient of an MD/PhD Studentship from the CIHR, and R.J. Ramirez is the recipient of a Research Traineeship Award from the Heart and Stroke Foundation of Canada. Dr Backx is a Career Investigator of the Heart and Stroke Foundation of Ontario. We are grateful for equipment support from the Tiffin Trust Fund, the Centre for Cardiovascular Research at the University of Toronto, and the Heart and Stroke/Richard Lewar Centre of Excellence. We also thank Robert Tsushima for providing myocytes for some of these studies.

Received September 14, 2001; revision received November 27, 2001; accepted November 28, 2001.

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