Modulation of Ca2+ Release in Cardiac Myocytes by Changes in Repolarization Rate
Role of Phase-1 Action Potential Repolarization in Excitation-Contraction Coupling
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 (ICa,L), suggesting that the effects of repolarization on excitation-contraction coupling occur primarily via changes in ICa,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 ICa,L. Isoproterenol application enhanced and largely resynchronized SR Ca2+ release, while it increased the magnitude and shortened the time to peak of ICa,L. Our data demonstrate that membrane repolarization modulates the recruitment and synchronization of SR Ca2+ release via ICa,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 β-adrenergic stimulation.
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 animals2–4⇓⇓ 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 (Ito), are also known to influence L-type Ca2+ current (ICa,L),12 intracellular Ca2+ transients,9,13⇓ and excitation-contraction (E-C) coupling.14 Therefore, reductions in Ito 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 β-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 ICa,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 β-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 β-adrenergic stimulation.
Materials and Methods
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 ICa,L
ICa,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 β-adrenergic stimulation were tested after ≈4 minutes of exposure to solution B plus 200 nmol/L ISO.
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.
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.
Repolarization Rate Modulates Ca2+ Release via Changes in ICa,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 ICa,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 ICa,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.
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 ICa,L during voltage ramps, the time to peak and full width at half-maximum ICa,L were also measured. Both the first latency of Ca2+ spikes and time to peak of ICa,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 ICa,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 ICa,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 ICa,L profile.
Effect of Altered Phase-1 AP Repolarization on SR Ca2+ Release
Ito is a critical determinant of early phase-1 AP repolarization. To examine the consequences of Ito changes and phase-1 repolarization on SR Ca2+ release, APs were generated by using a computer model in which Ito 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 Ito) and slow (low Ito) 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 ICa,L was reduced (P<0.001), whereas the time to peak and duration of ICa,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 (Afast=2.24±0.19, Aslow=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 (Latfast=8.41±0.72 ms, Latslow=22.4±3.7 ms; n=10), along with a 3.8-fold increase (P<0.05) in the standard deviation of latency (LSD,fast=5.0±0.7 ms, LSD,slow=19.1±5.0 ms; n=10; Figures 5C and 5D). The similarities in the trend of ICa,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.
Although freshly isolated rat cardiac myocytes are commonly used to study E-C coupling, it is possible that fundamental species differences in E-C coupling23,24⇓ 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 (Afast=2.50±0.11, Aslow=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 (Latslow=17.4±1.6 ms) versus rapid (Latfast=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 (LSD,fast=3.3±0.3 ms, LSD,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 β-Adrenergic Stimulation
β-Adrenergic receptor stimulation is known to influence SR Ca2+ release25–27⇓⇓ 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 β-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 6⇑B (right), ISO significantly (P<0.05) increased peak ICa,L and shortened the time to peak of the current (Table 2) in both rat and rabbit myocytes. Associated with these changes in ICa,L after ISO application was a significant increase in amplitude (Aslow+ISO=2.25±0.19 in rats, Aslow+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 (Latslow+ISO=11.53±1.6 ms in rats and 10.0±1.1 ms in rabbits) and standard deviation of latency (LSD,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 β-adrenergic stimulation is capable of enhancing E-C coupling sufficiently to reverse the asynchronous Ca2+ release that occurs when early repolarization is slowed.
Membrane Repolarization Modulates Ca2+ Release via Alterations in ICa,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 ICa,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 ICa,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 ICa,L · i (ie, open probability multiplied by i2).20 Thus, alterations in the time course and magnitude of ICa,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 ICa,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 β-adrenergic stimulation or ICa,L agonist (FPL).17 In failing rabbit myocytes, Litwin et al3 also demonstrated that a reduction of ICa,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 ICa,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 ICa,L and a shift in the time to peak of ICa,L. Although changes in overall AP duration after Ito 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 Ito 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 Ito 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 ICa,L. This relationship between Ito density, phase-1 AP repolarization, and ICa,L has also been shown theoretically in a recent canine AP computer model,12 in which selective reductions in Kv4.3-based Ito result in elevation of the AP notch and a decrease in peak ICa,L. Thus, reductions in Ito and in the rate of early repolarization might contribute to the slowed, blunted Ca2+ transient observed in failing human myocardium5 by decreasing trigger ICa,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 β-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 Ito is more important with respect to E-C coupling than is late repolarization of the AP.
β-Adrenergic Stimulation Promotes Synchronous Firing of Ca2+-Release Events
Recent studies have demonstrated that β-stimulation enhances E-C coupling27 and promotes synchronization of Ca2+ release in normal17 and failing3 myocardium. We also found that β-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 ICa,L triggered by the slow phase-1 AP was partially restored on β-adrenergic stimulation, with the exception of the width at half-maximum ICa,L and the total integrated ICa,L, which actually increase. β-Adrenergic stimulation may enhance SR Ca2+ release via increases in the ICa,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 ICa,L may contribute in part to the enhanced recruitment and synchronization of Ca2+ release observed in the present study, the modest increase in ICa,L amplitude and the discrepancy between the increase in width of ICa,L and Ca2+ release after β-adrenergic stimulation suggest that other mechanisms may also contribute to the observed effect. Regardless, the ability of β-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 β-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 ICa,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 Ito reduction in failing myocytes can be acutely overcome by β-adrenergic stimulation but may ultimately lead to impaired systolic and diastolic function due to β-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.
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.
Original received September 14, 2001; revision received November 27, 2001; accepted November 28, 2001.
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