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(Circulation. 1995;91:2510-2515.)
© 1995 American Heart Association, Inc.

Articles

Distinct Patterns of Calcium Transients During Early and Delayed Afterdepolarizations Induced by Isoproterenol in Ventricular Myocytes

Gaetano M. De Ferrari, MD; MariaCristina Viola, MS; Elvira D'Amato, MS; Renzo Antolini, MS; Stefano Forti, MS

From CMBM (S.F., M.C.V., R.A.), Centro Materiali e Biofisica Medica, Povo, Trento; Dipartimento di Fisica (E.D., M.C.V., R.A., E.D.), Università degli Studi di Trento, Povo, Trento; Centro di Fisiologia Clinica e Ipertensione (G.M.D.), Università degli Studi di Milano (Italy); and Divisione di Cardiologia (G.M.D), IRCCS Ospedale Maggiore Policlinico, Milano, Italy.

Correspondence to Stefano Forti, Biofisica Medica, CMBM-ITC, 38050 Povo, Trento, Italy.

	Abstract

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Background The relation between early afterdepolarizations (EADs) and changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is still unclear. In the present study, we compared spatiotemporal changes in [Ca²⁺]_i related to EADs and delayed afterdepolarizations (DADs) induced by isoproterenol.

Methods and Results Isolated patch-clamped guinea pig ventricular myocytes, loaded with fluo-3 acetoxymethyl ester (fluo-3 AM), were paced at 0.1 to 2 Hz. Isoproterenol (100 nmol/L) caused alterations in both phase 2 and phase 4 of the action potential (AP), consistent with EADs and DADs, respectively. During EADs (n=16), similar to driven APs, increases in [Ca²⁺]_i occurred simultaneously throughout the cell, whereas during DADs (n=25), they originated in discrete cell sites and propagated as a wave. This difference was confirmed by analysis of eight EADs and DADs coupled to the same beat. Ca²⁺ transients linked to EADs reached a peak relative fluorescence level (expressed as percentage of the maximal level reached during the last stimulated beat) that was always higher than that reached during the DADs (77±3% versus 64±2%, P<.001). Spatial heterogeneity of Ca²⁺ transients was assessed by the maximal time interval between peaks monitored in different cell regions; this time lag was always greater during DADs than during EADs (290 versus 40 milliseconds, P=.006).

Conclusions The present study had two main findings. First, even very modest notches occurring during the plateau of the AP may be accompanied by a marked secondary increase in [Ca²⁺]_i. Second, these Ca²⁺ transients occurring during EADs are synchronous throughout the cell and differ significantly from those observed under identical conditions during DADs.

Key Words: calcium • isoproterenol • myocytes • afterdepolarizations

	Introduction

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An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) accompanying diastolic depolarizations or delayed afterdepolarizations (DADs) has been demonstrated in several experimental preparations (for a recent review, see Lakatta¹ ). Also, cellular Ca²⁺ loading is known to favor the occurrence of DADs.² The suggested mechanism for the occurrence of DADs is a primary elevation of [Ca²⁺]_i, triggering a transient inward current.³ Recently, it has been suggested that increases in [Ca²⁺]_i may also be associated with the occurrence of early afterdepolarizations (EADs).⁴ However, although a potential role for L-type Ca²⁺ channels in the genesis of phase 2 EADs has been suggested,⁵ the relation between EADs and changes in [Ca²⁺]_i is still unclear.

The goal of the present study was to assess and compare the spatiotemporal changes in [Ca²⁺]_i related to EADs and DADs. Isolated voltage-clamped guinea pig ventricular myocytes, loaded with fluo-3 acetoxymethyl ester as a Ca²⁺ indicator, were used. Changes in fluo-3 fluorescence were monitored by using digital video microscopy. Isoproterenol was chosen to induce afterdepolarizations due to its capability to facilitate both EADs and DADs^{6 7} and to the relevant role of adrenergic stimulation in several important pathophysiological conditions.^{8 9} Preliminary results have been presented.¹⁰

	Methods

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Cell Isolation and Dye Loading
Male guinea pigs (weight, 250 to 300 g) were administered heparin (5000 U/kg IP), anesthetized with a combination of 8 mg/kg Zolazepanum and 12 mg/kg Tiletaminum, and quickly thoractomized. Ca²⁺-tolerant ventricular myocytes were dissociated from the heart by using an enzymatic method as previously described by De Young et al.¹¹

The myocytes were loaded with fluo-3 acetoxymethyl ester ([Fluo-3 AM] 5 µmol/L for 15 minutes) (lot 10A-1, Molecular Probes, Inc) at room temperature in absence of serum and in presence of Pluronic F-127 (BASF Wyandotte).

Electrophysiological Techniques
Membrane potentials of ventricular myocytes were measured by the whole-cell recording technique in current-clamp mode. The electrical signal was recorded with a patch-clamp amplifier (Axopatch-1C, Axon Instruments). The pipette filling solution was (in mmol/L): KCl 133, HEPES 5, Na₂ATP 5, GTP 0.4, phosphocreatine 5, MgCl₂ 3, pH 7.2, with KOH. The patch-electrode resistance was 1 to 3 M.

Fluorescence Digital Video Microscopy
Experiments were performed on a Zeiss IM-35 inverted microscope equipped for epifluorescence. Fluorescence from the cell was detected with a silicon-intensifier target camera (C2400-08 Hamamatsu). The video signal was digitized (8-bit, 256 gray levels) on a personal computer (Compaq Deskpro 386/33) with an image-processing board (MVP-AT Matrox), which allows optical monitoring during 2560 milliseconds by digitizing in real time (40-millisecond interval between two images) 64 frames (128x128 pixels, with 1 pixel corresponding to an area of 1.7 µm²).

Image Analysis
Fluo-3 AM was chosen as the Ca²⁺ indicator because of its large optical signal.¹² Although the lack of emission or excitation spectral shift on Ca²⁺ binding made it impossible to calibrate fluorescence signals in terms of absolute free Ca²⁺ concentration, providing a ratio over time (ie, ratio of the fluorescence level during the cell stimulation to the fluorescence level at rest) allowed the Fluo-3 AM signal to be normalized for path length and dye concentration.¹²

To better evaluate the magnitude and extent of the abnormal Ca²⁺ transients related to afterdepolarizations, we have expressed the amplitude of the fluorescence variation as a percentage of the maximal variation reached during the last stimulated beat and defined this as "relative fluorescence level." To visualize the fluorescence changes within the cell, mean percent fluorescence variations were measured in each frame of the sequence in 3x3–pixel areas (15.3 µm²) and plotted as a function of time. No area was chosen near the end of the myocyte to minimize distortion in the fluorescence signal during cell shortening or in the nuclear regions due to the different behavior of the Ca²⁺ changes in these areas compared with the cytosol.

Experimental Protocol
We used only rod-shaped, clearly striated ventricular myocytes that were well loaded with Fluo-3 AM and exhibited no spontaneous activity at rest. Cells were continuously superfused with Tyrode's solution ([in mmol/L]: NaCl 140, KCl 4, CaCl₂ 1.25, MgCl₂ 1, glucose 5.5, HEPES 10, pH 7.4, with NaOH), and all of the experiments were carried out at room temperature. To facilitate the development of EADs and DADs, isoproterenol (100 to 200 nmol/L) was added, and the cells were stimulated over a wide range of cycle lengths (500 to 10 000 milliseconds) for 30 or 60 seconds. Membrane potentials were registered throughout the entire train of pulses while fluorescent images were collected for 2560 milliseconds, starting from the last evoked AP, to monitor both elicited and spontaneous Ca²⁺ changes. Myocytes that showed a shortening >15% during the cell contraction, lateral motions, or swings were discarded to avoid marked distortions in the fluorescence measurements.

Statistical Analysis
Data are presented as mean±SEM. Normality of the distributions was assessed both graphically and with a Kolmogorov-Smirnov test. Differences between EADs and DADs linked to the same beat were assessed by paired t test or by Wilcoxon paired rank sum test for normally and nonnormally distributed variables, respectively. Differences in frequency were assessed by Fisher's exact test. A value of P=.05 was considered the limit for significance.

	Results

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The occurrence of an EAD and a DAD during isoproterenol infusion is shown in the recordings of Fig 1. Top traces show the last stimulated beat of a train of 30 APs at a cycle length of 1000 milliseconds; the middle and bottom traces show the electrical signal and the fluorescence signal (from three different sites of the myocyte), respectively, corresponding to the last AP. Concomitant with the DAD, the fluorescence signal showed the presence of a Ca²⁺ transient that, at variance with that of the AP, which was very synchronous throughout the cell, originated from a specific site of the myocyte (close to position a) and propagated toward the lower part of the cell as a wave. The speed of the Ca²⁺ wave was 117 µm/s, which is in agreement with that observed in other studies.^{1 2 3 4 5 6 7 8 9 10 11 12 13} The weak deflection in the trace b (marked by an asterisk) corresponds to a localized spontaneous Ca²⁺ release (focus), which did not spread to other regions of the cell.

View larger version (29K): [in this window] [in a new window]   Figure 1. Electrical and fluorescence signals during a delayed afterdepolarization (DAD) (A) and an early afterdepolarization (EAD) (B) (both indicated by arrows) induced by isoproterenol (100 nmol/L) and stimulation at 1 Hz. Middle, Last action potential trace is expanded (foot only in A), and the first derivative (thin line) is superimposed. Bottom, Normalized fluorescence signal from three different cell sites (a, b, and c). Note the difference both in dV/dt during plateau, facilitating detection of EAD in B, and in the secondary Ca2+ transient, synchronous in B but not in A. Maximal time interval between the peaks of the three transients was 880 milliseconds for the DAD and 40 milliseconds for the EAD.

We analyzed a total of 25 DADs that occurred after the last stimulated beat during isoproterenol infusion. The DADs always originated after complete repolarization of the cell and had a coupling interval (CI) ranging from 700 to 4400 milliseconds and an amplitude ranging from 2 to 10 mV. The CI decreased as the cycle length of the stimulation drive decreased (eg, see Fig 2A), which is in agreement with the expected behavior of DADs.¹⁴ In 19 of 25 cells (76%), two or more foci of Ca²⁺ transient were observed that gave origin to distinct waves spreading along the cell and disappearing after their collision. In 5 of 25 cells (20%), the DADs triggered a complete AP. This preferentially occurred at a stimulation cycle length of 1000 milliseconds and appeared to be favored by the presence of multifocal rather than monofocal Ca²⁺ transients (see also Fig 2B and 2C).

View larger version (24K): [in this window] [in a new window]   Figure 2. Tracings showing simultaneous presence of early afterdepolarizations (EADs) (arrows) and delayed afterdepolarizations (DADs) (closed circles) coupled to the same beat. A, Decrease in basic cycle length ([BCL] milliseconds) is accompanied by shortening of the coupling interval (CI) and an increased likelihood of triggered beats (asterisks); the EAD did not significantly change its CI but decreased its activation voltage and disappeared at a BCL of 500 milliseconds. B, Electrical signal and normalized fluorescence signal from four different cell sites of the last driven action potential at a BCL of 1000 milliseconds. Sites are evident in the cell profile. C, Twelve images in pseudocolor, taken as indicated by the vertical bars over the fluorescence signal. Ca2+ transient was synchronous during both the evoked action potential (image 2) and the EAD (image 4) but not during the DAD. A localized Ca2+ release (f1) originated in region a (image 7) and the DAD developed; when the wave moved away from the end of the cell and propagated toward the region b (image 8), the DAD decreased. While the wave reached region c, a second focus (f2) started in region d (image 9); this contributed to reach threshold and trigger an action potential (image 11). Site a was chosen for display, being the origin of the transient, despite the slight distortion of the signal during the driven contraction caused by its proximity to the cell border.

Infusion of isoproterenol often induced marked alterations in AP morphology and, especially, in Ca²⁺ transient, also during the plateau phase. A slowing of the repolarization process was observed that suggested the presence of an EAD (Fig 1B). In agreement with this interpretation are the following factors: (1) the absence of this phenomenon under control conditions and its appearance after ß-adrenergic stimulation⁶ ; (2) the marked alterations in Ca²⁺ transient, described below, that were always completely absent under control condition; (3) the disappearance of both the "notch" and the secondary Ca²⁺ transient at short drive cycle lengths (<600 to 750 milliseconds), also a feature consistent with EADs¹⁴ ; and (4) the observation of almost identical Ca²⁺ transients concomitant with actual membrane depolarizations during the plateau phase, induced by either isoproterenol or a depolarizing pulse (results not shown).

A total of 16 beats with EADs were analyzed. Although in 4 of these beats the presence of an EAD could be easily identified by inspection of the AP morphology, in 12 (75%), the notches corresponding to the EAD were of such limited amplitude that they could be recognized only by careful analysis of the first derivative of AP (Fig 1B; middle shows how the derivative of AP may facilitate the detection of an EAD). Despite the limited extent of the abnormality in AP, the fluorescence signal in correspondence of these notches always showed a marked alteration, consisting of a secondary Ca²⁺ transient originating much before the Ca²⁺ signal had returned to the diastolic level and reaching a maximal level often close to that reached during the previous excitation. Fig 1 (bottom two panels) illustrates the difference observed in the Ca²⁺ transient between APs with no EAD (left, A) and APs with phase 2 EADs (right, B). To further substantiate the abnormality in the Ca²⁺ transient, we compared the 16 beats with phase 2 EADs with 15 beats with a normal AP. A secondary increase in [Ca²⁺]_i (ie, originating during the descending phase of the transient related to the normal excitation-contraction coupling) was observed in 0 of 15 beats during normal APs and in 16 of 16 beats during an AP with a phase 2 EAD (P<.001). This secondary Ca²⁺ transient originated at a mean relative fluorescence level of 45.7±4.9 and reached a peak of 74.7±3.4. Furthermore, the duration of the Ca²⁺ transients observed during pacing at similar cycle lengths (an average of 2921±1015 and 3135±1038 milliseconds for beats with phase 2 EADs and with normal APs, respectively) was significantly longer in beats with phase 2 EADs (measured until return to a relative fluorescence level of 20%); the duration was 623±46 milliseconds versus 470±39 milliseconds in beats with normal APs (P<.02). The average duration of the Ca²⁺ transient was longer in beats with EADs at each cycle length studied (0.5, 1, 2, 5, and 10 seconds).

The spatial features of the Ca²⁺ transient observed during these phase 2 EADs were quite different from those observed during a DAD-linked transient. The transient appeared to occur synchronously throughout the cell, with no evidence of a propagating wave (see Fig 1, bottom). Linked to the secondary Ca²⁺ transient, the cells also showed an aftercontraction, originating before complete relaxation.

To further characterize the differences in the features of Ca²⁺ transients originating in phase 2 and phase 4 of the AP, we analyzed the subgroup of cells that showed, coupled to the same beat (the last of the train), both an EAD and a DAD, thus ensuring that the difference observed could not be due simply to a different level of cellular Ca²⁺ loading. The following data thus refer to the eight EADs and DADs that were observed coupled to the same beat. No EADs and two of eight DADs triggered an AP. One of these cells is represented in Fig 2. Resting membrane potential in these eight experiments was -79±2 mV. The average takeoff potential was 17±4 mV for EADs and -79±2 mV for DADs. The average CI was 352±19 milliseconds (range, 268 to 580 milliseconds) for EADs and 1784±351 milliseconds (range, 924 to 4090 milliseconds) for DADs. Because the dye technique does not allow reliable measurements of the absolute free [Ca²⁺], we analyzed the relative fluorescence level of the abnormal Ca²⁺ transients related to the afterdepolarizations. The Ca²⁺ transient linked to EADs originated at a mean relative fluorescence level of 54±4% and reached a peak of 77±3%, a level always higher than that reached during the DADs (64±2%, P<.001). To better quantify the spatial heterogeneity occurring during a Ca²⁺ transient linked to an afterdepolarization, we monitored the Ca²⁺ variations in three different regions separated by 30 µm along the longitudinal cell axis and assessed the time interval between the first and the last transient peak (see also Fig 1). This time lag between peaks was always greater during DADs than during EADs (an average of 290 versus 40 milliseconds, P=.006). Note that the values calculated for DADs were underestimated due to the frequent occurrence of multiple foci. A significant difference was also found if the beginning rather than the peak of the Ca²⁺ transient was analyzed.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study has shown the presence of important alterations in Ca²⁺ transients during superfusion of isolated guinea pig ventricular myocytes with isoproterenol. A marked increase in [Ca²⁺]_i accompanied alterations in both phase 2 and phase 4 of the AP, consistent with EADs and DADs, respectively. Two principal findings emerge from the present study. First, even very modest notches occurring during the plateau of the AP may be accompanied by a marked secondary increase in [Ca²⁺]_i. Second, these Ca²⁺ transients occurring during an EAD are synchronous throughout the cell and differ significantly from those observed under identical conditions during a DAD.

Relation With Previous Studies
The occurrence of a spontaneous release of Ca²⁺ from the sarcoplasmic reticulum during diastole, causing a DAD, has been well characterized.¹ Our findings concerning Ca²⁺ transients during a DAD are in good agreement with the observations that most often, Ca²⁺ release originates from a single localized area within the cell and propagates as a Ca²⁺ wave with a velocity of approximately 100 µm/s¹³ and that an increase in pacing rate increases the likelihood of multifocal rather than unifocal Ca²⁺ transients¹ and of triggered APs.^{1 2 15} The mechanism mediating the increased likelihood of triggered activity at faster rates is considered to be an increase in [Ca²⁺]_i. Elevated [Ca²⁺]_i levels have been suggested to play a significant role in the genesis of cardiac arrhythmias in a variety of conditions, such as acute myocardial ischemia^{16 17} and reperfusion¹⁸ and exposure to catecholamines¹⁹ and digitalis.²⁰ Thandroyen et al²¹ showed that an increase in [Ca²⁺]_i facilitates the initiation of the arrhythmia, presumably by favoring triggered activity, and that the arrhythmia itself induces a further rapid increase in [Ca²⁺]_i, thus creating a favorable setting for the perpetuation of the arrhythmia and its degeneration into fibrillation. This latter occurrence may be facilitated by cell-to-cell uncoupling that has been reported to result from increased [Ca²⁺]_i.

At variance with the accepted role of Ca²⁺ in the genesis of DADs, much less is known regarding the involvement of [Ca²⁺]_i during EADs. Szabo et al⁴ suggested in preliminary observations that increased [Ca²⁺]_i facilitates the induction of both EADs and DADs in the same cell by a potentially common mechanism and that EADs may be associated with prolonged Ca²⁺ transients.

Recently, Miura et al²² compared the spatial features of Ca²⁺ transients related to EADs and DADs occurring during superfusion with potassium-free Tyrode's solution. They also reported that transients associated with EADs were synchronous throughout the cell, whereas transients associated with DADs were heterogeneous. Compared with the report of Miura et al,²² the present study provides several additional insights into the behavior of [Ca²⁺]_i during EADs and DADs.

First, we used a relatively low concentration of isoproterenol (100 nmol/L), a setting that is more closely related to pathophysiological conditions than the complete absence of potassium. Specifically, EADs favored by adrenergic stimulation are believed to be the underlying mechanism of torsade de pointes ventricular tachycardia and sudden death in patients with the idiopathic long QT syndrome (LQTS).⁹ We have recently suggested that patients with LQTS who have recurrent malignant arrhythmias may have, even in baseline conditions, subthreshold EADs associated with secondary increases in [Ca²⁺]_i and with a prolonged or double-peaked contraction.²³ The findings of the present study are in agreement with this hypothesis.

Second, by comparing EADs and DADs coupled to the same beat, we have excluded that the different characteristics of the Ca²⁺ transients may have simply resulted from a different degree of Ca²⁺ loading of the sarcoplasmic reticulum.

Finally, we have provided a quantitative measure of the spatiotemporal inhomogeneity of the Ca²⁺ transient.

Mechanisms and Implications
Although the mechanism for diastolic Ca²⁺ release and related DADs has been well characterized,¹ the mechanism for EADs is still debated. Several different currents may be implicated in the genesis of EADs,²⁴ although January and colleagues^{5 25} have strongly suggested that depolarizations occurring during phase 2 of the AP may be due to the reopening of L-type Ca²⁺ channels within their window, following recovery after inactivation. The time resolution of the technique used in the present study (ie, one frame every 40 milliseconds) does not allow a reliable assessment of the exact sequence of events between the abnormalities in the AP and the increase in the fluorescence signal. In the case of DADs, the increase in [Ca²⁺]_i, caused by a spontaneous release from the sarcoplasmic reticulum, is known to precede, and actually to cause, through the activation of a transient inward current, the cellular depolarization.^{1 3} Our finding that the Ca²⁺ transient observed during phase 2 EADs has a completely different spatiotemporal behavior than the transient associated with DADs and, specifically, that it occurs simultaneously throughout the cell suggests that it is not due to a spontaneous release from the sarcoplasmic reticulum, a phenomenon known to be focal.¹ Rather, our finding is in agreement with the hypothesis of different ionic mechanisms for EADs and DADs²⁶ and specifically with a uniform inflow of Ca²⁺ through L-type channels during EADs.⁵ This initial influx of Ca²⁺ would then trigger a diffuse and synchronous Ca²⁺ release from the sarcoplasmic reticulum.²³ The marked increase in Ca²⁺ concentration thus would be the consequence of and follow the EAD. However, we cannot completely dismiss the alternative hypothesis that the same basic mechanism occurring at a more positive potential could give rise to a much faster Ca²⁺ wave, which appears synchronous due to the limited temporal resolution of the video system. Notably, preliminary data from ongoing experiments suggest that synchronization of Ca²⁺ transient appears to be more limited in subsequent depolarizations during multiple EADs or in phase 3 EADs.

During catecholamine infusion, barely detectable alterations in phase 2 of the AP may be coupled to marked changes in Ca²⁺ concentration, resulting in a secondary transient reaching a value close to that of the stimulated AP. This was a major finding of the present study. It suggests that the monitoring of both the derivative of AP and [Ca²⁺]_i may be of great value in detecting apparently subtle alterations during the plateau phase. The very modest difference in AP morphology between a beat that shows a normal Ca²⁺ transient and one that shows a marked secondary transient during phase 2, although surprising at first glance, is in good agreement with the finding in patients with LQTS. The contraction abnormality, believed to represent the mechanical equivalent of an EAD, can be completely abolished without significant changes being observed on surface ECG.²³

Conclusions
Results of the present study indicate that in isolated voltage-clamped guinea pig ventricular myocytes, low-dose isoproterenol (100 nmol/L) causes marked alterations in both systolic and diastolic Ca²⁺ transient. These alterations, occurring in the same cell and coupled to the same beat, are related to the development of EADs and DADs, respectively.

Marked increases in Ca²⁺ transients may occur during phase 2 of the AP, also in the presence of very modest changes in membrane voltage. These Ca²⁺ transients are synchronous throughout the cell and thus differ markedly from those observed during DADs, since the latter propagate as a wave from one or more localized foci. These findings are in agreement with the suggestion that depolarizations occurring during phase 2 of the AP may be due to reopening of L-type Ca²⁺ channels within their window.

Received February 9, 1995; revision received March 8, 1995; accepted March 8, 1995.

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