Distinct Patterns of Calcium Transients During Early and Delayed Afterdepolarizations Induced by Isoproterenol in Ventricular Myocytes
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.
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 DADs6 7 and to the relevant role of adrenergic stimulation in several important pathophysiological conditions.8 9 Preliminary results have been presented.10
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).
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 MΩ.
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 (128×128 pixels, with 1 pixel corresponding to an area of 1.7 μm2).
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 3×3–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.
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.
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.
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.
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⇓).
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 β-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.
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 ischemia16 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 colleagues5 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
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.
- Copyright © 1995 by American Heart Association
Lakatta EG. Functional implications of spontaneous sarcoplasmic reticulum Ca2+ release in the heart. Cardiovasc Res. 1992;26:193-214.
Capogrossi MC, Houser SR, Bahinski A, Lakatta EG. Synchronous occurrence of spontaneous localized calcium release from the sarcoplasmic reticulum generates action potentials in rat cardiac ventricular myocytes at normal resting membrane potential. Circ Res. 1987;61:498-503.
Berlin JR, Cannel MB, Lederer WJ. Cellular origins of the transient inward current in cardiac myocytes: role of fluctuations and waves of elevated intracellular calcium. Circ Res. 1989;65:115-126.
Szabo B, Marchi S, Scherlag BJ, Lazzara R. Simultaneous demonstration of early and delayed afterdepolarizations in Ca2+ loaded cardiac cells. J Am Coll Cardiol. 1989;13:185A. Abstract.
January CT, Riddle JM. Early afterdepolarizations: mechanism of induction and block: a role for L-type Ca2+ current. Circ Res. 1989;64:977-990.
Song Y, Thedford S, Lerman BB, Belardinelli L. Adenosine-sensitive afterdepolarizations and triggered activity in guinea pig ventricular myocytes. Circ Res. 1992;70:743-753.
Capogrossi MC, Suarez-Isla BA, Lakatta EG. The interaction of electrically stimulated twitches and spontaneous contractile waves in single cardiac myocytes. J Gen Physiol. 1986;88:615-633.
Zipes DP, Miyazaki T. The autonomic nervous system and the heart: basis for understanding interactions and effects on arrhythmia development. In: Zipes DP, Jalife J, eds. Cardiac Electrophysiology: From Cell to Bedside. Philadelphia, Pa: WB Saunders Co; 1990:312-330.
Schwartz PJ, Locati E, Priori SG, Zaza A. The long Q-T syndrome. In: Zipes DP, Jalife J, eds. Cardiac Electrophysiology: From Cell to Bedside. Philadelphia, Pa: WB Saunders Co; 1990:589-605.
Forti S, D’Amato E, De Ferrari GM, Viola MC, Antolini R. Distribution of intracellular calcium during afterdepolarizations in single voltage-clamped ventricular myocytes. Eur Heart J. 1994;15(suppl):1866. Abstract.
Minta A, Kao JPY, Tsien RY. Fluorescent indicators for cytosolic Ca2+ based on rhodamine and fluorescein chromophore. J Biol Chem. 1989;264:8171-8178.
Takamatsu T, Wier WG. Calcium waves in mammalian heart: quantification of origin, magnitude, waveform, and velocity. FASEB J. 1990;4:1519-1525.
Wit AL, Rosen MR. Afterdepolarization and triggered activity. In: Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE, eds. The Heart and Cardiovascular System. New York, NY: Raven Press; 1986:1149-1490.
Viola MC, Forti S, D’Amato E, Lasta S, Antolini R. Spontaneous intracellular calcium changes underlying oscillatory potentials in overdriven isolated ventricular myocytes. Eur Heart J. 1992;13(suppl):2311. Abstract.
Steenbergen C, Murphy E, Levy L, London RE. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res. 1987;60:700-707.
Marban E, Kitakaze M, Kusuoka H, Porterfield JK, Yue DT, Chacko VP. Intracellular free calcium concentration measured with 19F NMR spectroscopy in intact ferret hearts. Proc Natl Acad Sci U S A. 1987;84:6005-6009.
Sharma AA, Saffitz JE, Lee BI, Sobel BE, Corr PB. Alpha-adrenergic-mediated accumulation of calcium in reperfused myocardium. J Clin Invest. 1983;72:802-807.
Endoh M, Blinks JR. Actions of sympathomimetic amines on the Ca2+ transients and contractions of rabbit myocardium: reciprocal changes in myofibrillar responsiveness to Ca2+ mediated through α- and β-adrenoceptors. Circ Res. 1988;62:247-265.
Thandroyen FT, Morris AC, Hagler HK, Ziman B, Pai L, Willerson JT, Buja LM. Intracellular calcium transients and arrhythmia in isolated heart cells. Circ Res. 1991;69:810-819.
Miura M, Ishide N, Oda H, Sakurai M, Shinozaki T, Takishima T. Spatial features of calcium transients during early and delayed afterdepolarizations. Am J Physiol. 1993;256:H439-H444.
De Ferrari GM, Nador F, Beria G, Sala S, Lotto A, Schwartz PJ. Effect of calcium channel block on the wall motion abnormality of the idiopathic long QT syndrome. Circulation. 1994;89:2126-2132.
Hirano Y, Moscucci A, January CT. Direct measurement of L-type Ca2+ window current in heart cells. Circ Res. 1992;70:445-455.
Luo C-H, Rudy Y. A dynamic model of the cardiac ventricular action potential, II: afterdepolarizations, triggered activity, and potentiation. Circ Res. 1994;74:1097-1113.