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Effects of Na⁺/Ca²⁺ exchange induced by SR Ca²⁺ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21224

Submitted 25 March 2003 ; accepted in final form 15 August 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
In cardiac cells, evoked Ca²⁺ releases or spontaneous Ca²⁺ waves activate the inward Na⁺/Ca²⁺ exchange current (I_NaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to I_NaCa elicited by sarcoplasmic reticulum (SR) Ca²⁺ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of I_NaCa by Na⁺-free, Li⁺-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD₉₀) by 50% and caused SR Ca²⁺ overload. APD₉₀ was similarly abbreviated in myocytes exposed to the Na⁺/Ca²⁺ exchange inhibitor KB-R7943 (5 µM) or after inhibition of SR Ca²⁺ release with ryanodine (20 µM). In the absence of extracellular Na⁺, spontaneous SR Ca²⁺ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na⁺-containing solution, the potentiated intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients dramatically prolonged APD₉₀ and [Ca²⁺]_i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca²⁺ mimicked the effects of spontaneous [Ca²⁺]_i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca²⁺ release. These results suggest that Na⁺/Ca²⁺ exchange is a major physiological determinant of APD and that I_NaCa activation by spontaneous SR Ca²⁺ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca²⁺ overload.

early afterdepolarization; delayed afterdepolarization; flash photolysis; Ca²⁺ overload; triggered arrhythmia; ischemia-reperfusion arrhythmia; sarcoplasmic reticulum

As one of the complex influences that shape the cardiac action potential, Na⁺/Ca²⁺ exchange can have an adverse effect on contractility and arrhythmogenesis under pathophysiological conditions. For example, myocardial Ca²⁺ overload is characterized by waves of spontaneous SR Ca²⁺ release that propagate through the cytoplasm generating inward I_NaCa and delayed afterdepolarizations (DADs) (2, 3, 28, 38, 51, 58, 67). This mechanism is thought to underlie arrhythmias associated with myocardial ischemia-reperfusion (7, 49). A similar mechanism may also facilitate or trigger early afterdepolarizations (EADs) and long Q-T arrhythmias, although this possibility remains controversial (37, 48, 57, 73, 75). Earlier studies (26, 39, 40) suggested that EADs result solely from the reactivation or sustained opening of sarcolemmal L-type Ca²⁺ channels (window I_Ca) during prolonged action potentials. However, Ca²⁺ overload and spontaneous SR Ca²⁺ release have been implicated in the long Q-T syndrome and some reports (11, 25, 52, 72, 73) have suggested that spontaneous [Ca²⁺]_i transients can induce EADs in cells exposed to high doses of catecholamines. Recent evidence from perfused hearts exposed to the class III antiarrhythmic agent E4031 also showed a close association between [Ca²⁺]_i transients and EADs (9), implying that I_NaCa may be one of the main triggers for EADs rather than merely contributing to APD prolongation.

In the present study, we sought to clarify the role of I_NaCa during SR Ca²⁺ release on action potentials and afterdepolarizations in isolated guinea pig ventricular myocytes, which exhibit an action potential configuration and Na⁺/Ca²⁺ exchange activity similar to human myocytes (4, 5, 62). To evaluate the influence of inward I_NaCa on APD, I_NaCa was reversibly inhibited and SR Ca²⁺ loading was manipulated by substituting extracellular Na⁺ with Li⁺. This ion acts readily as a Na⁺ analog in sarcolemmal ion channels, but is not transported by the Na⁺/Ca²⁺ exchanger, and thereby blocks the efflux of cytsolic Ca²⁺ (15, 18, 35, 61). To examine directly the role of Ca²⁺ release on afterpolarization generation, the timing of SR Ca²⁺ release was precisely controlled by laser-flash photolysis of caged Ca²⁺, DM nitrophen [1-(2-nitro-4,5-dimethoxyphenyl)-1,2-diaminoethane-N,N,N,',N'-tetraacetate, 4 Na; (DMNP)] (12, 44). Our results using these techniques suggest that SR Ca²⁺ release and I_NaCa can cause large changes in the duration and kinetics of action potentials, eliciting both DADs and EADs. These findings support a role for I_NaCa as an arrhythmogenic mechanism with the capacity to alter the action potential configuration dramatically in cardiac ventricular cells.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Isolation of ventricular myocytes. Adult male Hartley guinea pigs (350 g) were euthanized by an intraperitoneal injection of pentobarbitone sodium (100 mg/kg) in accordance with guidelines of the Johns Hopkins University Animal Care and Use Committee. Myocytes were isolated by a modified method of Mitra and Morad (43). Briefly, the heart was removed and perfused retrogradely at 37°C for 5 min with nominally Ca²⁺-free Tyrode solution containing (in mM) 137 NaCl, 5 KCl, 1 MgCl₂, 10 D-glucose, and 10 NaHEPES (HEPES neutralized to pH 7.4 with NaOH). This was followed by recirculation of the same solution containing (U/ml) 300 collagenase (type I) and 1 protease (type XIV) for 10 min and then perfusion with enzyme-free Tyrode solution containing 0.2 mM CaCl₂ for a further 5 min to stop enzymatic digestion. The ventricles were cut radially, and myocytes were dispersed at room temperature for experiments within 8 h of isolation.

Evoked action potentials. Whole cell current-clamped myocytes, held at zero current, were superfused with Tyrode solution containing 2 mM CaCl₂. The ruptured patch-clamp method was employed to ensure that fluorescent indicators and caged compounds equilibrated fully with the cytosol and to prevent their intracellular compartmentalization. Action potentials were stimulated through the patch pipettes (resistance 2.5–5 M) by 5-ms depolarizing pulses of 1.5 nA or less, applied at 0.2–0.33 Hz. The electrode-filling solution contained (in mM) 75 K glutamate, 20 KCl, 0.5 MgCl₂, 10 MgATP, 5 Na₂-phosphocreatine, 0.1 NaGTP, 5 pyruvic acid, and 30 K HEPES (HEPES neutralized to pH 7.2 with KOH). Electrophysiological signals were stimulated, recorded, and analyzed with the use of pCLAMP software (version 8.1) coupled to an Axopatch amplifier (Axon Instruments; Foster City, CA). APD at 90% repolarization (APD₉₀) was determined for comparison. By following the reasoning of LeGuennec and Noble (33), we conducted the experiments at room temperature (25°C) to prolong APD₉₀ so that perturbations of the action potential plateau, in particular due to SR Ca²⁺ release activated by laser-flash photolysis of caged Ca²⁺, were readily resolvable. A previous report (53) indicates that the mammalian cardiac action potential is not substantially affected by temperature, other than in duration. Furthermore, EADs and DADs of similar characteristics have been reported at both body and room temperatures, suggesting that their occurrences are also insensitive to cooling (2, 11, 25, 40, 66).

Intracellular Ca²⁺ measurements. The Ca²⁺-sensitive fluorescent indicators indo-1 (60 µM) and fluo-3 (50–100 µM) were included in the patch pipette solutions to measure, respectively, whole cell or localized [Ca²⁺]_i. Indo-1 fluorescence was excited at 360 ± 8 nm with the use of a Nikon Diaphot microscope-based microphotometry system (PTI; S. Brunswick, NJ) and fluorescence signals were recorded at 405 ± 15 and 485 ± 12.5 nm to obtain the indo-1 fluorescence ratio (defined as F₄₀₅/F₄₈₅, where F₄₀₅ and F₄₈₅ are fluorescence intensities measured at the two emitted light wavelengths). All measurements were corrected for background fluorescence recorded before establishing whole cell current clamp. Action potentials and [Ca²⁺]_i transients were digitized at 1 kHz and low-pass filtered at 500 Hz. Confocal images were acquired using a Zeiss LSM 510 microscope with a Zeiss Plan-Neofluor x40 oil immersion objective (numerical aperture = 1.3), and the confocal pinhole was set to render spatial resolutions of 0.4 µm in the x-y axes and 0.9 µm in the z-axis. Fluo-3 was excited at 488 nm, and fluorescence was recorded at wavelengths >505 nm. The images were acquired in linescan mode, with the scan line oriented parallel to the long axis of the cell, containing 512 pixels/line (0.075 µm/pixel) that were scanned repeatedly at 2- or 3-ms intervals for 6 s. Photobleaching and laser damage to the cells were minimized by attenuation of the laser to 1% of its maximum power (25 mW) with the use of an acousto-optical tunable filter. Image acquisition was synchronized with current-clamp protocols, such that line scans were initiated 100 ms before the stimulation of an action potential. The precise timing of the stimulus was marked by a blanking light-emitting diode output into the transmitted light channel of the microscope. Confocal images were processed with the use of IDL software (Research Systems; Boulder, CO). Fluorescence signals were background subtracted, and expressed relative to resting fluorescence (F₀) as F/F₀.

Laser-flash photolysis of caged Ca²⁺. In experiments using caged Ca²⁺ to trigger intracellular Ca²⁺ release, the composition of the internal solution was modified by omitting Na₂-phosphocreatine and replacing it with 2 mM Na₄-DMNP (Calbiochem; San Diego, CA) plus 0.25–0.5 mM CaCl₂. In addition, 2 mM NaCl was included to give 10 mM total intracellular Na⁺, and reduced glutathione (3 mM) was added to protect against cytosolic damage due to photolysis byproducts (44). Ca²⁺ was released from DMNP by flashing the cells with a single pulse of UV light at 337 nm generated from a N₂ laser (model GL-3300, PTI). The laser energy (maximum = 2 mJ) was attenuated to about 300 µJ and delivered using a 400-µm-diameter UV-enhanced fiber-optic light guide (WPI; Sarasota, FL) positioned 100 µm from the myocyte of interest. Myocytes were loaded with fluo-3 (50 or 100 µM) to record changes in [Ca²⁺]_i using confocal microscopy as described above. In these experiments, a second light-emitting diode flash was used to indicate the timing of the laser pulse.

Chemicals and miscellaneous. Unless indicated otherwise, the chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Pentapotassium salts of fluo-3 and indo-1 were purchased from Molecular Probes (Eugene, OR). KB-R7943 (KBR, Tocris; Ellisville, MO) and ryanodine (Calbiochem) were dissolved in DMSO as 10 mM stock solutions and diluted with Tyrode solution to give final concentrations of 5 and 20 µM, respectively. For Li⁺ Tyrode solution, NaCl was substituted by equimolar LiCl, and the pH of HEPES was adjusted to 7.4 using LiOH instead of NaOH. Throughout this study, external solutions were changed rapidly (<1 s) using a concentration-clamp system. Data are expressed as means ± SE and were compared statistically using the appropriate paired or unpaired t-test. A P value of <0.05 is taken to indicate a statistically significant difference, and n indicates the number of cells studied unless indicated.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Influences of I_NaCa and SR Ca²⁺ release on guinea pig action potentials. To investigate the effects of I_NaCa on action potentials, ventricular myocytes were superfused with Na⁺ Tyrode solution before and after being switched to Li⁺ Tyrode solution as indicated schematically in Fig. 1A, top. In this cell, mean APD₉₀ shortened by 33% from 233 ms in control to 156 ms in Li⁺ Tyrode solution. In all cells, the rapid, reversible APD abbreviation due to Li⁺ was practically completed within a single stimulation interval (3 s) and was maintained until the cell was returned to Na⁺ Tyrode solution. During reversal of the effects of Li⁺, APD₉₀ prominently overshot the control value, decaying back over 10 stimulus intervals. The periods of APD abbreviation and overshoot coincided with gradual increases and decreases, respectively, in the peak amplitude of [Ca²⁺]_i transients indicated by indo-1 fluorescence (Fig. 1A, bottom). Similar changes occurred to a lesser extent in resting [Ca²⁺]_i. The progressive changes in [Ca²⁺]_i contrast strongly with the essentially instantaneous alterations in APD₉₀ (indicated by arrows in Fig. 1A), reflecting the dependence of [Ca²⁺]_i transient amplitude on progressive changes in SR Ca²⁺ loading. Figure 1B shows individual action potentials and fluorescence transients at higher resolution. Under control conditions, the action potential triggered a rather small [Ca²⁺]_i transient (Fig. 1, i). In contrast, the abbreviated action potential in Li⁺ Tyrode solution (Fig. 1, ii) evoked a [Ca²⁺]_i transient of roughly fivefold larger amplitude than control. This transient was followed by a spontaneous SR Ca²⁺ release, which is a characteristic sign of SR Ca²⁺ overload. We attribute this SR Ca²⁺ overloading to Ca²⁺ influx (I_Ca and reverse Na⁺/Ca²⁺ exchange) during action potentials, not compensated by Ca²⁺ efflux via forward Na⁺/Ca²⁺ exchange (65). After solutions were switched back to Na⁺ Tyrode solution, the potentiated [Ca²⁺]_i transients decayed in parallel with the overshoot of APD₉₀ (Fig. 1, iii). At the start of the decay, spontaneous [Ca²⁺]_i transients evoked "slow" action potentials that were initiated by gradual depolarizations and had smaller, less steep upstrokes than those observed during stimulated action potentials. Collectively, in six cells, mean APD₉₀ of 427 ± 56 ms decreased significantly to 207 ± 15 ms (a change of 52%) after solutions were switched to Li⁺ Tyrode solution, accompanied by a small but significant depolarization of the resting potential of 2.9 ± 0.2 mV (27). When Na⁺ was returned to the Ca²⁺-overloaded cells, the mean overshoot in APD was 59 ± 12% based on the APD₉₀ of the first and tenth action potentials after solutions were switched. Thus the results shown in Fig. 1 suggest that SR Ca²⁺ overload caused both spontaneous depolarizations and potentiated [Ca²⁺]_i transients, which, acting via inward I_NaCa, considerably prolonged the APD due to enhanced Ca²⁺ efflux.

View larger version (25K): [in this window] [in a new window]   Fig. 1. A, top: schematic representation of the solution protocol. Middle, time course of changes in 90% action potential duration (APD90) after exposure of a myocyte to control Na+-Tyrode and Li+ Tyrode solutions as shown above. Data are taken from a representative cell and numerals indicate individual action potentials shown in more detail in B. Bottom, changes in indo-1 fluorescence ratio at the peak of intracellular Ca2+ concentration ([Ca2+]i) transients and at rest between stimulations from the same cell. The arrows and dotted lines indicate that essentially stepwise changes in APD90 accompanied the switch to, and from, Li+ Tyrode solution, whereas the accompanying [Ca2+]i transients changed gradually. B: action potentials expressed as membrane potential (Em) and evoked [Ca2+]i transients (as indo-1 ratio) corresponding to the control condition (i), at the end of 30-s superfusion with Li+ Tyrode solution (ii), and immediately after Na+ Tyrode solution was returned (same cell) (iii). , Spontaneous sarcoplasmic reticulum (SR) Ca2+ releases and/or depolarizations. Bottom, time bar refers to all three traces.

To elucidate further the involvement of I_NaCa in E_m changes, Na⁺/Ca²⁺ exchange was inhibited either by eliminating SR Ca²⁺ release using 20 µM ryanodine or by using the antagonist KBR. Figure 2A, top, shows that exposing a cell to ryanodine caused rapid APD abbreviation and the substitution of Li⁺ for Na⁺ in the presence of ryanodine typically caused further APD shortening. Individual action potentials (Fig. 2A, bottom) indicate that ryanodine abbreviated the action potential plateau and slightly reduced the action potential amplitude. Mean APD₉₀ significantly decreased from 622 ± 65 to 366 ± 30 ms (41%) in the presence of ryanodine (n = 5), with a further decrease to 218 ± 10 ms (24%) after solutions were switched to Li⁺ Tyrode solution (plus ryanodine). Parallel results were obtained when I_NaCa was directly inhibited using 5 µM KBR (Fig. 2B) except that the time course of APD changes was slower than for ryanodine. Inhibition of Na⁺/Ca²⁺ exchange resulted in progressive abbreviation of APD₉₀, followed by further abbreviation after substitution of Li⁺ for Na⁺, suggesting that 5 µM KBR only partly inhibited Na⁺/Ca²⁺ exchange. Mean APD₉₀ in seven cells was 492 ± 62 mV and shortened to 332 ± 30 ms (32%) in the presence of KBR, with further abbreviation to 252 ± 18 mV (16%) after Li⁺ was exchanged for Na⁺. The total reductions in APD₉₀ due to ryanodine plus Li⁺ (65%) and KBR plus Li⁺ (48%) were quite similar to the inhibition produced by Li⁺ alone (i.e., 52%). It therefore appears that all three agents acted on APD primarily by inhibiting I_NaCa. Because Li⁺-containing solution may potentiate outward Na⁺/Ca²⁺ exchange in the presence of 10 mM intracellular Na⁺, the small further reduction in APD caused by Li⁺ in the presence of ryanodine or KBR might reflect the maximum influence of outward Na⁺/Ca²⁺ exchange on APD.

View larger version (19K): [in this window] [in a new window]   Fig. 2. A, top: time course of changes in APD90 after exposing a representative myocyte to Na+-Tyrode solutions before (control), after the addition of 20 µM ryanodine, and after the subsequent replacement of Na+ by Li+. Bottom: individual action potentials recorded from the same myocyte at the times indicated by numerals at top, shown in more detail. B, top: time course of changes in APD90 after exposure of a representative myocyte to Na+ Tyrode solution before (control), after the addition of 5 µM KB-R7943, and after the subsequent replacement of Na+ by Li+. Bottom, individual action potentials (as Em) recorded from the same cell at the times indicated by numerals at top, shown in more detail.

Effects on APD of [Ca²⁺]_i oscillations in Li⁺ Tyrode solution. Inhibition of Na⁺/Ca²⁺ exchange by Li⁺ caused SR Ca²⁺ overload made evident spontaneous [Ca²⁺]_i oscillations. After their onset, Ca²⁺ oscillations increased in frequency with the number of applied stimuli (Fig. 3A). Figure 3B shows that the mean oscillation frequency increased from 0.5 to 1.3 Hz over 10 stimulation intervals in Li⁺ Tyrode solution (n = 7). During this increase, the Ca²⁺ oscillations had no obvious effects on resting membrane potential, despite a maximum increase in the resting fluorescence ratio by a factor of two relative to control. This suggests that in guinea pig ventricular cells, other Ca²⁺-dependent currents were absent or had minimal effects on resting E_m. Figure 3C shows that spontaneous [Ca²⁺]_i transients nonetheless affected APD in Li⁺ Tyrode solution. This influence was quantified by calculating the difference in APD₉₀ between the longest and shortest action potentials in the presence of Li⁺ for each cell (n = 6). By this measure, the mean difference in APD₉₀ was 81 ± 11 ms (32 ± 3% of the longer duration action potentials). Careful examination of action potentials, such as those shown in Fig. 3C, reveals that [Ca²⁺]_i oscillations that coincided with the stimulation pulse caused the most APD abbreviation (dotted lines). Because I_Ca is the principal inward current able to affect APD during superfusion with Li⁺-Tyrode solution, this phenomenon appears to reflect cytosolic Ca²⁺-dependent inactivation of I_Ca (1, 21, 60). The dependence of APD on [Ca²⁺]_i changes during the normal action potential is therefore likely to be mediated by the Ca²⁺ sensitivity of both I_NaCa and I_Ca.

View larger version (23K): [in this window] [in a new window]   Fig. 3. A, top: action potentials expressed as Em and [Ca2+]i transients (bottom) expressed as indo-1 ratio from a representative cell. Left, at the start of superfusion with Li+ Tyrode solution. Right, at the end of superfusion with Li+ Tyrode solution. , Spontaneous [Ca2+]i transients. The time bar refers to all panels. B: dependence of the mean [Ca2+]i oscillation frequency on the duration of exposure of cells to Li+ Tyrode solution, as indicated by stimulus number after solutions were switched from control solution (stimulation frequency 0.33 Hz). C: action potentials (top) and [Ca2+]i transients (bottom) recorded from a cell during superfusion with Li+ Tyrode solution (Na+ free) at higher resolution (units same as in A). Dotted lines show the timing of stimulus pulses. Left, a spontaneous [Ca2+] transient arose before stimulation of the cell and the evoked action potential was abbreviated compared with the subsequent action potential (right) that was stimulated when the cell was quiescent. Time bar refers to all bottom panels.

[Ca²⁺]_i oscillations and afterdepolarizations. Figure 4 shows that a range of E_m depolarizations could be attributable to spontaneous oscillatory SR Ca²⁺ release immediately after reintroduction of Na⁺-containing Tyrode solution. Figure 4A shows a [Ca²⁺]_i oscillation that followed a stimulated action potential, eliciting a DAD that failed to reach the threshold for triggering a slow action potential. Fourteen similar responses were observed in six cells. More commonly, however, DADs exceeded the threshold for evoking slow action potentials (23 events). A typical example of this type is shown in Fig. 4B, in which a repetitive series of regularly timed Ca²⁺ oscillations and slow action potentials occurred immediately after the stimulated action potential. Uniquely, the stimulated action potential in Fig. 4B had an extra "dome" at the early plateau phase, suggesting that I_NaCa, due to a secondary SR Ca²⁺ release, was capable of further depolarizing the cell. This observation is supported by the occurrence of a secondary depolarization during phase 3 repolarization of the stimulated action potential shown in Fig. 4C, top. In this particular action potential, [Ca²⁺]_i was already elevated at the point of stimulation, as a result of a preceding spontaneous Ca²⁺ release, but the membrane potential was not strongly depolarized due to ongoing superfusion with Li⁺ Tyrode solution. During repolarization of the same action potential, the switch from Li⁺- to Na⁺-Tyrode solution was coincident with a secondary Ca²⁺ transient (Fig. 4C, bottom) and a large secondary depolarization. The secondary depolarization strongly resembles a phase 3 EAD as modeled by Luo and Rudy (37). Three similar events were recorded from other cells. A closer inspection of Fig. 4C reveals that this typical phase 3 EAD arose at an E_m of –28 mV and peaked at –10 mV. The upstrokes and peaks of the EAD and its associated [Ca²⁺]_i transient coincided, with no detectable delays. When considered with the fact that no further membrane depolarization (above –10 mV) occurred after [Ca²⁺]_i had begun to decay, these observations suggest that the EAD was caused by the underlying Ca²⁺ transient (not vice versa). For comparison, the two slow action potentials that followed the EAD in Fig. 4C were triggered by DADs and both had relatively fast upstrokes peaking at around +30 mV. It is also noticeable that [Ca²⁺]_i was increasing at the start of the phase 3 EAD in Fig. 4C (at –28 mV), whereas at the equivalent point during repolarization of the subsequent spontaneous action potentials, [Ca²⁺]_i was rapidly declining. Because the two spontaneous action potentials contained no phase 3 EADs, it follows that spontaneous [Ca²⁺]_i transients during repolarization may be the crucial trigger of phase 3 EADs. On the basis of these characteristics, it is deduced that voltage-gated sarcolemmal ion channels contribute relatively little to phase 3 EADs, which would otherwise have depolarized to a more positive potential, likely driven by Ca²⁺ equilibrium potential (+130 mV).

View larger version (23K): [in this window] [in a new window]   Fig. 4. A, top: action potentials and Em changes. Bottom, [Ca2+]i transients. Double-headed arrows indicate the electrical stimulus and solid circles indicate unstimulated events. Responses were recorded after solutions were switched to Na+-containing Tyrode solution after Ca2+ overloading was established by Li+ Tyrode solution. The action potential evoked a potentiated [Ca2+]i transient and a subsequent spontaneous SR Ca2+ release caused a small delayed afterdepolarization (DAD). B: labels and units are the same as in A. Superfusion with Na+ Tyrode solution produced a dome-like secondary depolarization in the stimulated action potential. Further spontaneous [Ca2+]i transients triggered a train of slow action potentials. C: labels and units are the same as in A. Stimulation was preceded by a spontaneous Ca2+ release that did not evoke a large diastolic depolarization, showing that Na+ was initially absent. A secondary Ca2+ release coincided with the return of Na+ to the bath, during action potential repolarization. This [Ca2+]i transient evoked an early afterdepolarization (EAD). Two subsequent spontaneous Ca2+ releases evoked slow action potentials. The time bar refers to all panels. Data from three different cells are presented.

To investigate the mechanisms underlying slow action potentials, the peak E_m during subthreshold DADs was compared with the potential at which the voltage slope increased during suprathreshold DADs. The latter is termed the "take-off" potential (TOP) for slow action potentials and is most evident in the second slow action potential shown in Fig. 4C. The mean TOP was –46 ± 1mV(n = 23 events), which is significantly more positive than the peak of subthreshold DADs of –62 ± 2mV(n = 15 events). This observation suggests that the membrane conductance underlying the faster depolarization phase of triggered slow action potentials was available at E_m above –46 mV, i.e., close to the activation threshold for voltage-gated Ca²⁺ channels (41).

Spatiotemporal relationships between [Ca²⁺]_i and E_m were examined more closely in myocytes imaged by confocal microscopy. Figure 5A shows a control action potential stimulated in Na⁺ Tyrode solution that elicited a synchronous fluorescence increase across the cell (Fig. 5A, middle). This Ca²⁺ release (Fig. 5A, bottom) occurred after a delay of 10 ms and peaked 80 ms after the beginning of the action potential upstroke (74). Compared with stimulated action potentials, spontaneous [Ca²⁺]_i oscillations in Li⁺ Tyrode solution produced much less synchronous linescan fluorescence changes, consistent with the propagation of cytosolic Ca²⁺ waves (at a frequency of 1 Hz). The inset in Fig. 5B, top, illustrates that these Ca²⁺ oscillations caused small "ripples" in the resting potential of the cell, visible only at high amplification. The mean amplitude of the voltage oscillations was found to be 1.0 ± 0.2 mV in 15 exposures to Li⁺ (n = 6), confirming the limited extent to which [Ca²⁺]_i oscillations affected resting E_m in the absence of Na⁺/Ca²⁺ exchange. After Na⁺ was returned to the superfusion solution, [Ca²⁺]_i oscillations at the resting potential triggered slow action potentials that had two distinct phases (Fig. 5C). First, an initial slow depolarization at the foot of the slow action potential appeared to be closely associated with the spontaneous wave of Ca²⁺ release. Second, a more rapid upstroke was associated with the more synchronous [Ca²⁺]_i change (see dotted line). Such results support the view that slow action potentials were initiated by Ca²⁺ waves, leading to subsequent activation of L-type Ca²⁺ channels to generate the fast upstroke and trigger synchronized SR Ca²⁺ release.

View larger version (46K): [in this window] [in a new window]   Fig. 5. A: action potential (top) and confocal line scan image (middle) recorded before exposure of a myocyte to Li+ Tyrode solution. Bottom, normalized spatially averaged fluo-3 fluorescence (F/F0). B: action potential and fluorescence changes displayed as above, elicited in Li+ Tyrode solution. Note the asynchronous appearance of Ca2+ waves in the linescan image (middle). Inset, small Em changes evoked by Ca2+ waves in Li+ Tyrode solution at higher resolution. C: signals (as in A) acquired after the cell was returned to Na+ Tyrode solution, when Ca2+ waves evoked slow action potentials. Note that the transition between a Ca2+ wave and synchronized [Ca2+]i transient coincided with the slow action potential upstroke (dotted line). , Spontaneous events; the time bar refers to all panels except the inset.

Induction of afterdepolarizations by photorelease of Ca²⁺. To demonstrate the involvement of [Ca²⁺]_i changes in EADs and DADs more conclusively, SR Ca²⁺ release was activated by photolysis of caged Ca²⁺ (DMNP) in cells imaged confocally. The spatially averaged [Ca²⁺]_i transients triggered by action potentials in cells coloaded with DMNP and fluo-3 were similar to those observed in cells not loaded with the caged compound (Fig. 6A). Figure 6B shows, in a myocyte that had submaximal evoked SR Ca²⁺ release, that flash photolysis of DMNP during action potential repolarization (at approximately –40 mV) caused the immediate appearance of a secondary Ca²⁺ transient superimposed upon the transient activated by stimulation. The secondary SR Ca²⁺ release due to caged Ca²⁺ photolysis unequivocally elicited a phase 3 EAD. Similar responses were observed in three other cells, clearly showing that photorelease of Ca²⁺ during the action potential plateau can interrupt repolarization, causing phase 3 EADs similar to those due to spontaneous SR Ca²⁺ releases (Fig. 4C). When a laser flash was applied earlier in the time course of the action potential (Fig. 6C), the smaller secondary Ca²⁺ transient triggered by DMNP photolysis was associated with an immediate acceleration of repolarization. Repolarization to negative potentials triggered a prolonged Ca²⁺ release during which a pseudoplateau developed at E_m –15 mV, followed by terminal repolarization as [Ca²⁺]_i declined. Finally, laser flashes applied at the resting potential evoked DADs (n = 20). In four cells, these DADs exceeded the threshold for stimulating action potentials, typified by Fig. 6D. The striking fact that these E_m changes closely mirrored [Ca²⁺]_i changes further suggests that I_NaCa is a major determinant of action potential morphology.

View larger version (36K): [in this window] [in a new window]   Fig. 6. A: control action potential (top), expressed as Em, linescan image (middle) and normalized spatially averaged fluo-3, fluorescence transient (bottom) from a cell loaded with DM nitrophen (DMNP). B: signals (see A) recorded during an action potential in which a laser-flash (arrow) photolyzed DMNP during repolarization. Photolytic release of Ca2+ from DMNP evoked a secondary [Ca2+]i transient and an EAD-like secondary depolarization. C: signals (as in A) showing that laser-flash photolysis of DMNP early in action potential phase 1 elicited a smaller Ca2+ release, just after the peak of the stimulated [Ca2+]i transient. Caged Ca2+ photolysis immediately initiated accelerated repolarization, and a subsequent prolonged elevation of [Ca2+]i introduced a pseudoplateau phase into the action potential. D: photolysis of DMNP after repolarization was complete evoked [Ca2+]i transient of similar amplitude to the stimulated [Ca2+]i transient. This photolytically evoked [Ca2+]i transient triggered an action potential with morphology that closely paralleled the [Ca2+]i waveform.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The major finding of the present study is that I_NaCa evoked by SR Ca²⁺ release could cause large changes in membrane potential such that during Ca²⁺ overload, a diversity of potentially arrhythmogenic effects originated from the same single mechanism. We observed that I_NaCa could account for up to 50% of normal APD in isolated guinea pig ventricular myocytes. In Ca²⁺ overload, spontaneous Ca²⁺ oscillations could evoke both DADs and EADs that disappeared as the level of SR Ca²⁺ receded, I_NaCa was inhibited, or SR Ca²⁺ release was abolished pharmacologically. The photolysis of caged Ca²⁺ during or after a stimulated action potential could also elicit, respectively, an EAD or DAD. Taken together, these findings strongly suggest that I_NaCa has major effects both on APD in guinea pig ventricular myocytes and on afterdepolarizations. Furthermore, our findings suggest that APD is reciprocally influenced by the effects of [Ca²⁺]_i on the inactivation of I_Ca, but is less affected by other Ca²⁺-dependent conductances.

Effects of I_NaCa on APD. During normal endothelial cell coupling, the amounts of Ca²⁺ influx and SR Ca²⁺ release depend on APD, which is itself modulated by [Ca²⁺]_i acting via a complex interplay of Ca²⁺-sensitive ionic mechanisms. Forward Na⁺/Ca²⁺ exchange is the predominant Ca²⁺ efflux mechanism in cardiac myocytes and is the most important regulator of [Ca²⁺]_i. It is thus quite expected that Ca²⁺ extrusion via this electrogenic mechanism may play a role in modulating APD. In the present experiments, we observed that inhibition of inward I_NaCa by substitution of Li⁺ for extracellular Na⁺ shortened the APD and, conversely, increasing inward I_NaCa by potentiating SR Ca²⁺ release during Ca²⁺ overload substantially lengthened the APD (Fig. 1). Moreover, APD abbreviation or prolongation developed within 1–2 stimulation intervals (3 s) of switch to or from Li⁺ Tyrode solution, respectively (see Fig. 1A). The absence of an appreciable lag indicates that the rapid alteration in the exchanger activity, instead of the gradual change in SR Ca²⁺ loading, was responsible for the APD changes (16, 65).

The notion that I_NaCa contributes to normal APD in guinea pig ventricular myocytes is further supported by the pharmacological elimination of Na⁺/Ca²⁺ exchange using KBR and by the inhibition of SR Ca²⁺ release using ryanodine (Fig. 2). Both of these agents mimicked the effect of Li⁺, abbreviating APD₉₀ by 30–40%. KBR may have differential effects on forward and reverse Na⁺/Ca²⁺ exchange in some species, but the available evidence suggests that this is not the case in guinea pig cells (29, 36). Despite other side effects of KBR in addition to inhibiting the exchanger (56, 70), and the fact that ryanodine may eliminate Ca²⁺ release-induced I_Ca inactivation (60, 64), the APD abbreviation caused by all three independent methods unequivocally suggests that Ca²⁺ release-mediated I_NaCa is a major determinant of APD in guinea pig ventricular myocytes. These findings are consistent with previous reports showing the effects on APD of eliminating Na⁺/Ca²⁺ exchange by Li⁺ substitution or by eliminating SR Ca²⁺ release with the use of EGTA, BAPTA-AM, ryanodine, or caffeine in cardiac cells of several species (2, 13, 27, 30, 33, 34, 47, 59).

Influences of other Ca²⁺-dependent mechanisms on APD. In contrast to our experimental results, a study in rat cardiac myocytes found a slight increase of 10–20% in APD when SR Ca²⁺ release was abolished (20). A simulation study also suggested that reducing Na⁺/Ca²⁺ exchange might actually cause APD prolongation in guinea pig cardiac cells (21). These findings indicate that in addition to Ca²⁺ fluxes via Na⁺/Ca²⁺ exchange, counteracting mechanisms such as I_Ca inactivation may significantly affect APD (1, 21, 60). Figure 3 shows that APD variations occurred in phase with spontaneous [Ca²⁺]_i oscillations in the presence of Li⁺. APD was shortest during action potentials evoked at the peak of the oscillatory Ca²⁺ transient, suggesting that an underlying variation in Ca²⁺ release-dependent inactivation of I_Ca was responsible. Consistent with this idea, flash photolysis of DMNP at an early phase of the action potential accelerated repolarization until E_m fell <0 mV (Fig. 6C). Moreover, APD may also be influenced by Ca²⁺-activated Cl^– or nonselective cation currents in some species (15, 31, 73). In the present study, Ca²⁺ oscillations evoked resting membrane depolarizations of about 1 mV in Li⁺ Tyrode solution (Fig. 5B) suggesting that the combined depolarizing effects of such Ca²⁺-dependent currents was negligible in guinea pig ventricular myocytes. It has to be emphasized, however, that the relative contribution of inward I_NaCa and I_Ca inactivation to APD during a Ca²⁺ release could be species and inotropic state dependent. For example, in species like rats, where APD is short, I_Ca inactivation is robust (60), and Na⁺/Ca²⁺ exchange activity is relatively low (62), I_Ca inactivation during SR Ca²⁺ release may have a greater influence on the AP configuration than in species with longer action potentials (20). Moreover, during steady stimulation when sarcolemmal Ca²⁺ fluxes are in balance, an increased I_Ca would lead to greater SR Ca²⁺ release and I_NaCa.

Afterdepolarizations evoked by SR Ca²⁺ release and I_NaCa. The chief focus of the present studies was to evaluate the possibility that inward I_NaCa can cause early and delayed afterdepolarizations. The rationale of superfusing myocytes with Li⁺ Tyrode solution was to increase SR Ca²⁺ loading sufficient to elicit spontaneous SR Ca²⁺ releases and to determine the influence of these [Ca²⁺]_i transients on APD. In Na⁺ Tyrode solution, SR Ca²⁺ releases occurred simultaneously with DADs, slow action potentials, and EADs (Fig. 4). These results are consistent with several previous studies showing the coexistence of afterdepolarizations when cardiac cells were exposed to certain pharmacological agents (11, 38, 52). We also observed marked kinetic differences between EADs and DADs. The typical EAD shown in Fig. 4C was in phase with a [Ca²⁺]_i transient, but the change in E_m during the EAD was considerably slower than in an action potential triggered by a DAD, and the peak potential was less positive. Such findings are consistent with a mechanism in which EADs are attributable mainly to spontaneous SR Ca²⁺ release and I_NaCa. At the resting membrane potential, slow depolarizations due to Ca²⁺ oscillations either progressed to the upstroke of a slow action potential when an apparent TOP was reached or subsided if the threshold was unattained. Confocal linescan images (Fig. 5) suggested that these initial slow depolarizations were related to Ca²⁺ waves traveling across the cell (8, 11, 71) because DADs were not observed when I_NaCa was inhibited by Li⁺ substitution or when Ca²⁺ waves were abolished by ryanodine. The step from DADs to slow action potentials likely involved additional activation of I_Ca because the TOP of slow action potentials was similar to the voltage threshold for Ca²⁺ channel activation, and the peak potential was highly positive, consistent with the driving force for Ca²⁺. This notion is supported by the abrupt synchronization of SR Ca²⁺ release with the slow action potential upstroke, which resembles Ca²⁺ release triggered by the L-type Ca²⁺ channel during an evoked AP. As far as we are aware, similar images of [Ca²⁺]_i changes during the transition from DADs to slow action potentials have not been reported previously.

Although I_NaCa driven by [Ca²⁺]_i is the well-accepted mechanism for DADs (2, 37, 51, 67, 73), our results also represent the first direct demonstration that SR Ca²⁺ release is capable of evoking EADs. This was evident clearly when the primary events in the activation of EADs were the precisely controlled photolysis of caged Ca²⁺ or unevoked SR Ca²⁺ release during the repolarization phase of the action potential. Nonetheless, earlier studies had dissociated the mechanisms of EADs and DADs, suggesting that EADs do not require Ca²⁺ elevation but rather are related to Ca²⁺ entry via the L-type Ca²⁺ channels (11, 37, 40). This hypothesis, which has led to the widely held view that window I_Ca is the major cause of EADs, is strongly supported by the work of January and colleagues (23, 26, 39), showing that the voltage dependence and pharmacological sensitivity of EADs is correlated with that of L-type Ca²⁺ current. However, a great diversity of agents evoke EADs in cardiac preparations (39, 66, 73, 75) and because EADs are categorized phenomenologically based on their timing of occurrence, regardless of the methods of initiation, it is possible that the various EADs may not all be explained by a single unifying mechanism such as I_Ca reactivation. For example, it is almost certain that window I_Ca cannot explain the initiation of EADs below –45 mV, the threshold voltage for Ca²⁺ channel activation (10, 48). Moreover, because EADs arise after widely variable delays and vary in occurrence from one action potential to the next, it seems plausible that an underlying random process, such as spontaneous SR Ca²⁺ release, is involved in their mechanism (10, 46, 48, 52, 66, 68, 72, 76). Consistent with this idea, the association of EADs with APD prolongation permits sufficient time for the recovery of SR Ca²⁺ release from refractoriness induced by the initial [Ca²⁺]_i transient triggered by the upstroke (63).

Despite our strong evidence that SR Ca²⁺ release can trigger EADs during Ca²⁺ overload, we cannot absolutely discount the involvement of L-type Ca²⁺ channels in the generation of EADs, especially under different physiological conditions and pharmacological manipulations. In fact, the kinetics of Ca²⁺ channels are strongly affected by SR Ca²⁺ release (1, 60), so it is possible that complex interactions among SR Ca²⁺ release, I_NaCa, and I_Ca are involved in shaping the characteristics of various forms of EADs. Hence, future experiments are required to further evaluate the involvement of I_NaCa in different EAD models.

Physiological and clinical implications. Afterdepolarizations are of great significance to arrhythmogenesis in cardiac cells, particularly in reperfusion-induced arrhythmias (7, 49), torsade de pointes, and human long Q-T syndrome (39, 40, 73, 75). In the present paper, we have shown that SR Ca²⁺ release acting via I_NaCa can be the primary underlying mechanism of both types of afterdepolarizations during Ca²⁺ overload. This mechanism is likely to be involved in catecholamine-induced arrhythmia (11, 25, 52, 72) and may contribute to arrhythmia induced by mechanical loading, which is known to increase Ca²⁺ transients and Ca²⁺ influx via mechanosensitive nonselective cation channels (20, 22, 55). The novel findings reported here could open new avenues for further research because the expression and density of I_NaCa have been convincingly shown to be upregulated in heart failure (9, 24, 50, 51). Studies in isolated spontaneously beating sinoatrial node cells have also shown that the chronotropic effect of -adrenergic stimulation is associated with changes in SR Ca²⁺ release (32) and I_NaCa was found to link [Ca²⁺]_i transients to membrane depolarizations, implying that a DAD-like mechanism plays a modulatory role in the pacemaker potential. Interestingly, ablation of inward rectifier K⁺ channels by gene therapy was recently reported to transform ventricular myocytes into pseudopacemaker cells (42). Our observation of short trains of regular slow action potentials during Ca²⁺ waves (e.g., Fig. 5B) raises the possibility that repetitive pacemaker-like activity could arise by DAD-like spontaneous SR Ca²⁺ release and I_NaCa, if the depolarized resting potential of pseudopacemaker cells permits Ca²⁺ entry and SR Ca²⁺ overload. Further experiments are necessary to test this possibility. Finally, the EAD-like events we induced by SR Ca²⁺ release were superimposed on repolarization rather than during a stable prolonged action potential plateau (75). Therefore, further experiments are in hand to examine the morphology and mechanism of Ca²⁺-dependent EADs during more prolonged action potentials.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-071835 and HL-63813 and an American Heart Association (National Center) Grant (to J. S. K. Sham).

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. S. K. Sham, Div. of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: jsks{at}welchlink.welch.jhu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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