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(Circulation Research. 2006;99:740.)
© 2006 American Heart Association, Inc.

Cellular Biology

Cardiac Alternans Do Not Rely on Diastolic Sarcoplasmic Reticulum Calcium Content Fluctuations

Eckard Picht, Jaime DeSantiago, Lothar A. Blatter, Donald M. Bers

From the Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Ill.

Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Chicago, Stritch School of Medicine, 2160 S First Ave, Maywood, IL 60153. E-mail dbers{at}lumc.edu

	Abstract

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Cardiac alternans are thought to be a precursor to life-threatening arrhythmias. Previous studies suggested that alterations in sarcoplasmic reticulum (SR) Ca²⁺ content are either causative or not associated with myocyte Ca²⁺ alternans. However, those studies used indirect measures of SR Ca²⁺. Here we used direct continuous measurement of intra-SR free [Ca²⁺] ([Ca²⁺]_SR) (using Fluo5N) during frequency-dependent Ca²⁺ alternans in rabbit ventricular myocytes. We tested the hypothesis that alternating [Ca²⁺]_SR is required for Ca²⁺ alternans. Amplitudes of [Ca²⁺]_SR depletions alternated in phase with cytosolic Ca²⁺ transients and contractions. Some cells showed clear alternation in diastolic [Ca²⁺]_SR during alternans, with higher [Ca²⁺]_SR before the larger SR Ca²⁺ releases. However, the extent of SR Ca²⁺ release during the small beats was smaller than expected for the modest decrease in [Ca²⁺]_SR. In other cells, clear Ca²⁺ alternans was observed without alternations in diastolic [Ca²⁺]_SR. Additionally, alternating cells were observed, in which diastolic [Ca²⁺]_SR fluctuations occurred interspersed by depletions in which the amplitude was unrelated to the preceding diastolic [Ca²⁺]_SR. In all forms of alternans, the SR Ca²⁺ release rate was higher during large depletions than during small depletions. Although [Ca²⁺]_SR exerts major influence on SR Ca²⁺ release, alternations in [Ca²⁺]_SR are not required for Ca²⁺ alternans to occur. Rather, it seems likely that some other factor, such as ryanodine receptor availability after a prior beat (eg, recovery from inactivation), is of greater importance in initiating frequency-induced Ca²⁺ alternans. However, once such a weak SR Ca²⁺ release occurs, it can result in increased [Ca²⁺]_SR and further enhance SR Ca²⁺ release at the next beat. In this way, diastolic [Ca²⁺]_SR alternans can enhance frequency-induced Ca²⁺ alternans, even if they initiate by other means.

Key Words: calcium • sarcoplasmic reticulum • alternans • excitation/contraction coupling

	Introduction

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Intracellular Ca²⁺ cycling in cardiomyocytes during excitation/contraction coupling (ECC) and Ca²⁺-induced Ca²⁺ release (CICR) is tightly controlled. The amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) via sarcoplasmic Ca²⁺ release channels (ryanodine receptors [RyRs]) is determined by the triggering L-type Ca²⁺ current (I_Ca), SR Ca²⁺ load,^1,2 RyR phosphorylation status,³ and metabolic factors.^4–6 Dysregulation of these processes underlie arrhythmogenesis during ischemia and heart failure and in certain inherited conditions.^7,8

Intracellular Ca²⁺ alternans is the cellular correlate of cardiac alternans, a beat-to-beat variation in cardiac contractility and repolarization at a constant heart rate. Cardiac alternans occurs during ischemia, acidosis, and heart failure and is a prominent risk factor for the development of ventricular arrhythmias.⁹ Numerous factors involved in ECC and CICR have been proposed to be involved in Ca²⁺ alternans, including alternating I_Ca trigger,¹⁰ SR Ca²⁺-ATPase (SERCA) activity,¹¹ RyR status,^12,13 temporal delay in Ca²⁺ movement between SR Ca²⁺ uptake and release sites,^14,15 and alternating SR Ca²⁺ load.^16,17 For details, see reviews published previously.^18–20

So far, measurements of SR Ca²⁺ load during alternans have had to rely on indirect approaches such as the measurement of caffeine-evoked cytosolic Ca²⁺ transients or sodium/calcium exchange (NCX) current. Changes in SR Ca²⁺ load can be measured under voltage-clamp conditions by integrating the Ca²⁺ influx during a depolarizing test pulse and by subtracting the NCX mediated Ca²⁺ efflux during the tail current.¹⁷ Using these techniques and alternans induced by reducing Ca²⁺ current, Diaz et al¹⁶ found small beat-to-beat diastolic SR Ca²⁺ content fluctuations and proposed this as the key mechanism underlying cardiac alternans. In alternating cat atrial myocytes, however, SR Ca²⁺ measurements using rapid application of caffeine following large and small Ca²⁺ transients, respectively, showed no diastolic SR Ca²⁺ load fluctuations.¹³ Whether diastolic SR Ca²⁺ content fluctuations occur during cardiac alternans has therefore remained controversial.

We tested the hypothesis that diastolic [Ca²⁺]_SR fluctuations are required for alternans to occur. During frequency-induced alternans, we used the low-affinity Ca²⁺ indicator Fluo5N to continuously and directly monitor intra-SR free Ca²⁺ ([Ca²⁺]_SR) in isolated rabbit ventricular cardiomyocytes.²¹ This technique allows the cells to remain self-regulated, a feature that is especially important during alternans, where the action potential (AP) waveform alternates on a beat-to-beat basis modifying cellular Ca²⁺ fluxes. We found that whereas diastolic [Ca²⁺]_SR fluctuations can occur during alternans, Ca²⁺ alternans can also readily occur without significant diastolic [Ca²⁺]_SR alternations. These results suggest that factors other than SR Ca²⁺ load, such as CICR restitution, are centrally involved in the mechanism underlying frequency-induced cardiac alternans.

	Materials and Methods

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Ventricular myocytes were isolated from New Zealand White rabbits and loaded with the membrane-permeable form of Fluo5N under conditions that promote dye accumulation in the SR as previously described.²¹ All experiments were performed at room temperature. For [Ca²⁺]_SR measurements, fluorescence was recorded on wide-field epifluorescence microscopes at an excitation wavelength of 488 nm and emission of >500 nm. When [Ca²⁺]_SR depletions and cytosolic Ca²⁺ ([Ca²⁺]_i) transients were measured simultaneously, cells were loaded additionally with Fura2/acetoxymethyl ester (Fura2/AM) and fluorescence was excited sequentially at 340, 380, and 488 nm and emitted light was detected at >500 nm. Twitches were either evoked by extracellular field stimulation or by stimulating pulses under current-clamp conditions. Cells were continuously superfused with a bath solution consisting of (in mmol/L) CaCl₂ 2, NaCl 140, KCl 4, MgCl₂ 1, HEPES 5, and glucose 10 (pH 7.4). APs were recorded under current clamp using the perforated patch technique. I_Ca was measured under voltage clamp in the ruptured patch configuration.

Data were analyzed offline with custom-made software. Diastolic [Ca²⁺]_SR refers to [Ca²⁺]_SR immediately preceding the beginning of the depletion. The alternans ratio (AR) of depletion amplitudes was calculated as described previously²² with modification to allow application during reloading of the SR: AR=1–2 S_n/(L_n–1+L_n+1), where S_n is the small depletion amplitude and L_n–1 and L_n+1 are the large depletion amplitudes preceding and following S_n, respectively.

Data are expressed as mean±SD. One-way ANOVA was used to test for statistical significance. An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

	Results

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[Ca²⁺]_SR Alternates in Phase With Shortening and [Ca²⁺]_i
When alternans was induced either under current-clamp conditions or by field stimulation, myocytes showed typical beat-to-beat variations in fractional shortening, AP waveform, and cytosolic Ca²⁺ ([Ca²⁺]_i) transient (Figure 1A and 1B). As expected, simultaneously measured intra-SR free [Ca²⁺] ([Ca²⁺]_SR) showed large depletions during large contractions and Ca²⁺ transients and small depletions during small contractions and Ca²⁺ transients. Therefore, the magnitude of [Ca²⁺]_SR depletions is closely related to the magnitude of the cytosolic Ca²⁺ transient and the shortening amplitude. Indeed, direct online measurements of [Ca²⁺]_SR are important to investigate the detailed relationship between diastolic [Ca²⁺]_SR and SR Ca²⁺ release amplitude during alternans. As intra-SR free Ca²⁺ is in rapid equilibrium with low-affinity SR Ca²⁺ buffers, the measured changes in [Ca²⁺]_SR also reflect changes in total SR Ca²⁺ content.

View larger version (29K): [in this window] [in a new window]   Figure 1. Electromechanical, [Ca2+]i, and [Ca2+]SR alternans in rabbit ventricular myocytes. A, Ca2+ alternans induced under current-clamp conditions. AP waveforms and fractional shortening show typical beat-to-beat alternans. Large contractions are associated with large [Ca2+]SR depletions and small contractions with small depletions. B, Ca2+ alternans induced by field stimulation. [Ca2+]i and [Ca2+]SR were measured simultaneously with Fluo5N and Fura2, respectively. Shortening alternated in phase with [Ca2+]i transients and [Ca2+]SR depletions. Signal-averaged traces are shown on the right.

We observed different patterns of diastolic [Ca²⁺]_SR during alternans in field-stimulated cardiomyocytes: alternans with regular diastolic [Ca²⁺]_SR fluctuations, alternans without diastolic [Ca²⁺]_SR fluctuations, and intermediate forms, in which diastolic [Ca²⁺]_SR fluctuations occurred interspersed by a number of twitches in which diastolic [Ca²⁺]_SR was unrelated to the following depletion amplitude.

Alternans With Diastolic [Ca²⁺]_SR Fluctuations
Approximately 50% of the cardiomyocytes studied showed regular diastolic [Ca²⁺]_SR fluctuations during alternans (13/28 cells). Figure 2 shows an example of Ca²⁺ alternans with regular diastolic [Ca²⁺]_SR fluctuations induced by field stimulation at 2.2 Hz. Diastolic [Ca²⁺]_SR before the next stimulus clearly fluctuated from beat to beat, ie, high diastolic [Ca²⁺]_SR was followed by large [Ca²⁺]_SR depletions and lower diastolic [Ca²⁺]_SR preceded smaller depletions. When stimulation frequency was gradually reduced to 1.5 Hz, [Ca²⁺]_SR depletions became uniform and diastolic [Ca²⁺]_SR fluctuations stopped (Figure 2A, with sections on an expanded time scale in Figure 2B).

View larger version (52K): [in this window] [in a new window]   Figure 2. Ca2+ alternans with distinct diastolic [Ca2+]SR fluctuations. A, Stable depletion alternans was present at 2.2 Hz, which ceased when the frequency was gradually reduced to 1.5 Hz. Bottom, Stimulation frequency. B, Regions a and b in A are shown on an expanded time scale. C, Relationship between diastolic [Ca2+]SR and the following depletion amplitude (individual data points and SD for stable conditions). Open symbols represent transitional depletions between stable alternans and regular depletions. D, Normalized SR Ca2+ release flux and duration of release (measured as d[Ca2+]SR/dt and time to nadir, respectively) for regular depletions and for the small and large alternating depletions. *P<0.01 vs no alternans, P<0.01 vs small depletions.

Figure 2C shows the relationship between diastolic [Ca²⁺]_SR and the immediately following depletion amplitude for the entire trace in Figure 2A. During regular depletions (without alternans) [Ca²⁺]_SR depletion amplitudes and diastolic [Ca²⁺]_SR were consistent, such that the data points clustered very tightly in a single area of the graph. This illustrates the small beat-to-beat variation and steady-state behavior with respect to the diastolic [Ca²⁺]_SR versus Ca²⁺ release relationship. During steady alternans, however, data points clustered with very little variation in 2 completely separate areas, where the large [Ca²⁺]_SR depletion was preceded by a higher diastolic [Ca²⁺]_SR and the small depletion, by a lower diastolic [Ca²⁺]_SR. Figure 2B and 2C also indicates the great sensitivity of these [Ca²⁺]_SR measurements, where a difference of 3% in diastolic [Ca²⁺]_SR signal is quite easily resolved during alternans (Figure I in the online data supplement).

The upper curve in Figure 2C, connecting large depletions during alternans along their transition to regular depletions has the monotonic positive slope expected from prior work studying the relationship between SR Ca²⁺ content and SR Ca²⁺ release.^1,2,21,23 That is, increased [Ca²⁺]_SR is associated with increased SR Ca²⁺ release. The additional lower limb (connecting regular depletions and small depletions during alternans) indicates that even though diastolic [Ca²⁺]_SR was higher preceding the small alternans beat, SR Ca²⁺ release was lower. This most likely reflects a change in either the trigger for SR Ca²⁺ release (I_Ca) or responsiveness of the RyRs to the trigger (eg, attributable to RyR refractoriness). Thus, whereas fluctuations in diastolic [Ca²⁺]_SR are occurring here in association with Ca²⁺ alternans, factors different from [Ca²⁺]_SR limit SR Ca²⁺ release during the small beats during alternans.

We assessed the maximal SR Ca²⁺ release flux based on the maximal rate of [Ca²⁺]_SR decline (–d[Ca²⁺]_SR/dt), analogous to the analysis of cytoplasmic Ca²⁺ transients. The duration of release was measured as time to nadir (Figure 2D). Normalized to regular depletions at 1.5 Hz, during alternans, the release flux was reduced to 54±10% during small depletions (P<0.01, n=26 depletions) and increased to 159±3% during large depletions (P<0.01, n=25). The duration of release was reduced to 82±7% (P<0.01, n=26) during small depletions and slightly, although significantly, reduced during large depletions. Furthermore, comparing small versus large depletions during alternans SR Ca²⁺ release flux and duration of release was less during small depletions (P<0.01).

We also examined whether diastolic [Ca²⁺]_SR was limited by the time between beats during alternans. When stimulation was stopped after a small or a large depletion, respectively, differences in diastolic [Ca²⁺]_SR remained. Figure 3A shows that during a pause after the small depletion, [Ca²⁺]_SR attained the same higher level as during diastole after small depletions in ongoing alternans. When the stimulation was paused after a large depletion (Figure 3B), resting [Ca²⁺]_SR stayed at the lower level typical of diastolic [Ca²⁺]_SR after the large depletions during alternans and therefore lower than after small depletions. This suggests that the diastolic [Ca²⁺]_SR during alternans is substantially controlled by the cellular Ca²⁺ available rather than being limited mainly by the diastolic interval.

View larger version (24K): [in this window] [in a new window]   Figure 3. [Ca2+]SR after stop of stimulation. Stable alternans with diastolic [Ca2+]SR fluctuations was induced by field stimulation at 1.2 Hz. Stimulation was then interrupted for 7 seconds either after the small depletion (A) or after the large depletion (B). Horizontal dashed lines indicate diastolic [Ca2+]SR after small depletions (upper line) or after large depletions (lower line). [Ca2+]SR remained elevated after the small depletion and remained decreased after the large depletion. Stimulation after the rest period resulted in a larger depletion than during alternans and was of similar amplitude for both cases.

The [Ca²⁺]_SR depletion after a 7-second rest in Figure 3A and 3B was potentiated, compared with depletions during alternans and was also similar in magnitude for both cases. This emphasizes that the time between beats can strongly impact fractional SR Ca²⁺ release at constant [Ca²⁺]_SR. This is consistent with prior work showing that enhanced fractional SR Ca²⁺ release during postrest potentiation over this time scale is attributable to a slow phase of RyR recovery rather than altered [Ca²⁺]_SR or I_Ca.²⁴

Alternans During Reloading of the SR
To investigate the impact of a wide range of SR Ca²⁺ load on cardiac alternans at constant stimulation frequency, we first emptied the SR by exposure to 10 mmol/L caffeine. Caffeine was then washed out and stimulation was resumed at a frequency that previously induced alternans. During stimulation, 3 distinct phases of SR reloading could be observed.

Initially, each stimulation led to a gradual increase in diastolic [Ca²⁺]_SR and depletion amplitude (beat 1 to 20 in Figure 4A, region a expanded in Figure 4Ba). During this phase, neither the depletion amplitude nor diastolic [Ca²⁺]_SR alternated (AR<0.1), but both gradually increased. As stimulation proceeded alternans of [Ca²⁺]_SR depletion amplitude and diastolic [Ca²⁺]_SR developed (AR rose from <0.1 to 0.85 during beat 21 to 99 in Figure 4A, with region b expanded in Figure 4Bb). The relationship between diastolic [Ca²⁺]_SR and depletion amplitude shows the expected monotonic increase during the initial phase but bifurcates when alternans begins (Figure 4C and 4D). The upper branch, representing large depletions during alternans, indicates increasing depletion amplitudes with higher diastolic [Ca²⁺]_SR. The lower branch, however, shows that for the same diastolic [Ca²⁺]_SR, less Ca²⁺ is released during the small alternating depletions than during the large depletions, indicating reduced releasability of SR Ca²⁺ at the small beats. The relationship eventually becomes very steep for both the large and the small depletions. During this phase, diastolic [Ca²⁺]_SR preceding large depletions further increases, leading to much larger depletion amplitudes, whereas diastolic [Ca²⁺]_SR preceding small depletions slightly decreases and is accompanied by very small depletion amplitudes. Finally, stable alternans developed (gray traces at the end of Figure 4A and in Figure 4Bc) represented by the tightly clustered data points at the ends of the bifurcated curves in Figure 4C and 4D.

View larger version (39K): [in this window] [in a new window]   Figure 4. [Ca2+]SR alternans during SR reloading. A, Cell stimulated at 2.0 Hz after complete depletion of the SR with caffeine. During SR reloading and development of alternans, 3 phases can be distinguished. (1) Initially, depletion amplitudes and diastolic [Ca2+]SR increase with each stimulation; no alternans. (2) Subsequently, depletion alternans and diastolic [Ca2+]SR fluctuations develop. Alternans ratio increases with each stimulation. (3) Finally, ongoing stable depletion alternans and fluctuating diastolic [Ca2+]SR. A, bottom, Alternans ratio. B, Regions marked as a, b, and c in A on an extended time scale. C and D, Relationship between diastolic [Ca2+]SR and following depletion amplitude in arbitrary units (A.U.) (C) and calibrated [Ca2+]SR (D), where [Ca2+]SR was calculated by using fluorescence in caffeine as Fmin and estimating Fmax based on steady-state diastolic [Ca2+]SR.21 x indicates stimulations 1 to 20, classical relationship between [Ca2+]SR and depletion amplitude; , stimulations 21 to 99, development of alternans during reloading, bifurcated relationship between diastolic [Ca2+]SR and depletion amplitude; , stimulations 100 to 165, stable alternans with stable diastolic [Ca2+]SR fluctuations.

Alternans Without Diastolic [Ca²⁺]_SR Fluctuations
In 20% of the myocytes, stable alternans was present without detectable diastolic [Ca²⁺]_SR fluctuations. Figure 5A shows an example where alternans was induced by electrical-field stimulation at 2 Hz. Diastolic [Ca²⁺]_SR did not vary systematically on a beat-to-beat basis, although pronounced depletion alternans occurred (AR=0.38). Figure 5B shows the relationship between the diastolic [Ca²⁺]_SR and the following depletion amplitude. Diastolic [Ca²⁺]_SR preceding the small and large depletions, respectively, varied only over a very small range and overlapped completely. The respective ranges of small and large [Ca²⁺]_SR depletion amplitudes were completely separated and did not overlap. The lack of diastolic [Ca²⁺]_SR alternans in this situation is not attributable to saturation of intra-SR Fluo5N, because reduction of extracellular [Na⁺] caused further increase in [Ca²⁺]_SR (supplemental Figure II).

View larger version (29K): [in this window] [in a new window]   Figure 5. Ca2+ alternans without alternating diastolic [Ca2+]SR depletions. A, Original trace at stimulation frequency of 2.0 Hz. B, Relationship between diastolic [Ca2+]SR and depletion amplitude. Complete overlap of diastolic [Ca2+]SR for the small (circles) and the large (triangles) depletions. C, Maximal fluorescence decline (d[Ca2+]SR/dt) and time to nadir normalized to the large depletion amplitude. *P<0.01 vs small depletions.

The maximal rate of fluorescence decline (d[Ca²⁺]_SR/dt) during the small depletion was reduced to 79±13% (n=40 depletions) in comparison with the large depletion (P<0.01, n=40). The duration of release (time to nadir) was not different between the small and the large depletions.

Intermediate Forms of Alternans
We also observed intermediate forms of alternans in 30% of the cardiomyocytes studied (10/28 cells). Diastolic [Ca²⁺]_SR fluctuations occurred in these cells, but with variable correlation between diastolic [Ca²⁺]_SR and [Ca²⁺]_SR depletion amplitude (Figure 6A). At some pulses, there was no variation in [Ca²⁺]_SR during alternans (), at others diastolic [Ca²⁺]_SR and depletion covaried (+, as in Figures 2 through 4) and in others there was no consistent correlation (*). Figure 6B shows the relationship between diastolic [Ca²⁺]_SR and the following depletion amplitude for this myocyte. Although mean diastolic [Ca²⁺]_SR was different between the small and the large depletion (P<0.01, n=95 depletions in each group), substantial overlap of diastolic [Ca²⁺]_SR occurred for individual depletions (striated area in Figure 6B). Depletion amplitudes were significantly different between the large and the small depletions (P<0.01), with no overlap occurring. Consistent with the other forms of alternans, d[Ca²⁺]_SR/dt and time to nadir were significantly smaller for the small depletions in comparison with large depletions (P<0.01).

View larger version (37K): [in this window] [in a new window]   Figure 6. Intermediate form of alternans. A, Original trace at stimulation frequency of 2.3 Hz. + indicates alternans with regular diastolic [Ca2+]SR fluctuations; , alternans without diastolic [Ca2+]SR fluctuations; *, alternans with variable preceding diastolic [Ca2+]SR levels. B, Relationship between diastolic [Ca2+]SR and depletion amplitude. Partial overlap of diastolic [Ca2+]SR for the small and the large depletions (shaded area). C, Maximal SR Ca2+ release flux (d[Ca2+]SR/dt) and duration of release (time to nadir) normalized to the large depletion. *P<0.01 vs small depletions.

SR Ca²⁺ Release Responsiveness Recovers More Slowly than I_Ca and [Ca²⁺]_SR
The implication from the foregoing is that the small beat during alternans is not dictated by lower [Ca²⁺]_SR but, rather, reflects a reduced fractional release caused by incomplete recovery of the ECC process. Prior work has shown that I_Ca typically does not alternate during alternans,^12,13,16,25 and we have confirmed that for our experimental conditions (supplemental Figure III). The experiment shown in Figure 7 examines the recovery of SR Ca²⁺ release under controlled conditions. Five AP clamps (1 Hz) were followed by 200 ms square test pulses to 0 mV from a holding potential of –80 mV at different rest intervals after the end of the last AP clamp (120 ms to 4 seconds). Figure 7A shows I_Ca (top), [Ca²⁺]_SR (middle), and the V_Clamp (bottom, only last AP clamp and test pulses shown). Our results show that I_Ca recovers much faster from inactivation than [Ca²⁺]_SR depletion amplitude, such that the gain of ECC ([Ca²⁺]_SR/I_Ca) recovers gradually toward a maximum (Figure 7B). This time course is consistent with both the refractoriness of RyR being responsible for the small [Ca²⁺]_SR depletions during alternans and also the large postrest potentiation of SR Ca²⁺ release demonstrated in Figure 3.

View larger version (20K): [in this window] [in a new window]   Figure 7. Restitution of SR Ca2+ depletions. Simultaneous measurement of ICa and [Ca2+]SR in a voltage-clamped and Fluo5N-loaded myocyte. A, ICa was elicited via 200-ms square pulses to 0 mV from a holding potential of –80 mV after 5 AP clamps at a frequency of 1 Hz (with Na+ current blocked by 30 µmol/L tetrodotoxin). ICa recovers faster than SR Ca2+ release. B, [Ca2+]SR depletion amplitude normalized for ICa trigger increases over time, indicating ECC restitution, independent of SR Ca2+ content.

	Discussion

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Given the importance of SR Ca²⁺ load for the regulation of Ca²⁺ release during ECC and CICR,²⁶ it is reasonable to assume that fluctuations of diastolic [Ca²⁺]_SR contribute to the mechanism underlying cardiac alternans. However, experimental results to date have been contradictory. Using rapid caffeine applications following large and small cytosolic Ca²⁺ transients in alternating cat atrial myocytes, Hüser et al¹³ did not find alternating diastolic SR Ca²⁺ loads. However, Diaz et al¹⁶ found small beat-to-beat diastolic [Ca²⁺]_SR fluctuations in alternating rat ventricular myocytes and proposed this as the general mechanism underlying cardiac alternans.^16,27 These conflicting results may either be accounted for because alternans can arise with or without diastolic [Ca²⁺]_SR fluctuations or because the indirect measures of SR Ca²⁺ load might not be able to detect a functionally important difference. Even with the occurrence of diastolic [Ca²⁺]_SR fluctuations, their significance for the induction and maintenance of electromechanical alternans remains elusive. Here we directly and continuously measured intra-SR free Ca²⁺ ([Ca²⁺]_SR) during frequency-induced alternans and identified different patterns of [Ca²⁺]_SR regulation.

Frequency-Induced Alternans With Diastolic [Ca²⁺]_SR Fluctuations
Regular beat-to-beat fluctuations in diastolic [Ca²⁺]_SR can occur during frequency-induced cardiac alternans (Figure 2A and 2B). This finding is consistent with predictions from mathematical models^28,29 and from some recent experimental results.¹⁶ Under specific conditions, alternans was suggested to occur because of the very steep relationship between [Ca²⁺]_SR and SR Ca²⁺ release at high SR Ca²⁺ load.^16,17,27 That is, small differences in [Ca²⁺]_SR can lead to substantial differences in the amount of SR Ca²⁺ released for a constant I_Ca trigger.^1,2,23 However, the conditions used by Diaz et al¹⁶ to induce alternans (small depolarizing steps from –40 to –20 mV in the presence of 6 mmol/L external Ca²⁺) lead to the initial activation of only a small fraction of SR Ca²⁺ release units, setting the stage for propagated CICR, and this may not apply to other forms of alternans. Indeed, this differs markedly from frequency-induced alternans described here. Consider the kinetics of cytosolic Ca²⁺ transients and [Ca²⁺]_SR depletions. At the large Ca²⁺ transients, Diaz et al¹⁶ saw consistently a biphasic [Ca²⁺]_i rise with an initial steep phase (attributable to Ca²⁺ release from the initially activated RyRs) followed by a second gradual SR Ca²⁺ release resulting from wavelike CICR propagation.^12,16 During frequency-induced alternans, we saw only monophasic SR Ca²⁺ release kinetics (for both large and small beats), suggesting that SR Ca²⁺ release is uniform within the cell. Furthermore, using confocal Ca²⁺ imaging, we observed spatially homogenous increase of cytosolic Ca²⁺ during frequency-induced alternans without propagated Ca²⁺ release (supplemental Figure IV). Reminiscent of [Ca²⁺]_SR depletion kinetics, cytosolic Ca²⁺ transient kinetics showed consistently a monophasic upstroke. Thus, the propagated Ca²⁺ release in ventricular myocytes during alternans studied by Diaz et al¹⁶ may apply to alternans induced by spatially limited initial activation of few SR Ca²⁺ release units (with high SR Ca²⁺ content). This mechanism might also come into play under other conditions where most Ca²⁺ channels are unavailable (eg, at very high frequencies). However, at the frequencies where alternans was already induced in the present study, Ca²⁺ transients were spatially uniform. Although frequency-induced alternans was studied here at room temperature (such that alternans occurs at relatively low frequencies), the underlying mechanisms may be conceptually similar in other forms of alternans induction (eg, cooling, metabolic inhibition, reduced I_Ca amplitude and reduced RyR sensitivity) and at 37°C.

Can [Ca²⁺]_SR Alternans Explain Frequency-Induced Ca²⁺ Transient Alternans?
To evaluate whether diastolic [Ca²⁺]_SR alternans can explain frequency-induced alternans, we examined the dependence of SR Ca²⁺ release on diastolic [Ca²⁺]_SR preceding each beat. Under normal conditions, an increase in [Ca²⁺]_SR causes an increase in the amount of SR Ca²⁺ released during cardiac ECC,^{1,2,17,21,26,30} attributable in part to the effect of [Ca²⁺]_SR on the gating of the RyR.^31–33 The result is a monotonic dependence of SR Ca²⁺ release on [Ca²⁺]_SR and total SR Ca²⁺ content, and the slope of this relationship increases at higher SR Ca²⁺ content. We find a very similar relationship for the large depletions during alternans, suggesting that RyR availability and I_Ca trigger is comparable to that of nonalternating depletions (Figure 2C and 4D). The small beats during alternans, however, fall clearly below this relationship (Figures 2C, 4C, 4D, 5B, and 6B) and even show an inverse dependence (or negative slope). This shows that the decrease in [Ca²⁺]_SR is not sufficient by itself to explain the reduced SR Ca²⁺ release at the small beat. Therefore, during the small alternating beats, factors other than SR Ca²⁺ load become dominant in limiting SR Ca²⁺ release, such as recovery of I_Ca or RyR after a prior activation. Although alternating peak Ca²⁺ influx caused by incomplete I_Ca recovery from inactivation could theoretically contribute to cardiac alternans,^10,29 we did not observe alternating I_Ca peak current during alternans (supplemental Figure III). This finding is consistent with previous studies that showed unaltered peak Ca²⁺ influx during cardiac alternans.^12,13,16,25 Thus, we consider RyR refractoriness a more likely factor limiting SR Ca²⁺ release at the small alternating beat.

A role for limited RyR recovery in frequency-induced cardiac alternans is emphasized by the decreased SR Ca²⁺ release flux during small depletions and by the finding that the postrest stimulus produces a much larger depletion than during alternans, despite unaltered [Ca²⁺]_SR (Figure 3). Prior work has shown that a slow phase of RyR recovery (for a given I_Ca and SR Ca²⁺ content) is responsible for postrest potentiation.^24,34 The unaltered [Ca²⁺]_SR after this rest, and alternans with constant diastolic [Ca²⁺]_SR, also rule out SR refilling as the limiting factor in the small alternans beat.

Recovery of RyRs from a process such as inactivation^35–37 is likely to be modulated by many ionic and metabolic factors in the microenvironment of the RyR.^4,5,38,39 CICR recovery time course lags substantially behind SR refilling and I_Ca recovery (Figure 7 and described previously^26,36). During frequency-induced alternans, there may be incomplete RyR recovery that results in reduced SR Ca²⁺ release. Then, at the following stimulation, RyRs will have recovered to a greater extent, resulting in a larger SR Ca²⁺ release.

Although fluctuations in diastolic [Ca²⁺]_SR are not required for alternans, they may be expected to accompany alternans. This is because cytosolic Ca²⁺ feeds back on sarcolemmal and SR Ca²⁺ fluxes.^16,17 With high SR Ca²⁺ release, as in the large alternans beat, integrated I_Ca is reduced via Ca²⁺-dependent inactivation and Ca²⁺ extrusion via NCX is increased. This reduces Ca²⁺ influx and enhances Ca²⁺ efflux such that diastolic [Ca²⁺]_SR is reduced for the next beat. Low cytosolic Ca²⁺, on the other hand, promotes Ca²⁺ influx via I_Ca and limits cytosolic Ca²⁺ removal via NCX resulting in an increased [Ca²⁺]_SR. Under normal conditions, this system can autoregulate steady-state Ca²⁺ transients, such that perturbations caused by altered RyR activity are damped out.^17,40,41 However, if the relationship between cytosolic [Ca²⁺]_i versus Ca²⁺ efflux from the cell were very steep^16,17 or if RyR availability fluctuated on a beat-to-beat basis, alternans (a bistable state) could occur. Thus, although diastolic [Ca²⁺]_SR alternations are secondary in initiating alternans described here, they can contribute to alternating SR Ca²⁺ release and amplify the extent of Ca²⁺ transient alternans.

Alternans Without Diastolic [Ca²⁺]_SR Fluctuations and Intermediate Forms
Although the appearance of alternans without diastolic [Ca²⁺]_SR alternans (Figure 5) clearly shows that diastolic [Ca²⁺]_SR fluctuations are not mandatory for alternans to occur, the discussion above suggests that they would be expected to accompany Ca²⁺ alternans. Why might that not always occur? A central factor is the feedback gain of the cytosolic Ca²⁺ on sarcolemmal Ca²⁺ fluxes. If the gain were low (ie, NCX-mediated Ca²⁺ removal is small compared with SR reuptake), then changes in [Ca²⁺]_SR during alternans would be limited. This would, for example, be the case with increased intracellular [Na⁺] or positive membrane potential during the AP plateau, both of which diminish the ability of NCX (versus SERCA) to extrude Ca²⁺ from the cytosol. Likewise, if SERCA activity were relatively high versus NCX (attributable to high SERCA levels or low phospholamban levels), it would favor minimal changes in [Ca²⁺]_SR and lower the feedback gain. This may explain why Hüser et al¹³ found no change in diastolic SR Ca²⁺ content during alternans in cat atrial myocytes. That is, in atrial myocytes, SR Ca²⁺ uptake is very fast, partly because atria have little phospholamban (which inhibits SERCA) and SERCA expression is higher than in ventricle.⁴²

As NCX, SERCA, and RyR function are dynamically regulated by metabolic and ionic conditions, different patterns of [Ca²⁺]_SR handling during alternans can occur. The example in Figure 6 shows that diastolic [Ca²⁺]_SR regulation can change within a few beats, resulting in an intermediate pattern between regular diastolic [Ca²⁺]_SR fluctuations and a more stable diastolic [Ca²⁺]_SR, in accordance with this notion.

In conclusion, the present study provides the first direct and continuous measurement of [Ca²⁺]_SR in isolated cardiomyocytes during cardiac alternans. We found that whereas diastolic [Ca²⁺]_SR fluctuations can occur during frequency-induced alternans, Ca²⁺ alternans can also readily occur without significant diastolic [Ca²⁺]_SR alternations. These results suggest that although [Ca²⁺]_SR may alternate in cardiac alternans, factors other than SR Ca²⁺ load, such as RyR restitution, are of major importance in frequency-induced cardiac alternans.

	Acknowledgments

 
We thank Jayme O’Brien and Karl Hench for excellent technical assistance.

Sources of Funding

This work was supported by NIH grants HL30077, HL 80101 (to D.M.B.), HL062231 (to L.A.B.) and by grants from the American Heart Association (a Grant-In-Aid to L.A.B. and Postdoctoral Fellowship to E.P.).

Disclosures

None.

	Footnotes

 
Original received April 3, 2006; revision received August 16, 2006; accepted August 22, 2006.

	References

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