Published online before print September 7, 2006, doi: 10.1161/01.RES.0000244087.36230.bf
© 2006 American Heart Association, Inc.
UltraRapid Communications |
Pacing-induced Heterogeneities in Intracellular Ca2+ Signaling, Cardiac Alternans, and Ventricular Arrhythmias in Intact Rat Heart
From the Departments of Molecular Pharmacology (G.L.A., J.A.W.), Biological Chemistry (G.L.A., J.A.W.), and Medicine (Division of Cardiology) (J.E.K., S.K., M.K., I.S.-W., A.H.K., J.A.W.) and The Feinberg Cardiovascular Research Institute (A.H.K., J.A.W.), Northwestern University Feinberg School of Medicine, Chicago, Ill.
Correspondence to J. Andrew Wasserstrom, Division of Cardiology (S203) Ward 3-105, 303 E Chicago Ave, Chicago, IL 60611. E-mail ja-wasserstrom{at}northwestern.edu
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Abstract |
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Optical mapping studies have suggested that intracellular Ca2+ and T-wave alternans are linked through underlying alternations in Ca2+ cycling-inducing oscillations in action potential duration through Ca2+-sensitive conductances. However, these studies cannot measure single-cell behavior; therefore, the Ca2+ cycling heterogeneities within microscopic ventricular regions are unknown. The goal of this study was to measure cellular activity in intact myocardium during rapid pacing and arrhythmias. We used single-photon laser-scanning confocal microscopy to measure Ca2+ signaling in individual myocytes of intact rat myocardium during rapid pacing and during pacing-induced ventricular arrhythmias. At low rates, all myocytes demonstrate Ca2+ alternans that is synchronized but whose magnitude varies depending on recovery kinetics of Ca2+ cycling for each individual myocyte. As rate increases, some cells reverse alternans phase, giving a dyssynchronous activation pattern, even in adjoining myocytes. Increased pacing rate also induces subcellular alternans where Ca2+ alternates out of phase with different regions within the same cell. These forms of heterogeneous Ca2+ signaling also occurred during pacing-induced ventricular tachycardia. Our results demonstrate highly nonuniform Ca2+ signaling among and within individual myocytes in intact heart during rapid pacing and arrhythmias. Thus, certain pathophysiological conditions that alter Ca2+ cycling kinetics, such as heart failure, might promote ventricular arrhythmias by exaggerating these cellular heterogeneities in Ca2+ signaling.
Key Words: calcium transients • calcium alternans • subcellular alternans • arrhythmias
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Introduction |
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One of the most important clues to the mechanisms responsible for repolarization alternans was derived from the fact that action potential duration (APD) alternans occurs at the cellular level in intact heart.1–3 It is now widely accepted that T-wave alternans (TWA) on the surface ECG reflects tissue repolarization alternans at the level of the whole heart. In contrast to a purely electrophysiological explanation involving ion channel kinetics,4,5 evidence suggests that APD and T-wave alternans are in fact associated with changes in intracellular Ca2+ dynamics.2,5–7 The link between alternations in intracellular Ca2+ dynamics and TWA has recently been summarized2 as possibly arising from underlying alternans in Ca2+ cycling. Intracellular Ca2+ release enters into an alternating pattern based on the balance between the dynamics of Ca2+ release, reuptake, and recovery rates that induce oscillations in APD as a result of Ca2+-sensitive conductances. Theoretically, a large contraction occurs as the result of a large release of Ca2+ from stores in the sarcoplasmic reticulum (SR), which would in turn cause a large inward Na/Ca exchange current (INCX) and a long APD. Because the large SR Ca2+ release would have the effect of temporary depletion of SR Ca2+ content, the next beat would activate a small Ca2+ release with a resulting small contraction and a small inward INCX contributing little to APD, which would then be short. Other Ca2+-sensitive currents may also be activated that could either prolong or abbreviate APD, depending on transmembrane potential and whether or not they are present in a given species and tissue type.6
The spatial organization of repolarization alternans has primarily been studied using optical mapping of voltage- and Ca2+-sensitive dyes.3,8–10 Despite its advantages in allowing simultaneous study of electrical activation across the entire left ventricle (LV), the low signal-to-noise ratio of these dyes requires that each detector element records from hundreds to thousands of cells. The result is that cardiac activity cannot be measured at the cellular level, and consequently the heterogeneities that might exist between cells will be missed. If there are in fact disparities in Ca2+ signaling in neighboring cardiac myocytes, this type of behavior will be of considerable importance because it is likely to contribute to electrical nonuniformities in small ventricular regions or even single cells, thus setting the stage for arrhythmias. However, the patterns of cellular Ca2+ release in LV have not been investigated; therefore, the development of Ca2+ cycling heterogeneities within microscopic regions of ventricle are not yet known. Their existence could provide further evidence that the substrate for arrhythmias might be present as an inherent property of the normal cell-to-cell variation in Ca2+ dynamics that is altered by increased rate. The goal of this study was to measure characteristics of Ca2+ signaling in individual myocytes in intact ventricle that could contribute to the development of pacing-induced alternans and resulting arrhythmogenesis.
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Materials and Methods |
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The methods used in this study involve modifications of Langendorff perfusion of intact rat heart, first loaded with the Ca2+-sensitive fluorescent dye fluo-4/acetoxymethyl ester (fluo-4AM), then paralyzed with cytochalasin D. Ca2+ transients were subsequently measured in individual myocytes on the left ventricular subepicardium using single-photon laser-scanning confocal microscopy. Because the details of our approach have not been described previously, we have included an extensive section in the online data supplement describing how these experiments were performed and analyzed.
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Results |
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Development of Synchronous Alternans
Figure 1A shows the region of the LV from which recordings were typically made, a 2D fluorescence intensity image of the left ventricular surface (Figure 1B) and the corresponding line-scan image (Figure 1C) from which high-resolution recordings of intracellular Ca2+ concentration were made. The square in Figure 1A indicates a typical recording site on the LV. Figure 1B shows an X-Y confocal frame-scan image of the cardiac surface with myocytes oriented in long bundles. The regions of bright fluorescence indicate the time and position of SR Ca2+ release transients during stimulation during the nearly 3 seconds required to construct this image.
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To measure Ca2+ transients simultaneously in a number of myocytes with high temporal and spatial resolution, the scan line was placed across 14 cardiac myocytes on the left ventricular epicardial surface (white arrow in Figure 1B). The X-t line-scan image to the right of the frame-scan (Figure 1C) shows how cytoplasmic Ca2+ transients are measured in individual myocytes. The location of each myocyte is indicated by the green horizontal lines (Figure 1B) during repetitive scanning along the line shown in Figure 1C. The average fluorescence for each scan from all cells is indicated in the intensity profile above and shows typical fluorescence intensity data, with the brightest intensity occurring at peak systole, followed by a fall in intensity during Ca2+ removal for the cytoplasm.
The normal behavior of cardiac myocytes at basal pacing rates (at BCL [basic cycle length]=500 ms) is shown in Figure 2A. This line-scan image shows 2 responses at the basal rate followed by a 10-second test train at BCL=260 ms, followed by the first response back at basal pacing. The last basal beat is followed by smaller transients in all myocytes and the ensuing test train demonstrates nominally uniform Ca2+ alternans in a large–small–large–small sequence until rapid pacing ends. Nearly all cells show some degree of alternans; however, there is a great degree of heterogeneity in the responses of individual myocytes. Figure 2B shows an expanded view of the last 4 rapid pacing beats and the first after return to basal rate. The variability in steady-state alternans magnitude from cell to cell is apparent in the profiles of 4 cells selected, ranging from nearly complete alternans (cells 2 and 12) to none at all (cell 5). All cells in alternans are in the same phase, demonstrating behavior termed "synchronous alternans." This form of alternans occurs over a range of BCLs such that the alternans ratio (AR=1–S/L, based on Ca2+ transient peak amplitude; where S indicates short and small transient and L, large and long transient) increases with decreasing BCL for each cell (Figure 2C). However, the BCL at which each cell develops alternans is variable, suggesting that the susceptibility to alternans onset differs between myocytes.
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The relationship between BCL and AR for each cell was fitted to a sigmoid and the threshold cycle length (effective cycle length at AR=0.20 [ECL20]) and the midpoint (ECL50) were calculated as demonstrated in Figure 3A. To determine which characteristics of Ca2+ transients might predict sensitivity of each cell to alternans development, the values for ECL20 and ECL50 were plotted as a function of a number of properties of the Ca2+ transients at the basal rate of 500 ms (Figure 3B through 3E). The only reliable predictors of vulnerability to alternans were related to transient duration (Figure 3B) and Ca2+ reuptake rate, as indicated by the transient decay time (Figure 3C). In contrast, there was no apparent relationship between vulnerability to alternans onset and rise time (Figure 3D), total Ca2+ released (Figure 3E), magnitude of release, or time-to-peak (data not shown).
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Development of Dyssynchronous and Subcellular Alternans
A different pattern of Ca2+ signaling among neighboring myocytes occurs at higher pacing rates. Immediately after acceleration of BCL from 500 to 230 ms (Figure 4A), a number of cells developed alternans that is out of phase with the majority of cells in the field. The left expanded segment below the complete image in Figure 4A (white bar, Figure 4B) shows that the initial responses exhibit disorganized behavior, particularly for cells 3 and 4 as indicated by their respective intensity profiles. The right image (Figure 4C) is a temporal expansion of the last 4 rapid pacing beats and the first after return to the basal rate (teal bar in Figure 4A). Several myocytes are out of phase with others in the immediate vicinity, including cells 2, 5, 8, 10, and 12, as indicated by their respective intensity profiles. This "dyssynchronous alternans" may remain stable for the duration of the test train or may last only for several beats and become synchronous with surrounding myocytes. However, note that myocytes both distant from one another and immediately adjoining may demonstrate dyssynchrony.
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Dyssynchrony among myocytes on the LV occurred with an overall incidence of 21% (119/575 myocytes in 31 sites from 18 hearts). These results demonstrate the very high degree of microscopic heterogeneity in Ca2+ cycling that occurs in a rate-dependent manner.
The recording in Figure 4B is a temporal and spatial expansion of cells 3 and 4 in the left lower image of Figure 4A (teal bar) and demonstrates a third distinct form of Ca2+ signaling that is evident in several cells in this image. Under these conditions, Ca2+ release also varies in different regions within both cells 3 and 4 during several cycles of rapid pacing. For example, during the forth cycle of the image shown in Figure 4D, the top parts of cells 3 and 4 show little Ca2+ release, whereas the bottom part shows prominent release. The opposite pattern of release then occurs in the fifth cycle. However, the spatial and temporal pattern of heterogeneous intracellular Ca2+ release changes throughout rapid pacing (Figure 4A). This intracellular alternans pattern illustrates a phenomenon known as "subcellular alternans."11–13 This figure demonstrates that the same conditions that promote dyssynchronous Ca2+ release between different myocytes simultaneously promotes heterogeneities in Ca2+ release within individual myocytes.
Subcellular Alternans in Intact Epicardium
To investigate subcellular alternans within individual myocytes of intact LV, we recorded line-scan images along the longitudinal axis of individual myocytes in the left ventricular epicardium (Figure 5). This approach allowed high spatial and temporal resolution Ca2+ imaging along myocyte length during stimulation. In addition, these hearts were stained with di-8-ANEPPS to outline the cell membranes, so that cell ends could be located during positioning of the line and during analysis. Figure 5A shows a 2D image of a single cell outlined by di-8-ANEPPS staining. The fluorescence intensity was high in this image because systole occurred during recording. The white box shows the cell outline, with the brightest portions appearing as lines along the cell ends (yellow). The scan line was placed from end to end (blue line, Figure 5B) as indicated by the dashed arrows showing the recording site. The top line-scan image in Figure 5C illustrates that this cell responded to a decrease from BCL=500 to 220 ms without development of Ca2+ alternans (mean whole cell intensity profile above the image). The middle (Figure 5C) illustrates that decreasing the BCL to 190 ms produced uniform large–small oscillations in successive Ca2+ transients, with the result being a whole cell alternans. However, faster pacing (BCL=180 ms, Figure 5D) induced subcellular alternans. In contrast to the lower rate, which showed only transient subcellular alternans (BCL=190 ms, Figure 5C), Ca2+ release never become uniform and subcellular alternans occurred throughout rapid pacing. The intensity profile demonstrates little alternans, owing to the fact that Ca2+ release involves nearly half of the cell and is out of phase with each successive beat in this example. Subcellular alternans was observed in 24/35 single myocytes (69%) in 4 hearts and was induced over the range of BCL from 220 to 130 ms in different cells.
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Note that subcellular alternans occurs in the same cellular regions over multiple cycles (Figure 5 and supplementary Video 1). Interestingly, both dyssynchronous whole cell and subcellular Ca2+ alternans can occur simultaneously in microscopic regions of LV (supplemental Video 2).
Alternans Subtypes Are Temperature Independent
It is well known that low temperature increases the likelihood of alternans development.3,14–16 Consequently, it is important to confirm that these patterns of inter- and intracellular Ca2+ alternans are also present at physiological temperature.
Figure 6 shows data from experiments in which temperature was maintained at 35°C following dye loading. Basal pacing rate was maintained at BCL=350 ms because of the higher spontaneous rate at higher temperature. Figure 6A shows that rapid pacing (BCL=200 ms) induced synchronous alternans among the different cells in the optical field. The 3 cells indicated at the right (cells 1, 4, and 6) demonstrate ARs varying from nearly 0 to approximately 0.90. Figure 6D summarizes data from both temperatures (data for 23°C are replotted from Figure 3B) and demonstrates the close relationship between vulnerability to alternans onset and transient duration at both temperatures. This relationship is shifted to shorter transient durations, reflecting the fact that durations are indeed shorter at higher temperature. However, the increase in the steepness of the relationship suggests an increased sensitivity of alternans development to increasing rate at physiological temperature.
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Figure 6B shows an example from the same heart in which cells in the optical field followed the stimulation at 140 ms for approximately 10 beats, after which a 2:1 block occurred that was characterized by dyssynchronous alternans. This is shown clearly in the intensity profiles for cells 16 and 17 (right). Approximately 26% of cells showed dyssynchronous alternans (23/90 myocytes in 6/7 sites in 3 hearts), an incidence rate that was close to that found at room temperature.
Figure 6C shows a longitudinal recording in a myocyte from a separate heart in which normal uniform activation of Ca2+ transients was evoked at 350 ms basal BCL (left line scan image). However, when BCL was reduced to 130 ms, subcellular alternans occurred in a manner similar to that found at room temperature. Overall, 9/14 (
64%) myocytes demonstrated subcellular alternans at 35°C, indicating a similar incidence rate to that observed during recording at the lower temperature.
Overall, there is a shift in their rate dependence of all 3 forms of alternans to shorter cycle lengths because Ca2+ cycling is highly sensitive to temperature, reflecting the shorter transient durations at 35°C. One result is an increase in rate sensitivity of synchronous alternans development with increasing rate at 35°C, so that more cells developed alternans with smaller changes in rate than at room temperature. Thus, hypothermia may have important influences on the exact stimulation rates at which the different alternans subtypes develop but is not in itself responsible for either incidence rates or the types of alternans that develop during rapid pacing. This observation poses a possible explanation for why alternans develops at slower pacing rates at lower temperatures and why most investigators choose to study alternans at lower temperatures.
Heterogeneity in Intracellular Ca2+ Signaling and Arrhythmias
Finally, we recorded Ca2+ transients in microscopic regions of LV in 12 rat hearts in which ventricular tachycardia (VT) could be induced by rapid pacing. The results of one experiment are shown in Figure 7A. The ECG recording at the top of the figure shows the last 8 beats of the induced VT caused by pacing at a BCL=120 ms, which was followed by a series of 7 beats of spontaneous VT. A fairly stable pattern of both dyssynchronous and subcellular alternans was present throughout the end of the test train and during the induced episode of VT. Subcellular alternans is apparent in the image of a single myocyte expanded below and is particularly noticeable in the intensity profiles of 2 selected regions of that single cell expanded at the bottom of Figure 7A.
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The characteristics of subcellular alternans during VT were investigated further in Figure 7B, which shows examples of subcellular alternans measured in stable spontaneous VT. The top image illustrates the variability in the spatial and temporal patterns of subcellular alternans in 2 complete cells and part of a third cell, whereas the bottom image illustrates the behavior within a single cell. Of 12 hearts in which VT was either induced or occurred spontaneously, 11 hearts demonstrated dyssynchrony within the site from which recordings were made. Longitudinal single-cell recordings were made in 9 of these hearts, and all demonstrated subcellular alternans in at least 1 cell in the visual field during VT. Typical examples of VT in multicellular sites of both rat and guinea pig heart (supplemental Videos 3 and 4, respectively) demonstrate simultaneous dyssynchrony and subcellular alternans during spontaneous VT. These movies demonstrate the highly complex Ca2+ dynamics that occur during arrhythmogenesis both among and within myocytes in microscopic regions of LV. At the present time, there are insufficient data to prove that the development of VT is mechanistically related to alternans under these experimental conditions. However, the occurrence of both dyssynchronous and subcellular alternans in nearly every heart that developed VT raises the possibility that these forms of heterogeneity may contribute to pacing-induced VT in this experimental model.
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Discussion |
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These results demonstrate that the degree of heterogeneity in Ca2+ signaling at the cellular level is greater than expected from previous studies in which cellular behavior could only be inferred and not recorded directly.3,8–10 Several previous reports have described the use of confocal microscopy in studying Ca2+ waves and transients in individual myocytes of the intact heart.17–22 We found that at low rates, alternans developed that was uniform in phase but not in magnitude. As pacing rate increases, dyssynchronous Ca2+ release between cells and subcellular alternans within individual myocytes contribute to heterogeneity of cellular Ca2+ signaling, the extent of which is clearly seen during pacing-induced VT. Because Ca2+ release affects membrane conductances and therefore the cardiac action potential, it is possible that the dispersion of action potential duration and of refractoriness is much greater at the microscopic level than previously considered. However, electrotonic influences will serve to smooth the disparities that might accompany the high degree of Ca2+ cycling heterogeneities that occur between neighboring myocytes. Thus, we might speculate that Ca2+ alternans may be most arrhythmogenic when increasing APD dispersion at the macroscopic level (in the form of discordant alternans), whereas subcellular alternans may serve as a protective mechanism at still higher rates by diminishing the impact of coordinated regional Ca2+ overload producing triggered arrhythmias.
Mechanism of Synchronous Alternans
There has been a great deal of study recently about how oscillations in SR Ca2+ release can produce Ca2+ alternans in isolated myocytes.23–25 One modeling study in particular7 provided strong theoretical support for a major role in alternans for the relationship between the diastolic SR Ca2+ load and its influence on Ca2+ release. A separate contribution to alternans development and magnitude might also arise from the degree of Ca2+-induced inactivation of the L-type Ca2+ current, which alternates with the level of cytoplasmic Ca2+ during Ca2+ alternans. However, these studies have largely ignored the role of Ca2+ transient duration in regulating the availability of Ca2+ for subsequent release with each cycle. Thus, a large and long (L) transient will be interrupted before SR Ca2+ content can recover at fast pacing rates, whereas small and short (S) transients will allow more complete recovery for the next cycle, which will then be large. One implication of this observation is that because Ca2+ transients in human heart failure26,27 and in animal models28–30 are prolonged, this effect could potentially increase the vulnerability to alternans onset.
Our results provide the first direct evidence that different cells in intact heart have different vulnerabilities to alternans development based on intrinsic properties of Ca2+ signaling within individual cells. These results are similar to recent observations in both dog31 and guinea pig32 and are consistent with the idea that the rate of Ca2+ cycling and, specifically, the duration of the Ca2+ transient might determine both the magnitude of and rate sensitivity to alternans. Moreover, this was the first demonstration of the variability in Ca2+ transients that exists at the microscopic level; previously, it has always been largely assumed that Ca2+ transients are uniform within each left ventricular region (eg, base versus apex) despite the fact that important differences exist between regions.1,3,9,10 Our observations demonstrate that this is not the case, and, furthermore, there is a high degree of heterogeneity in Ca2+ dynamics within microscopic regions of the LV at both low and physiological temperatures.
It is not yet clear how dyssynchrony develops, but the result is that neighboring groups of cells and, in many cases, immediately adjoining cells are capable of developing alternans that is out of phase. The result is a much greater cell-to-cell variability in Ca2+ signaling at the microscopic level than was previously recognized. It is not yet known how or even if these microscopic heterogeneities in Ca2+ signaling might contribute to regionally discordant electrical alternans. However, dyssynchrony could contribute to the substrate for reentrant arrhythmias, if its influence in the LV is sufficient in magnitude and distribution to contribute to a dispersion in refractoriness across the LV.
Subcellular Alternans, Cellular Ca2+ Heterogeneities, and Arrhythmias
It has only recently been shown that intracellular SR Ca2+ release can also go into oscillation in localized regions within individual myocytes.11,33 Part of the cell releases a large amount of Ca2+, whereas another cellular region released far less; the next activation produced the opposite pattern, causing a regional alternans within atrial cells but rarely in ventricular myocytes.12 A nearly identical phenomenon occurred during partial inhibition of ryanodine receptors (RyRs) in rat ventricular myocytes using low concentrations of tetracaine or acidosis.13
The mechanism for subcellular alternans is most probably similar to that underlying Ca2+ alternans in general. One cellular region with slow Ca2+ reuptake is unable to respond to 2 successive beats during rapid pacing and so requires an additional cycle to replenish stores for large releases, whereas another region, the transient of which can recover more quickly, is able to follow each stimulus. We have also recorded instances in which different cellular regions simply oscillate completely out of phase with one another. The result is regional L/S alternations that are out of phase at different sites within the cell on a beat-to-beat basis.
When we studied the incidence of rate-dependent subcellular alternans development, we found that this is not an unusual phenomenon in the intact heart. In fact, nearly 2/3 of myocytes in intact rat LV demonstrated subcellular alternans at rates that produced pacing-induced VT and again during episodes of spontaneous VT. These observations raise, for the first time, the possibility that nonuniform intracellular Ca2+ release occurs during rapid pacing and arrhythmias.
Finally, the presence of both dyssynchronous and subcellular alternans in nearly every heart that developed VT raises the possibility that alternans is involved in pacing-induced VT in this model. Conversely, if we had found that these forms of heterogeneity in Ca2+ signaling were absent during VT, it would be highly unlikely that they play any role in the arrhythmia. To our knowledge, these are the first direct measurements of Ca2+ signaling in single cells in intact heart during arrhythmias, and the results demonstrate an extraordinary heterogeneity in behavior at the level of individual myocytes during arrhythmias. Given that the electrical space constant far exceeds the behavior of individual myocytes, pacing-induced heterogeneities in ion channel activation and action potential in individual cells are unlikely to affect macroscopic electrical activity of the ventricular syncytium. The smoothing effect of electrical current spread across many cells would, if anything, filter the most drastic cell-to-cell differences in Ca2+ signaling and resulting discrepancies in ion channel and action potential activation, thus minimizing their influence in arrhythmia development. However, a reduced space constant such as occurs in disease states, where intercellular communication is reduced,34,35 would minimize protection against nonuniformities in refractoriness, possibly promoting the development of re-entrant arrhythmias.
In the context of the relationship between transmembrane potential (Vm) and Ca2+ signaling heterogeneities at the cellular level, it is important to note that there are several reports of subcellular changes in Vm that might be relevant to the intracellular heterogeneities in Ca2+ signaling observed here. Several studies have found that cardiac cells do not depolarize uniformly during field stimulation, suggesting that a cellular "action potential" is subject to local changes in ion channel activation kinetics.36–38 The fact that we found Ca2+ signaling heterogeneities within individual myocytes of intact heart raises the possibility that different cellular regions might demonstrate local action potential variability depending on activation and recovery kinetics of Ca2+ sensitive conductances (INCX, IClCa, among others). A role for this type of nonuniform electrical activation has not been identified in arrhythmogenesis as yet. Furthermore, it is not known if the heterogeneities in cellular Vm are also present in intact heart because these studies were performed in isolated myocytes. However, it is known that current flow from cell to cell depends on the uniformity of impedances during downstream conduction. Depolarizing ionic current as well as changes in resistance and capacitance between—and possibly within—myocytes can affect the spread and rise of depolarization.39 Thus it is possible that heterogeneities in cellular Ca2+ could also affect the rise time and shape of the action potential and the uniformity of propagation both between neighboring myocytes as well as within individual cells.
Lastly, it is worth mentioning that the observations of highly heterogeneous behavior in intact heart leading potentially to subcellular alternans have been described in theoretical studies published in recent years.40 This intracellular behavior is mathematically quite similar to that described for spatially discordant APD alternans in theoretical models41 across larger tissue regions. Alternatively, the behavior underlying subcellular alternans and possibly dyssynchronous alternans might best be described in terms of Turing-type instability within individual cells and between myocytes in small cardiac regions, respectively.42 It will be important in the future to analyze the cellular behavior observed here in terms of these theoretical considerations which could provide important insights into the basis for arrhythmia initiation and stabilization in the whole organ.
Limitations of This Study
One of the limitations of this study is that the arrhythmias were generated in hearts that were hypothermic. Low temperature affects ion channel behavior, intracellular Ca2+ cycling, and other factors that might contribute to arrhythmogenesis at nonphysiological temperatures. Ion channel activation is affected not only by a direct effect of cooling on kinetics but also reduced membrane fluidity, which alters channel environment in lipid membranes as well as membrane transport systems and therefore local transmembrane ionic balances. Conduction velocity is slowed because of reduced rapid Na+ current and gap junctional conductance. These factors will all contribute to arrhythmias at room temperature, making a definitive relationship between altered Ca2+ signaling and arrhythmias speculative at best.
One of the potential complicating factors in our interpretation of results is that cytochalasin D is required to prevent contraction. This agent is known to have direct effects on transients in addition to blocking contraction, raising the possibility that some of our observations about Ca2+ dynamics made in the presence of cytochalasin D might be influenced by its presence rather than occurring as a result of intrinsic properties of Ca2+ cycling in the heart. Cytochalasin D slows rise time and prolongs Ca2+ transient duration in rat ventricular myocytes43 but has little effect on dog heart (up to 80 µmol/L),44.45 Finally, the ability of cytochalasin D to slow Ca2+ transient kinetics was associated with a tendency to reduce the incidence and duration of reentrant ventricular arrhythmias in mouse heart.46 Overall, these data suggest that cytochalasin D is not affecting excitation/contraction coupling to a sufficient extent to explain the heterogeneities in Ca2+ signaling observed here.
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Acknowledgments |
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Sources of Funding
A.H.K. was supported by the Fannie Penikoff Trust and is a scholar of the Feinberg Cardiovascular Research Institute.
Disclosures
None.
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Footnotes |
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Original received May 5, 2006; resubmission received July 17, 2006; revised resubmission received August 15, 2006; accepted August 24, 2006.
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