CLINICAL PERSPECTIVE

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Original Articles

Multiple Defects in Intracellular Calcium Cycling in Whole Failing Rat Heart

J. Andrew Wasserstrom, PhD; Rohan Sharma, BS; Sunil Kapur, MD; James E. Kelly, BS; Alan H. Kadish, MD; C. William Balke, MD and Gary L. Aistrup, PhD

From the Departments of Medicine (Cardiology) (J.A.W., R.S., S.K., J.E.K., A.H.K., G.L.A.) and Molecular Pharmacology and Biological Chemistry (J.A.W., G.L.A.) and the Feinberg Cardiovascular Research Institute (J.A.W., A.H.K.), Northwestern University Feinberg School of Medicine, Chicago, Ill; and Departments of Physiology and Medicine (C.W.B.), University of Kentucky College of Medicine, Lexington, Ky.

Correspondence to J. Andrew Wasserstrom, PhD, Northwestern University Feinberg School of Medicine, Chicago, IL 60611. E-mail ja-wasserstrom{at}northwestern.edu

Received July 31, 2008; accepted March 4, 2009.

	Abstract

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Background— A number of defects in excitation-contraction coupling have been identified in failing mammalian hearts. The goal of this study was to measure the defects in intracellular Ca²⁺ cycling in left ventricular epicardial myocytes of the whole heart in an animal model of congestive heart failure (CHF).

Methods and Results— Intracellular Ca²⁺ transients were measured using confocal microscopy in whole rat hearts from age-matched Wistar-Kyoto control rats and spontaneously hypertensive rats at 23 months of age. Basal Ca²⁺ transients in myocytes in spontaneously hypertensive rats were smaller in amplitude and longer in duration than Wistar-Kyoto control rats. There was also greater variability in transient characteristics associated with duration between myocytes of CHF than Wistar-Kyoto controls. Approximately 21% of CHF myocytes demonstrated spontaneous Ca²⁺ waves compared with very little of this activity in Wistar-Kyoto control rats. A separate population of spontaneously hypertensive rat myocytes showed Ca²⁺ waves that were triggered during pacing and were absent at rest (triggered waves). Rapid pacing protocols caused Ca²⁺ alternans to develop at slower heart rates in CHF.

Conclusions— Epicardial cells demonstrate both serious defects and greater cell-to-cell variability in Ca²⁺ cycling in CHF. The defects in Ca²⁺ cycling include both spontaneous and triggered waves of Ca²⁺ release, which promote triggered activity. The slowing of Ca²⁺ repriming in the sarcoplasmic reticulum is probably responsible for the increased vulnerability to Ca²⁺ alternans in CHF. Our results suggest that defective Ca²⁺ cycling could contribute both to reduced cardiac output in CHF and to the establishment of repolarization gradients, thus creating the substrate for reentrant arrhythmias.

Key Words: arrhythmia • calcium • heart failure • sarcoplasmic reticulum

	Introduction

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Several defects in cardiac excitation-contraction (E-C) coupling have been identified in myocytes from failing hearts that could contribute to the mechanical and electrophysiological dysfunction causing pump failure and fatal arrhythmias. Studies in animal models and human hearts have shown that congestive heart failure (CHF) is associated with a loss of t-tubules,^1–4 leading to anatomic and functional dissociation between electrical activation of the sarcolemma and release of Ca²⁺ from sarcoplasmic reticulum (SR). Therefore, increasing numbers of Ca²⁺ release units become isolated from their normal trigger—the L-type Ca²⁺ channel—and are activated by Ca²⁺ diffusion from neighboring Ca²⁺ release units rather than by Ca²⁺ channels.⁵ The ensuing delay in activation of these "orphaned" ryanodine receptors (RyRs)⁵ may cause a poorly coordinated release of Ca²⁺ along the cell length, leaving a highly fractionated cellular wavefront during Ca²⁺ release.^2,4 In combination with a slowing in Ca²⁺ reuptake due to reduced activity of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase,⁶ reduced rates of Ca²⁺ cycling are likely to contribute to diminished cellular contractile performance in CHF. Interestingly, increased action potential (AP) amplitude alternans, T-wave alternans, and ventricular arrhythmias have been reported to be sensitive to altered Ca²⁺ cycling and are unrelated to AP duration (APD) restitution.⁷

Editorial see p 157

Clinical Perspective on p 223

Because all of these observations have been made in myocytes isolated from failing hearts, no information is available about how prevalent defective Ca²⁺ cycling is at the cellular level or what forms these defects may take in whole heart. Consequently, there is no basis on which to extrapolate defects in function in isolated myocytes to changes in behavior in the whole heart.

The goal of this study was to investigate how cellular Ca²⁺ dynamics are altered in a CHF model. In the spontaneously hypertensive rat (SHR) model, the development of spontaneous hypertension occurs at 2 to 3 months of age and compensatory mechanisms, primarily hypertrophy, slow the development of overt CHF until after 20 months. We tested the hypothesis that defects in intracellular Ca²⁺ cycling in heart failure are responsible not only for reduced contractile function but also for the development of both triggered and reentrant arrhythmias. The experimental approach involves the use of confocal microscopy to measure intracellular Ca²⁺ dynamics in left ventricular epicardial myocytes of whole hearts.⁸ We used this approach to determine whether defects in Ca²⁺ cycling are exhibited in myocytes of the intact epicardial surface (as opposed to isolated myocytes from various cardiac regions), what forms they take, if they are manifested as both triggered and spontaneous Ca²⁺ release and how uniform this defective behavior is in whole heart.

	Methods

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Briefly, hearts from male Wistar-Kyoto (WKY) and SHR rats (23 months old) were excised and maintained on a Langendorff apparatus. The heart was placed in an experimental chamber on the stage of a confocal microscope and loaded with 3 successive additions of fluo-4AM (Invitrogen, 10 to 15 µmol/L, 20 minutes each) in recirculating Tyrodes solution. Dye was then washed for 10 minutes with normal perfusate. Solution was then recirculated with cytochalasin-D (50 µmol/L) to block contraction for the remainder of the experiment and maintained at 25°C. Laser scanning of left ventricular epicardial sites was accomplished with an LSM510 laser scanning confocal microscope (Zeiss Instruments) and allowed measurements of intracellular Ca²⁺ transients in individual myocytes during rapid pacing protocols. The scan line was usually placed across the short axis of 10 to 20myocytes in a recording site on the epicardial surface using either a 25x (NA 0.82) or 40x (NA 1.2) water immersion objective. The scan line (512 pixels) was repeated at a sampling rate of 1.92 ms/line. Data were collected during pacing protocols that consisted of constant basal pacing (basic cycle length [BCL], 700 ms) of which the last 4 beats were recorded, followed by a 10-s train of rapid pacing at CLs ranging between 600 and 100 ms followed by a 4-s pause before resumption of basal pacing. Data analysis was performed on each myocyte from each image during each test train. Characteristics of basal Ca transients were measured using MatLab and pCLAMP8 software. Alternans ratio was measured as 1–(small/large) at the end of the 10-s test train. Restitution of SR Ca release was measured as the fractional recovery of the test beat compared with the last basal beat before the test interval. All data are presented as mean±SEM, and data were compared using paired Student t-tests. Differences between sample means were considered significant if P<0.05. Details of these methods are included in the online-only Data Supplement and have been published elsewhere.⁸

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

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Characteristics of Ca²⁺ Transients During Basal Pacing
Typical recordings of multicellular sites are shown in Figure 1A, in which basal pacing evoked good electrical coupling and simultaneous activation of Ca²⁺ transients in all cells of a WKY heart. All myocytes show rapid Ca²⁺ release in response to stimulation, and then decay of Ca²⁺ to resting levels as Ca²⁺ is removed from the cytoplasm. The average fluorescence for all cells is shown in the intensity profile plot above the image and indicates rapid rise and reuptake typical of normal heart. The profiles of 2 selected myocytes are shown to the right of the image and indicate normal Ca²⁺ transients typical of isolated myocytes and of normal whole heart. Also, small regions without fluorescence transients denote intercellular spaces that are either out of the focal plane or represent regions of connective tissue separating columnar cell bundles.

View larger version (65K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Linescan images showing intracellular Ca2+ transients of a multicellular site in control (WKY) and CHF (SHR) hearts. A, Transverse recording in WKY heart in which 15 myocytes are included in this recording site. Basal stimulation at BCL=700 ms evokes simultaneous activation of Ca2+ transients in all myocytes in which rapid release of stored Ca2+ is followed by slow reuptake. The average fluorescence intensity from all cells is shown above the image, giving a profile typical of intracellular Ca2+ transients. Cell borders are indicated by white-dashed lines in the image, and each cell is denoted by short thick lines to the left of each image. Intensity profiles of selected cells are shown to the right. B, Recording from a site with 15 myocytes in an SHR heart with profiles of selected cells at right.

 
The recording from the SHR heart (Figure 1B) displays several important differences in cell function and anatomy compared with normal heart. First, peak release was less and both the rising and falling phases of the transients are slower, giving smaller and longer transients. Second, there is increased heterogeneity between myocytes, particularly in transient magnitude and duration. Third, there are more and wider nonfluorescent regions in SHR, indicating the presence of more fibrous tissue and collagen deposition. Finally, spontaneous Ca²⁺ waves are present in several cells (arrows) that either precede or follow the regular stimulus. These Ca²⁺ waves are not present in all cells but occur in certain myocytes either spontaneously or after a delay following normal activation, indicating an apparent randomness of activation rather than the normal triggering expected from typical spontaneous Ca²⁺ release arising from Ca²⁺ overload. Overall, we found spontaneous Ca²⁺ waves in an average of 21.5±3.4% of myocytes in 15 sites with an overall incidence of 33 of 164 myocytes (20.1%, 3 SHR hearts) compared with 1 of 200 myocytes in 14 control sites (0.5%, 3 WKY hearts).

The average fluorescence profile (above) shows a slowing in both the rise and decay rates, typical of Ca²⁺ cycling defects in CHF. When transients from individual myocytes are examined, there is clearly a wide variability in behavior, reflecting some relatively normal transients, transients showing grossly delayed release and time to peak as well as spontaneous waves that affect both timing and magnitude of Ca²⁺ release between stimuli in ways that also contribute to the whole site Ca²⁺ cycling profile.

Analysis of the Ca²⁺ transient characteristics responding to basal stimulation is shown in Figure 2. There were decreases in peak magnitude and total amount of Ca²⁺ released, consistent with previous reports of reduced Ca²⁺ release in this model of CHF.⁵ Furthermore, there was a slowing of the maximal release rate (+dF/dt) and rise time as well as all characteristics relating to transient duration at 50% and 80% of recovery (TD₅₀ and TD₈₀) and decay time, also consistent with previous findings in isolated myocytes.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 2. WKY versus SHR Ca2+ transient characteristics. Summary of characteristics of Ca2+ transient at basal pacing (BCL=700 ms).

 
We further investigated the characteristics of cellular Ca²⁺ transients by analyzing intercellular variability within a recording site in the form of heterogeneity indices (HI; measured as SD within the recording site). These characteristics show significant differences in distribution within microscopic regions of the left ventricle (Figure 3). All properties that determine the overall duration of the transient (rise and decay times as well as TD₅₀ and TD₈₀) vary significantly more in SHR than in WKY hearts. Thus, not only are there significant differences in the magnitude of these properties, but also the cell-to-cell variability is greater in SHR than WKY controls.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 3. WKY versus SHR HI. Summary of HI for each of the Ca2+ transient characteristics during basal pacing.

 
Basis for Cell-to-Cell Variability in Ca²⁺ Transient Characteristics
It has been reported that myocytes from CHF hearts, including the SHR model, show defective E-C coupling in both the release and reuptake phases of the Ca²⁺ transient. Figure 4A shows that Ca²⁺ transients recorded during basal pacing along the long cell axis in 2 myocytes from a WKY heart rise rapidly to a sharp peak, then decline smoothly during Ca²⁺ reuptake, behavior that is fairly uniform for both myocytes.

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Longitudinal recordings of intracellular Ca2+ transients in cell pairs of control and CHF hearts. Scan line was placed along the long axis of 2 cells in WKY (A) and SHR (B) hearts. Average fluorescence intensity profiles for each cell are placed above or below each image. BCL=700 ms.

 
In contrast, the transients from the adjoining myocytes in the SHR heart (Figure 4B) show characteristics of defects in both release and reuptake. Regions of cell 1 show delayed release, a prolonged time to peak and highly fragmented Ca²⁺ reuptake such that cellular regions showing slow release are also slow to recover while those with rapid release recover quickly. These characteristics are also seen in the fluorescence profile (above) showing slow rise, long time to peak, and slow recovery of cytoplasmic Ca²⁺ levels during decline of the transient.

The adjacent cell 2 (Figure 4B) shows relatively normal E-C coupling with uniform rapid release throughout the cell as well as a normal pattern of Ca²⁺ reuptake. Note, however, that despite its apparently normal appearance, Ca²⁺ release is in fact slower as is the time to peak compared with WKY (Figure 4A), giving a more rounded rising phase and peak as seen clearly in the fluorescence profile below.

These data address why there is increased heterogeneity in CHF rats compared with controls. Immediately adjoining myocytes show greater intercellular variability in Ca²⁺ cycling in CHF than normal, thus accounting for the significantly greater HI for these parameters (Figure 3). The result is an overall slowing of Ca²⁺ release and prolongation of reuptake which reflects the average activity of a highly heterogeneous cell population with relatively "normal" transients immediately adjoining myocytes showing gross defects in Ca²⁺ handling.

Defective Ca²⁺ Handling During Rapid Pacing in CHF
Because Ca²⁺ transients contribute to inward currents and APD, it is important to know how local Ca²⁺ cycling is affected by increasing rate, especially under conditions known to be highly arrhythmogenic such as in CHF. The effects of increasing rate on Ca²⁺ transients in CHF are shown in Figure 5. The linescan image in Figure 5A shows transients activated at a site containing 10 myocytes interspersed with extensive fibrous tissue. All respond with fairly uniform transients to each stimulus delivered at basal pacing. Note that some—but not all—myocytes show signs of defective E-C coupling, with fractionated release and heterogeneous reuptake of Ca²⁺. For instance, the selected cells shown at expanded scale in Figure 5B show fairly normal transients (cell 9) in a cell adjoining a myocyte with clearly defective E-C coupling (cell 10), even at BCL=700 ms. However, the impairments in Ca²⁺ dynamics apparent in cell 10 are enhanced at a higher heart rate (BCL=390 ms, Figure 5C). The exaggerated fractionation of Ca²⁺ release causes a much greater delay in Ca²⁺ release in the cell center, which is consistent with a disruption of the t-tubule system and delay of voltage activation of orphaned RyRs in the cell core.⁵ The result is Ca²⁺ release similar to that observed in atrial myocytes⁹ showing the U-shaped activation pattern characteristic of Ca²⁺-induced Ca²⁺ release arising from Ca²⁺ diffusion from the sarcolemma inward (Figure 5B and 5C). Furthermore, the late release in the cell core during beat 1 (cell 10, Figure 5C) causes subcellular alternans as evidenced by a greatly diminished release of Ca²⁺ in beat 2, then a continued alternation of Ca²⁺ release on subsequent beats. The result is an unstable alternans pattern arising from subcellular alternans, especially apparent in the duration of the transient in cell 10 (profile below). Note that even cell 9 shows some alternans in intensity and, to a lesser extent, in duration on alternating beats (profile above).

View larger version (92K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Increasing heart rate promotes fractionation of intracellular Ca2+ transients in SHR. A, The linescan image of 10 myocytes in an SHR heart at BCL=700 ms and during rapid pacing (BCL=390 ms). B, An expanded scale image of 2 myocytes during basal pacing. C, Transients during rapid pacing.

 
Ca²⁺ Alternans Development in CHF
One of the characteristics of many human CHF patients is an increased sensitivity to the development of T-wave alternans,¹⁰ especially during exercise.^10–12 Because of the close association between Ca²⁺ and T-wave alternans, we investigated the possibility that SHR animals in CHF might also be more susceptible to Ca²⁺ alternans development during rapid pacing.

Figure 6A shows that decreasing BCL to 400 ms caused little if any Ca²⁺ alternans in a WKY heart at steady-state at the end of rapid pacing. When BCL was reduced to 240 ms, nearly all myocytes in the WKY heart showed alternans (Figure 6B). In contrast, a number of myocytes in the SHR heart showed alternans even at BCL=400 ms (Figure 6C) whereas all myocytes showed alternans at BCL=340 ms (Figure 6D). When the cycle length dependence of alternans ratio (AR=1 – small/large¹³) was plotted for all cells in 1 WKY site (Figure 6E), there was some variability between myocytes but the threshold for alternans was less than about 350 ms with a mean for the midpoint (estimated cycle length at 50% alternans, ECL₅₀) of 256±2.7 ms. The SHR site showed Ca²⁺ alternans at slower rates, with the threshold (20% alternans) for all cells beginning at about BCL=550 ms in this site with an average midpoint for all myocytes of BCL=395±4.2 ms. The summary data show that the midpoint for rate-dependent development of alternans was significantly greater in CHF (341±6.0 in SHR compared with 271±5.8 ms, P<0.001, n=116 myocytes in 8 sites in 3 hearts for SHR and 102 myocytes in 7 sites in 3 hearts for WKY), indicating a shift of 70 ms in alternans vulnerability in CHF. The threshold for alternans development for all sites (ECL₂₀) also occurred at significantly slower rates in CHF (414±10.5 in SHRs compared with BCL=340±13.2 ms in WKYs, P<0.001).

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Development of rate-induced Ca2+ transient alternans in whole heart of normal and CHF animals. A and B, Intracellular Ca2+ transients at 2 pacing rates in a WKY heart. C and D, Transients in an SHR heart. Note that alternans develops at longer BCL in the SHR heart than in the WKY heart (340 ms compared with 240 ms). E, Sigmoidal curves of alternans ratio as a function of BCL for each myocyte.

 
In addition to a greater rate-sensitivity to alternans development in CHF, there was also greater variability within each site in CHF hearts. The HIs for both the midpoint (21±2.4 ms in WKY compared with 42±7.2 ms in SHR, P<0.02, n=6 sites in 3 hearts in WKY and 8 sites in 3 hearts for SHR) and threshold (27±5.7 ms in WKY compared with 77±11.4 ms, P<0.001) of alternans development showed that there was greater intercellular variability for Ca²⁺ alternans development within each CHF site compared with normal hearts.

One of the determinants for the development of Ca²⁺ alternans is the rate of recovery of SR release. Figure 7A shows that individual myocytes in WKY rats demonstrated a rapid recovery of Ca²⁺ release with increasing interval, reaching nearly full recovery in most cases by 500 ms. In contrast, the rate of restitution in the SHRs (Figure 7B) was so slow that it was not possible to test intervals short enough to allow myocytes to show the full time course of restitution without inducing ventricular tachycardia. Consequently, it was not possible to quantify the restitution in most myocytes from CHF hearts. However, it is clear that there is a dramatic slowing of restitution of SR Ca²⁺ release in CHF and that this slowing contributes to the development of Ca²⁺ alternans at slower heart rates.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 7. WKY versus SHR Ca2+ transient restitution. Summary of rates of Ca2+ transient restitution in individual myocytes in normal (WKY; A) and CHF (SHR; B). Restitution of SR Ca2+ release was measured as fractional recovery of the transient magnitude following a progressively shorter interval of the test beat (S2) compared with the last basal beat at BCL=700 ms (S1). Each line represents the sigmoid fit to the fractional recovery (S2/S1) of each individual myocyte for all hearts.

 
Finally, the cycle-length dependency for ventricular tachycardia induction was shifted to much longer cycle lengths in SHR hearts (BCL=176±14.6 in WKYs compared with 365±33.0 ms in SHRs, P<0.001, n=4 to 5). In addition, the cycle-length threshold for intercellular discordant (or dyssynchronous⁸) alternans was significantly longer in SHR (260±14.6 ms compared with 427±19.6 ms, P<0.001, n=9 to 11 sites) with a similar increase in vulnerability to the development of subcellular alternans in SHR animals (287±7.5 compared with 468±17.7 ms, P<0.001, n=9 to 12 sites). These increases in rate sensitivity to both forms of complex Ca²⁺ cycling occurred with little change in overall incidence, with 9 of 14 (64%) of WKY sites compared with 11 of 15 (73%) of SHR sites demonstrating intercellular discordant alternans. Similarly, 10 of 14 sites (71%) of sites in WKY compared with 12 of 16 (75%) of sites in SHR developed subcellular alternans.

Triggered Ca²⁺ Waves
In addition to spontaneous Ca²⁺ waves, an additional and distinct type of Ca²⁺ release event was observed in nearly all sites studied in CHF hearts. Some myocytes showed extremely weak Ca²⁺ release during normal activation but periodic Ca²⁺ waves in those cells showed that a large Ca²⁺ release was possible in these cells. More importantly, these waves did not occur spontaneously but were in fact triggered in response to normal electrical stimulation. The single SHR myocyte shown in Figure 8A demonstrates weak Ca²⁺ transients in response to normal stimulation, but large Ca²⁺ waves occurred throughout the stimulation period (red arrows). This activity was abolished when stimulation was terminated and careful examination of the small transients evoked during rapid pacing shows that the central region of this cell was capable of small local transients from which Ca²⁺ waves originate and spread ("triggered Ca²⁺ waves"). This unique form of triggered Ca²⁺ release occurred in at least one myocyte in each of 11 of 20 sites (55%) in 3 SHR hearts. The image in Figure 8B shows transverse recordings of 2 neighboring myocytes from a failing heart in which cell 1 demonstrates nearly normal E-C coupling whereas the adjoining cell 2 shows triggered waves interspersed among very small and poorly coordinated Ca²⁺ transients.

View larger version (92K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Triggered waves in CHF. A, Longitudinal recording of a single SHR myocyte (BCL=400 ms). The ECG is shown above and the average fluorescence intensity profile is shown below the image. Red arrows indicate waves. Note that rapid pacing was initiated before this portion of the image which shows Ca cycling and triggered waves during the steady state. B, Recordings from 2 adjoining SHR myocytes in a transverse recording. Panel B shows linescan image at BCL=700 ms and at BCL=400 ms.

 
Supplemental Figure I shows that many myocytes in some sites demonstrate triggered Ca²⁺ waves during stimulation at basal pacing that are absent when pacing ceases. At the beginning of the movie, there are small Ca²⁺ release transients in all cells in the visual field even though many of the cells are also showing Ca²⁺ waves. When stimulation stops, all activity ceases for several seconds, followed by a few spontaneous waves in some cells immediately before pacing is restarted. In contrast, supplemental Figure II shows a movie from an age-matched WKY rat during basal pacing followed by a test train at BCL=360 ms for 10 s then a pause. Ca²⁺ cycling is unremarkable during rapid pacing and shows no alternans or waves during stimulation. Several cells show spontaneous waves during the pause after rapid stimulation which is typical of normal rat myocardium after rapid pacing.

These data demonstrate a novel form of defective SR Ca²⁺ release that occurs in addition to that previously described in CHF. This form of Ca²⁺ release is poorly coordinated between myocytes that are interspersed among normal neighbors, likely contributing to reducing contractile efficiency in whole heart. Moreover, the wave-like activity is likely to promote the triggered activity reported to develop in CHF.

	Discussion

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There have been numerous reports about defects in cellular Ca²⁺ handling in a variety of animal models of CHF and in human disease. Recent work has focused on the fractionation in the Ca²⁺ release wavefront that reduces the efficacy of the Ca²⁺ trigger for E-C coupling and occurs as the result of the loss of t-tubules in CHF.^1,2,4,5 The result is a population of "orphaned" RyRs in CHF, including that described in the SHR model, whose activation is delayed during the Ca²⁺ transient.⁵ All of these studies were performed in isolated myocytes so it has not been possible to get a picture of how frequent these cellular changes are or how cell-to-cell interactions reflect dramatic changes in function.

Our results demonstrate that this defect in E-C coupling has a number of consequences in whole heart. First, there is a high incidence of myocytes showing abnormal Ca²⁺ handling, ranging from mild slowing of release and reuptake to severe abnormalities in Ca²⁺ cycling. Second, there is an enormous variability between myocytes so that cells showing severe defects reside in the midst of myocytes showing only mild changes in Ca²⁺ cycling. Third, spontaneous Ca²⁺ waves are common in CHF myocytes, raising the likelihood of triggered arrhythmias. Fourth, the rate dependence for Ca²⁺ alternans is shifted to much slower heart rates, probably because of a slowing in restitution of SR Ca²⁺ release and which could be responsible for increased T-wave alternans and reentrant arrhythmias found in the setting of CHF. Finally, Ca²⁺ cycling may become so defective that Ca²⁺ overload in the few remaining viable Ca²⁺ release units triggers a small Ca²⁺ release that then causes Ca²⁺-induced Ca²⁺ release by diffusion along the cell length in the form of a triggered wave. Furthermore, these defects are distributed unevenly across the ventricle and contribute to diminished pump function and enhanced arrhythmogenic substrate. We speculate that the result is less efficient Ca²⁺ cycling and cardiac contraction and an increased likelihood of both triggered and reentrant arrhythmias generated at slower heart rates in CHF than normals.

Altered E-C Coupling in CHF
Many changes in E-C coupling have been reported in animal models and in patients with CHF. Ca²⁺ reuptake is slowed, possibly a result of reduced sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase2a expression.⁶ The Na-Ca exchanger is upregulated which may compensate for reduced sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase2a function^14,15 and increased RyR2 phosphorylation has been reported, although the extent and sites of this phosphorylation are controversial.^16,17 SR Ca²⁺ leak increases, possibly as a consequence of RyR2 phosphorylation.^16,18 There may also be increased Na⁺ influx and intracellular Na⁺ accumulation,^19–21 increasing internal Ca²⁺ and SR Ca²⁺ load. Finally, many models of CHF demonstrate disorganization of the t-tubule system which could provide an anatomic basis for fragmentation of SR Ca²⁺ release.^2–5

Previous studies in isolated cardiac myocytes from CHF in SHR animals have shown elevated RyR2 phosphorylation,¹⁸ an overall slowing of Ca²⁺ reuptake and transient duration and loss of t-tubules which leads to orphaned RyRs and loss of viable Ca²⁺ release units.⁵ We found that defective Ca²⁺ cycling is highly variable even among neighboring myocytes, with a severity sufficient in about 20% of myocytes to evoke spontaneous Ca²⁺ waves, even at slow heart rates. Furthermore, the problems in Ca²⁺ cycling are exaggerated at faster rates, which is a particular problem in CHF patients whose resting rates are typically higher than in normals. The result is reduced pump efficiency, increased T-wave alternans and increased likelihood of arrhythmias.²² Interestingly, there is also a recent report that AP amplitude alternans is associated with TWA and ventricular arrhythmias but not APD restitution, underscoring the link between Ca²⁺ cycling defects and arrhythmias that are independent of APD restitution.⁷ Finally, we found that defective Ca²⁺ release in the form of both triggered and spontaneous waves is more prevalent in CHF. It is known that Ca²⁺ waves promote triggered arrhythmias and we speculate that this occurs commonly in this patient population especially with their higher resting heart rates.

Both subcellular and intercellular discordant alternans developed during rapid pacing, but at much slower rates in CHF versus normals. We have reported that both forms of intracellular and intercellular Ca²⁺ transient heterogeneities develop in myocytes of normal heart during rapid pacing.⁸ Intercellular discordant alternans occurs when neighboring myocytes develop alternans that is out-of-phase between cells. This highly heterogeneous Ca²⁺ cycling behavior establishes large Ca²⁺ gradients between myocytes and is likely to contribute to alterations in electrical activity at the cellular level. Subcellular alternans has been found in isolated atrial myocytes during rapid pacing,²³ and in isolated ventricular myocytes under certain experimental conditions.²⁴ Subcellular Ca²⁺ alternans establishes large intracellular Ca²⁺ gradients, which could increase the likelihood of Ca²⁺ waves and triggered activity.^23,25 In the setting of CHF, where ultrastructural changes lead to dramatic deficiencies in E-C coupling, any additional influence that promotes Ca²⁺ waves and Ca²⁺ heterogeneities both within and between cells is likely to promote further electrical instabilities in the form of triggered and reentrant activity.

Basis for Triggered Activity in CHF: Spontaneous and Triggered Ca²⁺ Waves
One of our most interesting observations was that Ca²⁺ waves were not only spontaneous but were triggered during normal or rapid pacing in CHF. Spontaneous waves occurred in about 20% of myocytes and were not related to underlying cardiac activation. In contrast, over half of all sites studied showed a distinct population of myocytes with small transients that were interspersed with occasional large SR Ca²⁺ release events (waves) that in some cases involved the entire cell and in others involved only parts of the cell. The distinguishing characteristic of triggered waves is that they occur during regular stimulation and not during the quiescent interval after cessation of pacing when spontaneous Ca waves arise.

On the basis of our results, we propose a scheme to explain the triggering requirement for these waves; t-tubule disruption and resulting defects in activation of SR Ca²⁺ release allow little Ca²⁺ to be released during normal stimulation. Ca²⁺ accumulation then continues, leading to overload which is then responsible for Ca²⁺ waves in response to very small local triggering events. In fact, we found subcellular releases in certain cell regions that do not propagate as waves and involve parts of the myocyte in a manner similar to subcellular alternans.^8,25 However this behavior is activated, it seems to represent a novel form of defective Ca²⁺ cycling in the setting of CHF that has not been described previously.

Rate Dependent Effects on Defective Ca²⁺ Cycling in CHF
We also found that defective Ca²⁺ handling in CHF is exaggerated as cardiac rate increases. Fractionation of both Ca²⁺ release and reuptake show a type of use-dependent suppression of Ca²⁺ cycling which progressively decreases contractile efficiency and could cause the negative force frequency relationship in myocardium from CHF patients.²⁶ Fractionation of Ca²⁺ release has been reported in animal and human models of CHF, including SHRs, but without linking increased heart rate to pump failure and arrhythmias. Our results demonstrate that higher rates exaggerate the intracellular heterogeneities in SR Ca²⁺ release and reduce the efficacy of E-C coupling. We speculate that the increased SR Ca²⁺ load at higher heart rates can promote both spontaneous and triggered waves and therefore induce triggered arrhythmias. Thus, increased heart rate in the setting of CHF would both reduce contractile efficiency and promote triggered arrhythmias.

There is also increasing evidence that Ca²⁺ alternans may be responsible for T-wave alternans in ventricle^22,27 because a large Ca²⁺ release from the SR would activate more inward Na-Ca exchanger current and prolong APD than a small release. Thus, endocardial cells with long transient durations develop both Ca²⁺ and APD alternans at slower rates than epicardial myocytes^28,29 and myocytes in intact left ventricular epicardium with long transients develop alternans at slower rates than adjoining myocytes with shorter transients^8,30. The basis for Ca²⁺ alternans is currently thought to arise from the steepness of the relationship between SR Ca²⁺ load and resulting release.³¹ Therefore, the shift of alternans sensitivity to slower heart rates in CHF could be a result of at least 2 processes that affect SR Ca²⁺ load, which unfortunately cannot be measured directly in these experiments using a conventional rapid caffeine pulse. We have reported that the prolonged Ca²⁺ transients in CHF slow the recovery of Ca²⁺ release so that early activation would find less Ca²⁺ available for SR release, causing less release but allowing more time for recovery for the next beat in a small-large-small-large pattern.⁸ We speculate that, in addition to a prolongation of transient duration, the observed slowing in Ca²⁺ transient restitution could exaggerate this effect by further slowing SR recovery. Our results show that CHF is associated with both a prolongation of Ca²⁺ transients and a slowing in restitution, causing the development of an alternans pattern at slower than usual heart rates. The resulting APD alternans could therefore establish gradients of repolarization at slower heart rates in CHF, thus promoting reentrant arrhythmias.²⁷

	Acknowledgments

 
Sources of Funding

This work was supported in part by the Everett-O'Connor Trust.

Disclosures

None.

	References

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CLINICAL PERSPECTIVE

Experimental studies have suggested that defects of intracellular calcium cycling may play a crucial role in both pump failure and arrhythmogenesis, particularly in the failing heart. Therefore, understanding the characteristics of calcium handling in failing myocytes and their effects on the intact myocardium is potentially of great importance. We used the spontaneously hypertensive rat model of genetic hypertension which develops congestive heart failure (CHF) with aging. Our model uses a whole heart preparation, as opposed to myocytes isolated from failing hearts, because only with intact tissue can we directly relate cellular function to arrhythmia development. There were extensive changes in calcium cycling and increased cellular variability in calcium handling in CHF. We also found that cellular calcium alternans develops at slower heart rates in CHF, providing a possible basis for increased T-wave alternans development in CHF. Furthermore, we demonstrated greater heterogeneity in alternans development within microscopic regions of CHF myocardium, which could contribute to both local and regional dispersion of action potential duration and refractoriness, thus establishing the substrate for the development of reentrant arrhythmias. Finally, calcium waves, which have the potential to be highly arrhythmogenic, were observed to arise either spontaneously or during pacing (triggered waves) in CHF even at slow heart rates. In summary, our results suggest that defective calcium cycling takes several forms in failing heart, all of which could contribute to reduced cardiac output and to an increased susceptibility to both triggered and reentrant arrhythmias in CHF patients.

	Footnotes

 
The online-only Data Supplement is available at http://circheartfailure.ahajournals.org/cgi/content/full/CIRCHEARTFAILURE.108.811539/DC1.

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