© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Relative importance of SR load and cytoplasmic calcium concentration in the genesis of aftercontractions in cardiac myocytes
Cardiac Medicine, Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK
* Corresponding author. Tel.: +44-171-352-8121; fax: +44-171-351-8145 r.egdell{at}hotmail.com
Received 22 March 2000; accepted 5 June 2000
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Abstract |
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Objective: To determine whether calcium overload of the sarcoplasmic reticulum underlies drive train-induced aftercontractions in cardiac myocytes. Methods: Sarcoplasmic reticulum calcium contents were measured immediately prior to drive train-induced aftercontractions in isolated guinea pig cardiac myocytes, using caffeine application under voltage clamp conditions. Cell shortening during caffeine exposure and cell shortening during the final stimulated beat of the drive train and the delay between caffeine exposure and the onset of inward current were also used as indirect measures of sarcoplasmic reticulum load. Results: At the threshold for aftercontractions, all four measures of sarcoplasmic reticulum load showed interruption of the positive relationship between stimulation frequency and sarcoplasmic reticulum content, the sarcoplasmic reticulum being no more loaded prior to an aftercontraction than following subthreshold drive trains. Intracellular calcium concentration, estimated with the calcium-sensitive dye indo-1, was higher in cells showing aftercontractions than those not. Conclusions: We conclude that calcium overload of the sarcoplasmic reticulum does not underlie spontaneous calcium release in this situation and the primary trigger for spontaneous release may instead be raised cytoplasmic calcium concentration.
KEYWORDS Arrhythmia (mechanisms); Calcium (cellular); e–c coupling; Myocytes; SR (function)
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Myofilament activation in cardiac myocytes is dependent on intracellular calcium concentration ([Ca2+]i). The maintenance of tight control of [Ca2+]i is therefore critical for co-ordinated contraction and relaxation. Under certain conditions, believed to be pathological rather than physiological, calcium may be released spontaneously from the sarcoplasmic reticulum (SR) during the diastolic interval between stimulated contractions, leading to an ‘aftercontraction’ [1,2]. This phenomenon may lead to a number of effects detrimental to normal cellular function [1,3], including potentially arrhythmogenic delayed afterdepolarisations (DADs) [4].
The interventions which have been used experimentally to induce aftercontractions and DADs are ones which might be expected to raise [Ca2+]i. It has therefore become widely accepted that spontaneous release of calcium from the SR occurs during ‘calcium overload’ [5,6]. However, the precise nature of the events which link high cellular calcium loading with the generation of aftercontractions is unclear. Although the intracellular calcium fluxes prior to a spontaneous release are likely to be complex, they may be simplified into two main hypotheses. The factor which underlies spontaneous calcium release may be raised cytoplasmic [Ca2+], leading to calcium-induced calcium release from the SR, or it may be calcium overload of the SR itself, operating on the luminal side of the release channel. Analysis of cytoplasmic [Ca2+] immediately prior to a spontaneous calcium wave is more suggestive of a role for raised cytoplasmic [Ca2+] [7], while the response of calcium wave velocity to temperature changes also implicates cytoplasmic [Ca2+] [8]. The alternative hypothesis, that overload of the SR itself leads to spontaneous release, has the support of some experimental data from non-cardiac tissue [9], extrapolation of this to cardiac myocytes is sometimes assumed [10,11] and, indeed, such a factor has been built into computer models of cardiac myocyte Ca2+ handling [12,13]. However there have been few published accounts of direct measurements of SR load in association with spontaneous calcium release in cardiac myocytes. Using rapid cooling contractures in multicellular preparations, Bers and Bridge demonstrated that acetylstrophanthidin, a cardiotonic steroid, both increases SR load in a dose-dependent way and induces aftercontractions at higher doses [14], but no attempt was made to record SR loads prior to aftercontractions and compare them with loads below the aftercontraction threshold. A more convincing demonstration that SR overload itself underlies spontaneous release comes from the work of Diaz et al. [15], who used caffeine applications in unstimulated rat myocytes to identify a threshold SR load which differentiated between cells which did and did not show cyclical spontaneous releases. It is questionable, however, whether the findings from this model of SR release can be extrapolated to species such as guinea pig, rabbit and human with very different calcium-handling characteristics and which demonstrate aftercontractions when stimulated, not when quiescent. Here we describe experiments designed to measure SR load immediately prior to drive train-induced aftercontractions in guinea pig cardiac myocytes.
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2 Methods |
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The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Isolated guinea pig left ventricular myocytes were prepared by a previously described method [16]. Cells were impaled with high resistance microelectrodes to minimise cell dialysis and superfused with normal Tyrode solution, containing (mM) NaCl 140, KCl 5, CaCl2 2, MgCl2 1, glucose 10, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (Hepes) 10, pH adjusted to 7.4 using NaOH. Cell shortening was tracked with a video-based edge detector. Experiments were conducted at room temperature.
Cells were stimulated with 20-beat drive trains at various stimulation frequencies. Four different stimulation frequencies were investigated, 0.2 and 0.5 Hz (both below the threshold frequency for aftercontractions), threshold frequency (the lowest stimulation frequency that reproducibly induced an aftercontraction on three consecutive runs), and maximal stimulation frequency (the most rapid drive train compatible with successful stimulation). Membrane potential and cell shortening were recorded during the 20th stimulus and for 8 s thereafter to monitor aftercontraction and DAD activity.
2.1 Caffeine application
At a precise time following the termination of the drive train (see below for details of timings), the cell was voltage clamped to its resting membrane potential; 200 ms after this, the superfusate was switched to normal Tyrode containing 10 mM caffeine. The high caffeine concentration was delivered close to the cell via a delivery pipette placed on the cover slip upstream of the cell, in order to minimise dead space. In guinea pig, maintained caffeine application causes the SR to release all of its available calcium, which is then extruded almost entirely by the sodium–calcium exchanger [17], thereby generating an inward current [18]. Since one net positive charge moves across the membrane for every calcium ion extruded, the charge movement during such current flow allows a calculation to be made of the amount of calcium released from the SR [19]. In guinea pig the proportion of calcium extruded by mechanisms other than the sodium–calcium exchanger is known to be very small [16], and we have therefore not included any adjustment for this in our calculations. Charge movement occurring during caffeine application was measured by integration of the caffeine-induced current, relative to the stable baseline reached at the end of the caffeine application. This current level was sometimes different from that prior to caffeine application, possibly because of other small caffeine-induced conductances.
2.2 Data acquisition and analysis
Membrane potential, current signals and cell shortening were recorded simultaneously onto computer and magnetic tape. Acquisition direct to the computer was used to record current signals at higher resolution (digitisation rate 1.3 kHz), whereas the magnetic tape was used to record entire drive trains and caffeine applications. These were subsequently digitised and examined to ensure that no aftercontraction activity occurred during the drive train or between the drive train and caffeine application.
Data are presented (except where detailed) as mean±S.E.M. Groups were compared using Student's t-test, with a significance level of P<0.05.
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3 Results |
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Under our experimental conditions, threshold frequencies were 1.0±0.3 Hz (mean±S.D.), and maximal frequencies were 1.5±0.4 Hz.
3.1 Timing of caffeine delivery
Fig. 1A shows the final beat of a 20-beat drive train at 0.2 Hz. No aftercontraction activity is detected in the ensuing 8 s (final 2.5 s omitted for clarity). In order to standardise the delivery time of caffeine, following drive trains of 0.2 and 0.5 Hz, the solution switch was made at 1800 ms after the final stimulus of the drive train, in an attempt to deliver caffeine at approximately 1900–2000 ms after the final stimulus. In Fig. 1B the arrows mark the point at which the cell (same cell as Fig. 1A, same stimulation frequency) was voltage clamped to its resting membrane potential and, 200 ms later, the point at which the superfusing solution was switched to one containing 10 mM caffeine. Contraction associated with the release of calcium from the SR is evident in the cell shortening trace, accompanied by a small caffeine-induced inward current. Fig. 1C shows the final beat of a 20-beat drive train at maximal stimulation frequency in a different cell. An aftercontraction is evident about 2 s after the final stimulus, associated with a DAD of about 3–4 mV amplitude. For threshold and maximal drive trains, we were interested in recording SR load as close to the moment of spontaneous SR release as possible. The solution switch was therefore triggered 300 ms before the earliest evidence of aftercontraction or DAD activity on any of the three characterising records, in order to deliver caffeine approximately 100–200 ms before this point. Fig. 1D shows the final beat of a 20-beat drive train at maximal stimulation frequency in the same cell as Fig. 1C. In order to measure the SR load immediately prior to the expected aftercontraction, the voltage clamp and caffeine switch are timed a little earlier than in Fig. 1B. A larger inward current is seen associated with the caffeine application. Note the small outward current immediately upon voltage clamping, which was frequently seen and may represent sodium–potassium pump current, which is increased by the intracellular accumulation of sodium induced by these rapid drive frequencies. This offset of the baseline will have no effect on the integral of the caffeine-induced current and therefore on the calculation of SR load. By comparing the cell shortening traces of Figs. 1C,D, it is clear that caffeine-induced SR unloading occurred only a very short time before the expected spontaneous aftercontraction. Overall, the delay between the onset of caffeine-induced inward current and the earliest evidence of aftercontraction or DAD was 147.0±87.3 ms (n=29). Any record in which aftercontraction activity was evident prior to the delivery of caffeine was discarded.
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3.2 SR loads
SR loads differed between cells. However, SR loads measured at different times in the same cell were relatively consistent (see below), implying no systematic error in the method of calculation of SR load. In order that valid comparisons could be made, SR loads were normalised to the value obtained at 0.2 Hz.
Because of difficulty maintaining stable impalements in the face of multiple caffeine applications, most cells were subjected only to two caffeine applications. Fig. 2 shows representative results during caffeine applications following a drive train at 0.2 Hz and one other drive train frequency. Increases in SR load are seen following drive trains at 0.5 Hz and maximal frequency, compared with 0.2 Hz, but not following a drive train at threshold frequency.
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The mean results for normalised SR load are shown in Fig. 2D. Increasing the drive train frequency from 0.2 to 0.5 Hz led to a significant increase in measured SR load, as would be expected in a species with a positive force–frequency relationship. However, SR load measured at the threshold for reproducible aftercontractions was not significantly higher than that at 0.2 Hz. A further increase in the stimulation frequency to maximal once again led to an increase in SR load.
3.3 Caffeine-induced cell shortening
Although non-quantitative, the degree of cell shortening in response to caffeine application is another indication of SR calcium content. This was measured in those cells which were successfully tracked by the edge detection unit throughout the caffeine application. Representative traces are shown in Fig. 3A–C. Caffeine-induced cell shortening is greater after drive trains of 0.5 Hz and maximal frequency, but not at threshold frequency. Pooled data are illustrated in Fig. 3D. Normalised caffeine-induced cell shortening was significantly higher after drive trains of 0.5 Hz and maximal stimulation frequency than after drive trains of 0.2 Hz (n=26). In contrast, caffeine-induced cell shortening was no higher after threshold drive trains than after stimulation at 0.2 Hz.
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3.4 Stimulated cell shortening
Since the amplitude of cell shortening is a function of SR load, this was measured following the final stimulus of the 20-beat drive train for cells stimulated at the four stimulation frequencies. Representative paired traces are shown in Fig. 4A–C. Again comparisons have been made in different cells with the same cell following a drive train of 0.2 Hz. Increased cell shortening is apparent when cells are stimulated at 0.5 Hz and maximal frequency, but not at threshold frequency.
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The mean results for normalised cell shortening are illustrated in Fig. 4D. Once again an ‘N-shaped’ relationship is obtained, with significant increases in cell shortening at 0.5 Hz and at maximal stimulation rate compared with 0.2 Hz. Cell shortening at the threshold frequency for aftercontractions is not significantly different from cell shortening at 0.2 Hz.
An identical ‘N-shaped curve’ was obtained when the delay from the delivery of caffeine to the onset of the inward current was analysed (data not shown). This has previously been identified as another indirect measure of SR load [17].
3.5 SR loads compared at different times during the diastolic interval
The data presented above indicates that the SR immediately prior to an aftercontraction is no more loaded than following a subthreshold drive train (0.2 Hz). In addition, the consistent finding of an ‘N-shaped’ curve in Figs. 2D, 3D and 4D
implies that SR load may actually be lower prior to an aftercontraction than after subthreshold drive trains (0.5 Hz), although it should be noted that there is a statistically significant difference between the data for 0.5 Hz and threshold drive train frequency only in the caffeine-induced cell shortening data. This is contrary to what is expected of a species with a positive force–frequency relationship, i.e., that increasing stimulation frequency increases SR load. One possible explanation for this apparent discrepancy might be related to the timing of caffeine applications in our experiments. Whereas, following the subthreshold drive trains of 0.2 and 0.5 Hz the switch to caffeine solution was made 1800 ms after the final stimulus of the train, for the other two conditions the switch was made 300 ms before the earliest evidence of aftercontraction or afterdepolarisation activity on any of the characterising records. This led to significant differences in the timing of caffeine delivery. Following drive trains of 0.2 Hz, caffeine delivery (the onset of the caffeine-induced inward current) was 1980.1±5.4 ms after the final drive train stimulus, compared with 1945.3±7.0 ms following drive trains of 0.5 Hz, 3185.1±204.4 ms following drive trains at the threshold for aftercontractions, and 1706.8±122.7 ms following drive trains at maximal stimulation frequency. We considered the possibility that the SR might lose a proportion of its calcium load via rest decay during the diastolic interval prior to the delivery of caffeine. Although the half time of rest decay in guinea pig cardiac myocytes at room temperature is 24 s when stimulated at 0.5 Hz [17], it is possible that rest decay after higher stimulation frequencies is more rapid, possibly as a result of increased frequency of calcium sparks [20,21], and that this might explain the reduced SR loads measured following threshold drive trains. This was investigated by applying caffeine at 1800 ms following threshold drive trains and at 300 ms prior to the earliest aftercontraction or afterdepolarisation activity in the same cells. In nine cells so investigated, there proved to be no significant decline in SR calcium content between the end of the drive train and the onset of the aftercontraction (from 106.5±12.2 µmol/l accessible cell volume to 99.6±11.9 µmol/l accessible cell volume, P>0.5). This suggests that the precise timing of caffeine application during this interval has no influence on the load measured.
3.6 Double caffeine applications
Another possible explanation for the apparent interruption of the expected positive load–frequency relationship might be that caffeine fails to cause complete unloading of the available SR calcium under threshold conditions for aftercontractions. There may be a finite time required for calcium to recirculate from SR uptake sites to SR release sites. It is possible that, under certain conditions, not all the calcium ultimately available for release by caffeine is available at the time of the application. Previous work using rapid cooling contractures following caffeine application has demonstrated that no releasable calcium remains in the SR after caffeine application following stimulation at 0.5 Hz [17], but it is important to establish that this is still the case after the more rapid drive trains that lead to aftercontractions. To investigate this, we subjected seven cells to a protocol which involved double caffeine applications. Cells were driven at threshold frequency and caffeine applied 1800 ms following the final stimulus of the drive train. After 5 s, the superfusing solution was changed to one containing nominally zero sodium and calcium (LiCl substituted for NaCl in the superfusate, CaCl2 excluded, 1 mM EGTA added and pH adjusted to 7.4 with LiOH), in order to inhibit the sodium–calcium exchanger and thereby minimise calcium movement across the sarcolemma. Superfusion with this solution was continued for 5 s, in order to allow redistribution of calcium within the SR, between the putative functionally distinct compartments. Ten mM caffeine were then added in an attempt to elicit a second release of calcium from the SR. In all seven cells so investigated, the second caffeine application failed to induce a contraction. The apparent decrease in SR load at the threshold for aftercontractions is therefore unlikely to be an artifact caused by functional compartmentalisation within the SR.
3.7 Diastolic cytoplasmic calcium concentration
The above results suggest that, immediately prior to a spontaneous aftercontraction, the SR is no more loaded than after subthreshold drive trains. The trigger for spontaneous calcium release therefore is unlikely to be an effect of luminal SR calcium acting on the ryanodine receptor. We therefore investigated the other major hypothesis, that raised cytoplasmic [Ca2+] triggers spontaneous calcium release. We monitored cytoplasmic [Ca2+] using indo-1 (loaded in its esterified AM form at a concentration of 9.8 µM for 15 min at room temperature). Cells were field stimulated at 0.5 and 1.0 Hz for 20-beat drive trains. In the above experiments 0.5 Hz was below the threshold for aftercontractions, whereas 1.0 Hz was the mean threshold frequency for aftercontractions, but the SR loads at these two frequencies were not significantly different (Fig. 2). Indo-1 signal ratios were measured as absolute values and relative to a true resting level, recorded after 2 min of quiescence following each drive train. In Fig. 5A, representative traces of indo-1 ratio are illustrated from the final beat of 20-beat drive trains and the ensuing 8 s in two cells stimulated at 1.0 Hz, one of which demonstrated an aftertransient and aftercontraction. Bulk cytoplasmic [Ca2+] is greater throughout the systolic transient and post-train pause in the cell showing the aftertransient than in the other cell. Pooled data for all cells investigated in this way are shown in Fig. 5B. Indo-1 ratio was measured at 500-ms intervals from 1.0 to 8.0 s following the peak of the final stimulated calcium transient of the drive train. Twenty-two cells showed aftertransients and aftercontractions following 1.0 Hz drive trains, whereas 21 did not. The mean indo-1 ratio was significantly higher in those cells which demonstrated aftertransients throughout the post-train pause than in those cells which did not, at all points except 2.5 s. Data points occurring during aftertransients were excluded from this analysis. There was also a difference in indo-1 ratios after a 2-min rest period between cells showing and those not showing aftertransients at 1.0 Hz (0.71±0.05 vs. 0.55±0.05, P<0.05). Indo-1 ratios were also measured relative to 2-min resting levels, as a measure of residual cytoplasmic calcium loading during the immediate post-train period. This analysis was performed on those cells (n=18) which showed aftertransients at 1.0 but not at 0.5 Hz, in order to assess the role of bulk cytoplasmic [Ca2+] as a trigger for spontaneous SR calcium release. Representative traces are illustrated in Fig. 5C. In this cell, indo-1 ratio returned to near resting levels within 1 s of the peak of the stimulated transient, and was no different following drive trains of 0.5 Hz (trace without aftertransient) and 1.0 Hz (trace with aftertransient). Pooled data for this analysis are illustrated in Fig. 5D. The amplitudes of the stimulated transient were not significantly different between the two stimulation frequencies, consistent with the SR load data presented above. Cytoplasmic [Ca2+] following the final transient of the drive train were no different between the two stimulation frequencies throughout the post-train pause.
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4 Discussion |
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The events leading up to spontaneous release of calcium from the SR are obscure. However, the central role of high cellular calcium loading (sometimes referred to as ‘calcium overload’) in the induction of spontaneous release is beyond doubt. The question we have attempted to address in this paper is whether the influence of calcium overload is exerted within the lumen of the SR (i.e., SR calcium overload) or on the cytoplasmic side of the SR membrane (i.e., cytoplasmic calcium overload). These two possibilities are, of course, not mutually exclusive, but we aimed to assess which mechanism is the more important.
Four methods, one quantitative and three non-quantitative, of assessing SR load were utilised. For each method, the results at 0.2 and 0.5 Hz were consistent with the positive relationship between frequency and SR load expected of this species. However the results measured at the threshold frequency for aftercontractions were no higher than those at 0.2 Hz (Figs. 2–4). Experiments were performed to exclude the possibility that these findings might be caused by the timing of caffeine delivery during the post-drive train interval or by incomplete caffeine-induced calcium release from the SR at higher stimulation frequencies. We therefore conclude that the trigger for spontaneous release from the SR is not overload of the SR itself acting in some way from the luminal side of the SR membrane. In contrast the experiments illustrated in Fig. 5 suggest that cytoplasmic [Ca2+] predicts those cells which will and those which will not demonstrate aftertransients and aftercontractions at 1.0 Hz. It is therefore possible that raised cytoplasmic [Ca2+] is more important than SR calcium load in triggering aftercontractions. This is consistent with the findings of Wier and Hess, that aftercontractions, transient depolarisations and afterglimmers did not appear in canine Purkinje fibres exposed to cardiotonic steroids until there was a detectable rise in diastolic calcium levels, as measured with the calcium-sensitive bioluminescent protein aequorin [22]. A number of studies have suggested that cytoplasmic [Ca2+] is the critical determinant of propagation (as distinct from initiation) of calcium waves, as evidenced by examination of the leading edge of the wavefront [7], assessment of the thermodynamics of wave velocity [8] and, most recently, investigation of the effects of low-dose caffeine and thapsigargin application prior to focal caffeine application [23]. The process of wave initiation may not be very different from that of wave propagation. The initiating event in a calcium wave is believed to be a calcium spark. It has been shown that calcium sparks do not require a well-loaded SR to occur [24]. If unitary SR release events occur during the diastolic interval, the same factors which promote propagation of the established wave may promote initiation of the wave. Raised cytoplasmic [Ca2+] may trigger the transition from discrete sparks into propagating waves not only by triggering release itself but also by inhibiting diffusion of calcium away from spark sites, setting up the conditions necessary for calcium-induced activation of neighbouring clusters of SR calcium release channels. According to this scheme, cytoplasmic [Ca2+] assumes central importance in the triggering of aftercontractions, though SR content would still exert an influence by influencing the probability of spark occurrence. The finding of increased cytoplasmic [Ca2+] in those cells which show aftertransients at 1.0 Hz suggests that cytoplasmic [Ca2+] is indeed an important factor for triggering spontaneous release. However, the lack of any difference in bulk cytoplasmic [Ca2+] after sub- and suprathreshold drive trains in the same cells implies that this effect may be exerted at a local level, in the subsarcolemmal space.
Time for primary review 28 days.
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Acknowledgements |
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We thank Mr. P. O’Gara for his expert assistance with myocyte isolation. R.E. was supported by a fellowship from the Medical Research Council. A.D.S. was supported by a fellowship from the British Heart Foundation.
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