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Calcium sparks in human ventricular cardiomyocytes from patients with terminal heart failure
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Cell Calcium (2002) 31(4), 175–182 0143-4160/02/$ - see front matter © 2002, Elsevier Science Ltd. All rights reserved. doi: 10.1054/ceca.2002.0272, available online at http://www.idealibrary.com on
Calcium sparks in human ventricular cardiomyocytes from patients with terminal heart failure M. Lindner,1 M.C. Brandt,1 H. Sauer,2 J. Hescheler,2 T. Böhle1 D.J. Beuckelmann1 1Department 2Department
of Medicine III, University of Cologne, Cologne, Germany of Neurophysiology, University of Cologne, Cologne, Germany
Summary Cardiomyocytes from terminally failing hearts display significant abnormalities in e–c-coupling, contractility and intracellular Ca2; handling. This study is the first to demonstrate the influence of end-stage heart failure on specific properties of Ca2; sparks in human ventricular cardiomyocytes. We investigated the frequency and characteristics of spontaneously arising Ca2; sparks in single isolated human myocytes from terminally failing (HF) and non-failing (NF) control myocardium by using the Ca2; indicator Fluo-3. The Ca2; sparks were recorded by line-scan images along the longitudinal axis of the myocytes at a frequency of 250 Hz. After loading the sarcoplasmic reticulum (SR) with Ca2; by repetitive field stimulation (10 pulses at 1 Hz) the frequency of the Ca2; sparks immediately after stimulation (t:0 s) was reduced significantly in HF compared to NF (4.15<0.42 for NF vs. 2.81<0.20 for HF sparks s91, P0.05). This difference was present constantly in line-scan recordings up to 15 s duration (t:15 s: 2.75<0.65 for NF vs. 1.36<0.34 for HF sparks s91, P0.05). The relative amplitude (F/F0) of Ca2; sparks was also significantly lower in HF cardiomyocytes (1.33<0.015 NF vs. 1.19<0.003 HF, t:0 s) and during subsequent recordings of 15 s. Significant differences between HF and NF were also present in calculations of specific spark properties. The time to peak was estimated at 25.75<0.88 ms in HF and 18.68<0.45 ms in NF cardiomyocytes (P0.05). Half-time of decay was 66.48<1.89 ms (HF) vs. 44.15<1.65 ms (NF, P0.05), and the full width at half-maximum (FWHM) was 3.99<0.06 m (HF) vs. 3.5<0.07 m (NF, P0.05). These data support the hypothesis that even in the absence of cardiac disease, Ca2; sparks from human cardiomyocytes differ from previous results of animal studies with respect to the time-to-peak, half-time of decay and FWHM. The role of elevated external Ca2; in HF was studied by recording Ca2; sparks in HF cardiomyocytes with 10 mmol external Ca2; concentration. Under these conditions, the average spark amplitude was increased from 1.19<0.003 (F/F0, 2 mmol Ca2;) to 1.26<0.01 (F/F0, 10 mmol Ca2;). We conclude that human heart failure causes distinct changes in Ca2; spark frequency and characteristics comparable to results established in animal models of heart failure. A reduced Ca2; load of the SR alone is unlikely to account for the observed differences between HF and NF and additional alterations in intracellular Ca2; release mechanisms must be postulated. © 2002, Elsevier Science Ltd. All rights reserved.
INTRODUCTION End-stage human heart failure is characterised by severe myocardial contractile dysfunction, which could be related to several biochemical and pathophysiological alterations. Received 3 May 2001 Revised 8 October 2001 Accepted 9 January 2002 Correspondence to: Dr M. Lindner, University of Cologne, Department of Medicine III, Joseph-Stelzmann-Str. 9, D-50924 Cologne, Germany. Tel.: ;49 221 478 4475; fax: ;49 221 478 6490; e-mail: [email protected]
Because of its key function for cell contractility, intracellular Ca2; regulation and its changes appear to be the most important mechanism to characterise the underlying pathophysiology of heart failure. Gwathmey et al. [1,2] were the first to demonstrate changes of intracellular [Ca2;]i handling in papillary muscle strips from myocardium of patients with terminal heart failure. In single ventricular myocytes isolated from terminally failing myocardium, we have shown that [Ca2;]i transients are reduced, diastolic [Ca2;]i levels are increased and that the rate of diastolic decay of [Ca2;]i is slowed significantly [3]. The Ca2; uptake into isolated sarcoplasmic 175
176 M Lindner, MC Brandt, H Sauer, J Hescheler, T Böhle, DJ Beuckelmann
reticulum (SR)-vesicles was also found to be reduced in failing myocardium. This finding was associated with a reduction of the protein levels of the SR Ca2;-ATPase (SERCA-2a) and the m-RNA encoding for SERCA-2a [4–6]. We have demonstrated that the caffeine-induced Ca2; transients in isolated myocytes of terminal failing myocardium are reduced [7], indicating a reduced Ca2; load of the SR. These results were confirmed in a study by Pieske et al. [8] using rapid cooling contractures to estimate the SR Ca2; content. With the technique of confocal laser-scanning microscopy, Cheng et al. [9] were able to visualize in vitro single events of intracellular calcium release (Ca2; sparks) from the SR and to relate these observations to the concept of excitation-contraction coupling by Ca2; induced Ca2; release. It could be demonstrated further that the systolic Ca2; transient in a myocardial cell is the summation of a multitude of individual Ca2; sparks [10]. However, at present it is controversial whether Ca2; sparks indicate the opening of a single Ca2; release channel of the SR (ryanodine receptor) or if they comprise openings of clusters of ryanodine receptor channels [11,12]. Ca2; sparks can arise spontaneously or could be triggered by Ca2; entering the cell through the L-type Ca2; channel during voltage-clamp pulses [10,13–15]. Ca2; sparks were not only observed in single cardiomyocytes but also in rat cardiac trabeculae [16]. Despite these studies, indicating unanimously the important link between Ca2; sparks and myocardial contractile function, not much evidence could be established on the properties of Ca2; sparks under conditions of heart-failure. In recent years only two animal models of heart failure or cardiac hypertrophy were concerned with Ca2; spark characteristics in the presence of cardiac disease [17,18]. At present, no data are available on Ca2; sparks in any preparation of human cardiomyocytes. Therefore, the aim of the present study was to characterize the kinetic properties of Ca2; sparks in human cardiomyocytes and to compare these in non-failing hearts and those with end-stage heart failure.
METHODS Cell isolation Cells were prepared from six hearts of patients with terminal heart failure resulting from dilated cardiomyopathy (DCM, n:2) or ischaemic cardiomyopathy (ICM, n:4) undergoing transplantation. All patients received digoxin and diuretics, and were receiving vasodilator therapy (ACE inhibitors). Informed consent was obtained from all patients prior to organ explantation. Results were compared with cells isolated from human hearts without heart failure that could not be transplanted for technical Cell Calcium (2002) 31(4), 175–182
reasons (n:3). The cell isolation was started within 1 (explanted failing hearts) to 3 h (non-utilized donor hearts) after explantation. The isolation procedure has been described in detail [19]. A part of the left ventricular wall was excised together with its artery branch and the wall segment was perfused via this artery branch for 30 min with a nominally Ca2; free modified Tyrode’s solution (see below), followed by 40 min with the same solution additionally containing collagenase (type II, 70 mg/50 ml, Worthington) and protease (type XIV, 3 mg/50 ml, Sigma Chemicals). Finally, the enzyme was washed out for 15 min with modified Tyrode’s solution containing 100 M Ca2;. Cells were prepared from areas within the central portion of the myocardial wall. Chunks of well-perfused myocardium were placed into Tyrode’s solution containing 2 mM Ca2; and filtered through a nylon mesh. During experiments, the myocytes were stored at room temperature in Tyrode’s solution containing 2 mM Ca2;. The living cell yield was approximately 5–10% in failing as well as in control myocardium. Only cells with clear cross striations and without significant granulation were selected for experiments. These cells contracted on stimulation. Solutions and loading of cells with Fluo-3 The nominally Ca2; free Tyrode’s solution used during the isolation steps was composed of (in mM): NaCl, 135; KCl, 4.0; MgCl2, 1.0; glucose, 10; NaH2PO4, 0.33; NaHEPES, 10; pH adjusted to 7.3 with addition of NaOH. When indicated, this solution additionally contained CaCl2 at different concentrations. For measurements under conditions of high external [Ca2;], modified Tyrode’s solution with 10 mM Ca2; was used. Before spark measurements, the myocytes were loaded with the fluorescent Ca2; indicator Fluo-3 by 20 min incubation with 10 M Fluo-3 (acetoxymethylester, Molecular Probes, Eugene, OR, USA) at 37 ⬚C. During the Ca2; spark recordings, the cells were superfused at room temperature (22 ⬚C) with modified Tyrode’s solution containing 2 mM or 10 mM CaCl2. Confocal microscopy Experiments were carried out under steady-state conditions by loading the SR with Ca2; through field stimulation (10 pulses at 1 Hz). Ca2; measurements were performed using a Zeiss laser-scanning confocal microscope (LSM 410 coupled to an Axiovert 135 M inverted microscope, Carl Zeiss, Germany) that was equipped with an argon laser. The objective used was a Zeiss 25 oil immersion Plan-Neofluar with a numerical aperture of 0.8. The excitation wavelength of Fluo-3 by the argon laser was 488 nm. The laser was kept at the lowest level and light © 2002, Elsevier Science Ltd. All rights reserved.
Calcium sparks in human cardiomyocytes 177
intensity was further attenuated with a neutral-density filter in the excitation path (to 1%) in order to minimize photo-toxicity and -bleaching. Fluorescence was measured at wavelengths 515 nm. The line-scan mode was used for the analysis of Ca2; sparks. Each myocyte was scanned repetitively along a line in its longitudinal axis at a frequency of 250 Hz. The line-scan image was constructed by 512 lines vertically. The magnification was set by the objective and the hardware zoom factor (3) of the laser scanning microscope to give a pixel size of ~0.1 m2 (0.330.33 m). The confocal detector aperture was set to provide an axial (z) resolution of ~3 m measured as full width half-maximal amplitude (FWHM) in images of 0.5 m fluorescent microbeads (Molecular Probes). The images were analysed using Lsmdummy (Carl Zeiss, Germany), Origin 5.0 (Microcal Software Inc., Northampton, MA, USA) and IDL 5.0 software (Research Systems Inc., Boulder, CO, USA) on an IBM compatible pentium personal computer. Computer analysis The line-scan images were analysed using an automated spark-detection routine partially based on an algorithm published by Cheng et al. [20]. In order to keep bias to a possible minimum, all steps in the analysis were performed automatically. A program with window-based user-interface was created reading the initial line-scan images from hard-disk and setting up all parameters for spark-detection automatically. Human intervention was only requested to confirm the initial steps of the algorithm, such as detection of cell-edges, and the results presented at the end of the analysis. After different smoothing filter steps, the cell edges were detected by the analysis routine. Potential spark regions that exceeded the overall standard deviation of the fluorescence baseline were excised to calculate a corrected baseline containing background noise only. Then the image was normalized using the corrected fluorescence baseline. As described in more detail by Cheng et al. [20], potential spark regions were then detected as connected regions that exceeded a fluorescence threshold given by the sum of the cell-fluorescence baseline and its standard deviation multiplied with a given constant. Only those potential spark sites were accepted, that exceeded a normalized fluorescence intensity 3.2-fold. As in the algorithm by Cheng et al. [20], those regions greater than a 2-fold baseline fluorescence were used for further analysis. Spacial information on every spark site was handed over to a mathematical subroutine, calculating the maximum value, width, rise- and decay time and the FWHM. In order to exclude a possible influence of a fading background fluorescence intensity on the © 2002, Elsevier Science Ltd. All rights reserved.
normalized images, the total line-scans of up to 15 s duration were stored as separate images of 2.24 s duration. For all of these recordings, a separate analysis was performed, normalizing each image with its individually calculated array of average background-fluorescence intensity. During image-processing, representative images of the dataset were stored automatically after every major step by the algorithm (see Fig. 1). In the course of spark-detection, the sparks were superimposed over the normalized image, giving the user the chance to view every spark step by step and to identify potentially false detections. Finally, the complete set of analysis data was stored together with a set of images taken during the mathematical processing of the raw images. All spark sites as calculated by the program were presented to the user with the option to delete false spark detections. Statistical analysis Results were expressed as mean values
50 ms
178 M Lindner, MC Brandt, H Sauer, J Hescheler, T Böhle, DJ Beuckelmann
50 ms
50 µm
50 µm Fig. 1 Spontaneous Ca2; sparks after SR Ca2; loading by field stimulation (10 pulses at 1 Hz) in a non-failing (A, B and E) and a failing (C, D) cardiomyocyte. A and C show the original line-scans as recorded from the confocal laser scanner. B, D and E show the processed images, digitally filtered and normalized, being more comprehensible in terms of spark distribution within the image. The original data consists of 512 scan lines along the longitudinal axis (2.24 s duration). The scale bars indicate temporal and spacial dimensions. In A and C, the colour scale directly represents the fluorescence intensity ⌬F, depending primarily on local changes of the Ca2; flourescence within the primary line-scan recording. In B, D and E, the colour scale is adapted to the interval of relative ⌬F/F0 values resulting from normalization (B, E: ⌬F/F0 values from 0.58 to 1.61; D, ⌬F/F0 values from 0.90 to 1.22). E shows the spark sites as detected by the algorithm and accepted by the authors as yellow areas, rejected spark sites are coloured in white. The images were taken immediately after the end of stimulation (t:0 s). The Ca2; spark frequency was significantly higher in cardiomyocytes from non-failing myocardium.
Cell Calcium (2002) 31(4), 175–182
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Frequency and characteristics of Ca2; sparks in failing human cardiomyocytes For comparison, the same SR Ca2; loading protocol was applied to cardiomyocytes isolated from terminally failing hearts. Figures 1C and D show the line-scan image of a failing myocyte with only a few spontaneous Ca2; sparks. A total of 1503 Ca2; sparks in failing human cardiomyocytes were analysed. The time to peak (mean 25.75<0.88 ms) and the half-time of decay (mean 66.48<1.89 ms) and the FWHM (3.99<0.06 m) were significantly different to control cells (P0.05). The Ca2; spark frequency was significantly lower in cells from failing myocardium compared to controls (2.81<0.20 s91 for HF vs. 4.15<0.42 s91 for NF; P0.05 at t:0 s; 1.36<0.34 s91 for HF vs. 2.75<0.65 s91 for NF; P0.05 at t:15 s). The Ca2; spark amplitude was also decreased significantly. The mean fluorescence ratio (F/F0) during the first second of scanning was 1.19<0.003; at t:15 s it was 1.14<0.01 (mean 1.19<0.002). ; sparks in Frequency and characteristics of Ca2; failing cardiomyocytes under conditions of increased Ca2; load
Incubation of failing myocytes in 10 mM external [Ca2;] caused the Ca2; spark frequency to increase approximately 2.5-fold immediately after the end of stimulation. A total of 671 Ca2; sparks were detected. In non-failing myocytes elevation of external Ca2; to 10 mM caused Ca2; overload of the cells as indicated by the induction of Ca2; waves. Under these conditions, individual Ca2; sparks could not be differentiated adequately. Figure 2 shows a summary of Ca2; spark frequency in all three groups (failing, control and failing cells) in 10 mM external Ca2; (6.64<0.80 s91 after cessation of stimulation, t:0 s; 5.0<0.91 s91, t:13 s) over the entire time of observation, indicating a significant difference between groups. Also Ca2; spark amplitude increased in the state of increased Ca2; load, but did not reach the amplitude of Ca2; sparks in control myocardium in physiological Ca2; concentration (2 mM). Figure 3 summarizes the Ca2; spark amplitude calculations (as fluorescence ratio) for all three groups. A significantly higher Ca2; spark amplitude was present in control myocytes compared to cells from failing hearts. After incubation of cells in a high-Ca2; solution during the first 10 s after cessation of the SR-loading procedure, the Ca2; spark amplitude was increased significantly (mean F/F0 1.26<0.01 after cessation of stimulation at t:0 s; 1.19<0.01 at t:13 s). A further significant increase in the time to peak Ca2; could be detected (30.52<1.24 ms; P0.05) as well as in the full width at half maximum (FWHM 4.34<0.10 m; P0.05). The half-time of decay of Ca2; sparks was unchanged in an elevated external Ca2; solution (67.07<2.82 ms). © 2002, Elsevier Science Ltd. All rights reserved.
Fig. 2 Frequency of Ca2; sparks visualized in line-scan images after SR Ca2; loading. A significant difference in Ca2; spark frequency could be detected between all three groups investigated, i.e. non-failing myocardium (2 mM external Ca2;), failing myocardium ICM and DCM, 2 mM and 10 mM external Ca2;), during the full time of observation (15 s). The elevation of the external Ca2; to 10 mM resulted in an approximately 2.5-fold rise of the Ca2; spark frequency in failing myocardium.
Fig. 3 The Ca2; spark amplitude (fluorescence ratio F/F0) showed a decrease with time in all cells investigated. Significant differences in the Ca2; spark amplitude could be detected between failing (ICM and DCM) and non-failing myocardium in physiological Tyrode’s solution. By elevating the external Ca2; concentration to 10 mM, the Ca2; spark amplitude rose significantly in failing myocardium over the first 10 s, but not to the values as in non-failing cardiomyocytes in physiological Tyrode’s solution (2 mM Ca2;).
DISCUSSION This is the first study to report on the characteristics of Ca2; sparks in isolated human ventricular cardiomyocytes. The purpose of this study was to investigate if the Cell Calcium (2002) 31(4), 175–182
180 M Lindner, MC Brandt, H Sauer, J Hescheler, T Böhle, DJ Beuckelmann
Ca2; release mechanism of the SR, as indicated by the Ca2; spark characteristics, is altered in ventricular myocytes from patients with terminal heart failure, and if such alterations could explain the changes of intracellular [Ca2;]i handling that have been found in these cells [3,9]. According to the ‘local control theory’ of excitationcontraction coupling proposed by Stern [21], Ca2; induced Ca2; release is initiated locally by Ca2; influx through a single L-type Ca2; channel that activates a group of SR Ca2; release channels [12]. The Ca2; transient is then thought to be the summation of multiple local Ca2; releases during depolarization of the cell membrane [10]. Ca2; sparks are thought to indicate release of Ca2; from one or few of these local units of excitation-contraction coupling. In our experiments, cells from terminally failing myocardium not only showed a significantly lower Ca2; spark frequency compared to non-failing controls, but also spacio–temporal Ca2; spark characteristics were changed in the presence of heart failure. By inducing a higher Ca2; load in failing myocytes (10 mM external Ca2; concentration), the Ca2; spark frequency could be increased to values higher than in control cells under physiological conditions. The Ca2; spark amplitude was also increased significantly, but not to the extent as in control myocytes. However, the time to peak Ca2; increased even further with a higher Ca2; load, as well as the FWHM, which was already increased in failing myocardium. From these data, we conclude that the spacio–temporal alteration of Ca2; sparks in heart failure are not only due to a reduced Ca2; load of the sarcoplasmic reticulum. Spontaneous Ca2; sparks have been described in ventricular myocytes and in skeletal muscle cells of several other mammalian species. In the majority of studies on rat and guinea-pig cardiomyocytes [9,14–16], the time to peak was faster than in our experiments on human ventricular cells. Only Satoh et al. [22] measured a time to peak of approximately 15 ms (rat and rabbit ventricular myocytes) comparable to the values in our study. In our experiments, the half-time of decay of human myocardial cell Ca2; sparks was slower than in all other species described until now [9,14–16,22]. These findings demonstrate that even in the absence of cardiac disease Ca2; spark characteristics in human cardiomyocytes differ from those derived from animal studies. In an animal model of experimental cardiac hypertrophy and heart failure, Gomez et al. [17] showed unchanged temporal characteristics of voltage-clamp-induced Ca2; sparks. In this animal model of cardiomyocytes with clinical signs of heart failure, Ca2; transients were reduced and prolonged, but the L-type Ca2; current was unchanged. In the present study, we detected significant differences of temporal characteristics of Ca2; sparks Cell Calcium (2002) 31(4), 175–182
between non-failing and terminally failing myocytes. Furthermore, in contrast to our results on human cardiomyocytes, in which caffeine-induced Ca2; transients are reduced [7], Gomez et al. [17] showed that caffeineinduced Ca2; transients were unaltered in failing rat myocardium. Therefore, they concluded that alterations of intracellular Ca2; handling can only be explained by a change of the micro-architecture between the ryanodine receptor and the sarcolemmal L-type Ca2; channel, indicating an impaired Ca2; induced Ca2; release mechanism. The results of the present study also suggest an alteration of the Ca2; release mechanism, as the Ca2; spark characteristics (time to peak, half time of decay and FWHM) are significantly different in HF compared to NF. In isolated rat cardiomyocytes, Song et al. [23] showed that the Ca2; spark amplitude, but not the frequency, depends on the SR filling-state. In our study, however, both parameters are changed. Our analysis was performed with an equivalent computer routine as that described by their group [20]. A reduced number of L-type Ca2; channels (dihydropyridine receptors) could also impair SR Ca2; loading. However, measurements of dihydropyridine receptors showed that the number of Ca2; channels are unchanged in human heart failure [24,25]. Functional measurements of the L-type Ca2; current did not show any differences between control and failing myocardium [3], although these results could be frequency-dependent [26]. Results of studies trying to quantify SR Ca2; release channels (ryanodine receptors) are controversial. Brillantes et al. [27] demonstrated a reduction of mRNA levels encoding for the ryanodine receptor only in ischaemic cardiomyopathy compared to control myocardium. Go et al. [28] also found mRNA levels for the SR Ca2; release channels to be reduced in ischaemic and dilated cardiomyopathy, whereas other groups did not find any differences in mRNA expression [29], but showed an increase in ryanodine binding sites [30]. Electrophysiological single-channel recordings performed by Holmberg and Williams [31,32] indicated unimpaired characteristics of human Ca2; release channels in heart failure. A recent study by Ching et al. [33] gives evidence that besides the cytosolic Ca2; concentration luminal Ca2; regulatory mechanisms and the amount of intra-SR [Ca2;] are important for the SR Ca2; release. This might partially explain our data, especially the observed changes in Ca2; spark frequency, but not the persisting difference in Ca2; spark amplitude between failing and non-failing myocardium. Coupled gating between individual ryanodine receptors in skeletal muscle was described by Marx et al. [34]. A possible cause for changes in the excitation-contraction coupling and Ca2; spark characteristics could be the inhibition of coupled gating of the ryanodine receptors. Another group suggested that FKBP could also help in © 2002, Elsevier Science Ltd. All rights reserved.
Calcium sparks in human cardiomyocytes 181
the cooperative gating of multiple ryanodine receptors. Targeted knockout of the mouse FKBP12 gene produces severe cardiomyopathy with altered cardiac ryanodine receptor gating, whereas skeletal muscle function is not affected [35]. Our results support an alteration of coupled gating, as indicated by the characteristics of Ca2; sparks in heart failure. Recently, it could be shown that in failing human myocardium the type 2 ryanodine receptor polypeptides are protein kinase A hyperphosphorylated, resulting in a defective channel function due to increased sensitivity to Ca2; induced activation [36]. Discussions on the possible influence of alterations of ryanodine receptor activity on cardiac function are still controversial. From a mathematical model of net calcium fluxes, Eisner and colleagues [37–39] concluded that a decreased efflux through the calcium release channel would have only transient effects on systolic calcium, but no effects on cardiac contractility in a steady state. This model is based on experiments with different species (rat, ferret and guinea-pig) at room temperature and at 37 ⬚C. The Na/Ca exchange current is used as a tool to measure calcium fluxes. As the Na/Ca exchange current in human cardiac cells has not yet been characterized, further investigations will be required for a better understanding. In summary, our results support the hypothesis that although a major defect of excitation-contraction coupling in human terminal heart failure is a reduction of the SR Ca2; load, an additional alteration of the Ca2; release mechanism has to be postulated. ACKNOWLEDGEMENTS This research was supported by grants of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (01 KS 9502, Zentrum für Molekulare Medizin Köln, Projekt 4) and the M. & W. Boll Stiftung). Our special thanks go to Prof. DeVivie (Department of Cardiac Surgery, University of Cologne) and his colleagues for providing the myocardial tissue.
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31. Holmberg SRM, Williams AJ. Single channel recordings from human cardiac sarcoplasmatic reticulum. Circ Res 1989; 65: 1445–1449. 32. Holmberg SRM, Williams AJ. The calcium-release channel from cardiac sarcoplasmatic reticulum: function in the failing and acutely ischemic heart. Basic Res Cardiol 1992; 87 (Suppl. 1): 255–268. 33. Ching LL, Williams AJ, Sitsapesan R. Evidence for Ca2; activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex. Circ Res 2000; 87: 201–206. 34. Marx SO, Ondrias K, Marks AR. Coupled gating beetween individual skeletal muscle Ca2; release channels. Science 1998; 281: 818–821. 35. Shou W, Aghdasi B, Armstrong DL, et al. Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12. Nature 1998; 391: 489–491. 36. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 2000; 101: 365–376. 37. Diaz ME, Trafford AW, O’Neill SC, Eisner DA. Can changes of ryanodine receptor expression affect cardiac contractility? Cardiovasc Res 2000; 45: 1068–1069. 38. Yano M, Yamamoto T. Abnormal sarcoplasmic reticulum Ca2; release in heart failure. Cardiovasc Res 2000; 45: 1070–1071. 39. Eisner DA, Trafford AW, Diaz ME, Overend CL, O’Neill SC. The control of Ca release from the the cardiac sarcoplasmic reticulum: regulation versus autoregulation. Cardiovasc Res 1998; 38: 589–604.
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Cell Calcium (2002) 31(4), 175–182 0143-4160/02/$ - see front matter © 2002, Elsevier Science Ltd. All rights reserved. doi: 10.1054/ceca.2002.0272, available online at http://www.idealibrary.com on
Calcium sparks in human ventricular cardiomyocytes from patients with terminal heart failure M. Lindner,1 M.C. Brandt,1 H. Sauer,2 J. Hescheler,2 T. Böhle1 D.J. Beuckelmann1 1Department 2Department
of Medicine III, University of Cologne, Cologne, Germany of Neurophysiology, University of Cologne, Cologne, Germany
Summary Cardiomyocytes from terminally failing hearts display significant abnormalities in e–c-coupling, contractility and intracellular Ca2; handling. This study is the first to demonstrate the influence of end-stage heart failure on specific properties of Ca2; sparks in human ventricular cardiomyocytes. We investigated the frequency and characteristics of spontaneously arising Ca2; sparks in single isolated human myocytes from terminally failing (HF) and non-failing (NF) control myocardium by using the Ca2; indicator Fluo-3. The Ca2; sparks were recorded by line-scan images along the longitudinal axis of the myocytes at a frequency of 250 Hz. After loading the sarcoplasmic reticulum (SR) with Ca2; by repetitive field stimulation (10 pulses at 1 Hz) the frequency of the Ca2; sparks immediately after stimulation (t:0 s) was reduced significantly in HF compared to NF (4.15<0.42 for NF vs. 2.81<0.20 for HF sparks s91, P0.05). This difference was present constantly in line-scan recordings up to 15 s duration (t:15 s: 2.75<0.65 for NF vs. 1.36<0.34 for HF sparks s91, P0.05). The relative amplitude (F/F0) of Ca2; sparks was also significantly lower in HF cardiomyocytes (1.33<0.015 NF vs. 1.19<0.003 HF, t:0 s) and during subsequent recordings of 15 s. Significant differences between HF and NF were also present in calculations of specific spark properties. The time to peak was estimated at 25.75<0.88 ms in HF and 18.68<0.45 ms in NF cardiomyocytes (P0.05). Half-time of decay was 66.48<1.89 ms (HF) vs. 44.15<1.65 ms (NF, P0.05), and the full width at half-maximum (FWHM) was 3.99<0.06 m (HF) vs. 3.5<0.07 m (NF, P0.05). These data support the hypothesis that even in the absence of cardiac disease, Ca2; sparks from human cardiomyocytes differ from previous results of animal studies with respect to the time-to-peak, half-time of decay and FWHM. The role of elevated external Ca2; in HF was studied by recording Ca2; sparks in HF cardiomyocytes with 10 mmol external Ca2; concentration. Under these conditions, the average spark amplitude was increased from 1.19<0.003 (F/F0, 2 mmol Ca2;) to 1.26<0.01 (F/F0, 10 mmol Ca2;). We conclude that human heart failure causes distinct changes in Ca2; spark frequency and characteristics comparable to results established in animal models of heart failure. A reduced Ca2; load of the SR alone is unlikely to account for the observed differences between HF and NF and additional alterations in intracellular Ca2; release mechanisms must be postulated. © 2002, Elsevier Science Ltd. All rights reserved.
INTRODUCTION End-stage human heart failure is characterised by severe myocardial contractile dysfunction, which could be related to several biochemical and pathophysiological alterations. Received 3 May 2001 Revised 8 October 2001 Accepted 9 January 2002 Correspondence to: Dr M. Lindner, University of Cologne, Department of Medicine III, Joseph-Stelzmann-Str. 9, D-50924 Cologne, Germany. Tel.: ;49 221 478 4475; fax: ;49 221 478 6490; e-mail: [email protected]
Because of its key function for cell contractility, intracellular Ca2; regulation and its changes appear to be the most important mechanism to characterise the underlying pathophysiology of heart failure. Gwathmey et al. [1,2] were the first to demonstrate changes of intracellular [Ca2;]i handling in papillary muscle strips from myocardium of patients with terminal heart failure. In single ventricular myocytes isolated from terminally failing myocardium, we have shown that [Ca2;]i transients are reduced, diastolic [Ca2;]i levels are increased and that the rate of diastolic decay of [Ca2;]i is slowed significantly [3]. The Ca2; uptake into isolated sarcoplasmic 175
176 M Lindner, MC Brandt, H Sauer, J Hescheler, T Böhle, DJ Beuckelmann
reticulum (SR)-vesicles was also found to be reduced in failing myocardium. This finding was associated with a reduction of the protein levels of the SR Ca2;-ATPase (SERCA-2a) and the m-RNA encoding for SERCA-2a [4–6]. We have demonstrated that the caffeine-induced Ca2; transients in isolated myocytes of terminal failing myocardium are reduced [7], indicating a reduced Ca2; load of the SR. These results were confirmed in a study by Pieske et al. [8] using rapid cooling contractures to estimate the SR Ca2; content. With the technique of confocal laser-scanning microscopy, Cheng et al. [9] were able to visualize in vitro single events of intracellular calcium release (Ca2; sparks) from the SR and to relate these observations to the concept of excitation-contraction coupling by Ca2; induced Ca2; release. It could be demonstrated further that the systolic Ca2; transient in a myocardial cell is the summation of a multitude of individual Ca2; sparks [10]. However, at present it is controversial whether Ca2; sparks indicate the opening of a single Ca2; release channel of the SR (ryanodine receptor) or if they comprise openings of clusters of ryanodine receptor channels [11,12]. Ca2; sparks can arise spontaneously or could be triggered by Ca2; entering the cell through the L-type Ca2; channel during voltage-clamp pulses [10,13–15]. Ca2; sparks were not only observed in single cardiomyocytes but also in rat cardiac trabeculae [16]. Despite these studies, indicating unanimously the important link between Ca2; sparks and myocardial contractile function, not much evidence could be established on the properties of Ca2; sparks under conditions of heart-failure. In recent years only two animal models of heart failure or cardiac hypertrophy were concerned with Ca2; spark characteristics in the presence of cardiac disease [17,18]. At present, no data are available on Ca2; sparks in any preparation of human cardiomyocytes. Therefore, the aim of the present study was to characterize the kinetic properties of Ca2; sparks in human cardiomyocytes and to compare these in non-failing hearts and those with end-stage heart failure.
METHODS Cell isolation Cells were prepared from six hearts of patients with terminal heart failure resulting from dilated cardiomyopathy (DCM, n:2) or ischaemic cardiomyopathy (ICM, n:4) undergoing transplantation. All patients received digoxin and diuretics, and were receiving vasodilator therapy (ACE inhibitors). Informed consent was obtained from all patients prior to organ explantation. Results were compared with cells isolated from human hearts without heart failure that could not be transplanted for technical Cell Calcium (2002) 31(4), 175–182
reasons (n:3). The cell isolation was started within 1 (explanted failing hearts) to 3 h (non-utilized donor hearts) after explantation. The isolation procedure has been described in detail [19]. A part of the left ventricular wall was excised together with its artery branch and the wall segment was perfused via this artery branch for 30 min with a nominally Ca2; free modified Tyrode’s solution (see below), followed by 40 min with the same solution additionally containing collagenase (type II, 70 mg/50 ml, Worthington) and protease (type XIV, 3 mg/50 ml, Sigma Chemicals). Finally, the enzyme was washed out for 15 min with modified Tyrode’s solution containing 100 M Ca2;. Cells were prepared from areas within the central portion of the myocardial wall. Chunks of well-perfused myocardium were placed into Tyrode’s solution containing 2 mM Ca2; and filtered through a nylon mesh. During experiments, the myocytes were stored at room temperature in Tyrode’s solution containing 2 mM Ca2;. The living cell yield was approximately 5–10% in failing as well as in control myocardium. Only cells with clear cross striations and without significant granulation were selected for experiments. These cells contracted on stimulation. Solutions and loading of cells with Fluo-3 The nominally Ca2; free Tyrode’s solution used during the isolation steps was composed of (in mM): NaCl, 135; KCl, 4.0; MgCl2, 1.0; glucose, 10; NaH2PO4, 0.33; NaHEPES, 10; pH adjusted to 7.3 with addition of NaOH. When indicated, this solution additionally contained CaCl2 at different concentrations. For measurements under conditions of high external [Ca2;], modified Tyrode’s solution with 10 mM Ca2; was used. Before spark measurements, the myocytes were loaded with the fluorescent Ca2; indicator Fluo-3 by 20 min incubation with 10 M Fluo-3 (acetoxymethylester, Molecular Probes, Eugene, OR, USA) at 37 ⬚C. During the Ca2; spark recordings, the cells were superfused at room temperature (22 ⬚C) with modified Tyrode’s solution containing 2 mM or 10 mM CaCl2. Confocal microscopy Experiments were carried out under steady-state conditions by loading the SR with Ca2; through field stimulation (10 pulses at 1 Hz). Ca2; measurements were performed using a Zeiss laser-scanning confocal microscope (LSM 410 coupled to an Axiovert 135 M inverted microscope, Carl Zeiss, Germany) that was equipped with an argon laser. The objective used was a Zeiss 25 oil immersion Plan-Neofluar with a numerical aperture of 0.8. The excitation wavelength of Fluo-3 by the argon laser was 488 nm. The laser was kept at the lowest level and light © 2002, Elsevier Science Ltd. All rights reserved.
Calcium sparks in human cardiomyocytes 177
intensity was further attenuated with a neutral-density filter in the excitation path (to 1%) in order to minimize photo-toxicity and -bleaching. Fluorescence was measured at wavelengths 515 nm. The line-scan mode was used for the analysis of Ca2; sparks. Each myocyte was scanned repetitively along a line in its longitudinal axis at a frequency of 250 Hz. The line-scan image was constructed by 512 lines vertically. The magnification was set by the objective and the hardware zoom factor (3) of the laser scanning microscope to give a pixel size of ~0.1 m2 (0.330.33 m). The confocal detector aperture was set to provide an axial (z) resolution of ~3 m measured as full width half-maximal amplitude (FWHM) in images of 0.5 m fluorescent microbeads (Molecular Probes). The images were analysed using Lsmdummy (Carl Zeiss, Germany), Origin 5.0 (Microcal Software Inc., Northampton, MA, USA) and IDL 5.0 software (Research Systems Inc., Boulder, CO, USA) on an IBM compatible pentium personal computer. Computer analysis The line-scan images were analysed using an automated spark-detection routine partially based on an algorithm published by Cheng et al. [20]. In order to keep bias to a possible minimum, all steps in the analysis were performed automatically. A program with window-based user-interface was created reading the initial line-scan images from hard-disk and setting up all parameters for spark-detection automatically. Human intervention was only requested to confirm the initial steps of the algorithm, such as detection of cell-edges, and the results presented at the end of the analysis. After different smoothing filter steps, the cell edges were detected by the analysis routine. Potential spark regions that exceeded the overall standard deviation of the fluorescence baseline were excised to calculate a corrected baseline containing background noise only. Then the image was normalized using the corrected fluorescence baseline. As described in more detail by Cheng et al. [20], potential spark regions were then detected as connected regions that exceeded a fluorescence threshold given by the sum of the cell-fluorescence baseline and its standard deviation multiplied with a given constant. Only those potential spark sites were accepted, that exceeded a normalized fluorescence intensity 3.2-fold. As in the algorithm by Cheng et al. [20], those regions greater than a 2-fold baseline fluorescence were used for further analysis. Spacial information on every spark site was handed over to a mathematical subroutine, calculating the maximum value, width, rise- and decay time and the FWHM. In order to exclude a possible influence of a fading background fluorescence intensity on the © 2002, Elsevier Science Ltd. All rights reserved.
normalized images, the total line-scans of up to 15 s duration were stored as separate images of 2.24 s duration. For all of these recordings, a separate analysis was performed, normalizing each image with its individually calculated array of average background-fluorescence intensity. During image-processing, representative images of the dataset were stored automatically after every major step by the algorithm (see Fig. 1). In the course of spark-detection, the sparks were superimposed over the normalized image, giving the user the chance to view every spark step by step and to identify potentially false detections. Finally, the complete set of analysis data was stored together with a set of images taken during the mathematical processing of the raw images. All spark sites as calculated by the program were presented to the user with the option to delete false spark detections. Statistical analysis Results were expressed as mean values
50 ms
178 M Lindner, MC Brandt, H Sauer, J Hescheler, T Böhle, DJ Beuckelmann
50 ms
50 µm
50 µm Fig. 1 Spontaneous Ca2; sparks after SR Ca2; loading by field stimulation (10 pulses at 1 Hz) in a non-failing (A, B and E) and a failing (C, D) cardiomyocyte. A and C show the original line-scans as recorded from the confocal laser scanner. B, D and E show the processed images, digitally filtered and normalized, being more comprehensible in terms of spark distribution within the image. The original data consists of 512 scan lines along the longitudinal axis (2.24 s duration). The scale bars indicate temporal and spacial dimensions. In A and C, the colour scale directly represents the fluorescence intensity ⌬F, depending primarily on local changes of the Ca2; flourescence within the primary line-scan recording. In B, D and E, the colour scale is adapted to the interval of relative ⌬F/F0 values resulting from normalization (B, E: ⌬F/F0 values from 0.58 to 1.61; D, ⌬F/F0 values from 0.90 to 1.22). E shows the spark sites as detected by the algorithm and accepted by the authors as yellow areas, rejected spark sites are coloured in white. The images were taken immediately after the end of stimulation (t:0 s). The Ca2; spark frequency was significantly higher in cardiomyocytes from non-failing myocardium.
Cell Calcium (2002) 31(4), 175–182
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Calcium sparks in human cardiomyocytes 179
Frequency and characteristics of Ca2; sparks in failing human cardiomyocytes For comparison, the same SR Ca2; loading protocol was applied to cardiomyocytes isolated from terminally failing hearts. Figures 1C and D show the line-scan image of a failing myocyte with only a few spontaneous Ca2; sparks. A total of 1503 Ca2; sparks in failing human cardiomyocytes were analysed. The time to peak (mean 25.75<0.88 ms) and the half-time of decay (mean 66.48<1.89 ms) and the FWHM (3.99<0.06 m) were significantly different to control cells (P0.05). The Ca2; spark frequency was significantly lower in cells from failing myocardium compared to controls (2.81<0.20 s91 for HF vs. 4.15<0.42 s91 for NF; P0.05 at t:0 s; 1.36<0.34 s91 for HF vs. 2.75<0.65 s91 for NF; P0.05 at t:15 s). The Ca2; spark amplitude was also decreased significantly. The mean fluorescence ratio (F/F0) during the first second of scanning was 1.19<0.003; at t:15 s it was 1.14<0.01 (mean 1.19<0.002). ; sparks in Frequency and characteristics of Ca2; failing cardiomyocytes under conditions of increased Ca2; load
Incubation of failing myocytes in 10 mM external [Ca2;] caused the Ca2; spark frequency to increase approximately 2.5-fold immediately after the end of stimulation. A total of 671 Ca2; sparks were detected. In non-failing myocytes elevation of external Ca2; to 10 mM caused Ca2; overload of the cells as indicated by the induction of Ca2; waves. Under these conditions, individual Ca2; sparks could not be differentiated adequately. Figure 2 shows a summary of Ca2; spark frequency in all three groups (failing, control and failing cells) in 10 mM external Ca2; (6.64<0.80 s91 after cessation of stimulation, t:0 s; 5.0<0.91 s91, t:13 s) over the entire time of observation, indicating a significant difference between groups. Also Ca2; spark amplitude increased in the state of increased Ca2; load, but did not reach the amplitude of Ca2; sparks in control myocardium in physiological Ca2; concentration (2 mM). Figure 3 summarizes the Ca2; spark amplitude calculations (as fluorescence ratio) for all three groups. A significantly higher Ca2; spark amplitude was present in control myocytes compared to cells from failing hearts. After incubation of cells in a high-Ca2; solution during the first 10 s after cessation of the SR-loading procedure, the Ca2; spark amplitude was increased significantly (mean F/F0 1.26<0.01 after cessation of stimulation at t:0 s; 1.19<0.01 at t:13 s). A further significant increase in the time to peak Ca2; could be detected (30.52<1.24 ms; P0.05) as well as in the full width at half maximum (FWHM 4.34<0.10 m; P0.05). The half-time of decay of Ca2; sparks was unchanged in an elevated external Ca2; solution (67.07<2.82 ms). © 2002, Elsevier Science Ltd. All rights reserved.
Fig. 2 Frequency of Ca2; sparks visualized in line-scan images after SR Ca2; loading. A significant difference in Ca2; spark frequency could be detected between all three groups investigated, i.e. non-failing myocardium (2 mM external Ca2;), failing myocardium ICM and DCM, 2 mM and 10 mM external Ca2;), during the full time of observation (15 s). The elevation of the external Ca2; to 10 mM resulted in an approximately 2.5-fold rise of the Ca2; spark frequency in failing myocardium.
Fig. 3 The Ca2; spark amplitude (fluorescence ratio F/F0) showed a decrease with time in all cells investigated. Significant differences in the Ca2; spark amplitude could be detected between failing (ICM and DCM) and non-failing myocardium in physiological Tyrode’s solution. By elevating the external Ca2; concentration to 10 mM, the Ca2; spark amplitude rose significantly in failing myocardium over the first 10 s, but not to the values as in non-failing cardiomyocytes in physiological Tyrode’s solution (2 mM Ca2;).
DISCUSSION This is the first study to report on the characteristics of Ca2; sparks in isolated human ventricular cardiomyocytes. The purpose of this study was to investigate if the Cell Calcium (2002) 31(4), 175–182
180 M Lindner, MC Brandt, H Sauer, J Hescheler, T Böhle, DJ Beuckelmann
Ca2; release mechanism of the SR, as indicated by the Ca2; spark characteristics, is altered in ventricular myocytes from patients with terminal heart failure, and if such alterations could explain the changes of intracellular [Ca2;]i handling that have been found in these cells [3,9]. According to the ‘local control theory’ of excitationcontraction coupling proposed by Stern [21], Ca2; induced Ca2; release is initiated locally by Ca2; influx through a single L-type Ca2; channel that activates a group of SR Ca2; release channels [12]. The Ca2; transient is then thought to be the summation of multiple local Ca2; releases during depolarization of the cell membrane [10]. Ca2; sparks are thought to indicate release of Ca2; from one or few of these local units of excitation-contraction coupling. In our experiments, cells from terminally failing myocardium not only showed a significantly lower Ca2; spark frequency compared to non-failing controls, but also spacio–temporal Ca2; spark characteristics were changed in the presence of heart failure. By inducing a higher Ca2; load in failing myocytes (10 mM external Ca2; concentration), the Ca2; spark frequency could be increased to values higher than in control cells under physiological conditions. The Ca2; spark amplitude was also increased significantly, but not to the extent as in control myocytes. However, the time to peak Ca2; increased even further with a higher Ca2; load, as well as the FWHM, which was already increased in failing myocardium. From these data, we conclude that the spacio–temporal alteration of Ca2; sparks in heart failure are not only due to a reduced Ca2; load of the sarcoplasmic reticulum. Spontaneous Ca2; sparks have been described in ventricular myocytes and in skeletal muscle cells of several other mammalian species. In the majority of studies on rat and guinea-pig cardiomyocytes [9,14–16], the time to peak was faster than in our experiments on human ventricular cells. Only Satoh et al. [22] measured a time to peak of approximately 15 ms (rat and rabbit ventricular myocytes) comparable to the values in our study. In our experiments, the half-time of decay of human myocardial cell Ca2; sparks was slower than in all other species described until now [9,14–16,22]. These findings demonstrate that even in the absence of cardiac disease Ca2; spark characteristics in human cardiomyocytes differ from those derived from animal studies. In an animal model of experimental cardiac hypertrophy and heart failure, Gomez et al. [17] showed unchanged temporal characteristics of voltage-clamp-induced Ca2; sparks. In this animal model of cardiomyocytes with clinical signs of heart failure, Ca2; transients were reduced and prolonged, but the L-type Ca2; current was unchanged. In the present study, we detected significant differences of temporal characteristics of Ca2; sparks Cell Calcium (2002) 31(4), 175–182
between non-failing and terminally failing myocytes. Furthermore, in contrast to our results on human cardiomyocytes, in which caffeine-induced Ca2; transients are reduced [7], Gomez et al. [17] showed that caffeineinduced Ca2; transients were unaltered in failing rat myocardium. Therefore, they concluded that alterations of intracellular Ca2; handling can only be explained by a change of the micro-architecture between the ryanodine receptor and the sarcolemmal L-type Ca2; channel, indicating an impaired Ca2; induced Ca2; release mechanism. The results of the present study also suggest an alteration of the Ca2; release mechanism, as the Ca2; spark characteristics (time to peak, half time of decay and FWHM) are significantly different in HF compared to NF. In isolated rat cardiomyocytes, Song et al. [23] showed that the Ca2; spark amplitude, but not the frequency, depends on the SR filling-state. In our study, however, both parameters are changed. Our analysis was performed with an equivalent computer routine as that described by their group [20]. A reduced number of L-type Ca2; channels (dihydropyridine receptors) could also impair SR Ca2; loading. However, measurements of dihydropyridine receptors showed that the number of Ca2; channels are unchanged in human heart failure [24,25]. Functional measurements of the L-type Ca2; current did not show any differences between control and failing myocardium [3], although these results could be frequency-dependent [26]. Results of studies trying to quantify SR Ca2; release channels (ryanodine receptors) are controversial. Brillantes et al. [27] demonstrated a reduction of mRNA levels encoding for the ryanodine receptor only in ischaemic cardiomyopathy compared to control myocardium. Go et al. [28] also found mRNA levels for the SR Ca2; release channels to be reduced in ischaemic and dilated cardiomyopathy, whereas other groups did not find any differences in mRNA expression [29], but showed an increase in ryanodine binding sites [30]. Electrophysiological single-channel recordings performed by Holmberg and Williams [31,32] indicated unimpaired characteristics of human Ca2; release channels in heart failure. A recent study by Ching et al. [33] gives evidence that besides the cytosolic Ca2; concentration luminal Ca2; regulatory mechanisms and the amount of intra-SR [Ca2;] are important for the SR Ca2; release. This might partially explain our data, especially the observed changes in Ca2; spark frequency, but not the persisting difference in Ca2; spark amplitude between failing and non-failing myocardium. Coupled gating between individual ryanodine receptors in skeletal muscle was described by Marx et al. [34]. A possible cause for changes in the excitation-contraction coupling and Ca2; spark characteristics could be the inhibition of coupled gating of the ryanodine receptors. Another group suggested that FKBP could also help in © 2002, Elsevier Science Ltd. All rights reserved.
Calcium sparks in human cardiomyocytes 181
the cooperative gating of multiple ryanodine receptors. Targeted knockout of the mouse FKBP12 gene produces severe cardiomyopathy with altered cardiac ryanodine receptor gating, whereas skeletal muscle function is not affected [35]. Our results support an alteration of coupled gating, as indicated by the characteristics of Ca2; sparks in heart failure. Recently, it could be shown that in failing human myocardium the type 2 ryanodine receptor polypeptides are protein kinase A hyperphosphorylated, resulting in a defective channel function due to increased sensitivity to Ca2; induced activation [36]. Discussions on the possible influence of alterations of ryanodine receptor activity on cardiac function are still controversial. From a mathematical model of net calcium fluxes, Eisner and colleagues [37–39] concluded that a decreased efflux through the calcium release channel would have only transient effects on systolic calcium, but no effects on cardiac contractility in a steady state. This model is based on experiments with different species (rat, ferret and guinea-pig) at room temperature and at 37 ⬚C. The Na/Ca exchange current is used as a tool to measure calcium fluxes. As the Na/Ca exchange current in human cardiac cells has not yet been characterized, further investigations will be required for a better understanding. In summary, our results support the hypothesis that although a major defect of excitation-contraction coupling in human terminal heart failure is a reduction of the SR Ca2; load, an additional alteration of the Ca2; release mechanism has to be postulated. ACKNOWLEDGEMENTS This research was supported by grants of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (01 KS 9502, Zentrum für Molekulare Medizin Köln, Projekt 4) and the M. & W. Boll Stiftung). Our special thanks go to Prof. DeVivie (Department of Cardiac Surgery, University of Cologne) and his colleagues for providing the myocardial tissue.
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