Calcium Content of the Sarcoplasmic Reticulum in Isolated Ventricular Myocytes from Patients with Terminal Heart Failure

J Mol Cell Cardiol 30, 743–749 (1998)

Calcium Content of the Sarcoplasmic Reticulum in Isolated Ventricular Myocytes from Patients with Terminal Heart Failure Michael Lindner, Erland Erdmann and Dirk J. Beuckelmann Department of Medicine III, University of Cologne, 50924 Cologne, Germany (Received 22 January 1997, accepted in revised form 26 November 1997) M. L, E. E  D. J. B. Calcium Content of the Sarcoplasmic Reticulum in Isolated Ventricular Myocytes from Patients with Terminal Heart Failure. Journal of Molecular and Cellular Cardiology (1998) 30, 743–749. Systolic [Ca2+]i-transients have been shown to be depressed in isolated ventricular myocytes from patients with terminal heart failure compared to controls. Experiments were performed in human ventricular cells to investigate whether this reduced systolic [Ca2+]i-transient may be due to a decreased Ca2+-content of the sarcoplasmic reticulum (SR). Single myocytes were isolated from left ventricular myocardium of patients with terminal heart failure undergoing cardiac transplantation. These results were compared to those obtained from cells of healthy donor hearts that were not suitable for transplantation for technical reasons. [Ca2+]i-transients were recorded from isolated cells under voltage clamp perfused internally with the Ca2+indicator fura-2. The Ca2+-content of the SR was estimated by rapid extracellular application of caffeine (10 m) to open the Ca2+-release channel of the SR and comparison of the caffeine-induced [Ca2+]i-transients in cells from patients with heart failure and from controls without heart failure. Upon steady-state depolarizations to +10 mV (maximum of the Ca2+-current), [Ca2+]i-transients in cells from patients with heart failure were significantly smaller than in myocytes from undiseased hearts (333±26 v 596±80 n, P<0.05). Application of caffeine caused a [Ca2+]i-transient that was always larger than during depolarization. Caffeine-induced [Ca2+]i-transients were significantly smaller in cells from diseased hearts compared with controls (970±129 v 2586±288 n, P<0.01). A positive correlation was found between left ventricular ejection fraction and caffeine-induced [Ca2+]i-transients in these cells. It is concluded, that depressed [Ca2+]i-transients in myocytes from patients with heart failure may be caused by a decreased Ca2+-content of the SR possibly due to an altered Ca2+-ATPase activity in these hearts. It is not necessary to postulate an additional defect of the Ca2+-release function of the SR to account for the alterations of intracellular (Ca2+]i-handling.  1998 Academic Press Limited K W: Human ventricular myocyte; Sarcoplasmatic reticulum; Whole-cell clamp; Fura-2; Caffeine.

Introduction Intracellular [Ca2+]i-handling has been shown to be altered in myocardium of patients with severe heart failure (Gwathmey et al., 1987, 1990; Beuckelmann et al., 1992). In single ventricular cells isolated from myocardium of these patients whose disease was due to dilated or ischemic cardiomyopathy systolic [Ca2+]i-transients were reduced,

diastolic [Ca2+]i-levels were increased and the rate of diastolic decay of [Ca2+]i was significantly slowed (Beuckelmann & Erdmann, 1992; Beuckelmann et al., 1992). These changes are thought to be partially responsible for the altered contractility of these hearts in vivo. Experiments have provided evidence for an alteration of the Ca2+-uptake of the SR (Mercadier et al., 1990; Takahashi et al., 1992). Results concerning possible alterations of the SR

Please address all correspondence to: D. J. Beuckelmann, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany.

0022–2828/98/040743+07 $25.00/0

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Ca2+-ATPase protein have been controversial. In a recent publication, Schwinger et al. (1995) showed that the SR Ca2+-ATPase protein is unchanged, but that mRNA levels encoding for this enzyme are reduced in heart failure. However, Studer et al. (1994) found that the SERCA-protein was reduced. These controversial results are possibly due to methodological differences. A reduction of systolic [Ca2+]i-transients and the prolonged diastolic [Ca2+]i-decay can be explained by two alternative mechanisms. A reduction of the SR [Ca2+]i-uptake capacity would lead to a slower diastolic [Ca2+]i-decay and the SR Ca2+-content would be reduced. During the next beat the Ca2+ available to be released would then be reduced. This possible mechanism is supported by data of Pieske et al. (1996), who showed that post-restpotentiation, an indirect indicator for the SR calcium content, was reduced in failing myocardium. Alternatively, an alteration of the Ca2+-release mechanism would cause a reduced systolic [Ca2+]itransient. The slowed diastolic [Ca2+]i-decay would then be due to an SR Ca2+-overload with the Ca2+ATPase pumping against a very steep concentration gradient. In this case an increased SR Ca2+-content would be expected. Rapid cooling contractures have been used to estimate Ca2+-loading of the SR in multicellular preparations (Bers, 1987; Hryshko et al., 1989), as well as in single cells (Hryshko et al., 1989; Terracciano et al., 1995). However, another and easier methodological approach to estimate the SR calcium content is the use of caffeine. Caffeine opens the SR release channel (Rousseau and Meissner, 1989) and extracellular application of caffeine causes an instant release of Ca2+ from the sarcoplasmic reticulum into the cytoplasm. It has been shown that these caffeine-induced [Ca2+]i-transient can be used as a semiquantitative measure of the SR Ca2+-content (Bers, 1987; Hryshko et al., 1989; Varro et al., 1993; Terracciano et al., 1993). The purpose of the present study was to investigate whether the Ca2+-content of the sarcoplasmic reticulum is altered in ventricular myocytes from patients with severe heart failure, and if this may explain the alterations of systolic and diastolic intracellular [Ca2+]i-handling in these cells.

Materials and Methods Patients Cells were prepared from 23 hearts of patients with end-stage heart failure due to dilated cardio-

myopathy (DCM, n=14) or ischemic cardiomyopathy (ICM, n=9) undergoing transplantation. Patients’ mean age was 48±18 years, male:female was 17:6, cardiac index was 2.5±0.6 l/min/m2, ejection fraction was 22±8%. All patients received digoxin and diuretic and were under vasodilator therapy. Informed consent was obtained in all patients prior to organ explanation. Results were compared with cells isolated from five human hearts without heart failure that could not be transplanted for technical reasons.

Cell isolation The isolation procedure has been described in detail before (Beuckelmann et al., 1992). A part of the left-ventricular wall was excised together with its artery branch. The wall segment was then perfused via this artery branch for 30 min with a nomically Ca2+-free modified Tyrode’s solution (138 m NaCl; 4 m KCl; 1 m MgCl2; 10 m glucose; 0.33 m NaH2PO4; 10 m HEPES; pH 7.3 with addition of NaOH, 37°C), followed by 40 min with the same solution with added collagenase (type II, 70 mg/ 50 ml, Worthington) and protease (type XIV, 6 mg/ 50 ml, Sigma Chemicals). Finally, the enzyme was washed out for 15 min with modified Tyrode’s solution that contained 200 l Ca2+. Cells were prepared from areas within the central third of the myocardial width. Myocytes were disaggregated by mechanical agitation and, after filtering through a nylon mesh, stored in Tyrode’s solution containing 2.0 m Ca2+ at room temperature. The living cell yield was approximately 5–8%. Only cells with clear cross-striations without significant granulation or spontaneous contractions were selected for experiments.

Solutions and loading of cells with fura-2 After establishing whole-cell recordings, the electrode solution was allowed to exchange with the intracellular environment (5–10 min). Electrodes had resistances of 2.0–3.0 MX and were filled with 0.05 m fura-2 (Molecular Probes); 120 m Csaspartate; 10 m CsCl; 1 m MgCl2; 5 m NaCl; 10 m HEPES (cesium salt); and 2 m Mg-ATP; pH was 7.2 with addition of CsOH. Positive pressure to the electrode was never applied to avoid potential disintegration of intracellular structures during such procedure. Cells were superfused, at 37°C, with a modified Tyrode’s solution containing 2.0 m CaCl2; 140 m

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NaCl; 10 m CsCl; 1 m MgCl2; 10 m glucose; 10 m HEPES (Na+-salt); pH was 7.3 with addition of NaOH. Approximately 5 ll of the same solution to which caffeine 10 m had been added were applied to the surface of the myocyte through a second pipette with a 10-lm tip when indicated.

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[Ca ]i-measurements Experiments were carried out using standard wholecell recording techniques (Hamill et al., 1981), employing a patch-clamp amplifier model EPC-7 (LIST Instruments) with a 100-MX feedback resistor. For fluorescence recordings, u.v. light, emitted from a 75-W Xenon arc lamp, passed through 10nm interference filters (340 or 380 nm wavelengths) and was reflected by a dichroic mirror centered at 405 nm into the objective for excitation of the Ca2+-indicator in the cell. Fluorescence emitted from the cell passed through a 510–540 nm bandpass filter and was directed into a photomultiplier tube. An additional pipette was positioned near the electrode tip (diameter: 10 lm) for application of the caffeine solution. During fluorescence, recordings upon caffeine-application cells were held at −45 mV. All fluorescence recordings were filtered with a cut-off frequency of 120 Hz (Bessel), digitized (1 kHz), and stored for off-line analysis. [Ca2+]i-measurements through the use of a single wavelength of fura-2 fluorescence have been described before (Vranesic and Kno¨pfel, 1991). Since recordings had to be made at two wavelengths, two identical test pulses had to be given at both wavelength to +10 mV. The validity of the necessary assumption that during repetitive test pulses [Ca2+]i-transients are similar was demonstrated many times by recording fluorescence signals at the same wavelengths during successive depolarizations. [Ca2+]i-transients upon application of extracellular caffeine were calculated from fluorescence recordings using 340 nm as excitation wavelength. The linear relationship between fluorescence changes upon the last depolarizations to +10 mV upon excitation of the intracellular indicator at 340 and 380 nm was used to obtain a calibration for the calculation of [Ca2+]i from a single wavelength fluorescence recording. [Ca2+]itransients were calculated from these recordings using the equation of Grynkiewicz et al. (1985). Upon application of extracellular caffeine, fluorescence was recorded at 340 nm, and [Ca2+]i was calculated using the calibration as described above.

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Figure 1 Linear relation between fluorescence recordings upon excitation with 340 and 380 nm. Repetitive stimulation of a human ventricular myocyte to +10 mV. The corresponding fluorescence recording is shown in Figure 2 (au). Pulse duration was 200 ms. A linear relation could be demonstrated between fluorescence recordings during excitation at 340 and 380 nm while the cell was depolarized to +10 mV (data points from the last two depolarizations to +10 mV before caffeine application). m=0.92, r=0.80.

Figure 1 demonstrates the linear relation between fluorescence recordings during excitation at 340 and 380 nm while the cell was depolarized to +10 mV. Statistical analysis Results are expressed as mean values±standard errors. Mann–Whitney non-parametric analysis was used for statistical evaluation of the data and a P-value <0.05 was considered significant.

Results Thirty-eight cells from failing myocardium (13 cells from nine hearts with ischemic cardiomyopathy, and 25 cells from 14 hearts with dilated cardiomyopathy) and 14 cells from five non-failing donor hearts were studied in these experiments. The average size of the myocytes was estimated by measurements of cell capacitance assuming a specific capacitance of 1 lF/cm2 (188±22×10−6 cm2 in the non-failing myocardium, 219±27×10−6 cm2 in the failing myocardium; ..). Voltage- and caffeine-induced [Ca2+]i-transients – control A cell isolated from a control heart was stimulated six times at a frequency of 1 Hz to +10 mV at

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Figure 2 Voltage- and caffeine-induced [Ca2+]i-transients – control. A cell isolated from a control heart was stimulated six times at a frequency of 1 Hz to +10 mV at an alternating excitation wavelength of 340 and 380 nm. After the sixth pulse, the membrane potential was held at −45 mV and extracellular caffeine was applied during excitation of the cell at 340 nm (a). (b) depicts the calculated [Ca2+]i-transients upon stimulation to +10 mV and upon application of extracellular caffeine determined from the fluorescence recording (au) at 340 nm as shown in Figure 1. ∗, Shutter open; †, shutter closed.

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Figure 3 Voltage- and caffeine-induced [Ca2+]i-transients – heart failure. The same protocol as in Figure 2 was applied to a cell from a patient with terminal heart failure due to ischemic cardiomyopathy. Fluorescence changes upon depolarization to +10 mV and upon application of caffeine were markedly smaller than in control cells (a). [Ca2+]i-transients upon depolarization to +10 mV and on caffeine-application were significantly smaller than in the control cell (b). ∗, Shutter open; †, shutter closed.

age-clamp stimulation 596±80 n. alternating excitation wavelengths of 340 and 380 nm [Fig. 2(a)], to assure a steady-state level of SR Ca2+-loading. Cells that were stimulated with up to 12 times at the frequency of 1 Hz prior to caffeine application showed no further change of their [Ca2+]i-transients. [Ca2+]i-transients were maximal upon stimulation to this membrane voltage. After the sixth pulse, the membrane potential was held at −45 mV and extracellular caffeine was applied during excitation of the cell at 340 nm. Figure 2(b) depicts the [Ca2+]i-transients upon stimulation to +10 mV and upon application of extracellular caffeine calculated from the fluorescence recording at 340 nm, as described in Figure 1. During depolarization of the cell membrane to +10 mV, the [Ca2+]i-transient reached 1300 n. Upon caffeine application, [Ca2+]i increased to 3500 n. The average [Ca2+]i-transient upon volt-

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Voltage- and caffeine-induced [Ca2+]i-transients – heart failure When the same protocol as above was applied to a cell from a patient with terminal heat failure fluorescence changes upon depolarization to +10 mV, and upon application of caffeine were markedly smaller than in control cells [Fig. 3(a)]. The [Ca2+]i-transient [Fig. 3(b)] during the test pulse was 410 n. When caffeine was applied to the cell membrane, [Ca2+]i increased to 800 n. Results in myocytes isolated from hearts of patients with dilated cardiomyopathy were qualitatively similar. In accordance with previous experiments (Beuckelmann et al., 1992), average [Ca2+]i-transients were significantly reduced (333±26 nmol; P<0.05).

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Figure 4 Caffeine-induced [Ca2+]i-transients. Average of maximal [Ca2+]i-transients upon application of caffeine in patients with severe heart failure due to dilated cardiomyopathy and ischemic heart disease compared to controls. Mean values are shown ±... ∗, P<0.01.

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Caffeine-induced [Ca ]i-transients Figure 4 illustrates the average [Ca2+]i-transients upon application of caffeine in patients with severe heart failure due to dilated cardiomyopathy and ischemic heart disease compared to controls. The caffeine-induced [Ca2+]i-transients were significantly reduced in myocytes from patients with dilated and ischemic cardiomyopathy compared to controls (P<0.01). No significant difference could be observed between different types of heart failure.

Ejection fraction and caffeine-induced [Ca2+]i-transients Figure 5 depicts the relationship between the leftventricular ejection fraction measured prior to transplantation in ventriculograms and caffeineinduced [Ca2+]i-transients. A weak but significant positive correlation could be found. As ventriculograms were not done in the control group, only hearts from patients with heart failure were included in this analysis.

Discussion The purpose of this study was to investigate whether the Ca2+-content of the SR may be altered in ventricular myocytes from patients with heart failure, thus explaining the changes of intracellular [Ca2+]itransients which have been found in these cells. Our results indicate that after steady-state stimulation, application of caffeine elicits a smaller [Ca2+]i-transient in myocytes from patients with severe heart

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Figure 5 Ejection fraction and caffeine-induced [Ca2+]itransients. Relationship between the left ventricular ejection fraction measured invasively during heart catheterization prior to transplantation and caffeine-induced [Ca2+]i-transients. Average values for [Ca2+]i were used if more than one cell was measured in each patient. A weak but significant positive correlation between caffeineinduced [Ca2+]i-transients and ejection fraction could be found. r=0.76.

failure, due to dilated or ischemic cardiomyopathy compared to controls. Caffeine-induced [Ca2+]i-transients have been used as a measure for the Ca2+-content of the sarcoplasmic reticulum (Bers, 1987; Hryshko et al., 1989; Varro et al., 1993). However, some prerequisites are necessary to conclude that a smaller caffeine-induced [Ca2+]i-transient is indicative of a decreased Ca2+-content of the SR. Cellular and subcellular heterogeneity of [Ca2+]i in single ventricular myocytes under conditions of calcium overload of the cell was reported by several authors (Wier et al., 1987; Cheng et al., 1993; Cannell et al., 1994). However, we had no indication of a [Ca2+]i-overload of our cells under our experimental conditions. [Ca2+]i-transients upon caffeine-application may be influenced by an alteration of the activity of the Na+–Ca2+-exchanger of the cell membrane. Studer et al. (1994) reported the mRNA as well as the protein levels of the Na+–Ca2+-exchanger in terminally failing myocardium to be increased in comparison to non-failing human myocardium. These results were confirmed by Flesch et al. (1996). However, no data on the function of the Na+–Ca2+

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exchanger in intact human have yet been published. Furthermore, as Figures 2 and 3 show, the increase of [Ca2+]i upon caffeine-application was very rapid and the decay was slow. Therefore, the impact of an altered Na+–Ca2+ exchanger’s activity on peak [Ca2+]i-transients can be assumed to be small. However, the rate of decay of [Ca2+]i was not investigated systematically in this study. A further essential prerequisite for our interpretation is that application of extracellular caffeine completely opens the SR release channel for all Ca2+ to be released from the SR into the cytoplasm. O’Neill et al. (1990) measured [Ca2+]i-transients and caffeine concentrations in rat ventricular myocytes. They showed that a concentration of 1 mmol caffeine in the cell was sufficient to open the SR–Ca2+ channels and trigger a maximal [Ca2+]i-transient. Some data indicate that the caffeine-sensitivity of the SR release channel may be reduced in heart failure (D’Agnolo et al., 1992). In our present experiments, we used 5 ll of a solution with a caffeine concentration of 10 m through a pipette in close proximity to the cell surface, but without touching the membrane. Using such an experimental arrangement, the flow from this pipette was rather strong. Therefore, the concentration of caffeine during its application to the cell surface was close to 10 m: sufficiently high to open all SR release channels. A reduction of systolic [Ca2+]i-transients has been described before in isolated cells from patients with heart failure (Beuckelmann et al., 1992). Gwathmey et al. (1990) and Morgan (1991) did not observe these differences in the peak systolic Ca2+-transients between non-failing and terminally failing human myocardium in isometrically contracting ventricular muscle strip preparations, using aequorin as a Ca2+-indicator. However, Pieske et al. (1994) showed a decreased systolic [Ca2+]i-transients at higher stimulation frequencies in aequorin-loaded muscle strip preparations, indicating that alterations in systolic [Ca2+]i-transients in isometrically contracting preparations of patients with heart failure may only be observed at higher frequencies. Measurements of [Ca2+]i-transients above 1500 n are difficult to quantify, due to the relatively low dissociation constant of fura-2 (200 n). Therefore, Figure 5 should only indicate that there is a positive relation between the left ventricular ejection fraction and caffeine-induced [Ca2+]i-transients. Due to these methodological difficulties, it should not be concluded that this relationship is necessarily linear. In isolated ventricular myocytes, characteristics

and current density of the triggering Ca2+-current were found to be unchanged (Beuckelmann et al., 1991, 1992; Cohen and Lederer, 1993; Mewes and Ravens, 1994). Therefore, either an alteration of the SR release mechanism or a reduction of SR Ca2+-loading has to be postulated to account for the reduction of systolic [Ca2+]i-transients in heart failure. Our finding of a reduced Ca2+-content is in line with the finding of a reduced SR 45Ca2+-uptake from these hearts (Pieske et al., 1994; Schwinger et al., 1995). Most authors found the expression of the mRNA encoding for the SR Ca2+-ATPase to be significantly reduced (Mercadier et al., 1990; Takahashi, 1992; Studer et al., 1994; Schwinger et al., 1995). We recently showed that the rate of Ca2+ uptake into the sarcoplasmic reticulum in intact ventricular myocytes from patients with severe heart failure is profoundly reduced (Beuckelmann et al., 1995). Therefore, our present finding of a reduced SR Ca2+ content may be the result of an alteration of diastolic Ca2+ uptake into the SR. An additional defect of the release mechanism cannot be excluded by our study. However, one does not have to postulate such an additional defect to explain our findings of a reduced systolic [Ca2+]itransient. The alternative hypothesis that diastolic [Ca2+]i-decay would be impaired due to an SR Ca2+overload with the Ca2+-ATPase pumping against a very steep concentration gradient can be excluded by our experiments. Our results indicate that a major part of the alterations of systolic and diastolic [Ca2+]i-handling in heart failure can be explained by a reduced Ca2+reuptake from the cytoplasm into the sarcoplasmic reticulum. This would suggest that selectively enhancing Ca2+ uptake into the SR by pharmaceutical agents would be a promising pharmacological intervention in these patients.

Acknowledgments This research was supported by grants of the Deutsche Forschungsgemeinschaft (Be 1113/2-3) and the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie (01 KS 9502, Zentrum fu¨r Molekulare Medizin Ko¨ln, project 4). Our special thanks are due to Prof. DeVivie (Dept of Cardiac Surgery, University of Cologne) and to Prof. B. Reichart (Dept of Cardiac Surgery, University of Munich) and their colleagues for providing the myocardial tissue.

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