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Altered diastolic [Ca2+]i handling in human ventricular myocytes from patients with terminal heart failure
April 1995
Beuckelmann et al.
14. Helfenstein U. The use of transfer function models, intervention analysis and related time series methods in epidemiology. Int J Epidemiol 1991;20:808-15. 15. Box GEP, Jenkins GM. Time series analysis: forecasting and control. Oakland, Calif.: Holden-Day, 1976. 16. Bloomfield P. Fourier analysis of time series: an introduction. New York: Prentice-Hall, 1974. 17. Imperi GA, Lambert CR, Coy K, Lopez L, Pepine CJ. Effects of titrated beta blockade (metoprolol) on silent myocardial ischemia in ambulatory patients with coronary artery disease. Am J Cardiol 1987;60:519-24. 18. Frishman W, Charlap S, Kimmel B, Teicher M, Cinnamon J, Allen L, Strom J. Diltiazem, nifedipine, and their combination in patients with stable angina pectoris: effects on angina, exercise tolerance, and the ambulatory electrocardiographic ST segment. Circulation 1988;77:77486. 19. Mulcahy D, Cunningham D, Crean P, Wright C, Keegan J, Quyyumi A,
American Heart Journal
Park A, Fox. Circadian variation of total ischemic burden and its alteration with anti-anginal agents. Lancet 1988;2:755-9. 20. Stone PH, Gibson RS, Glasser SP, DeWood MA, Parker JD, Kawanishi DT, Crawford MH, Messineo FC, Shook TL, Raby K, ~urtis DG, Hoop RS, Young PM, Braunwald E. Comparison of propranolol, diltiazem, and nifedipine in the treatment of ambulatory ischemia in patients with stable angina. Circulation 1990;82:1962-72. 21. Deedwania PC, Carbajal EV, Nelson JR, Halt H. Anti-ischemic effects of atenolol versus nifedipine in patients with coronary artery disease and ambulatory silent ischemia. J Am Cell Cardiol 1991;17:963-9. 22. Theroux P, Baird M, Juneau M, Warnica W, Klinke P, Kostuk W, Pflugfelder P, Lavallee E, Chin C, Dempsey E, Grace M, Lalonde Y, Waters D. Effect of diltiazem on symptomatic and asymptomatic episodes of ST segment depression occurring during daily life and during exercise. Circulation 1991;84:15-22.
Altered diastolic [Ca2+]i handling in human ventricular myocytes from patients with terminal heart failure To investigate whether the slow diastolic decay of [Ca2+]i in myocardium of patients with heart failure is a result of alterations of the Ca 2t adenosine triphosphatase of the sarcoplasmic reticulum or the sarcolemma, [Ca2÷]~ transients were recorded in voltage-clamped ventricular cells isolated from hearts of patients with terminal heart failure or from undiseased donor hearts. To isolate the [Ca2t]i-reuptake function of the sarcoplasmic reticulum, myocytes were dialyzed via the patch pipette with Na÷-free solution and incubated in Ca2t-free and Nat-free solution to inhibit Nat/Ca 2÷ exchange. After superfusion with Ca2÷-containing, Nat-free medium, the sarcoplasmic reticulum was loaded with Ca 2t through repetitive voltage-clam p pulses to +10 mV. Under these conditions, [Ca2t]~ decay was significantly slower in myocytes from patients with heart failure (538 - 66 msec) than in controls (305 ± 16 msec; p < 0.05). After the addition of 10 mmol/L of caffeine, [Ca2t]~ levels did not show appreciable decay between two voltage-clamp pulses in diseased and undiseased myocytes. We conclude that diastolic decay of [Ca2+]i in ventricular myocytes from patients with terminal heart failure is partially the result of a decreased rate of Ca 2t reuptake by the sarcoplasmic reticulum. Sarcolemmal Ca 2t adenosine triphosphatase does not contribute significantly to cytoplasmic [Ca2÷]i removal during an individual heartbeat. (AM HEART J 1995;129:684-9.)
Dirk J. Beuckelmann, MD, Michael N~ibauer, MD, Carsten Krfiger, MD, and Erland Erdmann, MD Cologne, Germany
Alterations of diastolic relaxation of the myocardium is one of the prominent features of the heart in severe heart failure a n d hypertrophy. 13 Several mechaFrom the Department of Medicine III, University of Cologne. Supported by a grant of the Deutsche Forschungsgemeinschaft (Be 1113/ 2-2) and the Friedrich-Baur-Stiftung. Received for publication Feb. 17, 1994; accepted July 5, 1994. Reprint requests: Dirk J. Beuckelmann, MD, Department of Medicine III, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany. Copyright © 1995 by Mosby-Year Book, Inc. 0002-8703/95/$3.00 + 0 4/1/61205
684
nisms are responsible for this phenomenon. However, on the cellular level it has been shown 4 that delayed relaxation is caused by a slow diastolic decay of intracellular [Ca2+]i. It has been suspected that a reduced [Ca2+]i reuptake rate of the sarcoplasmic reticulum (SR) may be a major reason for the slow decline of [Ca2+]i and for the prolongation of diastolic relaxation in these patients. Results of experiments that were designed to measure the activity of the Ca 2+ adenosine triphosphatase (ATPase) of the SR in human beings have been conflicting. Some authors
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Intracellular [Ca2+]i-Handling Na+/Ca2+ exchange
Ca2 + - A T P a a e
Ca2+.channel
~::,!~,:;~;:~ii:i~:::~,:, : ~: i,i~:i:,i~: ?~:I, ,:~,/,:, ~i,~, ~
i
ADP
+ Pl
[Ca2+]i~t
J
~
/
rcOpal=m rectlcluU lm
contractile proteins ATP ADP + P|
Fig. 1. Scheme of intracellular [Ca2+]i handling in ventricular myocyte. Ca 2+ entering cell through voltage-activated Ca 2+ channel triggers release of Ca 2+ from SR. Intracellular [Ca2+]i increases and binds to troponin C, initiating contraction. Relaxation is initiated by separation of Ca 2+ from Ca 2+, binding contractile proteins and removal from cytoplasm by sarcolemmal Ca2+-ATPase, extrusion via Na+/Ca2+-ex change system, Ca 2+ reuptake by Ca2+-ATPase of SR, and binding to intracellular Ca 2+ buffers. ADP, Adenosine diphosphate.
r e p o r t e d an u n c h a n g e d Ca 2+ u p t a k e r a t e in severe h e a r t failure w h e n c o m p a r e d to the Ca 2+ u p t a k e rate in u n d i s e a s e d controls. 5 Others found t h e expression of the genes encoding for the SR Ca2+-ATPase to be significantly reduced, e' 7 During diastole, Ca 2+ is r e m o v e d f r o m the cytop l a s m of the m y o c y t e b y four m a i n m e c h a n i s m s (Fig. 1): (1) Ca 2+ r e u p t a k e b y the SR t h r o u g h an energyd e p e n d e n t Ca2+-ATPaseS; (2) t r a n s s a r c o l e m m a l ext r u s i o n by N a + / C a 2+ exchange 9' 10; (3) t r a n s s a r c o l e m m a l extrusion t h r o u g h an e n e r g y - d e p e n d e n t Ca 2+A T P a s e of the cell membrane11; a n d (4) the binding of Ca 2+ to intracellular p r o t e i n s like t r o p o n i n and calmodulin.12, 13 However, the binding of Ca 2+ to intracellular contractile proteins has already b e e n shown to be u n c h a n g e d in m y o c a r d i u m of p a t i e n t s with t e r m i n a l h e a r t failure. ~4 T h e r e f o r e the p u r p o s e of the p r e s e n t s t u d y was to investigate w h e t h e r alterations of Ca 2+ r e u p t a k e into the SR or Ca 2+ extrusion via the s a r c o l e m m a l Ca 2+ A T P a s e m i g h t be altered in intact m y o c y t e s of p a t i e n t s with severe h e a r t failure.
METHODS Patients. Cells were prepared from hearts of six patients with end-stage heart failure resulting from dilated cardiomyopathy (n = 3) or ischemic cardiomyopathy (n = 3) who
underwent transplantation. The mean patient age was 49 _+ 6 years; cardiac index was 2.3 -+ 0.2 L/min/m 2 and ejection fraction was 27% + 3%. All patients received digoxin and diuretics and were receiving vasodilator therapy. No catecholamines or ~-adrenoceptor blocking drugs were given during the 48-hour period before the operation. Informed consent was obtained before organ explantation. Results were compared with results from cells isolated from three normal human hearts without cardiac disease that could not be transplanted for technical reasons. Cell isolation. The isolation procedure has been described in detail. 4 A part of the left ventricular wall was excised with its artery branch. The wall segment was then perfused via this artery branch for 30 minutes with a nominally Ca2+-free modified Tyrode's solution (138 mmol/L of sodium chloride, 4 mmol/L of potassium chloride, 1 mmol/L of magnesium chloride, 10 mmol/L of glucose, 0.33 mmol/L of sodium biphosphate; and 10 mmol/L of HEPES pH 7.3, with addition of sodium hydroxide 37 ° C) followed by 40 minutes with the same solution with added collagenase (type II, 70 mg/50 ml, Worthington, Biochron KG, Berlin, Germany) and protease (type XIV, 6 mg/50 ml, Sigma Chemical, St. Louis). Finally, the enzyme was washed out for 15 minutes with modified Tyrode's solution that contained 200 ~mol Ca 2+. Because tissue digestion was maximal within the ventricular wall with the endocardial and epicardial layers almost undigested, cells were prepared from areas within the central third of the myocardial width. Ventricular cells were disaggregated by mechanical agita-
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,.w +
.o
5°°/ 0-J
I
I 400 ms
Fig. 2. Gradual loading of SR with Ca 2+ in myocyte from patient with heart failure. After Ca 2÷ depletion of SR, cell was repetitively stimulated to +10 mV. On first stimulation, [Ca2+]i increased only slightly. Further stimulation led to larger [Ca2+]i-transients. Every second recording is shown for clarity. After 17 pulses, spontaneous Ca 2+ release from SR was observed (last trace).
tion and, after being filtered, through nylon mesh, stored at room temperature in Tyrode's solution containing 2.0 mmol Ca 2+. The living cell yield was approximately 5 % to 8 %. Only cells with clear cross-striation without significant granulation or spontaneous contraction were selected for experiments. Cells that fulfilled these selection criteria contracted on field stimulation as judged by visual control. Solutions and stimulation protocol. Because selective blockers of the Na+/Ca 2+ exchanger are currently unavailable, experimental conditions had to be used that inactivated or blocked this Ca 2+ transport system by Na + removal inside and outside of the cell. 15 Cells were superfused at 35 ° C with a modified Tyrode's solution containing 2.0 mmol/L of calcium chloride, 140 mmol/L of sodium chloride; l0 mmmol/L of cesium chloride, I mmol/L of magnesium chloride, 10 mmol/L of glucose and 10 mmol/L of HEPES; pH was 7.3 with addition of cesium hydroxide (solution 1). Cs + was included to block K + currents. After whole-cell recording conditions were established, the solution was changed for 5 minutes to a low-calcium (50 tLmol of calcium chloride), but otherwise similar, solution to deplete the SR from Ca 2+ (solution 2). During this period, cells were held at a holding potential of -80 mV without stimulation. To inhibit Na+/Ca 2+ exchange, sodium chloride was replaced after this periodby equimolar amounts of Tetraethylammoniumchloride (TEA): 50 ttmol/L of calcium chloride, 140 mmol/L of TEA chloride, 10 mmol/L of cesium chloride, 1 mmol/L of magnesium chloride; 10 mmol/L glucose, and 10 mmol/L of HEPES; pH was 7.3 with the addition of cesium hydroxide (solution 3). During this period, cells were stimulated from a holding potential of -80 mV to -55 mV at a frequency of 0.1 Hz to monitor removal of Na + from the recording chamber. When Na + current cou!d no longer be recorded, Ca 2+ was added again to the Na+-free solution: 2.0 mmol/L of calcium chloride, 140 mmol/L of TEA chloride, 10 mmol/L of cesium chloride, 1 mmol/L of magnesium chloride, 10 mmol/L of glucose, and 10 mmol/L of HEPES; pH was 7.3 with addition of cesium hydroxide (solution 4). Cells were repetitively
stimulated at 0.5 Hz from -80 mV to +10 mV to gradually load the SR, and Ca 2+ entering the cell through the Ca 2+ current. When cells began ~o show signs of Ca 2+ overload (e.g., spontaneous release of Ca 2+ from the SR) 10 mmol/L of caffeine was added to solution 4 to release all Ca 2+ from the SR and render the SR function ineffective. After whole-cell recordings were established, the electrode solution was allowed to exchange with the intracellular environment. Electrodes had resistances of 2.0 to 3:0 M~2 and were filled with 0.05 mmol/L of fura-2 (Molecular Probes, Eugene, Ore.), 120 mmol/L of cesium aspartate, 10 mmol/L of cesium chloride, I mmol/L of magnesium chloride, 10 mmol/L of HEPES (cesium salt), and 2 mmol/L of magnesium adenosine triphosphate; pH was 7.2 with the addition of cesium hydroxide. Recording technique, Experiments were carried out b y using standard whole-cell recording techniques 16 in which a patch-clamp amplifier model EPC-7 (LIST Instruments, Darmstadt, Germany) was used with a 100 M~2 feedback resistor. For fluorescence recordings, ultraviolet light emitted from a 75 W xenon arc lamp passed through 10 nm 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 Ca 2+ indicator in the cell. Fluorescence emitted from the cell passed through the objective, and a 510 to 540 nm bandpass filter and was directed into a photomultiplier tube. Fluorescence and current recordings were digitized (1 kHz) and stored for off-line analysis. Analysis. The half-time of [Ca2+]i decay was determined by measuring the time of decay of [Ca2+]~ from a concentration of 500 nmol/L to the resting concentration at -80 inV. Mean values + SEM are shown. Mann:Whitney nonparametric analysis was used for statistical evaluation of the data, and a p value <0.05 was considered significant.
RESULTS Gradual loading of the SR with Ca 2+. After Ca2+ de-
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caffeine
r-
+ ¢o
.o.
°°°1 I''
3 0 0 ms
I
Fig. 3. Isolation of SR Ca2+-reuptake and Ca2+-extrusion via sarcolemma (cell from undiseased heart). First and tenth recording of [Ca2+]i on stimulation of cell to +10 inV. Half-time of [Ca2+]i decay from 500 mmol/L was 300 msec. After incubation of cell with 10 mmol/L of caffeine, [Ca2+]i was increased to approximately 800 mmol/L. During next 1.5 seconds, there was no significant decline of [Ca2+]i.
caffeine
A C
+
1 O-J 3 0 0 ms
Fig. 4. Isolation of SR Ca2+ reuptake and Ca2+ extrusion via sarcolemma (terminal heart failure). First and tenth recording of [Ca2+]i on stimulation of cell isolated from heart with terminal heart failure as a result of dilated cardiomyopathy on depolarization of membrane to +10 mV. Rate of decay of [Ca2+]i was markedly prolonged (600 msec). After addition of 10 mmol/L of caffeine, [Ca2+]i rose to 750 mmol/L. No decline of [Ca2+]i could be observed over 1.5-second period.
pletion of the SR (see Methods section), the voltageclamped myocyte was repetitively depolarized from a holding potential of -80 mV to +10 mV (Fig. 2). Na+/Ca 2+ exchange was inactivated through the use of Na+-free solutions inside and outside of the cell (see Methods section). On the first stimulation, [CaU+]i increased only slightly from the level reached after Ca2+-depletion and reintroduction of Ca 2+ in the extracellular solution, usually 200 to 300 nmol/L. This small increase presumably was caused by direct entry of Ca 2+ through the Ca 2+ current. Further stimulation led to increasingly larger [Ca2+]i transients as Ca 2+ entered the cell but could not be extruded through Na+/Ca 2+ exchange and was stored in the SR for release during subsequent beats. The increase in [Ca2+]i transients during subsequent de-
polarization is shown in Fig. 2. For clarity, only every second recording is shown. On the sixth stimulation, [Ca2+]i increased to approximately 500 nmol/L. This trace was used for quantitative analysis of the half time of [Ca2+]i decay used in Table I. After 17 pulses, spontaneous Ca 2+ release from the SR was observed. The number of pulses until spontaneous Ca 2+ release occurred was variable between cells. Isolation of SR Ca 2+ reuptake and Ca 2+ extrusion via
the sarcolemma. Fig. 3 shows the first and tenth recordings of [Ca2+]i on stimulation of a cell isolated from a control heart without heart failure from a holding potential of -80 mV to +10 inV. The half-time of [Ca2+]i decay from 500 nmol/L was 300 msec. After the cell was incubated with 10 mmol/L of caffeine, [Ca2+]i was increased to approximately 800
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Rate of decay of [Ca2+]i resulting from Ca2+ uptake into sarcoplasmic reticulum
T a b l e I.
Rate of [Ca2+ji decay H e a r t failure 6 hearts, 14 cells Control 3 hearts, 6 cells Significance
538 + 66 m s e c 305 +_ 16 m s e c p < 0.05
mmol/L. Under these conditions, the sarcolemmal Ca2+-ATPase was the only Ca 2+ transport system operating as a Ca 2+ removal system from the cytoplasm. However, during the next 1.5 seconds there was no significant decline of [Ca2+]i. When the same protocol was applied to a cell isolated from a heart with terminal heart failure resulting from dilated cardiomyopathy (Fig. 4), depolarization of the membrane to +10 mV again triggered a rise of [Ca2+]i to approximately 500 mmol/L. However, the rate of decay of [Ca2+]i was markedly prolonged (600 msec). After the addition of 10 mmol/L of caffeine, [Ca2+]i rose to 750 mmol/L. Again there was no decline in [Ca2+]i over the next 1.5 seconds. Ca 2+ reuptake of the SR. Table I depicts the rate of decay of [Ca2+]i resulting from Ca 2+ uptake of the SR. Values were obtained in 14 cells isolated from 6 hearts with terminal heart failure and in 6 cells isolated from 3 control hearts that had no heart disease. The rate of [Ca2+]i decay between these two groups was significantly different and was prolonged in myocytes from patients with end-stage heart failure. DISCUSSION
The aim of the present study was to characterize mechanisms underlying altered diastolic [Ca2+]i handling in patients with severe heart failure. A major mechanism of diastolic Ca 2+ removal from the cytoplasm at physiologic Na + concentrations is through Na+/Ca 2+ exchange. 17 Therefore prolongation of the action potential that has been demonstrated in these patients 4, is might contribute to the alterations in diastolic [Ca2+]i handling through its effect on the voltage-dependent Na+/Ca 2+ exchange. However, in voltage-controlled single myocytes isolated from patients with heart failure, diastolic decay of [Ca2÷]i is also significantly prolonged. 4 Theoretically this may be the result of reduced reuptake into the SR or reduced activity of Na+/Ca 2+ exchange. Altered activity of Ca 2+ reuptake into the SR has been shown in various animal models of cardiac hypertrophy and heart failure. 19:21In human heart failure, a reduced expression of genes encoding for SR
Ca2+-ATPase has also been reported. 6, 7 However, other authors could not find a difference in the Ca 2+ uptake of the SR from patients with heart failure resulting from dilated cardiomyopathy as compared to controls.5, 22In the present study, we have shown that the Ca 2+ reuptake of the SR is significantly slower in intact cells from patients with severe heart failure. In our experimental design, possible effects of the Na+/ Ca 2+ exchange system could be excluded because experiments were performed under Na+-free conditions. 15 Our findings suggest that Ca 2+ reuptake is significantly reduced in single myocytes isolated from failing human hearts. At the presenttime, however, we do not know for sure whether this is the result of a quantitative reduction of the number of pump molecules, whether some intracellular factor partially inhibits a normal number of pump molecules, or whether the SR Ca 2+ pump is structurally altered. Movsesian et al. 22a found evidence that the Ca2+-ATPase of the SR and phospholamban are unchanged as assessed by Western blot analysis. These differences may be caused by changes in the intracellular environment which, like the intracellular concentration of cyclic adenosin monophosphate, exhibit their effect on the intact cell but not on isolated proteins. BShm et al. 23 have shown that basal activity of intracellular adenylate cyclase activity is reduced. This reduced activity corresponds to a decrease of basal cyclic adenosine monophosphate content in myocardium of patients with heart failure. 24 Further investigations are needed to clarify whether there is a causal relation between delayed Ca 2+ uptake into the SR and reduction of the systolic [Ca2+]i transient. 4 However, our results show that relaxation abnormalities of the heart in severe h e a r t failure ~ire partially the result of a slowed Ca 2+ reuptake by the SR. The importance of the sarcolemmal Ca2+-ATPase for intracellular [Ca2+]i handling in human ventricular cells is largely unknown. Alterations of this Ca 2+ transport system have been demonstrated in a variety of animal models of heart failure. 21, 25, 26 [Ca2+]i did not decrease significantly from concentrations that are usually reached during systole 4 during the time course of a cardiac cycle. Therefore our results indicate that the Ca 2+ ATPase of the cell membrane does not contribute significantly to the cytoplasmic Ca 2+ removal during an individual heartbeat in myocytes from patients with heart failure or in undiseased hearts. In conclusion, alterations of diastolic [Ca2+]i handling in ventricular myocytes from patients with severe heart failure are partially caused by a delayed
Volume 129, Number 4 American Heart Journal
Ca 2+ uptake of the SR. The sarcolemmal Ca 2+ ATPase does not contribute significantly to the cytoplasmic Ca 2+ removal in the human ventricle. Further studies are necessary to clarify whether alterations of diastolic [Ca2+]i removal have any causal relation to the reduced systolic [Ca2+]i transient in heart failure. W e t h a n k J. N u s s e r for e x p e r t technical assistance a n d Prof. B. R e i c h a r t a n d his colleagues in t h e D e p a r t m e n t of Cardiac Surgery, U n i v e r s i t y of M u n i c h , for providing t h e myocardial tissue. REFERENCES
I. Gwathmey JK, Copelas L, MacKinnon R, Schoen FJ, Feldman MD, Grossman W, Morgan JP. Abnormal intracelinlar calcium handling in myocardium from patients with end-stage heart failure. Circ Res 1987;61:70-6. 2. Grossman W. Diastolic dysfunction and congestive heart failure. Circulation 1990;81(Suppl III):III-l-III-7. 3. Gwathmey JK, Warren SE, Briggs GM, Copelas L, Feldman MD, Phillips P J, Callahan M, Schoen F J, Grossman W, Morgan JP. Diastolic dysfunction in hypertrophic cardiomyopathy: effect on active force generation during systole. J Clin Invest 1991;87:1023-31. 4. Beuckelmann DJ, N~bauer M, Erdmann E. Intracellular calcium handling in ventricular myocytes from patients with terminal heart failure. Circulation 1992;85:1046-55. 5. Movsesian MA, Bristow MR, Krall J. Ca2+ uptake by cardiac sareoplasmic reticulum from patients with idiopathic dilated cardiomyopathy. Circ Res 1989;65:1141.4. 6. Mercadier J-J, Lompre A-M, Due P, Boheler KR, Fraysse J-B, Wisnewsky C, Allen PD, Komajda M, Schwartz K. Altered sarcoplasmic reticulum Ca2+-ATPase gene expression in the human ventricle during end-stage heart failure. J Clin Invest 1990;85:305-9. 7. Takahashi T, Allen PD, Lacro RV, Marks AR, Dennis AR, Schoen FJ, Grossman W, Marsh JD, Izumo S. Expression of dihydropyridine receptor (Ca2+ channel) and calsequestrin genes in the myocardium of patients with end-stage heart failure. J Clin Invest 1992;90:927-35. 8. Carafoli E. Membrane transport of calcium: an overview. In: Fleischer S, Fleischer B, eds. Methods in enzymology. Biomembranes. Vol. 157. San Diego: Academic Press, 1988:3-11. 9. Mullins LJ. A mechanism for Na+/Ca 2+ transport. J Gen Physiol 1977;70:681-95. 10. Caroni P, Carafoli E. The regulation of the Na+-Ca2+ exchanger of heart sarcolemma. Eur J Biochem 1983;132:451-60. 11. Jencks WP. How does a calcium pump pump calcium? J Biol Chem 1989;264:18855-8. 12. Robertson SP, Johnson JD, Potter JD. The time-course of Ca 2+ exchange with calmodalin, troponin, parvalbumin, and myosin in response to transient increases in Ca 2+. Biophys J 1981;34:559-69.
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13. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol 1983;245:C1-14. 14. Wankerl M, Bhhm M, Morano L Rfiegg JC, Eichhorn M, Erdmann E. Calcium sensitivity and myosin light chain pattern of atrial and ventricular skinned cardiac fibers from patients with various kinds of cardiac disease. J Mol Cell Cardiol 1990;22:t425-38. 15. Sipido KR, Wier WG. Flux of Ca 2+ across the sarcoplasmic reticulum of guinea-pig cardiac cells during excitation-contraction coupling. J Physiol (London) 1991;435:605-30. 16. Hamill OP, Maxty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflfigers Arch 1981;391:85-100. 17. Beuckelmann DJ, Wier WG. Sodium-calcium exchange in guinea-pig cardiac cells: exchange current and intracellular calcium. J Physiol (London) 1989;414:499-520. 18. Gwathmey JK, Slawsky MT, Hajjar RJ, Briggs GM, Morgan JP. Role of intracellular calcium handling in force-interval relationship of human ventricular myocardium. J Clin Invest 1990;85:1599-613. 19. Mead RJ, Peterson MB, Welty JD. Sarcolemmal and sarcoplasmic reticular ATPase activities in the failing canine heart. Circ Res 1971;29:1420. 20. De la Bastie D, Levitsky D, Rappaport L, Mercadier J-J, Marotte F, Wisnewsky C, Brovkovich V, Schwartz K, Lompre A-M. Function of the sarcoplasmic reticulum and expression of its Ca2+-ATPase gene in pressure overload-induced cardiac hypertrophy in the rat. Circ Res 1990;66:554-64. 21. O'Brien P J, Shen H, Weilar J, Mirsalimi M, Julian R. Myocardial Casequestration failure and compensatory increases in Ca-ATPase with congestive cardiomyopathy: kinetic characterization by a homogenate microassay using reabtime ratiometrlc indo-1 spectrofiuorometry. Mol Cell Biochem 1991;102:1-12. 22. Movsesian MA, Colyer J, Wang JH, Krall J. Phospholamban-mediated stimulation of Ca z+ uptake in sarcoplasmic reticulum from normal and failing hearts. J Clin Invest 1990;85:1698-702. 22a. Movsesian MA, Karimi M~ Green K, Jones LR. Ca2+-transporting ATPase, phospholamban, and calsequestrin levels in non-failing and failing human myocardium. Circulation 1994;90:653-7. 23. B6hm M, Gierschik P, Jakobs KH, Pieske B, Schnabel P, Ungerer M, Erdmann E. Increase of Gia in human hearts with dilated but not ischemic cardiomyopathy. Circulation 1990;82:1249-65. 24. Danielsen W, Von der Leyen H, Meyer W, Neumann J, Schmitz W, Scholz H, Starbotty J, Stein B, Dhring V, Kalmar P. Basal and isoprenaline-stimulated c-AMP content in failing versus non-failing human cardiac preparations. J Cardiovasc Pharmacol 1989;14:171-3. 25. Galindo J, Hudecki MS, Davis FB, Davis P J, Thacore HR, Pollina CM, Blas SD, Schoen M. Abnormal response to calmodulin in in vitro dystrophic chicken muscle membrane Ca2+-ATPase activity. Biochemistry 1988;27:7519-24. 26. Kuo TH, Tsang W, Wiener J. Defective Ca2+-pumping ATPase of heart sarcolemma from cardiomyopathic hamster. Biochim Biophys Acta 1987;900:10-6.
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14. Helfenstein U. The use of transfer function models, intervention analysis and related time series methods in epidemiology. Int J Epidemiol 1991;20:808-15. 15. Box GEP, Jenkins GM. Time series analysis: forecasting and control. Oakland, Calif.: Holden-Day, 1976. 16. Bloomfield P. Fourier analysis of time series: an introduction. New York: Prentice-Hall, 1974. 17. Imperi GA, Lambert CR, Coy K, Lopez L, Pepine CJ. Effects of titrated beta blockade (metoprolol) on silent myocardial ischemia in ambulatory patients with coronary artery disease. Am J Cardiol 1987;60:519-24. 18. Frishman W, Charlap S, Kimmel B, Teicher M, Cinnamon J, Allen L, Strom J. Diltiazem, nifedipine, and their combination in patients with stable angina pectoris: effects on angina, exercise tolerance, and the ambulatory electrocardiographic ST segment. Circulation 1988;77:77486. 19. Mulcahy D, Cunningham D, Crean P, Wright C, Keegan J, Quyyumi A,
American Heart Journal
Park A, Fox. Circadian variation of total ischemic burden and its alteration with anti-anginal agents. Lancet 1988;2:755-9. 20. Stone PH, Gibson RS, Glasser SP, DeWood MA, Parker JD, Kawanishi DT, Crawford MH, Messineo FC, Shook TL, Raby K, ~urtis DG, Hoop RS, Young PM, Braunwald E. Comparison of propranolol, diltiazem, and nifedipine in the treatment of ambulatory ischemia in patients with stable angina. Circulation 1990;82:1962-72. 21. Deedwania PC, Carbajal EV, Nelson JR, Halt H. Anti-ischemic effects of atenolol versus nifedipine in patients with coronary artery disease and ambulatory silent ischemia. J Am Cell Cardiol 1991;17:963-9. 22. Theroux P, Baird M, Juneau M, Warnica W, Klinke P, Kostuk W, Pflugfelder P, Lavallee E, Chin C, Dempsey E, Grace M, Lalonde Y, Waters D. Effect of diltiazem on symptomatic and asymptomatic episodes of ST segment depression occurring during daily life and during exercise. Circulation 1991;84:15-22.
Altered diastolic [Ca2+]i handling in human ventricular myocytes from patients with terminal heart failure To investigate whether the slow diastolic decay of [Ca2+]i in myocardium of patients with heart failure is a result of alterations of the Ca 2t adenosine triphosphatase of the sarcoplasmic reticulum or the sarcolemma, [Ca2÷]~ transients were recorded in voltage-clamped ventricular cells isolated from hearts of patients with terminal heart failure or from undiseased donor hearts. To isolate the [Ca2t]i-reuptake function of the sarcoplasmic reticulum, myocytes were dialyzed via the patch pipette with Na÷-free solution and incubated in Ca2t-free and Nat-free solution to inhibit Nat/Ca 2÷ exchange. After superfusion with Ca2÷-containing, Nat-free medium, the sarcoplasmic reticulum was loaded with Ca 2t through repetitive voltage-clam p pulses to +10 mV. Under these conditions, [Ca2t]~ decay was significantly slower in myocytes from patients with heart failure (538 - 66 msec) than in controls (305 ± 16 msec; p < 0.05). After the addition of 10 mmol/L of caffeine, [Ca2t]~ levels did not show appreciable decay between two voltage-clamp pulses in diseased and undiseased myocytes. We conclude that diastolic decay of [Ca2+]i in ventricular myocytes from patients with terminal heart failure is partially the result of a decreased rate of Ca 2t reuptake by the sarcoplasmic reticulum. Sarcolemmal Ca 2t adenosine triphosphatase does not contribute significantly to cytoplasmic [Ca2÷]i removal during an individual heartbeat. (AM HEART J 1995;129:684-9.)
Dirk J. Beuckelmann, MD, Michael N~ibauer, MD, Carsten Krfiger, MD, and Erland Erdmann, MD Cologne, Germany
Alterations of diastolic relaxation of the myocardium is one of the prominent features of the heart in severe heart failure a n d hypertrophy. 13 Several mechaFrom the Department of Medicine III, University of Cologne. Supported by a grant of the Deutsche Forschungsgemeinschaft (Be 1113/ 2-2) and the Friedrich-Baur-Stiftung. Received for publication Feb. 17, 1994; accepted July 5, 1994. Reprint requests: Dirk J. Beuckelmann, MD, Department of Medicine III, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany. Copyright © 1995 by Mosby-Year Book, Inc. 0002-8703/95/$3.00 + 0 4/1/61205
684
nisms are responsible for this phenomenon. However, on the cellular level it has been shown 4 that delayed relaxation is caused by a slow diastolic decay of intracellular [Ca2+]i. It has been suspected that a reduced [Ca2+]i reuptake rate of the sarcoplasmic reticulum (SR) may be a major reason for the slow decline of [Ca2+]i and for the prolongation of diastolic relaxation in these patients. Results of experiments that were designed to measure the activity of the Ca 2+ adenosine triphosphatase (ATPase) of the SR in human beings have been conflicting. Some authors
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Intracellular [Ca2+]i-Handling Na+/Ca2+ exchange
Ca2 + - A T P a a e
Ca2+.channel
~::,!~,:;~;:~ii:i~:::~,:, : ~: i,i~:i:,i~: ?~:I, ,:~,/,:, ~i,~, ~
i
ADP
+ Pl
[Ca2+]i~t
J
~
/
rcOpal=m rectlcluU lm
contractile proteins ATP ADP + P|
Fig. 1. Scheme of intracellular [Ca2+]i handling in ventricular myocyte. Ca 2+ entering cell through voltage-activated Ca 2+ channel triggers release of Ca 2+ from SR. Intracellular [Ca2+]i increases and binds to troponin C, initiating contraction. Relaxation is initiated by separation of Ca 2+ from Ca 2+, binding contractile proteins and removal from cytoplasm by sarcolemmal Ca2+-ATPase, extrusion via Na+/Ca2+-ex change system, Ca 2+ reuptake by Ca2+-ATPase of SR, and binding to intracellular Ca 2+ buffers. ADP, Adenosine diphosphate.
r e p o r t e d an u n c h a n g e d Ca 2+ u p t a k e r a t e in severe h e a r t failure w h e n c o m p a r e d to the Ca 2+ u p t a k e rate in u n d i s e a s e d controls. 5 Others found t h e expression of the genes encoding for the SR Ca2+-ATPase to be significantly reduced, e' 7 During diastole, Ca 2+ is r e m o v e d f r o m the cytop l a s m of the m y o c y t e b y four m a i n m e c h a n i s m s (Fig. 1): (1) Ca 2+ r e u p t a k e b y the SR t h r o u g h an energyd e p e n d e n t Ca2+-ATPaseS; (2) t r a n s s a r c o l e m m a l ext r u s i o n by N a + / C a 2+ exchange 9' 10; (3) t r a n s s a r c o l e m m a l extrusion t h r o u g h an e n e r g y - d e p e n d e n t Ca 2+A T P a s e of the cell membrane11; a n d (4) the binding of Ca 2+ to intracellular p r o t e i n s like t r o p o n i n and calmodulin.12, 13 However, the binding of Ca 2+ to intracellular contractile proteins has already b e e n shown to be u n c h a n g e d in m y o c a r d i u m of p a t i e n t s with t e r m i n a l h e a r t failure. ~4 T h e r e f o r e the p u r p o s e of the p r e s e n t s t u d y was to investigate w h e t h e r alterations of Ca 2+ r e u p t a k e into the SR or Ca 2+ extrusion via the s a r c o l e m m a l Ca 2+ A T P a s e m i g h t be altered in intact m y o c y t e s of p a t i e n t s with severe h e a r t failure.
METHODS Patients. Cells were prepared from hearts of six patients with end-stage heart failure resulting from dilated cardiomyopathy (n = 3) or ischemic cardiomyopathy (n = 3) who
underwent transplantation. The mean patient age was 49 _+ 6 years; cardiac index was 2.3 -+ 0.2 L/min/m 2 and ejection fraction was 27% + 3%. All patients received digoxin and diuretics and were receiving vasodilator therapy. No catecholamines or ~-adrenoceptor blocking drugs were given during the 48-hour period before the operation. Informed consent was obtained before organ explantation. Results were compared with results from cells isolated from three normal human hearts without cardiac disease that could not be transplanted for technical reasons. Cell isolation. The isolation procedure has been described in detail. 4 A part of the left ventricular wall was excised with its artery branch. The wall segment was then perfused via this artery branch for 30 minutes with a nominally Ca2+-free modified Tyrode's solution (138 mmol/L of sodium chloride, 4 mmol/L of potassium chloride, 1 mmol/L of magnesium chloride, 10 mmol/L of glucose, 0.33 mmol/L of sodium biphosphate; and 10 mmol/L of HEPES pH 7.3, with addition of sodium hydroxide 37 ° C) followed by 40 minutes with the same solution with added collagenase (type II, 70 mg/50 ml, Worthington, Biochron KG, Berlin, Germany) and protease (type XIV, 6 mg/50 ml, Sigma Chemical, St. Louis). Finally, the enzyme was washed out for 15 minutes with modified Tyrode's solution that contained 200 ~mol Ca 2+. Because tissue digestion was maximal within the ventricular wall with the endocardial and epicardial layers almost undigested, cells were prepared from areas within the central third of the myocardial width. Ventricular cells were disaggregated by mechanical agita-
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,.w +
.o
5°°/ 0-J
I
I 400 ms
Fig. 2. Gradual loading of SR with Ca 2+ in myocyte from patient with heart failure. After Ca 2÷ depletion of SR, cell was repetitively stimulated to +10 mV. On first stimulation, [Ca2+]i increased only slightly. Further stimulation led to larger [Ca2+]i-transients. Every second recording is shown for clarity. After 17 pulses, spontaneous Ca 2+ release from SR was observed (last trace).
tion and, after being filtered, through nylon mesh, stored at room temperature in Tyrode's solution containing 2.0 mmol Ca 2+. The living cell yield was approximately 5 % to 8 %. Only cells with clear cross-striation without significant granulation or spontaneous contraction were selected for experiments. Cells that fulfilled these selection criteria contracted on field stimulation as judged by visual control. Solutions and stimulation protocol. Because selective blockers of the Na+/Ca 2+ exchanger are currently unavailable, experimental conditions had to be used that inactivated or blocked this Ca 2+ transport system by Na + removal inside and outside of the cell. 15 Cells were superfused at 35 ° C with a modified Tyrode's solution containing 2.0 mmol/L of calcium chloride, 140 mmol/L of sodium chloride; l0 mmmol/L of cesium chloride, I mmol/L of magnesium chloride, 10 mmol/L of glucose and 10 mmol/L of HEPES; pH was 7.3 with addition of cesium hydroxide (solution 1). Cs + was included to block K + currents. After whole-cell recording conditions were established, the solution was changed for 5 minutes to a low-calcium (50 tLmol of calcium chloride), but otherwise similar, solution to deplete the SR from Ca 2+ (solution 2). During this period, cells were held at a holding potential of -80 mV without stimulation. To inhibit Na+/Ca 2+ exchange, sodium chloride was replaced after this periodby equimolar amounts of Tetraethylammoniumchloride (TEA): 50 ttmol/L of calcium chloride, 140 mmol/L of TEA chloride, 10 mmol/L of cesium chloride, 1 mmol/L of magnesium chloride; 10 mmol/L glucose, and 10 mmol/L of HEPES; pH was 7.3 with the addition of cesium hydroxide (solution 3). During this period, cells were stimulated from a holding potential of -80 mV to -55 mV at a frequency of 0.1 Hz to monitor removal of Na + from the recording chamber. When Na + current cou!d no longer be recorded, Ca 2+ was added again to the Na+-free solution: 2.0 mmol/L of calcium chloride, 140 mmol/L of TEA chloride, 10 mmol/L of cesium chloride, 1 mmol/L of magnesium chloride, 10 mmol/L of glucose, and 10 mmol/L of HEPES; pH was 7.3 with addition of cesium hydroxide (solution 4). Cells were repetitively
stimulated at 0.5 Hz from -80 mV to +10 mV to gradually load the SR, and Ca 2+ entering the cell through the Ca 2+ current. When cells began ~o show signs of Ca 2+ overload (e.g., spontaneous release of Ca 2+ from the SR) 10 mmol/L of caffeine was added to solution 4 to release all Ca 2+ from the SR and render the SR function ineffective. After whole-cell recordings were established, the electrode solution was allowed to exchange with the intracellular environment. Electrodes had resistances of 2.0 to 3:0 M~2 and were filled with 0.05 mmol/L of fura-2 (Molecular Probes, Eugene, Ore.), 120 mmol/L of cesium aspartate, 10 mmol/L of cesium chloride, I mmol/L of magnesium chloride, 10 mmol/L of HEPES (cesium salt), and 2 mmol/L of magnesium adenosine triphosphate; pH was 7.2 with the addition of cesium hydroxide. Recording technique, Experiments were carried out b y using standard whole-cell recording techniques 16 in which a patch-clamp amplifier model EPC-7 (LIST Instruments, Darmstadt, Germany) was used with a 100 M~2 feedback resistor. For fluorescence recordings, ultraviolet light emitted from a 75 W xenon arc lamp passed through 10 nm 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 Ca 2+ indicator in the cell. Fluorescence emitted from the cell passed through the objective, and a 510 to 540 nm bandpass filter and was directed into a photomultiplier tube. Fluorescence and current recordings were digitized (1 kHz) and stored for off-line analysis. Analysis. The half-time of [Ca2+]i decay was determined by measuring the time of decay of [Ca2+]~ from a concentration of 500 nmol/L to the resting concentration at -80 inV. Mean values + SEM are shown. Mann:Whitney nonparametric analysis was used for statistical evaluation of the data, and a p value <0.05 was considered significant.
RESULTS Gradual loading of the SR with Ca 2+. After Ca2+ de-
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caffeine
r-
+ ¢o
.o.
°°°1 I''
3 0 0 ms
I
Fig. 3. Isolation of SR Ca2+-reuptake and Ca2+-extrusion via sarcolemma (cell from undiseased heart). First and tenth recording of [Ca2+]i on stimulation of cell to +10 inV. Half-time of [Ca2+]i decay from 500 mmol/L was 300 msec. After incubation of cell with 10 mmol/L of caffeine, [Ca2+]i was increased to approximately 800 mmol/L. During next 1.5 seconds, there was no significant decline of [Ca2+]i.
caffeine
A C
+
1 O-J 3 0 0 ms
Fig. 4. Isolation of SR Ca2+ reuptake and Ca2+ extrusion via sarcolemma (terminal heart failure). First and tenth recording of [Ca2+]i on stimulation of cell isolated from heart with terminal heart failure as a result of dilated cardiomyopathy on depolarization of membrane to +10 mV. Rate of decay of [Ca2+]i was markedly prolonged (600 msec). After addition of 10 mmol/L of caffeine, [Ca2+]i rose to 750 mmol/L. No decline of [Ca2+]i could be observed over 1.5-second period.
pletion of the SR (see Methods section), the voltageclamped myocyte was repetitively depolarized from a holding potential of -80 mV to +10 mV (Fig. 2). Na+/Ca 2+ exchange was inactivated through the use of Na+-free solutions inside and outside of the cell (see Methods section). On the first stimulation, [CaU+]i increased only slightly from the level reached after Ca2+-depletion and reintroduction of Ca 2+ in the extracellular solution, usually 200 to 300 nmol/L. This small increase presumably was caused by direct entry of Ca 2+ through the Ca 2+ current. Further stimulation led to increasingly larger [Ca2+]i transients as Ca 2+ entered the cell but could not be extruded through Na+/Ca 2+ exchange and was stored in the SR for release during subsequent beats. The increase in [Ca2+]i transients during subsequent de-
polarization is shown in Fig. 2. For clarity, only every second recording is shown. On the sixth stimulation, [Ca2+]i increased to approximately 500 nmol/L. This trace was used for quantitative analysis of the half time of [Ca2+]i decay used in Table I. After 17 pulses, spontaneous Ca 2+ release from the SR was observed. The number of pulses until spontaneous Ca 2+ release occurred was variable between cells. Isolation of SR Ca 2+ reuptake and Ca 2+ extrusion via
the sarcolemma. Fig. 3 shows the first and tenth recordings of [Ca2+]i on stimulation of a cell isolated from a control heart without heart failure from a holding potential of -80 mV to +10 inV. The half-time of [Ca2+]i decay from 500 nmol/L was 300 msec. After the cell was incubated with 10 mmol/L of caffeine, [Ca2+]i was increased to approximately 800
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Rate of decay of [Ca2+]i resulting from Ca2+ uptake into sarcoplasmic reticulum
T a b l e I.
Rate of [Ca2+ji decay H e a r t failure 6 hearts, 14 cells Control 3 hearts, 6 cells Significance
538 + 66 m s e c 305 +_ 16 m s e c p < 0.05
mmol/L. Under these conditions, the sarcolemmal Ca2+-ATPase was the only Ca 2+ transport system operating as a Ca 2+ removal system from the cytoplasm. However, during the next 1.5 seconds there was no significant decline of [Ca2+]i. When the same protocol was applied to a cell isolated from a heart with terminal heart failure resulting from dilated cardiomyopathy (Fig. 4), depolarization of the membrane to +10 mV again triggered a rise of [Ca2+]i to approximately 500 mmol/L. However, the rate of decay of [Ca2+]i was markedly prolonged (600 msec). After the addition of 10 mmol/L of caffeine, [Ca2+]i rose to 750 mmol/L. Again there was no decline in [Ca2+]i over the next 1.5 seconds. Ca 2+ reuptake of the SR. Table I depicts the rate of decay of [Ca2+]i resulting from Ca 2+ uptake of the SR. Values were obtained in 14 cells isolated from 6 hearts with terminal heart failure and in 6 cells isolated from 3 control hearts that had no heart disease. The rate of [Ca2+]i decay between these two groups was significantly different and was prolonged in myocytes from patients with end-stage heart failure. DISCUSSION
The aim of the present study was to characterize mechanisms underlying altered diastolic [Ca2+]i handling in patients with severe heart failure. A major mechanism of diastolic Ca 2+ removal from the cytoplasm at physiologic Na + concentrations is through Na+/Ca 2+ exchange. 17 Therefore prolongation of the action potential that has been demonstrated in these patients 4, is might contribute to the alterations in diastolic [Ca2+]i handling through its effect on the voltage-dependent Na+/Ca 2+ exchange. However, in voltage-controlled single myocytes isolated from patients with heart failure, diastolic decay of [Ca2÷]i is also significantly prolonged. 4 Theoretically this may be the result of reduced reuptake into the SR or reduced activity of Na+/Ca 2+ exchange. Altered activity of Ca 2+ reuptake into the SR has been shown in various animal models of cardiac hypertrophy and heart failure. 19:21In human heart failure, a reduced expression of genes encoding for SR
Ca2+-ATPase has also been reported. 6, 7 However, other authors could not find a difference in the Ca 2+ uptake of the SR from patients with heart failure resulting from dilated cardiomyopathy as compared to controls.5, 22In the present study, we have shown that the Ca 2+ reuptake of the SR is significantly slower in intact cells from patients with severe heart failure. In our experimental design, possible effects of the Na+/ Ca 2+ exchange system could be excluded because experiments were performed under Na+-free conditions. 15 Our findings suggest that Ca 2+ reuptake is significantly reduced in single myocytes isolated from failing human hearts. At the presenttime, however, we do not know for sure whether this is the result of a quantitative reduction of the number of pump molecules, whether some intracellular factor partially inhibits a normal number of pump molecules, or whether the SR Ca 2+ pump is structurally altered. Movsesian et al. 22a found evidence that the Ca2+-ATPase of the SR and phospholamban are unchanged as assessed by Western blot analysis. These differences may be caused by changes in the intracellular environment which, like the intracellular concentration of cyclic adenosin monophosphate, exhibit their effect on the intact cell but not on isolated proteins. BShm et al. 23 have shown that basal activity of intracellular adenylate cyclase activity is reduced. This reduced activity corresponds to a decrease of basal cyclic adenosine monophosphate content in myocardium of patients with heart failure. 24 Further investigations are needed to clarify whether there is a causal relation between delayed Ca 2+ uptake into the SR and reduction of the systolic [Ca2+]i transient. 4 However, our results show that relaxation abnormalities of the heart in severe h e a r t failure ~ire partially the result of a slowed Ca 2+ reuptake by the SR. The importance of the sarcolemmal Ca2+-ATPase for intracellular [Ca2+]i handling in human ventricular cells is largely unknown. Alterations of this Ca 2+ transport system have been demonstrated in a variety of animal models of heart failure. 21, 25, 26 [Ca2+]i did not decrease significantly from concentrations that are usually reached during systole 4 during the time course of a cardiac cycle. Therefore our results indicate that the Ca 2+ ATPase of the cell membrane does not contribute significantly to the cytoplasmic Ca 2+ removal during an individual heartbeat in myocytes from patients with heart failure or in undiseased hearts. In conclusion, alterations of diastolic [Ca2+]i handling in ventricular myocytes from patients with severe heart failure are partially caused by a delayed
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Ca 2+ uptake of the SR. The sarcolemmal Ca 2+ ATPase does not contribute significantly to the cytoplasmic Ca 2+ removal in the human ventricle. Further studies are necessary to clarify whether alterations of diastolic [Ca2+]i removal have any causal relation to the reduced systolic [Ca2+]i transient in heart failure. W e t h a n k J. N u s s e r for e x p e r t technical assistance a n d Prof. B. R e i c h a r t a n d his colleagues in t h e D e p a r t m e n t of Cardiac Surgery, U n i v e r s i t y of M u n i c h , for providing t h e myocardial tissue. REFERENCES
I. Gwathmey JK, Copelas L, MacKinnon R, Schoen FJ, Feldman MD, Grossman W, Morgan JP. Abnormal intracelinlar calcium handling in myocardium from patients with end-stage heart failure. Circ Res 1987;61:70-6. 2. Grossman W. Diastolic dysfunction and congestive heart failure. Circulation 1990;81(Suppl III):III-l-III-7. 3. Gwathmey JK, Warren SE, Briggs GM, Copelas L, Feldman MD, Phillips P J, Callahan M, Schoen F J, Grossman W, Morgan JP. Diastolic dysfunction in hypertrophic cardiomyopathy: effect on active force generation during systole. J Clin Invest 1991;87:1023-31. 4. Beuckelmann DJ, N~bauer M, Erdmann E. Intracellular calcium handling in ventricular myocytes from patients with terminal heart failure. Circulation 1992;85:1046-55. 5. Movsesian MA, Bristow MR, Krall J. Ca2+ uptake by cardiac sareoplasmic reticulum from patients with idiopathic dilated cardiomyopathy. Circ Res 1989;65:1141.4. 6. Mercadier J-J, Lompre A-M, Due P, Boheler KR, Fraysse J-B, Wisnewsky C, Allen PD, Komajda M, Schwartz K. Altered sarcoplasmic reticulum Ca2+-ATPase gene expression in the human ventricle during end-stage heart failure. J Clin Invest 1990;85:305-9. 7. Takahashi T, Allen PD, Lacro RV, Marks AR, Dennis AR, Schoen FJ, Grossman W, Marsh JD, Izumo S. Expression of dihydropyridine receptor (Ca2+ channel) and calsequestrin genes in the myocardium of patients with end-stage heart failure. J Clin Invest 1992;90:927-35. 8. Carafoli E. Membrane transport of calcium: an overview. In: Fleischer S, Fleischer B, eds. Methods in enzymology. Biomembranes. Vol. 157. San Diego: Academic Press, 1988:3-11. 9. Mullins LJ. A mechanism for Na+/Ca 2+ transport. J Gen Physiol 1977;70:681-95. 10. Caroni P, Carafoli E. The regulation of the Na+-Ca2+ exchanger of heart sarcolemma. Eur J Biochem 1983;132:451-60. 11. Jencks WP. How does a calcium pump pump calcium? J Biol Chem 1989;264:18855-8. 12. Robertson SP, Johnson JD, Potter JD. The time-course of Ca 2+ exchange with calmodalin, troponin, parvalbumin, and myosin in response to transient increases in Ca 2+. Biophys J 1981;34:559-69.
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