Differences in mechanisms of SR dysfunction in

Am J Physiol Heart Circ Physiol 279: H709–H718, 2000.

Differences in mechanisms of SR dysfunction in ischemic vs. idiopathic dilated cardiomyopathy LUYI SEN,1 GUANGGEN CUI,1 GREGG C. FONAROW,1 AND HILLEL LAKS2 1 Division of Cardiology, Department of Medicine, and 2Division of Cardiothoracic Surgery, Department of Surgery, University of California Los Angeles Medical Center, University of California Los Angeles School of Medicine, Los Angeles, California 90095 Received 16 July 1999; accepted in final form 31 January 2000

Sen, Luyi, Guanggen Cui, Gregg C. Fonarow, and Hillel Laks. Differences in mechanisms of SR dysfunction in ischemic vs. idiopathic dilated cardiomyopathy. Am J Physiol Heart Circ Physiol 279: H709–H718, 2000.—We examined 1) contractile properties and the intracellular Ca2⫹ concentration ([Ca2⫹]i) transient in cardiac myocytes and 2) sarcoplasmic reticulum (SR) Ca2⫹ uptake and release function in myocardium from patients with end-stage heart failure caused by ischemic (ICM) vs. idiopathic dilated cardiomyopathy (DCM). The amplitude of cell motion was decreased 43 ⫾ 6% in ICM and 68 ⫾ 7% in DCM compared with that in normal organ donors (DN). Time to peak of shortening was increased 43 ⫾ 15% in DCM, but not in ICM. Prolongation of the relaxation time was more predominant in ICM. In DCM the systolic [Ca2⫹]i was decreased 27 ⫾ 9% and diastolic [Ca2⫹]i was increased 36 ⫾ 11%. In ICM the diastolic [Ca2⫹]i was increased 59 ⫾ 12% but the systolic [Ca2⫹]i was unchanged. A significant decrease of the ATP-dependent SR Ca2⫹ uptake rate associated with the reduction of the SR Ca2⫹-ATPase protein level was found in ICM. In contrast, the significant decrease in SR Ca2⫹ release rate was distinct in DCM. The large amount of Ca2⫹ retained in the SR associated with a significant decrease in the maximum reaction velocity and increase in the Michaelis-Menten constant in the caffeine concentration-response curve suggests a fundamental abnormality in the SR Ca2⫹ release channel gating property in DCM. We conclude that potentially important differences exist in the intracellular Ca2⫹ homeostasis and excitation-contraction coupling in ICM vs. DCM. The SR Ca2⫹ release dysfunction may play an important pathogenetic role in the abnormal Ca2⫹ homeostasis in DCM, and the SR Ca2⫹ uptake dysfunction may be responsible for the contractile dysfunction in ICM. calcium homeostasis; sarcoplasmic reticulum function

on human myocardial tissue obtained from the hearts of recipients undergoing transplantation for end-stage heart failure (14, 30). Because the sample sizes were relatively small in previous studies, in investigations of Ca2⫹ handling in the failing heart, ischemic cardiomyopathy (ICM) and idiopathic dilated cardiomyopathy (DCM) have frequently been grouped together (2, 10, 12, 14, 24, 30). Most recently, an observation of altered frequency dependence in Ca2⫹ release from the sarcoplasmic reticulum (SR) has been reported in DCM by Sipido et al. (29). No further comparison has been systematically made in the abnormalities of Ca2⫹ homeostasis and excitation-contraction coupling between these two major subgroups of DCM. Given the obvious differences in etiology, the potential exists for quantitative and/or qualitative differences in the important pathophysiological components of these two types of heart muscle disease (4, 5). The purpose of this study is to characterize the abnormalities of contractile dysfunction and Ca2⫹ homeostasis in myocardium from patients with end-stage heart failure caused by ICM vs. DCM. For the first time, we have been able to determine the contractile properties and intracellular Ca2⫹ transients in isolated ventricular myocytes and SR Ca2⫹ uptake and release functions in the myocardium of the same hearts. Our results indicate potentially important differences in intracellular Ca2⫹ handling between ICM and DCM. These differences presumably are related to the distinct pathophysiologies of two types of DCM. METHODS

failure is manifested by systolic and diastolic dysfunction. However, the underlying mechanism of contractile dysfunction remains poorly understood. Abnormal modulation of intracellular Ca2⫹ has been proposed as a major mechanism underlying the systolic and diastolic dysfunction that develops with heart failure in various cardiomyopathies (10). Several laboratories have performed studies

Patients. We analyzed data from 22 patients with endstage heart failure undergoing cardiac transplant surgery at the University of California Los Angeles Medical Center. The subjects included 11 patients with ICM and 11 with DCM. This study was approved by the Committee for the Protection of Human Subjects at the University of California Los Angeles Medical School. For each patient, the pretransplant medical record was reviewed to determine the clinical diagnosis as defined by clinical history, cardiac catheterization, ejection fraction and left ventricular end-diastolic dimension by

Address for reprint requests and other correspondence: L. Sen, Div. of Cardiology, Dept. of Medicine, UCLA Medical Center, UCLA School of Medicine, 10833 Le Conte Ave., 47-123 CHS, Los Angeles, CA 90095-1679 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

CARDIOMYOPATHY-INDUCED HEART

http://www.ajpheart.org

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SR DYSFUNCTION IN ISCHEMIC AND IDIOPATHIC CARDIOMYOPATHY

echocardiography, and hemodynamic parameters, such as the cardiac output and the pulmonary capillary wedge pressure. The diagnoses were further confirmed by the pathological findings from the examination of the explanted hearts. There were no significant differences in any of the clinical parameters recorded in this study between two major subgroups of patients (Table 1). All ICM and DCM patients were receiving digoxin, diuretics, and angiotensin-converting enzyme inhibitors. None of the patients had received a ␤-antagonist, a phosphodiesterase inhibitor, amiodarone, or a Ca2⫹ channel blocker within 4 wk of undergoing transplantation. All eight nonfailing donor hearts (DN) analyzed in this study were obtained from trauma victims and were free of cardiovascular pathology. The control hearts came from potential donors that were rejected as heart transplant donors because of size mismatch, blood group incompatibility, or underlying systemic disease. Clinical data for these individuals are reported in Table 2. None of patients included in this study were on dobutamine or dopamine at the time of harvest in donors or explantation in recipients. Cell isolation. On removal of the recipient’s diseased heart, transmural pieces of fresh tissue weighing ⬃5 g were excised rapidly from the left ventricular free wall near the apex and immediately placed into ice-cold cardioplegic solution. Care was taken to obtain samples from the same region of each heart. The regions free of scarring in the ICM patients were in the same region as the normal and DCM samples or from the closest region at the apex that was free of scar tissue. A coronary arterial branch was cannulated with polyethylene tubing for perfusion with media containing oxygenated warm (37°C) Krebs-Henseleit bicarbonate-buffered solution (pH 7.30) containing (mM) 118 NaCl, 4.7 KCl, 1.20 MgSO4, 1.20 KH2PO4, 25 NaHCO2, and 15 dextrose, 0.05% collagenase,

and 0.03% hyaluronidase (28). Distal superficial branches were ligated to obtain better perfusion of penetrating arteries. After 20 min of perfusion, the myocardium was removed from the perfusion setup, cut into 2-mm3 pieces, and placed in a flask containing 0.05% collagenase, 0.03% hyaluronidase, 0.001% trypsin, and 0.9 mM CaCl2 in Krebs-Henseleit buffer. The tissue fragments were shaken in an orbital shaking water bath at 37°C for 15 min. After they were washed twice, the cell suspensions were centrifuged through a Ficoll gradient to remove capillaries, blood cells, and other unwanted debris and then resuspended in 0.9 mM Ca2⫹ solution. Intracellular Ca2⫹ concentration and contractility measurements. Intracellular free Ca2⫹ concentration ([Ca2⫹]i) of single cardiac myocytes was measured using the Ca2⫹-sensitive fluorescent dye fura 2 (28). Cells attached to glass coverslips with collagen (Vitrogen 400) were incubated with 3 ␮M fura 2-AM for 10 min at room temperature and then washed for 5 min in HEPES-buffered medium to remove extracellular and bound dye. The glass coverslip with attached cells was placed in a specially designed chamber that allowed for continuous flow of 150 ␮l of perfusion medium over the monolayer (28). The chamber was placed on the stage of an inverted phase-contrast microscope (Nikon, Garden City, NY) enclosed in a Lucite box. The cells were perfused at a rate of 1 ml/min with a physiological solution containing (mM) 5 HEPES, 0.9 CaCl2, 4.0 KCl, 140 NaCl, 0.5 MgCl2, and 11 glucose (pH 7.35) at 37°C. Cells were electrically stimulated at 1.5 Hz with platinum electrodes. The microscope was attached to an instrument (Fluorolog 2, SPEX Industries, Edison, NJ) with excitation wavelengths set at 340 and 380 nm and emission wavelength set at 505 nm. The two excitation wavelengths were set to alternate 100

Table 1. Clinical characteristics of patients with end-stage heart failure Patient No.

Age, yr

Gender

Diagnosis

CI, l䡠 min⫺1䡠 m⫺2

PCWP, mmHg

LVEF, %

LVEDD, mm

Pathological Finding

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

64 66 59 61 68 57 45 69 57 43 51 56 45 54 23 43 64 46 15 29 51 54

M M F M M M M F M M F M F M M M M M F M M M

ICM ICM ICM ICM ICM ICM ICM ICM ICM ICM ICM DCM DCM DCM DCM DCM DCM DCM DCM DCM DCM DCM

2.6 1.4 2.5 1.5 1.6 2.9 1.8 1.3 1.5 1.4 1.8 1.6 1.7 2.6 1.4 1.6 2.1 1.3 1.5 1.4 2.4 1.5

20 38 30 16 25 30 38 31 40 30 17 27 32 17 28 21 32 35 36 34 19 26

12 20 12 15 16 25 18 17 10 15 20 13 15 19 10 17 10 9 16 14 18 20

78 64 70 84 70 63 62 60 90 87 68 73 61 70 98 73 66 77 93 62 62 84

ACVD, TILV ACVD, TILV ACVD, TILV ACVD, TILV ACVD, TILV ACVD, TILV ACVD, TILV ACVD, TILV ACVD, TILV ACVD, TILV ACVD, TILV CMG, FBS CMG, FBS CMG, FBS CMG, FBS CMG, FBS CMG, FBS CMG, FBS CMG, FBS CMG, FBS CMG, FBS CMG, FBS

ICM DCM

58 ⫾ 9 44 ⫾ 15

1.8 ⫾ 0.6 1.7 ⫾ 0.5

29 ⫾ 8 28 ⫾ 7

16 ⫾ 4 15 ⫾ 4

72 ⫾ 11 74 ⫾ 13

Medication

ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI, ACEI,

DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG, DIG,

DIU DIU DIU DIU DIU DIU DIU DIU DIU, NIT DIU DIU DIU DIU DIU DIU, NIT DIU DIU DIU DIU DIU DIU DIU

Values are means ⫾ SE for 8 men and 3 women for ischemic cardiomyopathy (ICM) and 9 men (M) and 2 women (F) for idiopathic dilated cardiomyopathy (DCM). CI, cardiac index; PCWP, pulmonary capillary wedge pressure; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end-diastolic dimension. Pathological finding indicates gross and histological findings from explanted heart specimens. ACVD, extensive ischemic change with severe artherosclerotic coronary artery disorder; TILV, transmural infarction of left ventricle; CMG, cardiomegaly without artherosclerotic coronary artery disease; FBS, myocardial fibrosis. ACEI, angiotensin-converting enzyme inhibitor; DIG, digoxin; DIU, diuretics; NIT, nitrates.

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SR DYSFUNCTION IN ISCHEMIC AND IDIOPATHIC CARDIOMYOPATHY

Table 2. Clinical and anamnestic data from nonfailing heart donors Gender

Age, yr

Diagnosis

Others

M M M M M M F M

27 24 16 29 35 47 43 50

Polytrauma; CB Polytrauma; CB Polytrauma; CB Trauma; CB Trauma; CB Trauma; CB Polytrauma; CB Polytrauma; CB

Nicotine abuse Obesity

HVB(⫹) HVB(⫹) Lung cancer

Diagnoses represent those that led to clinical death and other diagnoses in patients’ history. CB, intracerebral bleeding; HVB, hepatitis B virus.

times/s measurements and were stored in the separate memories of a microcomputer (Datamate, SPEX Industries). After equilibration, the fluorescence signal was continuously monitored. At the end of each experiment, the background autofluorescence from cells not loaded with fura 2 was subtracted from the original signals. In most cases the background signal was ⬍1% of the fluorescence signal from the fura 2-loaded cell. [Ca2⫹]i values are presented as 340 nmto-380 nm fluorescence ratios. Simultaneously, the single-cell contraction was monitored by a low-light-level silicone television camera attached to the microscope observation tube with a ⫻2 coupler. The television camera video output was connected to a video motion detector and displayed on a Panasonic television monitor. The television camera had an interlace defeat producing an image composed of 262 raster lines. The motion detector monitored a selected raster-line segment and provided the amplitude of a cell moving along the raster line at a sampling interval of 16 ms (⬃42 data points recorded during each contraction-relaxation cycle). Light-dark contrast at the edge of the cell provided a marker for measurement of the amplitude of motion. For caffeine-induced contraction, cells were equilibrated for 15 min, during which the cells were superfused with normal Tyrode solution and paced at 0.5 Hz before induction of caffeine contraction (1). The electrical stimulation was interrupted 5 s before the solution was switched to one containing 0 Na⫹ and 0 Ca2⫹ (with 1 mM EGTA). After superfusion with 0 Na⫹-0 Ca2⫹ solution for 15 s, various concentrations of caffeine were perfused from two directions to the center of the chamber at a rate of 2 ml/min for 3 s and then maintained at 1 ml/min. The desired caffeine concentration can be reached in ⬍2 s in the chamber. Only those cells in the center of the coverslip (⬃5 mm diameter area) were used. To determine the caffeine threshold, cells were first challenged with 20 mM caffeine. At this concentration of caffeine, all the SR Ca2⫹ is released (1, 23). After cells were washed with 0 Na⫹-0 Ca2⫹ solution and reloaded with Ca2⫹, they were challenged with stepwise-increasing concentrations of caffeine (from 0.1 mM at increments of 0.5 mM) until contracture was measured (caffeine threshold). The caffeine threshold concentration was defined as the lowest caffeine concentration that induced a contracture ⬎10% of the amplitude of contracture induced by 20 mM caffeine. Various concentrations of caffeine (0.1–20 mM) were used to determine EC50. EC50 was defined as the caffeine concentration at which half-maximal contraction was achieved. SR vesicles. The SR membrane vesicles were prepared from myocardium according to the method described previously (6). SR microsomes were first isolated by the homoge-

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nization of cardiac muscle and differential centrifugation. The subfraction of cardiac microsomes was isolated using a sucrose step gradient. Buffers used in the incubation of isolated SR of known Ca2⫹ activity were prepared using EDTA-buffered solutions. The sequential sections of the SR vesicle pellets from hearts of ICM and DCM were examined morphologically by transmission electron microscopy. The protein concentration of the SR microsomes was determined by the Lowry method with BSA as a standard (15). Western blot analysis of SR Ca2⫹-ATPase and ryanodine receptor. The amount of SR Ca2⫹-ATPase and ryanodine receptor-2 contained in a subfraction of SR microsomes was assessed using the Western blot technique (12, 25). Briefly, samples of 100 ␮g of protein of the particulate fraction were denatured by heating to 95°C in 5% SDS, 13% saccharose, 0.083 M dithiothreitol, 0.033 M Tris䡠HCl, pH 6.8, and 0.0033% bromphenol blue and subjected to a 7.5% SDSPAGE. Proteins were transferred to a Hybond-enhanced chemiluminescence nitrocellulose membrane for 2 h. For SR Ca2⫹-ATPase, the blot was incubated for 90 min with a 1:3,000-diluted mouse anti-dog cardiac Ca2⫹-ATPase monoclonal IgG1 antibody and then for 60 min with peroxidaseconjugated anti-mouse IgG secondary antibody. For ryanodine receptor, the blot was incubated overnight at 4°C with a 1:5,000-diluted pig anti-rabbit cardiac ryanodine receptor polyclonal antibody and then for 2 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG. The polyclonal antibody was shown to react with ryanodine receptor-1 and -2 at 4°C. The band densities were evaluated by densitometric scanning. Western blot analysis was also performed in the soluble fraction by using the same protocol. Because no immunoreactivity of SR Ca2⫹-ATPase or ryanodine receptor-2 was found in the soluble fraction, it can be assumed that the total amount of analyzed proteins was recovered from the particulate fraction. Analyzed protein levels were normalized against the total protein recovered from the SR vesicles and against ␤-myosin heavy chain (MHC) protein content. The linearity of the assay was checked by plotting the correlation of different amount of proteins (4–6 series dilutions for each sample) to the densitometric units for each series of blots. SR Ca2⫹ uptake rate. SR Ca2⫹ uptake was continuously monitored by measuring changes in the fura 2 signal from medium surrounding the vesicles (13). Fluorescence was measured in a 3-ml cuvette with a SPEX spectrofluorometer with dual-excitation monochrometers. Excitation wavelengths were set at 340 and 380 nm, and samples were alternately excited by means of a chopper. Emission was monitored at 510 nm. The spectrofluorometer was operated in the ratio mode (emission signal to reference signal) to compensate for any fluctuation in the intensity of the excitation source. The emission signal was integrated for 1 s. Ca2⫹ uptake was measured in buffer consisting of (mM) 100 KCl, 5 MgCl2, 20 HEPES, 2.5 oxalate, 0.625 ATP, and 5 creatine phosphate and 0.4 U/ml creatine phosphokinase. Oxalate was added to act as a Ca2⫹-precipitating anion inside the vesicles. The inclusion of a precipitating anion served to minimize any leakage of Ca2⫹ from the vesicles and to maintain the Ca2⫹ gradient across the vesicular membrane. The addition of 2.5 mM oxalate to buffers containing Ca2⫹ in the concentration ranges used in these experiments produced no change in the ratio 340/380, indicating that the product of their concentrations did not exceed their solubility product. Reactions were conducted at room temperature and pH 7.1. Fura 2 free acid was added to a final concentration of 2.5 ␮M. Reactions were initiated by the addition of Ca2⫹ to aliquots of

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vesicles (1–5 ␮g) previously equilibrated with buffer. Samples were mixed with a minimagnetic stirrer in the cuvette. The relationship between free Ca2⫹ concentration ([Ca2⫹]free) and fura 2 fluorescence was calculated from the following equation as discussed by Grynkiewicz et al. (9)

where ⌬[Ca2⫹]total is the decline in total Ca2⫹ (in ␮M) over the time interval (⌬t, in min) and vol and wt are the cuvette volume (in liters) and vesicle weight (in mg), respectively. If fura 2 is the only significant Ca2⫹ buffer in the sample solution, then [Ca2⫹]total can be calculated from [Ca2⫹]free as follows

duced by addition of caffeine to the aliquots of vesicles. Caffeine was quickly mixed (in seconds) into the aliquots by a minimagnetic stirrer in the cuvette. To validate the method, the hydrolysis and leakage of fura 2 from SR vesicles were examined. After SR vesicles were loaded with Mag-fura 2-AM, at the indicated times, aliquots were removed, and fluorescence in the supernatant was recorded at 340-nm excitation at 0 and 1 mM Ca2⫹. The time course of change in the ratio of extra-SR Ca2⫹-sensitive to -insensitive species of fura 2 was monitored. The leakage of dye from vesicles was very slow and linear with time. The change in the ratio caused by the hydrolysis and leakage was ⬍10% during 100 min. There was no significant difference in the rate of dye leakage from the vesicles isolated from DN and myopathic hearts. To evaluate the Ca2⫹-dependent SR transmembrane Ca2⫹ gradient, the ratio of fluorescence (340/380) was measured for the fura 2 signal from SR isolated from DN hearts over a range of Ca2⫹ activities (10 nM–10 mM). The mean Kd in eight preparations from DN hearts was 0.28 ␮M, which was the same as in preparations from myopathic hearts. The calibration of the kinetic parameters of Ca2⫹ release was the same as that of Ca2⫹ uptake. The velocity (V) of Ca2⫹ release by the vesicles (in nM 䡠 mg protein⫺1 䡠 s⫺1) was related to the changes in [Ca2⫹]free in the SR vesicles

[Ca 2⫹] total ⫽ [Ca 2⫹] free ⫹ [fura-Ca 2⫹]

V ⫽ ⫺(⌬[Ca 2⫹] free /⌬t) ⫻ (vol/wt)

where [fura-Ca2⫹] is the concentration of fura 2 bound to Ca2⫹. Likewise

where ⌬[Ca2⫹]total is the decline in total Ca2⫹ (in ␮M) over the time interval (⌬t, in min) and vol and wt are the cuvette volume (in liters) and vesicle weight (in mg), respectively. Materials. Hyaluronidase and trypsin were purchased from Sigma Chemical (St. Louis, MO), collagenase II and DNA from Worthington Biochemical (Malvern, PA), and fura 2 free acid and Mag-fura 2 from Molecular Probes (Eugene, OR). Statistical analysis. Statistical analysis was performed using Student’s two-tailed t-test and two-way ANOVA. Where appropriate, simple comparisons were made by paired t-test. Differences between more than two groups were assessed by ANOVA and Bonferroni’s correction, or Scheffe´’s F test was applied. Results (means ⫾ SD) are expressed as an index of dispersion of values around the mean.

[Ca 2⫹] free ⫽ K d ⫻ [(R ⫺ R min)/(R max ⫺ R)] ⫻ ␤ where R is the ratio of emission intensity at 340- to that at 380-nm excitation, Rmin and Rmax are minimum and maximum ratios, and Kd is the dissociation constant for fura 2 and Ca2⫹ (assumed to be 200 nM). Values for Rmin, Rmax, and ␤ were determined for the buffer conditions of the experiments and were 1.18, 29.05, and 10.6, respectively. The velocity (V) of Ca2⫹ uptake by the vesicles (in ␮M 䡠 mg protein⫺1 䡠 min⫺1) is related to the change in total Ca2⫹ concentration in the cuvette ([Ca2⫹]total) by V ⫽ ⫺(⌬[Ca 2⫹] total/⌬t) ⫻ (vol/wt)

[fura-Ca 2⫹] ⫽ [fura] total ⫺ [fura] free where [fura]total is the total dye concentration and [fura]free is the unbound dye concentration. Application of these equations was based on the assumption that ATP and creatine phosphate are not significant Ca2⫹ buffers and that Mg2⫹ does not bind significantly to fura 2 at the concentration range of Ca2⫹ used in the experiments. The Kd for the fura 2-Ca2⫹ complex is given as K d ⫽ [Ca 2⫹] free ⫻ [fura] free /[fura-Ca 2⫹] This expression for Kd allows for the calculation of [Ca2⫹]total in terms of known or measured parameters according to [Ca 2⫹] total ⫽ [Ca 2⫹] free ⫹ [fura] total ⫺ {(K d ⫻ [fura] total)/(K d ⫹ [Ca 2⫹] free)} The Ca2⫹ uptake kinetics can be described by the Hill equation n n V/V max ⫽ [Ca 2⫹] free /([K⬘ ⫹ [Ca 2⫹] free )

where n is the Hill coefficient, Vmax is the maximum uptake n velocity, and K⬘ ⫽ [Ca2⫹]0.5 . The velocities are reported as the averaged velocities, unless specified. SR Ca2⫹ release rate. The velocity of the SR Ca2⫹ release was determined by measuring the changes of the fura 2 fluorescence ratio in SR vesicles in response to caffeine exposure. Isolated SR vesicles were loaded with Mag-fura 2-AM, a membrane-permeable form (8). After SR vesicles were incubated for 15 min with Mag-fura 2-AM, organelles were washed twice and resuspended in buffer containing (mM) 100 KCl, 5 MgCl2, and 20 HEPES, pH 7.1. Fluorescence was measured in a 3-ml cuvette with the same spectrofluorometer and the same setting described above. The free Ca2⫹ activity inside SR vesicles was determined by measurement of the fluorescence ratio (340/380) in the fura 2-loaded SR vesicle. The velocity of Ca2⫹ release was estimated from the change in the fura 2 fluorescence ratio in-

RESULTS

Impairment of contractile function in ICM and DCM. Morphology of the cardiac myocytes from ICM, DCM, or DN was not distinguishable under light microscopy (Fig. 1A). Approximately 80% of cells exhibited rodshaped morphology with clear cross-striations and excluded trypan blue, and the isolated cell yield was identical for the three groups. The resting cell length was also not significantly different between DN and two subgroups (DN: 169 ⫾ 30 ␮m, n ⫽ 57 cell/8 hearts; ICM: 175 ⫾ 38 ␮m, n ⫽ 73 cells/11 hearts; DCM: 180 ⫾ 39 ␮m, n ⫽ 79 cells/11 hearts, 7–10 cells were measured/heart, P ⬎ 0.05). As shown in Fig. 1, B and C, the amplitude of cell motion normalized to the resting cell length (maximum length measured under phase-contrast microscopy) was significantly decreased 43 ⫾ 6% in ICM compared with that in DN (P ⬍ 0.01), and the decrease was more profound in DCM (68 ⫾ 7%, P ⬍ 0.001). The difference between ICM and DCM was statistically significant (P ⬍ 0.01). As shown in Fig. 1, B and D, time to peak of shortening was significantly prolonged in DCM (269 ⫾ 28 ms) compared with that in

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Fig. 1. Contractile amplitude of isolated cardiac myocytes from patients with end-stage heart failure caused by ischemic cardiomyopathy (ICM) and idiopathic dilated cardiomyopathy (DCM) compared with that from normal donor hearts (DN). A: cardiac myocytes isolated from patients with ICM and DCM and from DN. B: superimposed traces representing the amplitude of cell motion. Traces were recorded from cardiac myocytes from patients with ICM and DCM and from DN. One set of representative data is shown. Resting cell length was 169 ␮m for DN, 176 ␮m for ICM, and 187 ␮m for DCM. C: amplitude of cell motion recorded in cardiac myocytes from DN (n ⫽ 57 cells/8 hearts) and from patients with ICM (n ⫽ 73 cells/11 hearts) and DCM (n ⫽ 79 cells/11 hearts). Means ⫾ SD for the amplitude of cell motion were normalized by resting maximal cell length. D: time to peak of shortening and time to 50% of relaxation (t1/2) in cells from patients with ICM and DCM and from DN.

DN (190 ⫾ 10 ms, P ⬍ 0.001), but not in ICM (194 ⫾ 15 ms, P ⬎ 0.05). Time to 50% of relaxation (t1/2) was markedly increased in ICM (199 ⫾ 12 ms) compared with that in DN (125 ⫾ 11 ms, P ⬍ 0.001). In DCM, t1/2 was also significantly increased (169 ⫾ 19 ms) compared with that in DN (P ⬍ 0.01); however, the increment was much less than in ICM (P ⬍ 0.05). Decrease of the [Ca2⫹]i transient in ICM and DCM. To obtain further insights regarding [Ca2⫹]i handling and its association with contractile behavior, [Ca2⫹]i transients were measured simultaneously with the

amplitude of cell contraction. The diastolic [Ca2⫹]i was enhanced 49 ⫾ 9% in ICM and 31 ⫾ 7% in DCM compared with that in DN (P ⬍ 0.01). The systolic [Ca2⫹]i in DCM was reduced 39 ⫾ 9% compared with that in DN, but it was not changed in ICM (Fig. 2A). Therefore, the amplitude of the [Ca2⫹]i transient was reduced more significantly in DCM than in ICM (P ⬍ 0.01). As shown in Fig. 2B, time to peak [Ca2⫹]i transient was prolonged from 73 ⫾ 9 ms in DN to 108 ⫾ 27 ms in DCM (P ⬍ 0.05). The slope of the upstroke of the [Ca2⫹]i transient was reduced 48 ⫾ 9% in DCM com-

Fig. 2. Intracellular Ca2⫹ concentration ([Ca2⫹]i) transient in cardiac myocytes from patients with ICM and DCM compared with that from DN. A: superimposed traces of the [Ca2⫹]i transients recorded simultaneously with the amplitude of cell motion measurements from cardiac myocytes of DN and from patients with ICM and DCM. The systolic and diastolic [Ca2⫹]i manifested by the fura 2 fluorescence ratio (means ⫾ SD) were measured in cardiac myocytes from DN (n ⫽ 32 cells/8 hearts) and patients with ICM (n ⫽ 40 cells/11 hearts) and DCM (n ⫽ 45 cells/11 hearts). B: time to peak [Ca2⫹]i transient and t1/2 (means ⫾ SD).

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pared with that in DN (P ⬍ 0.05), but it was unchanged in ICM. In contrast, the downstroke of the [Ca2⫹]i transient was prolonged ⬎1.6-fold in ICM (t1/2 ⫽ 178 ⫾ 32 ms), but it was prolonged only 17% in DCM (t1/2 ⫽ 81 ⫾ 25 ms) compared with that in DN (t1/2 ⫽ 69 ⫾ 14 ms). Alterations in caffeine-induced single contraction in ICM and DCM. To further evaluate the intra-SR Ca2⫹ content and SR Ca2⫹ release function in intact cardiac myocytes, cell shortening and the [Ca2⫹]i transient were recorded during the steady-state twitch and caffeine-induced contraction. The caffeine threshold was not different in 28 cells/11 hearts (2–3 cells/heart) from ICM (0.27 ⫾ 0.07 mM) compared with 20 cells/8 hearts from DN (0.23 ⫾ 0.06 mM, P ⬎ 0.05), but it was significantly higher in 22 cells/11 hearts from DCM (2.24 ⫾ 0.68, P ⬍ 0.01). As shown in Fig. 3A, 1 mM caffeine could induce a single contraction with the peak amplitude 2.4-fold higher than twitch in DN, but the same concentration of caffeine could not induce cell contraction in DCM. In ICM the maximal caffeine responsiveness was significantly reduced; however, the EC50 (5.1 ⫾ 0.2 mM) was same as that in DN (5.0 ⫾ 0.1 mM, P ⬎ 0.05). In DCM, although the maximal response to 20 mM caffeine was reduced 27 ⫾ 5% compared with that in DN (P ⬍ 0.05), it was still 67 ⫾ 9% higher than in ICM. Not only was the caffeine threshold increased, the EC50 was also significantly higher (9.4 ⫾ 1.1 mM, P ⬍ 0.01) in DCM. As shown in Fig. 3B, time to peak of the single contraction induced by 5 and 10 mM caffeine in cells from ICM was not different from that in DN but was significantly reduced in DCM (P ⬍ 0.01). The relaxation was significantly prolonged in both subtypes of cardiomyopathy compared with that in DN (P ⬍ 0.01): t1/2 was prolonged 87 ⫾ 18% in ICM and 36 ⫾ 11% in DCM (P ⬍ 0.01).

Abnormalities of SR Ca2⫹ uptake in ICM and DCM. As shown in Fig. 4A, when 250 nM Ca2⫹ was present in the buffer, the averaged velocity of ATP-dependent Ca2⫹ uptake by the same amount of isolated SR vesicles (5 ␮g protein) was significantly decreased in ICM (0.87 ⫾ 0.25 ␮M 䡠 mg⫺1 䡠 min⫺1, n ⫽ 11, P ⬍ 0.01) but only slightly decreased in DCM (1.37 ⫾ 0.43 ␮M 䡠 mg⫺1 䡠 min⫺1, n ⫽ 11, P ⫽ 0.08) compared with that in DN (1.43 ⫾ 0.21 ␮M 䡠 mg⫺1 䡠 min⫺1). The curve of Ca2⫹ uptake velocity vs. pCa was significantly shifted to the right in SR vesicles from ICM compared with that from DN (n ⫽ 8, P ⬍ 0.05), but not from DCM (Fig. 4B). The Hill coefficient was 1.6 ⫾ 0.6 for ICM, 2.3 ⫾ 0.6 for DCM, and 2.4 ⫾ 0.5 for DN. As shown in Fig. 4C, SR Ca2⫹-ATPase protein levels obtained by Western blot analysis and normalized per total protein were significantly reduced by 34% in ICM and 16% in DCM compared with that in DN. A similar result was found when SR Ca2⫹-ATPase protein levels were normalized by ␤-MHC protein (in densitometric units of SR Ca2⫹ATPase per densitometric units of ␤-MHC). The differences between DN (15.7 ⫾ 2.3) and ICM (10.1 ⫾ 1.8), as well as DCM (13.1 ⫾ 3.1), were statistically significant (P ⬍ 0.05). SR Ca2⫹ uptake rate was significantly correlated with SR Ca2⫹-ATPase protein levels (data not shown). SR Ca2⫹ uptake rate normalized by SR Ca2⫹-ATPase protein level was not significantly different between DN and DCM but was still slightly decreased in ICM (P ⫽ 0.054; Fig. 4D). Abnormalities of SR Ca2⫹ release in DCM. As shown in Fig. 5A, the electron-microscopic morphology of the heavy SR vesicles from serial sections of the gradient centrifugation pellets was the same in DN, ICM, and DCM. Ryanodine receptor-2 protein levels quantified by Western blot analysis and normalized per total protein or normalized per ␤-MHC protein were not

Fig. 3. Amplitude of the caffeine-induced single contraction in cardiac myocytes from patients with ICM and DCM compared with that from DN. A: effects of different concentrations of caffeine on a single contraction. Data are presented as percent increase of the peak amplitude of a caffeine-induced single contraction from the peak amplitude of the twitch in the same cell. B: superimposed tracings of single contractions induced in cardiac myocytes from DN and patients with ICM and DCM by different concentrations of caffeine. C: percent increase of the time to peak of the contraction and t1/2 in a single contraction from twitch in the same cell induced by 10 mM caffeine. Values are means ⫾ SD.

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SR DYSFUNCTION IN ISCHEMIC AND IDIOPATHIC CARDIOMYOPATHY

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Fig. 4. Averaged velocity of Ca2⫹ uptake by sarcoplasmic reticulum (SR) vesicles isolated from the myocardium of patients with ICM and DCM compared with that from DN. A: ATP-dependent Ca2⫹ uptake by 5 ␮g of protein of SR vesicles isolated from patients with ICM and DCM and from DN. Signals from 3–5 recordings were averaged for each sample. Data (means ⫾ SD) were plotted every 20th s of the continuous recordings. B: Ca2⫹ concentration dependence of the SR Ca2⫹ uptake velocity in vesicles isolated from patients with ICM and DCM and from DN. Inset, typical fluorescence intensity recording made from a SR vesicle preparation from DN. C: Western blot analysis of SR Ca2⫹-ATPase protein levels normalized per total protein. D: SR Ca2⫹ uptake rate normalized by SR Ca2⫹-ATPase protein level.

significantly different in ICM and DCM compared with the level in DN (Fig. 5B). As shown in Fig. 5C, the initial intra-SR Ca2⫹ concentration was significantly higher in SR vesicles from DCM (1.38 ⫾ 0.21 ␮M/mg, n ⫽ 11) than from ICM (0.96 ⫾ 0.08 ␮M/mg, n ⫽ 11) and DN (0.98 ⫾ 0.11 ␮M/mg, n ⫽ 8, P ⬍ 0.05). The averaged velocity of SR Ca2⫹ release was decreased more than twofold in DCM compared with DN but was

unchanged in ICM. As shown in Fig. 5C, there were clearly two components in the SR Ca2⫹ release curve for DN and ICM. The velocity of the fast and slow component of the curve was determined by fitting one line to the steepest part of the fast component of the curve and another line to the steepest part of the slow component of the curve. The velocities of the fast and slow components of the SR Ca2⫹ release induced by 10

Fig. 5. Ca2⫹ release from SR vesicles isolated from the myocardium of patients with end-stage heart failure caused by ICM and DCM compared with that from DN. A: morphology of the heavy SR vesicles from patients with ICM and DCM and from DN was not different under electron microscopy. Transmission electron micrographs were taken from the top 10th section of the serial sections of the pellets obtained from the heavy SR vesicle gradient centrifugation procedure in ICM, DCM, and DN. B: protein level of ryanodine receptor-2 determined by Western blot analysis and normalized per protein in ICM and DCM was not different from that in DN. C: time course of caffeine (10 mM)-induced SR Ca2⫹ release normalized per ryanodine receptor-2 protein. Typical Ca2⫹ release curves recorded from a patient with DCM and from DN are superimposed. D: effect of graded concentration of caffeine on the averaged velocity of Ca2⫹ release from the SR vesicles isolated from patients with ICM and DCM compared with that from DN.

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mM caffeine were not significantly changed in ICM (4.34 ⫾ 0.54 and 1.68 ⫾ 0.25 nM 䡠 mg⫺1 䡠 s⫺1, respectively) compared with that in DN (4.36 ⫾ 0.31 and 1.69 ⫾ 0.21 nM 䡠 mg⫺1 䡠 s⫺1, respectively, P ⬎ 0.05). In DCM the biexponential velocity curve was singularized. The velocity of SR Ca2⫹ release was 1.85 ⫾ 0.18 nM 䡠 mg⫺1 䡠 s⫺1 in DCM, which was slower than the fast component observed in DN and ICM. The difference of the SR Ca2⫹ release rate normalized by ryanodine receptor-2 protein level between DN and myopathic (ICM or DCM) hearts remained the same as that normalized by SR protein level. As shown in Fig. 5D, the concentration-effect curve in DCM was significantly shifted to the right (P ⬍ 0.01), but not in ICM. The Michaelis-Menten constant of caffeine was increased from 2.4 and 2.5 mM in DN and ICM, respectively, to 3.1 in DCM (P ⬍ 0.01). The Vmax of the caffeine effect on velocity of SR Ca2⫹ release was reduced 37 ⫾ 9% in DCM compared with that in DN (P ⬍ 0.01) but was not decreased in ICM. DISCUSSION

The present study is the first to systematically characterize the SR function in two major subgroups of cardiomyopathy, ICM and DCM. The results presented here provide direct evidence that the fundamental abnormalities in SR Ca2⫹ release function are unique for DCM and the alteration in SR Ca2⫹ reuptake is distinct in ICM. Given the obvious differences in etiology, the potential exists for quantitative or qualitative differences in the important pathophysiological components of these two types of heart muscle disease. Idiopathic DCM. In DCM the amplitude of cell contraction was significantly decreased in association with a remarkable reduction in the amplitude of the [Ca2⫹]i transient, which was mainly due to the significant decrease in the peak systolic [Ca2⫹]i. These findings provide direct evidence that supports the hypothesis that the amount of Ca2⫹ cycling at physiological stimulation rates is significantly decreased; therefore, the systolic Ca2⫹ available for activation of contractile proteins in tension development is insufficient in DCM (11, 23). A similar observation has been reported by Sipido et al. (29), although the peak systolic [Ca2⫹]i was not compared statistically between DCM, ICM, and DN. Profound prolongation of the time to peak of cell motion and [Ca2⫹]i, in contrast to much less prolongation of the [Ca2⫹]i decline, indicates the characteristic systolic dysfunction in DCM (22). A high concentration of caffeine was able to restore the normal amplitude of cell contraction by releasing the large amount of Ca2⫹ retained in the SR. All these findings indicate that the reduction in SR Ca2⫹ release rate in DCM is mainly caused by alteration of Ca2⫹-induced Ca2⫹ release, rather than alteration of contractile proteins. Furthermore, the caffeine sensitivity of SR is lower in DCM than in DN, which is manifested by increased threshold and a rightward shift of the dose-response curve in caffeine-induced single contraction. It is pos-

sible that the lower caffeine affinity of SR in DCM is caused by a reduced Ca2⫹ loading of SR, which, in turn, is caused by a decreased Ca2⫹ uptake activity of SR (21, 31). However, the rate of Ca2⫹ uptake by SR vesicles was only slightly decreased (19) because of the decrease of SR Ca2⫹-ATPase expression (17). Greater alterations in the prolongation of relaxation and [Ca2⫹]i decay observed in a single-cell preparation could also be caused by the excessive Ca2⫹ retained in the SR. In contrast, the velocity of the caffeine-induced Ca2⫹ release from SR vesicles was remarkably and characteristically decreased in DCM, not in ICM. Additionally, the saturated concentration of caffeine could restore the normal contractile function in cardiac myocytes from DCM. Moreover, the SR Ca2⫹ content was significantly higher in DCM, although this measurement may not reflect the actual Ca2⫹ concentration in the SR vesicles, since some Ca2⫹ may be leaked during the vesicle isolation process (1). The findings in the caffeine-induced Ca2⫹ release in the isolated SR vesicles were complementary to the findings in the intact cell. More importantly, the abnormality of the results of caffeine-induced Ca2⫹ release in the SR vesicle preparations and in intact cells was consistent with the alterations in the kinetics of twitch amplitude and intracellular Ca2⫹ transients. These observations suggest a fundamental abnormality in the SR Ca2⫹ release channel in situ that, by itself, could result in significant insufficiency in the dynamic availability of Ca2⫹ for excitation-contraction coupling, regardless of the abnormality of such factors as the L-type Ca2⫹ channel and kinase-dependent phosphorylation, which may also be altered in DCM. On the other hand, these findings also indicate that the alteration of SR Ca2⫹ release in DCM is not due to the insufficient Ca2⫹ restored in the SR, because the luminal Ca2⫹ concentration was higher in DCM. More likely, it is caused by the abnormal gating mechanism of the SR Ca2⫹ release channel, whether the trigger was Ca2⫹ or caffeine. Expression of an altered Ca2⫹ release channel isoform might be responsible for the SR dysfunction in DCM. The protein level of the ryanodine receptor was not changed, and the density of the ryanodine binding was not altered (26). Ca2⫹ release channel function might be defective because of alterations in the structure of the channels, abnormal interactions with modulators, and defects in other proteins that may interact with the channel. In the present study the alterations in twitch and in caffeine threshold and the dose-response relation demonstrate the fundamental abnormality of the SR Ca2⫹ release channel gating property in DCM. The reduction in Vmax of the caffeine doseresponse relation and paradoxical alteration of the two components of the time course of Ca2⫹ release, on the other hand, suggest the abnormality of the channel kinetics. Directly characterizing the channel properties will be helpful in future studies. ICM. In ICM the amplitude of cell contraction was significantly reduced in association with a significant reduction in the amplitude of the intracellular Ca2⫹ transient. The reduction in the amplitude of the intra-

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SR DYSFUNCTION IN ISCHEMIC AND IDIOPATHIC CARDIOMYOPATHY

cellular Ca2⫹ transient was mainly due to the greatly increased diastolic Ca2⫹ concentration. It is possible that the increased diastolic [Ca2⫹]i could be mainly due to excessive Ca2⫹ uptake by mitochondria; therefore, compartmented Ca2⫹ was greatly increased. However, we found that the rate and amount of Mn-induced quenching of subcellularly compartmented dye were not different in ICM and DCM (unpublished observations). Additionally, the observation of the markedly slowed diastolic decline of [Ca2⫹]i in association with the increased relaxation time strongly suggests that the underlying abnormality of intracellular Ca2⫹ homeostasis in ICM is distinct from that in DCM. The significant reduction in the peak amplitude of caffeineinduced single contraction associated with a greatly prolonged relaxation, but normal time to peak of the single contraction, further indicates that the accumulation of Ca2⫹ by SR is the principal mechanism involved in the decreases of Ca2⫹ transient and contractile function in ICM. Although SR Ca2⫹ uptake in failing hearts has been previously evaluated by several other groups as well, no comparison has been made between the two major subgroups of cardiomyopathy (2, 19, 27). Hasenfuss et al. (11) reported a significant decrease in SR Ca2⫹ uptake rate in homogenized myocardium from 9 DCM and 5 ICM patients; however, Movsesian et al. (18) did not observe any change in Ca2⫹-calsequestrin in DCM. In the present study we compared the velocity and the kinetic parameters of Ca2⫹ uptake by the SR vesicles from ICM and DCM. A remarkable SR Ca2⫹ uptake dysfunction was observed in ICM by direct measurement of SR Ca2⫹ uptake velocity and kinetics. Additionally, the pCa-Ca2⫹ uptake velocity curve was shifted to the right and the Hill coefficient was significantly decreased. The changes of SR Ca2 uptake kinetic behavior could also account for the dominant SR Ca2⫹ uptake dysfunction in ICM and play an important role in the abnormal contractile function and Ca2⫹ homeostasis (19). Diminished SR Ca2⫹-ATPase activity was mainly due to the reduction of the amount of SR Ca2⫹-ATPase protein. When normalized by SR Ca2⫹-ATPase protein level, the alteration of the SR Ca2⫹ uptake rate was greatly, but not completely, eliminated (16). Therefore, alterations other than reduction of the amount of SR Ca2⫹-ATPase, such as decrease of enzyme activity, may also be in part responsible for the abnormality of SR Ca2⫹ uptake function in ICM. In contrast to DCM, caffeine-induced Ca2⫹ release from purified cardiac SR vesicles was not altered in ICM. Consistent with the previous observation that the ryanodine receptor density was not changed, we found that the ryanodine protein level analyzed by Western blot was also not changed in ICM (19). More likely, the diminished Ca2⫹ release from SR in intact left ventricular muscle strips observed previously by isometric force and heat measurements may be secondary to the insufficient Ca2⫹ in the SR Ca2⫹ pool as a result of the reduction of Ca2⫹ uptake by the SR (7, 20).

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The inconsistent findings of SR Ca2⫹ uptake rate in failing hearts, which included ICM and DCM (11), reported previously by several groups have been thought mainly to be caused by the relatively low sensitivity of SR Ca2⫹ uptake rate measured in purified SR vesicles compared with that in homogenates due to the vesicle isolation procedure. In the present study the oxalate-supported, ATP-dependent Ca2⫹ uptake by SR vesicles was continuously monitored using the Ca2⫹-selective fluorescent dye fura 2 (13). In 3 DN, 4 ICM, and 5 DCM, the SR Ca2⫹ uptake rate was also measured using the conventional radiometric method, and similar results were found, but with wider variability (unpublished observation). These findings indicate that the on-line measurement system not only allows determination of the kinetic parameter for Ca2⫹ uptake with smaller aliquots of vesicles but also has higher resolution and greater precision. The inconsistency of results in previous reports may have been caused by the methodology and, more likely, the different patient population. A larger study population with more uniform clinical characteristics is required in future studies to eliminate the limitation in previous studies and in the present study. In conclusion and summary, the present study demonstrates that although various types of end-stage heart muscle disease may exhibit common features, there are important pathophysiological differences due to the distinct differences in the pathogenesis. In ICM, a distinct SR Ca2⫹ uptake dysfunction resulted from the decreases of the SR Ca2⫹-ATPase gene expression and is responsible for the abnormal contractile function and Ca2⫹ homeostasis. In DCM, SR Ca2⫹ release dysfunction plays a pivotal role in the pathogenesis of abnormal Ca2⫹ homeostasis and systolic function. Expression of an altered Ca2⫹ release channel isoform might be responsible for the alteration of SR Ca2⫹ channel gating properties and the channel kinetics. This study was supported by a grant-in-aid from the American Heart Association, Greater Los Angeles Affiliate, and National Heart, Lung, and Blood Institute Grant HL-49126. REFERENCES 1. Bers DM and Berlin JR. Kinetics of [Ca]i decline in cardiac myocytes depend on peak [Ca]i. Am J Physiol Cell Physiol 268: C271–C277, 1995. 2. Beuckelmann DJ and Erdmann E. Ca2⫹ currents and intracellular [Ca2⫹]i transients in single ventricular myocytes isolated from terminally failing human myocardium. Basic Res Cardiol 87: 235–243, 1992. 3. Beuckelmann DJ, No¨bauer M, and Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85: 1046–1055, 1992. 4. Brillantes AM, Allen PD, Takahashi T, Izumo S, and Marks AR. Differences in cardiac calcium release channel (ryanodine receptor) expression in myocardium from patients with endstage heart failure caused by ischemic vs. dilated cardiomyopathy. Circ Res 71: 18–26, 1992. 5. Bristow MR, Anderson FL, Port D, Skerl L, Hershberger RE, Larrabee P, O’Connel JB, Renlund DG, Volkman K, Murray J, and Feldman AM. Differences in ␤-adrenergic neuroeffector mechanisms in ischemic vs. idiopathic dilated cardiomyopathy. Circulation 84: 1024–1039, 1991.

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