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Cardiovascular Research 1998 37(2):515-523; doi:10.1016/S0008-6363(97)00279-4
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Copyright © 1998, European Society of Cardiology

Calcium uptake by the sarcoplasmic reticulum, high energy content and histological changes in ischemic cardiomyopathy

Kanigula Mubagwa*, Peter Kaplan1, Bharati Shivalkar2, Marc Miserez, Veerle Leunens, Marcel Borgers3 and Willem Flameng

Laboratory of Experimental Cardiac Surgery, Katholieke Universiteit Leuven, KUL Campus Gasthuisberg, B-3000 Leuven, Belgium

* Corresponding author. Tel.: +32 (16) 347132; fax: +32 (16) 347139; e-mail: kanigula.mubagwa@med.kuleuven.ac.be

Received 7 July 1997; accepted 4 November 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Sarcoplasmic reticulum (SR) Ca2+ uptake, myocardial high energy content and histology were examined in different zones of hearts from patients with ischemic cardiomyopathy. Methods and Results: Unfractionated homogenates were prepared from left ventricular samples obtained in three zones of each heart: an infarct-remote zone, an outer peri-infarct zone, and an inner peri-infarct zone. Oxalate-supported Formula uptake was measured at 37°C using a filtration method. Maximum rate (Vmax) of uptake in absence or in presence of ryanodine was lower in inner peri-infarct (7.4±0.7 and 9.5±0.8 nmol min–1 mg–1 of protein, respectively; mean±SEM) and outer peri-infarct tissues (8.8±0.8 and 12.0±0.8 nmol min–1 mg–1) than in infarct-remote myocardium (12.7±2.1 and 15.8±2.2 nmol min–1 mg–1). The apparent affinity constants for Ca2+ (KCa) as well as the Hill coefficients were not different. Homogenate DNA (1.6±0.1, 1.6±0.1 and 1.7±0.1 mg/g of remote, inner peri-infarct and outer peri-infarct myocardium, respectively) and adenine nucleotides contents (ATP: 15±1.3, 14±0.8 and 15±1.0 µmol/g dry weight, respectively) were similar in all tissues. Fibrosis was increased in inner peri-infarct tissue (37±6%; vs. 13±2% and 12±2% in both remote and outer peri-infarct tissues, respectively), but the number of abnormal cells was not significantly different. Conclusion: The decrease of Ca2+ uptake in ischemic cardiomyopathy is not homogeneous in the ventricular wall, and reflects a decreased number/activity of SR Ca2+-ATPase, without altered Ca2+-affinity or increased Ca2+ leakage through ryanodine receptors.

KEYWORDS Sarcoplasmic reticulum; Calcium uptake; Ischemia; Infarction; Cardiac; Human


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Alterations of ventricular contraction and relaxation, as well as of the inotropic response to changes in heart rate [1]have been observed in heart failure. The mechanisms underlying the relaxation abnormality remain unclear but three major types of evidence support the hypothesis of a dysfunction of the sarcoplasmic reticulum (SR) in heart failure. (i) In tissues or myocytes isolated from end-stage failing hearts, high resting intracellular Ca2+ levels (Cai), low Cai transients, as well as an altered time course of these transients, have been reported [2–4]. (ii) SR vesicles from human or animal failing hearts show a depressed Ca2+ uptake rate [5, 6]. Similarly, the rate of Ca2+ loading in skinned muscles decreases with the severity of heart failure [7]. (iii) Finally, many studies have detected a decreased expression of SR Ca2+-ATPase and/or Ca2+-release channel mRNAs in heart failure ([8–10]; for review see Ref. [11]). However, there is still no consensus on myocardial SR function in heart failure. First, several studies (e.g. [12, 13]) failed to find any difference of Ca2+ uptake or ATPase in preparations from normal or failing human hearts. Second, it is still not clear whether the dysfunction of Ca2+ handling reported in failing hearts is characteristic of the syndrome of heart failure irrespective of its etiology or whether the underlying pathology also produces specific effects. The alterations in Cai transients seem to be common to idiopathic and ischemic failing hearts [3]; the decrease of Ca2+ uptake by SR vesicles has been obtained in idiopathic, ischemic, hypertrophic and other cardiomyopathies; similarly, the decrease of Ca2+-ATPase mRNA expression mentioned above has been reported in ischemic as well as in idio-myopathic hearts. In contrast, one study has found a decrease of gene expression for SR Ca2+-release channel in ischemic but not in idiopathic failing myocardium [10].

Except for a few studies (e.g., [4, 10, 13–15]) that used ischemic hearts, most investigations on SR function in heart failure have used hearts from idiopathic dilated cardiomyopathy. In the present study, we restricted our investigation to the etiologic group of ischemic cardiomyopathy, and compared the SR Ca2+ uptake of nonischemic and ischemic tissues obtained from the same hearts. It is expected that this approach will eliminate interindividual variations as well as effects induced by drugs used in the therapy, factors which necessarily complicate the comparison of diseased and nondiseased hearts. Since there is little information about the role of the SR Ca2+-release channel in the decrease of Ca2+ uptake observed in failing hearts, we carried out experiments in which the uptake was studied in the presence of ryanodine to block the channels. To investigate potential correlation between changes in SR function and energy state or structural changes, myocardial high energy content and histology were also examined in the samples used for Ca2+ uptake.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Patients
Hearts were obtained from 6 patients with severe coronary artery disease (‘ischemic cardiomyopathy’), who underwent cardiac transplantation. Tissues used were not needed for diagnostic or organ donation purposes and were to be discarded following excision. The study conforms with local institutional guidelines and with principles outlined in the Declaration of Helsinki. All patients received aspirin, angiotensin converting enzymes, cardiac glycosides and diuretics. Some also received nitrovasodilators or calcium antagonists. The pretransplantation diagnostic evaluation included electrocardiography at rest, coronary arteriography, ventricular function study by contrast ventriculography and/or by nuclear angiography using Tc-99m, and echocardiography (see Table 1Tables 2–4GoGo).


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  		Table 1 Characteristics of patients

 

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  		Table 2 Catheterization data

 

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  		Table 3 Echocardiographic data (2-D, M-mode and Doppler)

 

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  		Table 4 EGNA data

 
2.1.1 Electrocardiography
All patients had electrocardiographically proven infarction (Q or QS wave patterns) of the left ventricular anterior wall, and had experienced clinical symptoms of myocardial infarction before transplantation. Electrocardiographic localization of the infarct, along with other characteristics of the patients are presented in Table 1.

2.1.2 Coronary angiography and contrast ventriculography
All patients had a contrast angiography and ventriculography up to 3 months prior to transplantation. Coronary angiography was done with the Sones technique. Significant coronary artery disease was defined as >=80% stenosis of the coronary luminal diameter. After measurement of the left ventricular pressure, a biplane left ventriculography at 30° right anterior oblique projection was done. Calibration of the ventricular image was made with a grid filmed at the level of the left ventricular cavity. Wall motion was measured from the end-diastolic and end-systolic outlines of the left ventricle in right anterior oblique projection using routinely available software. A right heart catheterization was also performed to measure pressures (right ventricular, pulmonary and wedge pressures) and the cardiac output with the thermodilution technique.

2.1.3 Equilibrium gated nuclear angiography
The patients also underwent an equilibrium gated nuclear angiography during the pretransplant evaluation. Red blood cells were labelled with Tc-99m using an in vitro technique. After centrifugation and removal of the supernatant, the labelled cells were injected in the patient, and nuclear angiographic acquisition was made in the left anterior oblique projection (at 45° for 10 min, and twice at 70° for 5 min). A small field gamma camera (PHO/GAMMA V, Siemens) fitted with a high resolution parallel collimator was used and was connected to a computer. At 45° in the left anterior oblique projection there is minimum overlap of the left or the right ventricle, and the left ventricle is divided into 8 sectors. Global ejection fraction and sectorial ejection fraction of the anterior (A), posterolateral (PL), apical (AP), septal (S) and basal portions of the left ventricle were determined in this projection.

2.1.4 Echocardiography
2-D and M-mode echocardiography along with Doppler was performed in all patients for the measurement of global and regional function, as well as for the evaluation of valvular morphology and function.

2.2 Tissue procurement and handling
At the time of transplantation, cardiectomy was performed after cold cardioplegia (with the NIH-2 solution at 4°C) under extracorporeal circulation. The hearts were transported to the laboratory in cold (4°C) Tyrode solution containing 27 mM K+ and 50 mM glucose to arrest electrical and mechanical activity and to protect against hypoxia. The data obtained from functional and angiographic investigations mentioned above (Tables 1–4GoGoGo), as well as the macroscopic appearance of the heart after cardiectomy were used to define the infarcted zone and 3 regions outside the infarct from which tissue was procured. The infarct was located in the anterior or anterolateral part of the left ventricle, with the posterior part having the best preserved function. Samples corresponding to the ‘inner peri-infarct’ region were taken from the macroscopically non-infarcted border of the infarct. The ‘infarct-remote’ samples corresponded to the functionally best preserved basal segments, and the ‘outer peri-infarct’ zone corresponded to a region between the inner peri-infarct and the infarct-remote zones. From each of the three last zones, a transmural needle biopsy was taken and was kept in 3% glutaraldehyde buffered to pH 7.4 with 90 mM potassium phosphate. Transmural tissue samples (2 cmx2 cm; containing the sites of biopsy) were excised for Ca2+ uptake and other biochemical measurements. They were immediately immersed in liquid nitrogen and were stored at –180°C until use.

2.3 Ca2+ uptake studies
The methods used for Ca2+ uptake have been described in detail in a previous study from this laboratory [16]and are only briefly summarized here. After thawing, ventricular muscle was homogenized in 5 volumes of 20 mM imidazole (pH 7.0), 0.6 M KCl and 5 mM sodium azide (NaN3) using a Polytron homogenizer (Kinematica, Switzerland). The homogenate was filtered through two layers of cheesecloth and immediately assayed for Ca2+ uptake.

Ca2+ uptake by the homogenate was measured at 37°C with a filtration technique [17]. The final reaction medium contained (in mM): 100 KCl, 20 imidazole (pH 7.0), 10 K-oxalate, 5 MgCl2, 10 NaN3, 5 ATP, 0.5 EGTA, various amounts of CaCl2 (0.3 µCi/ml Formula ) to produce the desired free-Ca2+ concentration, 0.43 ryanodine, when added, and 0.6–1.0 mg/ml of protein. In order to measure Ca2+ uptake under conditions where little or no Ca2+ leak through SR release channels occurs [18]the homogenate was preincubated for 8 min, at 37°C, in a medium containing the drug. The Ca2+ uptake reaction was started by the addition of ATP, MgCl2, Formula , EGTA. Aliquots of the reaction medium were taken every min up to 4 min of reaction and were filtered by suction through Millipore filters (0.45 µm pore size), washed with (concentration in mM) 100 KCl, 20 imidazole (pH 7.0), 1 EGTA and 5 MgCl2. Radioactivity trapped on the filters was determined by liquid scintillation spectroscopy. The velocity of Ca2+ transport was determined by linear regression of the relationship calcium uptake vs. time. Parameters of Ca2+ transport (maximum velocity, Vmax; apparent dissociation constant for Ca2+, KCa; and Hill coefficient, n) were calculated by nonlinear least-square fitting of the experimental data to a Hill equation (see Table 5). Protein was determined by a modified Lowry method with included SDS, using bovine serum albumin as standard. To minimize variations due to daily conditions of the assay, tissues from the 3 zones of each heart were processed on the same day.


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  		Table 5 Parameters of cardiac Ca2+ uptake by the sarcoplasmic reticulum

 
2.4 Estimation of tissue cell mass and cell viability
Since the decrease of the rate of Ca2+ uptake (when expressed relative to the protein content) could be due to the presence of a lower number of cells associated with an increase in the degree of fibrosis, the tissue cell mass was roughly estimated by measuring the tissue DNA content, using a fluorometric method [19]. Briefly, an aliquot of the filtered homogenate used for Ca2+ uptake or protein determination was added to a freshly prepared solution containing (in mM): 50 NaH2PO4 (pH 7.4), 2000 NaCl, 2 NaEDTA and 1 µg/ml of the dye bisbenzimidine H33258 [GenBank] (Calbiochem). The mixture was incubated for 90 min in the dark at room temperature. Fluorescence intensity was read on a spectrofluorimeter with excitation and emission wavelengths set at 356 nm and 458 nm (5 nm slit width), respectively. Calf thymus DNA (Sigma) was used as standard.

In addition, in order to test the possibility that differences in Ca2+ uptake reflect in-vivo differences in high energy content and cell viability, myocardial contents of ATP and its metabolites were measured in whole tissues of the various zones. A piece of the same tissue procured for Ca2+ uptake studies was used. At the time of analysis, the frozen ventricular transmural specimen were lyophilized, then neutralized perchloric acid extracts of the tissues were prepared and were used for the determination of ATP and its metabolites, using high-performance liquid chromatography as described before [20].

2.5 Histology
Toluidine blue-stained semi-thin (2 µm) sections, and hematoxylin and eosin-stained semi-thick (6 µm) sections of the biopsies preserved in glutaradehyde were used for the assessment of structural changes. An analysis of the percentage of altered cells and of fibrosis was made. Cell alteration was defined as loss of cytoplasmic organelles and was determined by planimetry relative to the cellular surface section observed (see Ref. [21]). A minimum of 100 myocytes were evaluated per section and only cells with a visible nucleus were included. A score giving the relative number of cells showing various extents of loss of sarcomeres was obtained. A loss of >=10% of myofibrillar material was necessary for myocytes to be classified as ‘altered’ or ‘myolytic’.

2.6 Statistical analysis
All values are expressed as mean±standard error of the mean. Comparisons between tissue groups were made with ANOVA followed by the t-test, and a p<=0.05 indicated statistical significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Global and regional function
All patients suffered from chronic ischemic heart disease (see Tables 1 and 2Go). At the time of transplantation, the patients were in terminal heart failure (NYHA class III–IV; see Table 1), with increased cardiothoracic index and dilated left ventricle. In 5 patients, cardiac index (1.2±0.1 l/m2; patients 1–5 in Table 2) and global ejection fraction, as determined by contrast ventriculography, echocardiography and equilibrium gated nuclear angiography (26±2%, 33±2%, 20±1%, respectively; Tables 2 and 3GoTable 4), were low. In the same patients, an analysis of sectorial ejection fraction also showed decreased values (A: 7±1%; PL: 7±4%; AP: 12±5%; S: 8±5%), with only the basal portions showing significantly higher ejection fractions (42±6%; p<0.05 vs. other regions). Echocardiographically, there was hypo- to akinesia in the anterior portions and diffuse global hypokinesia in all patients (Table 3). In patient 6, cardiac index and ejection fraction were high despite heart failure due to accompanying interventricular communication and valve insufficiency.

3.2 Nonhomogeneous decrease of SR calcium uptake in the ventricular wall
Fig. 1, A and B, shows the rates of Ca2+ uptake (at different [Ca2+]) by the homogenates of remote, inner and outer peri-infarct tissues. The uptake in all tissues increased as a function of [Ca2+] and a maximum was obtained around pCa 5.3 (or 5 µM [Ca2+]). The uptake, measured either in the absence or in the presence of ryanodine, was lower in the peri-infarct zones compared to the infarct-remote zone. Fitting the data from each experiment with the Hill equation provided estimates of the maximal uptake rate (Vmax) and the apparent affinity for Ca2+ (KCa) (Table 5). Vmax in the presence of ryanodine was 16±2.2 nmol min–1 mg–1 and KCa was 0.4±0.1 µM in infarct-remote tissues. Relative to the value in remote tissues, Vmax in the presence of ryanodine was decreased to 60% and 76% in tissues obtained from inner and outer peri-infarct zones, respectively (Table 5; p<0.05 for Vmax in both tissues vs. in infarct-remote zone). The difference in Ca2+ uptake between inner and outer peri-infarct tissues was not statistically significant (p>0.2). KCa and Hill coefficient values were similar for all three types of tissues. The differences between Vmax of the various zones in the absence of ryanodine were qualitatively the same as those obtained in the presence of the drug (Table 5). The relative increase in Vmax produced by ryanodine (27±7%, 41±16% and 30±9%, in remote, outer and inner peri-infract zones, respectively) was not significantly different between tissues.


Figure 1
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  		Fig. 1 A: Ca2+-dependence of oxalate supported Ca2+ uptake by homogenates from infarct-remote (open circles; n=6), outer peri-infarct (filled circles; n=6) and inner peri-infarct (open squares; n=5) zones of human ventricular myocardium. Homogenates incubated in the absence of ryanodine. Values are means±SEM. B: Ca2+-dependence of oxalate supported Ca2+ uptake by homogenates in the presence of ryanodine (430 µM). Same samples as in A.

 
Tissue protein levels in the outer peri-infarct myocardium were decreased to 87±5% of the level in remote myocardium, and in the inner peri-infarct zone to 61±7% (126±2.0, 109±6.4 and 76±8.4 mg protein per g of tissue wet weight for remote, outer peri-infarct and inner peri-infarct zones, respectively; p<0.01 for value of inner peri-infarct vs. remote). However, DNA levels (Table 6) were similar in the homogenates of the various zones, suggesting that similar cellular masses were used in the uptake studies. Similarly, ATP levels and the total energy charge were not different between the three types of tissue (Table 6).


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  		Table 6 Homogenate DNA and tissue adenine nucleotide and levels

 
3.3 Histological findings
Table 7 shows the results of microscopic examination of the tissues and gives the degree of fibrosis as well as the percentage of cells showing more than 50% loss of sarcomere. The extent of fibrosis was higher in the inner peri-infarct zone (37±6%) compared to the outer peri-infarct and the remote zone (12±2% and 13±2%, respectively; p<0.05 vs. inner peri-infarct). The majority of cells in each zone had structural features similar to those in nonischemic hearts. However, two important structural changes were repeatedly observed in the biopsies. First, some myocytes showed variably decreased volume fraction of sarcomeres without showing signs of atrophy. The loss of contractile material in these cells was limited to the vicinity of the nucleus. Second, in the regions of myolysis there was an accumulation of glycogen, intermingled with numerous elongated and small mitochondria. In one patient a few myocytes showed signs of purely degenerative changes such as intracellular edema or abnormal lipid storage. The percentage of myolytic cells ranged between 12 and 25%, and there was no significant difference in the number of these cells between the various zones. There was no significant correlation between the degree of fibrosis (R=–0.19 and –0.15) or the number of abnormal cells (R=0.29 and 0.16) with the Vmax of Ca2+ uptake (in the presence or absence of ryanodine, respectively).


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  		Table 7 Histology data

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the present study, we wanted to investigate the effect of chronic ischemia on the ability of the SR to accumulate and store Ca2+. For this purpose, we used hearts from patients with left ventricular anterior wall infarction, who underwent transplantation due to terminal heart failure. Severe stenosis in the left anterior descendens artery and depression of regional contractile function, maximally localized in the antero-apical part of the left ventricle, were present in the patients. Usually, studies of cardiac SR function in diseased conditions compare normal and diseased hearts obtained from different individuals. This approach is complicated by the presence of variations due to the selection of different groups of subjects and by additional factors due to the fact that diseased and non-diseased hearts will have been exposed to different drugs or different humoral adaptive mechanisms. In addition, such studies do not allow a clear discrimination between the effect of heart failure and those due to the underlying etiological factor (e.g. chronic ischemia). The approach by which different zones of the same heart are compared allows to minimize differences due to genetic variations and to investigate the role of a given pathogenic mechanism. This is possible in ischemic cardiomyopathy (but not in idiopathic cardiomyopathy), since the infarcted and nonischemic myocardial zones (likely to be under the influence of the same humoral factors and of the same therapeutic agents) can be readily separated.

The results show that the rate of Ca2+ uptake is lower in peri-infarct tissues compared to the infarct-remote zone. Despite the small size of the sample, we are confident about these results since a comparison of infarct-remote and inner peri-infarct zones in 5 additional hearts in which a complete analysis could not be carried out confirmed the decrease of Ca2+ uptake in the peri-infarct zone (to 71±5% and 74±6% of the level in infarct-remote tissue, in the absence and presence of ryanodine, respectively; with unchanged KCa and Hill coefficient). Previous studies of Ca2+ uptake, carried out mainly in idiomyopathic hearts, detected a decreased Ca2+ uptake in failing hearts compared to normal hearts. In a study carried out in ischemic hearts [5], the SR Ca2+ uptake was also decreased compared to the uptake in non-ischemic hearts. The present study confirms these results and furthermore provides a kinetic analysis indicating that the decrease of Ca2+ uptake is due to a change in total number or activity of the Ca2+ ATPase molecules, without a change in its apparent affinity for Ca2+ or an increase of Ca2+ loss via SR Ca2+-release channels. Our study also documents a regional decrease of Ca2+ uptake in outer peri-infarct myocardium, that was not evidently infarcted. In this tissue, the extent of fibrosis was not different from the one found in the remote zone.

In the present study all measurements were carried out in a reaction medium of identical composition for all tissue types. This excludes a decrease in substrate availability for the SR Ca2+ pump among the mechanisms responsible for the observed decrease of Ca2+ uptake in the peri-infarct zones. However, an altered availability of Ca2+, Mg2+, ATP, ADP and inorganic phosphate could affect SR Ca2+ uptake in vivo. ATP contents of peri-infarct tissue were similar to the level observed in infarct-remote myocardium (see also Ref. [22]), suggesting that there is no depletion of the energy stores in chronically ischemic myocardium. Since acute ischemia causes a rapid decrease of ATP levels, the high ATP values also indicate that there was no extensive acute ischemic injury caused by the time needed for tissue procurement, transport and sampling.

Ca2+ transport in cardiac SR is stimulated by cAMP-dependent and by Ca2+-calmodulin-dependent phosphorylation of phospholamban [23, 24]. In human myocardium, during heart failure resulting from idiopathic dilated cardiomyopathy the phospholamban-mediated stimulation of SR Ca2+ uptake was not altered [12]and phospholamban was not qualitatively different from that of normal heart [13]. Altered phosphorylation of phospholamban is unlikely to be responsible for the decrease of Ca2+ uptake observed in the present study since we did not find any significant difference in KCa between peri-infarct and infarct-remote zones.

A decreased content in intracellular membrane structures has been observed with electron microscopy in ischemic hearts [22]. A decrease of Ca2+-ATPase mRNA expression and of the density of Ca2+-ATPase molecules was observed in human hearts during end-stage heart failure due to various cardiomyopathies, including ischemic cardiomyopathy [13, 25, 26]. These findings are consistent with the change in Vmax of Ca2+ uptake in our study.

In inner peri-infarct tissues, fibrosis was pronounced and made homogenization difficult. Protein concentration in homogenates from these tissues was lower, presumably as a consequence of a retention of a substantial fraction by the filtration used to prepare the homogenates. This difference in protein content is corrected for by expressing the Ca2+ uptake data per unit mass of protein in the homogenate. Despite this correction, artefactually abnormal values of Ca2+ uptake could result if filtration were to allow poor recovery of cellular material (hence of SR proteins) relative to extracellular material in the homogenates of inner peri-infarct tissues used for uptake studies. In order to assess the amount of cellular material used for Ca2+ uptake, we measured total DNA concentrations in the homogenates obtained after filtration. There was no difference in the DNA content between infarct-remote and peri-infarct myocardial zones. However, since the measured DNA comes from a variety of cell types in the myocardium, with myocytes probably constituting less than the numerical majority of them, it remains likely that in inner peri-infarct tissues there is a decrease in the number of myocytes, which may be replaced with other cell types, e.g. fibroblasts. Histological examination did not reveal any major difference in the percentage of abnormal cells, nor any specific infiltration by noncardiac cells in the ischemic tissues.

In conclusion, our results show a decreased Vmax with unchanged KCa of SR Ca2+ uptake in heart failure due to ischemic cardiomyopathy. Measurements carried out in the presence of ryanodine suggest that background activity of Ca2+-release channels is not affected by chronic ischemia. The regional heterogeneity in changes of SR function suggests a causative role of chronic ischemia in the regulation of SR function.

Time for primary review 21 days.


   Acknowledgements
 
We are grateful to Mr. P. Lemmens for assistance with HPLC, and to Mrs. Magda Mathijs for assistance with the literature.


   Notes
 
1 Permanent address: Department of Biochemistry, Comenius University, Martin, Slovakia. Back

2 Department of Cardiology, University of Leuven, Campus Gasthuisberg, Leuven, Belgium. Back

3 Janssen Research Foundation, Beerse, Belgium. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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