Author + information
- Received January 27, 1997
- Revision received June 10, 1997
- Accepted June 21, 1997
- Published online October 1, 1997.
- Dietmar Krüger, MDA,
- Abdolhamid Sheikhzadeh, MD, FACC, FESCA,*,
- Evangelos Giannitsis, MDA and
- Ulrich Stierle, MDA
- ↵*Dr. Abdolhamid Sheikhzadeh, Department of Cardiology, Medical University Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
Objectives. The aim of this study was to investigate the release kinetics of endothelin after induced short-lasting myocardial ischemia.
Background. Endothelin is an endothelium-derived vasoactive peptide. Unequivocal proof of its cardiac release in ischemic syndromes has not yet been demonstrated.
Methods. A coronary sinus study with atrial pacing was performed in 23 patients with coronary artery disease. Endothelin (ET), cardiac troponin-T (TnT), myoglobin (Mb) and creatine kinase (CK) samples were withdrawn from the coronary sinus and a peripheral vein before and 1, 5, 10, 30 and 45 min and 1, 2, 3 and 6 h after pacing. The appearance of angina pectoris, abnormal cardiac lactate metabolism and ST segment depression were further criteria for myocardial ischemia.
Results. In the study group, pacing stress induced severe ischemia (mean duration ± SD 6.1 ± 1.2 min), with a maximum of 0.34 ± 0.12-mV ST segment depression in 21 of 23 patients and angina pectoris in 22 of 23. The maximal cardiac lactate production was 42.8 ± 17.3% (p < 0.03). TnT and CK levels in the total group were normal; in 14 of 23 patients a transient elevation of Mb with a maximum after 3 h was detected (86.4 ± 27.1 μg/liter, p < 0.03). The ET concentrations increased significantly (p < 0.001) in the coronary sinus (from 4.6 ± 0.8 [baseline] to 12.9 ± 2.7 pg/ml at 1 min after cessation of pacing) and the peripheral vein, respectively (from 4.7 ± 0.7 [baseline] to 8.3 ± 2.1 pg/ml at 1 min). ET further remained elevated for 1 h with persisting higher coronary sinus than peripheral venous concentrations, indicating cardiac ET release. In a control group of 18 patients without heart disease, all variables were unchanged.
Conclusions. Short-lasting severe myocardial ischemia was associated with significant ET release of cardiac origin that lasted up to 1 h.
Endothelin (ET) is an endothelium-derived vasoactive mediator that was discovered in 1988 . Three isoforms of ET (molecular weight 2,492 daltons) have been described, each consisting of 21 amino acids and four cysteine residues that form two disulfide bonds [1, 2]. ET has multiple biologic actions, it contracts vascular and nonvascular smooth muscles in vitro, produces a sustained and long-lasting pressor response in vivo and exerts a positive inotropic effect on the cardiac muscle [2–4]. Increased plasma ET concentrations have been reported in a variety of cardiovascular disorders. ET is supposed to play a role in the pathophysiology of acute coronary syndromes.
A possible role of ET in myocardial infarction is suggested because circulating ET increases within hours after the onset of infarction in humans [5–8]. An ET increase in unstable angina and transient myocardial ischemia is discussed controversially [9–11]; however, normal levels have varied almost threefold from study to study, and this variation may reflect differences in sampling site as well as variations in the specificity of the ET antibodies employed for radioimmunoassay (RIA). ET achieves its highest concentrations at the site of its release . The coronary endothelium as a source for ET release after a myocardial ischemia has not yet been proved.
The aim of our study was to elucidate whether a brief period of myocardial ischemia not leading to myocardial infarction may increase plasma ET levels and to determine the kinetics of a possible cardiac ET release.
1.1 Coronary sinus study.
A coronary sinus lactate study with incremental atrial pacing was performed in 23 patients (15 men, 8 women; mean age ± SD 59.1 ± 5.6 years) with significant obstructive coronary artery disease and normal left ventricular function. Significant stenosis was defined as ≥70% diameter narrowing in an epicardial coronary artery. These patients had not been able to reach the predicted submaximal exercise level in the Bruce treadmill protocol because of unspecified fatigue or dyspnea or because of intermittent claudication in those with peripheral occlusive vascular disease. Exercise stress testing had also not been possible in four patients with an artificial leg. In all patients, significant stenosis was present in the ramus interventricularis anterior and in all other arteries classified as diseased. Seven patients presented with a one-vessel disease, 14 with two-vessel disease and 2 with three-vessel disease. The coronary sinus lactate study was performed to demonstrate or to exclude myocardial ischemia and to clarify the need for coronary revascularization.
The study was performed 1 day after the diagnostic cardiac catheterization in patients with stable sinus rhythm. Standard therapy consisted of aspirin (100 mg daily). No anti-ischemic acting drugs were given 48 h before the study. Patients with preexisting ST segment abnormalities—due, for example, to left or right bundle branch block, left ventricular hypertrophy or digitalis medication—were excluded from the study. At the beginning of the study all patients were free of symptoms and had no evidence of myocardial ischemia on a 12-lead electrocardiogram (ECG). A 7F Zucker catheter (USCI, Bard, Ireland) was positioned in the coronary sinus for incremental atrial pacing and blood sampling. The tip of the catheter was positioned ∼2 to ∼5 cm from the orifice of the coronary sinus to minimize the risk of admixture of right atrial blood in the samples. The correct position was confirmed by hand injection of dye. Pacing was performed for 3 min each at 100, 120, 140 beats/min and (twice) at 160 beats/min. A 12-lead ECG was obtained before pacing, after each 3-min pacing period and for 10 min at 1-min intervals after cessation of pacing.
Paired blood samples for creatine kinase (CK), cardiac troponin-T (TnT), myoglobin (Mb) and ET were withdrawn from the coronary sinus and an indwelling catheter in a vein of the forearm before and at 1, 5, 10, 30 and 45 min and 1, 2, 3 and 6 h after pacing. At the same times and at the end of every 3-min pacing interval, paired samples for lactate were withdrawn from the coronary sinus (cv) and an indwelling catheter in the radial artery (a). By definition, negative values stand for lactate extraction, positive values for lactate production. Myocardial ischemia was defined as a reduction of cardiac lactate extraction greater than −10% or a frank cardiac lactate production >0%. Pacing was stopped with the appearance of typical angina pectoris. The duration of myocardial ischemia was quantified by the persistence of angina and ST segment depressions in the 12-lead ECG after cessation of pacing .
Eighteen patients with angiographically normal coronary arteries served as a control group (13 men, 5 women; mean age 60.7 ± 4.9 years). All patients in the study and control groups gave written informed consent; the study was approved by the hospital ethics committee.
1.2 Laboratory analysis.
The assays of all laboratory variables were performed in blinded fashion without knowledge of whether the patient from whom the sample had been obtained was a member of the study group or of the control group.
1.3 Cardiac TnT.
Cardiac TnT was determined by means of a highly specific enzyme immunoassay (Boehringer Mannheim, Germany). According to the manufacturer, the mean immunoreactive cardiac TnT concentration in healthy control subjects is 0 to 0.5 μg/liter.
1.4 CK activity.
CK activity was measured by an N-acetylcysteine–activated, optimized ultraviolet test (Merck, Darmstadt, Germany). The upper limits of the reference interval of CK are 70 U/liter for women and 80 U/liter for men.
Mb concentrations were determined by a commercially available immunoturbidimetric assay (Behringwerke AG, Marburg, Germany). The upper limit of the reference interval was 75 μg/liter, the sensitivity of the assay had a cutoff value <50 μg/liter.
1.6 Plasma immunoreactive ET.
ET samples were collected into precooled EDTA–containing tubes (Sarstedt, Nümbrecht, Germany) and stored on ice. Samples were centrifuged at 2°C for 20 min (2,000 × g) within 30 min after collection and stored at −70°C for <7 days. Under these conditions no decrease in measured plasma ET concentrations was observed.
Plasma ET concentration was measured by an RIA (Nichols Institut Diagnostics B.V., Wijchen, Netherlands) as described earlier . Preliminary purification of ET was performed on silica C18cartridges after acidification of plasma. A competitive RIA was used with 125I-labeled tracer and a polyclonal rabbit antibody. Bound and free hormones were separated by a second antibody solid phase system followed by centrifugation. The ET-1 antibody exhibited cross reactivities of 52% with ET-2, 96% with ET-3 and 7% with big ET, respectively, but no cross reaction with unrelated peptides such as atrial natriuretic peptide, angiotensin-II, vasopressin or adrenocorticotropin. The sensitivity of the assay was 1 pg/ml and the intraassay and interassay coefficients of variation were 6.8% and 7.9%, respectively. According to the manufacturer, the mean ± SD ET concentration in healthy control subjects (peripheral venous blood) is 4.0 ± 0.67 pg/ml (range 2.54 to 5.29 pg/ml). Low and high concentration quality control samples were prepared by adding ET to EDTA plasma, stored in aliquots at −70°C and assayed in each batch. Recovery of ET was investigated by adding 5 pg/ml and 20 pg/ml ET-1 to EDTA plasma from healthy control subjects. Recovery rate of added ET was 96% and 98% for 5 and 20 pg/ml, respectively.
1.7 Statistical analysis.
The means of the baseline data of the Mb concentrations and cardiac lactate metabolism, respectively, were compared with the means of their maximal increase after pacing by a paired Student ttest (p < 0.05 was considered significant). The differences in values between the ET concentrations in the coronary sinus and peripheral venous blood before and at every sampling time after pacing were assessed by an alpha-adjusted Bonferroni analysis (multiple comparison analysis) preceded by a two-way analysis of variance with repeated measures (p < 0.005 was considered significant). A Pearson correlation analysis and a multiple linear stepwise regression analysis were performed to investigate the influence of the severity of myocardial ischemia (lactate metabolism, ST segment depressions, duration of angina pectoris) on the increase in ET (p < 0.05 was considered significant). Values are presented as mean value ± SD.
In the study group, pacing stress induced angina pectoris in 22 of 23 patients. The duration from the onset to the end of angina corresponded well with the persistence of significant ST segment depressions in the 12-lead ECG and cardiac lactate production after the end of pacing (6.1 ± 1.2 min). ST segment depressions were documented in 21 of 23 patients (maximum 0.34 ± 0.12 mV [range 0.2 to 0.6]) including the patient without angina pectoris. ST depressions were flat in 12 of 21 patients, and downsloping in 9 of 21. Pacing was stopped prematurely because of angina pectoris in 16 patients—at 120 beats/min in four, at 140 beats/min in two, at 160 beats/min within the 1st 3-min interval in five and within the last 3 min of the protocol in five.
Cardiac lactate production increased significantly from a normal lactate extraction <−10% to a maximum of 42.8 ± 17.3% (p < 0.03). Cardiac lactate metabolism (CLM) normalized within 10 min after cessation of pacing in all patients. CK and TnT remained unchanged in the total group. In 14 of 23 patients a transient and significant increase in Mb was documented (p < 0.03). The gradual increase in Mb in the latter group was measured 2 and 3 h after the end of pacing; at 6 h after the end of pacing, Mb had decreased to limits normal levels. For the total study group, the Mb peak was 86.4 ± 27.1 μg/liter (137.7% of the upper limit of normal). According to World Health Organization criteria, no patient had had a myocardial infarction. Nevertheless, pacing stress had induced severe short-lasting myocardial ischemia [12, 14].
The baseline concentrations of ET were 4.6 ± 0.8 pg/ml in the coronary sinus and 4.7 ± 0.7 pg/ml in the peripheral vein. A significant increase in ET was detected in the coronary sinus as well as in the peripheral venous blood with an instantaneous maximum after the end of pacing (p < 0.001) followed by a gradual decrease to the limits of normal within 1 h. Peak ET concentrations measured 1 min after the end of pacing were 12.9 ± 2.7 pg/ml (243.9 ± 51.0% of the upper limit of normal) in the coronary sinus and 8.3 ± 2.1 pg/ml (156.9 ± 39.7% of the upper limit of normal) in peripheral venous blood. The concentrations continued to remain higher in the coronary sinus than in the peripheral venous blood up to 2 h after the cessation of pacing, indicating a persisting cardiac release of ET (Table 1Fig. 1). Significant differences in mean ET levels were found in the coronary sinus between the baseline concentrations and the concentrations recorded 1, 5, 10 and 30 min after pacing, and in the peripheral venous blood between the baseline levels and the concentrations recorded 1, 5 and 10 min after pacing (p < 0.001). (Preliminary data reporting similar changes were published earlier ). There was a tendency to higher ET levels in patients with more accentuated myocardial ischemia (as measured by CLM, duration of angina pectoris and degree of ST segment depressions); however, the correlation was not significant by Pearson analysis although multiple linear stepwise regression analysis revealed a nearly significant partial correlation between the increase in coronary sinus ET and both CLM (p = 0.06) and the duration of angina pectoris (p = 0.07). In the control group all variables were unchanged, and no myocardial ischemia or ET increase was documented.
3.1 Role of ET in cardiovascular disorders.
The vascular endothelium of coronary arteries releases vasodilator and vasconstrictor agents that play an important role in the homeostasis of coronary blood flow. The human endothelium produces mainly ET-1, one of three isotypes (ET-1, ET-2, ET-3) discovered in 1988 from the supernatant of endothelial cells in culture . ET is the most potent vasoconstrictive peptide that exerts a long-lasting pressure response on isolated coronary arteries from animals and humans . It produces a marked increase in coronary vascular resistance in vivo .
Unlike other vasoactive peptides found in the coronary circulation, ET is not stored in subcellular vesicles but is dissolved in low concentrations in the cytoplasma. ET production is enhanced by several stimuli that cause increased transcription and translation of the pre-proendothelin gene [18, 19]. In vitro studies have shown that ET secretion is stimulated by vascular stretch and endothelial injury, shear stress, sympathetic activation, thrombin, an impaired release of endothelium-relaxing factor, endotoxin and hypoxia [20, 21]. ET as a product of vascular endothelium likely achieves its highest concentration and greatest action at the local vascular site of its release [1, 22]. The ET concentrations decrease rapidly because of biochemical breakdown or degradation after reuptake by endothelial cells. ET is thought to play an important role in cardiovascular regulation and pathophysiology. A twofold to fivefold increase in immunoreactive ET concentrations was reported in chronic heart failure and cardiogenic shock [23–26]. Release of ET was also found in patients with hypertension and chronic renal failure .
3.2 ET in myocardial infarction and ischemia.
It has been suggested that ET might contribute to myocardial ischemia and infarction. In several studies [8, 25, 28–30]elevated ET concentrations of 2 to 10 times normal levels were found in the early hours after myocardial infarction. ET concentrations were markedly higher in complicated infarctions reflecting depressed myocardial performance and recurrent myocardial ischemia . In the rat heart an endothelin antibody reduced experimentally induced infarct size .
In the study of Lechleitner et al. , no close correlation was found between peak ET levels and the markers of myocardial injury, suggesting that the stimulus of ET release is not myocardial necrosis itself. This observation supports the hypothesis that ET as a marker of endothelial perturbation is determined by the severity of myocardial ischemia. Thus, a possible association of ET with a transient myocardial ischemia was proposed, although the contribution of ET to the pathophysiology of unstable angina and myocardial ischemia is not yet well understood. Hypoxia in acute coronary syndromes is associated with a cascade of interactions between the atherosclerotic plaque, the endothelium and its local mediator substances, circulating platelets and coagulation factors .
Measurement of ET in induced ischemia in our study may help to elucidate whether the increase of ET is an indication of cell necrosis or is in some way related to the pathophysiology of acute coronary syndromes. The diagnostic procedures of cardiac catheterization and dye exposure do not modify ET concentrations in plasma [33, 34]. The baseline concentrations of ET in our study and control groups were similar and within the manufacturer’s normal range (2.54 to 5.29 pg/ml) and similar to levels found in other studies [28, 29]using the Nicols RIA. The laboratory markers of myocardial cell damage and the ECG showed no evidence of myocardial necrosis in our study. Exercise induced short-lasting frank cardiac lactate production, significant ST segment depressions and a transient increase of Mb characterizing severe myocardial ischemia in our study group. Myocardial ischemia was followed by or was associated with significant cardiac ET release. Owing to the small number of patients in our study group, statistical analyses failed to demonstrate a significant correlation between the severity of myocardial ischemia and ET increase. However, the multiple linear regression analysis data suggest that the results might have become significant in a larger study group.
The ET elevation persisted for 1 h with higher levels in the coronary sinus than in peripheral venous blood, despite the short half-life of ET. This finding suggests that the release of ET is an ongoing active process of cardiac origin. Our study allows a detailed description of the time course of cardiac ET release after well defined myocardial ischemia (Fig. 1). ET is released locally in the coronary endothelium. Our data also indicate that ET release may have a profound effect on coronary vascular tone due to elevated coronary levels, despite subthreshold concentrations in peripheral venous blood (Fig. 1; 45 min after cessation of pacing).
Conflicting results concerning ET release in unstable angina were reported in other studies, probably because of early sampling or interindividual variability of measurements. Ray et al. did not find increased peripheral venous ET concentrations in patients after rest angina pectoris with significant ECG changes. In their study, blood samples were withdrawn only once within a 4-h period after the onset of chest pain, and the severity and duration of myocardial ischemia were not quantified in detail. It is possible that their patients had only mild chest pain and that the time of blood sampling was too late to detect an ET increase. Similarly, Stewart et al. did not detect an ET increase in coronary venous blood in patients with unstable angina. However, at the time of blood sampling (4 to 24 h after an episode of chest pain with significant ECG changes), their patients had been free of symptoms and the ECG had normalized. Although the data in these two studies apparently contradict our findings, the most likely explanation for the observed lack of ET increase is the delay of several hours between the onset of chest pain and ECG changes and the collection of blood samples.
In accordance with our data, Qui et al. reported a significant, 1.5-fold increase in peripheral venous ET concentrations in patients with persisting angina pectoris in whom myocardial infarction had been ruled out. ET elevation was also measured in the coronary sinus of patients with coronary spastic angina associated with transient myocardial ischemia . Our results strongly support the cardiac endothelium as the source of ET release associated with or induced by short-lasting severe myocardial ischemia. Our study documents for the first time in detail unequivocal cardiac ET release kinetics and it provides a plausible explanation for the apparently conflicting results of earlier studies.
- creatine kinase
- cardiac lactate metabolism
- Received January 27, 1997.
- Revision received June 10, 1997.
- Accepted June 21, 1997.
- The American College of Cardiology
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