© 2006 European Society of Cardiology
Angiotensin II AT1 receptor density on blood platelets predicts early left ventricular remodelling in non-reperfused acute myocardial infarction in humans
a Department of Clinical Physiology, Medical Center of Postgraduate Education Warszawa, Poland
b Department of Cardiology, Wolski Hospital Warszawa, Poland
* Corresponding author. Department of Clinical Physiology, Medical Center of Postgraduate Education, Ul. Marymoncka 99, 01-813 Warszawa, Poland. Tel.: +48 22 834 03 67; fax: +48 22 864 08 34. E-mail address: maczmich{at}cmkp.edu.pl
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. Discussion5. Conclusions and future...References |
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Background: Renin—angiotensin-system activity, a principal factor determining ventricular remodelling after myocardial infarction (MI), is dependant on local angiotensin II concentration and angiotensin AT1 receptor (AT1R) density. The latter is regulated by systemic factors acting independently from angiotensin II concentration.
Objective: To test the hypothesis that AT1R density at the onset of MI determines post-MI ventricular remodelling.
Methods: In 48 patients with first acute MI who did not undergo reperfusion therapy, angiotensin AT1R density on blood platelets (reflecting cardiovascular AT1R density) was assessed 13±5 h after the onset of MI, using radioligand binding assay. Left ventricular end-systolic (LVESVI) and end-diastolic volume indices (LVEDVI) and ejection fraction (EF) were assessed by two-dimensional echocardiography as measures of ventricular remodelling.
Results: Predischarge LVESVI and LVEDVI positively and EF negatively correlated with AT1R density. Patients with AT1R density below median had significantly lower LVESVI (33.2±2.4 mL/m2), LVEDVI (70.0±2.8 mL/m2) and higher EF (52.8±2.3%) than patients with AT1R density above median (LVESVI=44.9±2.6, LVEDVI=81.3±3.9 mL/m2 and EF=44.9±2.6%, all p<0.01). In multivariate analysis, only AT1R density and infarct size were independent predictors of early post-MI ventricular dilation.
Conclusions: High density of AT1R at the onset of MI is a predictor of early left ventricular remodelling.
Key Words: Myocardial infarction • Remodelling • Renin angiotensin system • Heart failure • Angiotensin II
Received January 19, 2005; Revised March 25, 2005; Accepted June 30, 2005
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. Discussion5. Conclusions and future...References |
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Left ventricular (LV) remodelling after acute myocardial infarction (MI) is a process of progressive changes in left ventricle chamber size, shape, composition and resulting function [1]. In the early stages, ventricular dilatation can be beneficial as it maintains stroke volume through the Frank-Starling mechanism but the dilation eventually becomes detrimental as it exerts further demands on the surviving myocardium through increased wall tension. Indeed echocardiographic indices of left ventricular dilation and impaired systolic function have been shown to correlate with mortality in patients after myocardial infarction [2].
The process of LV remodelling is largely dependent on activity of renin-angiotensin system (RAS) [2]. Angiotensin II has been shown to activate matrix metalloproteinases [3], which participate in collagen breakdown causing slippage of necrotic myofilaments, leading to early infarct expansion, and to induce myocardial hypertrophy and increased collagen synthesis — landmarks of late remodelling [4].
The response to RAS activation depends on two factors: local angiotensin II concentration and angiotensin II receptor (AT1R) density [5]. Recently AT1R density has been shown to be regulated by a variety of systemic factors: cholesterol, cytokines, and glucocorticoids have been shown to increase while estrogens decrease AT1R density [6]. These factors act largely independently from angiotensin II concentration.
Thus the aims of our study were to verify the hypothesis that AT1R density at the onset of myocardial infarction influences early post-MI ventricular remodelling, and to characterize factors that influence AT1R density in patients with acute MI.
Therefore in the present study we evaluated AT1R density on blood platelets, as a marker of cardiovascular AT1R density, in patients with acute myocardial infarction, in relation to post-infarction echocardiographic indices of LV remodelling.
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2. Methods |
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2.1. Population and the study protocol
From July 2002 to February 2003, 48 consecutive patients out of 215 patients with first acute myocardial infarction (typical ECG changes, troponin T elevated significantly for myocardial infarction [>0.8 µg/L], <18 h from the onset of the chest pain) admitted to the coronary care unit of our institution, who did not receive reperfusion therapy (either due to lack of ECG criteria or due to lack of consent), were recruited into the study. Exclusion criteria were: age >80 years, arterial hypertension, history of myocardial infarction, history of heart failure, ventricular hypertrophy on echocardiography or ECG, cardiomyopathy, significant valvular disease, unstable clinical condition, treatment with ACE inhibitors, AT1 receptor blockers, steroids or hypolipemic drugs within 6 months prior to enrollment. During hospitalization, none of the patients had any episodes of instability requiring intervention. Diabetes mellitus was defined as two measurements of fasting plasma glucose
7.0 mmol/L or use of antidiabetic medication. The investigation conformed to the principles outlined in the Declaration of Helsinki. The study was approved by the local Ethics Committee. All patients signed an informed consent form. In all patients, blood was drawn immediately after admittance to the department, for evaluation of AT1R density on blood platelets and predischarge echocardiography was performed (on day 8±1).
2.2. AT1 receptor evaluation
AT1 receptor density was evaluated by radioligand binding assay, according to the method described previously [5,7]. Briefly, blood (20 mL) was drawn on ACD (anticoagulant citrate dextrose) and stored on ice. Platelet-rich supernatant was obtained by centrifugation (4 °C for 10 min) at 1100 xg. The platelet pellet was collected and resuspended in ACD (4:1 ratio) and centrifuged (4 °C for 15 min) at 2000 xg. The resultant platelet suspensions were used in the binding assay. The platelet suspensions contained small amounts of red cell and lymphocytes (<2%), but as has been shown [7] these cells do not possess specific angiotensin binding sites so they contributed only to nonspecific binding in our study.
Platelet aliquots (2x107 to 7x107) were incubated with increasing concentrations of 125I-angiotensin II (0.2 to 2 nmol/L). Nonspecific binding was assessed in the presence of 10 µmol/L losartan (generous gift from Merck, NJ, USA). Incubation was performed for 120 min at room temperature. Reaction was terminated by vacuum aspiration with ice-cold buffer containing 0.5% BSA and 10 mmol/L Tris-HCl through Millipore Multiscreen Assay System 0.45 µm filters (Millipore, Mass). The radioactivity was determined using a gamma-counter (Beckman Gamma 4000). For every patient a saturation binding curve was constructed and Bmax, number of specific binding sites, corresponding to the number of AT1R (expressed in receptors per one platelet), and Kd, receptor affinity, were calculated.
The intra-individual variability of this method in a group of healthy subjects (n=6) was 10.5±3.3% (data not shown).
2.3. Echocardiography
Left ventricular end-diastolic and end-systolic volumes were assessed by two-dimensional echocardiography on apical four- and two-chamber views. LV volumes and ejection fraction (EF) were calculated using the biplane (modified) Simpson's rule. Left ventricular end-diastolic volume index (LVEDVI) and end-systolic volume index (LVESVI) were derived from body surface area. Left ventricular mass index (LVMI) was calculated according to the previously described method [8]. Each echocardiographic examination was recorded on videotape and the parameters were measured by two blinded assessors, the final result was the mean of these two measurements.
2.4. Estimation of infarct size
Infarct size was estimated from a single troponin T measurement conducted 72±8 h after the onset of chest pain, which has been shown to reflect infarct size with high sensitivity and specificity [9].
2.5. Statistical analysis
Statistical procedures were performed using SSPS 6.0 software. For the comparison of relevant clinical baseline characteristics, 
2 test and two-sample Wilcoxon or Student's t test were used where appropriate. Group differences were tested by 2-way ANOVA. Correlations were calculated using the Pearson test. To evaluate predictors of LV dilatation at discharge multivariate regression analysis was used. Results are presented as mean±SEM (for those with normal distribution) or median (lower quartile; upper quartile) (for those with not normal distribution) and were considered statistically significant if P<.05.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. Discussion5. Conclusions and future...References |
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3.1. AT1 receptor density on blood platelets
In our population of 48 patients with acute MI, the mean AT1R density was 12.7±0.9 binding sites per platelet. Diabetic patients (n=9) had significantly higher AT1R density than non-diabetic subjects (18.3±1.8 vs. 11.1±0.3, respectively). AT1R density correlated with serum LDL cholesterol concentration (Fig. 1). Diabetic patients had disproportionately high AT1R density for their serum LDL cholesterol. The mean AT1R density/LDL cholesterol ratio was 0.079±0.010 for non-diabetic and 0.167±0.012 for diabetic patients (p<0.05), thus diabetic patients had twice the AT1R density per given LDL cholesterol concentration as compared to non-diabetic subjects. Regression analysis showed no other correlations with AT1R (age, sex, cigarette smoking, arterial blood pressure or heart rate, data not shown).
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The subjects were divided into two groups, according to baseline AT1R density: low AT1R density group (N=24; AT1R density below median, <12.7 binding sites/platelet, mean 7.5±0.8) and high AT1R density group (N=24; AT1R>12.7, mean 17.2±0.7). Despite differences in density, the ligand affinity was not significantly different between these two groups (Kd=3.0 nmol/L [95% CI 1.4 to 4.5 nmol/L] versus 3.6 nmol/L [95% CI 2.3 to 4.9 nmol/L]) for low and high AT1R group, respectively. Moreover ligand affinity did not correlate with AT1R density or serum LDL cholesterol (data not shown).
3.2. Clinical characteristics
LDL cholesterol was higher and diabetes mellitus was more prevalent in the high AT1R group. There were no other baseline differences between these two groups (Table 1). Specifically infarct size estimated by troponin T concentration, rate of anterior infarctions and STEMI did not differ between the two groups. There were no intergroup differences with regard to treatment received during hospitalization: none received thrombolytic therapy or primary PTCA. The percentage of patients receiving ACE-inhibitor, statin and spironolactone therapy was 80% vs. 87%, 86% vs. 91% and 8% vs. 13%, in the low-and high AT1R groups respectively (NS). In both groups 91% of patients received a beta-blocker and aspirin.
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3.3. Echocardiographic analysis
In the low AT1R group echocardiography was nonevaluable for two patients, in the high AT1R group two patients died before echocardiography was performed, therefore 22 patients underwent echocardiographic evaluation on day 8 in each group. Patients in the low AT1R group had significantly lower LVESVI, LVEDVI and higher EF than patients in the high AT1R group (Table 2). Echocardiographic parameters of ventricular dilation and contractile function showed positive (LVESVI and LVEDVI) and negative (EF) linear correlation with AT1R density (Fig. 2). Additionally multivariate regression analysis showed that AT1R density and troponin T concentration were the only significant independent predictors of both LVEDVI and LVESVI at discharge (Table 3).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. Discussion5. Conclusions and future...References |
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We have shown for the first time that in a population of patients with acute MI, angiotensin II AT1 receptor density on blood platelets is a strong predictor of early LV dilatation and impaired contractile function. Additionally we have shown that AT1R density correlates with blood LDL cholesterol concentration, confirming earlier results obtained in healthy subjects. For the first time we have shown that diabetic patients have significantly higher AT1R density than non-diabetic subjects. These results suggest that AT1R density is an important factor in the biology of post-MI ventricular remodelling.
4.1. Tissue AT1 receptor density
It has been shown that the sensitivity of resistance vessels to vasoconstrictor effects of angiotensin II is variable and depends on AT1R density [10]. Hypercholesterolemia has been shown to upregulate AT1Rs in rabbit aorta and potentiate angiotensin II induced contraction of these vessels [11], while pressor response to infusion of angiotensin II has been shown to correlate with AT1R density on human blood platelets [5], supporting the concept that final response to activation of the renin-angiotensin system depends on two major elements: concentration of angiotensin II and the density of AT1Rs on target tissues.
AT1Rs have been shown to be expressed in normal myocardium in both humans [12] and animals [13] although the relationship between their expression, MI and heart failure remains unresolved. It has been shown in animal experiments that AT1Rs are upregulated following myocardial infarction in the rat heart within 4 to 7 days [14,15], both on cardiomyocytes and fibroblasts. However, human studies report that AT1R density in explanted hearts from patients with end-stage heart failure is decreased by as much as 65% [16], which possibly reflects true downregulation related to highly increased levels of circulating angiotensin II in patients with end-stage heart failure. However, the process of upregulation of AT1R following MI, its triggers and its significance remain unknown.
It has been shown that human blood platelets possess AT1R [5,7]. Assuming 5-10 fmol angiotensin bound/mg protein for blood platelets, they have 10-20% of the receptor density reported for uterine smooth muscle (45 fmol/mg) [17] and vascular smooth muscle (57-81 fmol/mg) [18]. Additionally, AT1R density on blood platelets is regulated by the same factors that regulate density in other organs; salt loading has been shown to increase the number of AT1R while salt restriction had the opposite effect in animal studies of uterine and vascular smooth muscle cells [17] and brain [19]. Concurrently it has been shown that a high salt diet increases the number of AT1R on blood platelets in humans two fold [7]. It is known that hypercholesterolemia increases AT1R density in vascular smooth muscle in rabbits [11] and recently Nickenig at al. [5] showed that AT1R density on human blood platelets is closely correlated with LDL cholesterol and that LDL cholesterol reduction with statins is accompanied by a reduction in AT1R density.
These studies suggest that AT1Rs in all organs are regulated in a similar manner by a variety of systemic factors; however, no single study has ever examined the correlation between myocardial AT1R density and AT1R density in any other organ. Thus we chose to examine AT1R density on blood platelets and use it as a marker of cardiovascular AT1R density. This allowed us to estimate AT1R density in living subjects without performing invasive procedures.
4.2. AT1 receptors: correlation with risk factors
In our study, the mean AT1R density was approximately 30-40% higher than in two previous studies [5,7]. These differences need to be interpreted with caution, because the patient populations in these two studies were poorly characterized (no age or blood glucose data were available, and LDL cholesterol was only provided in one study). We can speculate that in our study, addition of a diabetic population could have increased the mean AT1R and that patients with myocardial infarction could be exposed to some unknown factors that increase AT1R density, possibly making them more prone to development of coronary artery disease and acute coronary syndromes.
We have confirmed earlier findings showing that AT1R density is linearly correlated with LDL cholesterol [5]. Additionally, for the first time we have shown that diabetic subjects have twice as many AT1R for their LDL cholesterol as non-diabetic subjects. It has previously been shown in animal models that both high glucose concentration and hyperinsulinemia lead to increased expression of AT1R. High glucose concentration increased AT1R expression in rabbit renal proximal tubules [20], streptozotocin treated rats had a 2-fold increase in renal cortex AT1R density [21] and incubation of isolated rat vascular smooth muscle cells with high glucose medium caused a 2.5-fold increase in AT1R mRNA [22], while incubation of rat cultured smooth muscle cells with insulin produced a 2-fold increase in AT1R density [23]. Additionally, in a rat model of hyperinsulinemia induced by dietary fructose, the vasoconstrictive potency of angiotensin II was increased [24].
4.3. AT1 receptors and post infarction ventricular remodelling
In the present study, we have shown that high AT1R density at the onset of myocardial infarction predicts early ventricular remodelling, showing a strong correlation with parameters of ventricular dilation (LVEDVI) and systolic function (EF) and LVESVI, but no correlation with ventricular mass was observed. Additionally, multivariate analysis showed that only AT1R density and troponin T level were independent predictors of early LV dilation, suggesting that it is unlikely that large infarcts contribute to remodelling through increased AT1R density.
Blood was drawn within 18 h from the onset of infarction pain (mean time 13 h) to assess AT1R density. Thus, we believe that AT1R density was not significantly changed by the index infarction or by the consequent neurohormonal changes and reflected pre-infarction levels, i.e. the AT1R level at which the patient had the myocardial infarction. Two lines of evidence argue against the possibility that AT1R density on blood platelets is affected by the index infarction. First, we would expect patients with large infarcts, high levels of circulating angiotensin II and pronounced remodelling to have low AT1R density as a result of reciprocal downregulation by angiotensin II, however we found the opposite in our study. Second, AT1R density in our population correlated with LDL cholesterol, as is true for healthy subjects [5].
To exclude the possible influence of preexisting conditions and treatments on ventricular remodelling, we excluded patients with hypertension, valvular heart disease, a history of myocardial infarction or heart failure, those taking statins which are known to downregulate AT1R or steroids which do the opposite, ACE inhibitors or angiotensin II receptor blockers. Additionally, we only recruited patients who did not receive reperfusion therapy, in order to obtain a group of patients with a propensity to aggressive remodelling and to exclude bias caused by a variable interval between the onset of pain and administration of the reperfusion therapy.
The high and low AT1R groups were very similar with regard to baseline characteristics, in particular patients had similar infarct size, measured by troponin T taken at 72 h (shown to be a good marker of infarct size) [9], and a similar rate of anterior infarction. This is important, since plasma renin activity and angiotensin II concentrations are elevated after myocardial infarction and the severity of this elevation correlates with infarct size [25]. The only significant differences were that LDL cholesterol (which is logical since LDL cholesterol is known to correlate with AT1R density) and diabetes (see previous section) were more prevalent in the high AT1R group. Similarly, patients from these two groups received comparable treatment, according to current guidelines.
4.4. Study limitations
We did not perform a baseline echocardiographic examination. But since we carefully excluded patients with preexisting abnormalities (e.g. LV hypertrophy, heart failure, significant valvular diseases and previous MI) we believe that at the onset of MI these were all fairly healthy people with normal left ventricle size and preserved LV function.
Additionally, diabetes and hypercholesterolemia can influence post-MI remodelling through other mechanisms, not related to AT1R density, however in our multivariate regression analysis neither diabetes nor hypercholesterolemia were correlated with left ventricular dilation.
Moreover we used AT1R density on blood platelets as an estimate of AT1R density on myocardial cells. Although numerous indirect observations suggest that these two are closely correlated, direct evidence of such correlation is lacking.
Finally, although we assessed AT1R density on blood platelets within a mean of 13 h from onset of myocardial infarction, we cannot exclude some minor influence of neurohumoral changes induced by the infarction itself on platelet AT1R receptor density.
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5. Conclusions and future directions |
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TopAbstract1. Introduction2. Methods3. Results4. Discussion5. Conclusions and future...References |
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We have shown show that AT1R density is a strong predictor of early post-myocardial infarction remodelling in humans. Additionally, LDL cholesterol and diabetes are associated with high AT1R density. We speculate that the detrimental effects of hypercholesterolemia, diabetes and possible other factors may be partially dependent on upregulation of RAS activity through increased AT1R density. Thus AT1R density on blood platelets can be considered a marker of cardiovascular risk related to activation of RAS.
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