The renin-angiotensin system (RAS), which is critically involved in cardiovascular homeostasis, has been a prime therapeutic target in cardiovascular diseases such as hypertension and heart failure. Recently a role for RAS in myocardial ischemia/reperfusion injury was suggested after studies in which angiotensin-converting enzyme (ACE) inhibitors reduced infarct size and reperfusion arrhythmias and improved postischemic myocardial functional recovery (1-3). However, the precise mechanisms behind these cardioprotective effects of ACE inhibition remain unclear. ACE inhibitors not only reduce the formation of angiotensin II, but they also attenuate the degradation of bradykinin, which has a myocardioprotective capacity (4).
Angiotensin II is the mediator of the function of RAS and exerts direct modulating effects on myocardial contractility and metabolism (5). It has been suggested that endogenous angiotensin II may exert a cardiotoxic effect (6). Yoshiyama et al. (7) claimed that angiotensin II may accelerate myocardial ischemic and reperfusion injury via the angiotensin II type 1 receptor. Thus angiotensin II may play a role in the development of myocardial injury caused by ischemia followed by reperfusion. This has, however, not been easy to prove until recently, when specific angiotensin II-receptor antagonists were made available. Two subtypes of angiotensin II receptors, type 1 and type 2, have been identified. The cardiovascular actions of angiotensin II are mainly mediated via the activation of the type 1 receptor, whereas the function of the type 2 receptor still remains to be determined, although evidence has suggested a possible role of the type 2 receptor in mediating central actions of angiotensin II and in regulating growth processes (8).
The aim of this study was to make a dose-response investigation of the effect of a potent angiotensin II type 1-receptor antagonist, candesartan (9), on myocardial injury after ischemia and reperfusion and to study whether the possible effect could be related to improved myocardial blood flow during reperfusion.
Pigs of either sex (25-30 kg) were premedicated with an intramuscular injection of 20 mg/kg ketamine hydrochloride and 0.1 mg/kg atropine. Anesthesia was induced with 20 mg/kg of intravenous sodium pentobarbital and maintained by a continuous infusion of 2-6 mg/kg/min throughout the experiment. The animals were intubated and mechanically ventilated by a respirator with oxygen-enriched air (Fio2 = 0.3). Respiratory rate and tidal volume were adjusted to keep arterial pH and pco2 within the physiologic range. Body temperature was maintained at 38.0-38.5°C. A 7F catheter was positioned in the right jugular vein for the administration of fluid. All the investigations were approved by the regional ethics committee for animal research and conform with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1985).
The pericardium was opened after sternotomy, and an adjustable snare was positioned around the distal third of the left anterior descending coronary artery (LAD). Myocardial ischemia and reperfusion were achieved by tightening and releasing this snare. Heparin (300 IU/kg) was given 5 min before LAD occlusion.
Blood sampling and recordings of arterial blood pressure were performed via a 7F catheter introduced through the right femoral artery. A 7F Micro-Tip pressure transducer catheter (Millar Instrument, TX, U.S.A.) was inserted through the right carotid artery and positioned in the left ventricle for recordings of the left ventricular pressure (LVP) and its first derivative (LVdP/dt). An ultrasonic flow probe (Transonic, Ithaca, NY, U.S.A.) was placed just proximal to the LAD snare for measurement of blood flow. Electrocardiogram (ECG; lead II) and all hemodynamic variables were continuously monitored on a multiple-channel physiological recorder (Grass Instruments, Quincy, MA, U.S.A.) and processed in a computer system (Acq Knowledge version 3.1.2 for Macintosh; BIOPAC System Inc., Santa Barbara, CA, U.S.A.).
Left ventricular regional function
Two pairs of 5-MHz ultrasonic crystals were implanted in ischemic and nonischemic areas of the left ventricle. The crystals were positioned in the midmyocardium, and each pair was inserted 10-15 mm apart and oriented parallel to the minor cardiac axis (10). End-diastolic and end-systolic segmental lengths were measured by a sonomicrometer (model 120-1000; Triton Technology, San Diego, CA, U.S.A.). The percentage of segmental shortening (%SS) was calculated as follows: Equation (1) where EDL equals end-diastolic length and was measured just before onset of LVdP/dt max; ESL equals end-systolic length and was measured 20 ms before peak negative LVdP/dt(11).
A 4F double-lumen catheter introduced through an 8F catheter into the coronary sinus via the left jugular vein was advanced to the great cardiac vein. The central lumen of the catheter was used for blood sampling from or retroinfusion of candesartan or vehicle into the ischemic/reperfused myocardium. The other lumen was used for inflating a balloon to occlude the vein during drug delivery to prevent regurgitation of the infusate into the systemic circulation during ischemia. The coronary venous retroinfusion technique was used to ascertain a high myocardial tissue concentration of candesartan/vehicle in the ischemic zone at the start of reperfusion. Details regarding this technique for drug administration were given elsewhere (12).
The LAD was occluded for 45 min, followed by 240 min of reperfusion. After 40 min of LAD occlusion (i.e., 5 min before reperfusion), the animals were randomly assigned to receive either candesartan, 0.2 μg/kg (Cand-0.2 group; n = 6), 2 μg/kg (Cand-2 group; n = 6), or 20 μg/kg (Cand-20 group; n = 6) or vehicle (0.1 μl/kg of 1N Na2CO3, Veh. group; n = 6) diluted in 300 ml saline retroinfused into the coronary vein. Retroinfusion of vehicle or candesartan lasted for 30 min. To select a proper dose range of candesartan to be tested in this study, pilot experiments were performed at our laboratory. Increasing doses of candesartan were retroinfused into the coronary vein to test the inhibition of the pressor response to subsequent intravenous administration of 300 ng/kg of angiotensin II. From the results of these pilot experiments, it was concluded that a dose of 0.2 μg/kg of candesartan did not exert any systemic angiotensin-blocking effect, whereas 2.0 μg/kg was a threshold dose, inhibiting the pressor response by <10%. The highest dose given in this study, 20 μg/kg, almost completely inhibited the angiotensin II-mediated pressor response.
No antiarrhythmic agents were used. In case of ventricular fibrillation, two DC shocks (maximum, 12 J each) were allowed for defibrillation.
Regional myocardial blood flow
In two separate groups of pigs (n = 6 in each) that received coronary venous retroinfusion of 20 μg/kg candesartan or vehicle (as described), regional myocardial blood flow was measured by means of radioactive microspheres (13). For the injection of microspheres, a 5F catheter was placed in the left atrium via the left auricle. Approximately 2 × 106 radioactive microspheres labeled with either 141Ce, 113Sn, 103Ru, 95 Nb, or 46Sc (DuPont, Boston, MA, U.S.A.) were suspended in 2 ml of saline and injected into the left atrium over a 10-s period followed by a 2-ml saline flush. A reference blood sample was collected from the arterial catheter at a constant rate of 13.5 ml/min with a syringe pump (model 940A; Harvard Apparatus, South Natick, MA, U.S.A.) starting 10 s before the microsphere infusion and maintained for 1 min after its termination. The same amount of saline was infused intravenously into the superior caval vein for blood-volume correction. Microspheres were injected before LAD occlusion, and at 5, 30, 120, and 240 min of reperfusion. Radioactivity in the tissue and reference blood samples were measured with a scintillation counter (LKB-Wallac 1282 CompuGamma; LKB-Wallac, Turku, Finland), which also performed the standard calculations (e.g., background subtraction) and correction of nuclide interaction by using the matrix method (13).
The myocardial area at risk was determined by injecting 30 ml of 2% Evans Blue into the left atrium after reocclusion of the LAD just before killing the animal. After excision of the heart, both atria and the right ventricle were removed. Thereafter the left ventricle was cut perpendicular to the apical-basal axis in ∼8-mm-thick slices. The slices were incubated with triphenyl tetrazolium chloride (TTC), which stains viable myocardium red, whereas unstained areas represent necrosis. The area at risk and the extent of necrosis were measured by planimetry (14). The planimetry was performed without the knowledge of group allocation.
Plasma candesartan concentration
Arterial blood was collected after 5, 25, 60, 120, and 240 min of candesartan retroinfusion. After immediate centrifugation, the plasma was removed and kept at −70°C until analysis. Plasma samples for candesartan determination were extracted at pH 1.5 into a diethyl ether/dichlormethane mixture. The organic phase was evaporated, and the residue redissolved. Candesartan was thereafter determined by reversed-phase liquid chromatography and fluorescence detection. The limit of quantification was 3 nM (1.2 ng/ml) with a coefficient of variation of <20% by using a plasma volume of 150 μl.
Drugs used were ketamine hydrochloride (Parke-Davis, Morris Plains, NJ, U.S.A.); sodium pentobarbitone (Nordvacc, Stockholm, Sweden); atropine sulfate and sodium heparin (KabiVitrum, Uppsala, Sweden); candesartan (Astra Hässle AB, Mölndal, Sweden). Candesartan was dissolved in 0.1N Na2CO3 solution and diluted in saline.
All data are presented as mean ± SEM. Comparison of data was performed by one-way analysis of variance (ANOVA) followed by Dunn's test. A p value of <0.05 was considered statistically significant. Data were analyzed by the use of InStat 2.01 statistic software program (GraphPad software, San Diego, CA, U.S.A.) on a Macintosh computer.
Three of 36 initially instrumented animals developed ventricular fibrillation during LAD occlusion before the start of drug administration (these animals belonged to the Cand-2, Cand-20, and Veh. groups, respectively). All pigs were successfully defibrillated by one DC shock each.
Hemodynamics and coronary blood flow
Hemodynamic data from the Cand-0.2, 2, 20, and Veh. groups are presented in Table 1. There were no significant differences between the groups throughout the study period. Table 2 presents hemodynamic data from the candesartan and vehicle groups in which tissue blood flow in the myocardium was determined. Again there were no differences between animals given candesartan and vehicle. Data presented in Tables 1 and 2 also demonstrate that the animals remained hemodynamically stable during the experimental period. Coronary blood flow (as measured by the flow probe around the LAD) was significantly higher in the Cand-20 than in the Veh. group during the initial reperfusion period (p < 0.05; Table 1).
Left ventricular regional function
Before LAD occlusion, %SS did not differ significantly between the four groups. Occlusion of the LAD resulted in a similar and severe depression of ischemic myocardial function in all groups. Reperfusion did not recover %SS in the Veh. group, whereas candesartan in the dosages of 2 and 20 μg/kg, but not of 0.2 μg/kg, enhanced functional recovery. However, only the 20 μg/kg group reached statistical significance at 240 min of reperfusion compared with 40 min of ischemia (p < 0.05; Fig. 1). The mean value (±SEM) of %SS was 32 ± 8% after 240 min of reperfusion in the Cand-20 group, which should be compared with 0.6 ± 14.3% after 240 min of reperfusion in the group receiving vehicle (p < 0.05; Fig. 1). There was no significant difference in %SS in the nonischemic area between groups.
Myocardial infarct size
The area at risk expressed as a percentage of the left ventricle was in the region of 15-16% in all groups. The infarct size as a percentage of the left ventricle or as a percentage of the myocardial area at risk was significantly smaller in pigs belonging to the Cand-2 and Cand-20 groups than in pigs in the Cand-0.2 or the Veh. group (p < 0.01; Fig. 2). In this respect, there was no difference between the Cand-0.2 and Veh. groups.
Myocardial tissue blood flow
After 5 min of reperfusion, the regional, ischemic subepi-, and subendocardial myocardial blood flow was significantly higher in the candesartan than in the vehicle group (candesartan, 322 ± 43 and 275 ± 74 ml/min/100 g tissue, vs. vehicle, 219 ± 51 and 160 ± 41 ml/min/100 g tissue, respectively; p < 0.05). These difference vanished during the continued period of reperfusion (Fig. 3). Furthermore, there was no sustained difference in ischemic blood flow between subepi- and subendocardial tissue in these two groups. There was no significant difference in nonischemic blood flow between the two groups at any time of reperfusion.
Plasma concentrations of candesartan are shown in Fig. 4. In pigs receiving a dose of 20 μg/kg, there were relatively high plasma concentrations of candesartan during the first 60 min after initiation of drug infusion. Pigs belonging to the Cand-2 and -0.2 groups had considerably lower plasma concentrations, and for these dosages, there was no obvious dose/plasma concentration relation.
The main finding of this study was that candesartan, a nonpeptide angiotensin II type I-receptor blocker, improved myocardial functional recovery and reduced infarct size after myocardial ischemia followed by reperfusion in a dose-related manner. This beneficial effect was achieved without any lasting effect on regional myocardial blood perfusion.
Many investigators have demonstrated that ACE inhibitors ameliorate myocardial injury induced by ischemia followed by reperfusion. This effect of ACE inhibition has partly been attributed to reduced degradation of kinins (15). In our study, candesartan was administered locally into the ischemic myocardium. At a dose without any hemodynamic effect, it reduced the infarct size by >50%. This indicates that local cardiac angiotensin II is involved in the development of myocardial injury. The damage may be initiated by activation of angiotensin II type 1 receptors, supporting the hypothesis that a reduction in angiotensin II levels may be a major contributor to the cardioprotective effects of ACE inhibitors. The lack of effect of the lowest dose of candesartan (0.2 μg/kg) supports a dose-dependent effect of candesartan, emphasizing a need for sufficient blockade of the angiotensin II type 1 receptors. Candesartan administration was initiated only during the last 5 min of ischemia to be completed during early reperfusion. During the ischemic period, the hemodynamic parameters were similar in the candesartan and vehicle groups. These facts favor the notion that candesartan protected the heart from reperfusion rather than from ischemic injury.
Our in vivo data are in general agreement with recent studies in isolated rat hearts in which the angiotensin II type 1-receptor antagonists losartan (16) and TCV-116 (7; the oral active prodrug of candesartan) improved the functional recovery after myocardial ischemia and reperfusion. It is, however, in contrast with some other studies performed in rabbits and dogs, in which no significant effect on infarct size was observed with losartan (17) and its active metabolite EXP 3174 (18). The reason for this discrepancy might be related to several factors such as differences in animal species, different binding characteristics of the compounds (competitive for losartan and noncompetitive for candesartan), and various times and routes of drug administration.
The regional myocardial blood flow was not measured during ischemia. Porcine hearts lack native collateral blood flow and have a negligible ability to develop new collaterals during acute ischemia. These facts, which are supported by a previous study in our own laboratory with a similar experimental model (19), make it unlikely that the retroinfusion of candesartan or vehicle during the last 5 min of ischemia changed the collateral blood flow. Thus ischemic blood flow, if it occurred at all, would have remained extremely low.
One possible mechanism for the cardioprotective effect of candesartan may obviously be inhibition of the direct cardiotoxic action of cardiac angiotensin II. Angiotensin II exerts important cardiac actions, including positive inotropism, promotion of myocardial hypertrophy, arrhythmogenic effects, and coronary vasoconstriction (20). It was recently reported that angiotensin II induces myocardial necrosis (21). In isolated rat hearts, exogenous angiotensin II, in amounts without any influence on basic cardiac function, accelerated myocardial ischemia/reperfusion injury, thereby supporting a direct cardiotoxicity (22). It is still unknown exactly how these effects are mediated. Binding of angiotensin II to type 1 receptors activates phospholipase C, an important mediator of cell signaling, that will provoke hydrolysis of inositol phosphates, followed by release of intracellular calcium. This will increase the susceptibility of the myocardium to ischemic and reperfusion injury (7). Blocking of this process may favorably influence myocardial resistance to reperfusion-induced damage.
Other mechanisms distinct from a direct angiotensin II-receptor blockade may also be involved. Blockade of the angiotensin II type 1 receptor has been shown to lead to an increase in angiotensin II release because of the removal of angiotensin negative feedback on renin secretion (8). Subsequently, the increased angiotensin II activates type 2 receptors in endothelial cells (23), resulting in enhanced synthesis and release of bradykinin, which has cardioprotective effects (24). Further studies with specific bradykinin antagonists would be valuable further to explore this possibility. There was a temporary enhancement of coronary flow by 20 μg/kg of candesartan at 5 min of reperfusion. This transient effect on myocardial perfusion during the early hyperemic phase probably does not contribute much to the cardioprotective effect of candesartan observed in this study. However, this enhanced vasodilatation may indicate an increased release of some vasodilating factors, such as nitric oxide or prostacycline or, indeed, bradykinin (25).
In addition, recent evidence suggested that angiotensin I and angiotensin II can be the substrates for the production of other biologically active peptides such as angiotensin-(1-7). Angiotensin-(1-7) can stimulate the release of the vasodilator prostaglandins and nitric oxide in intact animals and in cultured cells (26). Blockade of angiotensin II type 1 receptor could have led to an increased local level of angiotensin II and thus an enhanced formation of angiotensin-(1-7), which might have contributed to the beneficial effects of candesartan in our study.
Finally, angiotensin II has been reported to interact with the potent vasoconstrictor peptide endothelin-1 (ET-1; 27). These two peptides activate similar intracellular signaltransduction pathways. Thereby they can facilitate the vasoconstrictor activity of each other. Because endogenous ET-1 contributes to ischemia/reperfusion injury, as revealed by the beneficial effects of ET-receptor antagonists (28,29), there is a possibility that the beneficial effect of angiotensin II type 1-receptor blockade by candesartan on ischemia/reperfusion injury is partly mediated through the blockade of the interference of angiotensin II on ET-1. This mechanism also deserves further study.
In conclusion, local delivery of the angiotensin II type 1-receptor inhibitor candesartan into the ischemic myocardium just before reperfusion markedly protects against myocardial injury. This provides further evidence of the involvement of cardiac angiotensin II in the development of myocardial ischemia/reperfusion of value in the protection from myocardial injury caused by ischemia followed by reperfusion.
Acknowledgment: This study was supported by grants from the Swedish Heart and Lung Foundation, the Swedish Medical Research Council (grant 45521), and from Karolinska Institutet.
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