Opposite Effects of Angiotensin AT1 and AT2 Receptor Antagonists on Recovery of Mechanical Function After Ischemia-Reperfusion in Isolated Working Rat Hearts
Background Angiotensin II type 1 (AT1) receptor antagonists, when given over the long term, reduce the deleterious consequences of ischemia-reperfusion injury. Whether short-term administration of AT1 or angiotensin II type 2 (AT2) receptor antagonists is cardioprotective has not been investigated.
Methods and Results The effects of short-term administration of selective AT1 and AT2 receptor antagonists on the recovery of mechanical function during reperfusion after 30 minutes of global, no-flow ischemia were studied in left atrium–perfused isolated working rat hearts. Control hearts (n=8) showed incomplete recovery of left ventricular minute work (LV work) and cardiac efficiency during reperfusion to 51±15% and 61±19% of preischemic levels, respectively. Compared with control hearts, the selective AT2 receptor antagonist PD123,319 (0.3 μmol/L) given before ischemia (n=7) improved the recovery of LV work and efficiency to 82±4% and 98±7% of preischemic levels, respectively (P<.01). In contrast, the selective AT1 antagonist losartan (1 μmol/L) blocked the recovery of LV work and depressed efficiency to 0±0% and 1±0% (n=7) of preischemic levels, respectively (P<.01; n=7). Neither antagonist altered coronary vascular conductance.
Conclusions This is the first demonstration that short-term treatment with a selective AT1 versus AT2 antagonist exerts different effects on recovery of mechanical function after ischemia-reperfusion: the AT2 antagonist was cardioprotective, whereas the AT1 antagonist was not. These data suggest that AT2 antagonists and AT1 agonists may offer novel approaches for the treatment of mechanical dysfunction after ischemia-reperfusion.
Angiotensin II elicits several physiological effects that exacerbate IR injury.1 Consequently, the cardioprotective efficacy of strategies that decrease Ang II receptor stimulation has been investigated in models of IR injury. Although ACE inhibitors are beneficial during myocardial ischemia, their effects on reperfused myocardium remain controversial.2 A second strategy to reduce the effects of endogenous Ang II stimulation is the use of selective Ang II type 1 (AT1) and type 2 (AT2) receptor antagonists.3 Although long-term administration of AT1 antagonists reduces IR injury,4 the short-term effects of AT1 or AT2 antagonists on IR injury are unknown.The aim of the present study was to investigate the short-term effects of the selective Ang II AT1 and AT2 antagonists losartan and PD123,319, respectively, on the recovery of mechanical function during reperfusion of the ischemic left atrium–perfused isolated working rat heart.5 6 Because AT1 and AT2 receptors are expressed in both human and rat hearts,7 with little interspecies difference in drug affinity,8 we used the rat heart as our experimental model.
Experimental Animals and Preparation of the Isolated Working Heart
All animals were housed and treated in accordance with guidelines of the Canadian Council on Animal Care and the American Physiological Society. Male Sprague-Dawley rats (weight, 250 to 350 g) that had been fed ad libitum were anesthetized with sodium pentobarbital. Hearts were rapidly excised and placed in ice-cooled Krebs-Henseleit solution. With the use of an initial Langendorff perfusion for 10 minutes, with Krebs-Henseleit solution (pH 7.4, gassed with 95% O2/5% CO2) at a hydrostatic pressure of 60 mm Hg, extraneous tissue was removed and the pulmonary artery and left atrium were cannulated. The hearts were then switched to working mode by clamping the aortic inflow line and opening the left atrial inflow line by use of the method of Neely et al.5 During aerobic perfusion, atrial pacing was applied to the hearts at a frequency of 5 Hz (Grass S88 stimulator). Working hearts were perfused in a closed recirculating system at 37°C by use of an oxygenator with a large surface area in constant contact with a 95% O2/5% CO2 gas mixture. The perfusate (100 mL) consisted of a modified Krebs-Henseleit solution containing 2.5 mmol/L CaCl2, 11 mmol/L glucose, 1.2 mmol/L palmitate prebound to 3% BSA (Fraction V), 0.5 mmol/L lactate, and 100 μU·mL−1 insulin. Perfusions were performed at a constant hydrostatic left atrial preload (11.5 mm Hg) and afterload pressure (80 mm Hg). The oxygen content of the coronary effluent was measured continuously by use of an oxygen probe (YSI 5331) placed in the pulmonary artery outflow line and connected to an oxygen meter (YSI 5300). Heart rate (in bpm) and aortic systolic and diastolic pressures (Statham P23Db) were recorded on a Grass model 7D polygraph. Cardiac output and aortic flow were measured by use of ultrasonic flow probes (Transonic T206) placed in the left atrial inflow and aortic outflow lines, respectively. Coronary flow was calculated as the difference between cardiac output and aortic outflow. LV work was calculated as ([Systolic Pressure−Preload Pressure]×Cardiac Output)×0.133. Myocardial efficiency was calculated as LV work expressed as a percentage of the total potential work based on myocardial oxygen consumption (MV̇o2) in micromoles per minute per gram of dry heart tissue. CVC was calculated as the ratio of coronary flow to mean aortic pressure.
Hearts were randomly assigned to three groups: control (no drug), AT1 antagonist (losartan, 1 μmol/L), or AT2 antagonist (PD123,319, 0.3 μmol/L). These concentrations of antagonists have previously been demonstrated by radioligand binding to cause virtually 100% occupancy of the respective receptors.8 All hearts were perfused aerobically in the working mode for 50 minutes and then subjected to 30 minutes of global, no-flow ischemia (in the presence or absence of drug; Fig 1⇓). Hearts were not paced during global ischemia. After ischemia, the left atrial inflow was reestablished and pacing recommenced after 3 minutes of reperfusion. The drugs were added to the perfusate 5 minutes before the onset of ischemia and remained throughout the period of reperfusion.
The statistical tests used in the present study were ANOVA followed by Dunnett's test for the significance of differences between preischemic and reperfusion values within each group and two-way ANOVA with repeated measures followed by Dunnett's test for differences among the groups, with ANCOVA used for verification. Results are reported as mean±SEM. Statistical significance was set at P<.05.
During the initial period of aerobic perfusion, all parameters of cardiac function were stable and did not differ among the groups (Fig 2⇓; Table). During reperfusion of control hearts, LV work was significantly depressed and recovered partially to 51±15% (n=7) of the preischemic level (Table⇓; Fig 2⇓). Both MV̇o2 (58±15%, n=8) and efficiency (61±19%, n=8) were significantly depressed during reperfusion compared with preischemic levels. During reperfusion, CVC did not differ significantly from preischemic levels (Table⇓).
The presence of PD123,319 during ischemia and reperfusion (n=7) was associated with greater recovery of LV work to 82±4% (P<.01) (Table⇑; Fig 2⇑). Recovery of MV̇o2 and cardiac efficiency during reperfusion were also greater in PD123,319-treated hearts than in control hearts. Also, in PD123,319-treated hearts, MV̇o2 (87±7%), efficiency (98±7%), and CVC all recovered to levels that were not significantly different from those found during preischemic perfusion.
The presence of losartan during ischemia and reperfusion (n=7) completely prevented recovery of LV work (0±0% of preischemic levels) during reperfusion (Table⇑; Fig 2⇑). Losartan also depressed MV̇o2 and cardiac efficiency compared with controls (P<.01), the values being 22±10% and 11±1% of preischemic levels, respectively. There was no effect on CVC (P=NS).
Our results demonstrate radically different effects of short-term treatment with an AT1- versus an AT2-selective Ang II receptor antagonist on recovery of mechanical function after IR injury. This is the first report of enhanced recovery of mechanical function after IR with short-term treatment using the selective AT2 antagonist PD123,319. It is also the first report showing that short-term administration of the AT1-selective antagonist losartan before ischemia prevents recovery after IR.
It is well established that locally produced Ang II mediates its effects via Ang II receptor subtypes in the myocardium.9 Thus, the effects of PD123,319 and losartan are most likely mediated by antagonism of endogenous Ang II at AT2 and AT1 receptors, respectively.3 The lack of significant change in CVC with either antagonist in the present study provides further evidence that the responses are mediated by direct effects on the myocardium rather than via indirect effects on the coronary circulation. The effects of different antagonists or doses on arrhythmias and creatine kinase were not studied.
To date it has not been possible to identify any alteration in myocardial function that is mediated by AT2 receptors.10 Although functionality of AT2 receptors has been suggested in vascular smooth muscle,11 our finding of enhanced recovery of mechanical function after myocardial ischemia in the present study may represent the first documented role for AT2 receptors in the pathophysiology of IR in which the effects can be observed beyond the biochemical level. Our finding that short-term administration of the AT1 selective antagonist losartan markedly impaired recovery of function after IR was unexpected in view of the previously reported enhancement of recovery during reperfusion with long-term administration of the AT1 antagonist TCV-116.4 A possible explanation for the beneficial effect of long-term versus short-term losartan treatment on IR injury is that chronic AT1 antagonism might upregulate AT1 receptors. Moreover, our findings in the present study suggest that short-term treatment with an AT1-selective agonist might be cardioprotective against IR injury.
Our results might have profound implications for cardiovascular therapy. To date, the focus has been on AT1 receptor antagonism because of demonstrations that long-term treatment with AT1 antagonists reduces cardiac hypertrophy and associated failure.12 In contrast, our results suggest that short-term AT1 receptor agonism, rather than antagonism, and AT2 receptor antagonism might be cardioprotective in IR injury.
This study is the first demonstration that an AT2 antagonist improves the recovery of function after IR and suggests that short-term AT2 antagonism (or AT1 agonism) may be beneficial in the management of myocardial IR.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|CVC||=||coronary vascular conductance|
|LV work||=||left ventricular minute work|
This study was supported in part by a grant from the Medical Research Council of Canada, Ottawa, Ontario.
- Received August 22, 1996.
- Accepted September 12, 1996.
- Copyright © 1996 by American Heart Association
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