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J Am Coll Cardiol, 1999; 34:876-884
© 1999 by the American College of Cardiology Foundation
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EXPERIMENTAL STUDIES

EXP3174, the AII antagonist human metabolite of losartan, but not losartan nor the angiotensin-converting enzyme inhibitor captopril, prevents the development of lethal ischemic ventricular arrhythmias in a canine model of recent myocardial infarction

Joseph J. Lynch, Jr., PhD*, Gary L. Stump, BS*, Audrey A. Wallace, BS*, Carla A. Painter, BS*, Justina M. Thomas, BS{dagger}, Sandra E. Kusma, BS{dagger}, Robert J. Gould, PhD* and William Grossman, MD, FACC{ddagger},1

* Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania, USA
{dagger} Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania, USA
{ddagger} Department of Clinical Cardiovascular Research, Merck Research Laboratories, West Point, Pennsylvania, USA

Manuscript received December 22, 1998; revised manuscript received March 19, 1999, accepted May 10, 1999.

Reprint requests and correspondence: Joseph J. Lynch, Jr., WP46-300, Merck Research Laboratories, West Point, Pennsylvania 19486., USA


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
OBJECTIVES

The antiarrhythmic efficacies of the competitive angiotensin II (AII) antagonist losartan, losartan’s more potent noncompetitive AII antagonist human metabolite EXP3174 and the angiotensin-converting enzyme inhibitor captopril were assessed in a canine model of recent myocardial infarction.

BACKGROUND

Multiple hemodynamic and electrophysiologic effects of AII may contribute to cardiac electrical instability. In the recent Losartan Heart Failure Study, Evaluation of Losartan in the Elderly (ELITE), a 722-patient trial primarily designed to assess effects on renal function, an unexpected survival benefit was observed with losartan compared with captopril, with the lower mortality using losartan primarily confined to a reduction in sudden cardiac death.

METHODS

Intravenous losartan (1 mg/kg + 0.03 mg/kg/min), EXP3174 (0.1 mg/kg + 0.01 mg/kg/min), captopril (1 mg/kg + 0.5 mg/kg/h) or vehicle were infused in anesthetized dogs with recent (8.1 ± 0.4 days) anterior myocardial infarction. Electrolytic injury of the left circumflex coronary artery to induce thrombotic occlusion and posterolateral ischemia was initiated 1 h after the start of treatment.

RESULTS

Losartan, EXP3174 and captopril elevated plasma renin activities and comparably and significantly reduced mean arterial pressure. No significant electrocardiographic or cardiac electrophysiologic effects were noted with any treatment. Incidences of acute posterolateral ischemia-induced lethal arrhythmias were: vehicle, 7/9 (77%); losartan, 6/8 (75%); EXP3174, 2/8 (25%; p < 0.05 vs. vehicle control); captopril, 7/10 (70%). There were no among-group differences in time to onset of acute posterolateral ischemia or underlying anterior infarct size.

CONCLUSIONS

EXP3174, but not losartan nor captopril, reduced the incidence of lethal ischemic ventricular arrhythmia in this preparation. The antiarrhythmic efficacy of EXP3174 may be due to an attenuation of deleterious effects of local cardiac AII formed during acute myocardial ischemia or, alternatively, a non-AII-related activity specific to EXP3174. These findings suggest that in humans, metabolic conversion of losartan to EXP3174 may afford antiarrhythmic protection.

Abbreviations and Acronyms
  ACE = angiotensin-converting enzyme
  AI = angiotensin I
  AII = angiotensin II
  ANOVA = analysis of variance
  ELITE = Evaluation of Losartan in the Elderly
  IZ = infarct zone
  LCX = left circumflex
  NZ = noninfarct zone

Angiotensin-converting enzyme (ACE) inhibitors significantly reduce mortality in patients with chronic symptomatic heart failure and in survivors of myocardial infarction with left ventricular dysfunction (1–5). Early trials attributed the survival benefit of ACE inhibitors predominantly to a retardation of hemodynamic deterioration (1–3). Some recent trials have suggested a significant reduction in sudden, potentially arrhythmic death (5,6); however, sudden cardiac death still remains the most common mode of death in heart failure patients treated with ACE inhibitors (7). The classification of the exact mode of death in heart failure patients is complex. Sudden death in heart failure patients often is preceded by a severe worsening in hemodynamic status, and in the absence of direct documentation, a clear association between ventricular arrhythmias and sudden death is not possible. Therefore, whether or not ACE inhibitors afford survival benefit via antiarrhythmic protection remains uncertain (5,6,8-10). Further, clinical as well as preclinical in vivo studies have failed to demonstrate a consistent effect of ACE inhibitors on the frequency of ventricular arrhythmia (9).

Selective angiotensin II (AII) antagonists recently have become available for therapeutic use, and offer the potential of more specific and complete blockade of the deleterious actions of AII (11). Recently, the Losartan Heart Failure Study (Evaluation of Losartan in the Elderly [ELITE]) compared therapy with the prototype AII antagonist losartan with that of the ACE inhibitor captopril in patients with chronic symptomatic heart failure, a patient group in which ACE inhibitors including captopril have demonstrated a significant survival benefit (12). ELITE was a 722-patient trial primarily designed to assess effects on renal function. Although no difference was observed in renal function between treatment groups and hemodynamic status improved comparably in both treatment groups, an unexpected survival benefit was observed in the losartan group compared with the captopril group, with the lower mortality in the losartan group largely confined to a reduction in sudden cardiac death (12).

Few preclinical studies have assessed the antiarrhythmic potential of losartan (13–15). No preclinical studies to date have provided a direct comparison of the antiarrhythmic actions of the competitive AII antagonist losartan, losartan’s more potent noncompetitive AII antagonist human metabolite EXP3174 and an ACE inhibitor in vivo. Accordingly, the present study was designed to compare the antiarrhythmic efficacies of losartan, EXP3174 and the ACE inhibitor captopril in a canine model of recent myocardial infarction. In this experimental model, the occurrence of thrombotically induced acute posterolateral myocardial ischemia in the setting of anterior infarction results in a high incidence of sudden lethal ventricular arrhythmias, primarily ventricular fibrillation (16,17). In these studies, losartan, EXP3174 and captopril were administered intravenously starting 1 h before the acute ischemic triggering event at dosages eliciting significant elevations in plasma renin activity in order to assess their acute antiarrhythmic potentials. The use of the dog as a test species permitted the comparison of the antiarrhythmic activities of losartan and EXP3174 in the virtual absence of metabolic conversion.


   Methods
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All procedures related to the use of animals in these studies were reviewed and approved by the Institutional Animal Care and Use Committee at Merck Research Laboratories at West Point and conform with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996). The preparation and use of the canine model of recent anterior myocardial infarction in which lethal ventricular arrhythmias are precipitated by acute, thrombotically induced posterolateral myocardial ischemia was described originally by Patterson et al. (16). Modifications of this preparation as used in the present study have been described (17) and are summarized below.

Surgical preparation.   Male or female purpose-bred mongrel dogs (6.7 to 11.5 kg; 8.8 ± 0.2 kg; total n = 35) were preanesthetized with sodium thiamylal (5 mg/kg IV [intravenous]) and general anesthesia was induced with isoflurane. A left thoracotomy was performed in the fourth intercostal space, the pericardium incised and the heart suspended in a pericardial cradle. Anterior myocardial infarction was produced by a 2-h occlusion of the left anterior descending coronary artery followed by reperfusion. Surgical incisions were closed, and the animals were allowed to recover.

Experimental protocol, electrophysiologic testing and acute posterolateral myocardial ischemia.   Animals were studied at 5 to 14 (8.1 ± 0.4) days after surgically induced anterior myocardial infarction. Postinfarction dogs were anesthetized with alpha chloralose (80 to 100 mg/kg IV), and were ventilated with room air. Systemic arterial pressure was monitored via the cannulated left common carotid artery, and the right femoral vein was isolated and cannulated for test compound administration. The heart was reexposed via a left thoracotomy, and was suspended in a pericardial cradle. A bipolar electrode was sutured to the left atrial appendage for atrial pacing, and one bipolar plunge electrode per zone was sutured into the infarcted anterior region (infarct zone [IZ]) and into the noninfarcted posterolateral region of the left ventricle (noninfarct zone [NZ]) for the measurement of ventricular excitation thresholds and refractory periods. Lead II electrocardiogram was monitored continuously. After stabilization of the preparation, the following baseline parameters were measured: sinus heart rate, mean arterial pressure, electrocardiographic intervals including a rate-corrected QTc interval [QTc = (QT ms)/], a paced QT interval determined during 2.5-Hz atrial pacing, NZ and IZ ventricular excitation thresholds (2-ms pulse duration, 300-ms coupling interval) during 2.5-Hz atrial pacing and NZ and IZ ventricular relative and effective refractory periods (2-ms pulse duration at 2x and 10x ventricular excitation thresholds, respectively) during 2.5-Hz atrial pacing.

Immediately after baseline assessment, animals were entered in a randomized, blinded fashion into one of the following four treatment groups: losartan potassium salt, 1 mg/kg IV bolus + 0.03 mg/kg/min continuous IV infusion in saline vehicle (n = 8); EXP3174 HCl salt, 0.1 mg/kg IV bolus + 0.01 mg/kg/min continuous IV infusion in 50% PEG/D5W vehicle (n = 8); captopril, 1 mg/kg IV bolus + 0.5 mg/kg/h continuous IV infusion in saline vehicle (n = 10); and vehicle controls (total n = 9) comprised of matched volume bolus + continuous IV infusion subgroups of saline (n = 4) and PEG/D5W (n = 5) vehicles. All doses of test agents reflect amounts of free base compound. Dosing regimens for losartan, EXP3174 and captopril were based on previously published studies of these agents in dogs: losartan (18,19); EXP3174 (18,20); captopril (18,20–22). Before the initiation of treatment, and at 1, 2 and 4 h into the IV treatment infusions, blood samples were obtained for the measurement of plasma renin activity in all four treatment groups (GammaCoat [125I] Plasma Renin Activity Radioimmunoassay Kit; Incstar Corp., Stillwater, Minnesota) to confirm that the losartan, EXP3174 and captopril dosing regimens were pharmacodynamically active. Blood samples from the losartan and EXP3174 groups also were analyzed by HPLC with fluorescence detection (23) for the simultaneous determination of losartan and EXP3174 plasma concentrations in order to assess the extent of metabolic conversion of losartan to EXP3174.

At 1 h after the initiation of IV treatment infusion, heart rate, blood pressure, electrocardiographic and cardiac electrophysiologic parameters were redetermined. Immediately thereafter, the tip of a silver wire electrode was inserted through the wall and into the lumen of the proximal left circumflex (LCX) coronary artery. An anodal current of 200 µA was applied to the intimal surface of the coronary artery via this electrode, producing intimal injury, thrombus formation and ultimately acute posterolateral myocardial ischemia. Upon the development of lethal ischemic arrhythmias or at 3 h after the onset of acute posterolateral myocardial ischemia, the hearts were excised and wet thrombus mass in the LCX coronary artery determined. Anterior myocardial infarct size was determined by cutting the heart into 1-cm-thick transverse sections, which then were incubated in 0.4% triphenyltetrazolium chloride solution. Reaction with triphenyltetrazolium forms a red precipitate in viable tissue, whereas infarcted tissue remains pale. Infarct size was quantitated gravimetrically and was expressed as a percentage of total left ventricle. Note that continuous IV treatment infusions of losartan, EXP3174, captopril and vehicle continued throughout the period of LCX intimal injury and ensuing posterolateral myocardial ischemia.

Statistical analysis.   Data were expressed as mean ± SEM. Treatment effects on heart rate, mean arterial pressure, electrocardiographic and electrophysiologic parameters were analyzed using a two-way analysis of variance (ANOVA) comparing within treatment groups by repeated measure as well as among all treatment groups. If the two-way ANOVA indicated a significant change in a parameter among treatment groups, a two-tailed paired Student t test was used to detect significant differences within groups. Among-group differences in time to onset of acute posterolateral myocardial ischemia, LCX coronary artery thrombus mass and underlying anterior myocardial infarct size were analyzed using a one-way ANOVA. ANOVAs were performed using Statview v4.1 software (Abacus Concepts, Berkeley, California). Among-group differences in incidence of lethal ischemic arrhythmia were analyzed using a Fisher exact test.


   Results
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 Results
Discussion
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Table 1 summarizes the effects of vehicle, losartan, EXP3174 and captopril treatment on heart rate, mean arterial pressure and electrocardiographic intervals. Table 2 summarizes the effects of treatments on ventricular excitability and refractoriness in posterolateral NZ and anterior IZ. Neither the saline nor PEG/D5W vehicles altered these parameters in individual subgroups; hence, these vehicle data are grouped. In this chloralose-anesthetized postinfarction dog model, losartan, EXP3174 and captopril comparably and significantly reduced mean arterial pressure by 23.7 ± 5.1%, 20.3 ± 3.8% and 25.9 ± 5.4%, respectively (Table 1). Sinus heart rate, electrocardiographic intervals (Table 1) and NZ and IZ cardiac electrophysiologic parameters (Table 2) were not altered significantly by losartan, EXP3174 or captopril.


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  		Table 1 Effect of Intravenous Losartan (1.0 mg/kg + 0.03 mg/kg/min), EXP3174 (0.1 mg/kg + 0.01 mg/kg/min), Captopril (1.0 mg/kg + 0.5 mg/kg/h) or Vehicle on Sinus Heart Rate, Mean Arterial Pressure and Electrocardiographic Intervals in Postinfarction Dogs

 

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  		Table 2 Effect of Intravenous Losartan (1.0 mg/kg + 0.03 mg/kg/min), EXP3174 (0.1 mg/kg + 0.01 mg/kg/min), Captopril (1.0 mg/kg + 0.5 mg/kg/h) or Vehicle on Cardiac Electrophysiologic Parameters in Postinfarction Dogs

 
Table 3 summarizes plasma renin activities in the vehicle, losartan, EXP3174 and captopril treatment groups. Plasma renin activities were elevated maximally 5.2- and 5.3-fold in the captopril and losartan groups, respectively, and 3.2-fold in the EXP3174 group. Plasma renin activity was elevated maximally 1.6-fold in the vehicle control group. In the losartan treatment group, mean plasma losartan and EXP3174 concentrations were 13.1 µg/ml and 58 ng/ml, respectively, at 1 h into the losartan infusion, and 14.2 µg/ml and 78 ng/ml, respectively, at 2 h into the losartan infusion. Hence, the presence of EXP3174 in the losartan group was minimal (<1%) compared with that of parent losartan, indicating little metabolic conversion of losartan to EXP3174 in the dog. In the EXP3174 treatment group, mean plasma EXP3174 concentrations ranged between 4.8 and 6 µg/ml at 1 to 4 h into the EXP3174 infusion.


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  		Table 3 Plasma Renin Activities in Postinfarction Dogs Administered Intravenous Losartan (1.0 mg/kg + 0.03 mg/kg/min), EXP3174 (0.1 mg/kg + 0.01 mg/kg/min), Captopril (1.0 mg/kg + 0.5 mg/kg/h) or Vehicle

 
Table 4 summarizes the effects of vehicle, losartan, EXP3174 and captopril treatments on arrhythmic response to the development of secondary acute posterolateral myocardial ischemia. Figure 1 depicts the time course for the development of lethal ischemic arrhythmias after the development of acute posterolateral ischemia for the four treatment groups. Consistent with previous experience with this preparation (17,24), the incidence of lethal arrhythmias in the present vehicle control group was 7/9 (77%), with mortality rates of 3/4 in saline control and 4/5 in PEG/D5W control subgroups. All lethal arrhythmias in the vehicle control animals were ventricular tachycardias degenerating into ventricular fibrillation. In both the losartan and captopril groups, the incidences of lethal ischemic ventricular arrhythmias, 6/8 (75%) and 7/10 (70%), respectively, were comparable with that of the vehicle control group. All ischemic mortalities in the losartan and captopril groups were ventricular tachycardias degenerating into ventricular fibrillation. In contrast, the incidence of lethal ventricular arrhythmias in the EXP3174 group, 2/8 (25%), was significantly (p < 0.05) lower than that of the vehicle control group. Interestingly, one of the two arrhythmic mortalities in the EXP3174 group was bradyarrhythmia and arrest, while the second was ventricular tachycardia degenerating into ventricular fibrillation. Times to onset of acute posterolateral myocardial ischemia were equivalent in the vehicle, losartan, EXP3174 and captopril groups, suggesting no significant treatment effect on the process of thrombotic occlusion of the LCX coronary artery. Wet thrombus mass determined at the site of LCX coronary artery electrolytic lesion at the termination of the study, i.e., development of lethal ischemic arrhythmia or at 3 h after onset of posterolateral myocardial ischemia in surviving animals, did not differ significantly among the four treatment groups, again suggesting no significant treatment effect on thrombus formation. It is noteworthy that LCX thrombus mass tended to be greater in the EXP3174 group compared with the other treatment groups, reflecting the lower incidence of sudden ischemic lethal arrhythmias and therefore greater prevalence of animals in the EXP3174 group surviving to a later time point in the protocol. Underlying anterior myocardial infarct sizes did not vary significantly among the four treatment groups. The similarity in underlying anterior infarct sizes, particular in the losartan versus the EXP3174 treatment groups, precludes smaller infarcts and potentially less vulnerable myocardial substrates as a cause for the lower arrhythmic mortality in the EXP3174 treatment group.


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  		Table 4 Effect of Intravenous Losartan (1.0 mg/kg + 0.03 mg/kg/min), EXP3174 (0.1 mg/kg + 0.01 mg/kg/min), Captopril (1.0 mg/kg + 0.5 mg/kg/h) or Vehicle on the Incidence of Lethal Ischemic Arrhythmias Developing in Response to Secondary Acute Posterolateral Myocardial Ischemia in Postinfarction Dogs

 


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  		Figure 1 Survival of losartan (n = 8; 1 mg/kg IV bolus + 0.03 mg/kg/min continuous IV infusion), EXP3174 (n = 8; 0.1 mg/kg IV bolus + 0.01 mg/kg/min continuous IV infusion), captopril (n = 10; 1 mg/kg IV bolus + 0.5 mg/kg/h continuous IV infusion) and vehicle-treated (n = 9) dogs with previous anterior myocardial infarction expressed as a function of time after onset of thrombotically induced acute posterolateral myocardial ischemia.

 

   Discussion
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 Discussion
References
 
The renin-angiotensin system is activated in experimental animals and in humans after acute myocardial infarction (25,26) and in heart failure (27,28). By virtue of its direct positive inotropic, chronotropic and coronary vasoconstrictor actions, AII may promote ischemia in vulnerable myocardium, potentially destabilizing cardiac rhythm (29). Angiotensin II may further compromise the coronary oxygen supply/demand balance, exacerbate local ischemia and promote arrhythmia by increasing sympathetic tone through both a central effect as well as augmenting local (cardiac) neuronal and adrenomedullary catecholamine release (29,30). Angiotensin II also stimulates the expression and release of endothelin, a highly potent vasoconstrictor and arrhythmogenic cytokine, from endothelial cells (31,32). Several studies have demonstrated direct cardiac electrophysiologic effects of AII that may affect cardiac electrical stability. In isolated cardiac myocytes, AII facilitated or activated inward Ca2+ (33), Na+ (34) and Cl– currents (35) and increased the rapidly activating component (IKr) but decreased the slowly activating component (IKs) of outward delayed rectifier K+ current (36), and altered the kinetics of activation and inactivation of transient outward (Ito) K+ current (37). In isolated rat ventricular muscle strips, AII decreased refractoriness, an effect blocked by losartan (38). In conscious pigs with surgically induced myocardial infarction, intravenous AII decreased ventricular refractoriness and promoted electrically induced and spontaneous ventricular arrhythmia (39). In isolated ventricular cell pairs from normal rat hearts, AII decreased junctional conductance, an effect blocked by losartan (40). Similarly, in isolated ventricular cell pairs from normal and cardiomyopathic hamster hearts, AII decreased junctional conductance, and in the latter tissues caused complete electrical uncoupling, an effect reversed by losartan (41).

Antiarrhythmic efficacy of ACE inhibition and AII antagonism..   Given the multiple mechanisms by which AII may influence cardiac electrical activity and stability, considerable attention has focused on the potential for ACE inhibitors to afford antiarrhythmic protection. Large mortality trials with ACE inhibitors, however, have failed to conclusively demonstrate a favorable effect on arrhythmic mortality, and both clinical and in vivo preclinical studies have failed to consistently demonstrate a significant reduction in the incidence of ventricular arrhythmia (9,10). Interest in the antiarrhythmic potential of direct AII receptor antagonism recently has been heightened by the results of the Losartan Heart Failure Study ELITE. ELITE compared therapy with the prototype AII antagonist losartan with that of the ACE inhibitor captopril in patients with chronic symptomatic heart failure, a patient group in which ACE inhibitors including captopril have demonstrated significant survival benefit (12). ELITE was a 722-patient trial primarily designed to assess effects on renal function. Although no difference was observed in renal function between treatment groups, and hemodynamic status improved comparably in both treatment groups, an unexpected survival benefit was observed in the losartan group compared with the captopril group, with the lower mortality in the losartan group largely confined to a reduction in sudden cardiac death (12).

Any consideration of in vivo pharmacologic actions of losartan must include consideration of EXP3174, the active carboxylic acid metabolite of losartan formed in humans. Whereas losartan is a competitive, surmountable antagonist of AII, EXP3174 is a noncompetitive, insurmountable antagonist of AII with an intrinsic potency approximately 33-fold greater than that of losartan as determined by in vitro antagonism of AII-mediated contraction of isolated rabbit aortic strips (42). As noted previously, the present in vivo intravenous dosing regimens of losartan, EXP3174 and captopril were based on previous studies of these agents in dogs (18-22), and were intended to elicit comparable pharmacodynamic responses in this species. No attempt was made to target human clinical plasma concentrations with these dosing regimens. The formation of EXP3174 from losartan is highly species dependent. In humans, the oral bioavailability of losartan is 33%, with a time to peak plasma concentration of 1 h, a plasma clearance of 610 ml/min, a volume of distribution of 34 liters, and a terminal half-life of 2.1 h for parent compound (43). EXP3174 formed in humans after oral losartan administration displays a time to peak plasma concentration of 3.5 h, a plasma clearance of 47 ml/min, a volume of distribution of 10 liters and a terminal half-life of 6.3 h (43). As a result of its much lower plasma clearance, the AUC for EXP3174 formed after oral losartan administration is approximately fourfold greater than that of parent losartan, indicating EXP3174 to play an important role in the pharmacodynamic activity and duration of action of losartan in man (43). The rat is also an efficient metabolizer of losartan to EXP3174 (44). In contrast, EXP3174 plasma levels have been reported to be virtually undetectable after either intravenous or oral losartan administration to dogs (45). Likewise, in the present studies, the presence of EXP3174 in the losartan infusion group was minimal (<1%) compared with that of parent losartan. The dog, therefore, constitutes a useful species in which to compare the cardiac electrophysiologic and antiarrhythmic effects of losartan and EXP3174 in the virtual absence of metabolic conversion.

Preclinical antiarrhythmic assessments of losartan.   Few preclinical studies have assessed the cardiac electrophysiologic and antiarrhythmic effects of losartan, and no studies have simultaneously compared the antiarrhythmic activities of losartan and EXP3174. In an in vitro guinea pig ventricular wall preparation, losartan attenuated conduction delays and decreased the incidence and duration of arrhythmias during ischemic superfusion followed by reperfusion. Paradoxically, AII also tended to attenuate conduction delay during reperfusion and reduced the incidence of arrhythmia in this preparation (13). During nonischemic superfusion, losartan had no effects on cardiac conduction or refractoriness in the isolated guinea pig ventricular preparation (13). Losartan also has been reported to have no effect on junctional conductance in isolated ventricular cell pairs from normal rat hearts (40). In spontaneously hypertensive rats subjected to coronary artery occlusion and reperfusion, the intravenous administration of losartan immediately before coronary occlusion reduced the incidence of ischemic ventricular fibrillation (14). However, losartan also reduced developing myocardial infarct size in the latter study, suggesting that arrhythmia suppression may have been secondary to a reduction in severity of ischemia (14). In a similar study in dogs subjected to coronary artery occlusion and reperfusion, the intravenous infusion of losartan failed to reduce the incidence of ventricular tachycardia or ventricular fibrillation (15). There was no effect of losartan on ventricular refractoriness or ECG QTc interval in dogs in this study (15). A recent report describes the use of AII type Ia knockout mice to assess the role of AII in cardiac ischemia-reperfusion injury. When subjected to coronary artery occlusion followed by reperfusion, a significant reduction in the incidence of ventricular arrhythmia was observed in the angiotensin receptor knockout mice compared with wild-type controls, despite no difference in the extent of developing myocardial infarction (46). The latter observation suggests that AII may be an independent endogenous inducer of arrhythmia during ischemic-reperfusion injury, and that direct antagonism of the AII receptor may be useful in suppressing such arrhythmia (46).

Present studies.   The present canine model of recent anterior myocardial infarction (16) permits the assessment of the potential of pharmacologic interventions to affect cardiac electrophysiologic status of noninfarcted versus infarcted myocardium and to suppress the development of lethal ischemic ventricular arrhythmias. Susceptibility to the development of lethal ischemic ventricular arrhythmias has been shown to be strongly dependent on underlying anterior myocardial infarct size. In an early characterization study, a cohort of animals with an underlying anterior infarct size of 24.7 ± 1.7% of the left ventricle displayed a significantly higher incidence of lethal ischemic arrhythmias (73% and 93% incidence of lethal arrhythmias at 20 min and 24 h after the onset of posterolateral ischemia, respectively) compared with animals with an anterior infarct size of 5.3 ± 1.1% (13% incidence of lethal arrhythmias at both 20 min and 24 h after the onset of posterolateral ischemia) (47). Underlying anterior infarct sizes observed in the present treatment groups were comparable with that described for high-risk animals in the earlier characterization study (47). Further, the incidences of lethal ischemic arrhythmia in the present vehicle, losartan and captopril treatment groups were equivalent to that described for high-risk animals in the characterization study above (47), as well as to previous vehicle control group incidences of 80% to 85% in this laboratory (17,24), indicating that the present postinfarction dog model was at high risk for the development of ischemic lethal ventricular arrhythmia. Losartan, EXP3174 and captopril were infused in dosing regimens eliciting increases in plasma renin activity and decreases in mean arterial pressure. Quantitatively, the decreases in mean arterial pressure were comparable among treatments, whereas the increase in plasma renin activity achieved with EXP3174 was slightly less than those achieved with the losartan and captopril regimens. Electrocardiographic and cardiac electrophysiologic determinations measured during test agent infusion but before the onset of acute posterolateral myocardial ischemia revealed no significant effects with losartan, EXP3174 or captopril. Times to thrombotic occlusion of the LCX coronary artery, used as a trigger for acute posterolateral myocardial ischemia, as well as underlying anterior myocardial infarct sizes, likewise were similar among all treatment groups. The findings above indicate that the significant reduction in the incidence of lethal ischemic ventricular arrhythmias observed with EXP3174 but not losartan and captopril was not related to a suppression of peripheral vascular renin-angiotensin activity, peripheral hemodynamic activity or a direct cardiac electrophysiologic effect before acute myocardial ischemia.

Local cardiac AII generation.   One potential mechanism for the suppression of lethal ischemic ventricular arrhythmias with EXP3174 is an attenuation of a local adverse, arrhythmogenic effect of AII released during the acute myocardial ischemic trigger. Such a scenario, including the superior efficacy of EXP3174 compared with captopril in this study, is consistent with the existence and importance of local ACE-independent, AII generation in the heart. For example, it has been demonstrated that during acute myocardial ischemia induced by left anterior descending coronary artery occlusion in dogs, a greatly enhanced local production and release of AII into the anterior interventricular vein was inhibited by the serine protease inhibitor nafamostat and by chymostatin, but not by captopril (21). Sampling of cardiac interstitial fluid in dog heart has demonstrated >100-fold higher local cardiac concentrations of both angiotensin I (AI) and AII compared with plasma values, with the local interstitial concentration of AII insensitive to the intravascular administrations of both AI and captopril, despite entry of captopril into the cardiac interstitial compartment (22). These and other biochemical, immunohistochemical and molecular biologic findings (reviewed in reference 22) have led to the understanding that AII production and/or degradation in the intravascular and local interstitial space of the heart are compartmentalized and that AII generation in these compartments is mediated by different enzymatic mechanisms, with cardiac interstitial AII generated by a primarily ACE-independent pathway (22). A recent study utilizing isolated human atrial muscle strips has demonstrated AI-mediated tissue catecholamine release to be potently inhibited by EXP3174, indicating the catecholamine release to be due to local conversion of AI to AII, but conversely, was insensitive to captopril reflecting the functional significance of non-ACE-dependent AII generation in human cardiac tissue (48).

Specificity and potential mechanism of antiarrhythmic efficacy of EXP3174..   An important implication of the present study is that the antiarrhythmic efficacy displayed by EXP3174 in this preparation may not be common to all AII antagonists. Hypotheses addressing the superior antiarrhythmic efficacy of EXP3174 compared with losartan in the present study are speculative at this time. These hypotheses include the inherent nature of AII antagonism, i.e., noncompetitive insurmountable antagonism by EXP3174 versus competitive surmountable antagonism by losartan, with noncompetitive insurmountable antagonism potentially more efficacious in blocking the pro-ischemic and pro-arrhythmic effects of excessive local cardiac AII. Tissue compartment access also may differ for losartan and EXP3174, and therefore may contribute to their differential efficacies in this study. Finally, the existence of a non-AII-related activity specific to EXP3174 underlying the superior antiarrhythmic efficacy of this agent compared with losartan and captopril cannot be precluded. Future studies are required to further assess the relative antiarrhythmic activities of EXP3174 and losartan as well as other AII antagonists, and to explore the hypotheses noted above.

In summary, losartan’s noncompetitive AII antagonist human metabolite EXP3174, but not the parent competitive AII antagonist losartan nor the ACE inhibitor captopril, reduced the incidence of lethal ischemic arrhythmias in a canine model of recent anterior myocardial infarction. The antiarrhythmic efficacy of EXP3174 in this preparation was unrelated to inhibition of the peripheral renin-angiotensin activity and was not due to a direct cardiac electrophysiologic effect occurring before the acute ischemic trigger. These observations suggest that the antiarrhythmic efficacy of EXP3174 may be due to an attenuation of deleterious proarrhythmic effects of local cardiac AII formed during acute myocardial ischemia, or alternatively, a non-AII-related activity specific to EXP3174. These findings suggest that in humans, metabolic conversion of losartan to EXP3174 may impart antiarrhythmic protection superior to that provided by an ACE inhibitor, consistent with the findings of the Losartan Heart Failure Study ELITE (12). A further corollary of these findings, particularly the superior activity of EXP3174 relative to losartan, is that the antiarrhythmic efficacy displayed by EXP3174 in this preparation may not be common to all AII antagonists.


   Footnotes
 
1 Dr. Grossman’s present address is the Department of Medicine, Cardiology Division, University of California San Francisco School of Medicine, San Francisco, California. Back


   References
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 Abstract
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 Discussion
 References
 

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