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(Circulation. 1996;94:1752-1761.)
© 1996 American Heart Association, Inc.

Articles

Postischemic Antiarrhythmic Effects of Angiotensin-Converting Enzyme Inhibitors

Role of Suppression of Endogenous Endothelin Secretion

Friedrich Brunner, PhD; Walter R. Kukovetz, MD

the Institut fur Pharmakologie und Toxikologie, Karl-Franzens-Universitat Graz, A-8010 Graz, Austria.

Correspondence to Friedrich Brunner, PhD, Institut fur Pharmakologie und Toxikologie, Karl-Franzens-Universitat Graz, Universitatsplatz 2, A-8010 Graz, Austria. E-mail friedrich.brunner@kfunigraz.ac.at.

	Abstract

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Background ACE inhibitors improve reperfusion function in several animal models. We tested the hypothesis that ACE inhibitor–induced coronary protection and inhibition of reperfusion arrhythmias are mediated by suppression of cardiac endothelin-1 (ET-1) secretion and action.

Methods and Results The effects of two ACE inhibitors on ET-1 secretion and mechanical function during ischemia and reperfusion were studied in perfused rat hearts. Drugs were infused during the control (60 minutes), ischemic (60 minutes), and reperfusion (30 minutes) period. ET-1 appearing in coronary effluents and the interstitium was analyzed by radioimmunoassay. We observed (1) in hearts treated with ramiprilat (100 nmol/L) or captopril (5 µmol/L), a significant reduction of ET-1 secretion under all three experimental conditions and fewer ventricular extrasystoles during reperfusion; (2) increased ET-1 secretion and numerous tachyarrhythmic events in the presence of ACE inhibitor and a bradykinin B₂ receptor antagonist, icatibant (100 nmol/L); (3) an almost-complete suppression of reperfusion arrhythmias when an ET receptor antagonist, ie, SB 209670 (5 µmol/L) or PD 142893 (200 nmol/L), was infused together with ACE inhibitor and icatibant; and (4) SB 209670 alone to be equally antiarrhythmic as ACE inhibitors. Exogenous ET-1 (40 pmol/L) was proarrhythmic, whereas exogenous bradykinin (100 nmol/L) reduced ET-1 secretion and improved cardiac rhythm.

Conclusions ACE inhibitors suppress endogenous ET-1 secretion, which results in improved coronary function and stabilization of cardiac rhythm after ischemia in this model. Suppression of ET-1 results from both removal of endogenous angiotensin II and accumulation of endogenous bradykinin/nitric oxide. ET receptor antagonists may be prime antiarrhythmic drugs worthy of testing in cardiac patients, either alone or together with ACE inhibitors.

Key Words: endothelin • ischemia • reperfusion • angiotensin • arrhythmia • hemodynamics

	Introduction

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ACE inhibitors are important agents in the treatment of congestive heart failure and hypertension. In addition to these established indications, ACE inhibitors have been shown to be "cardioprotective" in several animal models of myocardial ischemia and reperfusion in which they reduced postischemic enzyme and norepinephrine release,¹ functional and metabolic damage,^{2 3} and reperfusion arrhythmias.^{4 5} The mechanisms of ACE inhibitor–induced vascular and myocardial protection (cardioprotection) are incompletely understood. Because the beneficial effect of ACE inhibition in the isolated ischemic heart was attenuated by a bradykinin receptor antagonist, cardioprotection was attributed to enhancement of the local actions of bradykinin,^{6 7 8} which may dilate coronary arteries and exert antiarrhythmogenic influences via stimulated production of prostaglandins and endothelium-derived relaxing factor.^{9 10} How these mediators effect cardioprotection, however, is not known.

The endothelium-derived polypeptide ET-1¹¹ belongs to a new class of cytokines that have a number of actions on blood vessels and the myocardium.¹² Of particular interest in this respect is electrophysiological evidence showing a direct arrhythmogenic action of very high (micromolar) concentrations of exogenous ET-1 in canine cardiac tissues.¹³ This prompted us to speculate that the cardioprotective action of ACE inhibitors may be mediated by a modulation of ET-1 synthesis and secretion. Several lines of evidence pointed in this direction. First, because acute myocardial infarction is accompanied by activation of the renin-angiotensin system and angiotensin II is a potent stimulator of ET-1 production in cultured endothelial cells,¹⁴ ACE inhibitors may reduce angiotensin-mediated ET-1 secretion and, therefore, arrhythmogenicity. Second, ACE inhibitors also slow the breakdown of bradykinin via inhibition of kininase II action, and bradykinin directly inhibits ET-1 secretion in cultured human endothelial cells.¹⁵ Third, as mentioned above, kinins may regulate the production of prostaglandins, some of which also have inhibitory effects on ET-1 secretion.¹⁶ All these actions would also tend to reduce ET-1–mediated cardiac arrhythmias. However, whether ACE inhibitors reduce plasma or tissue ET-1 levels and whether changes in ET-1 secretion correlate with ischemic and/or reperfusion cardiac function have not been studied.

Because ACE inhibitors reduce angiotensin II generation but also inhibit breakdown of bradykinin and because both these actions limit ET-1 generation, the present experiments were designed to test the hypothesis that the cardioprotective effects of ACE inhibitors are ultimately mediated by inhibition of ET-1 secretion and action. Cardiac ET-1 release in rat isolated, perfused hearts was determined under normoxic, ischemic, and reperfusion conditions following four different protocols: (1) in the absence of test drug (vehicle); (2) in the presence of an ACE inhibitor, ie, ramiprilat (carboxyl group–containing) or captopril (sulfhydryl group–containing); (3) in the presence of an ACE inhibitor together with the bradykinin B₂ receptor antagonist, icatibant⁹ ; and (4) in the presence of an ACE inhibitor, icatibant, and an ET-receptor antagonist, SB 209670¹⁷ (nonpeptidic) or PD 142893¹⁸ (peptidic; both are ET receptor subtype nonselective). Although similar results were expected, two chemically different ACE inhibitors and ET receptor antagonists were used to strengthen the conclusions reached. For the same purpose, the effects of exogenous ET-1 and bradykinin on cardiodynamics were also studied. Electrical, myocardial, and coronary functions were monitored under all experimental conditions. Following indications that endothelial cells may generate mature ET-1 from precursor peptide primarily within the cell¹⁹ and release it preferentially to the abluminal side, both vascular and interstitial rates of ET-1 secretion were determined.

	Methods

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Drugs and Chemicals
All materials used in preparation of perfusion buffer were of standard grade. Captopril (USP grade) was obtained from Research Biochemicals International. Ramiprilat, EXP 3174, and icatibant (HOE 140; D-Arg[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]bradykinin) were a gift from Prof G. Wiemer (Hoechst AG, Frankfurt/Main, Germany). PD 142893 (Ac-D-diphenyl-Ala-Leu-Asp-Ile-Ile-Trpx2 Na; lot number 7/V) was a gift from Dr A. Doherty (Parke-Davis Corp, Ann Arbor, Mich). Angiotensin II acetate and L-NNA were from Sigma Chemical Co. All materials for ET-1 radioimmunoassay have been described previously.²⁰ Drugs were freshly dissolved in perfusion buffer at the final concentrations given in the text.

Animals and Heart Perfusion
Sprague-Dawley rats (260 to 350 g) of either sex were anesthetized with diethyl ether, and the hearts were arrested in ice-cold Krebs-Henseleit perfusion medium and mounted within 2 minutes of thoracotomy. The experiments were performed in accordance with the Osterreichisches Tierversuchsgesetz (Austrian law on animal experimentation). Hearts were perfused retrogradely (Langendorff mode) at a rate of 9.0 mL·min^-1·g heart wt^-1 (fresh cardiac mass calculated from the body mass) with a modified Krebs-Henseleit bicarbonate buffer of the following composition (in mmol/L): NaCl 118, NaHCO₃ 25, KH₂PO₄ 1.2, KCl 4.8, MgSO₄ 1.2, CaCl₂ 2.0, and glucose 11, with the use of the ISOHEART perfusion system (Hugo Sachs Elektronik). Interstitial fluid (transudate) was collected from hearts mounted in an inverted, upside-down position as originally described by Wienen and Kammermeier²¹ and modified in this laboratory.²² The production of interstitial transudate in this model is the result of the lack of colloids in the perfusion buffer. Transudate flow rate was 50±5 µL·min^-1·g heart wet wt^-1, ie, 0.6% of total coronary flow. During ischemia (coronary flow: 1 mL·min^-1·g^-1), mean transudate flow was 17 µL·min^-1·g^-1 (1.7% of coronary flow), and on reperfusion, 160 µL·min^-1·g^-1 (1.8% of coronary flow). The transudate appearing on the surface of the ventricles was collected under a latex cap using slight suction and sampled in exchangeable vials. Venous effluent was collected from the pulmonary artery. Heart temperature was measured with a Physitemp probe (Physitemp Instruments) and was maintained at 37°C to 38°C during control and reperfusion conditions and at 34°C during low-flow ischemia. Cardiac parameters were monitored continuously and included CPP via a pressure transducer attached to the aortic cannula, coronary flow (via electromagnetic flowmeter Narcomatic RT 500, Narco Bio-Systems), and left ventricular peak systolic pressure via the left ventricular fluid-filled latex balloon attached to a second pressure transducer. The volume of the balloon was initially adjusted to achieve an LVEDP of 0 mm Hg. LVDP was calculated from the difference between left ventricular peak systolic pressure and LVEDP. All parameters were obtained on-line using the PLUGSYS data acquisition and control setup for circulatory studies (Hugo Sachs Elektronik) and recorded using a direct-writing polygraph apparatus (Thermo Array recorder TA 11, Gould Instrument Systems).

Experimental Protocol
All hearts were perfused for 15 minutes to establish stable perfusion conditions (equilibration period) followed by a period of 30 minutes over which basal ET-1 release was measured. Hearts were then perfused for 60 minutes under normoxic conditions (control), followed by perfusion at 0.9 to 1.0 mL·min^-1·g^-1 for an additional 60 minutes, resulting in global ischemia,²³ and finally by reperfusion for 30 minutes at normal flow (total duration of experiment: 195 minutes). This protocol was chosen in accordance with previous studies showing that low-flow ischemia, but not total global ischemia, stimulated ET-1 secretion in rat hearts.²⁴ The test substances angiotensin II (100 nmol/L), EXP 3174 (angiotensin II receptor antagonist at AT₁ subtype; active metabolite of losartan)²⁵ (100 nmol/L), ramiprilat (100 nmol/L), captopril (5 µmol/L), icatibant (100 nmol/L), SB 209670 (5 µmol/L), PD 142893 (200 nmol/L), phosphoramidon (10 µmol/L), bradykinin (100 nmol/L), and L-NNA (200 µmol/L) were added to the perfusion medium during the control, ischemic, and reperfusion phases, and coronary effluents and interstitial transudates were collected in intervals described below. Exogenous ET-1 (40 pmol/L) was infused, starting with the onset of reperfusion for a total of 6 minutes.

Evaluation of Ventricular Arrhythmias
An epicardial ECG was recorded throughout the experimental period with the use of two stainless steel electrodes attached directly to the base of the left and right ventricles, respectively. The signal was monitored on a Gould 1425 digital storage oscilloscope, stored on magnetic tape (TEAC XR 30 recorder, TEAC Corp), and transferred to the polygraph at a paper speed of 25 mm·s^-1. Arrhythmias were characterized according to the following features: (1) VES: single, couplet, or triplet ventricular responses, preceded by a shortened beat-to-beat interval; (2) VT: three or more consecutive, morphologically similar ventricular complexes occurring at a frequency of at least twice that of sinus rhythm with a recurring baseline voltage; and (3) VF: extremely rapid, bizarre activity of at least six or more ventricular responses without recurring amplitude or baseline voltage.²³ The incidence of VES as well as VT and/or VF was determined and is given for the intervals 0 to 3, 3 to 6, and 6 to 30 minutes of reperfusion.

Sample Collection and Processing and Measurement of ET-1 and LDH
The control (preischemic) period was divided into two intervals of 30 minutes each; coronary effluents were collected over 5 minutes at the end of each interval. The ischemic period was also divided into two intervals of 30 minutes each, but effluents were collected quantitatively due to the lower perfusate flow rate. On reperfusion, effluents were collected at 0 to 3, 3 to 6 and 20 to 30 minutes. Interstitial transudates were collected during the control period as two samples, each comprising 30 minutes, during ischemia as one sample (60 minutes), and during reperfusion as two samples, ie, from 0 to 15 and 15 to 30 minutes. Because results were similar for the two control and ischemic periods, respectively, only the data for the second interval of each period are shown in the figures. Coronary effluents and interstitial transudates were collected in chilled polypropylene containers spiked with BSA (final concentration 0.02%) and stored frozen at -20°C until they were processed.

The samples collected were worked up as follows: Effluents from individual hearts and transudates from three hearts were loaded onto preconditioned C2 cartridges (500 or 100 mg, INOVEX), the samples were chromatographed slowly, the cartridges were washed with H₂O, and ET-1 was eluted with 70% acetonitrile (2 mL). Eluates were frozen and freeze-dried at 0.1 to 1.2 millibars overnight. ET-1 recovery was checked in every assay using nonradioactive ET-1 dissolved in volumes of perfusion buffer corresponding to the actual effluent and transudate sample volumes and was 89±1.15% (500-mg cartridges) and 89±1.32% (100-mg cartridges), respectively (n=12 each). Recovery was corrected for 100% (factor: 1.1).

ET-1 was measured through radioimmunoassay using an antibody specific for ET-1 (RAS 6901, Peninsula Laboratories) essentially as described previously²⁰ except that samples and standards were incubated with anti–ET-1 antibody for 48 hours. Standard curves were made using 0.125 to 8 pg ET-1 (Peninsula) per tube. The IC₅₀ was 1.08±0.04 pg/tube (n=5), and the detection limit was 0.08 pg/tube. The intra-assay and interassay coefficients of variation were determined with 1 pg of ET-1 assayed four times in one run and in four different runs and were 4.5% and 5.4%, respectively.

LDH was measured according to standard techniques.

Presentation of Data and Statistical Analysis
Group data are presented as arithmetic mean±SEM. Statistical analysis was performed with the StatView II software (Apple Macintosh Corp). Changes in ET-1 secretion and hemodynamic parameters were subjected to a two-way ANOVA for repeated measurements to account for different treatments (control, ischemia, and reperfusion) and factors (vehicle and drugs). When a significant overall effect was detected, the Scheffe test was performed to compare single mean values. Arrhythmia incidences and durations per interval were compared using a two-tailed t test. All ET-1 secretion data are presented as secretion rates (pg·min^-1·g heart wet wt^-1). A value of P.05 was considered significant (denoted by an asterisk). P values of .01 were not indicated separately.

	Results

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Basal Characteristics of the Model, Effects of Exogenous Angiotensin II, and Angiotensin II Receptor Antagonism on ET-1 Secretion and Heart Function
The functional parameters of the preparation in the absence of added drugs are listed in Table 1A (vehicle). During low-flow perfusion resulting in global ischemia, heart rate, LVDP, CPP, and the index for diastolic stiffness, LVEDP, were reduced compared with preischemic control. After initiation of reperfusion at normal flow rate, cardiac function slowly returned; at the end of reperfusion (30 minutes), heart rate had recovered to 93% and LVDP had recovered to 74% of control, whereas CPP was increased by 1.8-fold (constant flow perfusion), and LVEDP was 13±2 mm Hg. Exogenous angiotensin II reduced postischemic LVDP recovery (-20%) and increased CPP (1.4-fold) and LVEDP (1.7-fold) compared with parameters in the absence of drug (Table 1B), whereas the AT₁ receptor antagonist EXP 3174 exerted protective effects during reperfusion as evident from a considerable reduction in CPP (-34%) and LVEDP (-62%) compared with vehicle (Table 1C).

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Parameters Under Control, Ischemic, and Reperfusion Conditions for the Various Experimental Protocols

The effects of exogenous angiotensin II and of blockade of AT₁ receptors by EXP 3174²⁵ on ET-1 secretion are shown in Fig 1. Basal rate of ET-1 secretion under normoxic conditions was 0.26±0.01 pg·min^-1·g^-1 and was unchanged over the entire experimental period. During ischemia, ET-1 secretion was reduced to 41% and increased up to twofold during reperfusion. In the presence of angiotensin II, release was enhanced 1.5- to 2-fold compared with vehicle, whereas in the presence of EXP 3174, ET-1 release was reduced under all conditions (-51%).

View larger version (19K): [in this window] [in a new window]   Figure 1. Basal secretion of ET-1 into coronary effluent under control (C), ischemic (I), and reperfusion (R) conditions (R-1, 0 to 3 minutes; R-2, 3 to 6 minutes; R-3, 20 to 30 minutes) and effects of angiotensin II (100 nmol/L) and of EXP 3174 (100 nmol/L) (AT1 receptor antagonist). The compounds were added to perfusate during the control, ischemic, and reperfusion phases. Heights of bars are mean values; brackets represent SEM; the number of hearts per group is given in the bars. *P<.05 compared with vehicle; the reduction of ET-1 secretion during ischemia and the increase during reperfusion (P<.05) compared with control are not indicated separately.

The basal rate of ET-1 secretion into interstitial transudate, ie, the abluminal release, was 0.01±0.001 pg·min^-1·g^-1 and was constant over the entire experimental period. Because the percent change of ET-1 secretion in ischemia (decrease) and reperfusion (increase) was similar to that for venous effluents (n=6), no secretion rates into transudates will be reported.

Effects of ACE Inhibition, Bradykinin Receptor Antagonism, and ET Receptor Antagonism on ET-1 Secretion and Heart Function
The effects of ramiprilat, either alone or together with icatibant, a bradykinin B₂ receptor antagonist, on ET-1 secretion are shown in Fig 2. Ramiprilat reduced ET-1 secretion during the ischemic and reperfusion periods compared with the vehicle, and the combination of ramiprilat and icatibant increased ET-1 secretion during all three experimental periods. In the combined presence of an ACE inhibitor, icatibant, and an ET receptor antagonist, SB 209670, ET-1 secretion was no different from the rate measured in the presence of ramiprilat plus icatibant alone (P>.05). Similar results were obtained with captopril, a sulfhydryl-containing ACE inhibitor tested either alone, in combination with icatibant, or with icatibant and a second ET receptor antagonist, PD 142893 (Fig 3).

View larger version (41K): [in this window] [in a new window]   Figure 2. Secretion of endogenous ET-1 into coronary effluent under control (C), ischemic (I), and reperfusion (R) conditions (R-1, 0 to 3 minutes; R-2, 3 to 6 minutes; R-3, 20 to 30 minutes). Hearts were perfused with vehicle, ramiprilat (100 nmol/L), ramiprilat and icatibant (100 nmol/L), or ramiprilat, icatibant, and SB 209670 (5 µmol/L). The compounds were added to perfusate during the control, ischemic, and reperfusion phases. Heights of bars are mean values; brackets represent SEM; the number of hearts per group is given in the bars. *P<.05 compared with vehicle; SB 209670 had no effect on ET-1 secretion (P>.05).

View larger version (37K): [in this window] [in a new window]   Figure 3. Secretion of endogenous ET-1 into coronary effluent under control (C), ischemic (I), and reperfusion (R) conditions (R-1, 0 to 3 minutes; R-2, 3 to 6 minutes; R-3, 20 to 30 minutes). Hearts were perfused with vehicle, captopril (5 µmol/L), captopril and icatibant (100 nmol/L), or captopril, icatibant, and PD 142893 (200 nmol/L). The compounds were added to perfusate during the control, ischemic, and reperfusion phases. Heights of bars are mean values; brackets represent SEM; the number of hearts per group is given in the bars. *P<.05 compared with vehicle; PD 142893 had no effect on ET-1 secretion (P>.05).

The corresponding hemodynamic parameters are shown in Table 1 (D through I). As expected, ramiprilat curtailed the postischemic rise in CPP, and icatibant aggravated it. In addition, icatibant provoked serious rhythm disturbances and an increase in LVEDP, effects that were completely antagonized by the blockade of ET receptors with SB 209670. Recovery of LVDP after a 30-minute reperfusion was 88% compared with preischemic control situations, ie, even higher than for vehicle (73%) (Table 1F). During control and ischemic phases, cardiodynamics was unaffected by inhibition of ACE but worsened quickly after infusion of icatibant, and these effects were also antagonized by ET receptor antagonists. Very similar changes in parameters were obtained with captopril, captopril plus icatibant, and the combination of captopril, icatibant, and PD 142893 (Table 1, G through I).

Effect of ACE Inhibition, Bradykinin Receptor Antagonism, and ET Receptor Antagonism on Reperfusion Arrhythmias
The effects of ramiprilat and other drugs on ischemia-induced disturbances of cardiac rhythm were investigated further by analyzing the ECG trace obtained under the different experimental conditions (Figs 4 and 5). In the absence of drug (Fig 4A), only VES, mostly composed of single and some couplet or triplet ventricular responses, occurred during reperfusion (no VT or VF). Ramiprilat (Fig 4B) reduced the number of extrasystoles, whereas the additional presence of icatibant (Fig 4C) provoked numerous periods of nonsustained VT and/or VF, mostly followed by nonsustained bradycardic events. The reperfusion proarrhythmic effect of icatibant, when administered alone, was indistinguishable from the effects of the combination with ramiprilat (445±45 versus 477±51 VES and 32±5 versus 34±5 incidences of VT/VF in 30 minutes, P>.05, n=4). All tachyarrhythmic ECG irregularities vanished completely in the additional presence of the ET receptor antagonist, SB 209670 (Fig 4D). The distribution of incidences of VES and of VF/VT over the entire reperfusion period is shown in Fig 5. All drugs were similarly effective in early and late reperfusion. The antiarrhythmic effect of ramiprilat was clearly due to suppression of ET-1 action as demonstrated by the impressive effect of SB 209670.

View larger version (25K): [in this window] [in a new window]   Figure 4. Representative tracings showing examples of ischemia-induced arrhythmias in hearts treated with vehicle (A), ramiprilat (100 nmol/L; B), ramiprilat and icatibant (100 nmol/L; C), and ramiprilat, icatibant, and SB 209670 (5 µmol/L; D). The compounds were added to perfusate during the control, ischemic, and reperfusion phases. Total global ischemia was produced by reducing coronary flow to 1-10th of normal flow. No arrhythmias occurred under baseline conditions (same for all groups, not shown). With the exception of icatibant, drugs had little effect on cardiac rhythm during the 60-minute control period (left). The tracings were taken at the end of the control period and at the beginning of the reperfusion period. For the occurrence of the different types of arrhythmias during the entire reperfusion period, compare with Fig 5.

View larger version (19K): [in this window] [in a new window]   Figure 5. Incidence of VES (A) and VT and/or VF (B) during reperfusion following infusion of vehicle, ramiprilat alone (100 nmol/L), ramiprilat plus icatibant (100 nmol/L), or ramiprilat plus icatibant and SB 209670 (5 µmol/L). The compounds were added to perfusate during the control, ischemic, and reperfusion phases. Heights of bars are mean values; brackets represent SEM; the number of hearts per group is given in or above the bars.

Effect of ET Receptor Antagonism and Inhibition of ET-1 Synthesis on Reperfusion Heart Function
The involvement of ET-1 in reperfusion arrhythmias was investigated further through inhibition of the effects of endogenous ET-1 using the receptor antagonist SB 209670 alone, ie, in the absence of an ACE inhibitor and icatibant (Fig 6 and Table 1J). The drug effectively reduced the incidence of reperfusion VES (-79%) compared with vehicle, and the reduction was similar throughout all three intervals of reperfusion. Thus, the occurrence of reperfusion VES was reduced by ET receptor antagonist to a similar degree (27±5 VES per 30 minutes) as with ramiprilat (39±5 VES per 30 minutes). In the combined presence of SB 209670 and angiotensin II, the incidence of VES was no different from that in the presence of SB 209670 alone, although angiotensin II in itself increased VES twofold compared with vehicle. Recovery of reperfusion LVDP was near complete (94% of preischemic control) and was considerably increased compared with vehicle and even better than that for ramiprilat and captopril (compare Table 1, J, D, and G). The effect of SB 209670 on reperfusion CPP was also pronounced, similar to that of an ACE inhibitor.

View larger version (22K): [in this window] [in a new window]   Figure 6. Incidence of VES during reperfusion following infusion of vehicle, SB 209670 alone (5 µmol/L), or SB 209670 plus angiotensin II (100 nmol/L). The compounds were added to perfusate during the control, ischemic, and reperfusion phases. Heights of bars are mean values; brackets represent SEM; the number of hearts per group is given in the bars.

A second approach consisted of inhibiting ET–converting enzyme, which releases ET-1 from the precursor peptide, big ET-1,²⁶ through the use of phosphoramidon (Table 1K and Table 2). The infusion of phosphoramidon effectively inhibits ET-1 production in this model as shown previously²² and led to a substantially reduced incidence of reperfusion VES as well as incidence and duration of episodes of VT/VF (Table 2). The stabilization of reperfusion cardiac rhythm occurred despite a significant increase in CPP (1.7-fold; Table 1K), which may be due to additional effects of this nonspecific inhibitor.

View this table: [in this window] [in a new window]   Table 2. Effect of Inhibition of ET-1 Synthesis on Arrhythmias During 30 Minutes of Reperfusion

Effect of ACE Inhibition, Bradykinin Receptor Antagonism, and ET Receptor Antagonism on LDH Release
Basal LDH release into interstitial transudate was 4.1 mU·min^-1·g^-1 at 45 minutes after mounting of hearts. The release rate decreased over the following control period to 1.5 mU·min^-1·g^-1 and increased to 92 and 30 mU·min^-1·g^-1 during reperfusion for 15 and 30 minutes, respectively (Fig 7). The increased release of LDH following ischemia is characteristic of ischemic damage in the isolated heart.²⁷ Ramiprilat reduced LDH secretion on reperfusion by 65% (15 minutes), whereas the additional presence of icatibant increased LDH secretion severalfold in all experimental phases, and this increase was completely abolished when ET receptors were blocked by SB 209670. Similar results were obtained when coronary effluents were analyzed. The same overall pattern of LDH release was also observed with captopril and in combination with icatibant and ET receptor antagonists (n=2 each; data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 7. LDH release into interstitial transudate during control (C), ischemic (I), and reperfusion periods (R-1, 0 to 15 minutes; R-2, 15 to 30 minutes) following infusion of vehicle, ramiprilat alone (100 nmol/L), ramiprilat plus icatibant (100 nmol/L), or ramiprilat plus icatibant and SB 209670 (5 µmol/L). The compounds were added to perfusate during the control, ischemic, and reperfusion phases. Heights of bars represent mean values for two hearts (maximal difference between hearts, 10%).

Effect of Exogenous ET-1 on Reperfusion Heart Function
In an attempt to reproduce arrhythmias with exogenous ET-1, the peptide (40 pmol/L) was infused with the onset of reperfusion, and the effects on cardiac rhythm were quantified (Fig 8). Within 6 minutes, ET-1 increased the number of VES twofold and the occurrence of VT eightfold compared with vehicle; in addition, the peptide induced events of VF (mean duration: 11 seconds in 6 minutes of reperfusion), which were absent in control hearts. After reperfusion, CPP was increased 1.6-fold and LVEDP was increased 1.5-fold, whereas LVDP was markedly reduced (-29%) compared with time-matched values for vehicle (all P<.05).

View larger version (15K): [in this window] [in a new window]   Figure 8. Incidence of VES (A) and duration of VT (B) during 6 minutes of reperfusion following infusion of vehicle or ET-1 (40 pmol/L) with the start of reperfusion. Heights of bars are mean values; brackets represent SEM; the number of hearts per group is given in the bars. *P<.05 compared with vehicle.

Effect of Exogenous Bradykinin and Nitric Oxide Synthase Inhibition on ET-1 Secretion and Heart Function
We tested whether the addition of bradykinin to the perfusate would lead to improvements in postischemic cardiodynamics and inhibition of ET-1 secretion comparable to ACE inhibitor effects (Table 1L and Fig 9). Preischemic control coronary and myocardial function was not affected, but on reperfusion, bradykinin significantly increased reperfusion LVDP (1.2-fold) and reduced the rise in CPP (-27%) compared with vehicle; LVEDP was not affected. These improvements in cardiac function were paralleled by a significant reduction in ET-1 secretion (-29%; Fig 9A) and a less frequent occurrence of VES (-88%; Fig 9B). Infusion of bradykinin together with the nitric oxide synthase inhibitor L-NNA not only antagonized these effects but also resulted in increased rates of ET-1 secretion and a 10-fold increase in the number of VES during the early phase of reperfusion (0 to 3 minutes) compared with vehicle (23-fold increase compared with bradykinin alone). Coronary and myocardial function also deteriorated in the presence of L-NNA, as evident from a lower LVDP (-17% in control phase, -23% in reperfusion) and greatly increased CPP (twofold in control phase, 1.9-fold in reperfusion) (Table 1M).

View larger version (25K): [in this window] [in a new window]   Figure 9. Effect of exogenous bradykinin (100 nmol/L) and L-NNA (200 µmol/L) on ET-1 secretion (A) (R-1, 0 to 3 minutes; R-2, 3 to 6 minutes; R-3, 20 to 30 minutes)) and on occurrence of reperfusion VES (B). The compounds were added to perfusate during the control, ischemic, and reperfusion phases. Heights of bars are mean values; brackets represent SEM; the number of hearts per group is given in or above the bars. *P<.05 compared with vehicle; #P<.05 compared with bradykinin.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we showed that the antiarrhythmic, coronary protective, and myocardial protective actions of ACE inhibitors in ischemic/reperfused rat hearts are in large part due to suppression of ET-1 secretion and action. This conclusion is supported by several sets of functional and biochemical data that combined provide a novel and coherent explanation for ACE inhibitor action.^{8 28 29}

Effects of Ischemia on Cardiac Angiotensin II, Bradykinin, and ET-1
Disturbances of cardiac rhythm are a consequence of reperfusion following experimental or clinical instances of myocardial ischemia.³⁰ Several investigators, using different ACE inhibitors, have demonstrated a reduction in both incidence and mean duration of malignant arrhythmias in a variety of different models. In hearts impaired by ischemia, the renin-angiotensin and kallikrein-kinin systems are activated, resulting in increased release of angiotensin II and kinins, mostly bradykinin (reviewed in Reference 31). ACE inhibitors inhibit the accumulation of angiotensin II and accumulate bradykinin in the myocardium. Consequently, ACE inhibitor–induced antiarrhythmic effects were attributed, among other causes, to inhibition of angiotensin II⁵ and preservation of bradykinin.³² However, accumulating evidence suggests that not only these two systems but also the ET-generating system are stimulated in myocardial ischemia, which results in enhanced release of ET-1 by endothelial cells and cardiomyocytes and increased coronary constrictor and negative inotropic effects (reviewed in Reference 33). In support, infusion of ET-1 directly into the coronary circulation of animals results in the development of myocardial infarction, with impaired ventricular functioning, precipitation of arrhythmias,³⁴ and lowering of the threshold for VF,³⁵ whereas the ET receptor antagonists BQ123 and BQ610 improved several aspects of functional recovery during reperfusion.³⁶ The present results are the first to show that the close link between the cardiac ET, renin-angiotensin, and kallikrein-kinin systems is of functional importance in the intact heart subjected to ischemia/reperfusion; this is shown by demonstrating that tachyarrhythmias are reduced or abolished by ACE inhibitor–mediated suppression of ET-1 action (Figs 5 and 6).

ACE Inhibition and Suppression of ET-1 and Arrhythmias
The antiarrhythmic effect of ramiprilat and captopril is probably the result of several interrelated actions. First, although not measured here, infusion of an ACE inhibitor for several hours probably reduced tissue levels of angiotensin II.³⁷ Because angiotensin II stimulates the release of ET-1 (Fig 1) via increased gene expression³⁸ and because of the direct proarrhythmic effect of ET-1 (Fig 8), ACE inhibition resulted in a marked antiarrhythmic effect because of effective antagonism of ET-1 synthesis. In keeping, angiotensin II worsened cardiodynamics and precipitated VES, as observed previously,⁵ whereas the AT receptor antagonist EXP 3174 significantly reduced ET-1 secretion (Fig 1) as well as the occurrence of VES. Conclusive evidence for the involvement of ET-1 in reperfusion arrhythmogenesis in the present model is based on the similar effectiveness of SB 209670 alone, compared with that of an ACE inhibitor, in suppressing VES following ischemia. Furthermore, the proarrhythmic effect of angiotensin II appeared to be mediated mostly by stimulated ET-1, as evident from a similarly reduced number of VES, both following application of SB 209670 alone and in the additional presence of angiotensin II (Fig 6). The tissue location and subtype of AT receptor, which mediates the stimulation of ET-1 synthesis, are not known. It could be the AT₁ receptor subtype, which was recently identified in rat coronary endothelial cells.³⁹ Alternatively, or additionally, an AT receptor localized on myocytes may be involved whose activation by angiotensin II results in a Ca²⁺-dependent transient inward current,⁴⁰ which could be a stimulus to myocardial ET-1 synthesis.

Second, exogenous bradykinin directly inhibits ET-1 secretion, probably involving nitric oxide as the active component.⁴¹ The fact that exogenous bradykinin reduced reperfusion VES and ET-1 secretion to a similar extent as ramiprilat (Figs 5 and 9) and the additional observation that L-NNA increased the incidence of VES similar to that seen by icatibant strengthen the concept that ACE inhibition resulted in rhythm stabilization by bradykinin/nitric oxide–mediated inhibition of postischemic ET-1 release and action. Furthermore, exogenous ET-1 in a very low concentration likely to be attained in cardiac tissues in vivo²² exerted the expected proarrhythmic effects, indicating that ET-1 is, indeed, involved in triggering reperfusion arrhythmias, probably by increasing the concentration of intracellular free Ca²⁺.⁴²

Third, because our results showed that bradykinin accumulation due to ramiprilat or captopril administration effectively reduced postischemic CPP (see Table 1), the direct coronary vasodilation due to bradykinin/nitric oxide probably contributed to rhythm stabilization, an effect that may also be of importance in vivo. Coronary vascular injury has been suggested to contribute to arrhythmogenesis and contractile dysfunction during reperfusion after ischemia.⁴³ ET-1 also potently contracts vascular smooth muscle; therefore, prevention of its synthesis by ACE inhibitors would also be expected to lessen vascularly induced arrhythmias.⁴⁴

Fourth, recent evidence suggests that certain actions of ET-1 may be mediated by secondary mediators, such as thromboxane A₂ and platelet activating factor. Both have been implicated in ET-1–induced mobilization of intracellular Ca²⁺ in vascular smooth muscle cells and in ST-segment elevation in rats.⁴⁵ Therefore, the positive effects of ACE inhibitors observed here may also have been due, in part, to attenuation of thromboxane A₂– and/or platelet activating factor–mediated deleterious effects on the coronary vasculature and the myocardium. Taken together, ACE inhibitors effectively reduced reperfusion arrhythmias in ischemic rat heart through suppression of ET-1 secretion (mostly by endothelial cells)²² and by inhibition of ET-1 action on cardiac myocytes and coronary smooth muscle. Such a mechanism may explain all present experimental observations and is illustrated in Fig 10.

View larger version (36K): [in this window] [in a new window]   Figure 10. Suggested mechanism for role of ET-1 in suppression of reperfusion ventricular arrhythmias by ACE inhibitors. Stimulatory effects (+), inhibitory effects (-), and receptor (Rec.; double bars) antagonism are indicated. ANG indicates angiotensin II; NO, nitric oxide; EXP, EXP 3174 (AT1 receptor antagonist); and icatibant (HOE 140; bradykinin B2 receptor antagonist).

ACE Inhibition, ET Receptor Antagonism, and LDH Release
Our conclusion of an involvement of ET-1 receptor activation in reperfusion arrhythmias is supported by measurements of LDH release under the various experimental conditions. Although the pattern of enzyme release following vehicle was characteristic of ischemic damage, it also closely paralleled rates of ET-1 release by the hearts in the three experimental groups, suggesting that reduction of tissue ET-1 levels (ACE inhibitor group) reduces ischemic damage to the myocardium and an increase (ACE inhibitor plus icatibant) exacerbates tissue damage. The complete reversal both of LDH release and reperfusion arrhythmias by ET receptor antagonists is strong evidence for a causal role of ET-1 in the generation of tachyarrhythmias. The mechanism involved in ET-1–induced increases in LDH release was investigated previously in cultured cells and is probably due to activation of the phosphoinositide cycle with consequent increased intracellular Ca²⁺ concentrations.⁴⁶

Measurement of ET-1 secretion rates into both coronary effluent and interstitial transudate indicated that the bulk of ET-1 of the rat heart is secreted to the luminal side of the coronary vessels and not to the interstitium. Therefore, the effects on cardiac rhythm observed here are probably mediated by luminal ET-1, ie, in an endocrine manner. The subtype of ET receptor⁴⁷ that mediates the rhythm-modifying effects is presently unknown and requires further research.

Conclusions and Clinical Implications
The present results show that the antiarrhythmic effect of ramiprilat and captopril following myocardial ischemia in isolated, perfused rat heart is mediated by suppression of ET-1 secretion and action, whereas a concentration of ET-1 representative of endogenous ET-1 induced proarrhythmic effects. These results suggest that the beneficial cardiovascular effects of ACE inhibitors ultimately depend on the effective inhibition of endogenous ET-1. It is presently unknown whether, in humans, ACE inhibitor actions also converge on suppression of ET-1; if so, the results of several clinical trials that demonstrated that different ACE inhibitors improved survival rate in patients with heart failure^{48 49} may in part have been due to suppression of ET-1 secretion. The latter is known to be enhanced following acute myocardial infarction. Therefore, the potential of ET receptor antagonists as antiarrhythmics, particularly in ischemic syndromes and in congestive heart failure, may be of great clinical relevance, and future experimental and clinical cardioprotection studies should consider examining the effects of ACE inhibitors as well as ET receptor antagonists.

	Selected Abbreviations and Acronyms

 

AT1 = angiotensin II subtype 1 CPP = coronary perfusion pressure ET-1 = endothelin-1 L-NNA = NG-nitro-L-arginine LDH = lactate dehydrogenase LVDP = left ventricular developed pressure LVEDP = left ventricular end-diastolic pressure VES = ventricular extrasystoles VF = ventricular fibrillation VT = ventricular tachycardia

	Acknowledgments

 
This work was supported by the Austrian Research Fund, project 11040 (Dr Brunner). This study is dedicated to Professor Ernst Mutschler, Frankfurt, on the occasion of his 65th birthday. The authors thank Dr K. Groschner and Dr G. Stark for stimulating discussions and G. Wolkart for expert technical assistance.

Received February 5, 1996; revision received March 28, 1996; accepted April 11, 1996.

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