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CARDIAC EFFECTS OF ANGIOTENSIN I AND ANGIOTENSIN II: DOSE–RESPONSE STUDIES IN THE ISOLATED PERFUSED RAT HEART
Pharmacological Research, Vol. 37, No. 1, 1998
CARDIAC EFFECTS OF ANGIOTENSIN I AND ANGIOTENSIN II: DOSE– RESPONSE STUDIES IN THE ISOLATED PERFUSED RAT HEART CRISTINA TRAQUANDI and EMMA RIVAU Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62-20157 Milan, Italy Accepted 11 No®ember 1997
We investigated the effects of angiotensin I Ž4 = 10y9 , 4 = 10y8 and 4 = 10y7 M. on myocardial contractility, heart rate and coronary perfusion in the isolated rat heart before and after inhibition of angiotensin-converting enzyme ŽACE. by captopril Ž4 = 10y4 M.. We also studied the post-ischaemic recovery of cardiac function in isolated hearts subjected to global myocardial ischaemia and reperfused with various doses of angiotensin II Ž1 = 10y9 , 1 = 10y8 and 1 = 10y7 M.. Angiotensin I significantly reduced coronary flow, the vasoconstrictor effect of a second identical dose was attenuated after inhibition of ACE with captopril. Angiotensin II reduced coronary flow to the same extent as angiotensin I at a concentration four times lower. Left ventricular developed pressure was reduced by angiotensin I and angiotensin II in a dose-dependent manner. Heart rate was not affected by angiotensin I and was significantly lowered by the highest doses Ž1 = 10y8 and 1 = 10y7 M. of angiotensin II. Post-ischaemic recoveries of vascular and contractile function were similar in control hearts and in hearts given angiotensin II during reperfusion. However, left ventricular end-diastolic pressure was increased by the highest dose Ž1 = 10y7 M. of angiotensin II throughout reperfusion compared with controls or hearts receiving lower doses ŽNS.. In conclusion the attenuated vasoconstrictor response to angiotensin I after captopril pre-treatment confirms the existence of an intracardiac renin]angiotensin system operative in ®itro. Our results also suggest that angiotensin II, at a high concentration, may play a negative role in relaxation in the ischaemic-reperfused injured heart. Q 1998 The Italian Pharmacological Society KEY
WORDS:
angiotensins, captopril, heart, ischaemia, rat, diastolic dysfunction.
INTRODUCTION Angiotensin II is the physiologically active product of the renin]angiotensin system. Its precursor, angiotensin I is transformed to angiotensin II mainly by a converting enzyme ŽACE. w1]5x. The presence of ACE and each component of the renin]angiotensin system, such as angiotensinogen, renin, angiotensin II receptors in cardiac tissue of mammals has been demonstrated at both mRNA and protein levels w4]7x. Experimental studies using different in ®i®o and in ®itro preparations have shown that angiotensin I and angiotensin II cause coronary constriction w8]13x, increase heart rate w4, 14x and contractility w9]11, 15, 16x. However, the positive cardiac inU
Corresponding author.
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otropic effect reported in the cat, hamster and rabbit has not been confirmed in the rat and other species w4, 8, 16x. At present there is no agreement about the role of angiotensin II in the setting of acute myocardial ischaemia. Some reports suggest that angiotensin II may be deleterious and contribute to the damage associated with ischaemia and reperfusion w17]19x, giving ground to the therapeutic use of ACE inhibitors in myocardial infarction. However, others have not confirmed these observations w8, 20x. The aim of this study was threefold: Ž1. to assess the dose]response profile of angiotensin I and angiotensin II on coronary and cardiac function using the isolated rat heart preparation; Ž2. to verify whether captopril, by inhibiting ACE activity, modifies the cardiac effects of angiotensin I as an indirect demonstration of the existence of an intracardiac renin]angiotensin system; Ž3. to investigate the Q1998 The Italian Pharmacological Society
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dose-related role of angiotensin II in the setting of acute myocardial ischaemiarreperfusion.
METHODS
Animals Adult male Sprague]Dawley rats were obtained from Charles River ŽCalco, Italy.. Rats were housed at room temperature Ž21]248C with 55 " 10% humidity.. Rats were fed standard animal chow and maintained on a 12:12-h light]dark schedule with the light on from 07:00 h. Procedures involving animals and their care were conducted in conformity with the institutional guidelines in compliance with National and International Laws and Policies ŽEEC Council Directive 86r609, OJ L 358,1, Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No 86-23, revised 1986..
Surgical preparation and perfusion technique Rats were anaesthetized with diethyl ether and 500 IU of sodium heparin was injected into a peripheral vein. Thirty seconds later, hearts were excised and placed in cold Ž48C. perfusion buffer until contraction ceased Žapprox. 15 s.. Each heart was then cannulated through the aorta.
Pharmacological Research, Vol. 37, No. 1, 1998
CaCl 2 1.4 and glucose 11.0. The perfusion fluid was filtered Ž5 m m pore size. before use and was continuously gassed with 95% oxygen plus 5% CO 2 ŽpH 7.4 at 378C.. After 5 min, an intraventricular balloon was introduced into the left ventricle through the mitral valve. The ultrathin compliant, but non-elastic balloon was constructed from plastic wrapping film Žcling-film. so as to match the internal dimensions of the left ventricle w23x. A suitable section of film was tied onto the end of a cut-off intravenous cannula which was connected to a small piece of rigid polythene tubing. The other end of polythene tubing was attached to the recorder via an appropriate pressure transducer, which had previously been calibrated against a known head of pressure. The balloon was then filled with saline and the volume of the balloon was gradually increased and decreased in an attempt to get the best possible fit to the contours of the ventricle. After 2 min the balloon volume was adjusted. Without distorting the shape of the left ventricle, left ventricular end-diastolic pressure was set in the range of 2]10 mm Hg. The balloon size remained unaltered from this point in the experiment onwards and thus, the heart contracted isochorically. After 30 min of controlled perfusion with bicarbonate buffer, values for cardiac function were recorded. Hearts which did not satisfy pre-defined inclusion criteria Žsee later. were then excluded.
Perfusion procedures Perfusion was carried out in the Langendorff mode at constant pressure w21x. Perfusion pressure was set at 80]90 cm H 2 0. These values were selected to correspond with the in ®i®o mean arterial blood pressure in rats w22x. Perfusion was with a bicarbonate buffer solution containing Žin mM.: NaCl 118.5, NaHCO3 25.0, KCl 4.8, MgSO4 1.2, KH 2 PO4 1.2,
Experimental protocol Study 1 Angiotensin I was dissolved in distilled water Žvehicle. and infused by a Harvard pump Žmod 975. through a side arm of the aortic cannula for 5 min. Infusion rate Žconstant flow. was set between 0.59 ml miny1 and 1.1 ml miny1 in order to have a final concentration of 4 = 10y9 , 4 = 10y8 , or 4 =
Fig. 1. Isolated rat hearts were perfused with bicarbonate buffer during the control period Ž30 min.. Vehicle or angiotensin I Ž4 = 10y9 , 4 = 10y8 or 4 = 10y7 M. was infused for 5 min, followed by a 15-min wash-out and 15 min infusion of captopril Ž80 m grml.. Coronary flow ŽCF., heart rate ŽHR., left ventricular developed pressure ŽLVDP. and enddiastolic pressure ŽLVEDP., were measured throughout the experiment Žsee arrows..
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10y7 M for each heart. After 15 min wash-out, 4 = 10y4 M captopril was infused for 15 min followed by a 5-min infusion of angiotensin I at the same concentrations as before. The heart was then perfused with bicarbonate buffer ŽFig. 1.. Study 2 Angiotensin II was dissolved in distilled water Žvehicle. and infused as described for study 1, at concentrations of 1 = 10y9 , 1 = 10y8 , or 1 = 10y7 M for 5 min. After a wash-out period of 10 min, hearts were subjected to 45 min of normothermic global ischaemia then reperfused. During the reperfusion period Ž45 min. hearts were infused with angiotensin II at the same concentrations as before ischaemia ŽFig. 2..
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8 ml miny1 , 220 b.p.m. and 90 mmHg. Any heart that was excluded was immediately replaced by another. During preliminary experiments Žstudy 1., we observed a dramatic reduction Žmore than 80% of pre-infusion value. of coronary flow and contractility after captopril. We therefore decided not to infuse angiotensin I a second time to those hearts where such alterations occurred. Eight hearts out of 21 were excluded for this reason. One heart in the control group was excluded because air bubbles entered the circuit.
Expression of ®ariables Variables measured Left ventricular systolic and end-diastolic pressures were obtained from high-speed recordings Ž25 mm sy1 . of the pressure trace and left ventricular developed pressure was calculated as the difference between these two ŽBattaglia Rangoni, eight channels, KO 380, Bologna I.. Heart rate was also derived from the pressure trace and coronary flow was measured by direct timed collection of coronary effluent. Incidence, magnitude, time to onset and time to peak of ischaemia-induced contracture were recorded. Coronary flow, heart rate, left ventricular developed pressure and left ventricular end-diastolic pressure were measured throughout the perfusion and reperfusion period ŽFig. 1..
Exclusion criteria Pre-defined exclusion criteria were applied before the start of the study and at 30 min of control perfusion. The minimum acceptable values for coronary flow, heart rate and left ventricular developed pressure in the isolated rat heart were, respectively,
Results are reported as mean " SEM. Control variables before infusion of angiotensin I and angiotensin II or before ischaemia are expressed as absolute values. Coronary flow, heart rate, left ventricular developed pressure recorded after angiotensin I and angiotensin II are expressed as percentages of baseline values. Post-ischaemic recovery of function is expressed as a percentage of the values recorded before ischaemia and after reperfusion. The magnitudes of ischaemic contracture and left ventricular end diastolic pressure are expressed as absolute values Žmm Hg..
Statistical analysis One-way analysis of variance ŽANOVA. was used for multiple comparisons when a significant F value was obtained; comparison of means was followed by Dunnett’s test. The Student’s t-test for paired data was used to compare cardiac function before and after the 2-min infusion of angiotensin I and angiotensin II. Differences are considered significant at P- 0.05.
Fig. 2. Isolated rat hearts were perfused with bicarbonate buffer during the control period Ž30 min.. Vehicle or angiotensin II Ž1 = 10y9 , 1 = 10y8 or 1 = 10y7 M. was infused for 5 min, followed by a 10-min wash-out before global normothermic ischaemia Ž45 min. and again during reperfusion Ž45 min.. Coronary flow ŽCF., heart rate ŽHR., left ventricular developed pressure ŽLVDP. and end-diastolic pressure ŽLVEDP., were measured throughout the experiment Žsee arrows.. Body weight ŽBW. was recorded at the start of the experiment and heart weight ŽHW. at the end.
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Table I Cardiac function in isolated rat hearts before (left column) and after 2 min (right column) angiotensin I (A I) or vehicle (mean" SEM) Group Control Ž n s 7.
BW (g)
HW (mg)
CF (ml miny 1 g y 1)
HR (b.p.m.)
LVDP (mmHg)
304 " 17
813 " 45
17.7" 1.9
17.1" 1.5
307 " 14
282 " 13†
104 " 3
105 " 3 101 " 3
A I 4 = 10y9 Ž n s 5.
M
287 " 8
775 " 14
16.1" 1.6
14.2" 1.6†
313 " 21
290 " 18†
104 " 2
A I 4 = 10y8 Ž n s 9.
M
294 " 13
837 " 38
17.6" 1.2
13.2" 1.1U †
317 " 9
290 " 15†
105 " 4
A I 4 = 10y7 Ž n s 7.
M
298 " 15
790 " 38
15.5" 0.7
9.4" 0.7U †
302 " 19
264 " 20†
104 " 3
95 " 3U † 92U † " 3
Abbre®iations: BW, body weight; HW, heart weight; HR, heart rate; CF, coronary flow; LVDP, left ventricular developed pressure. U P- 0.05 ®s control ŽANOVA" Dunnett’s test.; †P - 0.05 Žpaired t-test ..
RESULTS
Basal characteristics of hearts are shown in Tables I and II. No differences were observed in body weight, heart weight and cardiac function between groups.
Study 1 A dose-dependent reduction of coronary flow was observed during the 5-min infusion of angiotensin I. Maximal reduction of coronary flow, by approximately 20% and 40% compared to pre-drug values, was observed at the 2-min infusion of 4 = 10y8 M and 4 = 10y7 M angiotensin I wFig. 3ŽA.x ŽTables I and III.. Afterwards, coronary flow returned toward basal values, with a dose-dependent kinetics. During the wash-out period, coronary flow returned to predrug values. After captopril, the coronary flow response to a second 5-min infusion of 4 = 10y8 M and 4 = 10y7 M angiotensin I was attenuated, the area
under the time]effect curve was reduced by 34% and 41%, respectively, compared with the first infusion of angiotensin I wFig. 3ŽB. ®s Fig. 3ŽA.x. The maximal reduction of flow was observed after the 5-min infusion of 4 = 10y8 M and 4 = 10y7 M angiotensin I wFig. 3ŽB.x ŽTable III.. Left ventricular developed pressure was significantly reduced by approximately 10% in the first minutes, by 4 = 10y8 M and 4 = 10y7 M angiotensin I ŽFig. 4, Tables I and III.. Heart rate was significantly reduced in each group compared with pre-drug values ŽTable I.. However, the reduction was not due to angiotensin I, since the difference in the control group was similar to, or smaller than, that seen with any angiotensin I concentration.
Study 2 Angiotensin II caused a dose-dependent reduction of coronary flow. Maximal reduction, approximately 30]35% compared to pre-drug values, occurred at 1 and 2 min of angiotensin II 1 = 10y8 M and an-
Table II Cardiac function in isolated rat hearts before (left column) and after 2 min (right column) angiotensin II (A II) or vehicle (mean" SEM) Group Control Ž n s 4.
BW (g)
HW (mg)
CF (ml miny 1 g y 1)
HR (b.p.m.)
LVDP (mm Hg)
310 " 16
823 " 33
16.3" 1.2
15.5" 1.4
302 " 17
287 " 14
111 " 7
112 " 6 107 " 3
A II 1 = 10y9 Ž n s 5.
M
305 " 14
865 " 42
16.2" 1.0
14.2" 1.0†
298 " 13
276 " 12†
113 " 2
A II 1 = 10y8 Ž n s 5.
M
318 " 17
842 " 38
15.5" 0.4
11.1" 0.2U †
295 " 10
272 " 10†
113 " 4
97 " 4U †
A II 1 = 10y7 Ž n s 5.
M
316 " 14
859 " 45
15.5" 0.7
11.5" 0.6U †
301 " 5
273 " 13†
103 " 2
93 " 3U †
Abbre®iations: BW, body weight; HW, heart weight; HR, heart rate; CF, coronary flow; LVDP, left ventricular developed pressure. U P- 0.05 ®s control ŽANOVA" Dunnett’s test.; †P- 0.05 Žpaired t-test ..
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Table III Statistical comparison of mean coronary flow and developed pressure (reported in Figs 3–5) in isolated rat hearts Angiotensin I
Wash out
1 min
2 min
5 min
1 min
2 min
5 min
10 min
Data in Fig. 3ŽA. 4 = 10y9 M 4 = 10y8 M 4 = 10y7 M
NS - 0.05 - 0.05
NS - 0.05 - 0.05
NS - 0.05 - 0.05
NS - 0.05 - 0.05
NS NS NS
NS NS - 0.05
NS NS NS
Data in Fig. 3ŽB. 4 = 10y9 M 4 = 10y8 M 4 = 10y7 M
NS NS NS
NS NS NS
NS - 0.05 - 0.05
- 0.05 - 0.05 - 0.05
NS NS NS
NS NS NS
NS NS NS
Data in Fig. 4ŽA. 4 = 10y9 M 4 = 10y8 M 4 = 10y7 M
NS P s 0.05 P - 0.05
NS P- 0.05 P- 0.05
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS NS NS
Angiotension II Data in Fig. 4ŽB. 1 = 10y9 M 1 = 10y8 M 1 = 10y7 M
NS - 0.05 - 0.05
NS - 0.05 - 0.05
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS NS NS
Data in Fig. 5 1 = 10y9 M 1 = 10y8 M 1 = 10y7 M
NS - 0.05 - 0.05
- 0.05 - 0.05 - 0.05
- 0.05 - 0.05 - 0.05
- 0.05 - 0.05 - 0.05
NS - 0.05 - 0.05
NS NS - 0.05
NS NS NS
ŽANOVA and Dunnett’s test ®s control hearts ..
giotensin II 1 = 10y7 M infusion ŽFig. 5, Tables II and III.. At 5 min the flow was still reduced to the same extent Ž15%. with each dose of angiotensin II. Coronary flow returned very slowly toward pre-drug values during the 10-min Kerbs perfusion ŽFig. 5.. Contractility was significantly reduced 1 min and 2 min after 1 = 10y8 M and 1 = 10y7 M angiotensin II infusion wFig. 4ŽB.x ŽTables II and III. and the effect on left ventricular developed pressure was short-lasting. By 5 min, contractility had returned toward pre-drug values wFig. 4ŽB.x. The heart rate was sig-
nificantly reduced after each dose of angiotensin II ŽTable II.. Post-ischaemic vascular and cardiac function were significantly altered in control hearts and hearts given angiotensin II during reperfusion ŽTable IV.. Post-ischaemic recovery of coronary flow, heart rate and left ventricular developed pressure were similar in each group. At the reperfusion time left ventricular end-diastolic pressure rose steeply in each group ŽFig. 6.. The end-diastolic pressure values were highest
Fig. 3. Time-course of coronary flow Žpercentages of basal values. in isolated rat hearts after vehicle Žv]v. or ŽA. angiotensin I w4 = 10y9 M Ž`]`., 4 = 10y8 M w^]^x, 4 = 10y7 M ŽI]I.x and ŽB. 80 m g mly1 captopril and the same dose of angiotensin I w4 = 10y9 M Ž`]`., 4 = 10y8 M Ž^]^., 4 = 10y7 M ŽI]I.x.
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Fig. 4. Time-course of left ventricular developed pressure Žpercentages of basal values. in isolated rat hearts after vehicle Žv]v. or ŽA. angiotensin I w4 = 10y9 M Ž`]`., 4 = 10y8 M Ž^]^., 4 = 10y7 M ŽI]I.x and ŽB. angiotensin II w1 = 10y9 y8 y7 M Ž`]`., 1 = 10 M Ž^]^., 1 = 10 M ŽI]I.x.
Fig. 5. Time-course of coronary flow Žpercentages of basal values. in isolated rat hearts after vehicle Žv]v. or angiotensin II w1 = 10y9 M Ž`]`., 1 = 10y8 M w^]^x, 1 = 10y7 M ŽI]I.x.
consistently with time, in hearts given the highest dose of angiotensin II Ž1 = 10y7 M.. Magnitude, time to onset and time to peak of ischaemic contracture were similar in controls and hearts given angiotensin II ŽTable IV..
DISCUSSION In the present study we investigated the role of angiotensin I and angiotensin II on myocardial contractility, heart rate and coronary perfusion in the isolated rat heart. Angiotensin II has both direct and indirect actions on the heart and can modulate rate, contractility and coronary smooth muscle tone. Both angiotensin I and angiotensin II reduced the contractility of the heart and constricted coronary
arteries. On the isolated human atria muscle, angiotensin I had a positive inotropic effect which was equipment to angiotensin II w2x. In our preparation, angiotensin II was four times more potent than angiotensin I. The higher potency ratio has been confirmed in several experimental conditions but the actual ratio varies considerably Žfrom two to ten. w10, 24x. Contrasting results have been published on the inotropic effect of angiotensin II, depending on the models and species used. Angiotensin II induces a positive inotropic response in most species, which is not fully mediated by the b-adrenergic system w2, 4, 9, 10, 15, 16x. In the rat, however, angiotensin II had either no effect w4, 8, 16x or a negative inotropic effect w25x. We found angiotensin II induced a dosedependent negative inotropic response. Most studies used a single dose of either or both substances. We preferred to use three doses. The range of concentration was selected from studies reported to be active on coronary flow and contractility w8]12x. Our results confirm the coronary constriction response to angiotensin I and angiotensin II w8]12x. Other authors w26x, however, observed a coronary response in the isolated perfused rabbit heart consisting of transient vasoconstriction followed by dilatation, especially at a high concentration of angiotensin II. They suggested that the angiotensin II dilatation probably reflected rapid desensitization to the constrictor effect on the coronary arterial smooth muscle. All the effects of angiotensin II in the heart appeared to be mediated by the AT1 receptor subtype found on coronary endothelial and smooth muscle cells and on cardiomyocytes w26x. We do not know whether the marked reduction of the coronary flow is responsible for the negative inotropic response owing to a limited oxygen and metabolic substrate supply in the circulation. This possibility was raised by Fowler and Holmes in ex-
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Table IV Recovery of function (percentage of pre-ischaemic values) in isolated rat heart after 45 min ischaemia and reperfusion (mean" SEM) Group
Control Ž n s 4.
CF (%)
HR (%)
LVDP (%)
TTO (min)
Ischaemic contracture TTP (min) Peak (mm Hg)
68 " 3
77 " 7
47 " 3
8.5" 0.9
16.5" 1.7
55.3" 7.1
A II 1 = 10y9 Ž n s 5.
M
62 " 3
80 " 9
43 " 7
8.2" 0.7
16.4" 0.7
63.2" 5.1
A II 1 = 10y8 Ž n s 5.
M
69 " 3
76 " 7
39 " 7
7.6" 0.7
16.4" 1.3
59.6" 5.0
A II 1 = 10y7 Ž n s 5.
M
62 " 2
80 " 5
42 " 11
8.4" 1.0
17.0" 1.1
59.2" 4.5
Abbre®iations: A II, angiotensin II; CF, coronary flow; HR, heart rate; LVDP, left ventricular developed pressure; TTO, time to onset; TTP, time to peak.
Fig. 6. Time-course of left ventricular end-diastolic pressure Žmm Hg. during reperfusion in isolated hearts receiving vehicle Žv]v. or angiotensin II w1 = 10y9 M Ž`]`., 1 = 10y8 M Ž^]^., 1 = 10y7 M ŽI]I.x.
periments with a canine heart]lung preparation; they suggested that the coronary vasoconstrictor effect of angiotensin might limit its inotropic action w25x. Neves et al. found that, in the presence of ACE inhibition, w 125 Ixangiotensin II was still formed when w 125 Ixangiotensin I was infused again over a 10-min period, although an average of 43% reduction was seen. It is interesting to note that we observed a reduction in coronary flow when angiotensin I was infused again over 10 min. We found that, after captopril, coronary flow response to a second 5-min infusion of 4 = 10y8 M and 4 = 10y7 M angiotensin I was attenuated, the area under the time]effect curve Žmeasured by trapezoid rule. was reduced by 34% and 41%, respectively, compared with the first infusion of angiotensin I. Our data is in agreement with the metabolic data of Neves et al. w27x. These and other results confirms that ACE activ-
ity is present in the cardiac tissue w24, 27, 28x. Angiotensin I has often been used to estimate functional ACE activity in tissue and in the circulation because it requires conversion to angiotensin II to effect a response. Captopril is one of the many ACE inhibitors now available as ‘reference drug’. Being one of the first and most extensively studied ACE inhibitors, dose]response patterns have been characterised. Therefore, we selected a dose of captopril Ž80 m g mly1 , 4 = 10y4 M., already used by several investigators w8, 29, 30x and known to inhibit plasma ACE activity by 100% in the rat w31x. Even 4 = 10y4 M of captopril failed to completely suppress angiotensin I-mediated responses in the rat heart. Possibly this inability to abolish angiotensin I coronary responses was due to an intrinsic activity of angiotensin I Žindependent of conversion to angiotensin II. andror to the presence of an alternate enzymatic pathway for conversion to angiotensin II in the heart w32x. Angiotensin II formation in the rat heart appears to be primarily dependent on the converting enzyme w2, 5, 7x. However, other authors have indicated that, in humans, an ACE inhibitor-insensitive, soybean trypsin inhibitor-sensitive, plays an important role in the conversion of angiotensin I to angiotensin II w11x. Cardiac ventricular tissue in humans contains a dual enzymatic pathway for angiotensin II formation in which ACE-dependent angiotensin II formation is minor Žapprox. 10%. compared to major serine proteinase-dependent angiotensin II formation Žapprox. 80%. w2x. Our objective was to examine the role of angiotensin II on contractile and relaxant dysfunction associated with ischaemia and reperfusion. To achieve this we require two things. First, we need a model where contractile function can be conveniently evaluated. The isolated heart with global ischaemia is preferred because global contractile dysfunction is more easily measurable than regional. Second, we need a model in which moderate Ži.e.
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modulable. contractile dysfunction occurs. The duration of ischaemia we chose Ž45 min. achieves this. With longer ischaemia, irreversible injury may arise. With briefer ischaemia Žstunning. no injury may occur. We need a level of dysfunction that can be alleviated or exacerbated by a drug and can be detected statistically. The present model achieves this. We used multiple functional indices to assess the extent of tissue injury in the ischaemic heart, such as systolic Žcontractility . and diastolic Žrelaxation. function, heart rate, coronary flow and ischaemia-associated contracture. We preferred these to metabolic indices, such as enzyme leakage or depletion of high-energy phosphates, since, in our opinion, they are the ultimate arbiter of cardiac injury. The role of angiotensin II in acute myocardial ischaemia is not fully elucidated. There is clear evidence that the tissue RAS is activated during acute and chronic ischaemia w5, 17, 33, 34x and the ACE inhibition is clinically beneficial w4, 5, 17, 35x. In the experimental setting, exogenous angiotensin II, given before ischaemia to isolated rat hearts, had deleterious effects on coronary tone, post-ischaemic cardiac function and release of creatine kinase, these effects being abolished by a non-peptide angiotensin type 1 receptor antagonist w18x. Mochizuchi et al. tested the hypothesis that angiotensin II Ž10y8 M. directly impairs the preservation of contractile function in rabbit hearts during 30-min low-flow ischaemia and exacerbates myocardial stunning during reperfusion w33x. Angiotensin II directly impaired left ventricular diastolic relaxation during ischaemia and recovery. This effect was independent from that on coronary vascular tone and systolic function or magnitude of high-energy phosphate depletion. There are several potential mechanisms which might be responsible for the adverse effects of angiotensin II on diastolic function during low-flow ischaemia and subsequent reperfusion. Our results suggest it has an adverse effect on left ventricular diastolic relaxation during no-flow myocardial ischaemia and recovery. Transient myocardial ischaemia in patients with coronary artery disease is associated with a severe and reversible depression of myocardial relaxation and elevation of left ventricular end-diastolic pressure w36, 37x. In this isolated rat heart model, it is unlikely that the effects of angiotensin II were mediated by facilitation of sympathetic neurotransmitter release. However, a limitation of this study is that this indirect effect of angiotensin II may be more dominant in ®i®o. One clinical implication is that angiotensin II may play a contributory role in the development of congestive heart failure secondary to acute diastolic dysfunction during transient ischaemia. In such patients, an increase in angiotensin II through the systemic RAS or through ACE-specific or nonspecific cardiac angiotensin II-forming pathways
might promote adverse changes in both coronary vasomotor tone and diastolic relaxation. The adverse effects of angiotensin II during ischaemia may be particularly marked in the setting of cardiac disease, when the cardiac activation of angiotensin II appears to be enhanced. Such disease and local activation of angiotensin II may occur in a regional distribution in post-infarction remodelling associated with coronary artery disease in addition to the global hypertrophy that occurs in response to pressure overload. Thus, patients may be predisposed to ischaemic diastolic dysfunction mediated, at least in part, by the action of the RAS in the heart.
ACKNOWLEDGEMENTS This work was supported in part by the CNR ŽNational Research Council, Italy, No. 91.01277.PF70.. Cristina Traquandi is a ‘F. Ferraris, G. Bevilacqua’ Fellow. We wish to thank Judy Baggott for the English revision.
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13.
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Long-lasting ex ®i®o inhibition of rat vascular response to angiotensin 1. Methods Find Exp Clin Pharmacol 1994; 16: 633]8. Xiang JZ, Linz W, Becker H, Ganten D, Lang RE, Scholken B, Unger T. Effects of converting enzyme ¨ inhibitors. Ramipril and enalapril on peptide action and sympathetic neurotransmission in the isolated heart. Eur J Pharmacol 1985; 113: 215]23. Kobayashi M, Furukawa Y, Chiba S. Positive chronotropic and inotropic effects of angiotensin II in the dog heart. Eur J Pharmacol 1978; 50: 17]25. Ackerly JA, Tsai BS, Peach MJ. Role of converting enzyme in the responses of rabbit atria, aortas, and adrenal zona glomerulosa to wdes-Asp1x angiotensin I. Circ Res 1977; 41: 231]8. Ishihata A, Endoh M. Species related differences in inotropic effects of angiotensin II in mammalian ventricular muscle. Receptors, subtypes and phosphoinositide hydrolysis. Br J Pharmacol 1995; 114: 447]53. Linz W, Scholkens BA, Han YF. Beneficial effects of the converting enzyme inhibitor, ramipril, in ischaemic rat hearts. J Cardio®asc Pharmacol 1986; 8 (Suppl. 10): S91]9. Yoshiyama M, Kim S, Yamagishi H, Omura T, Tani T, Yanagi S, Toda I, Teragaki M, Akioka K, Takeuchi K, et al. Cardioprotective effect of the angiotensin II type 1 receptor antagonist TCV-116 on ischaemia-reperfusion injury. Am Heart J 1994; 128: 1]6. Yoshiyama M, Kim S, Yamagishi H, Omura T, Tani T, Takagi M, Toda I, Teragaki M, Akioka K, Takeuchi K, et al. The deleterious effects of exogenous angiotensin I and angiotensin II on myocardial ischaemia-reperfusion injury. Jpn Circ J 1994; 58: 362]8. Liu Y, Akihito T, Cohen MV, Downey JM. Pretreatment with angiotensin II activates protein kinase C and limits myocardial infarction in isolated rabbit hearts. J Mol Cell Cardiol 1995; 27: 883]92. Langendorff O. Untersuchungen am uberlebenden Saugetierherzen. Pflugers Arch 1895; 61: 291]332. Litchfield JB. Blood pressure in infant rats. Physiol Zool 1958; 31: 1]6. Curtis MJ, Macleod BA, Tabrizchi R, Walker MJA. An improved perfusion apparatus for small animal hearts. J Pharmacol Methods 1986; 15: 87]94. Holubarsch C, Hasenfuss G, Schmidt Schweda S, Knorr A, Pieske B, Ruf T, Fasol R. Angiotensin I and II exert inotropic effects in atrial but not in ventricular human myocardium. An in ®itro study under physiological experimental conditions. Circulation 1993; 88: 1228]37.
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25. Fowler NO, Holmes JC. Coronary and myocardial actions of angiotensin. Circ Res 1964; 14: 191]201. 26. Porsti I, Hecker M, Bassenge E. Dual action of ¨ angiotensin II on coronary resistance in the isolated perfused rabbit heart. Naunyn Schmiedeberg’s Arch Pharmacol 1993; 348: 650]8. 27. Neves LAA, Almeida AP, Khosla MC, Santos RA. Metabolism and angiotensin I in isolated rat hearts. Effect of angiotensin converting enzyme inhibitors. Biochem Pharmacol 1995; 50: 1451]9. 28. Meulemans AL, Andries LJ, Brutsaert DL. Does endocardial endothelium mediate positive inotropic response to angiotensin I and angiotensin II? Circ Res 1990; 66: 1591]601. 29. Van Gilst WH, De Graeff PA, Wesseling H, DeLangen CD. Reduction of reperfusion arrhythmias in the ischaemic isolated rat heart by angiotensin converting enzyme inhibitors: a comparison of captopril, enalapril and HOE 498. J Cardio®asc Pharmacol 1986; 8: 722]8. 30. Arad M, Shotan A, Horowitz LR. Effect of captopril on metabolic and hemodynamic alterations in global ischaemia and reperfusion in the isolated working rat heart. J Cardio®asc Pharmacol 1992; 19: 319]23. 31. Endoh M, Suzuki M, Katayama K. Kinetic studies of the pharmacologic response to captorpil in rats. II. Hypotensive effect and plasma angiotensin converting enzyme activity. J Pharmacobio-Dyn 1989; 12: 10]17. 32. Kinoshita A, Urata H, Bumpus FM, Husain A. Measurement of angiotensin converting enzyme inhibition in the heart. Circ Res 1993; 73: 51]60. 33. Mochizuki T, Eberli FR, Apstein CS. Exacerbation of ischaemic dysfunction by angiotensin II in red cell-perfused rabbit hearts. Effects on coronary flow, contractility, and high-energy phosphate metabolism. J Clin In®est 1992; 89: 490]8. 34. Youhua Z, Schouchun X. Increased vulnerability of hypertrophied myocardium to ischaemia and reperfusion injury. Relation to cardiac renin-angiotensin system. Chin Med J 1995; 108: 28]32. 35. Lee MA, Bohm M, Paul M. Tissue renin-angiotensin system. Their role in cardiovascular disease. Circulation 1993; 97 (Suppl. IV): 7]13. 36. Carroll JD, Hess OM, Hirzel HO. Exercise-induced ischaemia. The influence of altered relaxation on early diastolic pressures. Circulation 1983; 67: 521]8. 37. Bourdillon PD, Lorell BH, Mirsky I. Increased regional myocardial stiffness of the left ventricle during pacing-induced angina in man. Circulation 1983; 67: 316]23.
CARDIAC EFFECTS OF ANGIOTENSIN I AND ANGIOTENSIN II: DOSE– RESPONSE STUDIES IN THE ISOLATED PERFUSED RAT HEART CRISTINA TRAQUANDI and EMMA RIVAU Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62-20157 Milan, Italy Accepted 11 No®ember 1997
We investigated the effects of angiotensin I Ž4 = 10y9 , 4 = 10y8 and 4 = 10y7 M. on myocardial contractility, heart rate and coronary perfusion in the isolated rat heart before and after inhibition of angiotensin-converting enzyme ŽACE. by captopril Ž4 = 10y4 M.. We also studied the post-ischaemic recovery of cardiac function in isolated hearts subjected to global myocardial ischaemia and reperfused with various doses of angiotensin II Ž1 = 10y9 , 1 = 10y8 and 1 = 10y7 M.. Angiotensin I significantly reduced coronary flow, the vasoconstrictor effect of a second identical dose was attenuated after inhibition of ACE with captopril. Angiotensin II reduced coronary flow to the same extent as angiotensin I at a concentration four times lower. Left ventricular developed pressure was reduced by angiotensin I and angiotensin II in a dose-dependent manner. Heart rate was not affected by angiotensin I and was significantly lowered by the highest doses Ž1 = 10y8 and 1 = 10y7 M. of angiotensin II. Post-ischaemic recoveries of vascular and contractile function were similar in control hearts and in hearts given angiotensin II during reperfusion. However, left ventricular end-diastolic pressure was increased by the highest dose Ž1 = 10y7 M. of angiotensin II throughout reperfusion compared with controls or hearts receiving lower doses ŽNS.. In conclusion the attenuated vasoconstrictor response to angiotensin I after captopril pre-treatment confirms the existence of an intracardiac renin]angiotensin system operative in ®itro. Our results also suggest that angiotensin II, at a high concentration, may play a negative role in relaxation in the ischaemic-reperfused injured heart. Q 1998 The Italian Pharmacological Society KEY
WORDS:
angiotensins, captopril, heart, ischaemia, rat, diastolic dysfunction.
INTRODUCTION Angiotensin II is the physiologically active product of the renin]angiotensin system. Its precursor, angiotensin I is transformed to angiotensin II mainly by a converting enzyme ŽACE. w1]5x. The presence of ACE and each component of the renin]angiotensin system, such as angiotensinogen, renin, angiotensin II receptors in cardiac tissue of mammals has been demonstrated at both mRNA and protein levels w4]7x. Experimental studies using different in ®i®o and in ®itro preparations have shown that angiotensin I and angiotensin II cause coronary constriction w8]13x, increase heart rate w4, 14x and contractility w9]11, 15, 16x. However, the positive cardiac inU
Corresponding author.
1043]6618r98r010057]09r$25.00r0rfr970265
otropic effect reported in the cat, hamster and rabbit has not been confirmed in the rat and other species w4, 8, 16x. At present there is no agreement about the role of angiotensin II in the setting of acute myocardial ischaemia. Some reports suggest that angiotensin II may be deleterious and contribute to the damage associated with ischaemia and reperfusion w17]19x, giving ground to the therapeutic use of ACE inhibitors in myocardial infarction. However, others have not confirmed these observations w8, 20x. The aim of this study was threefold: Ž1. to assess the dose]response profile of angiotensin I and angiotensin II on coronary and cardiac function using the isolated rat heart preparation; Ž2. to verify whether captopril, by inhibiting ACE activity, modifies the cardiac effects of angiotensin I as an indirect demonstration of the existence of an intracardiac renin]angiotensin system; Ž3. to investigate the Q1998 The Italian Pharmacological Society
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dose-related role of angiotensin II in the setting of acute myocardial ischaemiarreperfusion.
METHODS
Animals Adult male Sprague]Dawley rats were obtained from Charles River ŽCalco, Italy.. Rats were housed at room temperature Ž21]248C with 55 " 10% humidity.. Rats were fed standard animal chow and maintained on a 12:12-h light]dark schedule with the light on from 07:00 h. Procedures involving animals and their care were conducted in conformity with the institutional guidelines in compliance with National and International Laws and Policies ŽEEC Council Directive 86r609, OJ L 358,1, Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No 86-23, revised 1986..
Surgical preparation and perfusion technique Rats were anaesthetized with diethyl ether and 500 IU of sodium heparin was injected into a peripheral vein. Thirty seconds later, hearts were excised and placed in cold Ž48C. perfusion buffer until contraction ceased Žapprox. 15 s.. Each heart was then cannulated through the aorta.
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CaCl 2 1.4 and glucose 11.0. The perfusion fluid was filtered Ž5 m m pore size. before use and was continuously gassed with 95% oxygen plus 5% CO 2 ŽpH 7.4 at 378C.. After 5 min, an intraventricular balloon was introduced into the left ventricle through the mitral valve. The ultrathin compliant, but non-elastic balloon was constructed from plastic wrapping film Žcling-film. so as to match the internal dimensions of the left ventricle w23x. A suitable section of film was tied onto the end of a cut-off intravenous cannula which was connected to a small piece of rigid polythene tubing. The other end of polythene tubing was attached to the recorder via an appropriate pressure transducer, which had previously been calibrated against a known head of pressure. The balloon was then filled with saline and the volume of the balloon was gradually increased and decreased in an attempt to get the best possible fit to the contours of the ventricle. After 2 min the balloon volume was adjusted. Without distorting the shape of the left ventricle, left ventricular end-diastolic pressure was set in the range of 2]10 mm Hg. The balloon size remained unaltered from this point in the experiment onwards and thus, the heart contracted isochorically. After 30 min of controlled perfusion with bicarbonate buffer, values for cardiac function were recorded. Hearts which did not satisfy pre-defined inclusion criteria Žsee later. were then excluded.
Perfusion procedures Perfusion was carried out in the Langendorff mode at constant pressure w21x. Perfusion pressure was set at 80]90 cm H 2 0. These values were selected to correspond with the in ®i®o mean arterial blood pressure in rats w22x. Perfusion was with a bicarbonate buffer solution containing Žin mM.: NaCl 118.5, NaHCO3 25.0, KCl 4.8, MgSO4 1.2, KH 2 PO4 1.2,
Experimental protocol Study 1 Angiotensin I was dissolved in distilled water Žvehicle. and infused by a Harvard pump Žmod 975. through a side arm of the aortic cannula for 5 min. Infusion rate Žconstant flow. was set between 0.59 ml miny1 and 1.1 ml miny1 in order to have a final concentration of 4 = 10y9 , 4 = 10y8 , or 4 =
Fig. 1. Isolated rat hearts were perfused with bicarbonate buffer during the control period Ž30 min.. Vehicle or angiotensin I Ž4 = 10y9 , 4 = 10y8 or 4 = 10y7 M. was infused for 5 min, followed by a 15-min wash-out and 15 min infusion of captopril Ž80 m grml.. Coronary flow ŽCF., heart rate ŽHR., left ventricular developed pressure ŽLVDP. and enddiastolic pressure ŽLVEDP., were measured throughout the experiment Žsee arrows..
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10y7 M for each heart. After 15 min wash-out, 4 = 10y4 M captopril was infused for 15 min followed by a 5-min infusion of angiotensin I at the same concentrations as before. The heart was then perfused with bicarbonate buffer ŽFig. 1.. Study 2 Angiotensin II was dissolved in distilled water Žvehicle. and infused as described for study 1, at concentrations of 1 = 10y9 , 1 = 10y8 , or 1 = 10y7 M for 5 min. After a wash-out period of 10 min, hearts were subjected to 45 min of normothermic global ischaemia then reperfused. During the reperfusion period Ž45 min. hearts were infused with angiotensin II at the same concentrations as before ischaemia ŽFig. 2..
59
8 ml miny1 , 220 b.p.m. and 90 mmHg. Any heart that was excluded was immediately replaced by another. During preliminary experiments Žstudy 1., we observed a dramatic reduction Žmore than 80% of pre-infusion value. of coronary flow and contractility after captopril. We therefore decided not to infuse angiotensin I a second time to those hearts where such alterations occurred. Eight hearts out of 21 were excluded for this reason. One heart in the control group was excluded because air bubbles entered the circuit.
Expression of ®ariables Variables measured Left ventricular systolic and end-diastolic pressures were obtained from high-speed recordings Ž25 mm sy1 . of the pressure trace and left ventricular developed pressure was calculated as the difference between these two ŽBattaglia Rangoni, eight channels, KO 380, Bologna I.. Heart rate was also derived from the pressure trace and coronary flow was measured by direct timed collection of coronary effluent. Incidence, magnitude, time to onset and time to peak of ischaemia-induced contracture were recorded. Coronary flow, heart rate, left ventricular developed pressure and left ventricular end-diastolic pressure were measured throughout the perfusion and reperfusion period ŽFig. 1..
Exclusion criteria Pre-defined exclusion criteria were applied before the start of the study and at 30 min of control perfusion. The minimum acceptable values for coronary flow, heart rate and left ventricular developed pressure in the isolated rat heart were, respectively,
Results are reported as mean " SEM. Control variables before infusion of angiotensin I and angiotensin II or before ischaemia are expressed as absolute values. Coronary flow, heart rate, left ventricular developed pressure recorded after angiotensin I and angiotensin II are expressed as percentages of baseline values. Post-ischaemic recovery of function is expressed as a percentage of the values recorded before ischaemia and after reperfusion. The magnitudes of ischaemic contracture and left ventricular end diastolic pressure are expressed as absolute values Žmm Hg..
Statistical analysis One-way analysis of variance ŽANOVA. was used for multiple comparisons when a significant F value was obtained; comparison of means was followed by Dunnett’s test. The Student’s t-test for paired data was used to compare cardiac function before and after the 2-min infusion of angiotensin I and angiotensin II. Differences are considered significant at P- 0.05.
Fig. 2. Isolated rat hearts were perfused with bicarbonate buffer during the control period Ž30 min.. Vehicle or angiotensin II Ž1 = 10y9 , 1 = 10y8 or 1 = 10y7 M. was infused for 5 min, followed by a 10-min wash-out before global normothermic ischaemia Ž45 min. and again during reperfusion Ž45 min.. Coronary flow ŽCF., heart rate ŽHR., left ventricular developed pressure ŽLVDP. and end-diastolic pressure ŽLVEDP., were measured throughout the experiment Žsee arrows.. Body weight ŽBW. was recorded at the start of the experiment and heart weight ŽHW. at the end.
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Table I Cardiac function in isolated rat hearts before (left column) and after 2 min (right column) angiotensin I (A I) or vehicle (mean" SEM) Group Control Ž n s 7.
BW (g)
HW (mg)
CF (ml miny 1 g y 1)
HR (b.p.m.)
LVDP (mmHg)
304 " 17
813 " 45
17.7" 1.9
17.1" 1.5
307 " 14
282 " 13†
104 " 3
105 " 3 101 " 3
A I 4 = 10y9 Ž n s 5.
M
287 " 8
775 " 14
16.1" 1.6
14.2" 1.6†
313 " 21
290 " 18†
104 " 2
A I 4 = 10y8 Ž n s 9.
M
294 " 13
837 " 38
17.6" 1.2
13.2" 1.1U †
317 " 9
290 " 15†
105 " 4
A I 4 = 10y7 Ž n s 7.
M
298 " 15
790 " 38
15.5" 0.7
9.4" 0.7U †
302 " 19
264 " 20†
104 " 3
95 " 3U † 92U † " 3
Abbre®iations: BW, body weight; HW, heart weight; HR, heart rate; CF, coronary flow; LVDP, left ventricular developed pressure. U P- 0.05 ®s control ŽANOVA" Dunnett’s test.; †P - 0.05 Žpaired t-test ..
RESULTS
Basal characteristics of hearts are shown in Tables I and II. No differences were observed in body weight, heart weight and cardiac function between groups.
Study 1 A dose-dependent reduction of coronary flow was observed during the 5-min infusion of angiotensin I. Maximal reduction of coronary flow, by approximately 20% and 40% compared to pre-drug values, was observed at the 2-min infusion of 4 = 10y8 M and 4 = 10y7 M angiotensin I wFig. 3ŽA.x ŽTables I and III.. Afterwards, coronary flow returned toward basal values, with a dose-dependent kinetics. During the wash-out period, coronary flow returned to predrug values. After captopril, the coronary flow response to a second 5-min infusion of 4 = 10y8 M and 4 = 10y7 M angiotensin I was attenuated, the area
under the time]effect curve was reduced by 34% and 41%, respectively, compared with the first infusion of angiotensin I wFig. 3ŽB. ®s Fig. 3ŽA.x. The maximal reduction of flow was observed after the 5-min infusion of 4 = 10y8 M and 4 = 10y7 M angiotensin I wFig. 3ŽB.x ŽTable III.. Left ventricular developed pressure was significantly reduced by approximately 10% in the first minutes, by 4 = 10y8 M and 4 = 10y7 M angiotensin I ŽFig. 4, Tables I and III.. Heart rate was significantly reduced in each group compared with pre-drug values ŽTable I.. However, the reduction was not due to angiotensin I, since the difference in the control group was similar to, or smaller than, that seen with any angiotensin I concentration.
Study 2 Angiotensin II caused a dose-dependent reduction of coronary flow. Maximal reduction, approximately 30]35% compared to pre-drug values, occurred at 1 and 2 min of angiotensin II 1 = 10y8 M and an-
Table II Cardiac function in isolated rat hearts before (left column) and after 2 min (right column) angiotensin II (A II) or vehicle (mean" SEM) Group Control Ž n s 4.
BW (g)
HW (mg)
CF (ml miny 1 g y 1)
HR (b.p.m.)
LVDP (mm Hg)
310 " 16
823 " 33
16.3" 1.2
15.5" 1.4
302 " 17
287 " 14
111 " 7
112 " 6 107 " 3
A II 1 = 10y9 Ž n s 5.
M
305 " 14
865 " 42
16.2" 1.0
14.2" 1.0†
298 " 13
276 " 12†
113 " 2
A II 1 = 10y8 Ž n s 5.
M
318 " 17
842 " 38
15.5" 0.4
11.1" 0.2U †
295 " 10
272 " 10†
113 " 4
97 " 4U †
A II 1 = 10y7 Ž n s 5.
M
316 " 14
859 " 45
15.5" 0.7
11.5" 0.6U †
301 " 5
273 " 13†
103 " 2
93 " 3U †
Abbre®iations: BW, body weight; HW, heart weight; HR, heart rate; CF, coronary flow; LVDP, left ventricular developed pressure. U P- 0.05 ®s control ŽANOVA" Dunnett’s test.; †P- 0.05 Žpaired t-test ..
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61
Table III Statistical comparison of mean coronary flow and developed pressure (reported in Figs 3–5) in isolated rat hearts Angiotensin I
Wash out
1 min
2 min
5 min
1 min
2 min
5 min
10 min
Data in Fig. 3ŽA. 4 = 10y9 M 4 = 10y8 M 4 = 10y7 M
NS - 0.05 - 0.05
NS - 0.05 - 0.05
NS - 0.05 - 0.05
NS - 0.05 - 0.05
NS NS NS
NS NS - 0.05
NS NS NS
Data in Fig. 3ŽB. 4 = 10y9 M 4 = 10y8 M 4 = 10y7 M
NS NS NS
NS NS NS
NS - 0.05 - 0.05
- 0.05 - 0.05 - 0.05
NS NS NS
NS NS NS
NS NS NS
Data in Fig. 4ŽA. 4 = 10y9 M 4 = 10y8 M 4 = 10y7 M
NS P s 0.05 P - 0.05
NS P- 0.05 P- 0.05
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS NS NS
Angiotension II Data in Fig. 4ŽB. 1 = 10y9 M 1 = 10y8 M 1 = 10y7 M
NS - 0.05 - 0.05
NS - 0.05 - 0.05
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS NS NS
Data in Fig. 5 1 = 10y9 M 1 = 10y8 M 1 = 10y7 M
NS - 0.05 - 0.05
- 0.05 - 0.05 - 0.05
- 0.05 - 0.05 - 0.05
- 0.05 - 0.05 - 0.05
NS - 0.05 - 0.05
NS NS - 0.05
NS NS NS
ŽANOVA and Dunnett’s test ®s control hearts ..
giotensin II 1 = 10y7 M infusion ŽFig. 5, Tables II and III.. At 5 min the flow was still reduced to the same extent Ž15%. with each dose of angiotensin II. Coronary flow returned very slowly toward pre-drug values during the 10-min Kerbs perfusion ŽFig. 5.. Contractility was significantly reduced 1 min and 2 min after 1 = 10y8 M and 1 = 10y7 M angiotensin II infusion wFig. 4ŽB.x ŽTables II and III. and the effect on left ventricular developed pressure was short-lasting. By 5 min, contractility had returned toward pre-drug values wFig. 4ŽB.x. The heart rate was sig-
nificantly reduced after each dose of angiotensin II ŽTable II.. Post-ischaemic vascular and cardiac function were significantly altered in control hearts and hearts given angiotensin II during reperfusion ŽTable IV.. Post-ischaemic recovery of coronary flow, heart rate and left ventricular developed pressure were similar in each group. At the reperfusion time left ventricular end-diastolic pressure rose steeply in each group ŽFig. 6.. The end-diastolic pressure values were highest
Fig. 3. Time-course of coronary flow Žpercentages of basal values. in isolated rat hearts after vehicle Žv]v. or ŽA. angiotensin I w4 = 10y9 M Ž`]`., 4 = 10y8 M w^]^x, 4 = 10y7 M ŽI]I.x and ŽB. 80 m g mly1 captopril and the same dose of angiotensin I w4 = 10y9 M Ž`]`., 4 = 10y8 M Ž^]^., 4 = 10y7 M ŽI]I.x.
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Fig. 4. Time-course of left ventricular developed pressure Žpercentages of basal values. in isolated rat hearts after vehicle Žv]v. or ŽA. angiotensin I w4 = 10y9 M Ž`]`., 4 = 10y8 M Ž^]^., 4 = 10y7 M ŽI]I.x and ŽB. angiotensin II w1 = 10y9 y8 y7 M Ž`]`., 1 = 10 M Ž^]^., 1 = 10 M ŽI]I.x.
Fig. 5. Time-course of coronary flow Žpercentages of basal values. in isolated rat hearts after vehicle Žv]v. or angiotensin II w1 = 10y9 M Ž`]`., 1 = 10y8 M w^]^x, 1 = 10y7 M ŽI]I.x.
consistently with time, in hearts given the highest dose of angiotensin II Ž1 = 10y7 M.. Magnitude, time to onset and time to peak of ischaemic contracture were similar in controls and hearts given angiotensin II ŽTable IV..
DISCUSSION In the present study we investigated the role of angiotensin I and angiotensin II on myocardial contractility, heart rate and coronary perfusion in the isolated rat heart. Angiotensin II has both direct and indirect actions on the heart and can modulate rate, contractility and coronary smooth muscle tone. Both angiotensin I and angiotensin II reduced the contractility of the heart and constricted coronary
arteries. On the isolated human atria muscle, angiotensin I had a positive inotropic effect which was equipment to angiotensin II w2x. In our preparation, angiotensin II was four times more potent than angiotensin I. The higher potency ratio has been confirmed in several experimental conditions but the actual ratio varies considerably Žfrom two to ten. w10, 24x. Contrasting results have been published on the inotropic effect of angiotensin II, depending on the models and species used. Angiotensin II induces a positive inotropic response in most species, which is not fully mediated by the b-adrenergic system w2, 4, 9, 10, 15, 16x. In the rat, however, angiotensin II had either no effect w4, 8, 16x or a negative inotropic effect w25x. We found angiotensin II induced a dosedependent negative inotropic response. Most studies used a single dose of either or both substances. We preferred to use three doses. The range of concentration was selected from studies reported to be active on coronary flow and contractility w8]12x. Our results confirm the coronary constriction response to angiotensin I and angiotensin II w8]12x. Other authors w26x, however, observed a coronary response in the isolated perfused rabbit heart consisting of transient vasoconstriction followed by dilatation, especially at a high concentration of angiotensin II. They suggested that the angiotensin II dilatation probably reflected rapid desensitization to the constrictor effect on the coronary arterial smooth muscle. All the effects of angiotensin II in the heart appeared to be mediated by the AT1 receptor subtype found on coronary endothelial and smooth muscle cells and on cardiomyocytes w26x. We do not know whether the marked reduction of the coronary flow is responsible for the negative inotropic response owing to a limited oxygen and metabolic substrate supply in the circulation. This possibility was raised by Fowler and Holmes in ex-
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Table IV Recovery of function (percentage of pre-ischaemic values) in isolated rat heart after 45 min ischaemia and reperfusion (mean" SEM) Group
Control Ž n s 4.
CF (%)
HR (%)
LVDP (%)
TTO (min)
Ischaemic contracture TTP (min) Peak (mm Hg)
68 " 3
77 " 7
47 " 3
8.5" 0.9
16.5" 1.7
55.3" 7.1
A II 1 = 10y9 Ž n s 5.
M
62 " 3
80 " 9
43 " 7
8.2" 0.7
16.4" 0.7
63.2" 5.1
A II 1 = 10y8 Ž n s 5.
M
69 " 3
76 " 7
39 " 7
7.6" 0.7
16.4" 1.3
59.6" 5.0
A II 1 = 10y7 Ž n s 5.
M
62 " 2
80 " 5
42 " 11
8.4" 1.0
17.0" 1.1
59.2" 4.5
Abbre®iations: A II, angiotensin II; CF, coronary flow; HR, heart rate; LVDP, left ventricular developed pressure; TTO, time to onset; TTP, time to peak.
Fig. 6. Time-course of left ventricular end-diastolic pressure Žmm Hg. during reperfusion in isolated hearts receiving vehicle Žv]v. or angiotensin II w1 = 10y9 M Ž`]`., 1 = 10y8 M Ž^]^., 1 = 10y7 M ŽI]I.x.
periments with a canine heart]lung preparation; they suggested that the coronary vasoconstrictor effect of angiotensin might limit its inotropic action w25x. Neves et al. found that, in the presence of ACE inhibition, w 125 Ixangiotensin II was still formed when w 125 Ixangiotensin I was infused again over a 10-min period, although an average of 43% reduction was seen. It is interesting to note that we observed a reduction in coronary flow when angiotensin I was infused again over 10 min. We found that, after captopril, coronary flow response to a second 5-min infusion of 4 = 10y8 M and 4 = 10y7 M angiotensin I was attenuated, the area under the time]effect curve Žmeasured by trapezoid rule. was reduced by 34% and 41%, respectively, compared with the first infusion of angiotensin I. Our data is in agreement with the metabolic data of Neves et al. w27x. These and other results confirms that ACE activ-
ity is present in the cardiac tissue w24, 27, 28x. Angiotensin I has often been used to estimate functional ACE activity in tissue and in the circulation because it requires conversion to angiotensin II to effect a response. Captopril is one of the many ACE inhibitors now available as ‘reference drug’. Being one of the first and most extensively studied ACE inhibitors, dose]response patterns have been characterised. Therefore, we selected a dose of captopril Ž80 m g mly1 , 4 = 10y4 M., already used by several investigators w8, 29, 30x and known to inhibit plasma ACE activity by 100% in the rat w31x. Even 4 = 10y4 M of captopril failed to completely suppress angiotensin I-mediated responses in the rat heart. Possibly this inability to abolish angiotensin I coronary responses was due to an intrinsic activity of angiotensin I Žindependent of conversion to angiotensin II. andror to the presence of an alternate enzymatic pathway for conversion to angiotensin II in the heart w32x. Angiotensin II formation in the rat heart appears to be primarily dependent on the converting enzyme w2, 5, 7x. However, other authors have indicated that, in humans, an ACE inhibitor-insensitive, soybean trypsin inhibitor-sensitive, plays an important role in the conversion of angiotensin I to angiotensin II w11x. Cardiac ventricular tissue in humans contains a dual enzymatic pathway for angiotensin II formation in which ACE-dependent angiotensin II formation is minor Žapprox. 10%. compared to major serine proteinase-dependent angiotensin II formation Žapprox. 80%. w2x. Our objective was to examine the role of angiotensin II on contractile and relaxant dysfunction associated with ischaemia and reperfusion. To achieve this we require two things. First, we need a model where contractile function can be conveniently evaluated. The isolated heart with global ischaemia is preferred because global contractile dysfunction is more easily measurable than regional. Second, we need a model in which moderate Ži.e.
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modulable. contractile dysfunction occurs. The duration of ischaemia we chose Ž45 min. achieves this. With longer ischaemia, irreversible injury may arise. With briefer ischaemia Žstunning. no injury may occur. We need a level of dysfunction that can be alleviated or exacerbated by a drug and can be detected statistically. The present model achieves this. We used multiple functional indices to assess the extent of tissue injury in the ischaemic heart, such as systolic Žcontractility . and diastolic Žrelaxation. function, heart rate, coronary flow and ischaemia-associated contracture. We preferred these to metabolic indices, such as enzyme leakage or depletion of high-energy phosphates, since, in our opinion, they are the ultimate arbiter of cardiac injury. The role of angiotensin II in acute myocardial ischaemia is not fully elucidated. There is clear evidence that the tissue RAS is activated during acute and chronic ischaemia w5, 17, 33, 34x and the ACE inhibition is clinically beneficial w4, 5, 17, 35x. In the experimental setting, exogenous angiotensin II, given before ischaemia to isolated rat hearts, had deleterious effects on coronary tone, post-ischaemic cardiac function and release of creatine kinase, these effects being abolished by a non-peptide angiotensin type 1 receptor antagonist w18x. Mochizuchi et al. tested the hypothesis that angiotensin II Ž10y8 M. directly impairs the preservation of contractile function in rabbit hearts during 30-min low-flow ischaemia and exacerbates myocardial stunning during reperfusion w33x. Angiotensin II directly impaired left ventricular diastolic relaxation during ischaemia and recovery. This effect was independent from that on coronary vascular tone and systolic function or magnitude of high-energy phosphate depletion. There are several potential mechanisms which might be responsible for the adverse effects of angiotensin II on diastolic function during low-flow ischaemia and subsequent reperfusion. Our results suggest it has an adverse effect on left ventricular diastolic relaxation during no-flow myocardial ischaemia and recovery. Transient myocardial ischaemia in patients with coronary artery disease is associated with a severe and reversible depression of myocardial relaxation and elevation of left ventricular end-diastolic pressure w36, 37x. In this isolated rat heart model, it is unlikely that the effects of angiotensin II were mediated by facilitation of sympathetic neurotransmitter release. However, a limitation of this study is that this indirect effect of angiotensin II may be more dominant in ®i®o. One clinical implication is that angiotensin II may play a contributory role in the development of congestive heart failure secondary to acute diastolic dysfunction during transient ischaemia. In such patients, an increase in angiotensin II through the systemic RAS or through ACE-specific or nonspecific cardiac angiotensin II-forming pathways
might promote adverse changes in both coronary vasomotor tone and diastolic relaxation. The adverse effects of angiotensin II during ischaemia may be particularly marked in the setting of cardiac disease, when the cardiac activation of angiotensin II appears to be enhanced. Such disease and local activation of angiotensin II may occur in a regional distribution in post-infarction remodelling associated with coronary artery disease in addition to the global hypertrophy that occurs in response to pressure overload. Thus, patients may be predisposed to ischaemic diastolic dysfunction mediated, at least in part, by the action of the RAS in the heart.
ACKNOWLEDGEMENTS This work was supported in part by the CNR ŽNational Research Council, Italy, No. 91.01277.PF70.. Cristina Traquandi is a ‘F. Ferraris, G. Bevilacqua’ Fellow. We wish to thank Judy Baggott for the English revision.
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