Long-term therapy with captopril has been demonstrated to slow the progress of clinical heart failure provided it is administered early in the course of the disease (1). In an experimental rat model of heart failure secondary to myocardial infarction, captopril was also reported to improve survival and attenuate the development of cardiac hypertrophy (2). The precise mechanism underlying this effect of captopril is not known. As an inhibitor of angiotensin-converting enzyme (ACE), captopril reduces the levels of angiotensin II and decreases arterial blood pressure (BP), thus reducing the workload of the heart (3). This may underline its ability to attenuate the development of cardiac hypertrophy. It has also been shown that captopril improves the responsiveness of the myocardium to β1-adrenoceptor stimulation in a rat model of heart failure secondary to myocardial infarction (4). Furthermore, when coadministered with isoprenaline, captopril, even in a dose too low to decrease BP (3,5) prevents β1-adrenoceptor downregulation (6).
We recently developed a model of norepinephrine (NE)-induced cardiac hypertrophy in rats which also involves β1-adrenoceptor downregulation (7). The aim of the present study was twofold: first to determine the effect of chronic administration of captopril on the development of cardiac hypertrophy and to relate any observed effect to changes in haemodynamic measurements and second to investigate whether captopril prevented β1-adrenoceptor downregulation. Two doses of captopril were examined, one equivalent to that used in the study of Maisel and co-workers (6), i.e., 52 μg kg-1 h-1 and one (250 μg kg-1 h-1) that is within the range of that reported to cause hypotension (5).
Implantation of osmotic minipumps
Male Sprague-Dawley rats weighing 270-360 g were anaesthetised with halothane (induced with 5% and maintained on 1-2% carried in a 70% nitrous oxide/30% oxygen mixture), and a small area (4 × 1.5 cm) on the back, ≈6 cm from the base of the tail, was shaved. A 3-cm lateral incision was made, and a subcutaneous pocket, reaching to the back of the neck, was made with blunt scissors. One or two osmotic minipumps (Alzet, model 2ML4, Alzet, Palo Alto, CA, U.S.A.) were inserted in the pocket, and the incision was closed with vycryl suture (Ethicon, W9121 2/0, Ethnor S.A., Neuilly, France). Minipumps were filled with vehicle (0.2% wt/vol ascorbic acid in saline), NE dissolved in vehicle, or captopril dissolved in saline. The rate of infusion was 2.34-2.38 μl h-1, and the NE concentration was such that it was delivered at 0.15 mg kg-1 h-1. Captopril was delivered at 52 or 250 μg kg-1 h-1. The number of rats implanted with miniosmotic pumps was as follows: vehicle, 8; NE alone, 9; captopril 52 μg kg-1 h-1, 6; NE plus captopril 52 μg kg-1 h-1, 6; captopril 250 μg kg-1 h-1, 6; and NE plus captopril 250 μg kg-1 h-1, 6. Body weights were measured before and during the 4 weeks postimplantation of the osmotic minipumps.
At 4 weeks postimplantation of the osmotic minipumps, rats were anaesthetised with sodium pentobarbital (60 mg kg-1 intraperitoneally, i.p.). Polythene cannulas were inserted in the right femoral vein for drug and anaesthetic administration and in the right femoral artery for measurement of arterial BP by a pressure transducer. A Millar microtip catheter pressure transducer (model No SPR 249, Millar Instruments, Texas, U.S.A.) was inserted in the left ventricle through the right carotid artery for measurement of left ventricular pressure. The trachea was cannulated, and the animals were artificially ventilated (volume 1.5 ml 100 g-1; rate 48 strokes min-1). Typical values for blood pH, PO2, and PCO2 were 7.3 ± 0.02, 81 ± 3, and 28 ± 4 mm Hg, respectively. Standard ECG leads were inserted subcutaneously. A precalibrated steel probe was inserted in the rectum to measure temperature, which was maintained at 37 °C with the aid of a heating lamp. A 15-min stabilisation period was allowed after the surgical procedure before any drugs were administered. At the end of the experiment, the animals were killed by an overdose of sodium pentobarbital.
Arterial and left ventricular BP and the ECG were measured by an Axon Cyberamp (Axon Instruments) and were continuously recorded on a Graphtec Linearecorder (Graphtec, Yokohama, Japan). An on-line data analysis system (John Dempster, Department of Physiology and Pharmacology, University of Strathclyde) recorded systolic, diastolic, and mean arterial BP, HR, and systolic and end-diastolic pressures. Two derived indexes of contracility [left ventricular dP dt-1max (the maximum rate of pressure generation) and dP dt-1max P-1 [the maximum rate of pressure generation divided by the left ventricular pressure at that timepoint, an index relatively unaffected by changes in afterload (8)] were calculated by computer analysis.
NE dose-response curves. Dose-response curves to NE (0.1-10 μg kg-1 intravenously, i.v.) were constructed (with sufficient time between individual doses to allow haemodynamic variables to return to preadministration levels). The protocol was terminated when a doubling of the contractile index. dP dt-1max, was attained.
Calcium chloride dose-response curves. After recovery from the dose-response curve to NE, we constructed dose-response curves to calcium chloride by infusing incremental doses (0.25-45 mg kg-1 min-1) until a stable response was achieved. The infusions were discontinued when arterial BP decreased after an increase in the dose of calcium chloride.
Ventricular weight determination. At the end of the protocol, the hearts were excised and rinsed in saline and the ventricles were separated from the atria. The right ventricular free wall was separated from the left ventricle and septum, and both were dried and weighed.
NE (± arterenol hydrochloride) and L-ascorbic acid were obtained from Sigma, Poole, Dorset. Sodium pentobarbital (Sagatal) and halothane were obtained from May & Baker, Dagenham, Kent; calcium chloride (Analar grade 1 M) was obtained from BDH, Poole, Dorset. Captopril was obtained from Bristol Myers Squibb. All drugs were dissolved in saline (0.9% sodium chloride) unless otherwise stated.
Statistical analysis of data
All data are expressed as mean ± SEM; n, indicates the number of animals. Student's t test or one-way ANOVA; (analysis of variance followed by a Bonferroni posttest) were used as appropriate to assess statistical significances between mean values; p < 0.05 was taken to denote statistical significance.
Effects of chronic administration of NE and/or captopril on body weight, ventricular weight, and mortality
Animals chronically treated with NE exhibited a slight (<5%) loss of body weight during the first 2 days postimplantation, whereas vehicle- and captopril-treated rats gained weight. Concomitant administration of either the low or high dose of captopril did not prevent the weight loss, but the weight loss was regained by ≈21 days postimplantation so that by 28 days there were no significant differences in body weight among the treated groups (Table 1).
NE pretreatment resulted in an increase in left ventricular and septal (LV&S) weights and ratios of LV&S weight to body weight but did not modify the weight of the right ventricle (Table 1). The degree of left ventricular hypertrophy was on the order of 20%. Chronic administration of captopril alone had no effect on heart weights; e.g., LV&S weight/body weight ratios were 0.444 ± 0.17 and 0.479 ± 0.16, respectively, in rats pretreated with the low and high dose of captopril. However, when administered concomitantly with NE, the high but not the low dose of captopril significantly attenuated the development of left ventricular hypertrophy (Table 1).
Only one death occurred during the pretreatment period-at 20 days postimplantation in the NE plus lowdose captopril group. The cause of death was not determined. All animals (including the animal that died) appeared healthy throughout the treatment period. At the end of the study, however, 5 of 12 animals treated with the high dose of captopril were noted to have pronounced enlargement of the large intestine.
Effect of chronic treatment with NE and/or captopril on basal haemodynamic variables
Table 2 summarises the haemodynamic variables measured in rats pretreated with NE and/or captopril for 28 days. NE clearly caused in increase in systolic but not diastolic arterial BP and also caused increases in HR and left ventricular systolic BP, and in the two indexes of contractility (dP dtmax-1 and dP dt-1max P-1).
Chronic treatment with captopril alone (52 μg kg-1 h-1) had no significant effect on any of the haemodynamic variables. However, the higher dose of captopril (250 μg kg-1 h-1) significantly reduced systolic and diastolic BP but did not modify the index of contractility (dP dt-1max P-1) or HR.
Concomitant administration of the lower dose of captopril with NE did not modify the effects of NE on basal haemodynamic variables (Table 2). In contrast, the higher dose of captopril prevented the effects of NE on systolic arterial BP and systolic left ventricular pressure but not on HR or dP dt-1max P-1(Table 2). In the group of rats coadministered NE and the higher dose of captopril, diastolic arterial BP was significantly lower than that in the vehicle pretreated group.
Effect of chronic treatment with NE and/or captopril on the acute response to NE
Figure 1 shows the effect of acute administration of NE in dP dt-1max in animals chronically pretreated with vehicle, NE, captopril (52 μg kg-1 min-1), or NE plus captopril. Acute administration of NE produced less marked increases in contractility in rats chronically treated with NE alone or in combination with captopril than in control or captopril-treated animals. A similar effect was noted with the higher dose of captopril, both on dP dt-1max and on the afterload independent index dP dt-1max P-1. Acute administration of NE also produced less marked increases in systolic BP, systolic left ventricular pressure, and dP dt-1max in animals pretreated with NE alone or NE combined with either dose of captopril. In contrast, diastolic arterial BP (Fig. 2) and HR (data not shown) were increased to a similar degree in control and NE-treated rats. Both doses of captopril led to an enhanced effect of acute NE administration on diastolic arterial BP, as is shown in Fig. 2 for the higher dose of captopril.
Effect of chronic treatment with NE and/or captopril on the acute response to calcium chloride
Infusion of incremental doses of calcium chloride in control animals produced slight but dose-dependent increases in all measured variables. Chronic administration of NE and/or captopril (low or high dose) did not significantly modify the effects of acute administration of calcium chloride. This is shown for rats treated chronically with NE and the higher dose of captopril in Figs. 3 and 4. In some of the rats pretreated with either NE alone or NE plus captopril, infusion of the higher doses of calcium chloride (20 and 40 mg kg-1 min-1) led to a marked decrease in arterial BP and in myocardial contractility (Fig. 3).
Chronic treatment of rats with NE (0.15 mg kg h-1) caused left but not right ventricular hypertrophy, the degree of which was on the order of 20%. This degree of hypertrophy is comparable to that reported by other investigators using similar doses of NE (9,10). The selective effect on the left ventricle is in accord with our previous results in this model (7) showing that doses of NE that increased left ventricular systolic and systolic arterial BP but not diastolic arterial BP increased left but not right ventricular mass. These data suggest that the hypertrophic stimulus in this model is not an NE-induced α-adrenoceptor vasoconstriction but rather a β1-adrenoceptor-mediated increase in myocardial contractility and subsequent increase in systolic left ventricular and arterial BP. The lack of effect of chronic administration of NE on diastolic arterial BP was confirmed in a previous study (7) and may be a consequence of reflex-induced mechanisms operating to offset the effect on the heart. Because the most pronounced haemodynamic effects of NE administration were an increase in left ventricular contractility and systolic left ventricular and arterial BP, the most likely stimulus for the left ventricular hypertrophy in this model change in either cardiac contractility or systolic hypertension. Isolated systolic hypertension may occur in humans and leads to cardiac hypertrophy (11,12).
Captopril pretreatment in a dose of 250 μg kg-1 h-1 for 28 days, but not of 52 μg kg-1 h-1 for the same period, significantly attenuated the development of left ventricular hypertrophy associated with chronic NE administration. This finding is similar to those of previous investigators who reported the ability of captopril (13) and other ACE inhibitors (14) to attenuate the left ventricular hypertrophy that develops after myocardial infarction. The beneficial effect of captopril in our study was apparent only with the higher dose studied, i.e., that which prevented the effect of chronic NE pretreatment in increasing systolic left ventricular and systolic arterial BP. Therefore, in this model, apparently it is the ability of captopril to prevent the NE-induced changes in systolic left ventricular and arterial BP that is responsible for its ability to attenuate the development of left ventricular hypertrophy. This is also in accord with results of a study in aging rats which showed that the ACE inhibitor perindopril decreased both systolic arterial BP and ventricular mass (15).
Chronic treatment with NE led to reduced effects of acutely administered NE on myocardial contractility and on arterial and left ventricular pressures, presumably due to a β1-adrenoceptor downregulation of NE, as previously reported (9,16). However, in the present study, the effects of acutely administered NE were studied at the end of a 4-week period of chronic administration of NE when plasma NE levels presumably are still increased. Captopril, at both doses studied, did not modify the β1-adrenoceptor downregulation induced by chronic treatment with NE because myocardial responses to acutely administered NE were reduced to the same extent in rats treated with NE and captopril as in rats treated only with NE. This observation conflicts with results obtained by van Wijngaarden and associates (4) in a rat model of heart failure secondary to myocardial infarction. This contradiction may be a result of the different models used (NE-induced hypertrophy vs. myocardial infarction) or of the higher doses of captopril used by van Wijngaarden and associates (equivalent to 4.2 mg kg-1 h-1). However, a less marked β1-adrenoceptor downregulation was reported by Maisel and co-workers (6), in guinea pigs treated chronically with isoprenaline that received the same dose of captopril (52 μg kg-1 h-1) used in the present study. Maisel and co-workers (6) suggested that isoprenaline caused β1-adrenoceptor downregulation in part by increasing NE release from sympathetic nerve terminals (through presynaptic β1-adrenoceptors) as well as by peripheral vasodilation leading to baroreflex activation and to neuronal NE release. Because angiotensin II also causes NE release from sympathetic nerves, it was argued that the ability of captopril to attenuate β1-adrenoceptor downregulation was explained by the consequent reduction in angiotensin II levels and thus by reduced NE release. In our experimental model, NE rather than isoprenaline was infused, and neuronal release of NE may have been of lesser importance. This may explain why captopril was apparently ineffective in preventing β1-adrenoceptor downregulation in our study.
Captopril has also been reported to diminish the response to acutely administered NE in pithed animals (5,17). However, we observed no such effect of captopril treatment in our anaesthetised animals, which is in agreement with the results of de Jong and colleagues (18) and further supports the view that captopril has such an effect in pithed rats only because plasma renin levels are markedly increased in that model.
Captopril did not modify the haemodynamic responses to infusion of calcium chloride which increases myocardial contractility as a consequence of the increased concentration gradient of calcium across the cardiac cell membrane and a resultant increase in calcium entry. That captopril did not modify calcium-induced changes in contractility implies that it has no direct effect on the cardiac contractile mechanisms.
Because downregulation of cardiac β-adrenoceptors, as a consequence of increased sympathetic nervous stimulation, is believed to play a role in the deterioration of cardiac function in patients with congestive heart failure (19), it has been suggested that part of the protective effect of captopril in heart failure is due to its ability to prevent such receptor downregulation (6). Our data do not support this suggestion and imply that any such effect of captopril may be dependent on the experimental model used to induce β-adrenoceptor downregulation. The protective effect of captopril that was most prominent in our experimental model was its ability to prevent cardiac hypertrophy, which is known to be an independent risk factor in the occurrence of sudden cardiac death (20,21).
Some (5 of 12) of the animals administered the large dose of captopril had a pronounced enlargement of the large intestine, which may have been due to blockade, by captopril, of the effect of angiotensin in stimulating intestinal motility (22). Consistent with this finding, a few clinical cases of severe constipation and bowel atony in patients receiving captopril have been reported (23).
We demonstrated that long-term administration of captopril prevents the development of NE-induced cardiac hypertrophy but does not influence NE-induced β-adrenoceptor downregulation, as assessed by changes in myocardial contractility induced by acutely administered NE.
Acknowledgment: This work was supported by an SERC studentship and by Bristol Myers Squibb.
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