Different Effects of Carvedilol, Metoprolol, and Propranolol on Left Ventricular Remodeling After Coronary Stenosis or After Permanent Coronary Occlusion in Rats
Background— Although carvedilol attenuates left ventricular (LV) remodeling in coronary occlusion-reperfusion, it is not known whether it attenuates ischemic LV remodeling because of coronary stenosis (CS) or permanent coronary occlusion (CO).
Methods and Results— We administered a vehicle, carvedilol, propranolol (2, 10, and 30 mg/kg per day, each), metoprolol (6, 30, and 90 mg/kg per day), or bunazosin (0.2 and 1 mg/kg per day), orally for 12 weeks to a total of 608 rats with CS or CO. In these groups and the sham (n=40), we assessed LV function by echocardiography, CS severity, myocardial blood flow and coronary flow reserve, serum ascorbyl free radical, and vitamin C. Both CS and CO increased LV end-diastolic and end-systolic diameters and decreased ejection fraction. The 4 agents failed to attenuate LV remodeling caused by CO. In contrast, the 3 β-blockers attenuated (P<0.01) or tended to attenuate the increase in LV end-diastolic diameters caused by CS. With similar blood pressure and heart rate lowering by 3 β-blockers, carvedilol additionally attenuated the increase in end-systolic diameters and improved ejection fraction. The CS reduced myocardial blood flow and coronary flow reserve, which was reversed by carvedilol without modifying the CS severity. Among the 4 agents, only carvedilol decreased ascorbyl free radical and increased vitamin C.
Conclusions— The effects of β blockade on ischemic cardiac dysfunction seem to depend on the different properties of the β-blockers and the doses used. Among the β-blockers tested, carvedilol provided potent cardioprotection for compromised ischemic but viable myocardium rather than for infarcted myocardium.
Received August 29, 2001; revision received December 17, 2001; accepted December 21, 2001.
Ischemic heart failure may have a worse prognosis and a different drug response than nonischemic heart failure.1,2⇓ However, there is a broad spectrum of ischemic heart disease, and it cannot be treated as a single entity. The influences of myocardial infarction and chronic ischemia attributable to fixed coronary stenosis on the development of left ventricular (LV) dysfunction and remodeling, as well as their modification by drugs, might be different.
Carvedilol, a β antagonist, improves the prognosis and prevents the progression of LV remodeling and dysfunction in heart failure of nonischemic origin.3 In heart failure of ischemic origin, carvedilol reduces cardiac events,4 attenuates cardiac dysfunction,5 and reduces mortality6 in acute infarction followed by reperfusion. However, it remains to be determined whether carvedilol attenuates LV remodeling or dysfunction associated with coronary stenosis. The present study was undertaken to assess whether carvedilol attenuates LV dysfunction and remodeling induced by chronic coronary stenosis as well as by coronary occlusion. In addition, to see whether the effects of carvedilol are attributable solely to lowering the heart rate (HR) and systolic blood pressure (SBP) or whether a blocking effect or antioxidant property may also be involved, the effects of propranolol, metoprolol, and bunazosin were also assessed in the same animal models.
The investigation conformed to the guidelines on animal experiments of Fukushima Medical University, the Japanese Government Animal Protection and Management Law (No. 115), and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).
Adult male Sprague-Dawley rats (290 to 310 g; n=699; CLEA Japan, Inc, Tokyo, Japan) were anesthetized by intraperitoneal 30 mg/kg sodium pentobarbital and were artificially ventilated. After a left thoracotomy (n=461, Figure 1), a stainless steel thread (275 μm in diameter) was placed on the epicardium along the left coronary artery (LCA), and the thread and LCA were ligated together followed by thread removal, as previously reported7 with minor modifications. During the brief coronary occlusion, the ST segment on ECG was elevated, and 15 animals with ST elevation persisting after thread removal were excluded from the study. In 198 animals, coronary ligation was performed at the equivalent site of the LCA, and surviving animals with persistent ST elevation (n=192) were used in the study.
The animals were returned to their cages, fed ad libitum, and killed 12 weeks later. We discontinued the preliminary studies treated with propranolol (90 mg/kg per day) and metoprolol (270 mg/kg per day) (n=15 each) in the coronary stenosis model because of high mortality rates, and the other rats (n=648, including 40 sham rats) were divided randomly into subgroups by treatments8–11⇓⇓⇓ (animal numbers shown in Figure 1), as follows: sham, inert vehicle administration in the coronary stenosis and occlusion models (V stenosis and occlusion groups), carvedilol administration (2, 10, and 30 mg/kg per day; C2, C10, and C30 stenosis and occlusion groups, respectively), metoprolol administration (6, 30, and 90 mg/kg per day; M6, M30, and M90 stenosis and occlusion groups, respectively), propranolol administration (2, 10, and 30 mg/kg per day; P2, P10, and P30 stenosis and occlusion groups, respectively), and bunazosin administration (0.2 and 1 mg/kg per day; B0.2 and B1 stenosis and occlusion groups, respectively). Within 24 hours after surgery, the drugs were administered daily by mouth with a cannula. The hemodynamic, echo, and microsphere studies were done 6 hours after the last administration of the drugs.
Hemodynamics in the Awake Animals
SBP and HR of awake animals were measured by the tail-cuff method before and 4, 8, and 12 weeks after surgery.
Before and 6 hours (except for the sham) and 4, 8, and 12 weeks after surgery, B- and M-mode echocardiography was obtained in anesthetized rats using a 10-MHz probe and Sonos 100 equipment (Hewlett Packard). LV end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD) were measured, then LV ejection fraction (LVEF) was calculated by the Pombo method. Among 648 rats, echocardiography was performed blindly in 531 and not blindly in 117.
HR, LV peak systolic and end-diastolic pressures (LVSP/EDP), and positive and negative LV dP/dt were measured by cardiac catheterization in anesthetized rats 12 weeks after surgery.
Twelve weeks after coronary stenosis, myocardial blood flow (MBF) (mL/min per wet g) and coronary flow reserve (CFR) (mL/min per wet g; maximal MBF by dipyridamole [10 mg/kg per min] minus basal MBF) were measured under anesthesia with colored microspheres (animal numbers shown in Figure 1). The risk areas (ischemic regions) were delineated by reoccluding the site of coronary stenosis followed by LV dye infusion at a pressure of 100 mm Hg.
Coronary Stenosis Severity
After the cardiac catheterization, rats (animal numbers shown in Figure 1) were sacrificed and fixed with 10% neutral buffered formalin perfused at a pressure of 100 mm Hg. In 5-μm-thick paraffin-embedded sections stained with elastica Van Gieson, cross-sectional areas (CSA) of LCA inner lumen were measured by the point-counting method of Weibel12 by light microscopy at ×400 magnification. The CSA at the stenotic site divided by the reference CSA located 50 sections proximal to the sections of stenotic area multiplied by 100 was considered the degree (%) of stenosis.
Blood samples were obtained from all rats of the groups (except rats for the microsphere study) at the time of cardiac catheterization. Plasma norepinephrine levels were measured by HPLC. Thiobarbituric acid reactive substance (TBARS) by fluorescence spectroscopy, ascorbyl free radical (AFR) (signal intensities relative to the standard signal intensities from manganese oxide including Mn2+ impurity) by ESR,13 and vitamin C by HPLC were measured in the sera of the sham (n=16), V (n=13), C2 (n=9), C30 (n=17), M6 (n=9), M30 (n=16), P2 (n=9), P30 (n=15), and B0.2 and B1 (n=9 each) stenosis groups (Table 1).
Data are expressed as mean±SEM. Statistical analysis was performed by the 2-way ANOVA with repeated measure. Statistics were presented in comparison with the sham, vehicle, and C30 groups (for intertreatment group comparisons). If F test results were <0.05, Scheffe’s post hoc test was performed. A value of P<0.01 was considered significant.
There were no significant differences in survival rates among the stenosis or occlusion groups.
Hemodynamics in the Awake Animals
Because SBP and HR measured by the tail cuff were similar between 4 and 12 weeks, only the data at 12 weeks are shown in Tables 1 and 2⇓. Both HR and SBP in the 3 β-blocker groups and SBP alone in the bunazosin groups were decreased in a dose-dependent manner.
Coronary Stenosis Severity, MBF, and CFR
The coronary stenosis severity (%) was similar among the 9 coronary stenosis groups examined (Table 1).
In risk areas, compared with the sham, basal MBF decreased in 10 stenosis groups but not in the C10 and C30 groups, and CFR decreased in 12 stenosis groups (Table 1). Compared with the nonrisk areas, MBF decreased in the risk areas of the V, C2, M6, M90, P2, P10, B0.2, and B1 stenosis groups and CFR decreased in all of the risk areas of 12 stenosis groups.
In the coronary stenosis groups, LVEDD (Figure 2, left) and LVESD (Figure 3, left) increased and LVEF (Figure 4, left) decreased in the V group at 4 to 12 weeks compared with the sham. Among the β blocker–treated groups, compared with the V group, the increase in LVEDD was not attenuated in the C2, M6, M90, P2, and P10 groups (Figure 2, left) but was attenuated in the C10 (at 8 to 12 weeks), C30 (at 4 to 12 weeks), and M30 (at 12 weeks) groups and tended to be smaller in the P30 group (Figure 2, left). Compared with the V group, the increase in LVESD was attenuated in the C10 (at 12 weeks) and C30 (at 4 to 12 weeks) groups (Figure 3, left). Consequently, the decrease in LVEF observed in the V group was attenuated only in the C30 group at 12 weeks (Figure 4, left).
Among the coronary occlusion groups, LVEDD (Figure 2, right) and LVESD (Figure 3, right) increased, and LVEF (Figure 4, right) decreased in all groups compared with the sham. These changes were greater than those in each treatment group with coronary stenosis (LVEDD in 11 groups except the V group and LVESD in 12 groups), and LVEDD, LVESD, and LVEF were not modulated by any treatment.
Because in the B0.2 and B1 stenosis and occlusion groups there were no significant changes in the LVEDD, LVESD, and LVEF compared with the corresponding V group at all time points measured, we show the data only at 12 weeks in these groups. LVEDD at 12 weeks was 8.1±0.2* and 7.5±0.1 mm, LVESD was 5.7±0.1* and 5.2±0.1* mm, and LVEF was 0.66±0.01* and 0.66±0.01* mm in the B0.2 and B1 stenosis groups, respectively. LVEDD at 12 weeks was 9.3±0.2* and 8.9±0.3* mm, LVESD was 7.2±0.2* and 6.8±0.2* mm, and LVEF was 0.53±0.01* and 0.56±0.01* mm in the B0.2 and B1 occlusion groups, respectively (*P<0.01 versus sham).
In rats with coronary stenosis (Table 1), compared with the sham, both LVSP and HR decreased in the C30, M30, M90, and P30 groups, whereas only LVSP decreased in the C10, P10, B0.2, and B1 groups. In addition, positive or negative LV dP/dt decreased in 11 stenosis groups except in the C10 group, and LVEDP increased in 10 groups but not in the C10 and C30 groups. Compared with the V group, LVEDP decreased, and positive or negative LV dP/dt increased in the C10 and C30 groups.
In rats with coronary occlusion (Table 2), compared with sham, LVSP decreased in 7 groups (not in the V, C2, M6, P2, and B0.2 groups), HR decreased in the C30, M30, M90, P10, and P30 groups, and positive or negative LV dP/dt decreased and LVEDP increased in all 12 groups.
Heart weights were greater in the V, P2, B0.2, and B1 stenosis groups than in the sham, and those of the C30 stenosis group were lower than in the V stenosis group (Table 1). Heart weights were greater in 9 coronary occlusion groups, except for the M30, P10, and P30 groups, compared with the sham (Table 2).
There were no differences in TBARS levels among the stenosis groups examined (Table 1). Serum AFR was higher in the stenosis groups examined, except for the C30 group compared with the sham, and was lower in the C30 stenosis group than in the V stenosis group. In contrast, serum vitamin C levels were lower in the V, M30, P2, P30, and B0.2 stenosis groups compared with the sham and were higher in the C30 stenosis group compared with the V stenosis group.
In the present study, carvedilol at relatively high doses attenuated the increase in LVEDD and LVESD and, at the highest dose, attenuated the decrease in LVEF in rats with coronary stenosis, whereas it did not attenuate them at any dose in rats with permanent coronary occlusion. In contrast, propranolol and metoprolol at doses showing similar effects as carvedilol on lowering HR and SBP in the resting state did not decrease LVESD and did not increase LVEF in rats with coronary stenosis, but they attenuated or tended to attenuate the increase in LVEDD. The SBP-lowering effect of bunazosin was similar to that of β blockade, but this blockade failed to attenuate LV dysfunction and remodeling. As far as we know, this is the first study to show that carvedilol preferentially attenuates LV remodeling and dysfunction induced by coronary stenosis rather than by coronary occlusion. These results suggest that the effect of β-blockers on myocardial ischemia and ischemia-related abnormalities might depend on the β-blockers used and the doses applied and that the presence of compromised ischemic but viable myocardium seems to be necessary for cardioprotection by a β blocker.
In the C10 and C30 stenosis groups, modulation of LV function and morphology is likely to be associated intimately with an increase in MBF and CFR. Because the coronary stenosis severity was similar among the 9 treatment groups examined and HR and LVSP, determinants of cardiac work, were not increased in both groups, the increases in MBF and CFR seem to be attributable to improvement of coronary microvascular dilating function. In addition, from the results with bunazosin, a significant contribution of a blockade to the effect on the C10 and C30 stenosis groups is unlikely. AFR was lower and vitamin C was higher in the C30 but not in the C2 stenosis group than in the V stenosis group. The level of AFR reflects the sum of radicals trapped by endogenous vitamin C, suggesting that the C30 treatment reduced oxygen radicals, thereby sparing the consumption of endogenous antioxidant. Thus, the antioxidant activity of carvedilol may have contributed in part to the improvement of coronary microcirculation as well as to the antiremodeling effect in our coronary stenosis model.
In the coronary occlusion model, compared with the V group, positive LV dP/dt in the C10 and C30 groups tended to increase (P<0.05), but LV remodeling was not attenuated by carvedilol. Carvedilol was reported to attenuate heart failure in which prior infarction was one of the causes.3,14⇓ The present study indicates that carvedilol, at doses effective for coronary stenosis–induced LV dysfunction, may fail to attenuate LV dysfunction in acute infarction without reperfusion.
The anti–heart failure effect of carvedilol in the coronary stenosis model was dose dependent. The C10 and C30 doses seem high when compared with human doses, suggesting that the HR- and SBP-lowering effects may be weaker in rats. Accordingly, we must be careful about the direct application of these data to clinical use.
The relative doses of β-blockers used were not determined by objective means, such as isoproterenol challenges. We compared the effects of 3 β-blockers at similar HR and SBP levels in the resting state. In humans,15 the greater anti–heart failure effect of carvedilol than of metoprolol likely corresponded to the greater β blocking action at peak exercise than in the resting state. Our data do not rule out the possibility that the potential differences in the β blocking action (not reflected at resting HR and SBP) between the agents may have contributed to the different antiremodeling effects. On the other hand, it is possible that in the β blocker groups treated with larger doses, a negative inotropic effect may have offset the improvement of LV function caused by the attenuation of ischemia. Additional studies using more precise determinations of comparable β blocking doses will need to be performed before concluding that carvedilol has qualitatively superior antiremodeling effects in the stenosis model.
Although bunazosin significantly decreased SBP in our long-term treatment, it did not increase plasma norepinephrine levels and HR. This may not be inconsistent with a previous observation16 in which bunazosin in a short experiment did not cause reactive tachycardia, decreased renal nerve activity, and increased preganglionic adrenal nerve activity, an index of central sympathoinhibitory activity. The mechanisms of these actions of bunazosin remain to be determined.
Our study has several limitations. First, β-blockers are used with incremental doses in humans,4 but we gave a constant dose to rats. This may have modified the drug’s effect. Second, we did not assess how daily dosing in our protocol affected the pharmaceutical effects of the agents with respect to differences in half lives, counteracting β receptor expression and postreceptor signaling.17 Third, in coronary occluded hearts, our results on the effect of metoprolol are not consistent with a previous study,10 probably partly because of different experimental design, doses, routes of drug delivery, or time of starting administration. Therefore, our results do not necessarily negate the beneficial effect of β blockade even in the infarcted heart. Fourth, the present study does not clarify how different degrees of coronary stenosis affect LV function and remodeling with and without treatment. Fifth, 117 animals were not tested in a blind fashion in the echocardiographic study. Sixth, MBF and CFR were not measured in awake animals, so the data may not reflect those obtained with intact sympathetic activity.
Compared with propranolol and metoprolol, carvedilol greatly attenuated LV remodeling and improved hemodynamics in rats with coronary stenosis. In contrast, at the same doses, it almost failed to do so in rats with coronary occlusion. Such effects of carvedilol may not be explained solely by the reduction of HR and SBP in the resting state and by α blocking action. Improvement of coronary circulation and reduction of free radicals might in part be involved in the antiremodeling effect of carvedilol. Our results show that carvedilol provides potent cardioprotection for compromised ischemic but viable myocardium rather than for infarcted myocardium
This study was supported by grant-in-aids for Scientific Research from the Japanese Ministry of Education, Science, and Culture (Nos. 09670733 and 11670696).
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