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1-adrenoceptor blockade promotes growth of arterioles and preserves coronary perfusion reserve in postinfarcted heart
Departments of 1Anatomy and Cell Biology and 2Internal Medicine and 3The Cardiovascular Center, University of Iowa Carver College of Medicine, and 4Department of Veterans Affairs Medical Center, Iowa City, Iowa
Submitted 10 October 2004 ; accepted in final form 26 January 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-blockade may also induce coronary growth in the post-MI heart. Transmural MI was induced in 12-mo-old male Sprague-Dawley rats by occlusion of the left anterior descending coronary artery. Bradycardia was induced by administration of the -adrenoceptor blocker atenolol (AT) via drinking water (30 mg/day). Three groups of rats were compared: 1) control/sham (C/SH), 2) MI, and 3) MI + AT. In the MI + AT rats, heart rate was consistently reduced by 25–28% compared with C/SH rats. At 4 wk after left anterior descending coronary ligation, infarct size was similar in MI and MI + AT rats (67.1 and 61.5%, respectively), whereas a greater ventricular hypertrophy occurred in bradycardic rats, as indicated by a higher ventricular weight-to-body weight ratio (3.4 ± 0.1 vs. 2.8 ± 0.1 mg/g in MI rats). Analysis of LV function revealed a smaller drop in ejection fraction in the MI + AT than in the MI group (
24 vs. 35%). Furthermore, in MI + AT rats, maximal coronary conductance and coronary perfusion reserve were significantly improved compared with the MI group. The better myocardial perfusion indexes in MI + AT rats were associated with a greater increase in arteriolar length density than in the MI group. Thus chronic reduction of heart rate induced with -selective blockade promotes growth of coronary arterioles and, thereby, facilitates regional myocardial perfusion in post-MI hearts.
myocardial infarction; angiogenesis; coronary circulation; resistance vessels; capillaries
Because scar tissue is not capable of contracting, LV function after MI is entirely dependent on the hypertrophied portion of the surviving LV myocardium. However, to accommodate an increased O2 demand in the surviving overloaded cardiac myocytes, an adaptation of the vascular bed is necessary. Two basic mechanisms allow the coronary vasculature to raise the myocardial oxygenation level as O2 demand increases: 1) augmentation of blood flow via dilation of the existing resistance vessels and 2) increase in the number of arterioles and capillaries via angiogenesis. Coronary dilation and, consequently, increased coronary flow have been found under baseline conditions in the hypertrophied LV myocardium of postinfarcted hearts (19, 20). This increased flow at rest, coupled with a decreased maximal coronary conductance, causes a reduction in coronary reserve (19, 20). Such a decrease in coronary reserve in the remaining LV myocardium after infarction suggests an inadequate compensatory angiogenesis (1). Therefore, the surviving LV myocardium of the postinfarcted heart remains vulnerable to the new episodes of ischemia.
To minimize the risk of cardiac damage during enhanced cardiac work in a hypertrophied post-MI heart, angiogenic therapy may be beneficial. The principal goal of such therapy is to protect the surviving cardiac myocytes from O2 deprivation by establishing a functional equilibrium between O2 demand and delivery. This can be accomplished by reduction of the myocyte O2 demand via negative chronotropic and inotropic actions or by augmentation of myocardial perfusion by means of coronary vascular growth. It appears that in clinical practice the effects described above might be easily achieved by a combined use of -blockers, a class of drugs designed to reduce heart rate and myocardial contractility via inhibition of sympathetic stimulation (26) and therapeutic angiogenesis (15, 38).
In recent years, new evidence has emerged indicating that chronic -blockade in patients suffering from heart failure, including that caused by MI, improves LV dysfunction and reduces the risk of sudden death (21, 24). It has been suggested that one of the key cardioprotective roles of -blockers under such conditions is the characteristic decrease in heart rate, leading to improved myocardial blood flow due to a lengthened diastolic perfusion time (4). This is consistent with an earlier animal study that revealed that heart rate reduction associated with -blockers was beneficial for regional redistribution of blood flow, an effect that could be prevented by atrial pacing (32). In addition, it was shown that only those -blockers with -selectivity caused a more favorable redistribution of flow in ischemic myocardium (8).
It has been known for at least two decades that bradycardia induced in animals by electrical pacing or pharmacological agents stimulates angiogenesis in normal (5, 7, 17, 18, 40–42), hypertrophied (41), and infarcted hearts (22). Thus it has been proposed that a chronic reduction of heart rate may be a way to stimulate angiogenesis in the heart after MI. Therefore, it seems reasonable to speculate that -blockers, especially with 1-selectivity, may facilitate angiogenesis of the coronary vasculature as a result of their negative chronotropic action on the heart. In concert with this supposition, it was previously demonstrated that prolonged -blockade with propranolol, a nonselective -blocker, could induce a significant increase in capillary density in the normal heart of young rabbits (35). The present study is the first to address the issue of the potential angiogenic effects of 1-selective blockade on the growth of coronary microvasculature in postinfarcted heart of middle-aged rats. We selected atenolol (AT), a -selective adrenoceptor blocker that is widely used in post-MI therapy, to test the hypothesis that 4 wk of reduction in heart rate by -selective blockade can stimulate coronary angiogenesis, especially the growth of arterioles, in the surviving LV myocardium of postinfarcted heart and, thereby, can minimize the decline in coronary perfusion reserve (CPR).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Surgical procedure.
Transmural MI was induced in animals by ligation of the left anterior descending coronary artery (LAD) as described in detail elsewhere (12, 22). Briefly, rats were anesthetized with a mixture of ketamine (100 mg/kg ip) and xylazine (10 mg/kg ip) and ventilated with a rodent ventilator (model 683, Harvard Apparatus). The thoracic cavity was opened, the heart was exteriorized, and the LAD was ligated near its origin with 5-0 Tycron suture. In sham-operated rats, the suture was pulled through the myocardium, but the LAD was not ligated. The heart was immediately interiorized, and the chest was closed in layers as the lungs were inflated. The rats were observed until they awakened, at which time they were returned to the Animal Care Unit. Standard postoperative care, including antibiotics, was provided. The mortality rate was 20%, with most deaths occurring within the first 24 h.
1-Blocker administration.
AT powder (Sigma, St. Louis, MO) was dissolved in drinking water (1 mg/ml). Each rat received 30 mg of AT per day based on the daily intake of drinking water, similar to that previously used in another laboratory to study the effect of AT on LV remodeling after MI in rats (33). Heart rate was monitored daily in conscious, unrestrained rats with the use of electrodes attached to an animal BioAmp differential amplifier and a PowerLab data-acquisition system (ADInstruments, Castle Hill, Australia). Only those rats that showed a reduction of heart rate >25% from the level detected in control rats (noted as bradycardic) were processed for further evaluation.
Experimental design. At 24 h after surgery, the rats were examined with transthoracic echocardiography to estimate the size of the akinetic (presumably ischemic) zone as well as to assess LV geometry and function. At 3 days after surgery, rats with a confirmed large ischemic zone (>30% of entire LV circumference; Table 1) were randomly assigned to one of two groups: 1) MI only (n = 10) and 2) MI treated with AT (MI + AT, n = 8). AT administration began 3 days after surgery, continued for 4 wk, and was terminated 24–48 h before the end of the experimental period, when echocardiographic (MI and MI + AT rats only) and myocardial perfusion analyses (all groups) were performed. Because data from sham-operated (SH, n = 6) and nonoperated control (C, n = 6) rats were similar, the two groups were combined (C/SH).
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After removal of the atria, the hearts were weighed and cut transversely into five parallel, 2-mm-thick slices from apex to base with a four-blade guillotine. In the MI hearts, ventricular slices with scar tissue were digitized, and images were used to estimate infarct size. One medial slice from each heart was taken for immunohistochemistry and morphometry. Anti-smooth muscle (SM) 
-actin and anti-laminin immunostaining, as well as labeling with BS-I lectin, was utilized to determine arteriolar length and capillary numerical densities as well as nucleated cardiac myocyte cross-sectional areas (CSAs). In all remaining slices, the right ventricular free wall was separated from the ring comprising the LV free wall and intraventricular septum. The myocardium from the septum and LV free wall was excised, weighed, and placed in a counting vial. In the MI hearts, myocardium from the LV free wall was excised under a dissecting microscope 1.5–2 mm from the edge of the scar tissue. Tissue samples, along with the reference blood samples collected during myocardial blood flow measurements, were sent to the BioPhysics Assay Laboratory (BioPAL, Worchester, MA) for microsphere counting.
Echocardiographic analysis. Details of the echocardiographic procedure have been described elsewhere (22, 45). Briefly, rats were lightly anesthetized with ketamine (50 mg/kg ip) and positioned supine. The chest was shaved, and acoustic coupling gel was applied. Two-dimensional short- and long-axis images of the LV were obtained with a clinical echocardiograph (Sequoia model 256, Acuson, Mountain View, CA) equipped with an 8.0-MHz sector-array transducer adapted for small rodents. Pulse-wave Doppler interrogation of mitral inflow was used to calculate heart rate. Planimetric measurements were used to estimate the size of the ischemic zone that demonstrated systolic akinesis, and this region was expressed as a percentage of the entire LV. LV mass and volume were calculated using the area-length method. From these measurements, LV parameters, including end-diastolic volume (EDV), volume-to-mass ratio, stroke volume, cardiac output, and ejection fraction, were calculated.
Hemodynamics and regional myocardial perfusion. Rats were anesthetized with a mixture of ketamine (100 mg/kg ip) and xylazine (10 mg/kg ip) and ventilated with a Harvard rodent ventilator. A polyethylene catheter (PE-50) attached to a blood pressure transducer was inserted into the right femoral artery, and systemic hemodynamic parameters, including heart rate and systolic, diastolic, and mean arterial pressure, were recorded using a bridge amplifier and a PowerLab data-acquisition system equipped with Chart version 4 software (ADInstruments). Then, polyethylene catheters were inserted into the LV via the right common carotid artery, the left femoral artery, and the left jugular vein, and LV systolic blood pressure and LV end-diastolic pressure were recorded.
Myocardial perfusion was determined using neutron-activated stable isotope-labeled microspheres (B-series, BioPAL). For each flow determination, 1 x 106 microspheres were infused into the LV. The catheter was then flushed with 0.4 ml of heparinized saline (50 U/ml), and an arterial reference blood sample was withdrawn from the left femoral artery by a programmable syringe pump (model NE-1000, New Era Pump Systems, Farmingdale, NY) at a constant rate (0.2 ml/min). The microspheres labeled with three different isotopes were used to determine myocardial perfusion once under the baseline condition and twice after maximal coronary dilation. Maximal coronary dilation was induced by infusion of dipyridamole (6 mg·kg–1·min–1 iv for 8 min) via the left jugular vein. The highest attained value was utilized as maximal flow. The mean arterial pressure was recorded via the right femoral artery during each microsphere infusion. Tissue samples from the heart, along with the reference blood samples, were prepared for microsphere counting according to the manufacturer's protocol (BioPAL).
From the measurements provided by BioPAL, myocardial blood flow was computed according to a standard formula: (Cm/Cr)· 
r·100 g(ml·min–1·100 g–1), where Cm is number of microspheres per gram of myocardium, Cr is number of microspheres in the reference blood sample, and r is withdrawal rate of the reference blood sample (ml/min). On the basis of these data, regional coronary resistance was calculated by dividing systemic mean arterial pressure by regional myocardial blood flow. Finally, myocardial perfusion at baseline and after maximal coronary dilation was expressed as coronary conductance, which is the reverse of coronary resistance (ml·mmHg–1·min–1·100 g–1). CPR was calculated as maximal coronary conductance divided by baseline coronary conductance.
Estimation of infarct size. The procedure for measurements of infarct size was described previously (22, 45). Briefly, digital images from each ventricular slice of the hearts were analyzed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). From each slice, the lengths of LV free wall (obtained at a midtransmural point) and its portion occupied by scar tissue were measured, and the size of the scarred portion was estimated per slice as the ratio of scarred fraction to length of the LV free wall. Subsequently, the mean of such ratios was calculated for each heart. Infarct size was expressed as a percentage of the LV free wall.
Immunohistochemistry and immunofluorescence microscopy.
One ventricular slice from each heart was fixed in 4% paraformaldehyde in 0.1 M PBS for 24 h at 4°C. The samples were washed overnight in PBS, cryoprotected in a graded sucrose series at 4°C, and then frozen in Tris-buffered saline (TBS)-tissue freezing medium (Triangle Biomedical Sciences, Durham, NC) by isopentane precooled with dry ice. Transverse 8.0-µm serial sections were cut with a cryostat and mounted on glass slides. The sections were incubated with 0.5% bovine serum albumin and labeled with primary antibodies. The primary antibodies used in this study were 1) monoclonal anti-smooth muscle (SM) -actin, clone 1A4 (1:600; Sigma) and 2) polyclonal antilaminin (1:30; Sigma). After they were labeled, the sections were washed in PBS, incubated with 10% normal goat serum, and stained with secondary antibodies and/or BS-I lectin. The secondary antibodies used for visualization of primary antibodies were FITC- or Cy3-conjugated goat anti-mouse or goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). BS-I lectin was conjugated with tetramethylrhodamine isothiocyanate (10 µg/ml; Sigma) or fluorescein (20 µg/ml; Vector Laboratories, Burlingame, CA). For controls, primary antibodies were omitted or substituted with 10% normal goat serum. The sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI, 10 mg/ml) and mounted using the ProLong antifade kit (Molecular Probes, Eugene, OR). Fluorescence images were captured on a computer using a microscope (Eclipse E-600, Nikon) equipped with a digital camera (model DXM 1200, Nikon). Final images were prepared using Adobe Photoshop software (Adobe Systems, San Jose, CA).
Morphometric and stereological analyses.
The sections cut from the ventricular slices, taken from at least five hearts per group, were labeled with anti-SM -actin antibody (a marker of arteriolar SM cells; Fig. 1A), BS-I lectin (a marker of capillaries; Fig. 1B), and/or anti-laminin antibody counterstained with DAPI (used to outline nucleated cardiac myocyte profiles; Fig. 1C) and then analyzed using Image-Pro Plus software (Media Cybernetics).
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-actin-positive outline and an external diameter between 6 and 50 µm), the randomly selected regions of myocardium in the LV free wall (excluding the area 2 mm from the edge of scar tissue in post-MI hearts) and septum were digitized using low-power magnification (x2 objective), and their areas were measured. The areas used in this analysis were 8–10.5 mm2 per region in each heart. With the use of systematic scanning of these regions under high-power magnification (x40 objective), every vessel profile (without respect to its sectioning plan) detected in the LV free wall and septum was captured and their morphometric parameters were measured. On the basis of these measurements, the length density (LV) was calculated as described previously (2): LV = (
a/b)/N·N/A (µm/µm3), where a and b represent long and short axes, respectively, of individual arterioles, N is total number of arteriolar profiles, and A is the total area in which arterioles were measured.
To calculate capillary numerical density (CD), the 7–10 randomly selected optical fields from epimyocardium and endomyocardium of the LV free wall and the LV endomyocardial region of the septum were digitized using high-power magnification. Only the areas that displayed cross-sectioned capillary and myocyte profiles were selected for evaluation. The areas used in this analysis were 0.35–0.55 mm2 per region in each heart. CD was calculated as N/A (counts/µm2), where N is number of cross-sectioned BS-I lectin-positive capillary profiles and A is total area of the myocardium in which capillaries were counted.
To estimate cardiac myocyte CSA, the same regions used for capillary counting were digitized on adjacent sections stained with an anti-laminin antibody and DAPI. Myocyte CSAs were determined only in those cells that showed a relatively circular shape and presence of a nucleus. On average, 250–400 nucleated myocyte profiles per region were analyzed in each heart.
Statistical analysis. Values are means ± SE. One-way ANOVA followed by Bonferroni's post test was used for multigroup comparison (C/SH, MI, and MI + AT). Paired t-test was used to compare intergroup differences, and an unpaired t-test was used to assess the difference between two groups. P < 0.05 was selected to denote significant differences.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-selective adrenoceptor blocker by 25–28% compared with C/SH rats and by 31–28% compared with MI rats. Thus our treatment protocol with AT produced a persistent bradycardia in the post-MI rats.
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1-blocker administration and 24 h after termination of AT treatment. At the end of the 4-wk experimental period, EDV was elevated by 58% in the MI group and 72% in the MI + AT group, whereas LV mass was reduced in the MI group by 17% and increased in the MI + AT group by 11% (Fig. 3). Despite these differences in LV EDV and mass, the two groups showed comparable increases in EDV-to-mass ratio, indicating quite proportional degrees of LV remodeling in both groups of rats (Table 1 and Fig. 3). Although AT-treated rats revealed a significant increase in stroke volume 4 wk after MI while it remained almost the same in the untreated MI rats, it appeared that such an increase could be in part attributed to the lower heart rate in the former. It remains unclear why heart rate in the MI + AT group was still reduced 24 h after 1-blocker administration was ceased, but it was obviously beneficial for cardiac output, which increased by 17% compared with the value 24 h after MI (Table 1). Most importantly, a smaller drop in ejection fraction was recorded in MI + AT rats (24 vs. 35% in MI rats; Fig. 3). These data suggest that chronic 1-blockade and subsequent bradycardia had no significant impact on LV remodeling in post-MI hearts but was able to minimize the decline in LV contractile performance.
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12% greater postmortem ventricular mass in the former (Table 2). To determine whether the increase in LV mass correlates with the growth of cardiac myocytes, CSAs of the nucleated myocyte profiles were measured on histological sections immunostained against laminin and counterstained with DAPI (Fig. 1C). CSAs of myocytes from the epimyocardial and endomyocardial regions of the LV free wall were significantly greater in MI and MI + AT than in C/SH rats but were similar for the three groups in the septum (see Table 5). These observations also document a relatively similar degree of expansion in the CSAs of myocytes remaining in the LV free wall of untreated and AT-treated post-MI rats. Thus an increase of the ventricular mass in bradycardic animals is not only due to the myocyte growth in diameter but also is likely a result of myocyte elongation, which is indicative of the eccentric hypertrophy of the heart.
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1-blocker administration in the MI + AT group was terminated 48 h before these measurements. The evaluation of systemic and LV hemodynamics conducted on deeply anesthetized and acutely instrumented rats before blood flow measurements revealed no significant differences in MI and MI + AT rats among analyzed parameters, including heart rate and mean arterial pressure, that could markedly affect the level of coronary blood flow at baseline (Table 3). Nevertheless, compared with C/SH rats, both post-MI groups of rats showed a marked decline in systolic blood pressure as well as in maximal systolic LV pressure. In addition, LV end-diastolic pressure was higher in MI + AT and MI than in C/SH rats, indicative of developing heart failure (Table 3). These findings obviously suggest that, at the beginning of blood flow analyses, neither of the two post-MI groups had hemodynamic parameters that differed to such an extent that significant miscalculations in the level of myocardial perfusion could result.
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Coronary conductance at baseline was elevated in the septum and LV free wall of MI rats, whereas it was similar in MI + AT and C/SH rats (Fig. 4). Maximal coronary conductance was significantly lower in MI than in C/SH rats, especially in the LV free wall, where coronary conductance was 35% lower in MI than in C/SH animals (Fig. 4). In contrast, maximal coronary conductance in the septum and LV free wall of the MI + AT rats was similar to that in C/SH rats and, hence, was also markedly higher than in untreated post-MI rats. To evaluate the changes in CPR, the differences between each baseline and maximal coronary conductance were calculated. CPR was markedly impaired in the septum as well as the LV free wall of MI rats. In contrast, in the MI + AT group, CPR remained similar to that in C/SH animals (Fig. 5). It was obvious that the significantly lower CPR in the untreated post-MI rats was in part due to the higher basal coronary blood flow (Table 4, Fig. 4). These findings clearly demonstrate a markedly improved myocardial perfusion and CPR in the surviving LV myocardium in post-MI rats that have undergone chronic reduction of heart rate induced by AT compared with untreated post-MI animals.
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Coronary arterioles were identified in histological sections by immunolabeling with an antibody against SM -actin (Fig. 1A). Although arteriolar length density was significantly higher in the septum in both post-MI groups than in C/SH rats, in the LV free wall only AT-treated animals demonstrated a marked increase (Fig. 6). Most importantly, the increase in arteriolar length density was significantly greater in the septum of MI + AT than MI rats. The evaluation of arteriolar length densities in relation to vessel diameters revealed that most of the expansion in MI + AT rats was due mainly to an increase in arterioles with an external diameter less than 35 µm (Fig. 7). These findings indicate that, in the surviving LV myocardium of the bradycardic animals, the coronary arteriolar bed enlarged to a much greater extent than in untreated post-MI rats.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-blocker AT can promote growth of coronary vessels in middle-aged (1-yr-old) post-MI rats. Our data support this hypothesis by showing that, after 4 wk of chronic bradycardia in post-MI rats, coronary conductance and coronary reserve are markedly improved. Such improvement in regional myocardial perfusion is associated with a significant growth of the coronary arteriolar bed in the surviving myocardium.
Bradycardia in postinfarcted heart promotes growth of resistance vessels.
In agreement with our latest study on alinidine-induced bradycardia in post-MI rats (22), we have documented significant arteriolar growth after chronic reduction of heart rate induced by 1-selective blockade with AT. Although we recognize that the mechanisms regulating growth and remodeling of the coronary vasculature are complex (29, 37), one such important mechanism is stretch. Because the reduced heart rate prolongs diastole, it enhances distension of the ventricular wall and, therefore, the time interval in which myocardial structures, including transmural vasculature, are under a relatively enhanced stretch. We propose that, in post-MI hearts, bradycardia aggravates the degree of stretch, because it operates in concert with the remodeling-associated dilation of the LV chamber. Consistent with this hypothesis, we previously showed that in vitro stretch of cardiac myocytes and endothelial cells upregulates vascular endothelial growth factor (44) and its Flk-1 receptor (43). These findings were consistent with our previous in vivo investigation that documented upregulation of vascular endothelial growth factor and basic fibroblast growth factor in the post-MI hearts treated with the bradycardic agent alinidine (22). Accordingly, we can assume that the augmented stretch-related mechanical forces in bradycardic hearts might trigger the more robust increase in stretch-associated release of angiogenic growth factors and, hence, might stimulate a greater coronary arteriolar growth under the condition of reduced heart rate.
Because a marked compensatory hypertrophy of surviving cardiac myocytes in the LV develops in response to the large intramural infarct (1, 30) as a result of myocyte overload, a growth of resistance vessels (i.e., arterioles) must occur to provide an adequate tissue perfusion. This supposition was validated by the results of our present and previous (22) studies, which demonstrated a marked arteriolar growth in post-MI hearts that underwent an eccentric hypertrophy. Moreover, these data are in concert with our previous work, which documented the induction of arteriolar development in volume-overloaded hearts, which also showed LV chamber remodeling typical of eccentric hypertrophy (10). These findings suggest that a greater development of the arteriolar bed in bradycardic post-MI rats might, to some extent, be triggered by more advanced growth in the ventricular mass detected in MI + AT animals.
In addition, we can propose that because of the evidence of some, more limited, arteriolar growth in the untreated post-MI rats, almost the same stimuli that triggered arteriolar growth in bradycardic heart, including mechanical stretch and myocardial hypertrophy, might also be effective in all post-MI hearts, only on more limited scale.
Most importantly, our present finding that the largest portion of the increase in arteriolar length density detected in post-MI hearts is due to formation of the smallest "terminal" arterioles (Fig. 7) is consistent with the concept that new arterioles most likely emerge from preexisting capillaries via capillary arteriolarization (28, 34, 39). Similar observations were previously reported in rats with MI that underwent pharmacological therapy (22, 45) or those subjected to volume overload (10). Furthermore, exercise training in swine also stimulated an increase in the small arterioles in the heart (39). On the basis of these observations, we suggest that greater expansion of the coronary arteriolar bed detected in MI + AT rats was in part due to more advanced arteriolar transformation of preexisting capillaries.
Is the cause of bradycardia and/or the type of experimental model important for induction of capillary growth? In the last few decades, several lines of evidence demonstrated that bradycardia induced by electrical pacing (5, 17, 18, 40, 41) or by the negative chronotropic agent alinidine (7, 42) stimulates angiogenesis in the adult myocardium of normal or volume-overloaded hearts. The results from these studies demonstrated that the long-term reduction in heart rate by 45% in rabbits (17, 18, 40, 41), 34% in pigs (5), and 28% in rats (7) was associated with an increased CD or a higher capillary-to-fiber ratio than in control animals. More recently, data from our laboratory also revealed that 3 wk of alinidine-induced bradycardia resulted in higher capillary length density in the surviving LV myocardium of young post-MI rats (22).
In this study, AT consistently reduced heart rate in the 12-mo-old post-MI rats by 25–28% compared with C/SH rats; thus the degree of bradycardia was similar to that achieved previously in younger (4-mo-old) post-MI rats by alinidine (7, 22). Surprisingly, in contrast to these studies, the bradycardic hearts of the MI + AT rats did not reveal any increase in capillary numerical density, even in the septum, where myocyte CSA enlargement was not detected. These contrasting data raised the following question: Might the method of inducing bradycardia to some extent be accountable for the difference between the present findings and data reported previously? One possible explanation is that, in contrast to alinidine-induced bradycardia, treatment with the 1-blocker AT not only lowers heart rate, it also markedly reduces myocardial contractility (negative inotropy) as well as afterload (systemic hypotension) and, therefore, decreases cardiac myocyte O2 consumption (11). Such a decrease in myocyte O2 consumption might inhibit capillary growth, because tissue PO2 is a strong determinant for induction and/or maintenance of capillary growth (9, 31). A second possible explanation is that because AT administration reduces myocardial blood flow (3, 13), it follows that shear stress and wall tension, the two vital mechanical stimuli needed for the activation of endothelial cells and angiogenesis (6), would also be diminished within a capillary network during an entire period of -blocker treatment. One additional explanation for the lack of capillary growth may be the use of 1-yr-old rats. A previous study showed a marked impact of advanced animal age on ability of capillaries to expand, in which 12 wk of exercise training stimulated capillary growth in 17-wk-old rats, whereas in two older age groups (31 and 94 wk) it did not (36). Finally, as noted above, capillary-to-arteriolar transformation may, to some extent, be responsible for the drop in capillary density in both post-MI groups of rats.
It is also important to note that in our previous studies (22, 42), where we showed a marked capillary growth in bradycardic hearts, we used 1-µm-thick plastic sections and measured the axial ratio of the capillary lumen. Such a method permitted computation of the capillary length density as an alternative to the less precise numerical density reported in this study. Because the degree of tissue shrinkage was much less in cryosections and the numerical density did not use the capillary lumen axial ratio as a multiplier, it should not be a surprise that capillary density values demonstrated in our present study were significantly different from those reported previously. Regrettably, the methodological limitation in this study has not permitted a more comprehensive analysis of adaptations that might occur in the capillary bed of post-MI rats with AT-induced bradycardia.
Coronary conductance and vasodilator reserve are improved in post-MI hearts after chronic bradycardia.
Previous studies of myocardial perfusion in rat hearts with large transmural MI have shown significantly reduced maximal coronary conductance and vasodilator reserve, especially in the region where myocyte hypertrophy was evident (19, 20). Consistent with these data, our present study has also documented markedly reduced levels of maximal coronary conductance and coronary perfusion (or vasodilator) reserve (CPR) in untreated post-MI rats compared with C/SH animals. In contrast, post-MI rats that had undergone 4 wk of continuous bradycardia caused by chronic 1-selective blockade with AT revealed no decline in maximal coronary conductance and CPR compared with C/SH animals after withdrawal of the drug. Most importantly, both perfusion indexes were significantly higher in MI + AT than in MI rats. This positive effect of chronic reduction in heart rate on myocardial perfusion was consistent with that documented previously in our laboratory in a model of alinidine-induced bradycardia in young post-MI rats (22).
Interestingly, our present findings as well as those of others (19, 20) indicate that a noticeable drop in CPR detected in untreated post-MI rats was, to a certain extent, due to a combined effect of higher coronary blood flow at baseline and lower coronary blood flow after maximal dilation. In view of the fact that, during blood flow measurements in our experiments, hemodynamic parameters, such as heart rate and mean arterial pressure (or perfusion pressure), were comparable among the three groups of rats at baseline as well as after maximal vasodilation, the alterations in coronary conductance must be attributed to other factors, such as O2/metabolic demand or the extent of arteriolar bed expansion.
The increase in basal myocardial perfusion in rats with large MI is not surprising, because the surviving myocardium experiences an increased afterload (or wall stress), which increases O2 demand in overloaded LV cardiac myocytes. In contrast, MI + AT rats demonstrated normal perfusion values at rest. The mechanism associated with the lower O2 demand in myocytes of bradycardic animals after withdrawal of AT is not evident at this time.
Although noticeable growth of coronary arterioles was documented in MI and MI + AT rats, the magnitude of arteriolar bed enlargement was less in MI than in MI + AT rats. Thus it is not unexpected that maximal coronary conductance was less in the MI than in the MI + AT rats. Consistent with this result is the fact that coronary minimal resistance was 43 and 27% higher in the LV free wall and septum, respectively, in the MI than in the MI + AT rats (Table 4). Most importantly, because maximal coronary conductance was similar in MI + AT and C/SH rats, it can be proposed that the aggregate arteriolar CSA per volume of LV myocardium is similar in these two groups. These results and our previous data (22) indicate that arteriolar development in bradycardic animals was able to fully prevent a deficit in myocardial perfusion and, therefore, to avert an imminent decline in CPR detected in untreated post-MI rats. Nevertheless, the fact that arteriolar growth in post-MI hearts always exceeded anticipated increases in maximal coronary conductance suggests some functional inadequacy of the newly formed vessels.
Could bradycardia in post-MI hearts minimize LV dysfunction by improving myocardial perfusion?
In recent years, it has become more obvious that -adrenoceptor blockade can reduce the symptoms of systolic dysfunction and diminish the risk of sudden death in patients suffering from cardiovascular events, including MI and heart failure (4, 21, 24). Several different mechanisms have been proposed to elucidate the beneficial effect of -blockers under such conditions (4, 23). Importantly, the most vital role of the -blockers (including AT) in restoration of LV function was ascribed to reduction in heart rate (14, 25). For instance, studies on the ischemic myocardium of conscious dogs revealed that AT-induced reduction in heart rate was associated with the concurrent improvement in coronary blood flow and contractile performance. Most importantly, when electrical pacing was used concurrently with AT treatment to increase heart rate to baseline levels, the improvement in blood flow as well as in cardiac function was eliminated (14, 32). In our present study, as well as in a previous study (22), we also found a dual beneficial role of chronic bradycardia 1) on LV contractile performance, as indicated by an attenuation of the drop in ejection fraction, and 2) on coronary perfusion indexes, as indicated by normalized CPR. In conclusion, we suggest that the bradycardia-related development of the coronary arteriolar bed and, consequently, a greater myocardial perfusion may play an essential role in the amelioration of LV dysfunction in the postinfarcted heart.
Conclusion.
Our data provide clear evidence that chronic bradycardia induced with -selective blockade promotes a greater arteriolar growth in the surviving LV myocardium of the postinfarcted heart that is adequate to restore the compromised myocardial perfusion and coronary reserve.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.001 vs. C/SH and MI rats.
