Materials and Methods

Animals—All experiments were conducted according to the Azabu University Animal Experiment Guidelines. Twenty-four Beagles from the Chugai Research Institute for Medical Science, Japan, were used for this study. Each dog was considered healthy on the basis of results of a physical examination, CBC, serum biochemical analyses, ECG, thoracic radiography, and echocardiography. The dogs were randomly assigned to 1 of 4 groups (6 dogs/group); 3 groups underwent PAB, and 1 group underwent a sham operation (thoracotomy only). Dogs that underwent PAB were further assigned to 1 of 3 treatment groups, in which they were administered candesartan (PABC group), enalapril (PABE group), or no treatment (PABNT group). At 60 days after surgery, all dogs were euthanatized via injection of an overdose of pentobarbital, IV, after which their hearts were immediately isolated for neurohormonal and histologic examinations.

Induction of RVH—Pulmonary arterial banding was performed in our laboratory as previously described.² Left fourth intercostal thoracotomy was performed to isolate the main pulmonary artery. A polyester tape was passed around the pulmonary artery just distal to the pulmonary valve. The outer circumference of the pulmonary artery was reduced to 60% of its original size in dogs of the PABNT, PABE, and PABC groups. A thoracotomy only was performed in dogs of the sham-operated group; the main pulmonary artery remained intact without any surgical manipulation in this group. On completion of surgery and as needed thereafter, bupivacaine^a (variable dose) was injected around the incision site and ketoprofen^b (2 mg/kg, SC, q 24 h) was administered for pain control in all dogs.

Drug administration—Dogs in the PABNT and sham-operated groups did not receive any drug treatment. Dogs in the PABE and PABC groups were treated with enalapril maleate^c (0.5 mg/kg, PO, q 24 h) and candesartan cilexetil^d (1.0 mg/kg, PO, q 24 h), respectively. Previous studies in healthy dogs⁵ and dogs with cardiac disease^6–8 revealed that this dosage of enalapril was clinically and pharmacologically efficacious. The dosage of candesartan was determined from data obtained in an unreported preliminary study performed by our group. Drugs were administered orally at 9 AM before feeding. Drug administration was started 1 day before PAB was performed and continued for 60 days after surgery. Duration of the experiment (ie, until 60 days after surgery) was determined on the basis of our previous study² findings, which indicated that RVH resulting from PAB reached a stable state in a 60-day period.

Echocardiography—Sixty days after PAB or a sham operation, an echocardiographic examination of each dog was performed; dogs were conscious during the examinations. The RV wall thickness just distal to the tricuspid valve was measured at the end of diastole in a short-axis view obtained from a right parasternal window. The wall thickness at 60 days after surgery was compared with the value recorded before study commencement; the final value was recorded as a percentage of the prestudy value.

Plasma renin-angiotensin system assessments—In all dogs, plasma renin activity, plasma ACE activity, and plasma AI and AII concentrations were measured before (baseline values) PAB or sham operation and 60 days later, before the euthanasia, as previously described.²

Histologic examinations—After euthanasia of each dog, the heart was removed for further assessments. An RV tissue sample was collected for measurements of ACE and chymase-like activities, immediately frozen by use of liquid nitrogen, and stored in a deep freezer at −80°C. Another RV tissue sample was collected for histologic examination, immersed in neutral-buffered 10% formalin, paraffin-embedded, and cut by use of a standard method; sections were stained with Masson trichrome stain. In RV tissue samples, collagenous fiber area and cardiomyocyte diameter were quantified to assess RV remodeling. The histologic images were uploaded into a computer. The collagenous fiber area was stained blue, but this coloration was converted to bright green by use of computer software^e; the green coloration was identified, and the collagenous fiber area was calculated by use of the computer. Interstitial and perivascular areas of collagenous fiber in both endocardium and epicardium were assessed because of variations in their distribution. Because the fibrous area surrounding vessels correlated with the cross-sectional area of corresponding vessels in our preliminary study, collagenous tissue areas were corrected by dividing cross-sectional area of vessels. Values of collagenous area were expressed as a percentage of the examined microscopic area. The methods to measure collagenous fiber areas and cardiomyocyte diameters in our laboratory were the same as those previously described.²

Measurements of tissue ACE and chymase-like activities—Tissue ACE and chymase-like activities in the free wall of the RV were assessed 60 days after PAB or sham operation by use of high-performance liquid chromatography, as previously described.²

Statistical analysis—All values were reported as mean ± SE. The differences among groups in all variables except for plasma renin activity, plasma ACE activity, and plasma AI and AII concentrations were analyzed via an ANOVA for repeated measures and a Fisher protected least significant difference test for the significance of differences among groups. A Student paired t test was used to compare values of plasma ACE activity, plasma renin activity, and AI and AII concentrations determined on the day before surgery and 60 days after surgery. For all analyses, a value of P < 0.05 was considered significant.

Results

RV wall thickness—Sixty days after surgery, RV wall thickness was measured echocardiographically in all dogs and recorded as a percentage of the prestudy value. The RV wall thickness in the PABNT (mean ± SE, 172.7 ± 10.3%), PABE (180 ± 4.6%), and PABC (135.0 ± 4.3%) groups was significantly greater than the wall thickness in the sham-operated group (105.5 ± 1.6%). Among the 3 PAB groups, RV wall thickness in the PABNT and PABE groups was greater than the thickness in the PABC group. Wall thickness in the PABNT and PABE groups did not differ significantly.

Cardiomyocyte diameter—Mean cardiomyocyte diameter was significantly greater in the PABNT group (35.4 ± 0.9 μm) and the PABE group (33.2 ± 0.6 μm), compared with the sham-operated group (28.5 ± 0.5 μm). This apparent hypertrophy was suppressed in the PABC group (mean diameter, 30.0 ± 0.5 μm); this value was significantly less than findings in the PABNT and PABE groups but did not differ significantly from the value in the sham-operated group.

Collagenous fiber area—Sections of RV tissue were examined via light microscopy to assess collagenous fiber areas (Figure 1). Collagenous fiber areas in both the interstitial and perivascular areas of the epicardium and endocardium were significantly increased in the PABNT group, compared with findings in the sham-operated group (Figure 2). This fibrosis was suppressed in the PABC group but not in the PABE group. Data obtained from dogs in the PABNT and PABE groups did not differ significantly.

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Representative photomicrographs illustrating interstitial collagenous fiber areas in sections of the RV epicardium in 4 dogs that underwent PAB and received treatment with candesartan cilexetil (1 mg/kg, PO, q 24 h [PABC group]), enalapril maleate (0.5 mg/kg, PO, q 24 h [PABE group]), or no treatment (PABNT group) or underwent thoracotomy only and received no treatment (sham-operated group). Treatments were commenced the day before surgery, and tissue samples were collected 60 days after surgery. Collagenous fiber areas were stained blue by use of Masson trichrome stain, but the color was converted to bright green by use of image processing software. A—Section from a dog in the sham-operated group. B—Section from a dog in the PABNT group. C—Section from a dog in the PABE group. D—Section from a dog in the PABC group. Notice that the collagenous fiber area in sections from the PABNT and PABE group dogs was typically greater than areas in sections from the sham-operated and PABC group dogs. Bar = 100 μm.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1574

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Mean ± SE interstitial and perivascular collagenous fiber areas (%) in endocardium (A and C, respectively) and epicardium (B and D, respectively) in dogs in the sham-operated, PABNT, PABE, and PABC groups (6 dogs/group). *Values in the 2 groups indicated by a bracket were significantly (P < 0.05) different. SO = Sham operated.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1574

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Circulating renin-angiotensin system variables—At 60 days after surgery, mean plasma ACE activity in the PABE group was significantly reduced, compared with the baseline value (Table 1). Plasma renin activity and AI concentration in the PABE group were significantly increased, compared with their respective baseline values. In the PABC group, plasma renin activity and AI and AII concentrations were significantly greater than the values at baseline.

ACE and chymase-like activities in RV free wall—At 60 days after surgery, mean ACE activity in RV tissue did not differ significantly among groups (sham-operated group, 1.85 ± 0.26 mU/g of tissue; PABNT group, 2.69 ± 0.22 mU/g of tissue; PABE group, 1.97 ± 0.32 mU/g of tissue; and PABC group, 2.80 ± 0.67 mU/g of tissue). Mean chymase-like activity in the PABE group (54.87 ± 10.11 nmol/min/g) was significantly greater than the value in the sham-operated (20.71 ± 2.14 nmol/min/g) and PABNT (18.70 ± 2.79 nmol/min/g) groups. Chymase-like activity in the PABC group (32.59 ± 9.63 nmol/min/g) was not significantly different from the value in the PABE group. Values in the PABNT, PABC, and sham-operated groups did not differ significantly.

Discussion

The tissue renin-angiotensin-aldosterone system is one of the major components in the regulation of cardiac remodeling and ventricular function. Tissue AII increases the amount of collagenous fibers and induces remodeling of cardiac tissues through activation of transforming growth factor-β1, mitogen-activated protein kinase, and phospholipase.

Angiotensin II is formed from AI in a reaction that is catalyzed by 2 major enzymes: ACE and chymase. The expressions of these 2 enzymes differ during the development of heart disease.^2,9 In addition, there is an important species difference in the expression of AIIforming enzymes.¹⁰ Therefore, a predominant AII-forming pathway should be considered to effectively prevent cardiac tissue remodeling and hypertrophy. Our previous study² in dogs with experimentally induced RVH revealed that ACE was activated in the acute stage of RVH development and that both ACE and chymase activities increased in the late stage. Results of the present study indicated that an AII type 1 receptor blocker but not ACE inhibitor suppressed development of myocardial hypertrophy and cardiac fibrosis in dogs with experimentally induced RVH. However, in an investigation by Weinberg et al,¹¹ ACE inhibitor suppressed myocardial hypertrophy development in rats with systemic pressure overload. The discrepancy in results of that investigation and findings of the present study could be explained by a species difference in the expression of chymase or perhaps by a different stage of the disease.

Chymase activates matrix metalloproteinase under conditions of increased myocardial stress, thereby providing another pathway through which chymase may induce cardiac tissue remodeling, in addition to the production of AII.¹² However, in the present study, chymase-like activity was not increased in the PABNT group dogs despite the development of RVH, and administration of candesartan successfully prevented RVH development in dogs that underwent PAB. Mechanical stress can not only activate extracellular signal-regulated kinases and stimulate increases in phosphoinositide production but also induce cardiac hypertrophy without the involvement of AII. It has been reported¹³ that left ventricular pressure overload induces marked cardiac hypertrophy in angiotensinogen gene–deficient mice that do not produce AII. In those mice with left ventricular pressure overload, development of cardiac hypertrophy was significantly attenuated by administration of candesartan. Moreover, candesartan inhibited activity of stretch-induced extracellular signal-regulated kinases, which suggests that it worked as an inverse agonist of the AII type 1 receptor.¹³ This might possibly be the mechanism by which candesartan attenuated the development of RVH without increasing chymase-like activity in the dogs of the present study.

In the PABE group dogs, chymase-like activity was increased at 60 days after surgery, compared with the values in the sham-operated and PABNT group dogs. Such activity could be caused by a negative feedback system triggered by decreased plasma AII concentration. This could be one of the mechanisms underlying myocardial fibrosis and cardiac tissue hypertrophy in the PABE group, although such a hypothesis is uncertain because tissue AII concentration was not measured at the same time. However, it is still possible that increased chymase activity was involved in the development of myocardial fibrosis because increases in chymase activity induce signal transduction, which results in the development of cardiac remodeling, even without an increase in the tissue AII concentration.¹² It has to be kept in mind that treatment with an ACE inhibitor would enhance cardiac tissue remodeling if it resulted in increased chymase activity.

It is known that the expression of AII type 1 receptor mRNA in the myocardium increases in rats with systemic pressure overload.¹⁴ Dell'Italia et al¹⁵ reported that the expression of AII type 1 receptor mRNA was upregulated when an ACE inhibitor was administered to dogs with volume overload. These data suggest that the AII type 1 receptor density could have been increased in the dogs with experimentally induced RVH in the present study and that treatment with enalapril further increased the receptor density, which would be another possible reason why enalapril failed to suppress cardiac tissue remodeling. This hypothesis seemed to be proved by the data from the dogs in the PABC group, which had less myocardial remodeling than the dogs in the PABE group.

Although the importance of AII type 1 receptor–mediated effects on myocardial remodeling is well known, the roles of AII type 2 receptors, bradykinin, endothelins, growth hormone, insulin-like growth factor-1, and cytokines have also been recognized.^16–18 Further studies to investigate the importance of other pathways in promoting myocardial remodeling are warranted.

In the PABE group, the significant increase in plasma AI concentration and decrease in plasma ACE activity detected at 60 days after surgery, compared with the baseline values, can be attributed to the effects of enalapril. These changes indicated that enalapril was absorbed and effectively blocked ACE.¹⁹ In addition, increases in plasma renin activity and AI and AII concentrations were evident at 60 days after surgery in the PABC group. These changes were to be expected if candesartan was effectively absorbed.²⁰ Therefore, it appears that enalapril and candesartan were effectively absorbed in the dogs of the present study, allowing us to rule out malabsorption of enalapril as a reason for its failure to suppress cardiac tissue remodeling in the PABE group.

Although assessments of circulating renin-angiotensin system variables suggested effective ACE suppression by enalapril in the dogs in the PABE group, tissue ACE activity in the RV free wall was not significantly decreased as result of treatment. In a study²¹ of humans with chronic heart failure who were treated with enalapril, there was a discrepancy between tissue ACE activity and serum ACE activity; this discrepancy could be dose related.²² In addition, it has been reported²³ that tissue affinity differs among ACE inhibitors. Enalapril was considered an ACE inhibitor with low tissue affinity in the vasculature, compared with trandolapril. Further investigation is needed to determine whether or not the failure of enalapril efficacy is a drug class effect.

One of the limitations of the present study is that tissue AII concentration was not measured. Assessments of tissue AII could have determined the role of renin-angiotensin-aldosterone system in cardiac tissue remodeling, especially in the PABE group.

Although application of PAB in dogs resulted in hemodynamic changes similar to those associated with pulmonary stenosis and induced RVH and cardiac tissue remodeling, the data obtained from dogs with experimentally induced RVH and dogs with naturally occurring pulmonary stenosis likely differ somewhat. However, a previous investigation³ by our group revealed that changes in tissue chymase and ACE activities were similar to those detected in the dogs with experimentally induced RV pressure overload in the present study. Right ventricular hypertrophy attributable to pulmonary stenosis often poses a problem because it causes a dynamic RV outflow tract obstruction even after a stenotic lesion has been relieved via an intervention such as balloon valvuloplasty. Treatment with candesartan should be considered if such intervention is delayed for some reason, although other hemodynamic effects such as hypotension should also be monitored. In the field of veterinary cardiology, administration of β-adrenoceptor blockers would be the first choice of medical treatment in dogs with pulmonary stenosis. Further investigation is needed into determine whether β-adrenoceptor blockers, candesartan, or a combination of those drugs would be more effective in terms of cardiac tissue remodeling and long-term prognosis in dogs with pulmonary stenosis.