Follow-up study of morphology and cardiac function in rats undergoing induction of supravalvular aortic stenosis

Abstracts

OBJETIVO: Caracterizar, evolutivamente, o modelo experimental de hipertrofia ventricular esquerda (HVE) por indução de estenose aórtica supravalvar em ratos jovens. MÉTODOS: Ratos Wistar foram submetidos a toracotomia para colocação de clipe ao redor da aorta torácica (grupo EAo, n=12). Animais controle foram submetidos à mesma cirurgia, porém sem a colocação do clipe (grupo C, n=12). A função cardíaca foi analisada por ecocardiogramas seriados após 6, 12 e 21 semanas. Outros grupos foram utilizados para avaliação histológica e quantificação da hidroxiprolina miocárdica (HOP: 2, 6, 12 e 21 semanas). RESULTADOS: A EAo promoveu precocemente HVE concêntrica e aumento progressivo da concentração de HOP. À microscopia óptica, observou-se hipertrofia dos miócitos e da camada média dos ramos intramurais das artérias coronárias. A função sistólica foi supranormal no período 6 semanas (porcentagem de encurtamento - EAo6:70,3±10,8; C6: 61,3±5,4; p<0,05), apresentando-se reduzida apenas no último período. A partir de 12 semanas, verificou-se disfunção diastólica (E/A - EAo12: 4,20± 3,25; C12: 1,61±0,16; p<0,05). CONCLUSÃO: A indução de EAo promove HVE concêntrica e fibrose miocárdica. A função cardíaca, dependendo do período de análise, encontra-se melhorada, normal ou deprimida. O modelo é adequado e potencialmente útil para estudos sobre a fisiopatologia e tratamento nas diferentes fases evolutivas da hipertrofia cardíaca.

hipertrofia miocárdica; ecocardiograma; função ventricular; estenose aórtica; ratos

ORIGINAL ARTICLE

Follow-up study of morphology and cardiac function in rats undergoing induction of supravalvular aortic stenosis

Henrique Barbosa Ribeiro; Katashi Okoshi; Antonio Carlos Cicogna; Edson Antonio Bregagnollo; Maria Aparecida Marchesan Rodrigues; Carlos Roberto Padovani; Flávio Ferrari Aragon; Elenize Jamas; Marina Politi Okoshi

Faculdade de Medicina de Botucatu, UNESP. Botucatu, SP - Brazil

^{Correspondence}

ABSTRACT

OBJECTIVE: To characterize the follow-up of an experimental model of left ventricular hypertrophy (LVH) induced by supravalvular ascending aortic stenosis in young rats.

METHODS: Wistar rats were submitted to thoracotomy and aortic stenosis was created by placing a clip on the ascending aorta (AoS group, n=12). Age-matched control animals underwent a sham operation (C group, n=12). Cardiac function was analysed by echocardiograms performed 6, 12, and 21 weeks after aortic banding. Myocardial morphological features and myocardial hydroxyproline concentration (HOP) were evaluated 2, 6, 12, and 21 weeks after surgery in additional animals.

RESULTS: Aortic banding promoted early concentric LVH and a progressive increase in HOP. Under light microscopy, we observed myocyte hypertrophy and wall thickening of the intramural branches of the coronary arteries due to medial hypertrophy. Cardiac function was supranormal after 6 weeks (percentage of fractional shortening - EAo₆: 70.3±10.8; C₆: 61.3±5.4; p<0.05), and depressed in the last period. Diastolic dysfunction was detected after 12 weeks (ratio of early-to-late filling velocity - EAo₁₂: 4.20±3.25; C₁₂: 1.61±0.16; p<0.05).

CONCLUSION: Ascending aortic stenosis promotes concentric LVH with myocardial fibrosis and minimal histological changes. According to the period of evaluation, cardiac function may be improved, normal, or depressed. The model is suitable and useful for studies on pathophysiology and treatment of the different phases of cardiac hypertrophy.

Key words: myocardial hypertrophy, echocardiogram, ventricular function, aortic stenosis and rats.

Cardiac hypertrophy is an important adapting mechanism that occurs as a response to chronic hemodynamic overload, allowing the heart to maintain its basic functions during overloaded conditions ^1,2. However, left ventricular hypertrophy is a risk factor for the development of congestive heart failure and sudden death ³.

Several experimental models have been proposed for the study of left ventricular hypertrophy due to pressure overload. Among them, the induction of renovascular stenosis ⁴, of abdominal aorta stenosis ^5,6, or thoracic aorta stenosis ⁷, or even the development of animals genetically modified, such as spontaneously hypertensive rats ^8-12. However, several of these animal models have limitations for use in experimental studies. For example, the induction of renovascular stenosis or abdominal aortic stenosis, in rats, in addition to leading to systemic blood hypertension and left ventricular hypertrophy, can also promote systemic activation of the adrenergic nervous system, and of the renin-angiotensin-aldosterone system ^13-15. The intense neuro-hormonal activation is associated with the presence of important cardiac lesions, such as myocardial necrosis, peripheral arteritis, and reparative fibrosis ^4,6. Thus, these models may be very aggressive in the myocardium and, therefore, less representative of the chronic lesions that develop gradually in humans with chronic pressure overload. On the other hand, spontaneously hypertensive rats develop a gradual increase in systemic blood hypertension at 1 month of age, and, at 3 months, they already have established left ventricular hypertrophy ^16,17. However, these rats have a long period of stable myocardial hypertrophy, without heart failure, which usually occurs by 20 months of age ^9,11,18. Therefore, although in this model cardiac alterations are similar to those occurring in human hypertensive heart disease ^9,16, the prevention studies of heart failure are expensive, because the animals have to be kept in captivity for a long period.

More recently, the model of ascending aorta stenosis has been used to promote the gradual development of left ventricular hypertrophy in young rats ^7,19-25. The animals, 3 to 4 weeks after delivery, undergo median thoracotomy to place a band, with an internal diameter of approximately 0.6 mm, around the thoracic aorta, 2 to 3 mm above its root. Immediately after the thoracic banding, the diameter of the artery is maintained; with animal growth, the vessel diameter is maintained and aortic stenosis progressively appears. The rats develop left ventricular hypertrophy precociously, which is associated, in the short term, with improvement in the systolic function of the heart. Then, the animals start to experience depression of mechanical heart performance, and develop cardiac decompensation, 20 weeks after the induction of aortic stenosis ^7,20,26-28. In this model of left ventricular hypertrophy, no systemic activation of the sympathic nervous system or of the renin-angiotensin-aldosterone system occurs ^7,21,26,29. Probably because of this fact, histological evaluation, performed 21 weeks after aortic stenosis induction, did not demonstrate the presence of important cardiac lesions, such as peripheral arteritis, myocardial necrosis, or extensive fibrosis ^7,26.

The advantages of this experimental model are the gradual development of left ventricular hypertrophy, absence of severe anatomical lesions in the myocardium, and the low cost of the maintenance of animals because of the short period necessary to develop left ventricular hypertrophy and heart failure. In the several studies performed with this model ^{7,19-21,26,30,31}, different periods were used for the morphological or functional analysis of the heart, or both, and no studies exist regarding simultaneous and follow-up evaluations of myocardial histology and of cardiac function after induction of aortic stenosis.

The objective of this study was to characterize the follow-up of the experimental model of left ventricular hypertrophy because of supravalvular aortic stenosis, in young Wistar rats, using morphologic and functional evaluation of the heart. Myocardial morphology was analyzed, using optical microscopy, 2, 6, 12, and 21 weeks after the induction of aortic stenosis and cardiac function was assessed, in vivo, using echocardiographic study, performed 6, 12, and 21 weeks after banding of the ascending aorta. In addition to that, the collagen content was quantified using serial determination of hydroxyproline concentration in the left ventricle.

Methods

Male Wistar rats, weighing 90 to 100g, were used from our biotery. Animals were kept in cages with 4 rats per box, at 23^oC, with luminosity cycles of 12h and fed with Purina rat ration and water ad libitum. The protocol used was approved by the Ethical Committee for Animal Research of the Faculdade de Medicina de Botucatu, UNESP.

Rats underwent median thoracotomy after being anesthetized with ketamine hydrochloride (50 mg/kg intramuscular) and xylidine hydrochloride (10 mg/kg intramuscular). Next, the ascending aorta was dissected and a silver band with an internal diameter of 0.6 mm was placed at approximately 3 mm of the aortic root. During surgery, the rats were manually ventilated with positive pressure. Control animals underwent the same surgery; however, a band was not implanted.

Rats were divided into 2 groups: control (C) and aortic stenosis (AoS). For morphologic and biochemical evaluation (hydroxyproline dose), 5 to 7 animals from each group were sacrificed 2, 6, 12, and 21 weeks after AoS induction. The subgroups formed were given the following designations: C₂, C₆, C₁₂, C₂₁, and AoS₂, AoS₆, AoS₁₂, and AoS₂₁.

The other groups of rats (C and AoS) were formed only for functional study. Because this evaluation was performed in vivo and without later sacrifice of the animals, the same rats were assessed in 3 experimental periods: 6, 12, and 21 weeks after the induction of AoS (n= 12 for the C and AoS groups). Due to the difficulty in obtaining adequate echocardiographic images in low-weight rats, the functional study was not performed in the 2-week period after AoS induction.

Rats were weighed and anesthetized with sodium pentobarbital (50 mg/kg intraperitoneal) then underwent median thoracotomy to remove the heart, which was rapidly washed in saline solution. Next, the right ventricle (RV) and left ventricle (LV) were dissected and weighed separately. From the central part of the left ventricle, a 2- to 3-mm thick annulus was cut, taking all the extension of its wall. The material was immersed in neutral and tamponade formalin 10% for 48 hours at 4^oC. After this period, the tissue was washed, dehydrated, and imbedded in paraffin. The histological cuts 5- to 7-mm thick were dyed with hematoxylin-eosin and were analyzed with optical microscopy.

Morphometric analyses were performed using a VCR attached to a Leica microscope connected to the computer equipped with an analysis program (Image-Pro Plus 3.0, Media Cybernetics, Silver Spring, Maryland, USA). In left ventricle transverse sections, sectional areas (SA) of at least 50 cardiac fibers, whose nucleus was clearly identified in the center of the cell, were measured.

Myocardial concentration of hydroxyproline was assessed in tissue obtained at the tip of the left ventricle, according to the methods described by Switzer ³², using a previously described technique used in other studies at our laboratory ^33-35.

The level of ventricular hypertrophy was assessed by the ratio between humid weight of the right and left ventricle and the body weight of the animals.

Rats received anesthesia with ketamine hydrochloride (50 mg/kg) and xylidine hydrochloride (1 mg/kg), administered intramuscularly. A trichotomy was performed in the anterior region of the thorax, and the animals were placed in the left lateral decubitus position for the performance of echocardiography ³⁶ with the Sonos 2000 from Hewlett-Packard Co., equipped with an electronic transducer with a 7.5 MHz frequency.

To measure cardiac structures, M-mode images were obtained with an ultrasound beam guided by the bi-dimensional mode, with the transducer positioned at the smaller parasternal axis. An image of the left ventricle was obtained with the M-mode cursor positioned right below the mitral valve plane at the papillary muscle level ^22,37,38. Aorta (AO) and left atrium (LA) images were obtained with the M-mode cursor positioned at the level of the aortic valve plane. Images obtained were recorded afterwards with the UP-890 printer from the Sony Co.; cardiac structures were measured manually with the help of a pachymeter.

Left ventricle diastolic diameter (LVDD) and systolic diameter (LVSD) were measured at the period of the cardiac cycle when their values were maximum and minimum, respectively. Diastolic thickness of the posterior left ventricle wall (LVDT) was measured at the same period of the cardiac cycle when left ventricle diastolic diameter was assessed. Aortic root diameter (AO) was obtained in the period immediately prior to the opening of the aortic valve. The left atrium (LA) was assessed when its diameter was at its maximum ^22,38. From the dimensions above described, we obtained: relative left ventricle thickness (RLVT), LA/AO, LVDD/BW, and LA/BW.

Left ventricle systolic function was assessed by using the shortening percentage (DD): [(LVDD – LVSD)/LVDD] X 100, and the diastolic function analyzed by the ratio between initial filling flow velocity (E wave) and of the atrial contraction (A wave) of the transmitral flow.

One day after the performance of echocardiography, corresponding to 21 weeks, the animals were sacrificed while under anesthesia with sodium pentobarbital. At this time, signs of heart failure were identified, according to Conrad et al ³⁹ and Cicogna et al ^11,18 as tachypnea, pleuropericardial effusion, ascites, and the presence of left atrial thrombus.

Regarding the statistical analysis, numerical data are expressed as mean ± standard deviation. Study of the morphometric variables and of the myocardial concentration of hydroxyproline was performed with analysis of variance (ANOVA) for the factorial scheme 2X4, for the entirely casual model, completed with the Tukey test of multiple comparisons. Results obtained in the echocardiographic study were analyzed with analysis of variance of the multivariate profiles for dependent groups (MANOVA). Significance level was considered 5%.

Results

Corporal parameters are provided in table I. Body weight was similar between groups AoS and C in the period 2 weeks after surgery. Significant variations occurred between the groups: after 6 weeks body weight was greater in C; after 12 and 21 weeks, it was lower in the C groups in comparison with that in the AoS groups. Left ventricle weight was always greater in the AoS groups compared with that in the C groups. In the evolvement analysis, left ventricle weight increased significantly between 2 and 6 weeks in the 2 groups, C and AoS, and did not change in the other period of analysis. Right ventricle weight was greater in the AoS groups than in the C groups only in the 21-week period. In the evolvement analysis, RV weight had the same pattern as that of the left ventricle weight in the C groups and demonstrated the following results in the AoS groups: AoS₂<(AoS₆=AoS₁₂ )<AoS_21. LV/BW ratio was greater in AoS in the 4 periods assessed. In the C groups, the ratio was greater in C₂ than at other times and, in rats with AoS it had the following pattern: AoS₂>AoS₆> (AoS₁₂=AoS₂₁). The RV/BW ratio was greater in the AoS group than in C only in the last period of evaluation. In the C groups, this ratio was greater in C₂ than in the other periods and, in AoS groups, it was similar in the 2- and 21-week periods, both greater than at 6 and 12 weeks.

Of the 12 animals in the AoS group, which evolved for 21 weeks, 3 had signs of heart failure, including tachypnea, pleural effusion, and ascites.

In the left ventricle histological evaluation, C groups had normal morphology. In the AoS groups, progressive hypertrophy of the myocytes was observed at 2-week follow-up. Intramural branches of the coronary artery had medium lower hypertrophy that was discreet after 2 and 6 weeks, becoming evident after 12 weeks (fig. 1). Myocardial fibrosis, interstitial fibrosis, or peripheral arteritis was not observed in all periods studied.

The cross sectional area of left ventricle myocytes was significantly greater in the AoS groups than in the C groups, in all periods studied. In both groups, a progressive increase of the myocyte cross sectional areas occurred during the evolvement process (fig. 2).

Myocardial concentration of hydroxyproline was greater in the AoS groups than in C groups, at 6 weeks of evolvement. In the C groups the concentration was similar in all periods of evaluation, and in AoS, a progressive increase in their values occurred with the evolvement of the hypertrophic process (fig. 3).

Mean and standard deviations of the left ventricle, left atrium, and aorta measures are presented in table II. LVDD was similar in the C groups and AoS in the 3 periods assessed and was greater in the 12- and 21-week periods, compared with that at 6 weeks, in both groups. When normalized for BW, the variable had similar patterns in the C and AoS groups, with a progressive reduction in their values during evolvement assessment (P<0.05). LVSD was smaller in AoS₆ than in C₆. In the C group, this parameter did not change in the experimental period, and in the AoS group, the 21-week period was significantly greater than that at 6 weeks. LVDT and LVDT/LVDD were greater in the AoS group in the 3 periods assessed and did not change with the age of the animal or the evolvement of the hypertrophic process. LA diameter was greater in the AoS group in the 3 periods, and its greatest value was in AoS₂₁ rather than in AoS₆ and AoS₁₂. AO diameter was greater in AoS₁₂ than in C₁₂. The LA/AO ratio was greater in group AoS in the 3 periods studied. In the 2 groups of animals, LA/AO did not change during evolvement, and LA/BW was greater in period 6 than in the 12- and 21-week periods.

Means and standard deviation of the heart rate (HR), and the systolic and diastolic function indexes are demonstrated in table III. Heart rate was greater in the AoS group in the 12- and 21-week periods, compared with the same period in group C. In group C, HR was lower in C₂₁ than in C₆, and in AoS this variable did not occur in the 3 periods analyzed. DD was greater in AoS₆ than in C₆. In group C, this index did not change with age, and in the AoS group a decrease occurred in its value in the 21-week period compared with that in the 6- and 12-week periods. E/A ratio was greater in AoS₁₂ and AoS₂₁ compared with that in groups C and AoS_6. In the control group, E/A did not change with age.

Discussion

In this study, we assessed the cardiac function, morphology, and development of myocardial fibrosis in young rats undergoing induction of supravalvular aortic stenosis in different periods of follow-up.

AoS induction rapidly encourages left ventricle hypertrophy, which is evident in the early period of evaluation, with 2 weeks of follow-up. After 6 weeks, left ventricle weight or ventricular wall diastolic thickness did not increase, which could suggest that, after aortic stenosis induction, left ventricular hypertrophy occurs precociously and then it becomes stable. However, morphometric evaluation demonstrated that the values of the cross sectional area of the myocytes increased progressively until 12 weeks, demonstrating the presence of additional hypertrophy during the evolvement of the process.

Echocardiographic study allowed the definition of left ventricular hypertrophy as concentric, characterized by increased wall thickness with normal or reduced dimensions of the ventricular cavity. According to several authors ^7,26, animals start to develop heart failure in the 21st week after the induction of aortic stenosis. Thus, it would be expected that eccentric hypertrophy would be found during the last period of study. Because only 25% of our rats had heart failure after the 21 weeks of evolvement, it is probable that the final evaluation was performed when most animals were in the compensated phases of left ventricular hypertrophy and, therefore, still having concentric hypertrophy. However, the increase in right ventricle weight in the last assessment period demonstrates that the change from compensated left ventricular hypertrophy to heart failure may have already occurred. Histological analysis of the myocardium confirmed the findings of other authors ^7,26, who did not find relevant tissue lesions, such as myocyte necrosis, extensive areas of myocardial fibrosis, or peripheral arteritis, after 21 weeks of aortic stenosis induction. In our study, in addition to myocyte cell hypertrophy, we observed the presence of vascular remodeling characterized by medium-layer hypertrophy in the intramyocardial arteries, which was discreet in the first assessment periods and became evident after 12 weeks of follow-up. Thickening of the medium layer of the arteries is usually found in human beings with systemic hypertension ⁴⁰. This finding may be due to hypertrophy or hyperplasia of the smooth muscle, or both; to an increase in water and/or collagen content of the vascular wall; and thickening of the basal membrane ⁴⁰.

Although this model is not associated with severe fibrosis ⁷, myocardial concentration of hydroxyproline was greater in the AoS groups at 6 weeks, and it increased progressively during the evolvement of the hypertrophic process. In the present study, we did not perform morphometric quantification of the collagen content and, therefore, we were not able to state whether the increase was due to the interstitial collagen or perivascular. Weinberg et al ²⁶, using picrosirius red dye, verified the presence of a moderate increase in interstitial and perivascular collagen 21 weeks after the induction of aortic stenosis.

The analysis of left ventricle systolic function demonstrated that in the initial phases of the hypertrophic process the shortening is increased. A decrease in contractile function of the chamber occurred over time, evidenced by the progressive increase in LVSD and by the reduction of DD. These indexes were significantly different between the periods of 6 and 21 weeks after aortic stenosis. It is possible to detect the 3 evolvement phases of systolic function during the hypertrophic process in this model: initially, the presence of supranormal performance, which is followed by unchanged cardiac performance, and, finally, by depression of the contractile capacity of the heart. Similar results were observed by other authors who also assessed cardiac function sequentially with echocardiography ^7,22.

Diastolic dysfunction may be detected in the 12th week of evolvement by the E/A ratio, which was significantly greater in the AoS groups than in the respective C groups. After this period, additional variation was not observed in the values of this ratio. However, the increase in LA diameter, verified in the 6th week, demonstrates that diastolic dysfunction may have occurred earlier. Similar results were observed during the serial study of left ventricle function with echocardiography ²². E/A increase reflects the restrictive pattern of ventricular filling alteration. Currently, this filling model has been attributed, in great part, to the increase in left atrial pressure and also to the increase in passive stiffness of the ventricular chamber, which may be due to the increase in wall thickness or interstitial fibrosis, or both of these ²².

In conclusion, induction of supravalvular aortic stenosis in young rats promotes early development of concentric left ventricular hypertrophy with cardiac function initially supranormal, followed by diastolic dysfunction in the 12th week of evolvement. Reduction of systolic ventricular performance occurred only in the late period after 21 weeks of AoS induction. Cardiac function alterations are followed by myocardial fibrosis without expressive histological lesions. These characteristics demonstrate that the model is suitable and potentially useful for studies on physiopathology and treatment of cardiac hypertrophy due to chronic pressure overload both in the compensated stage and during transition to heart failure.

Acknowledgments

For the valuable technical support of Vitor Marcos de Souza, José Carlos Georgette, and Mário Augusto Dallaqua and to the support of CNPQ/PIBIC.

Received 12/5/02

Accepted 2/23/03

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Publication Dates

• Publication in this collection
  29 Jan 2004
• Date of issue
  Dec 2003

History

• Received
  12 May 2002
• Accepted
  22 Mar 2003