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Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, 18618-00 São Paulo, Brazil
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Retinoic acid (RA) plays a role in regulating cardiac geometry and function throughout life. The aim of this study was to analyze the cardiac effects of RA in adult rats. Wistar rats were randomly allocated to a control group (n = 18) receiving standard rat chow and a group treated with RA (n = 14) receiving standard rat chow supplemented with RA for 90 days. All animals were evaluated by echocardiography, isolated papillary muscle function, and morphological studies. Whereas the RA-treated group developed an increase in both left ventricular (LV) mass and LV end-diastolic diameter, the ratio of LV wall thickness to LV end-diastolic diameter remained unchanged when compared with the control group. In the isolated papillary muscle preparation, RA treatment decreased the time to peak developed tension and increased the maximum velocity of isometric relengthening, indicating that systolic and diastolic function was improved. Although RA treatment produced an increase in myocyte cross-sectional area, the myocardial collagen volume fraction was similar to controls. Thus our study demonstrates that small physiological doses of RA induce ventricular remodeling resembling compensated volume-overload hypertrophy in rats.
hypertrophy; echocardiography; papillary muscle; collagen
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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VITAMIN A IS A GENERIC TERM encompassing any compound that possesses the biological activity of retinol, whereas the term retinoids refer both to retinol and its natural metabolites, and to many synthetic analogs having structural similarities to retinol, such as retinoic acid (RA). RA is a pleiotropic regulatory compound that modulates the structure and function of a wide variety of inflammatory, immune, connective tissue cells, and others.
Experimental studies suggest that RA plays a role in regulating cardiac structure and function throughout life. During early stages of cardiogenesis, excess RA produces congenital defects related to abnormal folding and septation of the outflow tract and cardiac chambers (20). In the adult rat, excess of RA signaling (overexpression of RA receptor or retinoid X receptor) results in cardiomyocyte abnormalities and dilated cardiomyopathy (6, 7, 21). In contrast, vitamin A-deficient animals and receptor knockout models have thin myocardial walls and the embryos develop a generalized edema associated with heart failure (9). In vitro data suggest that RA suppresses both morphological alterations and changes in gene expression typically associated with hypertrophy induced by endothelin, phenylephrine, and angiotensin II (22-24). However, it is not known whether small physiological doses of RA have any effect in adult animals. Accordingly, the following studies were designed to test the hypothesis that RA modest supplemented in vitamin A-replete adult rats is associated with an adverse myocardial remodeling.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal model and experimental protocol. All experiments and procedures were performed in conformance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of the Faculdade de Medicina de Botucatu, Universidade Estadual Paulista.
Six-week-old male Wistar rats (n = 32; 252.9 ± 11.3 g) were randomly divided into two groups. One group (n = 18) received a standard rat chow and water ad libitum. The standard diet contains 12,000 U/kg of vitamin A. The other group received the same diet, supplemented with 0.3 mg/kg of all trans-RA per diet (Sigma; St. Louis, MO) milled in feed, formulated so that each rat ingested ~24 µg · kg
1 · body
wt day1 of RA. Rats were maintained on these
dietary regimens for 90 days. All animals were housed in individual
cages in a room maintained at 23°C on a 12:12-h light-dark cycle.
Before death, all animals were weighed and evaluated with the use of
transthoracic echocardiography. The exams were performed with the use
of a commercially available echocardiogram (Sonos 2000, Hewlett-Packard
Medical Systems; Andover, MA) equipped with a 7.5-MHz phased-array
transducer. Imaging was performed with the use of a 60° sector angle
and 3-cm imaging depth. Rats were lightly anesthetized by intramuscular
injection with a mixture of ketamine (50 mg/kg) and xylazine (1 mg/kg).
After the chest was shaved, the rats were placed in left lateral
recumbancy. Targeted two-dimensional M-mode echocardiograms were
obtained from short-axis views of the LV at or just below the tip of
the mitral-valve leaflets, and at the level of aortic valve and left
atrium. M-mode images of the LV, left atrium, and aorta were recorded
on a black-and-white thermal printer (model Up-890MD, Sony) at a sweep
speed of 100 mm/s. All tracings were manually measured with a caliper
by the same observer according to the leading-edge method recommended by the American Society of Echocardiography (18).
Measurements represented the mean of at least five consecutive cardiac
cycles. LV end-diastolic dimension (LVEDD) and posterior wall thickness were measured at maximal diastolic dimension, and the end-systolic dimension (LVSD) was measured at the point of maximal anterior motion
of posterior wall. LV systolic function was assessed by calculation of
the fractional shortening index [(LVEDD 
LVSD)/LVEDD × 100]. Left atrium was measured at its maximal diameter and aorta at
end of diastole. Echocardiographic LV mass was calculated by using the
standard cube function formula (12, 15). The velocity of
diastolic flow through the mitral valve (E and A wave velocities) was
obtained in the apical four-chamber view. The E/A ratio was used as an
index of LV diastolic function.
After the echocardiographic evaluation, in vitro papillary muscle
functional studies were performed using methodology previously described (2-4). Briefly, the heart was quickly
removed from the anesthetized rat and placed in oxygenated
Krebs-Henseleit solution at 28°C. A papillary muscle (trabecular
carneae) was carefully dissected from the LV, mounted between two
spring clips, and placed vertically in a chamber containing
Krebs-Henseleit solution at 28°C and oxygenated with a mixture of
95% O2-5% CO2 (pH 7.38). The composition of
the Krebs-Henseleit solution was as follows (in mmol/l): 118.5 NaCl,
4.69 KCI, 2.52 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 5.50 glucose, and 25.88 NaHCO3 (11).
The lower spring clip was attached to a force transducer (model
12OT-20B, Kyowa) by a thin steel wire (1/15,000 in.), which passed
through a mercury seal at the bottom of the chamber. The top spring
clip was connected by a thin steel wire to a rigid lever arm attached
to a micrometer stop for adjustment of muscle length. The lever arm was
made from magnesium with a ball-bearing fulcrum and a lever arm ratio
of 4:1. A displacement transducer (model 7 DCDT-050, Hewlett-Packard)
was mounted above the short end of the lever arm. Preparations were
stimulated 12 times/min with 5-ms square-wave pulses through the
parallel platinum electrodes, at voltages which were ~10% greater
than the minimum stimulus required to produce a maximal mechanical
response. After a 60-min period, when the preparation was permitted to
contract under light loading conditions, the papillary muscle was
loaded to contract isometrically and stretched to the apices of their
length-tension curves. The following parameters were measured during
the isometric contractions: peak developed tension (DT;
g/mm2), resting tension (RT; g/mm2), time to
peak tension (TPT; ms), maximum rate of tension development (+dT/dt; g/mm2/s), maximum rate of tension
decline (dT/dt; g/mm2/s), and time from peak
tension to 50% relaxation (RT1/2; ms). At the end of each
experiment, the muscle length at the maximum length was measured and
the muscle between the two clips was blotted dry and weighed. Muscle
cross-sectional area was calculated from the muscle weight and length
by assuming cylindrical uniformity and a specific gravity of 1.04.
Morphological study.
After the papillary muscle (s) was isolated, the LV, RV, and atria
(left + right) were separated and weighed. To obtain the wet-to-dry ratio, fragments of liver (
= 0.57 g), lung ( = 0.31 g), and LV
myocardium ( = 0.19 g) were weighed before
and after they were placed in an oven at 60°C for 48 h. Eight
animals in the RA group and 12 from the control group were used for
histological evaluation, with the morphometric analysis of the
myocardium performed as described before (13). Transverse
sections of LV were fixed in 10% buffered formalin and embedded in
paraffin. Five-micron-thick sections were stained with hematoxylin and
eosin or the collagen-specific stain picrosirius red (Sirius red F3BA
in aqueous saturated picric acid). Myocyte cross-sectional area was
determined for a minimum of 100 myocytes per hematoxylin and
eosin-stained cross section. The measurements were obtained from
digitized images (×400 magnification) collected using a video camera
attached to a Leica microscope and computerized image analysis software
(Image-Pro Plus 3.0, Media Cybernetics; Silver Spring, MD). The myocyte
cross-sectional area was measured with a digitizing pad, and the
selected cells were transversely cut with the nucleus clearly
identified in the center of the myocyte. Interstitial collagen volume
fraction was determined for the entire picrosirius red stained cardiac
section by using digitized images captured under polarized light (×200 magnification). The components of the cardiac tissue were identified according to the color level: red for collagen fibers, yellow for
myocytes, and white for interstitial space. The collagen volume fraction was calculated as the sum of all connective tissue areas divided by the sum of all connective tissue and myocyte areas. On the
average, 35 microscopic fields were analyzed per heart with a ×20
lens. Perivascular collagen was excluded from this analysis.
Statistical analysis. Comparisons between groups were made by Student's t-test when there was a normal distribution. When data showed a nonnormal distribution, comparisons between groups were made with the Mann-Whitney U test. Data are expressed as means ± SD or medians (including the lower quartile and upper quartile). Data analysis was carried out with SigmaStat for Windows version 2.03 (SPSS; Chicago, IL). The level of significance was considered to be P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The animals entered the study with the same initial weight (control = 254 ± 11 g, RA supplemented group, RA = 250 ± 12 g; P = 0.39) and ended with comparable weights (control = 415 ± 29 g, RA = 419 ± 28 g, P = 0.70). The average weight gains of the two groups were: control = 160 ± 23 g and RA = 168 ± 21 g (P = 0.34).
Echocardiographic studies.
Table 1 summarizes the echocardiographic
data. The RA group showed an increased LV mass (P = 0.02) and LVEDD (P = 0.02) were significantly increased
in the RA-treated group, whereas the LV wall thickness-to-LVEDD ratio
was not significantly different between RA and control group. All other
echocardiographic structural and functional data were similar in both
groups.
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In vitro functional study.
Table 2 summarizes the data obtained from
the isolated papillary muscle studies. There was no significant
difference in papillary muscle cross-sectional area between the control
and RA groups. Values for TPT (P < 0.001) and RT
(P = 0.08) were lower and dT/dt (P = 0.004) and +dT/dt (P = 0.097) were higher in the RA group when compared with the control
group. Regarding the other variables, values in the RA supplementation
group were not different from those seen in the control group.
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Morphological study.
Data illustrating the influence of RA supplementation on heart weight
and organ wet weight-to-dry weight ratios are shown in Table
3. The values for LV weight, RV weight,
atrial weight, LV weight-to-body weight ratio, RV weight-to-body weight
ratio, and atrial weight-to-body weight ratio were significantly higher in the RA-treated group, whereas no differences were observed between
groups with regard to the wet weight-to-dry weight ratios for liver,
lung, and LV tissues.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of this investigation was to examine the cardiac effects of chronic RA supplementation in normal rats. Our results demonstrate that RA supplementation induces myocardial hypertrophy, ventricular enlargement, and enhanced myocardial function. These findings suggest that vitamin A derivatives (RA) may have an important role in the adult heart comparable to that reported in the embryonic heart.
The major finding of the present study was that the administration of RA resulted in comparable increases in atrial and left and right ventricular mass. In addition, the echocardiographic study demonstrated a significant LV dilatation as indicated by the modest increase in LVEDD. These alterations are consistent with myocardial remodeling, which has been referred in the literature as the cardiac changes that follow myocardial injury or chronic hemodynamic overload (16, 17). Myocardial remodeling is induced in response to both chronic pressure and volume overload; however, the structural and morphological changes in the myocardium differ depending on the inciting cause. Chronic pressure overload typically produces concentric myocardial hypertrophy and interstitial fibrosis, which is associated with ventricular dysfunction and the progression to heart failure (5, 14). In contrast, the eccentric hypertrophy induced by chronic volume overload is not typically associated with fibrosis, and the mechanisms underlying the ventricular dysfunction are not well understood (1). However, the hypertrophic response induced by chronic volume overload is initially characterized by increased mass in all four cardiac chambers and a proportional increase in both wall thickness and LV diameter, with systolic function being preserved or even improved, reflecting a compensated state (5). Thus the morphological changes induced by RA supplementation are consistent with the remodeling induced in the compensated stage of chronic volume overload.
The second major finding identified in this study was that animals
treated chronically with RA developed relevant alterations in
myocardial functional variables. The isolated papillary muscle preparation allows a direct analysis of myocardial function without the
influence of the indirect effects of RA on the heart (i.e., hemodynamic
load and heart rate). The isometric contraction data (e.g., RT and
+dT/dt) indicate that RA supplementation resulted in an
improved ability to develop force. Importantly, TPT was lower in RA
group than in control animals, which indicated that the treatment
resulted in a faster contraction. Likewise, the alterations in
dT/dt and RT1/2 suggest that relaxation was improved.
Taken together, the remodeling is interpreted as a compensated physiological hypertrophy, given the increases in cardiac mass, myocyte cross-sectional area, and LVEDD; preserved mass to volume ratio; normal interstitial collagen density; and preserved or improved cardiac function. In this regard, it may be pointed out that the RA receptors share a high degree of homology and interaction with thyroid hormone receptors (8) and can regulate the thyroid hormone signal transduction in a manner similar to triiodothyronine (10). If we assume that RA acts on cardiac cells in the same fashion as thyroid hormones, we would anticipate that vitamin A replete rats supplemented with physiological levels of RA might have cardiac remodeling similar to that occurring in hyperthyroidism, which is characterized by myocardial hypertrophy and maintained or enhanced cardiac function (19). Therefore, two potential mechanisms that may contribute to the myocardial remodeling in RA-treated rats should be considered. First, an indirect action mediated by RA, such as induction of a volume overload, or alternatively, a direct action of RA on myocardial cells, such as thyroid hormone receptor stimulation. Further studies are needed to address the possible role of thyroid receptors in the response to RA in this experimental model.
Others have studied the cardiac effects of RA, but the results are still poorly understood. In pathological conditions, RA treatment suppressed the myocardial hypertrophy induced by endothelin (23), phenylephrine (24), and angiotensin II (22). However, transgenic mice with overexpression of RA receptors, the animals developed dilated cardiomyopathy associated with depressed cardiac function and development of congestive heart failure (6, 21). The reasons for these disparities are not clear, but the different animal models utilized, differences in the dosage of RA, and differing pathological and/or physiological conditions existing during exposure of different developmental periods, could contribute to these discrepancies in the cardiovascular response to RA.
In conclusion, our study indicates that small doses of RA induce physiological hypertrophy with a normal interstitial collagen matrix, a proportional increase in cardiac mass and volume, and improved cardiac function, resembling a compensated volume-overload-induced ventricular remodeling in rats.
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ACKNOWLEDGEMENTS |
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This study was supported by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo (São Paulo, Brazil).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. A. R. de Paiva, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, 18618-00 São Paulo, Brazil (E-mail: paiva{at}fmb.unesp.br).
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.
First published February 6, 2003;10.1152/ajpheart.00646.2002
Received 23 July 2002; accepted in final form 5 February 2003.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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