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Am J Physiol Heart Circ Physiol 284: H2242–H2246, 2003. First published February 6, 2003; 10.1152/ajpheart.00646.2002.

Ventricular remodeling induced by retinoic acid supplementation in adult rats Sergio Alberto Rupp de Paiva, Leonardo Antonio Mamede Zornoff, Marina Politi Okoshi, Katashi Okoshi, Luiz Shiguero Matsubara, Beatriz Bojikian Matsubara, Antonio Carlos Cicogna, and Alvaro Oscar Campana Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, 18618-00 Saõ Paulo, Brazil Submitted 23 July 2002; accepted in final form 5 February 2003

De Paiva, Sergio Alberto Rupp, Leonardo Antonio Mamede Zornoff, Marina Politi Okoshi, Katashi Okoshi, Luiz Shiguero Matsubara, Beatriz Bojikian Matsubara, Antonio Carlos Cicogna, and Alvaro Oscar Campana. Ventricular remodeling induced by retinoic acid supplementation in adult rats. Am J Physiol Heart Circ Physiol 284: H2242–H2246, 2003. First published February 6, 2003; 10.1152/ajpheart.00646.2002.—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 RAtreated 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

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 VITAMIN A IS A GENERIC TERM

Address for reprint requests and other correspondence: S. A. R. de Paiva, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, 18618-00 Saõ Paulo, Brazil (E-mail: [email protected]). H2242

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. MATERIALS AND METHODS

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 ␮g 䡠 kg⫺1 䡠 body wt day⫺1 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 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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RETINOIC ACID AND CARDIAC REMODELING

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 fourchamber 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 (RT ⁄ ; 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 1 2

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liver (x៮ ⫽ 0.57 g), lung (x៮ ⫽ 0.31 g), and LV myocardium (x៮ ⫽ 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. RESULTS

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. 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

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Table 1. Echocardiographic data

HR, beats/min LVEDD, mm LVSD, mm LVWT, mm LVWT/LVEDD LA, mm Ao, mm FS, % E/A LVM, g

Table 3. Data of heart weight, heart weight-to-body weight ratio, and wet-to-dry weight ratio in liver, lung, and heart

Control (n ⫽ 18)

Retinoic Acid (n ⫽ 14)

P Value

282 ⫾ 41 7.89 ⫾ 0.51 3.43 ⫾ 0.43 1.48 ⫾ 0.1 0.19 (0.18–0.19) 5.49 ⫾ 0.54 3.98 ⫾ 0.27 56.5 ⫾ 4.3 1.58 ⫾ 0.36 0.82 ⫾ 0.15

270 ⫾ 20 8.31 ⫾ 0.43 3.50 ⫾ 0.46 1.54 ⫾ 0.1 0.19 (0.17–0.20) 5.66 ⫾ 0.33 3.92 ⫾ 0.19 58.0 ⫾ 4.0 1.73 ⫾ 0.27 0.94 ⫾ 0.12

0.367 (t) 0.021 (t) 0.681 (t) 0.123 (t) 0.790 (MW) 0.302 (t) 0.494 (t) 0.327 (t) 0.204 (t) 0.020 (t)

Values are means ⫾ SD or median (lower quartile ⫺ upper quartile); n, no. of rats. LVEDD, left ventricular (LV) end-diastolic diameter; LVSD, LV end-systolic diameter; LVWT, LV wall thickness; LA, left atrium; Ao, aorta; EF, ejection fraction; FS, fractional shortening; E, velocity of the E wave in the curve of diastolic mitral flow (cm/s); A, velocity of the A wave in the curve of diastolic mitral flow (cm/s); LVM, LV mass; t, Student’s t-test; MW, Mann-Whitney U test.

variables, values in the RA supplementation group were not different from those seen in the control group. 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. The RA-treated group had an increased myocyte cross-sectional area relative to the control group (Figs. 1 and 2, P ⫽ 0.012). However, the interstitial collagen volume fraction was similar in both groups (Figs. 1 and 3, P ⫽ 0.39). DISCUSSION

BW, g LVW, g LVW/BW, g/kg RVW, g RVW/BW, g/kg AW, g AW/BW, g/kg Liver, w/d Lung, w/d LV, w/d

Control (n ⫽ 18)


P Value

415 ⫾ 29 0.76 ⫾ 0.1 1.84 ⫾ 0.18 0.22 ⫾ 0.04 0.52 ⫾ 0.09 0.09 ⫾ 0.01 0.23 ⫾ 0.04 3.2 ⫾ 0.09 4.6 ⫾ 0.8 4.2 ⫾ 0.2

419 ⫾ 28 0.85 ⫾ 0.06 2.03 ⫾ 0.14 0.26 ⫾ 0.04 0.62 ⫾ 0.09 0.11 ⫾ 0.01 0.26 ⫾ 0.03 3.2 ⫾ 0.06 4.9 ⫾ 0.6 4.2 ⫾ 0.06

0.700 (t) 0.010 (t) 0.004 (t) 0.008 (t) 0.004 (t) 0.009 (t) 0.005 (t) 0.228 (t) 0.198 (t) 0.650 (t)

Values are means ⫾ SD; n, no. of rats. BW, body weight; RV, right ventricular; AW, atrial weight (left ⫹ right); w/d, wet-to-dry weight ratio.

tation 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 purpose of this investigation was to examine the cardiac effects of chronic RA supplementation in normal rats. Our results demonstrate that RA supplemenTable 2. Data from isolated papillary muscle preparations

CSA, mm2 DT, g/mm2 RT, g/mm2 TPT, ms ⫹dT/dt, g 䡠 mm⫺2 䡠 s⫺1 ⫺dT/dt, g 䡠 mm⫺2 䡠 s⫺1 RT ⁄ , ms 1 2

Control (n ⫽ 18)


P Value

0.99 ⫾ 0.22 7.42 ⫾ 1.37 1.08 ⫾ 0.33 169 ⫾ 14 73.5 ⫾ 13.0 22 (18–25) 224 ⫾ 30

0.98 ⫾ 0.15 7.84 ⫾ 1.26 0.87 ⫾ 0.29 147 ⫾ 13 83.1 ⫾ 18.6 26 (25–28) 205 ⫾ 31

0.87 (t) 0.38 (t) 0.08 (t) ⬍0.001 (t) 0.097 (t) 0.004 (MW) 0.086 (t)

Values are means ⫾ SD or median (lower quartile ⫺ upper quartile); n, no. of rats. CSA, cross-sectional area; DT, peak developed tension; RT, resting tension; TPT, time-to-peak tension; ⫹dT/dt, maximum velocity of isotonic shortening; ⫺dT/dt, maximum velocity of isotonic relengthening; RT ⁄ time from peak tension to 50% relaxation. 1 2


Fig. 1. Myocyte cross-sectional area (A) and collagen volume fraction (B) in the control and retinoic acid groups. Data are means ⫾ SD. *P ⬍ 0.05, Student’s t-test.

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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

Fig. 2. Representative hematoxylin and eosin-stained left ventricular (LV) cross-sections from control (A) and retinoic acid-supplemented (B) rat.

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 AJP-Heart Circ Physiol • VOL

Fig. 3. Representative picrosirius red-stained LV section visualized with the use of polarized light from control (A) and retinoic acidsupplemented (B) rat.

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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. This study was supported by a grant from the Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (Saõ Paulo, Brazil). REFERENCES 1. Brower GL and Janicki JS. Contribution of ventricular remodeling to pathogenesis of heart failure in rats. Am J Physiol Heart Circ Physiol 280: H674–H683, 2001. 2. Cicogna AC, Padovani CR, Okoshi K, Aragon FF, and Okoshi MP. Myocardial function during chronic food restriction in isolated hypertrophied cardiac muscle. Am J Med Sci 320: 244–248, 2000. 3. Cicogna AC, Padovani CR, Okoshi K, Matsubara LS, Aragon FF, and Okoshi MP. The influence of temporal food restriction on the performance of isolated cardiac muscle. Nutr Res 21: 639–648, 2001. 4. Cicogna AC, Robinson KG, Conrad CH, Singh K, Squire R, Okoshi MP, and Bing OHL. Direct effects of colchicine on myocardial function. Studies in hypertrophied and failing spontaneously hypertensive rats. Hypertension 33: 60–65, 1999. 5. Cohn JN, Ferrari R, and Sharpe N. Cardiac remodeling concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. J Am Coll Cardiol 35: 569–582, 2000. 6. Colbert MC, Hall DG, Kimball TR, Witt SA, Lorenz JN, Kirby ML, Hewett TE, Klevitsky R, and Robbins J. Cardiac compartment-specific overexpression of a modified retinoic acid receptor produces dilated cardiomyopathy and congestive heart failure in transgenic mice. J Clin Invest 100: 1958–1968, 1997. 7. Colbert MC, Kirby ML, and Robbins J. Endogenous retinoic acid signaling colocalizes with advanced expression of the adult smooth muscle myosin heavy chain isoform during development of the ductus arteriosus. Circ Res 78: 790–798, 1996. 8. Drvota V. Downregulation of thyroid hormone receptor subtype mRNA levels by retinoic acid in cultured cardiomyocytes. Biochem Biophys Res Commun 242: 593–596, 1998.


9. Dyson E, Sucov HM, Kubalak SW, Schmidschonbein GW, Delano FA, Evans RM, Ross J Jr, and Chien KR. Atrial-like phenotype is associated with embryonic ventricular failure in retinoid-X receptor-␣⫺/⫺ mice. Proc Natl Acad Sci USA 92: 7386–7390, 1995. 10. Higueret P, Pallet V, Coustaut M, Audouin I, Begueret J, and Garcin H. Retinoic acid decreases retinoic acid and triiodothyronine nuclear receptor expression in the liver of hyperthyroidic rats. FEBS Lett 310: 101–105, 1992. 11. Krebs HA and Henseleit K. Untersuchungen u¨ber die harnstoff-bildung im turko¨rper. Hoppe Deylers Z Physiol Chem 210: 33–66, 1932. 12. Litwin SE, Katz SE, Weinberg EO, Lorell BH, Aurigemma GP, and Douglas PS. Serial echocardiographic-Doppler assessment of left-ventricular geometry and function in rats with pressure-overload hypertrophy–chronic angiotensin-converting enzyme-inhibition attenuates the transition to heart-failure. Circulation 91: 2642–2654, 1995. 13. Matsubara LS, Matsubara BB, Okoshi MP, Cicogna AC, and Janicki JS. Alterations in myocardial collagen content affect rat papillary muscle function. Am J Physiol Heart Circ Physiol 279: H1534–H1539, 2000. 14. Matsubara LS, Matsubara BB, Okoshi MP, Franco M, and Cicogna AC. Myocardial fibrosis rather than hypertrophy induces diastolic dysfunction in renovascular hypertensive rats. Can J Physiol Pharmacol 75: 1328–1334, 1997. 15. Pawlush DG, Moore RL, Musch TI, and Davidson WR. Echocardiographic evaluation of size, function, and mass of normal and hypertrophied rat ventricles. J Appl Physiol 74: 2598– 2605, 1993. 16. Pfeffer JM, Pfeffer MA, and Braunwald E. Influence of chronic captopril therapy on the infarcted left-ventricle of the rat. Circ Res 57: 84–95, 1985. 17. Pfeffer MA and Braunwald E. Ventricular remodeling after myocardial infarction–experimental observations and clinical implications. Circulation 81: 1161–1172, 1990. 18. Sahn DJ, DeMaria A, Kisslo J, and Weyman AE. The Committee on M-Mode Standardization of the American Society of Echocardiography. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 58: 1072–1083, 1978. 19. Seely EW and Williams GH. The heart in endocrine disorders. In: Heart Disease: A Textbook of Cardiovascular Medicine (6th ed.), edited by Braunwald E, Zipes DP, and Libby P. Philadelphia, PA: Saunders, 2001, p. 2151–2171. 20. Shenefel RE. Morphogenesis of malformations in hamsters caused by retinoic acid–relation to dose and stage at treatment. Teratology 5: 103–118, 1972. 21. Subbarayan V, Mark M, Messadeq N, Rustin P, Chambon P, and Kastner P. RXR alpha overexpression in cardiomyocytes causes dilated cardiomyopathy but fails to rescue myocardial hypoplasia in RXR alpha-null fetuses. J Clin Invest 105: 387– 394, 2000. 22. Wang HJ, Zhu YC, and Yao T. Effects of all-trans retinoic acid on angiotensin II-induced myocyte hypertrophy. J Appl Physiol 92: 2162–2168, 2002. 23. Wu JM, Garami M, Cheng T, and Gardner DG. 1,25 (OH)(2) vitamin D-3 and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes. J Clin Invest 97: 1577–1588, 1996. 24. Zhou MD, Sucov HM, Evans RM, and Chien KR. Retinoiddependent pathways suppress myocardial-cell hypertrophy. Proc Natl Acad Sci USA 92: 7391–7395, 1995.

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