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Angiology

Experimental Cardiac Hypertrophy Induced by Oral Administration of Mineralocorticoid and Saline in Rats
Christos Voucharas, Georgios Tagarakis, Antigoni Lazou, Filippos Triposkiadis and Nikolaos Tsilimingas ANGIOLOGY 2012 63: 416 originally published online 17 October 2011 DOI: 10.1177/0003319711423529 The online version of this article can be found at: http://ang.sagepub.com/content/63/6/416

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Experimental Models
Angiology 63(6) 416-419 The Author(s) 2012 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003319711423529 http://ang.sagepub.com

Experimental Cardiac Hypertrophy Induced by Oral Administration of Mineralocorticoid and Saline in Rats
Christos Voucharas, MD, PhD1,2, Georgios Tagarakis, MD, PhD1, Antigoni Lazou, PhD2, Filippos Triposkiadis, MD, PhD1, and Nikolaos Tsilimingas, MD, PhD1

Abstract We suggest a new, easily applicable way of myocardial hypertrophy induction in rats. Forty randomized age-matched male Wistar rats were divided into 2 groups of 20 each: a control group and an orally administered fludrocortisone/salt group. Myocardial hypertrophy was estimated by measuring body weight, heart-weight-to-body-weight ratio and ventricular free wall thickness. Moderate myocardial hypertrophy without heart failure was established in fludrocortisone/salt group in 4 weeks. Our method is effective and low cost, and it provides a model of hypertrophic heart. Keywords heart, hypertrophy, hypertension, corticoid, fludrocortisone, rat

Introduction
Moderate cardiac hypertrophy is a common state of many physiological and pathological conditions in humans: exercise, pregnancy, hypertension, heart valve disease, and myocardial infarction.1 As a result, experimental models producing myocardial hypertrophy are of interest. Several models have been proposed and a variety of animals have been used. Several authors have described cardiac hypertrophy induced by drugs (mineralocorticoid, catecholamine, thyroid hormone, and nitro-L-arginine methyl-ester), aortic or pulmonary artery constriction, renal ischemia, exercise, hypoxia, chronic pacing, and arteriovenous fistula.2-7 Genetic hypertensive experimental animals are mainly formed by murines (as Smirk and Halls New Zealand spontaneous hypertensive rats SHR strain, Dahls salt sensitive rats, Okamoto and Aokis Japanese SHR strain, Bianchis Milan strain, Zamirs Sabra strain, and the Vincents Lyon strain).8 These are good models of experimental hypertrophy and are widely used, but they are not always easily available to all laboratories.8,9 Additionally, considering the multifactorial nature of hypertension and the heterogeneity of the experimental animals, each genetic hypertensive rat strain corresponds to specific variants.10 Invasiveeven the minimally invasivetechniques11 of hypertrophy induction are frequently associated with high animal mortality, up to 20%. Hypertensive cardiac hypertrophy induced by concurrent administration of deoxycorticosterone acetate (DOCA) injected

subcutaneously twice a week and saline per os, over 4 weeks, is a well-established method.12-14 Parenteral potency is more than oral potency and the dose delivery is accurate. However, subcutaneous administration of DOCA is somehow invasive, needs extra manipulation of the animals, and may produce stress and anxiety or even local irritation and infection at the point of injection. The desoxycorticosterone implant technique has the same restrictions mentioned above.15 Instead, we suggest daily oral administration of a synthetic mineralocorticoid (fludrocortisone acetate [FCA]) and saline. Our method is effective, easily applicable, low cost, and has minimal mortality.2-7

Methods
All animals were treated according to the Guidelines for the Care and Use of Laboratory Animals stated in the Greek law (160/1991) based on European Union regulations (European
1 Department of Cardiovascular and Thoracic Surgery, School of Medicine, University of Thessaly, Larissa, Greece 2 Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece

Corresponding Author: Christos Voucharas, Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, Sourmenon 10, 54636 Thessaloniki, Greece Email: voucharas@gmail.com

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Voucharas et al Commission Directive 86/609/EEC). The experimental protocol was approved by our Institutional Ethical Committee.

417 adrenocortical steroid with very potent mineralocorticoid properties and moderate glucocorticoid activity; it is used only for its mineralocorticoid effects. The chemical name is 9-fluoro11b,17,21-trihydroxypregn-4-ene-3,20-dione21-acetate. Fludrocortisone acetate is available for oral administration as tablets providing 0.1 mg of the drug. Fludrocortisone acetate is indicated as partial replacement therapy for primary and secondary adrenocortical insufficiency in Addison disease and for the treatment of salt-losing adrenogenital syndrome and chronic postural hypotension.16-18 The main side effects are sodium and water retention, hypertension, hypokalemia, headache, muscle weakness, fatigue, increased susceptibility to infection, impaired wound healing, increased sweating, hirsutism, dyspepsia, peptic ulcer, osteoporosis, insomnia, depression, weight gain, decreased carbohydrate tolerance, changes to the menstrual cycle, cataract, glaucoma, and increased intracranial pressure.17-19 Pharmacokinetic and pharmacodynamic properties of the drug have been studied in humans, dog, rat, monkey, and guinea pig after intravenous (iv) and intraduodenal administration. Qualitatively, the physiologic action of FCA is similar to that of hydrocortisone. However, the effects of FCA, particularly on electrolyte balance but also on carbohydrate metabolism, are considerably heightened and prolonged. Mineralocorticoids act on the distal tubules of the kidney to enhance the reabsorption of sodium ions from the tubular fluid into the plasma; they increase the urinary excretion of both potassium and hydrogen ions. In small oral doses, FCA produces marked sodium retention and increased urinary potassium excretion. It also causes a rise in blood pressure, apparently because of the effects on electrolyte levels. In larger doses, FCA inhibits endogenous adrenal cortical secretion, thymic activity, and pituitary corticotropin excretion; promotes the deposition of liver glycogen; and, unless protein intake is adequate, induces negative nitrogen balance. Fludrocortisone acetate is rapidly and completely absorbed after oral administration. The approximate plasma half-life of FCA is 3.5 hours or more and the biological half-life is 18 to 36 hours. In rats, most of the dose is excreted in the bile. It is likely that excretion into the bile is balanced by reabsorption in the intestine and some is excreted with the feces.20,21

Experiment Protocol
Hypertensive myocardial hypertrophy was established to 20 randomly selected male Wistar rats (8 weeks of age and weighing 150-200 g) by concurrent oral administration of FCA and saline for 4 weeks. Another group of 20 male rats of 8 weeks acted as the control group. These animals drank tap water. All animals from both groups had free access to standard laboratory rat food for 4 weeks. Rats were housed in individual cages. All animals (both normal and hypertrophic heart groups) showed a normal pattern of growth and at 12 weeks of age weighed 200 to 250 g. The myocardial hypertrophy group were given 12.5 mL of a salt solution with FCA (the water solution that the rats drank contained 0.9% NaCl, 0.2% KCl, 2.54 mEq % Mg2, and 0.002 mg % FCA) to drink instead of water for the first half of every day and a free quantity of a salt solution (the final concentration in the solution was 0.9% NaCl, 0.2% KCl, and 2.54 mEq % Mg2) for the rest of the day, in order to ensure a standard corticoid intake of 25 105 mg/animal per d. Four weeks later, animals were anesthetized by intraperitoneal injection of sodium pentothione (100 mg/kg). Body weight was measured. Heparin was delivered intravenously (300 IU/kg) through the femoral vein so that clots would not remain in heart cavities. The pericardium, the pleural cavities, and the peritoneal cavity were explored for effusions. The hearts were excised and perfused with normal saline through the aorta. The lungs and the livers were also excised. The organs were weighed after lightly blotted with filter paper. Then, hearts were sliced horizontally in the middle of the long axis. Photographic records of the crosssection of the sliced hearts were obtained. The degree of myocardial hypertrophy was estimated by measuring the wet heart weight and the body weight and the heart-weight-to-body-weight ratio (HW/BW) was calculated. The thickness of both the left and the right ventricle free wall was measured with the help of the photographic captures. The lung-weight-to body-weight ratio (LuW/BW), as well as the liver-weight-to-body-weight ratio (LiW/BW) was also calculated in order to estimate the differences in organ growth between the 2 groups, suggesting evidence for cardiac failure.

Results Statistical Analysis

Values were expressed as the mean + standard error of the mean (SEM). The unpaired t test was used to compare BW, HW/BW ratio, LuW/BW ratio, LiW/BW ratio, and the ventricular free wall thickness between the 2 groups. Data in every separate group passed the normality test. A 2-tailed P < .05 was considered significant. The animal mortality was zero. Body weight was similar between normal and hypertrophic heart rats, but HW/BW ratio showed clear evidence of hypertrophy (approximately 45% in excess) when compared with controls (Table 1). Myocardial growth in the hypertrophic heart group was partially due to the reduction in the ventricular chamber, so ventricular hypertrophy was both concentric and eccentric. Left ventricle free wall was approximately 30% thicker in the hypertrophic heart group than in the control group, while right ventricle free wall was 28.4% thicker (Table 2). There was no evidence of heart failure: no observed cavity effusions in the hypertrophic group and no significant

Pharmacology of FCA
Fludrocortisone acetate (Institute for pharmacological research and technology, IFET, Pallini Attikis, Greece) is a synthetic

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418
Table 1. Comparison of Body Weight and Organ Weight/Body Weight Ratios of Control and Hypertrophic Heart Groupa Control (n 20) BW (g) HW/BW ( 1000) LuW/BW ( 1000) LiW/BW ( 1000) 224.1 4.27 5.50 43.87 + 2.79 + 0.13 + 0.06 + 0.34 Hypertrophic (n 20) 228.2 6.20 5.44 44.07 + 1.95 + 0.18 + 0.04 + 0.27 P .24 <.0001 .40 .64

Angiology 63(6) cost, has zero mortality, and can provide matched groups of both hypertrophic and normal heart from the same animal strain. Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The authors received no financial support for the research, authorship, and/or publication of this article.

Abbreviations: BW, body weight; LuW, lung weight; LiW, liver weight; SEM, standard error of the mean. a Values are expressed as the mean + SEM.

References
Table 2. Left and Right Ventricle Free Wall Thickness of the Hypertrophic and the Normal Heart Groupa Control (n 20) LV free wall thickness, mm RV free wall thickness, mm 4.41 + 0.23 1.58 + 0.09 Hypertrophic (n 20) 5.74 + 0.25 2.02 + 0.11 P .0004 .003 1. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev. 1999;79(1):215-262. 2. Taylor PB, Tang Q. Development of isoproterenol-induced cardiac hypertrophy. Can J Physiol Pharmacol. 1984;62(4):384-389. 3. Taylor Q, Taylor PB, Helbing RK. Catecholamine induced hypertrophy. Can J Cardiol. 1987;3(6):311-316. 4. Hasenfuss G. Animal models of human cardiovascular disease, heart failure and hypertrophy. Cardiovasc Res. 1998;39(1):60-76. 5. Bregagnollo EA, Rodrigues MAM, Montenegro MR, Curi PR, Tucci PJ. Temporal evolution of structural and functional parameters of cardiac hypertrophy induced in Wistar rats by abdominal aorta constriction. Arq Bras Cardiol. 1986;46(1):9-17. 6. Bing OHL, Matsushita S, Fanburg BL, Levine HJ. Mechanical properties of rat cardiac muscle during experimental hypertrophy. Circ. Res. 1971;28(2):234-245. 7. Schlensak C, Sarai K, Gilden HP, Beyersdorf F. Pulmonary banding with a novel percutaneously, bidirectionally adjustable device. Eur J Cardiothorac Surg. 1997;12(6):931-933. 8. Trippodo NC, Frohlich ED. Similarities of genetic (spontaneous) hypertension. Man and rat. Circ Res. 1981;48(3):309-319. 9. Doggrell SA, Brown L. Rat models of hypertension, cardiac hypertrophy and failure. Cardiovasc Res. 1998;39(1):89-105. 10. Folkow B, Hallback M. Physiopathology of spontaneous hypertension in rats. In: Genest J, Koiw E, Kuchel O, eds. Hypertension Physiopathology and Treatment. New York, NY: McGraw-Hill; 1977:507-529. 11. Martins AS, Aguilera NW, Matsubara BB, Bregagnollo EA. Experimental myocardial hypertrophy induced by a minimally invasive ascending aorta coarctation. Bra J Med Biol Res. 2001; 34:413-415. 12. Baxter GF, Yellon DM. Regression of left ventricular hypertrophy and susceptibility to reperfusion-induced arrhythmias after DOCA-salt hypertension in the rat. Cardioscience. 1992;3(4): 245-250. 13. Speechly-Dick ME, Baxter GF, Yellon DM. Ischaemic preconditioning protects hypertrophied myocardium. Cardiovasc Res. 1994;28(7):1025-1029. 14. Yoshida K, Kim-Mitsuyama S, Wake R, et al. Excess aldosterone under normal salt diet induces cardiac hypertrophy and infiltration via oxidative stress. Hypertens Res. 2005;28(5):447-455. 15. Blank SD, Lahorra JA, McDonald RS, et al. Superior recovery of hypertrophied rat myocardium after cardioplegic arrest. Ann Thorac Surg. 1998;65(2):390-396.

Abbreviations: LV, left ventricle; RV, right ventricle; SEM, standard error of the mean. a Values are expressed as the mean + SEM.

difference in LuW/BW ratio and LiW/BW ratio between the 2 groups (Table 1).

Discussion
Pressure overload is a classical model for studying cardiac hypertrophy. Chronic mineralocorticoid and high salt intake leads to pressure overload and hypertension, which in turn leads to myocardial hypertrophy.12-14,22-25 In corticoid/salt model, hypertrophy is both concentric and eccentric, similarly to hypertrophy in humans.26 While taking care of the animals, it should be kept in mind that sudden withdrawal of corticosteroids after prolonged therapy can lead to acute adrenal insufficiency, hypotension, and death. We adopted the method of oral administration of corticoid instead of subcutaneous injection in order to avoid additional anxiety and stress to animals because of the injection and to avoid topical hematomas or infection. Animals drank about 20 to 25 mL of water or saline daily, so during the first half of the day a standard dose of FCA and salt was guaranteed. Magnesium and potassium were added in FCA/salt solution to counteract the ion loss caused by the corticoid intake. On the basis of the 45% increase in HW/BW ratio in the hypertrophic heart group, the 30% increase in left ventricle wall thickness, and the absence of congestive heart failure, our model can be classified as moderate (or compensated) cardiac hypertrophy.26,27 We describe a model of myocardial hypertrophy induction in rats. The method is efficient and easily applicable, is low

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Voucharas et al
16. Rayner B. Primary aldosteronism and aldosterone-associated hypertension. J Clin Pathol. 2008;61(7):825-831. 17. Oki K, Yamane K. Therapies for adrenal insufficiency. Expert Opin Pharmacother. 2007;8(9):1283-1291. 18. Hahner S, Allolio B. Management of adrenal insufficiency in different clinical settings. Expert Opin Pharmacother. 2005;6(14): 2407-2417. 19. Freeman R. Current pharmacologic treatment for orthostatic hypotension. Clin Auton Res. 2008;18(suppl 1):14-18. 20. Genard P, Palem-Vliers M. Effect of 6-dehydro-DOCA and 6dehydro-9 alpha-fluorocortisol acetate on the excretion of sodium and potassium in the rat. J Steroid Biochem. 1985;23(5A):673-675. 21. Igarashi Y. Synthetic mineralocorticoid, clinical application of fludrocortisone acetate (Florinef). Nippon Rinsho. 1994;52(3): 779-786.

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22. Baxter GF, Yellon DM. Changes in myocardial collagen content after chronic DOCA-salt hypertension in the rat. Med Sci Res. 1992;20:527-529. 23. Diamond JA, Phillips RA. Hypertensive heart disease. Hypertens Res. 2005;28:191-202. 24. Prisant LM. Hypertensive heart disease. J Clin Hypertens. 2005;7: 231-238. 25. Callens-el Amrani F, Snoeckx L, et al. Anoxia-induced changes in ventricular diastolic compliance in two models of hypertension in rats. J Hypertens. 1992;10(3):229-236. 26. Friberg P, Folkow B, Nordlander M. Structural adaptation of the rat left ventricle in response to changes in pressure and volume loads. Acta Physiol Scand. 1985;125(1):67-79. 27. Hart G. Cellular electrophysiology in cardiac hypertrophy and failure. Cardiovasc Res. 1994;28(7):933-946.

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