ABC | Volume 115, Nº1, July 2020

Original Article Oliveira-Junior et al Effects of losartan in obesity Arq Bras Cardiol. 2020; 115(1):17-28 in experiments of 20, 8 30, 11 33, 13 and 35 weeks. 16 Other investigations have reported impaired cardiac contraction, showed by in vitro papillary muscles analysis of obese rats in experimental models with15 weeks of diet. 7,17,18 There are also reports of unchanged cardiac function after 20, 9 30, 19 and 32 14 weeks of dietary intervention. Therefore, cardiac performance should be further studied in high-fat diet-induced obesity experiments. Obesity regards greater activity of the renin-angiotensin- aldosterone system (RAAS). 11,20,21 High levels of angiotensin-II (Ang-II) coupling to receptors type I (AT 1 ) exert a vasoconstrictor and a trophic effect on myocardium, stimulating several intracellular signaling cascades and multiple physiological responses. 21-23 RAAS activation is the main mechanism responsible for blood pressure disorders and cardiac remodeling in obesity; these effects were attenuated after AT 1 antagonism. 16,11,21,24 However, when considering the in vitro analysis of the papillary muscle, the association between RAAS activation and ventricular remodeling in obesity models based on high-fat diet administration is scarcely studied. The in vitro preparation of papillary muscle allows myocardial contractile capacity measurements in terms of shortening and force generation, despite changes in load, heart rate and heart geometry; such conditions modify mechanical performance in vivo . 7,13,17,19 Using inotropic and lusitropic maneuvers, myocardial performance may also be studied to identify changes in contraction and relaxation that could not be observed under baseline conditions. The most used maneuvers are post-pause potentiation, extracellular [Ca 2+ ] elevation and beta-adrenergic stimulation. 7 From this perspective, the objective of this study was to assess the influence of AT 1 blockade on cardiac morphology and performance using in vitro papillary muscle analysis in rats with saturated high-fat diet induced obesity. The initial hypothesis is that obesity is associated with changes in myocardial functional performance, sustained under different stimulation conditions; these responses are attenuated by AT 1 receptor antagonism. Methods Animal and experimental design Male Wistar rats (n=48), aged 30 days-old were used from the Animal Center of São Paulo State University – UNESP – Botucatu/SP, Brazil. The sample size definition was based on a previous study, 19 developed with a similar experimental model and functional analysis of the isolated papillary muscle. Firstly, animals were divided into two groups: control (CO), treated with control diet (2.9 kcal/g), and obese (OB), fed with high-fat diet with a predominance of saturated fatty acids (3.6 kcal/g). 9 The following ingredients were used for both dietary preparations: corn bran, soybean bran and hulls, dextrin, and palm and soybean oils, plus vitamin and mineral supplementation. In terms of saturated/unsaturated fatty acids content, 9,16 while the control diet presented 61.6/38.4%, the high-fat diet showed 64.8/35.2%. After 16 weeks, the animals were allocated into four groups: CO, OB, CL and OL. For another four weeks, while CO and OB continued to receive their respective diets, CL and OL also received losartan in drinking water (30 mg/kg/day). 11 The animals were kept in individual cages at 22±2°C (room temperature), 55±5% humidity, and 12 hours light/dark lighting cycles. The experimental protocol was reviewed and approved by the Ethics Committee on Animal Experiments of the Botucatu Medical School (protocol 1000/2013). Cardiovascular study The cardiovascular study involved systolic blood pressure (SBP) measurement, cardiacmorphologyassessment, echocardiographic functional analysis and in vitro papillary muscle study. SBP and echocardiogram analysis were performed at 16 and 20 weeks of the experiment. SBP was obtained by plethysmography 26 using a sphygmomanometer (Narco Bio-Systems®, model 709-0610 - International Biomedical, Austin, TX, USA). For echocardiography, the animals were anesthetizedwith amixture of ketamine hydrochloride (50mg/kg) and xylidine hydrochloride (1 mg/kg) administered intramuscularly. After trichotomy in the anterior thorax, each animal was positioned in the left lateral position. For cardiac geometry analysis, one-dimensional images (M-mode) were obtained with the ultrasound beam adjusted in the two-dimensional mode, keeping the transducer on the parasternal position and smaller axis. Left ventricle (LV) imaging was obtained by positioning the M-mode cursor below the mitral valve plane at the papillary muscles level. 27 Aortic and left atrial images were obtained with the M-mode cursor positioned at the aortic plane level. Images were recorded on a printer (model UP-890, Sony Co.). Cardiac structures were measured manually with a caliper. During the maximum ventricular cavity diameter, LV diastolic diameter (LVDD), LV posterior wall diastolic thickness (LVDT), and interventricular septum (IVDT) were measured. The LV systolic diameter (LVSD) was assessed during the minimum cavity diameter. The left atrium (LA) was measured at its maximum diameter. LV weight (LVW) was estimated according to the following formula: LVW = [(LVDD+LVDT+IVDT) 3 - (LVDD) 3 ] x 1.04. The ratio between LVDD and tibia length was also considered. LV systolic function was assessed by posterior wall shortening velocity (PWSV) and percentage of eendocardium fractional shortening (%ES) = [(LVDD-LVSD)/LVDD]. The diastolic function was analyzed by the following indexes: 1) ratio between the initial filling flow velocity peaks (E wave) and the atrial contraction (A wave) of the transmitral flow (E/A); 2) E wave deceleration time (EDT); 3) isovolumetric relaxation time (IVRT); 4) early mitral annulus diastolic displacement velocity peak (E’) and late mitral annulus diastolic displacement velocity peak (A’) obtained by tissue Doppler; and 5) ratio between the waves E and E’ (E/E’). All measurements were performed by the same expert according to the American Society of Echocardiography 28 procedures, using an echocardiograph (General ElectricMedical Systems, Vivid S6, Tirat Carmel, Israel), equipped with a multifrequency electronic transducer (5-11.5 MHz). 18