ABC | Volume 111, Nº3, September 2018

Original Article Pinotti et al Fasting/refeeding cycles and myocardial remodeling Arq Bras Cardiol. 2018; 111(3):400-409 Table 1 – Ingredient composition of the Labina rat experimental diet Ingredient (g/kg) Starch 397.5 Dextrinized Corn Starch 132.0 Sucrose 100.0 Carbohydrates 629.5 Casein 200.0 L-Cysteine 3.0 Choline bitartrate 2.5 Protein 205.5 Soy oil 70.0 Fat 70.0 Fiber 50.0 Vitamin mix 10.0 Mineral mix 35.0 Total 1000 and attenuated the structural injuries in papillary muscles caused by CR in normotensive rats. 24 Nevertheless, it is not yet clear whether fasting/refeeding cycles are able to promote similar effects and/or reverse the cardiac damage induced by food restriction in SHR rats. 7-12 Thus, the objective was to investigate the fasting/refeeding approach on myocardial remodeling and function. In addition, the current study was designed to bring information regarding the mechanisms underlying the participation of Ca 2+ handling and β -adrenergic system. Our hypothesis is that fasting/refeeding condition would attenuate the myocardial injury caused by food restriction and would contribute to normal cardiac remodeling in SHR rats without alterations in the Ca 2+ handling and β -adrenergic system. Methods Animal model and experimental protocol Sixty-day-old male SHR were distributed into three groups: control (C, n = 7); food-restriction (R 50 , n = 7); and fasting/ refeeding cycles (RF, n = 7). The C group was fed Labina rat chow containing 7.0% fat, 20.55% protein, 62.95% carbohydrate, 5.0% fiber and 4.5% moisture (Agribands, Brazil), and water was provided ad libitum . Table 1 shows the ingredient composition of Labina rat chow . The R 50 group received 50% of the amount of food consumed by the C group. The RF group was submitted to cycles of 50% food restriction and refeeding ad libitum weekly. All rats were maintained on this dietary regimen for 90 days and were then euthanized. All animals were housed in individual cages in a room maintained at 23ºC with a 12-hour light/dark cycle and were weighed once a week. Initial and final body weights (IBW and FBW, respectively), the ratios between left and right ventricular weights to final body weight (LVW/FBW and RVW/FBW, respectively) and papillary muscle cross-sectional area (CSA) were also measured. All experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and were approved by the ethics committee of Botucatu School of Medicine, UNESP, São Paulo, Brazil. Systolic blood pressure Systolic blood pressure evaluation was assessed by the non-invasive tail-cuff method with a Narco BioSystems Electro-Sphygmomanometer (International Biomedical, Austin, TX, USA) at the beginning and after the end of the experimental protocol. The average of two pressure readings was recorded for each animal. Isolated muscle performance Cardiac intrinsic contractile performance was evaluated by studying isolated left ventricular (LV) papillary muscle as described previously. 9,10,12 Isometric contraction parameters, including peak of developed tension (DT, g/mm 2 , defined as peak isometric tension minus resting tension), resting tension (RT, g/mm 2 ), time to peak tension (TPT, ms), peak isometric tension development rate (+dT/dt, g/mm 2 /s) and maximum tension decline rate (-dT/dt, g/mm 2 /s), time from peak tension to 50% relaxation (RT 50 , ms) were determined. The isotonic parameters were percentage of shortening (PS, %), time to peak shortening (TPS, ms), maximum shortening velocity (-dL/dt, ML/s) and maximum relaxation velocity (+dL/dt, ML/s). The mechanical behavior of the papillary muscle was evaluated under baseline conditions at 1.25 mM [Ca 2+ ] and after the following inotropic maneuvers: increase in extracellular Ca 2+ concentration from 0.625 to 1.25, 2.5 and 5.2 mM, and β -adrenergic stimulation with 0.01, 0.1 and 1.0 µM isoproterenol. The parameters used to characterize papillary muscle were as follows: length (mm), weight (mg) and CSA (mm 2 ). Muscle length (ML) at peak DT was defined as L max in vitro and measured with a Gartner cathetometer (Chicago, IL, USA). To compare the mechanical function between different muscle lengths, isometric and isotonic parameters were normalized to CSA and L max . Morphological study For the ultrastructural study (three animals per group), small pieces of the LV papillary muscle were fixed in Karnovsky’s fixative in 0.12 M phosphate, pH 7.2, for 1-2 hours and were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 2 hours. 25 After dehydration in a graded ethanol series, the samples were embedded in epoxy resin. Ultrathin sections were cut from selected areas with a diamond knife, double‑stained with uranyl acetate and lead citrate, and examined using a Philips EM 301 electron microscope. The LV myocyte CSA was measured using a compound microscope attached to a computerized imaging analysis system (Image‑Pro Plus 3.0, Media Cybernetics, Silver Springs, MD, USA). Statistical analysis Statistical analyses were performed using SigmaStat 3.5 software (SYSTAT Software Inc., San Jose, CA, USA). Normally distributed variables from general characteristics and 401