ABC | Volume 115, Nº2, August 2020

Original Article Basilio et al. Effects of intermittent fasting on the heart Arq Bras Cardiol. 2020; 115(2):184-193 kinase (JNK) and p38 kinase (p38K), which regulate the gene transcription of several messengers involved in survival, apoptosis, cell differentiation and cardiac remodeling. 13,14 Regular exercise training is widely used as a procedure of health promotion and prevention of different cardiovascular conditions. 15,16 However, different experimental studies have reported controversial results, showing that exercise training intervention did not affect or even reduced myocardial performance. 8,9,17-19 Moreover, potential effects of treadmill training on cardiac remodeling in IF models have yet to be clarified. Therefore, the aim of this study was to analyze the influence of IF and exercise training on functional fitness and morphological and molecular parameters of myocardial remodeling. Our initial hypothesis is that exercise training increases functional fitness and attenuates myocardial remodeling induced by IF. Methods The experimental protocol was reviewed and approved by the Animal Ethics Committee of the Federal University of Mato Grosso do Sul (CEUA/UFMS; Protocol 615/2014), and was in accordance with the guidelines of the Brazilian Society of Animal Experimentation (COBEA). Animals and Experimental Design Male Wistar rats ( Rattus norvegicus albinus ; n=60), aged 30, obtained from the Animal Center of the Federal University of Mato Grosso do Sul (UFMS), Campo Grande/MS, Brazil, were used. The sample size was based on a previous study 16 and considered the probability of exercise refusal and/or escape instinct during the test. 20,21 The animals were randomly divided into four groups: control, intermittent fasting (IF), exercise training (ET) and exercise training plus intermittent fasting (ETI). The control and ET groups were fed daily ( ad libitum ) a standard commercial diet (Nuvilab®, Brazil), while IF and ETI received a similar dietary intervention every other day (intermittent fasting). Besides nutritional support, animals of the ET and ETI groups were also submitted to a treadmill running protocol (Table 1), according to previous studies. 16,20 Five weekly exercise sessions of physical exercise were held and the experimental period lasted 12 weeks. The animals were kept in cages with two to three experimental units per box at an ambient temperature of 22±2 ºC, humidity of 55±5%, 12- hour light/dark cycle and free access to water. Determination of Functional Fitness Functional fitness was analyzed at the end of the experiment using a multistage incremental test, according to previous studies. 21,22 The test was started with a 5-minute warm-up at a speed of 5 m/min. After an interval of 1 min, each animal was submitted to progressive effort at an initial speed of 6 m/min, followed by increments of 3 m/min, lasting 3 min. The protocol was interrupted when the animal had reached exhaustion or when coordination between steps was difficult. 21 For the evaluation of lactate levels, 25 µl of blood was collected from the animal’s tail at rest and after each exercise stage. The blood samples were immediately transferred to Eppendorf tubes containing 50 µl of 1% sodium fluoride (NaF), refrigerated, and stored in a freezer (-20 °C) until the time of analysis. Lactate was measured in an YSI 150 Sport electrochemical analyzer (Yellow Springs Instruments®, Ohio, USA), with standard error of the measurement of ± 2%. The results are reported in mmol/L. The anaerobic lactate threshold (LT) was determined by graphically plotting lactate concentrations during the test. The LT was determined from the time of linearity breaking as a function of load increase, obtained by visual inspection. Functional capacity was assessed by the speed at the lactate threshold (SLT), distance covered and blood lactate concentration at the lactate threshold (LacLT) and at exhaustion (LacE), determined during the exercise test. In addition, for a more detailed analysis of lactate kinetics, the relative variation (%) in lactate levels was also obtained from the LacLT and LacE values. Metabolic Characterization For analysis of glycemia, the animals were fasted for 8–12 hours and blood samples were collected from the caudal artery to measure glucose at baseline. Next, a 20% glucose solution (Glucose Monohydrate, Merck, São Paulo, Brazil) was administered intraperitoneally at a dosage of 2 g/kg. Blood glucose levels were then evaluated after 15, 30, 60, 90, 120 and 180 minutes. 7,10 Glucose was measured with an Accu- Chek Go glucose meter (Roche Diagnostic Brazil Ltda., São Paulo, Brazil). 23,24 Nutritional Characterization Nutritional characterization consisted of the measurement of food intake, calorie intake, and feed efficiency. Food intake was measured daily and calorie intake was calculated from food intake × energy density of the diet. 23 Body weight (BW) was measured once a week using a digital scale. BW gain was obtained as the difference between initial and final BW. To analyze the ability to convert ingested energy into BW, feed efficiency was calculated as the ratio between total BW variation (g) and total calorie intake (kcal). 23,24 After the experimental period, the animals were fasted for 8 hours and anesthetized by intraperitoneal administration of ketamine hydrochloride (50 mg/kg; Dopalen®, Sespo Indústria e Comércio Ltda — Vetbrands Division, Jacareí/SP, Brazil) and xylazine hydrochloride (10 mg/kg; Anasedan®, Sespo Indústria e Comércio Ltda — Vetbrands Division, Jacareí/ SP, Brazil). After euthanasia by decapitation, thoracotomy and median laparotomy were performed in order to remove heart and white adipose tissue from the retroperitoneal and Table 1 – Description of the treadmill exercise protocol Period Speed (m/min) Duration (min) Week 1–3 10 40–60 Week 4–6 15 40 Week 7–9 18 35 Week 10–12 19 15–25 185