ABC | Volume 111, Nº2, August 2018

Original Article Rodrigues et al Physical exercise and calcium regulation Arq Bras Cardiol. 2018; 111(2):172-179 Methods Experimental animals SHR and normotensive Wistar rats, 16 weeks of age, obtained from the central vivarium of the Biological Science and Health Center of Viçosa Federal University, were divided into four groups (of 13 animals each): sedentary normontensive (SN); trained normotensive (TN), sedentary hypertensive (SH) and trained hypertensive (TH). Sample size was determined by convenience. In each group, 8 animals were used for cardiomyocyte isolation, and 5 for analysis of gene expression. All procedures were carried out following the ethical principles of the Brazilian Society of Laboratory Animal Science (COBEA, Colégio Brasileiro de Experimentação Animal ), and approved by the Ethics Committee on Animal Experimentation of Viçosa Federal University (CEUA-UFV; approval number 29/2014). The animals were housed in group cages (4 animals per cage) and allowed free access to water and chow at controlled temperature (mean of 22°C) and lighting (12:12 h light–dark cycle). Protocol of treadmill exercise training and stress test Before the training was initiated, animals were adapted to the treadmill during a 5-day period, 10min/day, 0 o inclination at 5 m/min. After 48 hours, all animals were subjected to an incremental treadmill test for establishment of the maximal running velocity (MRV), starting at 5 m/min, 0 o inclination and increments of 3m/min every 3 minutes until exhaustion. Exhaustion was defined as the time point when the animals could not run at the predetermined speed and, at this point, the test was stopped. The TN and TH groups were subjected to an exercise training program for 8 weeks, 5 days/week (from Monday to Friday). The training started with treadmill running at 5-6m/min and 0 o inclination for 10 minutes on the first day. In the first week, exercise duration was increased in 5 minutes per day and the intensity was maintained. In the second week, duration continued to be increased in 5 minutes/day, but intensity was increased by 2% of MRV per day, so that from the first day of the third week to the end of the eighth week, the animals ran at 60% of MRV (~18-22 m/min) during a 60-minute period per day. MRVs of each animal were determined before the training started and at the end of the fourth week in both TN and TH groups for measurement of the time to exhaustion (TTE) and definition of the training intensity. Forty-eight hours after the last training session, the tests were repeated in all animals for analysis of the effects of the physical training on running capacity. During the experimental period, animals of the sedentary groups (SN and SH) were placed on the treadmill 3 days/week (Mondays, Wednesdays and Fridays), 10 minutes/day, 0 o inclination at 5 m/min to subject them to similar conditions of the trained groups. Systolic arterial pressure (SAP) was measured in the beginning and in the end of the experimental period, i.e., 48 hours after the last exercise training session. The measurements were taken in the mornings, without anesthesia, by tail-cuff plethysmography (LE 5001, Panlab, Harvard Apparatus, Spain). Three measurements were performed, and the intermediate value used for analysis. Isolation of cardiomyocytes Forty-eight hours after the last exercise session, isolation of LV myocytes was performed as described by Locatelli et al. 15 Briefly, the animals were weighed and euthanized by cervical dislocation. The heart was excised, weighed, cannulated and perfused in the Langendorff's mode with the isolation solution of the following composition (in mM): 130 Na+, 5.4 K+, 1.4 Mg+, 140 Cl−, 0.75 Ca 2+ , 5.0 Hepes, 10 glucose, 20 taurine and 10 creatine, pH7.3, at room temperature. The heart was then perfused with the calcium-free solution containing 0.1 mM ethylene glycol-bis (ß-aminoethyl ether)-N, N, N’, N’‑tetraacetic acid (EGTA), for 4 to 6 min. The perfusion was then changed to a solution containing 1.0 mg/mL collagenase type II (Worthington, USA) and 0.1 mg/mL protease (Sigma‑Aldrich, USA) for 10-15 min. All the solutions were oxygenated (O2 100% - White Martins, Brazil) and maintained at 35°C. After perfusion, ventricles were separated from the atria and weighed. The ventricles were placed in a flask containing 5.0 mL of enzymatic solution (collagenase + protease). The flasks were briefly shaken for 5 min in a water bath at 37°C. Next, tissues were removed from the flasks and the remaining content centrifuged (3,000 rpm for 30s). The supernatant was removed, and the cardiomyocytes suspended in the isolation solution and stored in refrigerator (5 o C) until being used. Measurements of cell contractility Contractility of isolated myocytes was measured by evaluation of cell length using the motion edge detector (Ionoptix, Milton, MA-USA) mounted on an inverted microscope (Nikon Eclipse - TS100, Japan), as previously described. 15 Briefly, myocytes were placed in a chamber with a glass coverslip base and bathed with a buffer solution containing (mM) 136.9 NaCl; 5.4 KCl; 0.37 NaH2PO4; 0.57 MgCl2; 5.0 Hepes = 5; 5.6 glucose and 1.8 CaCl 2 (pH = 7.4 at room temperature). Cells were visualized on a monitor with a camera (Myocam, Ionoptix, at 240 Hz) attached to a microscope using an image detector system (Ionwizard, Ionoptix). External stimulation was applied at 1.0 Hz (20V) for 5 minutes at room temperature (~25 o C) via platinum electrodes and an electric field stimulator (Myopacer, Ionoptix). Motions of myocyte longitudinal borders were captured by the motion edge detector (Ionwizard, Ionoptix) and stored for posterior analysis. Only myocytes in good conditions, with clear borders and striated sarcomere, relaxed at rest and without voluntary contractions were selected for analysis. Myocyte contractions were analyzed as previously described. 15 Measurement of intracellular Ca 2+ transients Intracellular Ca 2+ transients in isolated cardiomyocytes were measured using fluorescence imaging (Ionoptix, USA), mounted on an inverted microscope (Nikon Eclipse – TS100, USA) equipped with oil immersion objective lens (S Fluor, 40x, Nikon, USA), as described by Natali et al. 16 Briefly, the myocytes were 173