ABC | Volume 110, Nº3, March 2018

Original Article Rodrigues et al Aerobic exercise training in β 1ARKO mice Arq Bras Cardiol. 2018; 110(3):256-262 left ventricular (LV) myocytes in β 1 AR knockout ( β 1 ARKO) mice. We hypothesized that MCAE training positively affects mechanical properties of LV myocytes from β 1 ARKO mice. Methods Experimental animals A cohort of 4- to 5-month-old male wild type (WT) and congenic β 1 ARKO mice in the C57Bl6/J genetic background were studied. Mice were maintained in cages under a 12:12-h light-dark cycle in a temperature-controlled room (22°C), with free access to water and standard rodent diet. WT and β 1 ARKO mice were randomly assigned into one of the following groups by using the simple random sampling: WT control (WTc), WT trained (WTt), β 1 ARKO control ( β 1 ARKOc) and β 1 ARKO trained ( β 1 ARKOt). The sample size was defined by convenience. All groups initiated the experimental period with eight animals, however, during the cardiomyocyte isolation procedure, some animals/hearts were lost. Thus, the final number of animals in each group is specified in figures and table. Body weight (BW) was measured every week. The experimental protocols were approved by the Ethics Committee for Animal Use at the Vi osa Federal University (protocol #59/2012) in accordance with the Guide for the Care and Use of Laboratory Animals/2011. Exercise training protocol and graded treadmill exercise test MCAE was performed on a motor treadmill (Insight Equipamentos Científicos, Brazil) 5 days/week (Monday to Friday), 60 min/day, for 8 weeks. Over the first week, the duration and running speed of exercise were progressively increased from 10 minutes and 10% of the maximal speed until 60 minutes and 60% of the maximal speed, achieved during a graded treadmill exercise test. At the end of the fourth week of aerobic exercise training, graded treadmill exercise tests were repeated to readjust the running speed. This intensity was maintained during the rest of the training period. During the training period, animals from the untrained groups were handled every day and subjected to a short period of mild exercise (5 min, 0% grade, 5 m/min, 3 days/week). The exercise capacity estimated by total distance run was evaluated using a graded treadmill exercise protocol for mice (Panlab/Harvard Apparatus, Spain), as described previously. 18 Briefly, after being adapted to the treadmill for 1 week (10 min/day, 0% grade, 0.3 km/h), mice were placed in the exercise streak and allowed to acclimatize for at least 30 minutes. The graded treadmill exercise test began at 6 m/min with no grade and increased by 3 m/min every 3 minutes until fatigue, which was defined as when the test was interrupted because the animals could no longer keep pace with the treadmill speed. The graded treadmill exercise test was performed in WT and β 1ARKO untrained and exercise-trained groups before and after the exercise training period. Cardiomyocyte isolation Forty-eight hours after the last exercise training session, mice were weighed and killed by decapitation, and their hearts were removed quickly. Left ventricular myocytes were enzymatically isolated as described previously. 19 Briefly, hearts were mounted onto a Langendorff system and perfused with calcium-free HEPES-Tyrode solution for 6 minutes with the following composition (in mM): 130 NaCl, 1.43MgCl 2 , 5.4 KCl, 0.4 NaH 2 PO 4 , 0.75 CaCl 2 , 25 HEPES, 22 glucose, 0.01 μg/ml insulin, 0.1 EGTA, pH 7.4, at 37°C. Afterwards, the hearts were perfused for 7-10 minutes with a solution containing 1 mg/ml collagenase type II (Worthington, USA) and CaCl 2 (0.8 μM). The digested heart was then removed from the perfusion apparatus and the heart and left ventricle were carefully weighed. Left ventricle was cut into small pieces and placed into conical flasks with collagenase-containing solution. The cells were dispersed by agitating the flasks for periods of 3 minutes at 37°C. Single cells were separated from the non-dispersed tissue by filtration. The resulting cell suspension was centrifuged and resuspended in HEPES-Tyrode solution containing CaCl 2 (2.5 and 5 μM, subsequently). The isolated cells were stored in HEPES-Tyrode solution containing 10 μM CaCl 2 at room temperature until use. Only calcium-tolerant, quiescent, rod‑shaped cardiomyocytes showing clear cross‑striations were studied. The isolated cardiomyocytes were used within 2‑3 hours of isolation. Cell contractility measurement Cell contractility was evaluated as described previously. 20 Briefly, the isolated cells were placed in a chamber with a glass coverslip base mounted onto the stage of an inverted microscope (Nikon Eclipse, TS100). The chamber was perfused with HEPES-Tyrode solution plus 10 μM CaCl 2 at 37°C. Steady-state 1-Hz contractions were elicited via platinum bath electrodes (Myopacer, Field Stimulator, IonOptix) with 5-ms voltage pulses and an intensity of 40 V. The cells were visualized on a personal computer monitor with a NTSC camera (MyoCam, IonOptix) in partial scanning mode. The image was used to measure cell shortening (our index of contractility) in response to electrical stimulation using a video motion edge detector (IonWizard, IonOptix). The cell image was sampled at 240 Hz. Cell shortening was calculated from the output of the edge detector using an A/D converter (IonOptix, Milton, MA). Cell shortening (expressed as percentage of resting cell length) and the velocities of shortening and relaxation were calculated. Statistics Datawere subjected toShapiro-Wilkor Kolmogorov‑Smirnov normality tests as appropriate. Paired t test was used to compare initial and final BW in each group. The comparisons among groups of the values of BW, heart weight (HW), left ventricular weight (LVW) and ratios, as well as cell contraction were made using a two-way ANOVA followed by Tukey test using software SigmaPlot®, 12.5 version (Systat Software, San Jose, CA). Data are presented as means ± SD. A statistical significance level of 5% was adopted. Numbers of mice, hearts, and myocytes used are given in the relevant table and figure legends. Results Table 1 shows BW and LVW. The initial BW of β 1 ARKO animals was higher as compared to their respective control 257