ABC | Volume 111, Nº2, August 2018

Original Article Rodrigues et al Physical exercise and calcium regulation Arq Bras Cardiol. 2018; 111(2):172-179 Figure 1 – (A) Time to exhaustion of normotensive and hypertensive before (pre) and after (post) training. (B) Running velocity during training sessions. TN: trained normotensive; TH: trained hypertensive. Data are expressed as mean ± SD of 8 animals in each group. * compared with TN (pre); # compared with TH (pre) (p < 0.05). 60 40 20 0 TN (pre) TH (pre) TN (post) TH (post) *# *# TN TH Time to exhaustion (min) 1 2 3 4 5 6 7 8 Weeks 35 30 25 15 20 A B Running velocity (m/min) incubated with calciumprobe (5 µM for 10minutes) (Fura‑2AM, ThermoFisher, Waltham, USA). The ratio of fluorescence emission at 510 nm in response to excitation wavelengths of 340 nm to that in response to 380 nm wavelengths was used as concentration index of intracellular Ca 2+ transients. The myocytes were electrically stimulated (Myopacer, Field Stimulator, Ionoptix, USA) by a pair of platinum electrodes with a 0.2ms (20V) supra-threshold pulse, frequency of 1 Hz at room temperature (~25 o C). Parameters of intracellular Ca 2+ transients were analyzed using the IonWizard software (IonWizard, 6.3, IonOptix, Milton, USA). Gene expression analysis For analysis of gene expression after euthanasia, LV samples were collected and stored at -80 o C. LV total RNA was isolated in 1 mL of Trizol (Invitrogen) following the manufacturer’s recommendations and stored at -70 o C. RNA samples were diluted 1:100 with water and analyzed by spectrophotometry at 260-280 nm. Analysis of the miRNA-214 gene expression was performed using the Taqman MicroRNA Assays (Applied Biosystems) following the manufacturers’ recommendations. Gene expression quantification was performed in two stages: first, complementary DNA (cDNA) was obtained from reverse transcription of the total RNA sample using a stem-looped primer for reverse transcription to detect the miRNA analyzed and the TaqMan®MicroRNA Reverse Transcription Kit; second, by real-time polymerase chain reaction, PCR products were amplified from the cDNA samples previously obtained using the TaqMan® MicroRNA Assay and the TaqMan® Universal PCR Master Mix II. The U6 snRNA normalizer was used as control, and analysis was performed using the ABI 7500 Real Time-PCR Systems (Applied Biosystems). Statistical analysis Analysis of variance (ANOVA) assumptions regarding homogeneity of variances between the groups and normality of observations were checked and no violations of the assumptions were detected. The variables had normal and continuous distribution. The Levene’s test, the chi-square test and the Kolmogorov test were used for analysis. Comparisons between initial and final SAP and TTE in each group were performed by the paired student’s t-test. Two-way ANOVA followed by Tukey post-hoc test was used for comparisons between the four groups. Analysis was performed using the SigmaPlot software (Systat Software, Inc., San Jose, CA, USA) and significance level was set at 5%. Results are expressed as mean ± standard deviation (SD). Results The physical training program increased TTE of both normotensive and hypertensive animals (Figure 1A). Figure 1B shows running velocity of these animals during training. As compared with normotensive animals, hypertensive animals showed lower body weight (BW), similar LV weight (LVW), higher LVW/BW, whereas TH animals showed higher ventricular weight (VW)/BW (Table 1). Physical training had no effect on these parameters. Results of SAP are presented in Figure 2. As compared with pre-training values, although the physical training program had no effect on SAP in normotensive animals, a reduction in SAP was observed in hypertensive animals (Figure 2A). Cell contractility data are depicted in Figure 3. Contraction amplitude was not affected by hypertension or by physical training (Figure 3 A); and time to peak contraction did not change in any of the groups (Figure 3B). However, physical training reduced time to relaxation by 50% (Figure 3C) in normotensive, but not in hypertensive animals. Figure 4 shows that after training, [Ca 2+ ] i amplitude did not change in normotensive animals, but increased in the TH group as compared with SH animals. In addition, physical exercise decreased the time to peak [Ca 2+ ] i in TN animals as compared with the SN group, but had no effect 174