Constant monitoring was given to ensure the safety of the mice [17]. Sedentary mice were placed GSK 3 inhibitor in water tanks for 5 min daily to mimic the water stress. Twenty four hours after the last session of swimming exercise mice were killed by decapitation and the trunk blood
was rapidly collected into chilled polypropylene tubes containing guanidine thiocyanate and centrifuged, as described previously [42]. Simultaneously, the heart was excised and dissected onto ice. Left ventricle (LV) was weighted and divided in three transversal portions: base, median and apex. Base and apex were snapped frozen and the median segment of LV was processed for histology. Total blood and LV segments were kept in −80 °C until assayed.
Total RNA from the apex segment of the LV was prepared using TRIzol reagent (Invitrogen, San Diego, CA), treated with DNAse, and reverse transcribed with MML-V (Moloney murine leukemia virus) (Invitrogen) [43]. The endogenous HPRT – hypoxanthine guanine phosphoribosyltransferase (internal control), collagen I, collagen III, fibronectin, angiotensin converting enzyme (ACE), ACE 2 and AT1 receptor cDNA were amplified using specific primers (Table 1) and SYBR green reagent (Applied Biosystems) in an ABI Prism 7000 platform (Applied Biosystems). The relative comparative CT method of Livak and Schmittgen was applied to compare gene expression levels between groups, using the equation Alanine-glyoxylate transaminase 2−ΔΔCT[29]. Median LV segment was fixed EPZ015666 mouse in Bouin solution (4% at 4 °C) for 24 h, washed with water and maintained in 70% ethanol overnight. Subsequently, tissue was embedded in paraffin, sectioned (5 μm) and stained with hematoxylin and eosin. Images from 3 slides of each animal were captured and cardiac fibers from at least 150 cells of each group were measured using LC Evolution/Olympus
Bx50 using a 40× objective. Cardiomyocytes were analyzed only from longitudinal fibers with well defined central nucleus. The segment from the base of the LV was homogenized in 4 mol/L of guanidine thiocyanate/1% trifluoroacetic acid (vol/vol; 5 ml for each tissue) in water and then processed as described previously [8] and [36]. Blood and LV peptides were extracted onto bond-elut phenylsilane cartridges (Varian) and Ang-(1–7) and Ang II immunoreactivity (ir) was measured by radioimmunoassay, as described previously by Botelho et al. [7]. Data are expressed as mean ± SE. Comparisons between two observations in the same animal or two groups were performed by Student t-test paired or not paired, respectively. Differences among more than 2 groups were assessed by two-way ANOVA followed by the Bonferroni test. The statistical analysis was performed with GraphPad Prism software (version 4.0), and the level of significance was set at p < 0.05. As observed in Table 1, sedentary WT mice gained weight during the 6 weeks period (36.0 ± 0.