ABC | Volume 115, Nº1, July 2020

Original Article Silva-Bertani et al. Mechanism of decreased heart collagen I in obesity Arq Bras Cardiol. 2020; 115(1):61-70 Morphological study The hearts were removed and dissected following euthanasia and thoracotomy. Atria, left and right ventricles weights, and their respective relations with final body weight were determined to evaluate the presence of cardiac remodeling (i.e., presence or absence of hypertrophy). Myocardial protein levels of collagen type I, TIMP-1, TIMP- 2, and leptin Left ventricular tissue was analyzed by Western Blot to quantify protein levels of collagen type I, TIMP-1, TIMP-2, and leptin. Six samples were used in each group to ensure that all samples were analyzed in the same electrophoresis run in order to avoid inter-gel variations. Briefly, frozen left ventricle samples were homogenized using a Polytron device (Ika Ultra TurraxTM T25 Basic, Wilmington, USA) in a lysis buffer containing 10 mM Tris, pH 7.4, 100 Mm NaCl, 1 mM EDTA, 1 Mm EGTA, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, and phosphatase and protease inhibitors (Sigma-Aldrich). The homogenate was centrifuged at 4ºC for 20 minutes at 12,000 rpm. The supernatant was collected, and total protein content was determined by the Bradford Method. Samples (50 µg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in polyacrylamide gels (6% or 10% depending on molecular protein weight). After electrophoresis, proteins were electro-transferred to nitrocellulose membrane (BioRad Biosciences; NJ, USA). The blotted membrane was then blocked (5% nonfat dry milk, 10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, and 0.1% Tween 20) for 2 hours at room temperature and incubated with specific antibodies overnight at 4ºC. Subsequently, the blotted membrane was incubated for 1.5 hours at room temperature with peroxidase-conjugated anti-rabbit or anti- mouse secondary antibody (1:10,000 dilution), and then incubated with enhanced chemiluminescence (Amersham Biosciences, NJ, USA) and detected by autoradiography. Quantification analysis of the blots was performed using Scion Image software (Scion, based on NIH Image). Mouse monoclonal antibodies to collagen type I (1:10,000), TIMP- 2 (1:1,000), and leptin (1:1,000) and rabbit monoclonal antibodies to TIMP-1 (1:1,000) and β -actin (1:1,000) were obtained from Abcam (Cambridge, USA) and Cell Signaling (Danvers, USA), respectively. Targeted bands were normalized to the expression of cardiac β -actin. Myocardial metalloproteinase-2 activity Myocardial MMP-2 activity was determined as reported by Tyagi et al. 26 . Six samples were used in each group to ensure that all samples were analyzed in the same electrophoresis run in order to avoid inter-gel variations. In brief, left ventricular tissues were homogenized in a buffer containing the following: Tris 50 mM, pH 7.4, NaCl 0.2 M, Triton-X 0.1% and CaCl2 10 mM. The homogenate was centrifuged at 4ºC for 20 minutes at 12,000 rpm. The supernatant was collected, and total protein content was determined by the Bradford Method (Bradford 1976). Samples were diluted in application sample buffer consisting of 0.5 M Tris, pH 6.8, 100% glycerol, and 0.05% bromophenol blue. The samples were loaded into the wells of 8% SDS-polyacrylamide containing 1% gelatin. Electrophoresis was carried out in a Bio-Rad apparatus at 80 V for 2 hours. The gel was removed and washed two times with 2.5% Triton-X-100 and then washed with 50 mM Tris pH 8.4. The gel was then incubated at 37°C overnight in an activation solution consisting of 50 mM Tris, pH 8.4, 5 mM CaCl2 and ZnCl2. Staining was performed for 2 hours with 0.5% Coomassie blue, and destaining was performed in 30% methanol and 10% acetic acid until clear bands were observed over a dark background. The gels were photographed, and the intensity of gelatinolytic action (clear bands) was analyzed in UVP, UV, and a White Darkhon image analyzer. Statistical analysis Prior to statistical analysis, all data were tested for normality using the Shapiro-Wilk test. Results were expressed as mean ± standard deviation and submitted to Student’s t-test for independent samples. Pearson correlation was used to evaluate the association between the variables collagen I, MMP-2, TIMP, and leptin. All statistical analyses were performed using SigmaStat for Windows (Version 3.5). The level of significance considered was 5 % ( α = 0.05). Results Animal general characteristics The general animal characteristics are displayed in Table 1. Final body weight; deposits of epididymal, retroperitoneal, and visceral fat; total BF; and AI were significantly higher in the obese group than in the control group. During the experimental period, animals in the obese group ate less food and calories than those in the control group; however, the feed efficiency was higher in obese animals. Metabolic and endocrine profiles The metabolic and endocrine profiles are summarized in Figure 1. Long-term obesity induced by high fat led to significant metabolic and hormonal changes. There was a significant increase in the glucose AUC, as well as in insulin and leptin levels in the obese group, compared to control. The obese animals presented increased AUC, serum insulin, and HOMA-IR. The serum measurements of glucose, triacylglycerol, total cholesterol, HDL, and LDL were similar between groups. Systolic blood pressure and cardiac morphological profile Table 2 shows that SBP was higher in the obese animals, and there were no significant differences between the groups for any of the studied parameters concerning morphological profile, except for the right ventricle, suggesting that obesity did not trigger left ventricular hypertrophy. 63