ABC | Volume 114, Nº2, February 2020

Original Article Stefani et al. DNA damage and heart failure Arq Bras Cardiol. 2020; 114(2):234-242 Table 1 – Body mass, morphometric cardiac characteristics, infarcted area and pulmonary and hepatic congestion of sham-operated rats and rats with left ventricular dysfunction Variables Sham CHF Initial Body Mass (g) 330.25 ± 17.24 328.29 ± 18.12 Final Body Mass (g) 400.50 ± 29.61 356.38 ± 32.23 Infarcted Area (%) --- 36.39 ± 8.11 MM/BM (mg/g) 2.56 ± 0.08 3.29 ± 0.46* LV/BM (mg/g) 1.88 ± 0.21 2.36 ± 0.47* RV/BM (mg/g) 0.58 ± 0.12 1.40 ± 1.01* Pulmonary Congestion (%) 65.67 ± 9.34 87.31 ± 3.36* Hepatic Congestion (%) 70.46 ± 1.05 71.43 ± 1.07 Values are presented in mean ± SD; n = 6 for all groups. Sham, sham-operated rats; CHF: Chronic heart failure rats; MM/BM: Myocardial mass-to-body mass ratio; LV/BM: left ventricle mass-to-body mass ratio; RV/BM: right ventricle mass-to-body mass ratio. * p < 0.05 compared to the Sham group. of 25 mV and 300 mA for 15 minutes in alkaline buffer solution (pH > 10.0). Then the plate was neutralized, stained with silver nitrate, rinsed and kept at room temperature to dry for later analysis. The slides of each animal were made in duplicate and a positive control of DNA damage with hydrogen peroxide (30 µl/slide). The analysis was conducted under an optical microscope with a 20x increase by quantifying the size of the comet’s tail in 50 to 100 cells, according to the lengths, diameters, radii and dimensions of individual comets. Percentages of tail DNA, tail moment and Olive tail moment were used as damage quantification parameters. Quantification of DNA Damage All the parameters presented in the results session regarding SCGE were calculated by the software CASP (CASP Labs®, Poland). 20 The percentage of DNA in the tail, tail moment and Olive tail moment formulas are available for consultation in the supplementary data. Tail moment is characterized as the product of tail length and the percentage of DNA in the tail. The Olive tail moment, which is another parameter for DNA damage, comprises the product of the distance (relative to the x-axis) between the center of gravity of the head with the center of gravity of the tail of the comet and the percentage of tail DNA. Sample Size and Statistical Analysis For a minimum difference of 23 arbitrary units of tail moment of ± 4 SD, it was possible to determine minimum statistical difference of two groups with three animals each. 21 In our investigation, we decided to use six animals in each group. Data are presented in mean ± SD. The normality test of Shapiro‑Wilk was used to assess variables distribution. For comparisons between groups, an unpaired Student’s t test and a two-way analysis of variance were performed among different tissues with a Tukey’s post hoc test. A significance of 5%was considered. Statistical analysis was carried out by using SigmaPlot, version 12.0 for Windows, and graphics were created by GraphPad Prism, version 5.0 for Windows. Results Morphological Parameters The animals showed no difference regarding neither initial nor final body mass. Animals submitted to MI showed mean infarction area of 36%. It was possible to observe higher ratio of myocardial mass, right ventricle and left ventricle-to-body mass compared to sham group, indicating cardiac remodeling in both ventricles. Regarding the congestion in the lungs and liver, higher rates of the former were only observed in the CHF group (Table 1). Hemodynamic Parameters When compared to the sham group, lower mean blood pressure was observed in the CHF group. SBP and DBP, as well as HR showed no difference in relation to the control group (Table 2). Regarding ventricular pressure variables, LVSP and higher LVEDP was observed in the CHF group, when compared to the sham one (Table 2). The maximal positive derivative of ventricular pressure (+dP/dt max ) showed alterations in the CHF group, presenting lower values, as well as maximal negative derivative of ventricular pressure (-dP/dt max ), which showed lower values when compared to the control group (Table 2). DNA Damage Parameters Higher values of DNA damage were observed in all variables (% tail DNA, tail moment and Olive tail moment) in the CHF group, in all analyzed tissues (Table 3). DNA damage can be observed in the formation and frequency of comets in left ventricle, pulmonary, diaphragmatic, gastrocnemius and soleus cells (Figure 1). Despite DNA damage being remarkably higher in CHF rats in all tissues when compared to other tissues in the same pathologic condition, it was observed higher damage in soleus compared to gastrocnemius and left ventricle in CHF group. The difference between tissue DNA damage in sham-operated animals and CHF ones can be observed in Figure 2. 236