ABC | Volume 114, Nº2, February 2020

Original Article Stefani et al. DNA damage and heart failure Arq Bras Cardiol. 2020; 114(2):234-242 chronicity of the syndrome, the objective of this study was to evaluate DNA damage in different tissues, such as left ventricle, lungs and skeletal muscles (diaphragm, gastrocnemius and soleus) in rats affected by the condition. Methods Animals There was a selection of 12 male Wistar rats (100 days old, from 300 to 330 g) from the Animal Breeding Unit of Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA, Brazil). The animals were housed in groups of three animals per cage, which received food and water ad libitum in an specific room maintained at 22°C under a 12:12-hour light-dark cycle. The handling of the animals obeyed Law No. 11.794 of 10/08/2008, Law No. 6.899 of 07/15/2009, and Resolution Nº. 879 of 02/15/2008 (CFMV), as well as other provisions applicable to the use of animals for research. The experiment complied with resolutions of the National Council on Animal Experimentation, the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Washington, D.C., 1996), as well as of the Ethical Principles in Animal Experimentation of the National Animal Experimentation Control Council (CONCEA). This study was approved by CEUA/UFCSPA, under protocol number 114/13. Induction of Myocardial Infarction (MI) The animals were anesthetized with xylazine (12 mg/kg ip) and ketamine (90mg/kg ip), intubated and artificially ventilated. The ligation of the left coronary artery was performed. Sham operations were performed as described elsewhere. 11 After the surgeries, the animals received one injection of cetoprophane (5.4 mg/kg ip) every 6 hours – completing 48 hours – and penicillin (70,000 units/ml ip). The surgeries were performed by one surgeon. Post-surgery mortality rate was 15%. After MI induction, 6 weeks of recovery were designated, which was necessary for the animals to develop CHF. In order to document the animals that developed heart failure, we used different variables to characterize this syndrome, such as the presence of left atrial trombi, thoracic effusions, pulmonary congestion, left and right ventricular hypertrophy to body weight. 12 Heart Failure Condition Those animals that presented left ventricular-end diastolic pressure (LVEDP) higher than 15.0 mmHg, as well as increased right ventricle weight to body weight ratio (> 0.8 mg/g) and the presence of pulmonary congestion were considered positive to CHF. 12–14 Hemodynamic Evaluation After the sixth week, the animals were anesthetized with xylazine (12 mg/kg i.p.) and ketamine (90 mg/kg i.p.). A polyethylene catheter (PE-50) was placed into the right carotid artery. Arterial pressure was recorded and the catheter was positioned into the left ventricle to perform ventricular pressure recording. Data were registered by a pressure transducer (strain-gauge, Narco Biosystem Miniature Pulse Transducer RP-155, Houston, Texas, USA), coupled to a pressure amplifier. Pressure analogical signals were digitalized by a data acquisiton system (CODAS-Data Acquisition System, Akron, Ohio, USA) with a sampling rate of 2,000 Hz. These data were used to determine diastolic blood pressure (DBP), systolic blood pressure (SBP), mean blood pressure (MBP), heart rate (HR), left ventricular systolic pressure (LVSP), LVEDP and left ventricular maximum positive and negative dP/dt (+dP/dt max , -dP/dt max ), as previously described. 15 Animals that presented LVEDP higher than 15 mmHg in hemodynamic evaluation were considered with left ventricular dysfunction. 13 Tissue Collection The animals were euthanized through intravenous infusion overdose of the anesthetic pentobarbital (80 mg/kg i.p.). 16 After that, the lungs, the diaphragm, the right gastrocnemius, the right soleus and the heart were removed. The left ventricle was separated from the right one for the comet assay. All samples were stored at -80°C for posterior analysis. Determination of Infarct Size, Cardiac Hypertrophy and Pulmonary and Hepatic Congestion The hearts were removed and weighted, without blood within the chamber and without atria. The size of the infarct area was determined by planimetry. 17 To evaluate cardiac hypertrophy, organ mass was expressed as a proportion of body mass (tissue mass/body mass - mg/g). 18 Animals with right ventricle hypertrophy (i.e. right ventricle mass-to-body weight ratio > 0.80 mg/g) were considered as rats that developed heart failure. 12 To determine pulmonary and hepatic congestion, the lungs and liver of each animal were removed, weighted and dehydrated (80°C) for 48 hours, and then weighted again to evaluate water percentage. Single Cell Gel Electrophoresis (SCGE) Single Cell Gel Electrophoresis (SCGE) was performed in alkaline conditions (pH > 13.0). 19 All procedures were performed avoiding any direct incidence of light. For the assay, a cell suspension of the tissue (left ventricle, lungs, diaphragm, right gastrocnemius and right soleus) was primarily carried out in PBS buffer (pH = 7.40) with standard and gentle manual homogenization. This step required the observation of the density of cells that would be used in each slide. Neubauer’s chamber was used to count approximately 7.3 x 10 5 cells/slide. The suspension of cells (40 μl) was added to agarose of low melting point (90 μl). After gently mixed, this material was carefully superimposed over a slide previously covered with a thin agarose gel layer with a coverslip, and kept in a humid chamber at 4°C for 10 minutes, in order to further secure the suspension of tissue cells in the gel. Then, the coverslip was carefully removed and the slide was conditioned in a vertical cuvette containing lysis solution for at least 1 hour at 4°C. The following step consisted in the unfolding of the cells, for 30 minutes in an alkaline buffer (pH > 10.0). Thereafter, it was followed by the process of electrophoresis, where lysed cells contained in the agarose gel were subjected to a voltage 235