ABC | Volume 114, Nº6, June 2020

Original Article Yurre et al. Evaluation of the cardiac effects of WSMoL Arq Bras Cardiol. 2020; 114(6):1029-1037 Action potential In order to perform intact cardiac action potential (AP) records, a Langendorff retrograde perfusion system was used tomaintain the hearts functional ex vivo for hours, as previously described. 31,32 To avoid tissue damage by the formation of blood clots, animals were injected intraperitoneally with Na + - heparin 15 min before euthanization by cervical dislocation. Hearts were rapidly removed, cannulated by the aorta, and perfused continuously with an oxygenated Tyrode solution containing the following, (in mM): NaCl 140, KCl 5.3, CaCl 2 2, MgCl 2 1, NaPO4H2 0.33, HEPES 10, and glucose 10. The pH was calibrated to 7.4 with NaOH at 32 ºC. To decrease mechanical contraction, the hearts were perfused with Tyrode containing 4 mM of Blebbistatin (Selleckchem, Houston, TX, USA). To record electrical signals, borosilicate glass (10-40 MΩ) microelectrodes were used. These microelectrodes were filled with 3 M KCl solution and inserted into a holder (MEH1SF12, World Precision Instrument [WPI], Sarasota, FL, USA) embedded in a micromanipulator (MM33 links, WPI) connected to the input of a pre-amplifier (Electro 705, WPI). The microelectrodes were placed on the surface of the left ventricle and the reading of the microelectrode was set to zero. Amplified signals were digitalized (NI USB 6281, National Instrument) and analyzed with a homemade program in LabView (kindly developed and provided by Dr. Ariel Escobar, University of California, Merced, CA, USA). The parameters analyzedwere action potential duration (APD) at 30% and 90% repolarization (APD 30 and APD 90 , respectively). Isolation of mice heart mitochondria Isolation of mitochondria from the hearts was adapted from the protocol described by Affourtit et al. 33 with minor modifications. The hearts were rapidly dissected and rinsed in ice-cold Chappell-Perry (CP) buffer containing the following (in mM): KCl 100, Tris-HCl 50, EGTA 2 at pH 7.2). The hearts were weighed, minced with razor blades and washed 4 to 5 times with CP buffer. The tissue was subsequently incubated for 5 min with CP buffer supplemented with 0.5% albumin, 5 mM MgCl2, 1 mM ATP, and 125 U/100 mL protease type VIII, at a proportion of 1 mL/100 mg of tissue. The hearts were then homogenized (Ultra-turrax homogenizer [IKA®, Campinas, SP, Brazil], low setting, 3 s, 3 times) and the resultant homogenate was centrifuged. The supernatant was centrifuged and the pellet was washed and resuspended into ice-cold CP buffer and finally centrifuged. The final mitochondrial pellet was resuspended into a small volume of CP buffer. The protein dosage of the obtained preparation was performed by the method described by Lowry et al. 34 . The isolated mitochondrial preparations were subjected to high resolution respirometry to measure the fluxes of oxygen consumption. High resolution respirometry For the analyses of the oxygen consumption, isolated mitochondria were used. The experiments were performed on a high-resolution O2k-respirometer (Oroboros Instruments, Innsbruck, Austria, EU) at 37°C with mitochondrial respiration media (MIR05) containing the following (in mM): EGTA 0.5, MgCl 2 3, K-MES 60, taurine 20, KH 2 PO 4 20, HEPES 20, sucrose 110 and 1 g/L fat free BSA at pH 7.1. The protocol used to evaluate mitochondrial function was adapted from Pesta and Gnaiger, 35 consisting of sequential addition of multiple substrates and inhibitors, namely, the following: 5 mM pyruvate, 2.5 mM malate, 10 mM glutamate, 100 μM adenosine 5’-diphosphate (ADP), 1mMADP, 10mM succinate, 0.2 μg/mL oligomycin, and 2 μMantimycin A. Respiratory control ratio (RCR) was calculated by the oxygen flux after addition of succinate in the presence of ADP, divided by the flux after oligomycin. The maximal phosphorylative capacity of electron transport system (OXPHOS) was calculated by the oxygen consumption following addition of succinate minus residual oxygen consumption (ROX), which was estimated after the addition of antimycin A. The non-specific leak of protons was determined by oxygen flux insensitive to oligomycinminus ROX. Another distinct protocol was performed by changing the sequence of the substrates in order to calculate the electron leakage, the ratio of hydrogen peroxide (H 2 O 2 ) production by O 2 flux. The order of titration of this protocol was the following: 5mMpyruvate, 2.5mMmalate, 10mMglutamate, 10 mM succinate, 1 mM ADP, and 0.2 μg/mL oligomycin. Data were analyzed in DatLab 5 software (Oroboros Instruments) and expressed in pmol O 2 /mg/s. Mitochondrial H 2 O 2 production Mitochondrial H 2 O 2 was measured by monitoring the resorufin appearance rate at 563/587 nm (excitation/ emission) in a fluorescence spectrophotometer (Varian Cary Eclipse, Agilent Technologies, Santa Clara, CA, USA). The same concentration of isolated mitochondria used in the oxygen consumption experiments was added in 2 mL of MIR05 supplemented with 5.5 μM Amplex red, 2 U/mL peroxidase, and 40 U/mL superoxide dismutase. The assays of H 2 O 2 production were performed at 37 ºC, and the substrates, inhibitors, and uncouplers were added every 2 min in the following order: 5 mM pyruvate, 2.5 mM malate, 10 mM glutamate, 10 mM succinate, 1 mM ADP, 0.2 μg/mL oligomycin, 2 titres of 0.5 μM carbonyl cyanide- 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 2 μM antimycin A. The data generated in arbitrary units of fluorescence were analyzed in Origin Pro-8 software (Origin Lab Corporation, Northampton, MA, USA) and normalized to pmol of H 2 O 2 /mg/min from standard calibration curves of H 2 O 2 performed in the presence of the same number of isolated mitochondria for each experiment. Statistical analysis Values are expressed as mean ± SD or median (with interquartile range) . In order to compare the results between CNTRL andWSMoL, unpaired Student’s t test was used, when appropriate. On the other hand, data showing non-Gaussian distribution (Kolmogorov-Smirnov test) were compared by the Mann-Whitney test. Differences between variables were considered significant when p value was < 0.05. All analyses were performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). We did not use statistical methods to predetermine sample size. Samples sizes were estimated on the basis of sample availability and previous experimental studies of the cardiovascular system. 29,30 1031