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 WSMoL has emerged as a potential antibacterial drug and as an immunomodulatory agent. Some plant lectins are non-toxic to mammals, 21,22 but others have been reported to be harmful when ingested or administered by other means, such as intraperitoneal injection. 23 Thus, as one of the steps needed to define the safety of WSMoL, this study evaluated this protein’s possible cardiotoxic effects. Methods Plant material and lectin isolation Moringa oleifera seeds were collected in Recife (Pernambuco, Brazil) with the authorization (no. 38690) of the Chico Mendes Institute for Biodiversity Conservation (ICMBio, acronym in Portuguese) and stored at −20 ºC. A sample of the collected material was stored as a voucher specimen (number 73345) at the Dárdano de Andrade Lima herbarium of the Agronomy Institute of Pernambuco. The access was recorded (A6CAB4C) in the National System For The Management Of Genetic Heritage And Associated Traditional Knowledge (SisGen, acronym in Portuguese). WSMoL was isolated from seed powder according to the protocol previously described by Coelho et al. 5 Briefly, proteins were extracted in distilled water, and, after filtration and centrifugation, the extract was treated with ammonium sulfate at 60% saturation 24 for 4 h at 28 °C. After another centrifugation, the precipitate was resuspended in water and dialyzed for 8 h against distilled water (4 h) and 0.15 M NaCl (4 h). The dialyzed fraction (100 mg of proteins) was loaded onto a chitin column equilibrated with 0.15 M NaCl (20 mL/h flow rate) and WSMoL was eluted with 1.0 M acetic acid. The isolated lectin was dialyzed against distilled water with three liquid changes for eluent elimination. Carbohydrate-binding activity of the lectin was monitored during the purification process by the hemagglutinating activity assay according to the method described by Paiva and Coelho. 25 Animals Adult male C57BL/6 mice were used and maintained at the Carlos Chagas Filho Biophysics Institute (IBCCF, acronym in Portuguese) of the Federal University of Rio de Janeiro (UFRJ, acronym in Portuguese) under controlled conditions of constant temperature (23 °C), in a standard light/dark cycle of 12h/12h with free access to food and water. All experiments were performed in accordance with the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation, and the the applied protocols received approval from the Committee on Ethics in Animal Research of UFRJ , under protocol number DFBCICB041. The mice were used for experiments for 21 days. Experimental conditions The animals were separated into two experimental groups: CNTRL (control group) and WSMoL (animals treated with WSMoL). Several studies by our group have extensively performed experiments with WSMoL using concentrations ranging from 10 μg/ml to 0.2 mg/ml 5-12 in order to test several biological effects of WSMoL. In the present study, in order to test its cardiotoxicity of this purified protein, a 10 times higher concentration of WSMoL was used. Thus, the animals of the WSMoL group were treated with the lectin (purified protein) via gavage, at a concentration of 5 mg/kg body weight (equivalent to 2 mg/ml) for 21 days. The animals in the CNTRL group were treated with milli-Q water via gavage for 21 days. Cardiac hypertrophy In order to evaluate the existence of possible cardiac hypertrophy, the mice’s hearts were weighed, and data were normalized by calculating the heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) ratios. 26,27 After weighing, the animals were euthanized by cervical dislocation. Subsequently, the hearts were extracted, washed with phosphate buffered saline (PBS), dried to remove liquid excess, and weighed. The length of the tibia was measured with a caliper. Fasting glucose, intraperitoneal glucose tolerance test, and intraperitoneal insulin tolerance test Fasting blood glucose (FBG) concentrations were determined from tail vein blood using an automated glucometer (Contour TM TS Bayer). For intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT), mice were fasted for 6 h and 4 h, respectively. After the fasting period, animals received intraperitoneally 2 g/kg of glucose for IPGTT or 0.5 IU/kg of insulin for IPITT, 28 and FBG levels were monitored 0, 15, 30, 60, 120 min after injection from a tail snip. The area under the curve (AUC) was calculated using all the time points, discounting baseline glucose values from each animal. Electrocardiography and echocardiography In order to assess the cardiac electrical activity in vivo , an electrocardiogram (ECG) recording was carried out in conscious animals using a noninvasive method, 29 namely, two subcutaneous electrodes were implanted under isoflurane anesthesia in the right and left forepaws, corresponding to ECG lead I. At the time of registration, the electrodes were connected by flexible cables to a homemade DC‑coupled differential amplifier (kindly provided by Dr. Ariel Escobar, University of California, Merced, USA) using a 500 Hz low‑pass filter and an acquisition frequency of 1 kHz. The signal was digitized using Digidata 1440A (Axon Instruments, San José, CA, USA) and recorded using a Labview-based acquisition program (National Instruments, Austin, TX, USA). The durations of the following intervals were analyzed: PR, RR, QRS, and QJ. Cardiac function was evaluated by in vivo echocardiography (ECHO) using the Vevo 770 High-Resolution Imaging System (VisualSonics, Toronto, Canada) coupled to a 30 MHz transductor, under isoflurane anesthesia. Images were acquired in bidimensional mode and analyzed by a blinded investigator. Left ventricular end-diastolic volume, end-systolic volume, ejection fraction, and fractional area change were calculated using Simpson’s method. In brief, these parameters of cardiac function were evaluated in a long parasternal axis view and four high-temporal resolution B-mode short-axis images, taken at different ventricular levels, as described previously. 30 1030