ABC | Volume 113, Nº5, November 2019

Original Article Vasconcelos et al. Antiarrhythmic effects of d-limonene Arq Bras Cardiol. 2019; 113(5):925-932 They were maintained under standard temperature conditions (22 ± 2 o C) on a 12-hour light/dark schedule (lights on at 06 am) and were fed a standard diet (Labina®) with water ad libitum . In each set of experiments, rats were divided into two groups: control with 4 or 6 animals, and DL treated, with 4 or 6 animals. All experimental protocols were previously approved by the Animal Care and Ethics Committee of the Universidade Federal de Sergipe. Effects of DL on Hemodynamic parameters in vivo Rats were anesthetized with ketamine and xylazine (80 mg/Kg and 10 mg/Kg; i.p., respectively), and polyethylene catheters were implanted in the abdominal aorta and inferior vena cava via left femoral artery and vein, respectively. After insertion and fixation by cotton threads, the catheters were tunneled subcutaneously and exteriorized through an incision in the posterior cervical region of the animal. The incisions were closed and the animals were allowed a 24‑hour post‑operative recovery period. Mean arterial pressure (MAP) and heart rate (HR) were recorded through a pressure transducer (Edwards LifeSciences, Irvine, CA, EUA) coupled to an amplifier (FE221, Bridge Amp; ADInstruments, Bella Vista, NSW, Australia) After post-operative recovery period, MAP and HR were recorded before (baseline values) and after IV bolus administration of DL (1, 5, 10, 20, and 40 mg/kg, i.v.) or vehicle to obtain dose-response curves. The DL curve (n = 6) was compared with the vehicle curve (n = 6). In vivo electrocardiographic measurements Animals from both groups, control (n = 4) and DL treated (n = 4), were heparinized (200 I.U, i.p.), anesthetized with an i.p. injection of ketamine (80 mg/kg) and xylazine (10 mg/kg), and the vena cava was cannulated as previously described. The rats were kept in the supine position with spontaneous breathing and were administrated vehicle or DL (10 mg/kg). The volume injected through the cannula was the same for all concentrations (200 µL). To record the electrocardiographic signal, the electrodes were placed under the skin in the DII lead arrangement. The electrocardiographic parameters observed were the PR interval, QT interval, duration of QRS complex and HR. The QT interval was corrected by Bazett's formula modified for rats. 13 Isolated perfused heart (Langendorff technique) experiments Animals, from both groups, control (n = 4) and DL treated (n = 4), were heparinized (200 I.U., i.p.) and after 15 min the heart was removed and mounted in an aortic perfusion system of the Langendorff type, on a constant flow (10 mL/min). 14 The hearts were continuously perfused with Krebs-Henseleit solution (in mM: 120 NaCl, 5.4 KCl, 1.2 MgCl 2 , 1.25 CaCl 2 , 2 NaH 2 PO 4 , 27 NaHCO 3 , 11 glucose), previously filtered through a cellulose acetate membrane (0.45 µm), pH was adjusted to 7.4 and oxygenated (95% O 2 + 5% CO 2 ) and maintained at 37 ± 0.1 o C (Haake F3, Berlin, Germany). Electrocardiographic (ECG) heart signals were captured using three electrodes (Ag/AgCl/NaCl, 1 M) that were placed inside the chamber close to the heart. The signals were amplified, digitalized (PowerLab 4/35 ADInstruments, USA) and stored in a computer. Left ventricular development pressure (LVDP, mmHg) and HR (bpm) were measured using a water-filled balloon introduced into the cavity of the left ventricle. This device was coupled to a pressure transducer (FE221, Bridge Amp, ADInstruments, USA) and an amplifier (PowerLab 4/35, ADInstruments). The system was calibrated using a column of mercury. Time of peak (ms), which is defined as the time necessary to achieve the peak of maximal ventricular contraction, and relaxation time, were also analysed. Evaluation of the antiarrhythmic effect of DL The antiarrhythmic effect of DL was determined the in vivo animal model using (S)-(−)-Bay K8644, an L-type calcium channel (LTCC) agonist. Arrhythmia was induced by administration of (S)-(−)-Bay K8644 (1mg/kg, i.v.) in the femoral vein of both groups, control (n = 4) and DL treated (n = 4). DL (10 mg/kg) was administered 10 min prior to (S)-(−)-Bay K8644. 15 Cardiac arrhythmias were observed for 20 min by analysis of the ECG recording. For a better analysis, the total Figure 1 – Chemical structures of the d-limonene (DL) (FW: 136.24). H 2 C CH 3 CH 3 926