ABC | Volume 113, Nº5, November 2019

Original Article Vasconcelos et al. Antiarrhythmic effects of d-limonene Arq Bras Cardiol. 2019; 113(5):925-932 5 mg/kg, produced transitory and non-significant hypotension without altering HR (Figure 2B – left graph). On the other hand, DL, at doses of 10, 20, and 40 mg/kg, produced an intense and persistent bradycardia (-33.0 ± 21; -57.0 ± 19, and -53.0 ± 30 %; n = 6; p < 0.05, respectively) (Figure 2B – right graph) which persisted up to 19 ± 12 min after administration. This bradycardia was associated to hypotension only in the doses of 20 and 40 mg/kg (-53.0 ± 6, and -28.0 ± 18 %; n = 6, p < 0.05, respectively). Figure 3A shows ECG representative tracings for the vehicle (top) and 10 min after i.v. administration of 10 mg/kg of DL (bottom). As can be seen, DL significantly decreased HR (p < 0.05) and increased the QTc (Figures 3B and E). However, DL did not change the PRi and QRS complex duration (Figures 3C and D). In the experiment on Langendorff system, DL (10 ‑3  M) evoked a significant reduction in LVDP compared to the control (Figure 4A). Average data showed that the LVDP decreased from 25.2 ± 11.4 mmHg to 5.9 ± 6.1 mmHg (n = 5, p < 0.05, Fig 4B). DL also promoted a reduction of HR from 228.6 ± 19.1 to 196.0 ± 20.8 bpm (n = 4, p < 0.05, Figure 4C). Regarding the temporal course of the contraction and relaxation phases, our results showed that DL did not alter the time to peak contraction of the left ventricle (Figure 4D) but increased the relaxation time from 129.0 ± 3.0 to 193.5 ± 37.8 ms (n = 4, p < 0.05, Figure 4E). In in vivo model of arrhythmias induced by Bay K8644, DL significantly reduced the incidence of ventricular arrhythmias, such as VPB, VT and VF, and the total arrhythmia index from 15.3 ± 6.1 a.u to 4.0 ± 4.5 a.u (n = 4, p < 0.05) (Figure 5B). Furthermore, DL was able to prevent the sinus tachycardia evoked by Bay K8644 (300.0 ± 46.1 for 93.0 ± 17.4 bpm, n = 4, p < 0.05). Figure 3 – In vivo effects of DL (10 mg/kg) on electrocardiographic parameters (ECG) before (vehicle) and after DL perfusion. Original traces (A) and bar chart of DL effect on heart rate (B), PR interval (PRi) (C), QRS duration (D), and corrected QT interval (QTc) (E). Data are mean ± SD and were analysed by the unpaired Student T test. * p < 0.05 vs vehicle, n = 4. 250 80 50 40 20 0 25 20 15 10 5 0 200 100 150 50 0 100 150 50 0 Vehicle Vehicle DL DL Vehicle DL Vehicle DL Vehicle PRi (ms) QRS (ms) QTc (ms) Heart rate (bpm) DL 316 ms 475 ms 119 ms 87 ms RR RR QRS QTc QTc PR 3 mV 3 mV * * A B C D E Discussion In conscious rat, the intravenous administration of DL at lower doses (1 and 5 mg/kg) was able to produce a non‑significant hypotension, without altering HR. On the other hand, DL at higher doses (10, 20, and 40 mg/kg) produced intense and persistent bradycardia, which persisted up to almost 20 min. Similar effects have been found for other monoterpenes such as 1,8-cineol 17 terpinen-4-ol, 18 and y(–)- α -bisabolol. 19 In order to understand the DL-induced bradycardic effect better, we investigated any changes in the electrocardiographic profile at a dose of 10 mg/kg. This dose was chosen because it was the lowest dose able to produce bradycardia without producing any significant effect on MAP (see Figure 2B). The administration of 10 mg/kg of DL (bottom). significantly decreased HR and increased the QTc. However, DL did not change the PRi and QRS complex duration. Corroborating the data observed in the hemodynamic experiments, we observed in the ECG that DL also promoted bradycardia by increasing QTc. As shown in the literature, calcium channels blockers can reduce cardiac conduction velocity and cause bradycardia. 20,21 These effects may or may not be associated with QTc prolongation. 22 The direct effect of DL on the cardiac muscle was evaluated in an isolated heart mounted on a Langendorff system. In this experiment, DL or vehicle was perfused through the aorta to record LVDP. The volume of blood in a rat is approximately 7 mL to each 100 g of body weight, 23 therefore, the DL concentration after administration of 10 mg/kg in the in vivo experiments is approximately the same as the perfusion with 10 -3 M of DL in the ex vivo experiments. As shown, the perfusion with DL evoked a significant reduction in LVDP. Interestingly, these results are very similar to that 928