ABC | Volume 113, Nº5, November 2019

Original Article Vasconcelos et al. Antiarrhythmic effects of d-limonene Arq Bras Cardiol. 2019; 113(5):925-932 observed in vivo , where DL also promoted a reduction of HR. 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. As known, the recapture of calcium by sarco/endoplasmic reticulum calcium ATPase (SERCA) is a pivotal step for myocardial relaxation in rats 24 . Our data show that DL delayed the relaxation of cardiac muscle, which can be associated to minor recapture of calcium by SERCA, but this assumption will still need to be elucidated. Our ex vivo results showed that DL decreased pacemaker activity and promoted negative inotropism. It is well known that drugs that block LTCC in cardiac muscle decrease pacemaker activity and result in bradycardia. Furthermore, calcium antagonists decrease myocardial force generation (negative inotropy). These results show that DL presents a similar action to an LTCC blocker. In smooth muscle, De Sousa et al. 25 showed that the relaxant effects of DL in guinea pig tracheas and rat aortas were independent of the endothelium and probably caused by direct action on calcium dynamics. LTCC blockers are classified as class IV antiarrhythmic drugs. The antiarrhythmic properties of LTCC blockers are related to their ability to decrease the firing rate of aberrant pacemaker sites within the heart and to their ability to decrease conduction velocity in the AV node that can help to block reentry mechanisms, which can cause supraventricular tachycardia. Indeed, we decided to investigate the possible antiarrhythmic effect of DL using an in vivo model with arrhythmia induced by Bay K8644, an LTCC agonist. In all the animals tested, Bay K8644 increased HR and inducing arrhythmias such as VPB, VT and VF. As showed, DL significantly reduced the incidence of ventricular arrhythmias and was able to prevent the sinus tachycardia evoked by Bay K8644. 15 Similarly to our results, another study showed that verapamil, an LTCC antagonist, was able to prevent the enhancement of total arrhythmia scores induced by Bay K8644. 15 These results provide some pharmacological evidence that DL significantly attenuates cardiac arrhythmias evoked by Bay K8644, probably via inhibition of LTCC, however, further experiments should be performed to clarify this assumption. Conclusion The results showthatDLhas in vivo and in vitro anti‑arrhythmic properties. Furthermore, DL induced bradycardia associated with hypotension in healthy rats ( in vivo ), corroborating the results in an isolated heart ( in vitro ), which DL promoted bradycardia and reduction of left ventricular pressure. We, therefore, consider this monoterpene to be a promising substance to the development of a new therapeutic agents to treat cardiovascular diseases, such as cardiac arrhythmias. Figure 4 – Ex vivo effects of DL perfusion (10 -3 M) on cardiac parameters.A) Representative traces of left ventricular development pressure (LVDP) recorded in the control situation (top panel) and after perfusion of 10 -3 M DL (bottom panel); Bar chart graphic of DL effect on LVDP B), heart rate C), Time of peak D), and Relaxation time E). Data are mean ± SD and were analysed by unpaired Student T-test. * p < 0.05 vs vehicle, n = 4. Vehicle Vehicle LVDP (mmHg) Time to peak (ms) Relaxation time (ms) Heart rate (bpm) DL 10 –3 M DL 10 –3 M Vehicle DL 10 –3 M Vehicle DL 10 –3 M Vehicle DL 10 –3 M 2.0 s 0.2 s 20 mmHg 20 mmHg 50 250 200 150 100 * * 50 0 250 200 150 100 50 0 150 100 50 0 40 30 20 10 0 A B C D E 929