Electrocardiographic profile of guinea pig heart submitted to Ginkgo biloba extract and its terpenoids

SANTOS, A.C.O.; SOUZA, J.A.S.; CONDE-GARCIA, E.A.; SOUZA, A.A.; MENEZES-FILHO, J.E.R.; OLIVEIRA, E.D.; VASCONCELOS, C.M.L.* Laboratório de Biofísica do Coração, Departamento de Fisiologia Centro de Ciências Biológicas e da Saúde, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil. *Carla Vasconcelos, PhD, Cidade Universitária Prof. “José Aloísio de Campos”, Av. Marechal Rondon, s/n, Jardim Rosa Elze, CEP 49.100-000, São Cristóvão, Sergipe, Brasil. Tel.: +55 79 21056642, E-mail addresses: carlamlv@ufs.br, carlamlv@hotmail.com

Ginkgo biloba may be of great value in cases of age-related mental dysfunction (Kleijnen & Knipschild, 1992;Zhang et al., 2000;Maclennan et al., 2002) including senility, dementia (Le Bars et al., 1997), and diminished memory (Keenedy et al., 2000).Numerous preclinical studies have shown the neuroprotective effects of the standard extract EGb761 and support the notion that it may be effective in the treatment and prevention of neurodegenerative disorders such as Alzheimer's disease 2 .Anti-oxidative, anti-apoptosis, antiinflammation, protection against mitochondrial dysfunction, amyloidogenesis and amyloid beta peptide (Aβ) aggregation, ion homeostasis, modulation of phosphorylation of tau protein and even induction of growth factors are possible mechanisms of action (Cheng, 1996).
Several authors reported that therapeutic properties presented by EGb seems to be associated to its vasorelaxant action involving activation of the cellular nitric oxide pathway, antagonism of the platelet activating factor (PAF), anti-oxidant effect (Diamond et al., 2000), and/or the inhibition of Ca 2+ influx through the L-type Ca 2+ channel (Satoh & Nishida, 2004).Ginkgolide B (GB) could antagonize PAF receptors, preventing thus the ischemical damage of myocardial cells (Keeskemeti & Balogh, 2001).Experimental results obtained with GB revealed that it prevented cardiac arrhythmias induced by the ischemiareperfusion protocol (Wainwright et al, 1989;Koltai et al., 1989).
Wa s v e r i f i e d t h a t E G b ( 1 2 0 m g ) administrated during 7 days to healthy volunteers did not modify ECG parameters neither the heart rate, nor systolic or the diastolic blood pressures 16 .However, it was reported a case of a patient that used EGb and presented ventricular arrhythmias that only disappeared after discontinuing EGb (Cianfrocca et al., 2002).EGb and bilobalide (BB), used at high concentrations, usually produced cardiac arrhythmias and/or sinus arrest in rats (Satoh & Nishida, 2004).Recently, was reported that EGb in high concentrations reduced the contraction force in rat heart and decreased of mitochondrial respiratory function (Baliutyte et al., 2012).However, the authors don´t elucidate what was the compound bioactive responsible for this effect.Such findings encourage us to study the effect of EGb and some of its majoritaria terpenoids (ginkgolide A, ginkgolide B and bilobalide) on the eletrocardiographic parameters in guinea pig isolated heart, which can be responsible of the EGb effect.

Animals
The experiments were carried out on the guinea pig (Cavia porcellus) heart of male adult animals (300 to 500 g) of both genders.Animals had free access to food and water Ad libtum and were kept at room temperature (27 ± 3 o C) with 12 h of light-dark cycles.The research project was previously approved by the Ethical Animal Research Committee of the Federal University of Sergipe, according to the protocol #33/07.

Electrocardiographic parameters and heart rate measurements
The following electrocardiographic parameters were measured: PR interval (PRi), QT interval (QTi), and QRS complex duration (QRS).They were evaluated during the control situation (Tyrode + DMSO 0.5 %), test (EGb, GA, GB or BB diluted in DMSO 0.5 %), and washout (Tyrode + DMSO 0.5 %).The effect Ginkgo biloba constituents on the heart rate was studied on isolated heart allowed to beat spontaneously.To accomplish that, successive R-R intervals were determined during control, test, and washout (Calculate and WindaqEx softwares from Dataq, Akron, Ohio, USA).

Statistical analysis
Data were analyzed by using ANOVA one-way followed by the 'post hoc' test of Tukey.Differences were significant when p < 0.05.Results are presented as mean ± standard deviation.

RESULTS
The electrocardiographic (ECG) changes promoted by EGb (10 mg/ml), GA (50 µM), GB (70 µM), and BB (70 µM) on the PR interval (PRi) are illustrated in Fig. 1A.PRi is measured from the beginning of the P wave to beginning of the QRS complex and represent the time conduction of electrical impulse through atrioventricular node (AV node) (Azevedo, 1999).We results revealed that slow concentration of EGb (0.1 and 1.0 mg/ml) not evoked significant alteration on the ECG profile (data not shown).However, 10 mg/ml EGb was able to alter ECG parameters increasing significantly the PRi at 21 ± 2.0 % (p < 0.001).Furthermore, we also verified that of terpenoids tested only GA (20 ± 5.0 %, p < 0.001) and BB (13 ± 0.39 %, p<0.001) were able to increased this interval.On the other hand, the opposite was observed for GB that slightly decreased PRi at 6.0 ± 0.39 % (p < 0.001).It was found PRi changes promoted by EGb and GB were not restored to the control situation after the washing procedure.
Beyond evaluate PRi also we determined QT interval (QTi) in heart perfused by both EGb or terpenoids.The QTi is a measure of the time between the start of the Q wave and the end of the T wave in the heart's electrical cycle and represents electrical depolarization and repolarization of the left and right ventricles 22 .Ours results showed that the  16%, n = 6, p < 0.001), and from 218 ± 0.14 ms to 130 ± 0.73 ms (40 %, n = 6, p < 0.001), respectively.Moreover, unlike behavior was observed by EGb increasing QTi from 211 ± 0.11 ms to 223 ± 0.9 ms (6.0 %, n = 4, p < 0.001).These effects disappeared after removing Gingko biloba constituents from the perfusion fluid (Fig. 1B).QRS complex duration was not altered by EGb or by the terpenoids tested.Fig. 2A show ECG traces of spontaneous beating heart in control situation (top trace), with EGb (middle trace) and after washout (bottom trace).This results show that EGb induced increased of RR interval from 386 ms to 500 ms (29 %).This effect was partially reversible after 20 minutes of washout (426 ms).Fig. 2B summarizes the heart rate decrease promoted by both EGb and GB from 147 ± 15 bpm to 106 ± 22 bpm (28 %, n=4, p<0.05) and from 158 ± 18 bpm to 138 ± 21 bpm (13%, n=5, p<0.05), respectively.This bradycardia disappeared 20 minutes after initiating the washout.Neither GA nor BB modified the heart rate.
In isolated heart allowed to beat spontaneously, EGb (10 mg/ml) did not induce arrhythmia, but in preparations electrically it prolonged the PRi leading to a Wenkebach second degree AV block as seen in Fig. 3A.The tracing shows the PRi increasing progressively before appearing a complete AV block.This type of arrhythmia was observed in 40 % of hearts prefunded with EGb.GA (50 mM) on spontaneously beating heart induced the appearance of ventricular extrassystole (Panel B) and also was elicited third degree AVB with a multifocal ventricular rhythm (Panel C).BB evoked a third degree AVB as can be seen in panel D and Wenkebach periods (data not shown).The arrhythmias produced by EGb, GA, and BB disappeared during washout.

DISCUSSION
Pervious study evidenced that high concentration of Ginkgo biloba extract was able to elicit electrocardiographic changes in rat heart A B (Baliutyte et al., 2012).However, no correlation was provided between extract effect and its constituents.
It is known that the main biological effects of the extract of G. biloba are produced by the terpenoids present in its constitution.Furthermore, as EGb is a mixture of its constituents, the final pharmacological response of EGb would be result of complex interaction between the constituents probably by synergism action.Thus, we decided to compare the electrocardiographic effects of the extract with those presented by their major constituents on guinea pig isolated heart.One of the parameters of ECG measured was PR interval.This interval is the time (in seconds) from the beginning of the P wave (onset of atrial excitation) to the beginning of the QRS complex (onset of ventricular excitation).Is important to emphasize that PRi evaluate principally the electrical conduction through the atrioventricular (AV) node.
We results showed that EGb induced PRi interval.This findings corroborating with other studies (Baliutyte et al., 2012), that observed PRi increased induced by this extract.Furthermore, both GA and GB also induced PRi increased suggesting that both GA and BB can be responsible by enhance PRi induced by GBE.The increased PRi, also know as AV block of first degree, produced by EGb, GA, and BB showed that such compounds have a potential arrhythmogenic effect.This phenomenon occurs because they depressed the electrical conduction through the AV node.It´s well established that activity of calcium channels in AV nodal cells plays an important role for electrical impulse conduction (Conde-Garcia, 2002).The magnitude of the increase of L-type calcium current (I Ca,L ) determines the velocity of depolarization during the upstroke of an action potential in nodal cells, and thus the conduction velocity by AV nodal (Schram, 2002).Several authors showed that EGb (1 mg/ ml) markedly inhibited the I Ca,L in guinea pig and rat ventricular myocytes submitted to a voltage-clamp experiments (Satoh, 2003;Satoh & Nishida, 2004).The reduction of I Ca,L can be responsible for the AV block of first degree induced by EGb observed in our experiments.On the other hand, it´s was verified GA (Satoh & Nishida, 2004), GB (0.1-3 mM), and BB (1 mM) (Satoh, 2003;Satoh & Nishida, 2004) enhanced the I Ca,L and the delayed rectifier K + current (I K ).In cardiac muscle, it was evidenced that intracellular calcium increase can promote cellular decoupling by diminish the conductance of nexi (Burt & Spray, 1988).The ability of terpenoids to block calcium channels has been extensively in recent years (Magyar, 2004;Cerqueira et al., 2011).
Several authors use extracts plant at high concentration for studies in vitro and in vivo (Circosta et al., 2005;Menezes et al., 2007).This fact can be justified due the slow percentage of majoritarian compounds present in this extracts.Even at low concentrations, these constituents are extensively studied and present various biological effect (Singh et al., 2010).It´s was observed that changes in environmental conditions and other factors can influence in plant metabolism inducing difference in the levels of this majoritarian compound (Satoh, 2005).Was used 1 mg/ml of Ginkgo biloba extract in isolated ventricular cardiomyocyte submitted to patchclamp technique (Satoh & Nishida, 2004) and in the present study we used 0.1, 1.0 and 10 mg/ml because the experiments was carried out in isolated organ.In the present study, 10 mg/ml, this extract contains only approximately 3.5% of terpenoids of which are 1.8 % of ginkgolides and 1.69% of bilobalide.In recent study, authors use several concentration of Ginkgo biloba, but not show the concentration of extract in grams (mass unity), difficultly the comparison with our research (Baliutyte et al., 2012).
As noted in our experiments, the QTi was only increased by EGb.This result reflects an increase in the action potential duration (APD) probably by potassium channel block.In fact, it was demonstrated in guinea pig and in rat ventricular myocytes that EGb (1 mg/ml) prolonged the APD (Satoh, 2004;Satoh & Nishida, 2004).Simultaneously, these authors showed that EGb markedly inhibits delay rectifying K + current (I K ) and also the inward rectifying K + current (I K1 ) what could explain the prolonged QTi observed in our results.Unlike of extract, GA, GB, and BB decreased the QTi.This could be reflecting a shortening of the APD.Satoh & Nishida (2004) demonstrated in the guinea pig ventricular cardiomyocytes that GB (1 mM) and BB (3 mM) were able to shorten the APD and also increased I K .We have observed that those terpenoids shorten QTi that could be due to increase of I K .
In spite of the GA decrease the QTi, studies performed on the guinea pig ventricular cardiomyocytes showed that GA (1-3 mM) prolonged the APD, a fact that should increase the QTi in the ECG (Satoh & Nishida, 2004).This finding indicates that the prolonging QTi by EGb can't be attributable to the isolated action of a single terpenoid but to a synergistic action of its constituents which individually have an effect opposite to that shown by the extract.
We results showed that both EGb and GB were able to reduce pacemaker activity of the sinus node.It´s was demonstrated that EGb and BB can changes the pacemaking activity (Satoh, 2005).This author reported that EGb (0.03 mg/ml) and BB (0.3 mM) promote a negative chronotropic effect.Furthermore, EGb (0.03 mg/ml) decreased the slope of slow diastolic depolarization (SDD) (action potential phase 4) of sinus nodal cells and depressed their spontaneous activity in a concentration-dependent fashion.Moreover, at high concentration, EGb and BB often cause dysrhythmias and/or a sinus arrest (Satoh & Nishida, 2004).In sino-atrial nodal cells, submitted to patch-clamp techniques, it´s was demonstrated that the following currents were decreased by EGb (0.03 mg/ml) and BB (0.3 mM): a) I Ca,L ; b) I f , funny current (hyperpolarization-activated inward current); and c) I K .These results indicated that both EGb and BB can alter the cardiac ionic channel functioning in rat sino-atrial nodal cells (Satoh & Nishida, 2004).
Our findings showed that in guinea pig isolated heart EGb, GA and BB depress the conduction through atrioventricular node, leading to a several degrees of AVB (1st, 2nd, 3rd degrees).
Ours results indicate that these compounds would can have potential arrythmogenic confirming the clinical findings observed in patients making use of extracts of Ginkgo biloba.In addition, Ginkgo biloba effects on mammalian myocardium are due synergic actions of its major constituents.

FIGURE 2 .
FIGURE 2. Effects of EGb and its terpenoids on the guinea pig heart rate.Panel A shows ECG registers of the heart beating spontaneously in control (top trace), EGb (middle trace) and washout (bottom trace).Panel B represents the summary of the effects of EGb, GA, GB, and BB on the guinea pig rate (n = 5, *p < 0.001, 34 ± 0.1 o C).

FIGURE 3 .
FIGURE 3. Cardiac arrhythmias induced by EGb and its constituents in guinea pig isolated heart.Panel A: EGb (10 mg/ml) elicited Wenkebach periods characterized by PRi increased following atrioventricular block (AVB).The values in milliseconds relate the measurements of PRi.Third degree AVB with multifocal ventricular rhythm (Panel B) and isolated ventricular extrassystole (Panel C) induced by GA (50 mM) in spontaneously beating (on) guinea pig heart.Panel D: third degree AVB induced by BB (70 mM) on guinea pig isolated heart.S: stimulus artifact; P: p wave; Q: q wave (34 ± 0.1 o C).