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Pharmacological evaluation of R(+)-pulegone on cardiac excitability: role of potassium current blockage and control of action potential waveform.

ABSTRACT

Introduction: R(+)-pulegone is a ketone monoterpene and it is the main constituent of essential oils in several plants. Previous studies provided some evidence that R(+)-pulegone may act on isolated cardiac myocytes. In this study, we evaluated in extended detail, the pharmacological effects of R(+)-pulegone on cardiac tissue.

Methods: Using in vivo measurements of rat cardiac electrocardiogram (ECG) and patch-clamp technique in isolated myocytes we determinate the influence of R(+)-pulegone on cardiac excitability.

Results: R(+)-puIegone delayed action potential repolarization (APR) in a concentration-dependent manner ([EC.sub.50] = 775.7 [+ or -] 1.48, 325.0 [+ or -] 1.30, 469.3 [+ or -] 1.91 [micro]M at 10, 50 and 90% of APR respectively). In line with prolongation of APR R(+)-pulegone, in a concentration-dependent manner, blocked distinct potassium current components (transient outward potassium current ([I.sub.ss]), rapid delayed rectifier potassium current ([I.sub.Kr]), inactivating steady state potassium current ([I.sub.ss]) and inward rectifier potassium current ([I.sub.K1])) ([EC.sub.50] = 1441 [+ or -] 1.04; 605.0 [+ or -] 1.22, 818.7 [+ or -] 1.22; 1753 [+ or -] 1.09 [micro]M for [I.sub.to], [I.sub.Kr], [I.sub.ss] and [I.sub.K1], respectively). The inhibition occurred in a fast and reversible way, without changing the steady-state activation curve, but instead shifting to the left the steady-state inactivation curve ([V.sub.1/2] from -56.92 [+ or -]0.35 to -67.52 [+ or -] 0.19 mV). In vivo infusion of 100 mg/kg R(+)-pulegone prolonged the QTc(~40%)and PR(~62%) interval along with reducing the heart rate by ~26%.

Conclusion: Taken together, R(+)-pulegone prolongs the APR by inhibiting several cardiomyocyte [K.sup.+] current components in a concentration-dependent manner. This occurs through a direct block by R(+)-pulegone of the channel pore, followed by a left shift on the steady state inactivation curve. Finally, R(+)-pulegone induced changes in some aspects of the ECG profile, which are in agreement with its effects on potassium channels of isolated cardiomyocytes.

Keywords:

Pulegone

Myocyte

Heart

Electrocardiogram

Action potential

Potassium current

Introduction

Pharmacological characterization of essential oils and/or its constituents is becoming an area of vital interest mainly to delineate their potential toxic and therapeutic effects. R(+)-pulegone ((R)-2-isopropyIidine-5-methyl-cyclohexanone) (Fig. 1) is a ketone monoterpene and constitutes more than eighty percent of the essential oil of Mentha pulegium (also known as pennyroyal) (Gordon et al. 1982). The latter was reported to have relaxant effects on rat vascular smooth muscle (Guedes et al. 2004), isolated myometrium (Soares et al. 2005), isolated trachea and urinary bladder (Soares et al. 2012). Also organic extracts of Mentha pulegium have antispasmodic activity on rat ileum strips (Estrada-Soto et al. 2010). Despite the wide range of substances that comprise essential oils, usually only from one to three of their constituents account for between 20 and 70% of all them. These substances often determine the general biological activity of the essential oils they comprise (Ipek et al. 2005). Indeed, many studies have identified hepatotoxic effects in R(+)-pulegone containing essential oils (Anderson et al. 1996; Mizutani et al. 1987a, b; Thomassen et al. 1988).

[FIGURE 1 OMITTED]

Recently, the relaxant effects of R(+)-pulegone have been attributed to changes in membrane ion fluxes (Estrada-Soto et al. 2010; Soares et al. 2012). In fact, it was verified that R(+)-pulegone causes negative inotropism in mammalian myocardium that was related to blockage of L-type [Ca.sup.2+] current (de Cerqueira et al. 2011), which led to impaired cardiomyocyte mechanical function. The proper control of electrical properties of heart cells is of paramount important to their function. Thus, disturbing such equilibrium usually has a deleterious impact on cardiac physiology (Bassani et al. 2004; Bers 2008). Additionally, modulation of electrical activity of cardiac myocyte by natural occurring chemical compounds may present a therapeutic application, especially in the context of cardiac arrhythmias (Li et al. 2008). Furthermore, a more comprehensive pharmacological characterization of their effects may provide additional research tools to investigate ion channel function (de Araujo et al. 2011).

Despite the previous study conducted by (de Cerqueira et al. 2011) showing that R(+) pulegone impairs the cardiac contractility by reducing [Ca.sup.2+] and [K.sup.+] currents, so far there is no information about the effect of this drug on distinct [K.sup.+] channel subtypes expressed in cardiac myocytes. Additionally, it is not know the implications of those cellular effects on the in vivo cardiac electrophysiology profile. Here we describe and quantify the effects of R(+)-pulegone on the action potential (AP) parameters and on distinct [K.sup.+] current subtypes that contribute to the AP waveform in isolated cardiac myocytes. Furthermore, we investigated if these alterations can account for changes on the in vivo electrocardiographic (ECG) profile of rat heart.

Materials and methods

Chemicals and reagents

R(+)-Pulegone, protease type XXIII, porcine pancreas insulin, bovine serum), Cd[Cl.sub.2],TEA-Cl, NMDG, CsCl, NaCl, KC1, HEPES; EGTA, and K-aspartate were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Dimethyl sulphoxide (DMSO), Mg[Cl.sub.2], NaHC[O.sub.3], Ca[Cl.sub.2], Na[H.sub.2]P[O.sub.4], NaOH, and D-glucose, were bought from Vetec (Rio de Janeiro, Brazil) or Merck (Darmstadt Germany). Collagenase type II was acquired from Worthington Biochemical Co. (Freehold, NJ, USA). Ketamine Chloridrate and (Cetamin[R]) and Xilazina Chloridrate (Xilazin were bought from Syntec (Sao Paulo, Brazil). Heparin was a gift from Cristalia Laboratories Farmaceuticos.

Animals

Adults (250-350g) Wistar rats from both sex were used. The animals were maintained in a temperature controlled room on a 12-h light-dark cycle, with free access to food and water. All the experiments were performance according to Animal Research ethics committee of Federal University of Minas Gerais, Brazil and the European community guidelines.

Myocyte isolation

Adult ventricular myocytes were enzymatically isolated as described by (Shioya 2007). Myocytes were freshly isolated and stored in Tyrode' solution until they were used for experiments (within 6-8 h). Only calcium-tolerant, quiescent, rod-shaped myocytes showing clear cross striations were studied.

Patch clamp experiments

Whole-cell recordings were obtained using an EPC-9.2 patchclamp amplifier (HEKA Electronics, Rheinland-Pfalz, Germany), at room temperature (23-25[degrees]C). After the establishment of the whole-cell configuration, the cells were maintained for 3-5 min at rest to allow equilibration between the pipette solution and the intracellular medium. Current records were filtered at 2.9 kHz and digitally sampled at 2-10 kHz. Patch pipettes had tip resistances between 1.0 and 2.0 M[OMEGA], and myocytes presenting series resistance above 8.0 M[OMEGA] were not used in the analysis. During recording of the action potential (AP) and [K.sup.+] currents, cardiomyocytes were maintained in a Tyrode solution, containing (in mM) 140 NaCl, 5.4 KC1, 0.5 Mg[Cl.sub.2], 0.33 Na[H.sub.2]P[O.sub.4], 1.8 Ca[Cl.sub.2], 5 HEPES and 11 glucose, pH set at 7.4 with NaOH. Pipettes were filled with an internal solution containing (in mM) 130 K-aspartate, 20 KC1, 5 NaCl, 2 Mg[Cl.sub.2], 10 HEPES, and 5 EGTA, pH set at 7.2 with KOH.

To investigate the effects of R(+)-pulegone on the action potential, the holding potential was set to -80 mV. APs were elicited by short pulses (3-6 ms) of 1 nAcurrent at 1 Hz frequency. After 50 stable control pulses, R(+)-pulegone (from 0.01 to 3 mM) was perfused for 3 minutes, before washing out the drug. The dose-response curve was fitted according to the following equation;

y = A1 [A2 - A1]/[1 + [10.sup.-(log IC50-x)p]] (1)

where, A1 and A2 are the bottom and top asymptotes, logIC50 is the 'x' drug concentration which half of the biological effect was observed, and p is the Hill coefficient.

To investigate the effect of R(+)-pulegone on cardiac potassium currents it was applied a biphasic pulse, from a holding potential of -80 to -140 mV (for 2 s) following a depolarizing pulse to +50 mV (for 4 s), every 15 s. This protocol was used to investigate the effect of R(+)-pulegone on the inward and outward components of potassium current. Myocytes were perfused with a modified Tyrode solution, in which the NaCl was replaced by N-metyl-Dglucamine (NMDG) (to abolish sodium current), and 100 [micro]M Cd[Cl.sub.2] (to block L-type calcium current) was added. After the steady-state was achieved, R(+)-pulegone (between 0.01 and 3 mM) was applied until its steady state effect at a given concentration was achieved, followed by washout. The relative effect of each concentration was normalized and fitted by Eq. (1). The distinct subtypes of outward potassium currents were kinetically isolated: the transient outward K current ([I.sub.to], measured at the peak of the depolarizing pulse), delayed rectifier K current ([I.sub.Kr]/[I.sub.Ks], measured 500 ms after the peak of the depolarizing pulse), and the steady state non inactivating K current ([I.sub.ss], measured at the end of the depolarizing pulse) (Xu et al. 1999). To investigate the action curve of the potassium currents, cells were stepped from -120 mV to +70 mV (for 3 s) from a holding potential of -70 mV in steps of 10 mV, every 15 s. The protocol was followed before, during and after washing out the 1.1 mM R(+)-pulegone. Data points were fitted according to Eq. (2).

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (2)

where [G.sub.max] is the maximal conductance; [V.sub.m] is the test membrane potential. E; is the electrochemical equilibrium potential for the ion; [V.sub.0.5] is the membrane potential where 50% of the channels are activated and S is the slope factor

To investigate the voltage dependence of [I.sub.to] for inactivation, it was applied a two steps protocol. Cells were stimulated at a range from -70 mV to +20 mV (for 500 ms) in steps of 5 mV every 15 s, leaving from a holding potential of-70 mV. Immediately after each pre-conditioning pulse, a depolarizing test pulse at 50 mV (500 ms) was applied. The protocol was repeated before, during and after washing out 1.1 mM R(+)-pulegone. The currents evoked from the test pulse at +50 mV, relative to each pre-conditioning pulse, were then normalized to their respective maximum value and fitted according to a modified Boltzmann equation, described in Eq. (2).

Time dependent recovery from inactivation of [I.sub.to] was investigated by performing a two-step voltage clamp protocol, in which a first, activated and inactivating step of 50 mV (500 ms) was followed by a test pulse to 50 mV (500 ms), with a variable time between them (from 10 to 250 ms with steps of 10 ms from 10 to 100 ms and steps of 25 ms from 125 to 250 ms) keeping the cell at a holding potential of -70 mV. Test pulses were then normalized to their respective inactivating pulse (P2/P1). Data points were plotted as a function of two step-pulses interval and best fitted by a mono-exponential function described in Eq. (3)

y = [y.sub.0] + [Ae.sup.t/[tau]] (3)

where [y.sub.0] is the no inactivated current, A is the contribution of the inactivated component, t is time, and [tau] is the time constant. For all the recordings it has to be applied a -20 mV junction potential correction.

Electrocardiographic experiments

Animals were heparinized (200 U) and anesthetized with 80 mg/kg ketamine and 10 mg/kg xylazine, by the intra-peritoneal route. The rats were kept in the supine position with spontaneous breathing. The right jugular vein was cannulated for administration of vehicle (0.5% DMSO) and different concentrations of R(+)-pulegone (from 0.1 to 100 mg/kg). The volume injected through the jugular vein was the same for all concentrations (200 [micro]L). To record the ECG, three stainless steel electrodes were subcutaneously implanted. The ECG signals were amplified, digitized (DATAQ DI400, DI 205, Windaq PRO) and stored in a computer. The heart rate, PR interval (PRi), QT interval (QTi) and duration of the QRS complex were measured in 10 consecutive beats. As QTi varies inversely with the heart rate, it was corrected for the heart rate using Bazett's formula (QTc = QT/[square root]/RR) (Roguin 2011).

Statistics

Data were analyzed by Student's t-test or one way ANOVA following Tukey's post-test (as indicated in the figure legends). All tests were two tailed and p<0.05 was set as the significance level. The [EC.sub.50] was estimated using the Elill-Langmuir equation to fit the experimental points. All the results are expressed as the means [+ or -] Standard Error of the Means (S.E.M). Data analysis was carried out using Graph Pad Prism 5.0 (GraphPad Software, CA, USA) and Microsoft Excel 2007.

Results

R(+)-pulegone prolongs the AP in isolated ventricular myocyte in a concentration-dependent manner

In order to evaluate the effects of R(+)-puIegone on the electrical properties of cardiac myocytes, we first studied its effect on the action potential waveform of isolated ventricular myocytes. Fig. 2A shows that R(+)-pulegone had a dose-dependent effect on prolonging the time required for action potential repolarization (APR). Besides, as indicated by the dotted line, R(+)-pulegone induced hyperpolarization of the resting membrane potential. Fig. 2 (left panel) shows the time course of R(+)-pulegone effect on the time to reach 90% of APR and also on the resting membrane potential (right panel). Importantly, both effects were reversible upon washout suggesting that R(+)-pulegone interact to the cell membrane constituents in a non-covalent fashion.

The results shown in Fig. 2C indicate the dose-dependent effects of R(+)-pulegone on the time to reach 90% of APR (left) and on resting membrane potential (right). The [EC.sub.50] values were 775.7 [+ or -] 1.48, 325.0 [+ or -] 1.30 and 469.3 [+ or -] 1.91 [micro]M at 10, 50 and 90% of the APR (n = 6-10), respectively. The efficacy ([E.sub.max]) was determined as the maximum response achievable by the drug, and was 220.45 [+ or -] 31.35, 96.40 [+ or -] 17.75 and 66.62 [+ or -] 8.01 % at 10, 50 and 90% of the APR (n = 6-10), respectively. Furthermore, the [EC.sub.50] for the effect of R(+)-pulegone on the resting membrane potential was 971.4 [+ or -] 1.37 [micro]M (n = 6-10). The dose-dependent effect was also observed in both, the maximal rate of AP depolarization ([EC.sub.50] = 778.0 [+ or -] 7.70 [micro]M and [E.sub.max] = 41.54 [+ or -] 3.33%) and AP overshoot ([EC.sub.50] = 518.1 [+ or -] 1.37 [micro]M and [E.sub.max] = 33.23 [+ or -] 2.93%).

Differential effect of R(+)-pulegone on cardiomyocyte potassium currents

It is well known that the repolarization phase of cardiomyocyte AP is influenced by several subtypes of [K.sup.+] currents, each one having a distinct contribution (Nerbonne and Kass 2005). As R(+)-pulegone increases the time to APR, this effect could be attributed to a concentration-dependent effect of this drug on the [K.sup.+] currents. Four different subtypes of potassium current were isolated using a biphasic pulse; the inward rectifier [K.sup.+] current ([I.sub.K1]), transient outward [K.sup.+] current ([I.sub.to]), delayed rectifier [K.sup.+] current ([I.sub.Kr]), and the steady state non inactivating [K.sup.+] current ([I.sub.ss]). The outward components of [K.sup.+] current were isolated using an exponential adjustment, as proposed by (Xu et al. 1999). Fig. 3A depicts representative recordings before and during 1.1 mM R(+)-pulegone exposure. The [K.sup.+] current subtypes are indicated by arrows. The time course of the [K.sup.+] currents blockage (Fig. 3A, inset, similar to all studied [K.sup.+] current components) indicates that R(+)-pulegone takes ~45 s to attain its steady state effect, which was completely reversible within ~45 s after washing R(+)-pulegone off. We determined the dose-response curve of R(+)-pulegone for the distinct [K.sup.+] currents, as plotted in the Fig. 3B. The [IC.sub.50] values were 1441 [+ or -] 1.04, 605.0 [+ or -] 1.22, 818.7 [+ or -] 1.22 and 1753 [+ or -] 1.09 [micro]M for [I.sub.to], [I.sub.Kr], [I.sub.ss] and [I.sub.K1] blockade, respectively.

A set of mechanisms may be involved in the observed reduction of the distinct cardiac [K.sup.+] currents by R(+)-pulegone. To evaluate whether this reduction was related to changes in the fraction of channels available for opening at a given potential, we performed experiments to define the underlying mechanisms accounting for the voltage dependent behavior of the [K.sup.+] currents blockage. Fig. 4 summarizes the results. Fig. 4A provides representative records of the current evoked at tested potentials, before (top) and during (bottom) infusion of 1.1 mM R(+)-pulegone. Fig. 4B (top) shows that R(+)-pulegone reduced [I.sub.to] current density over a range of membrane potential (from +40 to +70 mV) ([sup.*]p< 0.05, n = 10). Similar results were obtained for the other aforementioned [K.sup.+] current components [I.sub.Ks] and [I.sub.ss] (data not shown). In order to investigate if the voltage dependent activation was altered, the current at a given potential was normalized by the maximal current (Fig. 4B, bottom). Adjusting the experimental data using Eq. (1) (see methods), we did not observe any difference in the [V.sub.1/2] and slope factor comparing before and after drug exposure (p > 0.05, n = 10).

[FIGURE 2 OMITTED]

R(+)-pulegone left-shifted the voltage dependence curve for steady-state inactivation of the transient outward If current

Despite the fact that R(+)-pulegone did not change the [K.sup.+] current voltage dependent activation curve, it may differentially modulate its inactivation component. It is known that [K.sup.+] current activation and inactivation are controlled by distinct biophysical mechanisms (Osteen et al. 2010). In rodents, [I.sub.to] is the main potassium current involved in AP repolarization (Nerbonne and Kass 2005) and it has a fast inactivation when compared to other [K.sup.+] currents expressed in cardiac tissue. Thus, changing [I.sub.to] inactivation properties and its recovery from inactivation may be one of the mechanisms underlying R(+)-pulegone effect. Fig. 5A shows representative recordings, before (upper panel), and after (bottom panel) 1.1 mM R(+)-pulegone exposure. As depicted in Fig. 5B, R(+)-pulegone left-shifted the [I.sub.to] inactivation curve (gray). According to Fig. 5C, R(+)-pulegone shifted the [V.sub.1/2] from -56.92 [+ or -] 0.35 mV to -67.52 [+ or -] 0.19 mV (n = 7, p < 0.05). However, R(+)-pulegone had no effect on the slope factor (K) (p > 0.05). On the other hand, the recovery from inactivation was not changed as indicated in Fig. 6. Fig. 6A shows representative recordings before (upper panel) and after (bottom panel) R(+)-pulegone exposure. Fig. 6B depicts the normalized current recorded from the test pulse (P2) divided by the preconditioning pulse (PI), before (black curve) and after R(+)-pulegone exposure (gray curve). The curve was adjusted using Eq. (3). The time constant ([tau]) was unchanged by R(+)-pulegone (25.2 vs. 24.4 ms, before and after R(+)-pulegone exposure, respectively, p > 0.06, n = 6).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

R(+)-pulegone modulates the electrocardiographic profile in rats

In vivo ECG measurements were performed to correlates the cellular to the whole organ effects of R(+)-pulegone. It is well known that the QT interval measured on the ECG is an indicative of action potential duration of ventricular myocytes (Shimizu and Antzelevitch 1997). Fig. 7 A shows representative ECG recordings before (top) and after (bottom) administration of 100 mg/kg R(+)-pulegone. Fig. 7B summarizes the observed changes in ECG parameters. At 100 mg/kg, R(+)-pulegone prolonged the PR and QTc intervals by around 62 and 40%, respectively (p<0.05, n = 7). On the other hand, the heart rate declined in a dose-dependent manner, with significant effects occurring at 10 mg/kg (17.72 [+ or -] 7.03%, p<0.05, n = 7) and 100mg/kg (26.30 [+ or -] 2.75%, p<0.05, n = 7) of R(+)-pulegone. However, the QRS complex amplitude remained unchanged for all tested doses (p > 0.05, n = 7).

Discussion

In this study, we correlated the effects of R(+)-pulegone on cardiac muscle excitability by measuring the electrophysiological parameters in isolated cardiomyocytes and in the ECG of rats. Such correlation is relevant since cardiac function requires proper control of the electrical properties of cardiomyocytes, which relies on the control of ion channel function. Aberrant changes on ion channel activity may contribute to the genesis of cardiac arrhythmia resulting in a decline of cardiac function (George 2013; Shimizu and Antzelevitch 1997, 1999). This concept drives the efforts to identify novel drugs that can modulate ion channel activity and eventually be applied in the clinical control of cardiac arrhythmias. Based on that, we investigated in details the effects of a natural occurring compound, R(+)-pulegone, on the electrical properties of the heart. In brief, we found that R-pulegone in a concentration dependence manner caused (1) lengthening of the AP duration, (2) reduction of distinct subtypes of the K+ currents, (3) negative shift in the voltage dependent inactivation of [I.sub.to], (4) prolongation of the QT and PR intervals, and attenuation of heart rate.

It is described in the literature that several natural occurring compounds can block different types of potassium channel. For instance cyclovirobuxine-D and dauricine inhibit HERG [K.sup.+] channel, preferentially when the channel is found on its open state (Zhao et al. 2012). Interestingly, Shensong Yangxin, a mix of 12 distinct natural occurring compounds reduces and left-shifts the voltage dependent of the inactivation of [I.sub.to] (Li et al. 2007), which is similar to the R(+)-pulegone effect on the very same [K.sub.+] current. We found that R(+)-pulegone left-shifts the voltage dependent inactivation curve of [I.sub.to], although it has no effect on both the recovery from inactivation, and voltage dependent activation of [I.sub.to]. R(+)-pulegone also acted in a reversible manner, which indicates a non-covalent interaction to the channel. Taken together, these results suggest at least two mechanisms involved in the blockage of Ito by R(+)-pulegone: direct channel modulation and left shift in the inactivation curve.

In another study anandamide, an endogenous cannabinoid receptor agonist had similar effects to those found for R(+)+ pulegone. In both cases, it was observed the drug was able to block [I.sub.to] current, reducing the peak current amplitude and left-shift the inactivation curve with no effect on its slope (Li et al. 2012). Furthermore, anandamide activated the ATP-sensitive [K.sup.+] current (KATP), which is known to induce membrane hyperpolarization in cardiac myocyte and cardioprotection upon its activation (Baczko et al. 2004). It was shown that anandamide was not able to blockage [I.sub.K1], which is an important component involved in the control of resting membrane potential in cardiac myocytes (Dhamoon and Jalife 2005; Hibino et al. 2010). Thus, we may speculate that R(+)-pulegone may activate KATP channels and induce membrane hyperpolarization (Li et al. 2012).

R(+)-Pulegone prolonged the QTc interval in the ECG. This QTc interval prolongation is in agreement with the lengthening of action potential duration due to decline in [K.sup.+] current density in the presence of R(+)-pulegone. Slowing heart rate was also observed under administration of R(+)-Pulegone and it is consistent with [Ca.sup.2+] channel blockage and resting potential hyperpolarization. In fact, the latter would lead to increased threshold to trigger an action potential. In line with the present hypothesis a previous study showed that R(+)-pulegone was able to block L-type [Ca.sup.2+] channels (de Cerqueira et al. 2011).

[FIGURE 7 OMITTED]

R(+)-pulegone also prolonged the PR interval. This interval is characterized as the time necessary for the electrical impulse to be conducted from the sinoatrial node (SAN) to the ventricles. Prolongation of the PR interval is interpreted as 1st degree atrioventricular (AV) block, which can result from delays in impulse conduction from the atria to the AV node and/or the His-Purkinje system (Bexton and Camm 1984). Typically the depolarization phase of the action potential in cardiac cells is mediated by increases in [Na.sup.+] influx, mainly through the Nav 1.5, the cardiac subtype of [Na.sup.+] channel (Terrenoire et al. 2007). On the other hand, conduction through the SAN and AV node is partially determined by [Ca.sup.2+] influx specifically via Cav1.2 channels, whose pore domain patency determines the L-type [Ca.sup.2+] current (Maltsev and Lakatta 2008). Thus, a possible explanation for the observed prolongation of the PRi is that R(+)-pulegone reduces the [Ca.sup.2+] current, which might reduce the conduction through the AV node (Yamamoto et al. 1998). Furthermore, [Na.sup.+] channel blockage may lead to PR interval prolongation (Pugsley et al. 1998). In fact, we observed a reduced in the maximal rate for depolarization during R(+)-pulegone application, which is an indicative of Na+ channel blockage. Thus, the molecular mechanism involved in the prolongation of PRi by R(+)-pulegone is only partially elucidated.

Overall, this study demonstrates that R(+)-pulegone increases the AP duration in a concentration-dependent manner, specially due to blockage of distinct subtypes of K+ currents on isolated ventricular cardiomyocytes. We also demonstrate that the present cellular effects may be responsible for the changes induced by R(+)-pulegone on in vivo electrophysiological parameters studied in the ECG of rats.

ARTICLE INFO

Article history:

Received 23 February 2014

Received in revised form 10 April 2014

Accepted 11 May 2014

Acknowledgment

CAPES, FAPEMIG, CNPq and FAPITEC/SE.

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Artur Santos-Miranda (b), Antonio Nei Gondimb (c), Jose Evaldo Rodrigues Menezes-Filho (d), Carla Marina Lins Vasconcelos (d), Jader Santos Cruz (b,1), Danilo Roman-Campos (3,*,1)

(a) Departamento de Biofisica, Universidade Federal de Sao Paulo/Escola Paulista de Medicina, Sdo Paulo, Brazil

(b) Departamento de Bioqulmica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Minas Gerais, Brazil

(c) Laboratorio Laboratorio de Biofisica e Farmacologia do Corapao, Departamento de Educapao--Campus XII, Universidade do Estado da Bahia, Guanambi, Bahia, Brazil

(d) Laboratorio de Biofisica do Corapao, Departamento de Fisiologia, Universidade Federal de Sergipe, Aracaju, Sergipe, Brazil

(1) These authors contributed equally to this work.

* Corresponding author. Tel.: +55 11 5576 4848 branch 2350.

E-mail addresses: drcbio@gmail.com, drcampos@unifesp.br (D. Roman-Campos).

http://dx.doi.org/10.1016/j.phymed.2014.05.007
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Author:Santos-Miranda, Artur; Gondim, Antonio Nei; Menezes-Filho, Jose Evaldo Rodrigues; Vasconcelos, Carla
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Aug 27, 2014
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