Antônio Nei Santana Gondim

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca2+ current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2±75.7 μg/ml. Nifedipine, a well known L-type Ca²+ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1±3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4±56.2 μg/ml. Whole-cell L-type Ca²+ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I(Ca,L) by 32.6±9.2% and at 250 μg/ml it decreased by 89.3±7.4%. Thus, inhibition of L-type Ca²+ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.

Functional Cross-Talk Between Aldosterone and Angiotensin-(1-7) in Ventricular Myocytes

High serum levels of aldosterone have been linked to the development of cardiac disease. In contrast, angiotensin (Ang)-(1-7) was extensively shown to possess cardioprotective effects, including the attenuation of cardiac dysfunction induced by excessive mineralocorticoid activation in vivo, suggesting possible interactions between these 2 molecules. Here, we investigated whether there is cross-talk between aldosterone and Ang-(1-7) and its functional consequences for calcium (Ca2+) signaling in ventricular myocytes. Short-term effects of aldosterone on Ca2+ transient were assessed in Fluo-4/AM-loaded myocytes. Confocal images showed that Ang-(1-7) had no effect on Ca2+ transient parameters, whereas aldosterone increased the magnitude of the Ca2+ transient. Quite unexpectedly, addition of Ang-(1-7) to aldosterone-treated myocytes further enhanced the amplitude of the Ca2+ transient suggesting a synergistic effect of these molecules. Aldosterone action on Ca2+ transient amplitude was mediated by protein kinase A, and was related to an increase in Ca2+ current (ICa) density. Both changes were not altered by Ang-(1-7). When cardiomyocytes were exposed to aldosterone, increased Ca2+ spark rate was measured. Ang-(1-7) prevented this change. In addition, a NO synthase inhibitor restored the effect of aldosterone on Ca2+ spark rate in Ang-(1-7)-treated myocytes and attenuated the synergistic effect of these 2 molecules on Ca2+ transient. These results indicate that NO plays an important role in this cross-talk. Our results bring new perspectives in the understanding of how 2 prominent molecules with supposedly antagonist cardiac actions cross-talk to synergistically amplify Ca2+ signals in cardiomyocytes.

R(+)-pulegone impairs Ca2+ homeostasis and causes negative inotropism in mammalian myocardium

The present study aimed to investigate the inotropic effects of R(+)-pulegone, a monoterpene found in plant species belonging to the genus Mentha, on the mammalian heart. In electrically stimulated guinea pig atria, R(+)-pulegone reduced the contractile force (~83%) and decreased the contraction time measured at 50% of the maximum force amplitude (CT(50)) from 45.8 ± 6.2 ms to 36.9 ± 6.2 ms, suggesting that R(+)-pulegone may have an effect on Ca(2+) homeostasis. Nifedipine (40 μM), taken as a positive control, showed a very similar profile. To explore the hypothesis that R(+)-pulegone is somehow affecting Ca(2+) handling, we determined concentration-response curves for both CaCl(2) and BAY K8644. R(+)-pulegone shifted these curves rightward. Using isolated mouse ventricular cardiomyocytes, we measured whole-cell L-type Ca(2+) current and observed an I(Ca,L) peak reduction of 13.7 ± 2.5% and 40.2 ± 2.9% after a 3-min perfusion with 0.11 and 1.1mM of R(+)-pulegone, respectively. In addition, the intracellular Ca(2+) transient was decreased (72.9%) by 3.2mM R(+)-pulegone, with no significant changes in [Ca(2+)](i) transient decay kinetics. Moreover, R(+)-pulegone at 1.1mM prolonged the action potential duration at 10, 50, and 90% of repolarisation. The lengthening of the action potential duration may be attributed to the substantial blockade of the outward K(+) currents caused by 1.1mM of R(+)-pulegone (90.5% at 60 mV). These findings suggest that R(+)-pulegone exerts its negative inotropic effect on mammalian heart mainly by decreasing the L-type Ca(2+) current and the global intracellular Ca(2+) transient.

Geraniol Blocks Calcium and Potassium Channels in the Mammalian Myocardium: Useful Effects to Treat Arrhythmias

Geraniol is a monoterpene present in several essential oils, and it is known to have a plethora of pharmacological activities. In this study, we explored the contractile and electrophysiological properties of geraniol and its antiarrhythmic effects in the heart. The geraniol effects on atrial contractility, L‐type Ca2+ current, K+ currents, action potential (AP) parameters, ECG profile and on the arrhythmia induced by ouabain were evaluated. In the atrium, geraniol reduced the contractile force (~98%, EC = 1,510 ± 160 μM) and diminished the positive inotropism of CaCl2 and BAY K8644. In cardiomyocytes, the ICa,L was reduced by 50.7% (n = 5) after perfusion with 300 μM geraniol. Moreover, geraniol prolonged the AP duration (APD) measured at 50% (n = 5) after repolarization, without changing the resting potential. The increased APD could be attributed to the blockade of the transient outward K+ current (Ito) (59.7%, n = 4), the non‐inactivation K+ current (Iss) (39.2%, n = 4) and the inward rectifier K+ current (IK1) (33.7%, n = 4). In isolated hearts, geraniol increased PRi and QTi without affecting the QRS complex (n = 6), and it reduced both the left ventricular pressure (83%) and heart rate (16.5%). Geraniol delayed the time to onset of ouabain‐induced arrhythmias by 128%, preventing 30% of the increase in resting tension (n = 6). Geraniol exerts its negative inotropic and chronotropic responses in the heart by decreasing both L‐type Ca2+ and voltage‐gated K+ currents, ultimately acting against ouabain‐induced arrhythmias.

Aqueous leaf extract of Averrhoa carambola L. (Oxalidaceae) reduces both the inotropic effect of BAY K 8644 on the guinea pig atrium and the calcium current on GH3cells

It was previously showed that aqueous leaf extract (AqEx) of Averrhoa carambola depresses the guinea pig atrial inotropism. Therefore, experiments were carried out on guinea pig left atrium and on pituitary GH3 cells in order to evaluate the effect of AqEx on the cellular calcium influx. The atrium was mounted in an organ chamber (5 mL, Tyrode, 27 ± 0.1 oC, 95 % O2, 5 % CO2), stretched to 10 mN, and paced at 2 Hz (0.5 ms, 400 V) and GH3 cells were submitted to a whole cell voltage clamp configuration. In the atrium, the AqEx (1500 µg/mL) shifted to the right the concentration-effect curve of the positive inotropic effect produced by (±) BAY K 8644, an L-type calcium channel agonist. The AqEx increased EC50 (concentration required to promote 50% of the maximum effect) of the inotropic effect of BAY K 8644 from 7.8 ± 0.38 to 115.1 ± 0.44 nM (N = 3; p < 0.05). In GH3 cells assayed with 500 µg/mL of AqEx, the L-type calcium inward current declined 30 % (from 282 to 190 pA). Nevertheless, the extract did not change the voltage correspondent to the peak current. These data suggest that, at least in part, the negative inotropic effect of AqEx on the guinea pig atrium is due to a reduction of the L-type calcium current.

Aqueous fraction from Costus spiralis (Jacq.) Roscoe leaf reduces contractility by impairing the calcium inward current in the mammalian myocardium

ETHNOPHARMACOLOGICAL RELEVANCE Brazilian folk medicine uses infusion of Costus spiralis leaf to help people to treat arterial hypertension and syndromes of cardiac hyperexcitability. AIM OF THE STUDY Evaluate the aqueous fraction (AqF) effect on atrial contractility and investigate its mechanism of action. MATERIALS AND METHODS The AqF effect on the cardiac contractility was studied on isolated electrically driven guinea pig left atria. Atropine and tetraethylammonium (TEA) were employed to investigate whether potassium contributes for the inotropic mechanism of the AqF. The role of calcium in this effect was also studied. This was done by analysing the AqF effect on the Bowditchs phenomenon, as well as by studying whether it could interfere with the concentration-effect curve for CaCl(2), isoproterenol, and BAY K8644. Mice isolated cardiomyocytes were submitted to a whole-cell patch-clamp technique in order to evaluate whether the L-type calcium current participates on the AqF effect. Furthermore, the intracellular calcium transient was studied by confocal fluorescence microscopy. RESULTS AqF depressed the atrial contractile force. It was the most potent fraction from C. spiralis leaf (EC(50)=305 ± 41 mg/l) (crude extract: EC(50)=712 ± 41; ethyl acetate: EC(50)=788 ± 121; chloroform: EC(50)=8,948 ± 1,346 mg/l). Sodium and potassium content in the AqF was 0.15 mM and 1.91 mM, respectively. Phytochemical analysis revealed phenols, tannins, flavones, xanthones, flavonoids, flavonols, flavononols, flavonones, and saponins. Experiments with atropine and TEA showed that potassium does not participate of the inotropic mechanism of AqF. However, this fraction decreased the force overshoot characteristic of the Bowditchs phenomenon, and shifted the concentration-response curve for CaCl(2) (EC(50) from 1.12 ± 0.07 to 7.23 ± 0.47 mM) indicating that calcium currents participate on its mechanism of action. Results obtained with isoproterenol (1-1,000 pM) and BAY K8644 (5-2000nM) showed that AqF abolished the inotropic effect of these substances. On cardiomyocytes, 48mg/l AqF reduced (∼23%) the L-type calcium current density from -6.3 ± 0.3 to -4.9 ± 0.2 A/F (n=5 cells, p<0.05) and reduced the intracellular calcium transient (∼20%, 4.7 ± 1.2 a.u., n=42 cells to 3.7 ± 1.00 a.u., n=35 cells, p<0.05). However, the decay time of the fluorescence was not changed (control: 860 ± 32 ms, n=42 cells; AqF: 876 ± 26 ms, n=35 cells, p>0.05). CONCLUSIONS The AqF of C. spiralis leaf depresses myocardial contractility by reducing the L-type calcium current and by decreasing the intracellular calcium transient. Despite the lack of data on the therapeutic dose of AqF used in folk medicine, our results support, at least in part, the traditional use of this plant to treat cardiac disorders.

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

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(+)-pulegone delayed action potential repolarization (APR) in a concentration-dependent manner (EC50=775.7±1.48, 325.0±1.30, 469.3±1.91 μM 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(to)), rapid delayed rectifier potassium current (I(Kr)), inactivating steady state potassium current (I(ss)) and inward rectifier potassium current (I(K1))) (EC50=1441±1.04; 605.0±1.22, 818.7±1.22; 1753±1.09 μM for I(to), I(Kr), I(ss) and I(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 (V1/2 from -56.92±0.35 to -67.52±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(+) 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.

(-)-Terpinen-4-ol changes intracellular Ca2+ handling and induces pacing disturbance in rat hearts

Abstract (‐)‐Terpinen‐4‐ol is a naturally occurring plant monoterpene and has been shown to have a plethora of biological activities. The objective of this study was to investigate the effects of (‐)‐terpinen‐4‐ol on the rat heart, a key player in the control and maintenance of arterial blood pressure. The effects of (‐)‐terpinen‐4‐ol on the rat heart were investigated using isolated left atrium isometric force measurements, in vivo electrocardiogram (ECG) recordings, patch clamp technique, and confocal microscopy. It was observed that (‐)‐terpinen‐4‐ol reduced contraction force in an isolated left atrium at millimolar concentrations. Conversely, it induced a positive inotropic effect and extrasystoles at micromolar concentrations, suggesting that (‐)‐terpinen‐4‐ol may have arrhythmogenic activity on cardiac tissue. In anaesthetized animals, (‐)‐terpinen‐4‐ol also elicited rhythm disturbance, such as supraventricular tachycardia and atrioventricular block. To investigate the cellular mechanism underlying the dual effect of (‐)‐terpinen‐4‐ol on heart muscle, experiments were performed on isolated ventricular cardiomyocytes to determine the effect of (‐)‐terpinen‐4‐ol on L‐type Ca2+ currents, Ca2+ sparks, and Ca2+ transients. The arrhythmogenic activity of (‐)‐terpinen‐4‐ol in vitro and in vivo may be explained by its effect on intracellular Ca2+ handling. Taken together, our data suggest that (‐)‐terpinen‐4‐ol has cardiac arrhythmogenic activity. Graphical abstract Figure. No Caption available.

Dissection of the Effects of Quercetin on Mouse Myocardium

Quercetin is a plant flavonoid with several biological activities. This study aimed to describe quercetin effects on contractile and electrophysiological properties of the cardiac muscle as well as on calcium handling. Quercetin elicited positive inotropism that was significantly reduced by propranolol indicating an involvement of the sympathetic nervous system. In cardiomyocytes, 30 μM quercetin increased ICa,L at 0 mV from −0.95 ± 0.01 A/F to −1.21 ± 0.08 A/F. The membrane potential at which 50% of the channels are activated (V0.5) shifted towards more negative potentials from −13.06 ± 1.52 mV to −19.26 ± 1.72 mV and did not alter the slope factor. Furthermore, quercetin increased [Ca2+]i transient by 28% when compared to control. Quercetin accelerated [Ca2+]i transient decay time, which could be attributed to SERCA activation. In resting cardiomyocytes, quercetin did not change amplitude or frequency of Ca2+ sparks. In isolated heart, quercetin increased heart rate and decreased PRi, QTc and duration of the QRS complex. Thus, we showed that quercetin activates β‐adrenoceptors, leading to increased L‐type Ca2+ current and cell‐wide intracellular Ca2+ transient without visible changes in Ca2+ sparks.

The positive inotropic effect of the ethyl acetate fraction from Erythrina velutina leaves on the mammalian myocardium: the role of adrenergic receptors

We studied the effects of ethyl acetate fraction (EAcF) obtained from Erythrina velutina leaves on mammalian myocardium.