Carla Maria Lins de Vasconcelos

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.

Cardioprotective Action of Ginkgo biloba Extract against Sustained β-Adrenergic Stimulation Occurs via Activation of M2/NO Pathway

Ginkgo biloba is the most popular phytotherapic agent used worldwide for treatment of several human disorders. However, the mechanisms involved in the protective actions of Ginkgo biloba on cardiovascular diseases remain poorly elucidated. Taking into account recent studies showing beneficial actions of cholinergic signaling in the heart and the cholinergic hypothesis of Ginkgo biloba-mediated neuroprotection, we aimed to investigate whether Ginkgo biloba extract (GBE) promotes cardioprotection via activation of cholinergic signaling in a model of isoproterenol-induced cardiac hypertrophy. Here, we show that GBE treatment (100 mg/kg/day for 8 days, v.o.) reestablished the autonomic imbalance and baroreflex dysfunction caused by chronic β-adrenergic receptor stimulation (β-AR, 4.5 mg/kg/day for 8 days, i.p.). Moreover, GBE prevented the upregulation of muscarinic receptors (M2) and downregulation of β1-AR in isoproterenol treated-hearts. Additionally, we demonstrated that GBE prevents the impaired endothelial nitric oxide synthase activity in the heart. GBE also prevented the pathological cardiac remodeling, electrocardiographic changes and impaired left ventricular contractility that are typical of cardiac hypertrophy. To further investigate the mechanisms involved in GBE cardioprotection in vivo, we performed in vitro studies. By using neonatal cardiomyocyte culture we demonstrated that the antihypertrophic action of GBE was fully abolished by muscarinic receptor antagonist or NOS inhibition. Altogether, our data support the notion that antihypertrophic effect of GBE occurs via activation of M2/NO pathway uncovering a new mechanism involved in the cardioprotective action of Ginkgo biloba.

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.

(-)-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.

Negative inotropism of terpenes on guinea pig left atrium: structure-activity relationships

Abstract The aim of this work was to evaluate the pharmacological effect of seven structurally related terpenes on the contractility of cardiac muscle. The effect of terpenes was studied on isolated electrically driven guinea pig left atrium. From concentration–response curves for inotropic effect were determined the EC50 and relative potency of such terpenes. Our results revealed that all terpenes, except phytol, showed ability to reduce the contractile response of guinea pig left atrium. Further, relative potency was directly related to the number of isoprene units and to the lipophilicity of the compounds. For example, sesquiterpenes farnesol and nerolidol showed higher relative potency when compared with the monoterpenes citronellol, geraniol and nerol. We can conclude that most of the evaluated terpenes showed a promising negative inotropism on the atrial muscle. Future studies are necessary to investigate their action mechanism.

Resistance exercise mediates remote ischemic preconditioning by limiting cardiac eNOS uncoupling

BACKGROUND Currently viewed as a complementary non-pharmacological intervention for preventing cardiac disorders, long-term aerobic training produces cardioprotection through remote ischemic preconditioning (RIPC) mechanisms. However, RIPC triggered by acute exercise remains poorly understood. Although resistance exercise (RE) has been highly recommended by several public health guidelines, there is no evidence showing that RE mediates RIPC. Hence, we investigated whether RE induces cardiac RIPC through nitric oxide synthase (NOS)-dependent mechanism. METHODS AND RESULTS Acute RE at 40% of the maximal load augmented systemic nitrite levels, associated with increased cardiac eNOS phosphorylation, without affecting nNOS activity. Using an experimental model of myocardial infarction (MI) through ischemia-reperfusion (IR), RE fully prevented the loss of cardiac contractility and the extent of MI size compared to non-exercised (NE) rats. Moreover, RE mitigated aberrant ST-segment and reduced life-threatening arrhythmias induced by IR. Importantly, inhibition of NOS abolished the RE-mediated cardioprotection. After IR, NE rats showed increased cardiac eNOS activity, associated with reduced dimer/monomer ratio. Supporting the pivotal role of eNOS coupling during MI, non-exercised rats displayed a marked generation of reactive oxygen species (ROS) and oxidative-induced carbonylation of proteins, whereas RE prevented these responses. We validated our data demonstrating a restoration of physiological ROS levels in NE + IR cardiac sections treated with BH4, a cofactor oxidatively depleted during eNOS uncoupling, while cardiac ROS generation from exercised rats remained unchanged, suggesting no physiological needs of supplemental eNOS cofactors. CONCLUSION Together, our findings strongly indicate that RE mediates RIPC by limiting eNOS uncoupling and mitigates myocardial IR injury.