Resumo

No presente trabalho investigou-se o efeito inotrópico do acetato de eugenil (AE), bem como sua ação sobre a corrente de Ca²⁺ do tipo L (I_Ca,L). Os experimentos de contratilidade foram realizados em átrio esquerdo isolado de cobaia exposto às concentrações crescentes da droga (1 a 5.000μM). O AE reduziu a força de contração atrial (IC₅₀=558±24,06μM) de modo dependente de concentração. O efeito do AE sobre a I_Ca,L também foi avaliado em cardiomiócitos ventriculares isolados de camundongos, utilizando-se a técnica de “patch-clamp”. O AE apresentou um efeito inibitório (IC₅₀=1.337±221μM) sobre os canais de Ca²⁺ sensíveis à voltagem (Ca_V1.2). Em conclusão, o AE apesenta efeito cardiodepressor que se deve, pelo menos em parte, à diminuição da entrada de Ca²⁺ nos cardiomiócitos.

Palavras-chave
Acetato de Eugenil; Contração Miocárdica; Syzygium Aromaticum, Ratos

Introduction

Cardiovascular diseases are a public health problem being among the leading causes of death worldwide.¹1. Wang H, Naghavi M, Allen C, Barber RM, Bhutta ZA, Carter A, et al. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980–2015: a systematic analysis for the Global Burden of Disease Study 201. The lancet. 2016; 388(10053):1459-544. In this perspective, there is a growing interest in the search for new substances with pharmacological actions on the cardiovascular system, including drugs of natural origin.

The Syzygium aromaticum (L.) Merr. & L.M. Perry, popularly known as clove, produces several chemical compounds that have a wide range of biological effects. Eugenol (Figure 1A) is the most abundant bioactive compound found in clove essential oil, followed by eugenyl acetate (EA) (Figure 1B).²2. Khalil AA, urRahman U, Khan MR, Sahar A, Mehmoodac T, Khan M. Essential oil eugenol: sources, extraction techniques and nutraceutical perspectives. RSC Advances. 2017;7(52):32669-81.

Studies have shown that eugenol has cardiodepressor activity in rats³3. Damiani CEN, Moreira CM, Zhang HT, Creazzo TL, Vassall DV. Effects of eugenol, an essential oil, on the mechanical and electrical activities of cardiac muscle. J Cardiovasc Pharmacol. 2004;44(6):688-95. and guinea pigs⁴4. Sensch O, Vierling W, Brandt W, Reiter M. Effects of inhibition of calcium di and potassium currents in guinea-pig cardiac contraction: comparison of β-caryophyllene oxide, eugenol, and nifedipine. Br J Pharmacol. 2000; 131(6):1089-96. probably due to the inhibition of the L-type Ca²⁺ current (I_Ca,L). In addition, eugenol acts as cardioprotector.⁵5. Choudhary R, Mishra KP, Subramanyam C. Interrelations between oxidative stress and calcineurin in the attenuation of cardiac apoptosis by eugenol. Mol Cell Biochem. 2006;283(1-2):115-22.

Although several studies have adressed the pharmacological properties of eugenol on mammalian heart, so far there is no information on the action of EA on the cardiomyocytes. Thus, this study describes the effects of EA on atrial contractility and its inhibitory action on I_Ca,L.

Methods

Animals

For contractility experiments guinea pigs (Cavia porcellus, both sexes, 400-600g) were used. For electrophysiological studies, adult male C57Bl/6J mice were used. All procedures were approved by the Ethics Committee for the Use of Animals (CEUA) of the University of the State of Bahia (license: 03/2017).

Experimental Protocols

Evaluation of EA Inotropic Effect

The left atrium from guinea pig was maintained in a modified Tyrode's solution (10 mL, 36.5±0.5°C) with the following composition (in mM): 140 NaCl; 5.4 KCl; 0.5 MgCl₂; 0.33 NaH₂PO₄; Glucose; 5 HEPES and 1.8 CaCl₂ (pH=7.4), aerated with oxygen (99.9%). The atrium was stretched to attain a resting tension of 1gF and it was electrically stimulated (2Hz, 100V, 15ms). The contractile force was captured by an isometric transducer. Signals were digitized (512Hz) and stored on a computer. The left atria were subjected to increasing concentrations of EA (1-5,000μM, 3 to 5 minutes for each tested concentration).

Dimethyl sulfoxide (DMSO) was used to make the stock solution of EA (obtained from Sigma-Aldrich).

Investigation of EA Effect on the L-type Calcium Current.

Ventricular cardiomyocytes from C57Bl/6J mice were isolated as previously described.⁶6. Shioya T. A simple technique for isolating healthy heart cells from mouse models. J Physiol Sci. 2007;57(6):327-35. The composition of the internal solution (in mM) was: 120 CsCl, 10 HEPES, 5 EGTA, 20 TEA-Cl, and 5 NaCl (pH=7.2; CsOH). Modified Tyrode was used as external solution. To measure L-type Ca²⁺ current (I_Ca,L) patch-clamp technique in whole-cell voltage-clamp mode was used.^7,8 Cells were maintained at a resting membrane potential of −80mV, then the sarcolemma was subjected to a pre-pulse to −40mV (50ms) followed by a pulse to 0 mV (300ms, 0.1Hz). The I_Ca,L amplitude was measured by the difference between the current at the end of the test pulse (0mV) and the peak current. The cells were exposed to EA (10-3,000 μM, 2-3 minutes for each concentration). The signals were digitized (5kHz) and stored on the computer.

Statistical Analysis

The results are expressed as mean ± standard error of the mean and were statistically analyzed using the two-tailed “t” test (significance level: p<0.05).

Results

EA Effect on the Left Atrial Contractility

Representative traces in Figure 1C show that EA (700 μM) reduced the amplitude of atrial contraction by approximately 60% when compared to the control. This effect was partially reversed (approximately 75%) after removing the drug (washout). In Figure 1D, it is possible to observe the EA concentration-effect curve on contractility (n = 4). EA presented an IC₅₀ (concentration that induces half of the maximum effect) of 558±24.06μM and a maximum effect = 100%.

EA Action on I_Ca,L

Figure 2A depicts a representative trace of the I_Ca,L measured in healthy isolated cardiomyocytes. Figure 2B shows the time course of the EA effect on the I_Ca,L. In Figure 2C, it is possible to see representative traces of I_Ca,L in the control (absence of the drug), and in the presence of 10, 700, and 3,000 μM of EA. Figure 2D demonstrates that exposure to EA reduced the amplitude of I_Ca,L in a concentration-dependent manner (IC₅₀=1,337±221μM).

Discussion

In our study we show that EA reduces the contraction force of guinea pig left atrial myocardium in a concentration-dependent manner. It was also observed that EA inhibits L-type Ca²⁺ channels (Ca_V1.2) in isolated cardiomyocytes from mice.

The EA showed a cardiodepressor effect on atrial inotropism. Although there are no data in the literature that demonstrate the negative inotropic effect of AE, information from its analogues, such as eugenol, is found. Similarly to the drug investigated here, Eugenol reduced the contraction force of ventricular cardiomyocyte from guinea pig⁵5. Choudhary R, Mishra KP, Subramanyam C. Interrelations between oxidative stress and calcineurin in the attenuation of cardiac apoptosis by eugenol. Mol Cell Biochem. 2006;283(1-2):115-22. and rat,⁴4. Sensch O, Vierling W, Brandt W, Reiter M. Effects of inhibition of calcium di and potassium currents in guinea-pig cardiac contraction: comparison of β-caryophyllene oxide, eugenol, and nifedipine. Br J Pharmacol. 2000; 131(6):1089-96. corroborating the data in our present study.

The cardiomyocyte contraction correlates with the increase in cytosolic Ca²⁺ concentration which is determined by the Ca²⁺ influx through Ca²⁺ channels present in the sarcolemma, as well by the amount of Ca²⁺ released by the sarcoplasmic reticulum (SR) in the process called excitation-contraction coupling. The membrane depolarization leads to the opening of the L-type Ca²⁺ channels mainly during the plateau phase of action potential leading to an inward Ca²⁺ current. This Ca²⁺ entry stimulates the release of Ca²⁺ stored in SR, a process known as Ca²⁺-induced Ca²⁺ release, which contributes to cardiac contractility.⁷7. Eisner DA, Caldwell JL, Kistamás K, Trafford AW. Calcium and excitation-contraction coupling in the heart. Circ Res. 2017;121(2):181-95. Thus, the mechanisms that alter the intracellular Ca²⁺ handling are involved in the control of cardiac contractility.

In order to explain the negative inotropic effect of EA on the cardiac muscle, the drug effect on the I_Ca,L amplitude was investigated in isolated cardiomyocytes. The findings described here show that EA reduces the peak of I_Ca,L. This effect may be associated with the activation of receptors that modulate I_Ca,L, and/or direct action of EA on Ca_V1.2. The reduction of I_Ca,L is able to explain the decrease of contractility induced by EA, since it would lead to a reduction in the release of Ca²⁺ from SR. Although additional mechanism may also be involved.

Sensch et al.,⁴4. Sensch O, Vierling W, Brandt W, Reiter M. Effects of inhibition of calcium di and potassium currents in guinea-pig cardiac contraction: comparison of β-caryophyllene oxide, eugenol, and nifedipine. Br J Pharmacol. 2000; 131(6):1089-96. studying the pharmacological properties of EA analogue eugenol, demonstrated that this substance depresses the force of atrial contraction by reduction Ca²⁺ influx in cardiomyocytes. In these experiments, it was observed that eugenol has an IC₅₀ of 127μM, a value lower than the IC₅₀ observed for AE (1,337μM). These data suggest that blocking effect of eugenol on Ca²⁺ channels is more potent than EA.

Conclusion

Eugenyl acetate has a cardiodepressor effect that can be explained, at least in part, by the inhibition of Ca_V1.2.

Acknowledgement

FAPESP 2014/09861-1 and PICIN/UNEB.

Referências

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