Isopulegol isomers block L-type calcium channel and attenuate the cardiac hypertrophy in rats: PKA and ERK 1/2 pathway

Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe - FAPITEC/SE Introduction: Cardiac hypertrophy (CH) is characterized by the remodeling of cardiac muscle associated with change in its contractile and electrical function. Isopulegol is an alcoholic monoterpene with antioxidant, anxiolytic and anticonvulsant activities. Objectives: To investigate the effects of (+)-isopulegol ((+) - ISP) and (-)-isopulegol ((-)-ISP) isomers on intracellular calcium signaling and cardiac hypertrophy induced by isoproterenol (ISO). Methods: The contractile effects of isomers were evaluated in rat left atrium mounted and ventricular cardiomyocyte of rat by shortening fraction using edge detection system (Ionoptix). In ventricular cardiomyocyte were evaluated the L-type calcium current (ICa,L) by patch-clamp technique in ‘whole-cell’ configuration and intracellular calcium transient by confocal microscopy (FLUO 4AM) was investigated in control situation and after incubation with 100 μM of both isomers. The docking was done to analysis the interaction of isomers with the L-type calcium channel. Cardiac hypertrophy was induced by administration of ISO (4.5 mg/kg, 7 days, i.p.). Four groups of animals were evaluated: 1) control (saline 0.9% + DMSO 0.1%), 2) isoproterenol (ISO), 3) ISO + (-)-ISP (50 mg/kg) and 4) ISO + (+)-ISP (50 mg/kg). In hypertrophic and treated animals were evaluated morphometric and electrocardiographic parameters, biochemical markers (LDH, CPK and CK-MB), oxidative stress (TBARS, SOD, CAT e GPx), inflammatory mediators (TNF-α and IL1-β), expression of proteins (PKA C-α, CAMKII, α/β CAMKII, ERK ½ total, p-ERK ½, SERCA, sarcalumenin, PP1γ e NCX. Results: showed that (-)-ISP (EC50 = 533.0 ± 53.80 μM) and (+)-ISP (EC50 = 1836 ± 165.71 μM) reduced the atrial contractility, of concentration dependent manner. In ventricular cardiomyocytes, after incubation with 100 μM of (-)-ISP and (+)-ISP was observed reduction of shortening fraction (40% and 28%) and ICa,L (68% and 59%), respectively. In addition, (-)-ISP e (+)-ISP reduced the intracellular calcium transient in 21% and 22%, respectively. The docking revealed the interaction of (-)-ISP and (+)-ISP with the L-type calcium channel with energies -65.84 Kcal/mol and -63.09 Kcal/mol. Hypertrophic animals presented increase of heart weight/body weight ratio (5.12 ± 0.09 mg/g, p < 0.05) as well as heart weight/tibia length ratio (364.20 ± 13.74 mg/cm, p < 0.05) in compared to control (3.52 ± 0.11 mg/g; 246.2 ± 6.33 mg/cm) and were attenuated with treatment with (-)-ISP (4.26 ± 0.11 mg/g and 299.40 ± 7.45 mg/cm) and (+)-ISP (4.42 ± 0.03 mg/g and 318.10 ± 3.24 mg/cm). (-)-ISP and (+)-ISPs were able to prevent electrocardiographic changes (increase of QRS, QTc and intrinsicoid deflection) and increase of serum levels of LDH, CPK and CPK-MB of hypertrophic animals. In addition, the treatment of hypertrophic animals with (-)-ISP and (+)-ISP the oxidative promoted decrease of TBARS and increase of SOD, CAT and GPx. Furthermore, inflammatory mediators TNF-α (in 59% and 40%) and IL1-β (in 34% and 55%) were reduced in the two groups treated with (-)-ISP and (+)-ISP, respectively. Treatment of animals with (-)-ISP and (+)-ISP decreased the overexpression of proteins involved in cardiac hypertrophy (PKA, ERK1/2, NCX), as well as prevented the decrease of SERCA and sarcalumelin expression. Conclusion: The isopulegol isomers block L-type calcium channels reducing the intracellular calcium transient in the ventricular cardiomyocyte exhibiting cardioprotective effect in isoproterenol-induced cardiac hypertrophy model Introdução: A hipertrofia cardíaca (HC) é caracterizada pelo remodelamento do tecido cardíaco acompanhada de alteração na sua função contrátil e elétrica. O isopulegol é um monoterpeno alcoólico com atividades antioxidante, ansiolítica e anticonvulsivante. Objetivos: Investigar os efeitos dos isômeros (+)-isopulegol ((+)-ISP) e (-)-isopulegol ((-)-ISP) na sinalização do cálcio intracelular e hipertrofia cardíaca induzida por isoproterenol (ISO). Métodos: Os efeitos contráteis dos isômeros foram avaliados em átrio esquerdo montado em cuba para órgão isolado e em cardiomiócito ventricular de rato pela fração de encurtamento usando sistema de detecção de bordas (Ionoptix). Em cardiomiócito ventricular isolado, foram avaliados a corrente de cálcio tipo-L (ICa,L) pela técnica de ‘patch-clamp’ na configuração ‘whole-cell’ bem como o transiente intracelular de cálcio usando a miscroscopia confocal (sonda FLUO 4AM) em situação controle e após a incubação com 100 μM dos isômeros do ISP. O docking foi feito para avaliar a interação dos isômeros com o canal para cálcio tipo-L. A HC foi induzida pela administração de ISO (4,5 mg/kg, 7 dias, i.p). Foram avaliados 4 grupos de animais: 1) controle (salina 0,9% + DMSO 0,1%), 2) isoproterenol (ISO), 3) ISO + (-)-ISP (50 mg/kg) e 4) ISO + (+)-ISP (50 mg/kg). Nos animais hipertróficos e tratados, foram avaliados os parâmetros morfométricos, eletrocardiográficos, marcadores séricos bioquímicos (LDH, CPK e CK-MB), estresse oxidativo (TBARS, SOD, CAT e GPx), mediadores inflamatórios (TNF-α e IL-1β), expressão de proteínas (PKA C-α, CAMKII, α/β CAMKII, ERK½ total, p-ERK½, SERCA, sarcalumelina, PP1γ e NCX). Resultados: o (-)-ISP (CE50 = 533,0 ± 53,80 μM) e o (+)-ISP (CE50 =1836 ± 165,71 μM) reduziram a contratilidade atrial, de modo dependente de concentração. Em cardiomiócito ventricular, após a incubação com 100 μM de (-)-ISP e (+)-ISP, foi observado tanto redução da fração de encurtamento (40% e 28%) quanto redução da ICa,L (em 68% e 59%), respectivamente. Além disso, os (-)-ISP e (+)-ISP reduziram o transiente intracelular do cálcio em 21% e 22%, respectivamente. O docking revelou a interação do (-)-ISP e (+)-ISP com o canal para cálcio do tipo-L com energias -65,84 Kcal/mol e -63,09 Kcal/mol, respectivamente. Os animais hipertróficos apresentaram aumento na relação peso do coração/peso corporal (5,12 ± 0,09 mg/g, p