Protective Effects Of Anandamide On Heart And The Underlying Mechanisms

Endogenous cannabinoids (endocannabinoids), which are synthesized from lipid precursors in plasma membranes, are signaling lipids consisting of amides and esters of long chain fatty acids. Anandamide (N-arachidonoylethanolamide, AEA) is one of endocannabinoids that are derivatives of arachidonicacid, which are involved in many physiological and patho-physiological processes such as neurobehavior, gastrointestinal function, stress and anxiety, and cardiovascular functions. At least two types of cannabinoid receptors, the CB1 and CB2 has been found and cloned depending on their pharmacological effect and gene sequences. These two receptors are widespread in many tissues including cardiac myocyte. It has been shown that cannabinoids (CBs) are importantly involved in regulating cardiovascular function. For example, endocannabinoids have been shown to tonically suppress cardiac contractility in spontaneously hypertensive rat. Anandamide has been proved to protect the heart from adrenaline-induced arrhythmias or arrhythmias induced by ischaemia/reperfusion. Similarly, anandamide can limit the damage induced by ischaemia/reperfusion in rat isolated hearts through different mechanisms. However, the mechanism underlying for the anti-arrhythmias effects and anti-ischaemia–reperfusion injury of anandamide are not clear.The purpose of this study was to explore the protective effects of anandamide on rat heart and underlying mechanisms using electrophysiological, functional, immune histochemistry and flow cytometric techniques. Our study consisted of two parts: (1) To investigate the antiarrhythmic effect of anandamide in rat and the electrophysiological mechanism. (2) To investigate the cardiac protection of anandamide against ischemia/reperfusion injury in rat and the underlying mechanism. ⅠElectrophysiological mechanism for the antiarrhythmic effect of anandamide in ratObjective: Anandamide, one of endocannabinoids, has been reported to have anti-arrhythmia effect. The aim of present study was to study the electrophysiological mechanism for antiarrhythmic effect of anandmide.Methods: Arrhythmias were observed by using heart ischaemia/reperfusion experiment in vivo. Action potential (AP) (evoked) was recorded by using intracellular recording technique in rat cardiac papillary muscles. Also, L-type Ca2+ current and different K+ currents (including transient outward potassium current (Ito), steady-state outward potassium current (Iss), inward rectifier potassium current (IK1), ATP sensitive potassium current (IKATP)) were measured and analysised by using whole-cell patch-clamp recording technique in isolated rat ventricular myocytes.Results:(1) Anandamide (1μMkg-1) suppressed the ischaemia/reperfusion-induced ventricular arrhythmias in vivo heart.(2) In ventricular papillary muscles, anandamide (1, 10, 100 nM) decreased the AP duration (APD) in a concentration-dependent manner. Furthermore, 100 nM anandamide decreased action potential amplitude (APA), action potential overshoot (OS), and maximal rising velocity of depolarization in phase 0 (Vmax) in partially depolarized papillary muscles. These effects were abolished by AM251 (100 nM), a selective antagonist for CB1 receptor, but not AM630 (100 nM), a CB2 receptor antagonist. Also, a agonist of L-type Ca2+ channel Bay K8644 (0.5μM), a K+ channel blocker tetraethylammonium chloride (TEA, 20mM) and the nitric oxide synthase inhibitor L-NAME (1 mM) had no effect on anandamide-induced shorten of AP duration.(3) In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased L-type Ca2+ current concentration-dependently and shifted the current-voltage relationship curve of Ca2+ current upword. Anandamide (100 nM) shifted steady-state inactivation curve to the left while shifted the recovery curve to the right. Blockade of CB1 receptors with AM251 (100 nM), but not CB2 receptors with AM630 (100 nM), eliminated the effect of anandamide on L-type Ca2+ current.(4) In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased Ito and increased IKATP in a concentration-dependent manner, but had no effects on Iss and IK1. Anandamide shifted steady-state inactivation curve of Ito to the left, and shifted the recovery curve of Ito to the right. Blockade of CB1 receptors with AM251 and CB2 receptors with AM630, did not eliminate the inhibition effect of anandamide on Ito. Blockade of CB2 receptors, but not CB1 receptors, eliminated the augmentation effect of anandamide on IKATP.Conclusion: These data suggest that anandamide has a significant antiarrhythmic effect through activation of CB1 receptor, acceleration of inactivation and slowing of recovery from inactivation in L-type Ca2+ channel as well as decrease of L-type Ca2+ current, shorten of APD. Also anandamide augments IKATP through CB2 receptor, suppresses Ito through a non-CB1 and non-CB2 receptors pathway, acceleration of inactivation and slowing of recovery from inactivation in ventricular myocytes.ⅡCardiac protection of anandamide against ischemia/reperfusion injury and underlying mechanism in ratObjective: The aim of this study was to investigate the effects of anandamide (AEA) on cardiac function injury, infarct size and apoptosis induced by ischemia-reperfusion (I-R) in rat myocardium and underlying mechanism.Methods: In Langendorff isolated rat heart, cardiac function was recorded before and after 30 min global ischemia followed by 180 min reperfusion. At the end of reperfusion, the infarct size of the heart was measured by triphenyltetrazolium chloride (TTC) staining. TUNEL labeling and flow cytometric techniques were used for the measurement of apoptosis in cardiomyocytes of rat. Intracellular calcium ([Ca2+]i) was detected by confocal microscopy and represented by relative fluorescent intensity ((FI-FI0) /FI0, %).Results: (1) Anandamide (1, 10, 100 nM) displayed a better recovery of cardiac function and reduction of infarct size during reperfusion after ischemia in a concentration-dependent manner. The protective effects of anandamide were completely abolished by mitochondrial permeability transition pore (MPTP ) opener atractyloside (20μM), CB1 receptor antagonist AM251 (1μM) and CB2 receptor antagonist AM630 (1μM).(2) Anandamide significantly decreased the TUNEL-positive cells and apoptosis rate detected by flow cytometry. Compared with I/R group, Bax protein expressions of the myocardium decreased after administration of anandamide, while Bcl-2 protein expressions and Bcl-2/Bax ratio increased. The effects of anandamide on apoptosis were completely abolished by MPTP opener atractyloside (20μM), CB1 receptor antagonist AM251 (1μM) and CB2 receptor antagonist AM630 (1μM).(3) Anandamide (1, 10 and 100 nM) reduced [Ca2+]i of cardiomyocytes in normal Tyrode's solution, Ca2+-free Tyrode's solution and simulated ischemic solution in a concentration-dependent manner. In normal Tyrode's solution, inhibition of anandamide (100 nM) on [Ca2+]i was canceled by pretreatment with CB1 receptor antagonist AM251 (100nM) , but not by CB2 receptor antagonist AM630 (100nM). While in simulated ischemic solution, pretreatment of cells with AM630, not AM251, abolished the inhibitory effect of anandamide (100 nM) on [Ca2+]i. The augment of [Ca2+]i induced by L-type Ca2+ channel agonist Bay K8644 was inhibited by anandamide (100nM) in normal Tyrode's solution. The ryanodine-induced [Ca2+]i increase was markedly inhibited by anandamide (100nM) in Ca2+-free Tyrode's solution.Conclusion: Anandamide can promote the recovery of left ventricular function, reduce myocardial infarct size and inhibit the apoptosis induced by I-R through activation of both CB1 receptor and CB2 receptor, decrease of [Ca2+]i, and inhibition of MPTP open of rat ventricular myocytes. The decreasing effect of anandamide on [Ca2+]i may be related with inhibition of Ca2+ influx from L-type Ca2+ channel and releasing of Ca2+ from sarcoplasmic reticulum. The depression effect of anandamide on apoptosis may be related with increase of Bcl-2/Bax proteins expression in myocardium.