Mechanisms of cardiac ryanodine receptor inhibition by anti arrhythmic drugs

Mehra, Divya Rajendra

Title: Mechanisms of cardiac ryanodine receptor inhibition by anti arrhythmic drugs
Creator: Mehra, Divya Rajendra
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2014
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: The cardiac ryanodine receptors (RyR2) are the calcium release channel in the sarcoplasmic reticulum (SR). Mutations in RyR2 or calsequestrin are known to cause Catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmia occurs in periods of emotional stress or exercise. Previous work showed that a Class I anti arrhythmic drug, flecainide blocked RyR2, thus reducing the spontaneous Ca²⁺ release that causes arrhythmia in CPVT. Tetracaine is a classical RyR2 blocker and it might be expected that block of RyR2 via tetracaine could potentially be a solution to arrhythmia due to SR overload. However, contrary to this tetracaine is pro-arrhythmic. This project investigates the mechanisms of RyR2 inhibition by anti arrhythmic drugs with a view to identifying inhibitory mechanisms that are antiarrhythmic. I investigated RyR2 inhibition including nine Class I agents-Na⁺ channel blockers, Class II-β blocker carvedilol, Class IV-Ca²⁺ channel blocker verapamil and K201 (JTV519). RyR2 was isolated from sheep heart, incorporated in lipid bilayers and investigated by single-channel recordings in presence of diastolic Ca²⁺ (100 nM cytoplasmic) and systolic Ca²⁺ (100μM cytoplasmic). All drugs showed inhibition from both cytoplasmic and luminal sides of the membrane consistent with the ability of the drugs to permeate through the bilayer. Two inhibition modes on RyR2 with distinct kinetics were detected, 1) induction of brief closed events with a mean duration of ~ 0.5-4 ms referred to as the fast block, 2) induction of long closed events with a mean duration of ~ 20-600 ms referred as the slow block. Binding rates for both forms of block were proportional to concentration and unbinding rates were concentration independent, consistent with bimolecular binding. Drug binding was strongly voltage-dependent (more potent at positive membrane potential) consistent with movement of the cation form of the drug in the trans membrane electric field. All drugs showed fast block RyR2 but they varied substantially in their potency. The association rates of the drugs fell into two broad classifications. Group A: Flecainide, propafenone, quinidine, encainide, verapamil, K201 and carvedilol have a fast association (exceeding a threshold of 2μM^-1s^-1). The association rates for Group B: mexiletine, procainamide, disopyramide, pilsicainide and tocainide lie below this threshold. Drugs in Group A were seen in previous studies to reduce SR Ca²⁺ release while those in Group B did not. Moreover, the potency of drug block of RyR2 correlated with their IC₅₀ for Ca²⁺ wave supression, spontaneous Ca²⁺ wave rate and the proportion of animals with ventricular tachycardia after exercise. Fast block for Class I drugs and K201 was to a substate exhibiting 20-50% of full open state conductance. Procainamide and verapamil caused a blocked state, with conductance indistinguishable from baseline. K201 causes two substates with conductances of 40 and 30 % of the open state (+40 mV). Kinetics of block was consistent with substate (or substates in the case of K201) due to binding of one molecule and complete block by a second molecule. Only flecainide, carvedilol, K201 and verapamil showed slow block. The slow block was amplified by conditions that cause lower levels of channel open probability such as the case with 1 mM Mg²⁺ or Ca²⁺ in the cytoplasm. The binding rate for slow block increased proportionally with channel closed probability indicating a high preference for the closed state of the channel. The duration of the slow block seen for carvedilol, verapamil and K201 (~ 40 ms) was very different to that for flecainide (500 ms). Class I agents are use-dependent blockers of the Na⁺ channel as they preferentially bind to these channels in their inactivated (closed) state. Activation of Na⁺ channels (i.e. their use) leads directly to their inactivation. This work shows that flecainide, K201, verapamil and carvedilol also bind to the RyR2 in its closed state. However, in the physiological context, use of the RyR2 (i.e. Ca²⁺ release) depends on the channel being open so that use of the RyR2 results in loss of drug effect even though in both cases, the drug binds preferentially to the closed channel. The term inverse-use dependence may be used to describe drugs in Group A with respect to their RyR2 inhibition. This study illustrates two mechanisms: fast and slow block of RyR2 that are specific for each class of drug. Our results show that Mg²⁺, at physiological concentrations, makes flecainide, K201, carvedilol and verapamil a more potent inhibitor of RyR2 by inducing the slow inhibition mechanism. At +40 mV, slow mechanism for all four agents have ~ two-fold lower IC₅₀(41, 10, 17 and 45μ, respectively) than the fast mechanism (70, 24, 30, 90μM, respectively). Differences lie in the off rate of flecainide compared to tetracaine, a classical RyR2 blocker. Flecainide off rate is 2 s^-1 and tetracine is 20 s^-1. This could have a direct implication on the control of RyR2-mediated SR Ca²⁺ release and on how the release is terminated on a beat basis given the self-regenerating nature of Ca²⁺ induced Ca²⁺ release.
Subject: ryanodine receptors; flecainide; anti arrhythmic drugs; lipid bilayer; calcium signalling
Identifier: http://hdl.handle.net/1959.13/1045214
Identifier: uon:14428
Language: eng
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