The single channel RyR2 measurements and the effects of hydantoin derivatives on channel function

Ashna, Amir

Title: The single channel RyR2 measurements and the effects of hydantoin derivatives on channel function
Creator: Ashna, Amir
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2020
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: In failing heart, an elevated diastolic Ca²⁺ leak from the sarcoplasmic reticulum can be normalised by the RyR2 inhibitor, dantrolene without inhibiting Ca²⁺ release during systole or affecting Ca²⁺ release in normal healthy hearts. Unfortunately, dantrolene is hepatotoxic and unsuitable for chronic long-term administration. Since dantrolene, phenytoin, ethotoin, primidone, and mephenytoin belong to the hydantoin class of compounds, I tested the hypothesis that dantrolene and other hydantoin derivatives have similar inhibitory effects on RyR2 using single channel recording of RyR2 activity in artificial lipid bilayers. RyR2 was isolated from sheep heart, incorporated in lipid bilayers and investigated by single-channel recordings in presence of diastolic Ca²⁺ (0.1 μM cytoplasmic) and systolic Ca²⁺ (0.1 mM cytoplasmic). Among hydantoin derivatives dantrolene, phenytoin, and ethotoin could inhibit RyR2 activity, while primidone and mephenytoin had no inhibitory effect on RyR2 channels from sheep heart in the presence and absence of CaM at +40mV. Phenytoin produced a reversible inhibition of RyR2 channels from sheep and human failing hearts. It followed a hyperbolic dose-response with maximal inhibition of ~ 50%, Hill coefficient ~ 1 and IC₅₀ ranging from 10 to 20 μM. It caused inhibition at diastolic cytoplasmic [Ca²⁺] but not at the much higher Ca²⁺ levels that arise in the dyadic cleft during systole. Notably, phenytoin inhibited RyR2 from failing human heart but not from healthy heart indicating that phenytoin may selectively target defective RyR2 channels in humans. Interestingly, IC₅₀ values for phenytoin inhibition of sheep RyR2 from single channel recordings is 3-fold lower than its IC₅₀ for Na⁺ channels (IC₅₀ = 58 μM) and much lower than its IC₅₀ for L-type and T-type Ca⁺² channels (IC₅₀ ~ 100-200 μM). Thus, the amount of phenytoin required for RyR2 inhibitory purposes is lower than therapeutic levels used for the treatment of epilepsy. Phenytoin and dantrolene had similar dependencies on cytoplasmic [Ca²⁺]. Increasing cytoplasmic [Ca²⁺] from end-diastolic [Ca²⁺] of 0.1 µM cytoplasmic to systolic [Ca²⁺] of 100 µM cytoplasmic caused reduction of RyR2 inhibition by phenytoin and dantrolene from 50% inhibition at 0.1 μM to approximately no inhibition at 100 μM [Ca²⁺]. Even in the presence 1 mM Mg²⁺ phenytoin could not inhibit RyR2 at the systolic condition and produced no significant inhibition of RyR2, which again indicates that phenytoin like dantrolene is a diastolic inhibitor of RyR2 activity, and it has no significant effect on the activity of RyR2 at systolic [Ca²⁺]. In addition, dantrolene and phenytoin inhibition of RyR2 showed no dependence of gating parameters on membrane voltage. Ethotoin was found to be the strongest diastolic inhibitor of sheep RyR2 among the hydantoin derivatives tested, with maximal inhibition of ~70%, Hill coefficient ~ 1, IC₅₀ from 0.5 to 0.8 μM at +40 mV and -40mV in the absence of CaM, respectively (p = 0.2), indicating that inhibition of RyR2 by ethotoin is independent of CaM and voltage. Thus, ethotoin may be highly effective in decreasing diastolic Ca²⁺ leak from the SR. It inhibits RyR2 activity from failing human heart without affecting healthy human heart. The IC₅₀ of ethotoin to inhibit RyR2 was more than 100-fold lower than the therapeutic concentrations of ethotoin used for the treatment of partial epilepsy (74-245 μM). These findings indicate that use of ethotoin to inhibit the RyR2 leak in failing human hearts will produce less adverse side effects than the therapeutic ethotoin concentrations used for the treatment of epilepsy. Thus, ethotoin is a more human-safe alternative to dantrolene for therapies against leaky RyR2. Ethotoin inhibited RyR2 channels with maximal inhibition of ~50% at +40mV in the absence of FKBP12.6 and CaM indicating that inhibition of RyR2 by ethotoin is independent of CaM and FKBP12.6. To conclude, phenytoin and ethotoin could effectively inhibit RyR2 mediated release of Ca²⁺ in a manner paralleling that of dantrolene. It was found that there is no difference between phenytoin and ethotoin-mediated inhibition of sheep RyR2 and RyR2 from failing human heart. Moreover, the IC₅₀ of phenytoin and ethotoin in human RyR2 are lower than for serum levels used clinically to inhibit Na⁺ channels in treatment of epilepsy, opening the possible use of phenytoin and ethotoin as human-safe therapies against heart failure.
Subject: heart function; phenytoin; ethotoin; dantrolene; heart failure
Identifier: http://hdl.handle.net/1959.13/1514230
Identifier: uon:56828
Language: eng

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