- Title
- Effects of calmodulin on the function of cardiac and skeletal ryanodine receptors
- Creator
- Oo, Ye Wint
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2016
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Dantrolene is a muscle relaxant that has been used clinically for the treatment of the malignant hyperthermia (MH) response to volatile anaesthetics such as halothane. Interestingly, it has been recently found to protect against heart failure and arrhythmias caused by spontaneous Ca²⁺ release. Assays of Ca²⁺ release in intact myocytes and cell homogenates indicate that dantrolene inhibits Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. However, its mechanism of action has remained controversial because single channel studies did not find any effect of dantrolene on the RyR Ca²⁺ release channel. Realizing that the physiological RyR binding partner calmodulin (CaM) readily dissociates from RyRs during incorporation into lipid bilayers, and that endogenous CaM is absent during single channel recordings, it was hypothesized in this thesis that CaM was required for dantrolene inhibition. RyR1 and RyR2 from rabbit skeletal muscle or sheep and human hearts were isolated and incorporated into artificial lipid bilayers and their open probability (Pₒ) was measured using single channel recordings. In the presence of exogenous 100 nM CaM (100 nM cytoplasmic [Ca²⁺] + 2 mM ATP), dantrolene caused an allosteric inhibition of the RyR1 (rabbit skeletal muscle) and the RyR2 (sheep heart) in single channel assays showing a maximum relative Pₒ (Emax) of 52 ± 4 % of control values. In saponin-permeabilized mouse cardiomyocytes supplemented with 100 nM CaM, dantrolene similarly reduced Ca²⁺ wave frequency (IC₅₀ = 0.42 ± 0.18 µM, Emax= 47 ± 4 %) and amplitude (IC₅₀ = 0.19 ± 0.04 µM, Emax= 66 ± 4 %). Together, the results demonstrate that CaM is essential for dantrolene inhibition of RyR1s and RyR2s and that its absence explains why dantrolene inhibition in single channels has not been previously observed. This study also presents the first demonstration of dose-dependent dantrolene inhibition of RyR2s with an IC₅₀ of 0.16 ± 0.03 µM in the presence of exogenous 100 nM CaM. The study also aimed to characterize dantrolene inhibition on RyR1s in the presence of exogenous CaM. The effects of dantrolene on RyR1s differentially regulated by CaM were measured at various cytosolic and luminal Ca²⁺ concentrations. It was then tested whether dantrolene could restore the channel activity affected by activating substances such as ATP, halothane and the interdomain domain destablising peptide DP4.which mimics the effects of inherited mutations underlying MH susceptibility. Dantrolene inhibited the RyR1 with identical hyperbolic dose-responses with an IC₅₀ of 0.20 ± 0.02 µM and a maximum Pₒ of 50 ± 2 % of controls in the presence of exogenous 100 nM CaM (100 nM cytoplasmic [Ca²⁺] + 2 mM ATP). In the absence of dantrolene, the RyR1 has a bell-shaped cytoplasmic Ca²⁺ activation curve with half activation at 1.7 ± 0.4 µM (Hₐ = 1.3 ± 0.2), half inhibition at 0.22 ± 0.01 mM (Hi = 2.7 ± 0.5) and a peak Pₒ = 0.93 ± 0.04. Dantrolene reduced Pₒ in sub-activating and inhibiting [Ca²⁺] but failed to reduce the peak Pₒ. Dantrolene significantly inhibited RyR1 activity at luminal [Ca²⁺] of 0, 100 and 1000 µM in the presence of exogenous CaM regardless of the trans-membrane voltages. Dantrolene caused a 50-60% reduction in Pₒ of RyR1s activated by cytoplasmic Ca²⁺ alone (100 nM) or in conjunction with ATP (2mM), halothane (5 mM) or DP4 (10 µM). Dantrolene equally inhibited RyR1 activity (50-60%) in the absence and presence of 20 and 50 µM cytoplasmic Mg²⁺. To conclude, dantrolene inhibited the RyR1 activity by decreasing RyR1 sensitivity to Ca²⁺ activation and increasing sensitivity to Ca²⁺ inhibition. Because dantrolene decreased channel activity both in the absence and presence of modulators such as halothane and DP4, this suggests the action of dantrolene inhibition of RyR1 activity could be independent of halothane and DP4. Regulation of RyR1s by Ca²⁺, Mg²⁺ and ATP is well characterised. However, there is minimal data on these regulation processes in the presence of CaM. Therefore, the effects of CaM on cytosolic Ca²⁺ activation and inhibition of the RyR1, as well as its regulation by luminal Ca²⁺ and inhibition by cytoplasmic Mg²⁺ were investigated in this thesis using single channel recordings. In addition, the effects of CaM on RyR1s altered by ATP, halothane and DP4 were tested. CaM increased the Pₒ of RyR1s to ~150% of control at a cytoplasmic [Ca²⁺] of 0.1 µM, whereas it reduced the Pₒ of RyR1s to 50 % of control at 1 µM and concentrations higher than 500 µM. Notably, CaM failed to have any impact on RyR1 activity at cytoplasmic [Ca²⁺] of 10-100 µM. CaM increased the Pₒ of RyR1 by ~140% at 0 and 100 µM luminal [Ca²⁺] whereas it was inhibited by 25-35% at 1000 µM luminal [Ca²⁺] regardless of the voltage. At cytoplasmic [Ca²⁺] of 0.1 µM, CaM equally activated RyR1s by 150-200% in the absence and presence of 20 and 50 µM cytosolic Mg2+. ATP did not affect CaM activation at cytoplasmic [Ca²⁺] of 0.1 µM. However, CaM inhibited the channel activity only in the absence of ATP at cytoplasmic [Ca²⁺] of 100 µM. CaM activation of RyR1s was not affected by halothane and DP4.
- Subject
- skeletal muscle; myocytes; heart; ryanodine receptor; calmodulin
- Identifier
- http://hdl.handle.net/1959.13/1332736
- Identifier
- uon:26929
- Rights
- Copyright 2016 Ye Wint Oo
- Language
- eng
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