Mechanisms of calcium handling during a malignant hyperthermia event. Rocky Hyun Suk Choi M.S., Molecular Biosciences & Bioengineering B.S., Molecular Biology A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine Abstract Malignant hyperthermia (MH) is a clinical syndrome of the skeletal muscle that presents as a hypermetabolic response to volatile gas anaesthetics such as halothane, isoflurane, and to the depolarizing muscle relaxant succinylcholine. Genetic predisposition mainly arises from mutations in the gene for skeletal muscle ryanodine receptor (RyR1); a 2+ calcium channel that is tightly regulated to release Ca for muscle contraction. MH susceptible RyR1 are more prone to open by RyR1 agonist, resulting in the uncontrolled release of Ca2+ from the sarcoplasmic reticulum (SR). The hypermetabolic state is due to the excessive consumption of adenosine triphosphate (ATP) as the muscle works to clear the high cytosolic Ca2+. Dantrolene has been used to great effect by clinicians in treating MH. However the mechanism of its action remains highly contested. This thesis examines Ca2+ handling in muscle fibers where RyR1 activity is changed due to mutations. RyR1 activity was also acutely changed by altering the cytoplasmic environment and by treating the muscle fiber with varying RyR1 modulators. The release of SR Ca2+ was monitored using fluorescent Ca2+ indicators in mechanically skinned fibers isolated from rat, toad and human muscle susceptible to MH. The results show that the inhibitory effect of dantrolene is dependent on cytosolic concentrations of 2+ 2+ Mg ([Mg ]cyto), an endogenous negative regulator of the RyR1. Furthermore, it was demonstrated in human muscle susceptible to MH that dantrolene was ineffective at 2+ 2+ reducing halothane-induced Ca release in the presence of resting levels of [Mg ]cyto (1 2+ mM). These results suggest that raised [Mg ]cyto, a condition which is likely to arise during an MH event, works synergistically with dantrolene to inhibit Ca2+ release from the RyR1. It was also demonstrated that halothane induced Ca2+ release took the form of a repetitive Ca2+ wave in human MH, and toad muscle. A close examination of the release mechanism in human MH muscle revealed that the release of SR Ca2+ and the mechanism underlying the propagation of the Ca2+ wave was not due to Ca2+-induced- Ca2+-release (CICR). These results were contrasted to the observation made in toad skeletal muscle which showed the involvement of CICR. However, it was found that both luminal SR and cytosolic Ca2+ plays an important role in the mechanisms involved in overactive Ca2+ release. The demonstration of the Mg2+ dependence on dantrolene inhibition of the RyR1 emphasized the importance of studying the RyR1 under near physiological conditions. Utilizing a novel approach by trapping Ca2+ indicator in either the SR or the transverse tubule (t-system) of mechanically skinned rat skeletal muscle fibers, it was determined if i | P a g e either compartment would be able to report changes in RyR1 activity. The activity of the RyR1 was experimentally manipulated by exposing the preparation to varying concentrations of Mg2+, RyR1 agonists (halothane, caffeine), and RyR1 antagonist (dantrolene, tetracaine). Though it is well known that RyR1 activation will deplete the SR of Ca2+, it is only a relatively recent finding that activation of the RyR1 also will deplete the t-system of Ca2+ via a store-operated-Ca2+-entry (SOCE) mechanism. It was found that 2+ 2+ the free [Ca ]SR remained comparable over the broad range of [Mg ]cyto tested. 2+ Therefore, it was concluded that the free [Ca ]SR was not able to report changes in RyR1 2+ activity. In contrast, an increase in RyR1 activity (induced by low-Mg or pharmacological 2+ 2+ agonist) resulted in the depletion of the [Ca ]t-system. Importantly, the degree of [Ca ]t- system depletion was shown to be dependent on the activity of the RyR1. Utilizing this novel approach the Mg2+ dependence on RyR1 inhibition by dantrolene was again demonstrated. ii | P a g e Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. iii | P a g e Publications during candidature Choi RH, Koenig X & Launikonis BS. (2017). Dantrolene requires Mg2+ to arrest malignant hyperthermia. Proceedings of the National Academy of Sciences 114, 4811-4815. Conference abstracts R.H. Choi, and B.S. Launikonis (2017). Dantrolene requires Mg2+ to arrest malignant hyperthermia. Oral presentation at the Australian Physiological Society annual meeting for 2017 in Melbourne, Victoria, Australia. R.H. Choi, X. Koenig, and B.S. Launikonis (2017). Dantrolene requires Mg2+ to arrest malignant hyperthermia. Poster presentation at the Gordon Research conference (Muscle: Excitation contraction coupling). R.H. Choi, X. Koenig, T.R. Cully and B.S. Launikonis (2016). Exercise partially reverses the changes in calcium handling in skeletal muscle with age. Oral presentation as a representative of the University Of Queensland School of Biomedical Science at The Otago School of Medical Sciences Postgraduate Symposium for 2016 in Dunedin, New Zealand R.H. Choi, X. Koenig, T.R. Cully and B.S. Launikonis (2016). Integral role of Mg2+ in observing the inhibitory effect of dantrolene. Poster presentation at the Australian Physiological Society annual meeting for 2016 in Adelaide, SA, Australia. R.H. Choi, X. Koenig, T.R. Cully and B.S. Launikonis (2016). The transverse tubule Ca2+ handling properties are a sensitive detector of ryanodine receptor activity. Poster presentation at the Gage Muscle Conference 2016 in Canberra, NSW, Australia. R.H. Choi, X. Koenig, T.R. Cully and B.S. Launikonis (2015). The effect of intracellular [Mg2+] on Ca2+ handling by the sarcoplasmic reticulum. Oral presentation at the Australian Physiological Society annual meeting for 2015 in Hobart, TAS, Australia. R.H. Choi, T.R. Cully and B.S. Launikonis (2014). Effect of changing the [Mg2+] on ryanodine receptor leak activity in rat skeletal muscle. Poster presentation at the Australian Physiological Society annual meeting for 2014 in Brisbane, QLD, Australia. iv | P a g e 2+ R.H. Choi, T.R. Cully and B.S. Launikonis (2014). Effect of changing the [Mg ]cytoplasm on ryanodine receptor leak activity in rat skeletal muscle. Poster presentation at the Australian Physiological Society annual meeting for 2014 in Brisbane, QLD, Australia. v | P a g e Publications included in this thesis Choi RH, Koenig X & Launikonis BS. (2017). Dantrolene requires Mg2+ to arrest malignant hyperthermia. Proceedings of the National Academy of Sciences 114, 4811-4815. Publication citation – incorporated as Chapter 3. Contributor Statement of contribution Rocky H Choi (Candidate) Conception and design (70%) Analysis and interpretation (70%) Drafting and production (20%) Xaver X Koenig Conception and design (15%) Analysis and interpretation (15 %) Drafting and production (20%) Bradley S Launikonis Conception and design (15%) Analysis and interpretation (15%) Drafting and production (60%) vi | P a g e Contributions by others to the thesis Dr. Robert Fassett and Dr. Claire Hyam (Stewart) performed the muscle biopsy used in this thesis. Crystal Seng and Jeremy Briggs helped perform the experiments necessary to determine the relative halothane evaporation rate. Statement of parts of the thesis submitted to qualify for the award of another degree None Research Involving Human or Animal Subjects Animal ethics was approved by the School of Biomedical Science at The University of Queensland (Chief investigator: Dr. Bradley Launikonis; Title: Calcium regulation in healthy and dystrophic skeletal muscle; AEC approval Number: SCMB/032/14/ARC, approval duration: 17-March-2014 to 17-March-2017). The use of human muscle biopsy was approved by the Human Ethics Committee at The University of Queensland (Chief investigator: Dr. Bradley Launikonis; Title: Examining Functional and Structural Changes of Skeletal Muscle in Inherited Muscly Myopathies; approval number: 2014000826; approval duration: 31-July-2019). vii | P a g e Acknowledgements I would like to express my sincere appreciation to my Principal Supervisor, Dr. Bradley Launikonis, for giving me an opportunity to join his research laboratory and for the constant guidance and support throughout my doctoral studies. I would also like to thank Dr. Peter Noakes and Dr. Gregory Monteith for their time, support, mentorship, and encouragement during each major thesis milestone examination.