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Associated Malignant Hyperthermia Mechanisms underlying the phenotypic diversity in RYR1- associated Malignant Hyperthermia VIKAS KAURA Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Medicine and Health Leeds Institute of Medical Research at St James’s January 2020 The candidate confirms that the work submitted is his/her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2020 The University of Leeds and VIKAS KAURA i Acknowledgements First and foremost, I would like to thank my supervisors Professor Philip Hopkins, Dr. Marie-Anne Shaw and Professor Paul Allen for their help, guidance and continued support throughout my PhD, as well as during the application for my clinical research training fellowship. I am grateful for their patience, encouragement and providing constructive feedback, particularly when I needed to move on from an experiment that was not working. I would also like to thank the Medical Research Council and the British Journal of Anaesthesia for jointly funding my clinical research training fellowship, without which I would have been unable to pursue this Doctorate. Thank you to all the members of the MH Unit both past and present who have been there as a helping hand, teaching me where needed, looking after my cells on the occasions I was away, and for the general laughs particularly during the more testing times of the research. Thanks, in particular to Catherine Daly who helps the cogs in the MH unit run smoothly and has always been there when I needed help. Xiaochen Liu for the laughs and assistance, Rachel Dodds in refreshing my molecular biology skills, Essam Ali for working on the letter together, Leon Chang for the statistics chat, Garima Singh for some of the imaging, Dr Jonathan Bilmen and Dr Pawan Gupta for their support. Adam Davison for advice whilst I established the live-cell imaging system, as well as Adam and Liz Straszynski for conducting the FACS purification. Thank you to Dr Jose Miguel Eltit for advice whilst I was developing the manganese quench technique, and Dr Alan Merritt for the HEK constructs. A huge thank you to my immediate family for their steadfast support throughout my life, also the Wong family for all their help and encouragement through this PhD, and for looking after Riaan when I was away to the lab or working on the thesis. Finally, I would like to thank my dear wife Christine for her unconditional support and encouragement throughout this process, putting up with me and my absence from home during this doctorate, and enabling me to reach my aspirations. I dedicate this to you, our parents and Riaan. ii Abstract Malignant hyperthermia (MH) is a potential fatal hereditary skeletal muscle disorder that occurs upon exposure to certain anaesthetic agents. Susceptibility is predominantly conferred by variants in the RYR1 gene encoding the type 1 ryanodine receptor (RYR1). A common feature observed in MH susceptible patients and in animal models, is a RYR1-leak dependent elevation in the intracellular Ca2+ 2+ concentration ([Ca ]i) in the non-triggered state. However, it is not fully understood whether extracellular Ca2+ entry plays an important role in maintaining this elevated 2+ resting [Ca ]i. It is also becoming increasingly evident that RYR1 variants can produce different MH phenotypes, for example the variant p.G2434R (c.7300G>A) is the major MH variant found globally, but has a weaker clinical phenotype relative to rarer variants such as p.R2454H (c.7361G>A). Live-cell calcium imaging was used to examine different aspects of the molecular mechanisms underlying RYR1-associated MH. The first was to use HEK293 cells to explore the caffeine sensitivity of novel potentially pathogenic RYR1 variants. Next skeletal muscle myotubes derived from MH patients with p.G2434R or p.R2454H, were found to have an enhanced sensitivity to caffeine, and a TRPC3/6 mediated increased sarcolemmal cationic influx relative to non-susceptible controls. There was no significant difference between the two MH associated RYR1 variants. Myotubes from a novel MH mouse model containing the equivalent variant to human p.G2434R, were also found to have an elevated sensitivity to RYR1 agonists. This model has further confirmed that the TRPC3/6 mediated enhanced cationic entry is conserved between the two species, and this entry can be reduced to control levels by blocking the RYR1-dependent leak. Taken together, the data presented in this thesis furthers our understanding of the molecular mechanisms that underlie the perturbed Ca2+ handling observed in MH tissue, and provides new avenues of research into MH and related RYR1 disorders as well as a potential target for novel therapies. iii Table of Contents Acknowledgements…...............................................................................................i Abstract.....................................................................................................................ii Table of Contents.....................................................................................................iii List of Tables…..……..............................................................................................xii List of Figures………..............................................................................................xiii List of Abbreviations ............................................................................................xvi 1 Introduction………................................................................................................1 1.1 Malignant Hyperthermia................................................................................1 1.1.1 Clinical Manifestations………………………………………..…...……….…1 1.1.2 Epidemiology………………………………………………………..…………2 1.1.3 Diagnosis of MH………..……………………………………………………..3 1.1.3.1 Clinical Grading Scale…………………………………….………….…3 1.1.3.2 IVCT…………………………..………………………………………..…3 1.2 Genetics of MH…………………………………………………………..………..7 1.2.1 Inheritance……………………………………………………………..………7 1.2.2 Phenotypic Diversity……………….…………………………………….……8 1.2.3 Molecular Genetics of the Susceptibility to MH……………….……….…..9 1.2.3.1 Genetic Testing for MH…………………..……………………..….…..9 1.2.3.2 Investigating Novel Variants………………….…………………..…..10 1.2.3.3 Characterising RYR1 Variants………………………………..………11 1.2.4 Related Skeletal Muscle Disorders…………………………………….….12 1.2.4.1 Exertional Heat Illness…………..…………………………………....12 1.2.4.2 Central Core Disease…………..…………………………………..…12 1.3 Treatment of MH and Related Disorders…………….………………....……13 1.4 Skeletal Muscle…………………………………………….………...……..……14 1.4.1 Muscle Regeneration……………………..…………………..…..…..….…14 1.4.2 Physiology………………………………...…………………………….…....15 1.4.3 RYR1………………………………………………………………...…..……16 1.4.4 RYR1 Regulatory Molecules and Proteins………………………….…….18 iv 1.4.4.1 Small Molecules…………….…………………………….….………..18 1.4.4.2 Proteins…………………………...……...……………………....…….19 1.4.5 Cav1.1………………………….…………………………………….….……20 1.4.6 STAC3……………………………………………………….………..….…..22 1.4.7 TRPC Channels………………………………………….……………..…...22 1.4.8 Extracellular Calcium Entry Mechanisms………………………...…...….23 1.4.8.1 ECCE………………………………………………………………..…..23 1.4.8.2 SOCE…………………………………………………….....…………..24 1.4.8.3 RCaE……………………………………………………………...………25 1.4.9 MH and Ca2+ Handling……….………………………………….……….…26 1.5 Thesis Aims and Objectives…………………………………….….………….27 1.5.1 Aims…………………………………………….…………………………..…27 1.5.2 Objectives………………………………………………………..…………...28 2 Methods.…………………………………………………………………………….…..29 2.1 Generation of p.G2435R Mice……………………………………….…..…….29 2.2 General Cell Culture Methods………………………………..…………….…30 2.2.1 Isolation of Human Myoblasts…………………………………….….….…30 2.2.2 Isolation of Mouse Myoblasts………………………………………………31 2.2.3 Matrix Coating of Plates………………………………………...…………..32 2.2.4 Differentiation of Human and Mouse Myoblasts…………………...…….32 2.2.4.1 Live Cell Calcium Imaging Experiments…………………………….32 2.2.4.2 Immunocytochemistry Experiments………………………………....32 2.2.4.3 RT-PCR Experiments………………………………………………....32 2.2.5 Passage and Cryostorage of Cells……………………………......……....33 2.2.6 Cell Counting and Viability Assays………………………………………...33 2.3 General Molecular Biology Methods……………………………….………...34 2.3.1 DNA Extraction……………………........….……………………………......34 2.3.2 PCR and Agarose Gel Electrophoresis……….....…………..…….…......34 2.3.3 Direct PCR…………………………………………………….………..........35 2.3.4 Sanger Sequencing…………………………………....……………...........35 v 2.4 General Bacterial Methods……………...…....…..…..…..…..…...................36 2.4.1 Bacterial Growth………..............…………………...……...……………....36 2.4.2 Plasmid Maxi Preparation………………………………………….............36 2.4.3 Production of Glycerol Stocks………….........………………………….....36 2.5 Immunocytochemistry…………………………………….…………...............37 2.6 Live-Cell Calcium Imaging…………………………………………….............38 2.6.1 Perfusion System………………………………….………..……...............38 2.6.2 Experimental Solutions…………………………..……………..................39 2.6.2.1 Buffers………………………………………………...........................39 2.6.2.1.1 Imaging Buffer……………………………................................39 2.6.2.1.2 Manganese Buffer……………………………....………...........39 2.6.2.2 Drugs…………………………………………………..…...................39 2.6.2.2.1 Caffeine and Other Compounds……………………...............39 2.6.2.2.2 KCl………………………………………...................................40 2.6.2.2.3Halothane………………………………………..........................40
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