THIOL SIGNALLING IN SKELETAL MUSCLE AGEING Neil T Smith Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy February 2018 1 Abstract An age-related loss of muscle mass is associated with increased frailty in the elderly. The effect is felt at both a national scale, with an increased budgetary demand for health services directed towards the ageing population, and by the individual where reduced mobility significantly reduces their quality of life. It is unclear whether all skeletal muscle types are affected in the same manner. This thesis considered how thiol signalling, facilitated through reactive thiol groups on cysteine amino acids, may affect skeletal muscle ageing as it is crucial for normal intracellular function. Several studies have identified reactive oxygen species (ROS) as crucial signalling molecules in healthy muscle and various proteins can detect and respond to changes in their concentration. The cysteines are evolutionarily conserved in functionally important locations and have a direct impact on protein function, affecting either its active site or conformation. In healthy muscle, proteins can quickly and efficiently respond to changes in ROS concentrations via this mechanism whereas in aged muscle these responses appear to be impaired. The quadriceps and soleus muscles were selected because of their differing primary metabolic pathways and physiology, reflecting fast and slow twitch muscle respectively. This enabled determination of age related changes to the redox proteome between two different skeletal muscles. They are hypothesised to age differently and to determine this, adult (12 months) and old (24 months) tissue were subjected to a deep proteomics investigation, elucidating changes to the global proteome of ageing mouse muscle as well as using differential labelling of reduced and reversibly oxidised cysteine residues to identify redox-susceptible locations on individual proteins. Prior to this a proteomics study had not analysed changes to the redox proteome between two skeletal muscle tissues before. Analysis of the quadriceps label free results identified changes to redox protein abundance such as a significant increase in Protein Disulphide Isomerase, crucial to disulphide bond formation and breakage. HSC70, important for protein folding, was significantly decreased with age. Differential labelling of specific cysteine residues demonstrated Cys46 increased in its reduced form with age in PARK7. Furthermore, many changes observed in the label free analysis highlighted cytoskeletal proteins as those primarily affected. The soleus label free results demonstrated significant decreases in abundance of a number of mitochondrial proteins involved in the electron transport chain such as NAD(P)H dehydrogenase and ATP Synthase. One example of differential labelling highlighted ATP Synthase Cys101 as becoming increasingly reduced with age. This increase in a reduced redox state of cysteines was observed across a range of other mitochondrial proteins, possibly indicating a negative impact on energy metabolism in the soleus with age. A successful preliminary study considered the effect of stretching C2C12 mouse skeletal muscle cells in vitro. A protocol for testing the effect of mechanical stretching on C2C12 cells was optimised with a future goal of producing replicable in vitro proteomics data and thereby reducing the requirement for animal tissue. The studies in this thesis identified various redox proteome changes in quadriceps and soleus muscle with age. This data will provide a basis for a targeted analysis of musculoskeletal proteins with a view to a better understanding of musculoskeletal ageing and its impact via the proteome. 2 Doctor of Philosophy Declaration I hereby declare that this dissertation is a record of work carried out in the Institute of Ageing and Chronic Disease at the University of Liverpool during the period from September 2013 to June 2016. The dissertation is original in content except where otherwise indicated. February 2018 ………………………………………………….. Neil T. Smith 3 Acknowledgements Many warm thanks to my primary supervisor Dr Brian McDonagh for his support and guidance throughout the PhD and write up. I am indebted to him for the opportunity and the experience. Many thanks to Professor Malcolm J Jackson as my secondary supervisor for his help and advice throughout my three years and during the thesis writeup. Thanks to Dr. Aphrodite Vasilaki as my secondary supervisor for her advice throughout the three years. Thanks to the Biotechnical and Biological Sciences Research Council Doctoral Training Programme for their financial support and to the Institute of Ageing and Chronic Disease for providing the equipment, reagents and space for my work. I would like to extend my thanks to all who have helped and supported me throughout my time in IACD. To Dr Gareth Nye, many thanks for the provision of quadriceps and soleus muscle and for his friendship, advice and good chats throughout the three years. Miss Niamh Horton and Dr Ian Li for their wonderful offerings of help, perspective and laughs, sometimes all at once! Heartfelt thanks to Mr Stephen Beard whose love and support during the write up and beyond has been immeasurable. Warm thanks to Dr Hannah Barrow for the friendship and encouragement at the start of this journey. Finally, Mr G. Roebuck. I am forever grateful for his inspiration and encouragement at school, where this adventure began. “I asked the question for the best reason possible, for the only reason, indeed, that excuses anyone for asking any question - simple curiosity.” Oscar Wilde, The Picture of Dorian Gray 4 Dedication This work is dedicated to my family: Mr Jeremy Smith, Mrs Moira Smith and Miss Gillian Smith. Thanks to their unwavering support, no challenge is insurmountable. 5 LIST OF ABBREVIATIONS: ALDH Aldehyde dehydrogenase (mitochondrial) ALDOA Fructose bisphosphate aldolase A AMPK Adenosine monophosphate kinase CASQ1 Calsequestrin 1 ENO1 α-Enolase ENO3 β-Enolase GAPDH / G3p Glyceraldehyde 3-Phosphate Dehydrogenase GPX1 Glutathione peroxidase 1 GRP78 78 kDa Glucose-regulated protein GSH Glutathione GSSG Glutathione (reduced) GST-k/GST-μ/GST-π Glutathione S-Transferase κ / μ / π HS71A/HSPA1A Heat shock 70 kDa protein H2O2 Hydrogen peroxide MYH Myosin heavy chain MYL Myosin light chain NAD Nicotinamide adenine dinucleotide NADP Nicotinamide adenine dinucleotide phosphate Nicotinamide adenine dinucleotide phosphate NAD(P)H (reduced) NO Nitric oxide NOS Nitric oxide synthase NOX NAD(P)H oxidase P70S6K Ribosomal protein s6-kinase β 1 PARK7 Parkinson disease protein 7 PDI/PDIA1/P4HB Protein disulphide isomerase PRDX Peroxiredoxin PYGM Glycogen Phosphorylase RNS Reactive Nitrogen Species ROS Reactive Oxygen Species SIRT1 Succinate Dehydrogenases SOD1/Cu-Zn SOD Superoxide Dismutase 1 SOD2/Mn-SOD Superoxide Dismutase 2 SRXN Sulfiredoxin TBS Tris-Buffered Saline TXN Thioredoxin TXNIP Thioredoxin Interacting Protein TXNR Thioredoxin Reductase 6 Publications Results from this study were published in: Journal of Proteomics “Redox responses are preserved across muscle fibres with differential susceptibility to aging” (Neil T. Smith et al., 2018, https://doi.org/10.1016/j.jprot.2018.02.015). This work was presented at the following scientific meeting: SFRR-Europe 2016; Budapest, Hungary under the title “Redox proteomics of mouse skeletal muscle ageing”. 7 TABLE OF CONTENTS Abstract 2 Doctor of Philosophy Declaration 3 Acknowledgements 4 Dedication 5 List of Abbreviations 6 Publications 7 List of Figures 13 List of Tables 17 CHAPTER 1 INTRODUCTION 18 1.1 Physiology of Skeletal Muscle 19 1.2 Energy Metabolism 22 1.3 Reactive Oxygen Species in Skeletal Muscle 29 1.4 The Importance of Cysteine in Redox Signalling 33 1.5 Skeletal Muscle Ageing 38 1.6 Thesis Aims and Hypothesis 39 CHAPTER 2 EXPERIMENTAL METHODS 41 2.1 Chemical Reagents 42 2.2 C45BL/6 Mice: Quadriceps and Soleus Muscles Dissection 42 2.3 Skeletal Muscle Homogenisation for Western Blotting 42 8 2.4 Proteomics Sample Preparation and Analysis 43 2.4.1 Proteomics Samples Preparation: Muscle Homogenisation 43 2.4.2 Proteomics Sample Preparation: Differential Labelling 43 2.4.3 Tandem Mass Spectrometry and Label Free Relative Quantification 44 2.4.4 Targeted Analysis of Differential Cysteine Labelling 45 2.4.5 Proteomic Data Analysis 46 2.5 SDS-PAGE and Western Blotting 50 2.5.1 Determining Protein Concentration – Bradford Assay 50 2.5.2 Western Blotting 51 2.6 Analysis of Carbonylation 54 2.7 Analysis of Glutathionylation 54 2.8 Enzyme Activity Assays 54 2.8.1 Aconitase Enzyme Activity Assay 54 2.8.2 Thioredoxin and Thioredoxin Reductase Enzyme Activity Assays 55 2.9 Cell Culture 56 2.9.1 Media Preparation 56 2.9.2 C2C12 Myoblast Culture, Differentiation and Storage 56 2.9.3 C2C12 Myotube Stretching 58 2.10 Analysis of Succinate Dehydrogenase in Mouse Skeletal Muscle 59 2.11 Software and Statistics 61 9 CHAPTER 3 STUDY OF THE QUADRICEPS MUSCLE 64 3.1 Introduction 65 3.2 Aims and Hypothesis 67 3.3 Results 67 3.3.1 Muscle Weights 67 3.3.2 Succinate Dehydrogenase Activity of the Quadriceps Muscle 68 3.3.3 Global Label Free Proteomics Data Analysis 69 3.3.4 Western Blotting and Enzyme Activity Assays 75 3.3.5 GOrilla Pathway Analysis 94 3.3.6 STRING Analysis of Significantly Changed Quadriceps Proteins 98 3.3.7 Redox Changes in Mouse Quadriceps with Age 100 3.3.8 Comparison of Differentially Labelled Proteins 107 3.3.9 STRING Analysis of Differentially