EFFECTS OF CARBOHYDRATE AVAILABILITY ON FATIGUE AND FATIGUE PRE-CONDITIONING IN MOUSE FDB MUSCLE By Erik Hesse A thesis submitted to the Faculty of Graduate and Post-Doctoral Studies of the University of Ottawa in partial fulfillment of the requirements of the Degree of Masters of Science Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Erik Hesse‚ Ottawa‚ Canada‚ 2019 ABSTRACT To prevent damaging ATP depletion during periods of intense activity‚ intrinsic mechanisms within skeletal muscle are activated and lead to myoprotection; a process known as muscle fatigue. It has been proposed that the primary mechanism of fatigue is a submaximal sarcoplasmic reticulum Ca2+ release and decreased force generation‚ however‚ what triggers this mechanism remains controversial. It is possible that glycogen may act as a trigger as studies have repeatedly shown a direct correlation between glycogen content at the beginning of activity and time to fatigue. In previous studies‚ a fatigue bout and/or period of fasting to deplete glycogen was used. However, this leaves investigators to differentiate between the effects of glycogen depletion methodology causing a metabolic stress and effects of glycogen itself. One objective of this M.Sc. project was to produce a low glycogen model without a prior metabolic stress that could forgo these limitations. It was extended to differentiate between the role of glycogen and extracellular glucose during fatigue as well as fatigue pre-conditioning (FPC)‚ a phenomenon in which fatigue resistance increases for about 2 hours after a first fatigue bout. During a single‚ first fatigue bout (one contraction every sec for 3 min) a mean decrease in glycogen from 95 to 20 μmol/g dry wt. had no effect on the decrease in 2+ 2+ tetanic [Ca ]i‚ i.e. the [Ca ]i during tetanic contractions‚ whereas removing glucose from the 2+ physiological solution led to a 46% greater decrease in tetanic [Ca ]i than when glucose was present. During a subsequent fatigue bout (i.e. FPC) a greater amount of glycogen was used as glycogen content was 27% greater than prior to the first fatigue bout. When glycogen and/or glucose was limited‚ FPC was abolished. It is concluded that extracellular glucose is critical to prevent fatigue. Additionally, whereas glycogen is important for FPC‚ it appears to be much less important during a first fatigue bout initiated in absence of any prior metabolic stress. ii TABLE OF CONTENTS ABSTRACT.................................................................................................................................... ii TABLE OF CONTENTS ................................................................................................................... iii LIST OF FIGURES .......................................................................................................................... v LIST OF ABBRVIATIONS .............................................................................................................. vii ACKNOWLEDGEMENTS ................................................................................................................ x CHAPTER 1: INTRODUCTION......................................................................................................... 1 Muscle contraction ............................................................................................................... 2 Energy metabolism during exercise ........................................................................................ 4 Skeletal muscle fiber types .................................................................................................... 6 Fatigue ................................................................................................................................. 8 Role of metabolites during fatigue.......................................................................................... 8 Decreased Ca2+ released for the SR during fatigue ............................................................... 10 Membrane excitability and SR Ca2+ release during fatigue .................................................. 13 Glycogen: more than just glucose storage............................................................................ 16 The glycogen molecule .......................................................................................................... 16 Glycogen and skeletal muscle fatigue ................................................................................... 18 Current methods in the study of glycogen and fatigue ........................................................ 22 Fatigue pre-conditioning ..................................................................................................... 24 CHAPTER 2: METHODS AND MATERIALS ..................................................................................... 27 Animals and muscles ........................................................................................................... 27 Single fiber preparation ...................................................................................................... 27 Physiological solution ......................................................................................................... 28 2+ [Ca ]i measurements .......................................................................................................... 28 Force measurements........................................................................................................... 30 Stimulation and fatigue protocol ......................................................................................... 30 Glycogen determination...................................................................................................... 31 iii Statistical analysis ............................................................................................................... 32 CHAPTER 3: RESULTS .................................................................................................................. 33 Single muscle fiber glycogen content ................................................................................... 33 2+ Effect of carbohydrate availability on [Ca ]i during fatigue .................................................. 35 2+ Unstimulated [Ca ]i during fatigue ....................................................................................... 39 Variability among individual fibers........................................................................................ 39 Effect of glucose availability on force during fatigue ............................................................ 42 Effect of carbohydrate availability of FPC ............................................................................ 44 Glycogen content during FPC ................................................................................................ 44 Tetanic force during FPC ....................................................................................................... 46 Unstimulated force during FPC ............................................................................................. 46 2+ Tetanic and unstimulated [Ca ]i during FPC ......................................................................... 48 Variability in fatigue kinetics between single muscle fibers ................................................. 52 CHAPTER 4: DISCUSSION ............................................................................................................ 60 Glycogen content in single muscle fibers ............................................................................. 61 Single fibers vs. muscle bundles in the study of muscle fatigue ............................................ 62 Insulin only effects muscle fatigue under extreme carbohydrate limitation .......................... 64 Low glucose not low glycogen lowers fatigue resistance during a single fatigue bout ........... 65 Extracellular glucose availability is critical in maintaining fatigue resistance....................... 66 Without a prior metabolic insult glycogen does not affect muscle fatigue .......................... 69 Fatigue pre-conditioning ..................................................................................................... 72 The least fatigue resistant single fibers experience the greatest effect of FPC .................... 73 Glycogen supercompensation and FPC ................................................................................. 74 Carbohydrate availability is a central component of FPC ..................................................... 75 Role of glycogen: FAT1 vs. FAT2 ............................................................................................ 77 Conclusions ........................................................................................................................ 78 APPENDIX I ................................................................................................................................ 80 iv LIST OF FIGURES Figure 1-1. Relationship between force and intracellular Ca²⁺ concentration during muscle 11 fatigue (Westerblad & Allen, 1993). Figure 3-1. Insulin is necessary to maintain normal glycogen content while a lack of 34 extracellular glucose depleted glycogen reserves in single FDB fibers. Figure 3-2. Example of an original record of 360/380 ratio signals obtained from a fatigue 36 bout of a fiber loaded with fura-2 AM. Figure 3-3. The lack