U Ottawa

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L'Universite canadienne Canada's university

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FACULTE DES ETUDES SUPERIEURES ET POSTOCTORALES

TTTTT

u Ottawa

L'UniveraiUi conadicnrtc Canada's university

FACULTY OF GRADUATE AND POSDOCTORAL STUDIES

Maria Benkhalti M.Sc. (Cellular and Molecular Medicine) Department of Cellular and Molecular Medicine facultOC^ITDI^

The Effect of the KATP Channel on Energy Metabolism in Skeletal Muscle During Fatigue

TITRE DE LA THESE / TITLE OF THESIS

Dr. J-M. Renaud

CO:DlRWfuT(CO^

EXAMINATEURS (EXAMINATRICES) DE LA THESE/THESIS EXAMINERS

Dr. J. DaSilva Dr. J. Cote

Gary W. Slater

Le Doyen de la Faculte des etudes superieures et postdoctorales / Dean of the Faculty of Graduate and Postdoctoral"Studies

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THE EFFECT OF THE KATP CHANNEL ON ENERGY METABOLISM IN SKELETAL MUSCLE DURING FATIGUE

By

Maria Benkhalti

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

? Maria Benkhalti, Ottawa, Canada, 2009

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ACKNOWLEDGEMENTS

I first and foremost would like to thank my supervisor, Jean-Marc Renaud for his help and guidance. Your mentoring has gone beyond the sole purpose of this project. I also need to thank the members of my advisory committee, Mary-Ellen Harper and Jocelyn Cote for their valuable questioning and advice. A special thank you to my colleague Zhen Li for her inspirational work; there is still so much more to discover! Many thanks to all the rest of my lab colleagues: Krystyna, Patrick, Louise, Samir, and Simon, your help and daily laughter made a difference.

The deepest thank you to my parents. Words cannot express my gratitude for your constant support and always believing in me. You have sacrificed so much to allow your daughters achieve their best; I hope I make proud! Shout out to the greatest sisters one could wish for, Rita and Sofia; thanks for keeping it real les poches! Thanks to my new sister, Shuran, and my new parents for all the support, it kept me going and I hope that we will now have more time to know each other better.

Lastly, I need to thank my amazing husband, Shaunpal. You have been there for me every step of the way from far or from close, through laughter and tears. Thank you for understanding that your new wife be constantly on the computer with no time for simple life! I truly owe you.

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ABSTRACT

KATP channels are found on the sarcolemma where they affect muscle contractility and thus, energy demand. They are also found in the mitochondria where they affect ATP production. The overall objective was to determine how the KATP channel affects skeletal muscle metabolism during fatigue, elicited with one tetanic contraction every sec for 3 min. Exposing Kir6.2"/" flexor digitorum brevis (FDB) muscles, which have no sarcolemmal KATP channels, to the KATP channel blocker, glibenclamide, or the opener, pinacidil, did not affect lactate production during the three min fatigue. However, DMSO, used to dissolve glibenclamide and pinacidil caused a decrease in lactate content during the last two min of fatigue, an effect not observed in its absence of DMSO. It is therefore possible that DMSO also influence how glibenclamide and pinacidil affect lactate content during the last two minutes of fatigue. Further studies were then restricted to a comparison between wild type and Kir6.2_/" FDB. The amount of glucosyl units entering glycolysis, from glycogen breakdown and glucose uptake, was the same between wild type and Kir6.2~/_ FDB, while lactate production was much less in Kir6.2/_ FDB following fatigue. It is suggested that the absence of KATP channel activity in Kir6.2"/_ FDB results in an increased oxidative capacity in which more pyruvate enters the Krebs cycle than in wild type FDB. This suggestion is supported by the fact that the oxygen uptake is greater in Kir6.2_/" than wild type mice during a 24 hour period.

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ABBREVIATIONS

ABS: ATP-binding site ADP: adenosine diphosphate AMP: adenosine monophosphate AMPK: 5'-AMP-activated protein kinase ANOVA: Analysis of variance AP: action potential ATP: adenosine-5'-triphosphate CaMK: Ca2+-calmodulin-dependent protein kinase CoA: coenzyme-A DHPR: Dihydropyridine receptor DMSO: dimethyl sulfoxide EC coupling: excitation-contraction coupling EDL: extensor digitorum longus ETC: electron transport chain FADH: flavin adenine dinucleotide FDB: flexor digitorum brevis FFA: free fatty acids G-l-P: glucose-1-phosphate G-6-P: glucose-6-phosphate GAPDH: glyceraldehyde 3-phosphate dehydrogenase GLUT: glucose transporter

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IMP: inosine monophosphate IMTG: intramyocellular triacylglycerol KATP channel: ATP-sensitive potassium channel Kir: potassium inward rectifier ml: milliliter mM: millimolar mV: millivolt n: number of samples NADH: nicotinamide adenine dinucleotide NBD: nucleotide-binding domain NBF: nucleotide-binding fold ?C: degrees Celsius PCr: phosphocreatine PGK: phosphoglycerate kinase pHi: intracellular pH P;: inorganic phosphate PIP2: phosphatidylinositol-4, 5-biphosphate RER: respiratory exchange ratio RQ: respiratory quotient RYR: Ryanodine receptor S.E.: standard error SR: sarcoplasmic reticulum SUR: sulfonylurea receptor

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