«by BLAKE R. ZELICKSON VICTOR M. DARLEY-USMAR, COMMITTEE CHAIR JOHN C. CHATHAM YABING CHEN LOUIS J. DELL’ITALIA AIMEE LANDAR C. ROGER WHITE A ...»
ASSESSMENT OF MITOCHONDRIAL STRESSORS ON CELLULAR
BLAKE R. ZELICKSON
VICTOR M. DARLEY-USMAR, COMMITTEE CHAIR
JOHN C. CHATHAM
LOUIS J. DELL’ITALIA
C. ROGER WHITE
A DISSERTATIONSubmitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy
ASSESSMENT OF MITOCHONDRIAL STRESSORS ON
BLAKE R. ZELICKSON
MOLECULAR AND CELLULAR PATHOLOGY
Keywords: Mitochondrial Function, Extracellular Flux, 4-Hydroxynonenal, Nitric Oxide, Hypoxia, Reserve Capacity iii
The completion of this dissertation and the research within would not have been possible without the help of several people throughout my tenure in graduate school.
First and foremost, I would like to thank my mentor, Victor Darley-Usmar. In the four and a half years I spent in your lab, you helped me to grow not only as a scientist, but also as a person. You always took the time to figure out the best way to push me to succeed, and I am truly grateful for all of the support, encouragement, patience, and even the “bollockings” you have given me over the years. I can only hope to one day be as good of a mentor to someone else as you have been to me.
To my “lab family,” both past and present, thank you so much for everything that you’ve done for me over the years. To Michelle Johnson, my “lab mom,” your proficiency in all things science is always impressive and beyond compare. You always seem to know how to do every possible kind of assay, as well as the secrets of how to make them work. But more importantly, you were always willing to take the time out of your absurdly busy day to share your expertise with me. To Gloria Benavides, my “lab wife,” I have thoroughly enjoyed working and traveling with you over the past few years. We made an excellent team, and I can’t think of anyone with whom I would have rather spent all those countless hours running up to eight Seahorse experiments per day. You helped me to become more patient, understanding, and an all-around better person, and for that I
in-crime for the past four and a half years. You taught me all the basics of bench-work and were always willing to discuss my data and give me advice, even when it meant that you would have to stay late as a result. We have been through a lot together, and I will definitely miss all of our chats and life-coaching sessions. To Sam Giordano, my “little lab sister,” I enjoyed acting as your pseudo-mentor while you were rotating through our lab, and maybe even a little bit afterwards as well. To Balu Chacko, I have really enjoyed all of our scientific discussions. You know so much about so many different things, and always seem to have a new and interesting perspective about what different results could mean. To Brad Hill, I loved working with you during your short time at UAB. I learned so much about how to be successful in science from you, and your never-ending energy and enthusiasm continue to amaze me. To Joo Yeun Oh, the Korean ninja, you were a constant source of entertainment in the lab. In addition, you are probably one of the most selfless and giving people I have ever met, and I will always be in awe of all that you do. To Mi Jung Chang, never in my entire life have I witnessed anyone do as much work as you managed to do on a daily basis. I will miss working with you and your help with my Korean, even if I only know two phrases. To Sarah Owusu, I really enjoyed working with you for the year that you were here. We have so much in common, and it was really great to have a partner in the avoidance of all things unkosher.
Thanks also to Colin Reily, Philip Kramer, Elena Ulasova, Yaozu Ye, Brian Dranka, and Jessica Perez for helping to make it so that I never dreaded going to work, even when I knew it would be a really long day. I have enjoyed working with you all.
You have more enthusiasm for science than I have ever seen before, which inspires me to be more enthusiastic and interested as well. I have always been impressed by how you manage to juggle being a clinician and a researcher, and I hope to be able to achieve even a percentage of the success that you have in your career. I would also like to thank several members of the Dell’Italia lab for all of their support throughout all of our collaborations. To Doug Gladden, thanks for always being willing to isolate myocytes whenever they were needed, even if you sometimes decided that they didn’t need to be counted.
Working with you has been interesting and fun to say the least, and I will miss all of our random conversations and your unique perspective on things. To Eddie Bradley, rarely have I seen such impressive skills as I have witnessed observing you perform various surgeries or procedures on mice and rats. To Pam Powell, thanks for always being willing to perform immunohistochemistry for me, even when I came to you with ideas that could have potentially kept you busy for months at a time.
To Aimee Landar, thank you for always being willing to share your expertise with me whenever I needed it. I don’t know anyone who can troubleshoot a Western blot as well as you can. Thank you also to the members of the Landar lab, both past and present, for all of their support over the years. To Stephanie Wall, I have enjoyed interacting with you whenever I got the chance. Trying to get you to be just a little bit less nice was always a great source of entertainment for me. To Fen Zhou, thank you so much for ordering antibodies for me so quickly when I decided last minute that I needed to probe for just one more thing. Thanks also to Anne Diers and Praveen Vayalil, my fellow investigators into bioenergetics in hypoxia.
the years. Between scheduling meetings, getting reimbursements, figuring out what exactly the training grant would pay for, and helping me track down Victor, you made my time in graduate school go so much more smoothly than it ever could have without you.
To Nicole Newton, you were always an incredibly valuable source of information.
Thank you so much for always having answers to my one million questions, particularly as I was preparing to graduate. To Rakesh Patel, thank you for all of your advice over the years. You always knew what my best course of action was, and probably helped me avoid a lot of grief in the process. To my committee, John Chatham, Yabing Chen, Lou Dell’Italia, Aimee Landar, and Roger White, thank you so much for your ideas regarding my research and for always pushing me to look for additional explanations for my data.
Last, but certainly not least, I would like to thank my family for everything that they have done for me throughout my entire life. To my parents, thank you so much for always supporting and believing in me, even when it was not deserved. You are the smartest and hardest working people I know, and I can only hope to live up to the example that you have set for me. To my grandparents, thank you for all the faith you have had in me. You make me strive to succeed in everything that I do in the hopes that I can one day deserve how proud you always are of me. To my brothers, aunts, uncles, and cousins, thank you as well for your encouragement over the years. I could never have made it this far without your constant support.
“Truly great friends are hard to find, difficult to leave, and impossible to forget.”
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
CHAPTER 1 INTRODUCTION
Role of the Mitochondrion in Physiology
Mitochondrial ROS Production
Importance of mtDNA Haplotype
Role of the Mitochondrion in Pathology
Diseases Associated with Mitochondrial Dysfunction
Alcoholic Liver Disease
Ischemia/Reperfusion in the Vasculature
Cardiac Volume Overload
Role of Nitric Oxide in Alcohol-Induced Hepatotoxicity
Chronic Alcohol Consumption Causes Mitochondrial Dysfuntcion
Materials and Methods
Mitochondrial Enzyme Activity Assays
Hepatocyte Density for Measurement of Mitochondrial Bioenergetics
Alcohol Induces Characteristics of Liver Toxicity in Hepatocytes
Hepatocytes Exhibit Bioenergetic Defect in Response to Alcohol Exposure
Chronic Alcohol Increases Hepatocyte Sensitivity to Nitric Oxide
Alcohol Exacerbates Bioenergetic Alterations Induced by Hypoxia and Nitric Oxide
Alcohol-Induced Hypoxia in the Liver in iNOS-Dependent...............59 Discussion
3 HYPOXIA/REOXYGENATION ALTERS THE RESPONSE OF
CELLULAR BIOENERGETICS TO 4-HYDROXYNONENALIN ENDOTHELIAL CELLS
Materials and Methods
Measurement of Oxygen Consumption in Endothelial Cells..............77 Mitochondrial Function Assay
Exposure to Hypoxia and Reoxygenation
Oxygen Dependence of Endothelial Cell Mitochondrial Function
Reactive Lipid-Induced Alterations to Endothelial Cell Bioenergetics
Oxygen-Dependence of Endothelial Cell Bioenergetic Alterations Induced by HNE
Exacerbation of Reoxygenation-Induced Loss of Mitochondrial Function by HNE
4 MITOCHONDRIAL HAPLOTYPE DETERMINES SUSCEPTIBILITY
TO CARDIOMYOCYTE DYSFUNCTION RESULTING FROMCARDIAC VOLUME OVERLOAD
Effects of Acute Volume Overload on the Cardiomyocyte Mitochondrion
Elucidating Role of mtDNA Haplotype on Susceptibility to Volume Overload
Materials and Methods
Generation of Mitochondria:Nuclear Exchange (MNX) Mice
Aortocaval Fistula Surgery
xi Mitochondrial Enzyme Activities
Echocardiography and Hemodynamic Measurements
Transmission Electron Microscopy
Comparison of C57BL/6 and C3H/HeN Mitochondrial Phenotypes
Bioenergetic Response of C57BL/6 and C3H/HeN Mice to Volume Overload
Morphometry and Left Ventricular Function of Mice after Volume Overload
Volume Overload-Induced Alterations in Bioenergetic Function
Alterations in Desmin Organization in Cardiomyocytes after Volume Overload
Effect of Volume Overload on Mitochondrial Morphology..............136 Discussion
Reserve Capacity and the Cellular Response to Pathological Stresses
Alcohol-Induces Loss of Mitochondrial Function
Role of Nitric Oxide in Alcohol-Dependent Hepatotoxicity.............149 Alcohol Consumption Alters Hepatocellular Response to Hypoxia
Hypoxic Signaling as a Mediator of Alcohol-Induced Bioenergetic Alterations
Effect of Hypoxia/Reoxygenation on Bioenergetic Function
Endothelial Cell Mitochondrial Function is Altered in Hypoxia
Reoxygenation Induces Progressive Loss of
Susceptibility to Bioenergetic Alterations is Mediated by mtDNA Haplotype
Bioenergetic Responses to Volume Overload in C57BL/6 and C3H/HeN Mice
Effect of mtDNA on Changes in Morphometry and Function by Volume Overload
Volume Overload-Induced Alterations in Mitochondrial Function in MNX Mice
Loss of Desmin Mediates Bioenergetic Alterations Induced by Volume Overload
Volume Overload-Induced Changes in Cardiomyocyte Morphology