Congratulations Brooke Ahern, PhD
On Friday, April 23, 2021 Brooke Ahern (Jon Satin Lab) successfully defended her dissertation and earned her doctoral degree. Congratulations Dr. Ahern!
RAD MODULATION OF THE L-TYPE CALCIUM CHANNEL CONFERS SYSTOLIC ADVANTAGE IN THE HEART
Doctoral Committee
Dr. Jon Satin, Department of Physiology, Mentor
Dr. Douglas A. Andres, Department of Molecular & Cellular Biochemistry
Dr. Brian Delisle, Department of Physiology
Dr. Ahmed Abdel-Latif, Department of Internal Medicine - Cardiology
Dr. John McCarthy, Department of Biology
Dr. Sanda Despa, Department of Pharmacology & Nutritional Sciences, Outside Examiner
Abstract
Heart failure is a major public health problem and a leading cause of mortality. This clinical condition affects populations of all ages, and is the result of various cardiomyopathies. Almost half of these patients suffer specifically from heart failure with reduced ejection fraction; these hearts have decreased performance due to a failure of the heart to contract with sufficient force to meet demand. While there are therapies available to increase contractility, none of these enhance contraction without also further promoting pathological signaling and remodeling.
Under normal physiological conditions, the body elevates cardiac output through the fight-or-flight response. This response activates b-adrenergic receptors (b-AR) at the level of individual cardiomyocytes, which leads to enhanced calcium handling in order to increase contraction. One of the major targets of b-AR downstream signaling is the L-type calcium channel (LTCC). The influx of calcium through the LTCC (ICa,L) provides the trigger for calcium induced calcium release from the sarcoplasmic reticulum in order to produce a contraction; LTCC activity is significantly increased when b-ARs are activated. However, b-ARs are chronically activated in heart failure, leading to pathological remodeling and further development of heart failure. This has served as a foundation to establish dogma that increasing ICa,L in a manner that reflects b-AR activation necessarily promotes pathology. Because b-AR signaling is a principle physiological mechanism to increase cardiac output, understanding this pathway and how to increase calcium safely is critical to successfully treating heart failure. Discovering a mechanism to increase cardiac output downstream of b-AR signaling would be ideal so as to preserve the fight-or-flight response while also boosting cardiac performance in order to meet demand.
The mechanism by which LTCC activity is increased under b-AR signaling, known as modulation, has been a major focus of study for many years; however, it remains unknown. The LTCC is a heteromultimeric protein complex, and is inhibited by an endogenous small monomeric GTPase called Rad. Studies in heterologous expression systems show overexpression of Rad blocks calcium current through the LTCC; absence of Rad yields a significant increase in calcium current. Whole-body Rad knock out mouse models demonstrate calcium current that mirrors calcium current stimulated by b-AR signaling; however, this also promoted significant growth of the heart. To investigate the effect of Rad deletion without contributions from non-cardiac tissue, a cardiomyocyte-restricted inducible Rad knock out mouse model was created. The work of this dissertation utilizes this mouse to better understand the mechanism by which Rad inhibits the LTCC by studying the effects on channel function, cellular calcium handling, and overall cardiac structure and function in the absence of Rad.
Using an array of methods and techniques, the studies in this dissertation establish Rad as a critical target of the LTCC to respond to b-AR stimulation. When Rad is depleted specifically from cardiomyocytes, ICa,L is increased in a safe, stable manner that mirrors LTCC modulation, both in sinoatrial node and in the ventricle. This regulation is governed specifically by the C-terminus of Rad. Elevated ICa,L in the absence of Rad promotes enhanced calcium handling and increased cardiac output without progression to heart failure, and occurs independently of b-AR activation. Enhanced calcium cycling in the absence of Rad is balanced by accelerated inactivation of the LTCC so as to promote positive inotropy without instigating arrhythmogenesis. This allows for cardiac protection under conditions of pressure-overload induced heart failure. In summary, the work of this dissertation supports Rad deletion specifically from cardiomyocytes as an ideal positive inotrope for heart failure treatment due to the novel mechanism to increase ICa,L in a manner that preserves structure, function, and the fight-or-flight response within the heart.
Acknowledgements
Completing this dissertation work would not have been possible for me without the help and guidance from my support system. I would like to acknowledge the following people who have helped me succeed and grow as a student and individual.
First, thank you to my mentor, Dr. Jonathan Satin. Thank you for challenging me daily to become a better scientist, critical thinker, writer, electrophysiologist, and person. Your passion for all things cardiac and calcium quickly convinced me to switch mindsets from neuroscience to studying the mechanisms of the heart, and I am very grateful for that. Thank you for seeing my potential, and helping me to succeed and grow these past five years.
I would also like to express sincere gratitude for the members of my committee: Dr. Doug Andres, thank you for pushing me to think differently (not always as an electrophysiologist) and to see both science and life from a different perspective. Dr. Brian Delisle, thank you for always providing a listening ear and a sounding board for my projects and my life; also, thank you for teaching me how to patch-clamp – a technique that has taught me the true meaning of patience and perseverance. Dr. Ahmed Abdel-Latif, thank you for providing helpful feedback on my work from a clinical perspective, and giving me career advice as I have tried to navigate my future. Dr. John McCarthy, thank you for your support of my work, my love for space, and for allowing me to be an honorary member of your lab. And to Dr. Sanda Despa, thank you for serving as my outside examiner and taking the time to review my dissertation.
Thank you to the American Heart Association for awarding me a pre-doctoral fellowship that funded me for two years. I am sincerely grateful for the time and service by volunteers within that wonderful organization that provided the financial support so I could pursue my passion for understanding Rad. I am also very grateful for the NIH T32 that I was given my first year within the department and the work from faculty within the department to have that available for students.
I have also been blessed to share the lab with two amazing women, Bryana Levitan and Andrea Sebastian. Thank you Bryana (aka lab mom) for your support and teaching me what you have learned about the heart and about life. Thank you Andrea (the science fairy) for your constant support in all of my experiments, for all of the therapy sessions while isolating cells or making solutions, and for being a great friend. I am a much better person for knowing both of you.
I was also very lucky to join the best department on campus, the Physiology Department. The faculty and staff made each day great – thank you to all those that have given me advice over the years, shared a laugh or taught me both in and out of the classroom. I also appreciate that we had the best lunches, best seminar treats (always had Diet Coke!), and the best holiday parties. I will cherish the fun I had with all of you.
To all of the friends I have made in graduate school: Ben Shaw, Beth Oates, and Sarah Sternbach – the original dream team of first year and the reason I survived. Ryan Cloyd, Brandon Farmer – the wonderful additions to the crew. Our adventure lunches, Mario Kart, parties, therapy sessions and of course the pet therapy from all of you have helped me maintain my sanity throughout the years here. Words do not truly express my gratitude; thank you guys for being a major part of my support system. To Taylor Valentino, Yuan Wen, Ivan Vechetti, and Brooks Mobley – thank you for all of your support while I figured out my life and career, teaching me, letting me vent, always providing chocolate and Diet Coke, and giving me honorary member status in the lab. I am lucky to have been able to call your lab, as well as the Satin lab, home. To those that I have met in graduate school but have not mentioned thank you for supporting me! I have been very blessed to meet many wonderful people, and call them friends.
To Kate Holbrook and Trent Simmons – friends who have known me for a long time and have been critical support for much of my life. Thank you for the late night phone calls, the supportive texts, and for being the best cheerleaders even though you both are so far away. I would not have gotten this far without you two.
And last, but certainly not least, I want to thank my family. To my wonderful parents, David and Heidi Ahern – thank you for always pushing me to be the best I can be. You have always believed in me, encouraged me to follow my passions, and given me confidence when I doubted myself. I cannot express in words how grateful I am for your sacrifices so that I could make it this far. To my amazing grandparents, Donald and Abbey Schluter and Rel Ahern, who have always been the most supportive of anything I do in life, especially in graduate school. Thank you for always being there, whether in person, in spirit, or on Facetime; I am so grateful to have you in my life. And to the best sister anyone could ask for, Alexandra Ahern – thank you for being my best friend through everything, for understanding all aspects of my life (including getting a PhD), and for inspiring me daily to be better than I was the day before. Thank you for always being the ideal example in my life.
To those that I have not mentioned but have helped me, given me advice, and made my life better, thank you!
