SKI Activates Hippo Signalling to Modulate Cardiac Fibroblast Function and Activation by Natalie M. Landry A thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology & Pathophysiology Rady Faculty of Health Sciences University of Manitoba Copyright © 2020 by Natalie Mireille Landry II Abstract Even with ongoing improvements in cardiac patient treatment and outcomes, current therapies for cardiac fibrosis only alleviate the symptoms of heart failure, rather than treating the underlying cause of the disease. Furthermore, the ability to study cardiac fibroblast activation and pro-fibrotic signalling in vitro is complicated by the cells’ phenotypic plasticity and sensitivity to mechanical input. Our lab has previously established that SKI, an endogenous TGF-β1 signalling inhibitor, halts fibroblast activation and deactivates the myofibroblast phenotype. We have also determined that conventional cell culture does not lend itself to accurate physiological studies of primary cardiac fibroblasts, in vitro. The thesis presented herein establishes a method of maintaining primary cardiac fibroblasts in a resting state in two-dimensional cell culture. In addition, this improved technique is applied to test the hypothesis that SKI is a multi-functional protein that can act independently of TGF-β to specifically activate Hippo signalling, and in turn inhibit the cardiac myofibroblast phenotype. In order to study cardiac fibroblast activation in vitro, we developed cell culture conditions which better maintain the native phenotype of primary cells by limiting mechanical, hormonal, and nutritional input. Both rat and mouse primary cardiac fibroblasts could be maintained in a quiescent state for up to ten days post-isolation, which is sufficient for most commonly-used cellular and molecular assays. Using this optimized method, we first sought to determine the role of the primary nuclear effectors of the Hippo pathway, Yes-Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ, or WWTR1), in cardiac fibroblast activation. Constitutively-active forms of YAP and TAZ were overexpressed in primary rat cardiac fibroblasts, and demonstrated their ability to overcome external stimuli to promote fibroblast activation and pro-fibrotic gene expression. However, III TAZ demonstrated greater activation of the Collagen 1αI and 3α1 promoters. Subsequently, YAP and TAZ expression were examined in a rat post-myocardial infarction (post-MI) model of fibrosis. It was found that TAZ expression increases and translocates to the nucleus, while YAP expression was relatively unchanged in the infarcted left ventricle. Taken together, these data suggest that TAZ has a greater role in the pathogenesis of cardiac fibrosis. Ensuing studies used first-passage (P1) primary cardiac myofibroblasts to examine the effects of SKI on YAP/TAZ signalling. Overexpression of SKI induced the degradation of endogenous TAZ, but not YAP. Moreover, loss-of-function studies suggested that the Large Tumor-Suppressor kinase 2 (LATS2) is required for SKI’s specific effects on TAZ, but not its homolog, LATS1. To better understand the SKI-TAZ link, and whether it results from direct interaction with the Hippo pathway, the human cardiac fibroblast-specific SKI interactome was elucidated by using BioID2 and novel interactors were identified by tandem mass spectrometry. We identified the focal adhesion-associated protein, LIMD1, as a mediator between LATS2 and SKI. Additionally, knockdown of Limd1 in myofibroblasts resulted in the proteasomal degradation of TAZ, but not YAP, recapitulating the TAZ-specific effects of SKI overexpression. Overall, our data suggest that LIMD1 is an important component of the regulatory effects between SKI and TAZ/LATS2, and presents a novel point of crosstalk among pro- and anti-fibrotic pathways. This study is the first report of the role of TAZ-Hippo signalling in post-MI remodelling and fibrogenesis using optimized in vitro and in vivo models of fibrotic disease. The findings described herein postulate that the SKI-LIMD1-TAZ signalling axis as a novel and selective therapeutic target for consideration in future treatments for cardiac fibrosis. IV Acknowledgements First and foremost, I must thank my supervisor, Dr. Ian Dixon, for his support and encouragement during my graduate studies. I am so grateful to have had an advisor that allowed me to take risks and “follow the data” with respect to my project. I will miss our morning chats, armchair quarterback analyses of Blue Bombers games, and hearing about your latest adventures in astrophotography. I am proud to have been part of your lab, and even prouder to call you a friend and colleague. Many thanks are extended to my advisory committee members, Drs. Barbara Triggs- Raine and Andrew Halayko, who have been invaluable sources of insight through every iteration of my project. Thank you for never letting me rest on my laurels and challenging me to better myself as a scientist. I am grateful to have had you both as mentors during the formative years of my academic career. I must also acknowledge Dr. Peter Cattini, whose unwavering support from day one has truly been invaluable in my success as a graduate student. I would like to thank the members of the Dixon lab, past and present. A special thanks to Sunil Rattan, whose technical expertise and steadfast friendship I consider to be second to none. To my “work wife,” Krista Filomeno, whose legendary sneezes and wicked sense of humour made long days in the lab feel much shorter. I am also indebted to the incomparable Dr. Mark Hnatowich, whose meticulousness and supernatural skills in everything DNA-related made all the difference in completing my project. I will especially miss your ability to conjure up the most interesting conversation topics and your devilish sense of humour. I consider you a dear friend, and will continue to hope that one day, your “Math and Cheese” dreams will come true. I must also thank Dixon lab members, Drs. Matthew Zeglinski, Morvarid Kavosh, Shivika Gupta, Jared Davies, and Rebeca de Oliveira Camargo for their friendship and support throughout my time at ICS. Finally, I am eternally grateful to all the summer students that helped me complete (too) many Western blots and qPCR assays for my project: Sarah Foran, Thomas Meier, Simon Meier, Nikita Sarangal, Ken Xing, Jooyeon Seung, and Claire Meier. As we say in French, “mille mercis, mes amis”! I would be remiss to not recognize my extended ICS family for all their help during my graduate studies. To all the PI’s who have mentored and encouraged me to better my skills as a scientist and communicator, I thank you for your dedication to student development and to the culture of success at ICS. To the members of Dr. Elissavet Kardami’s lab: thank you for all your technical help and your comradery. A special thanks to Dr. Navid Koleini, whose friendship I will always hold dear, and whose infectious laugh was always a welcome sound in the hallways of the Albrechtsen Research Centre. I am also thankful to all the friendships I have made with the other graduate students of ICS. A special thanks to the members of the ICS “Cheese Club,” Matthew Guberman and Cameron Eekhoudt—thank you for all the great conversations, laughs, and I wish you all the best in your future endeavours. I must also thank my other work family, the team at the Youth BIOlab Jeunesse. To Steve Jones: thank you for taking a risk on a shy French-Canadian when you hired me nine V years ago and giving me the opportunity to better myself as a scientist, communicator, and teacher. To Meghan Kynoch, I am so thankful for your friendship and positive attitude, even when things get tough. I am so grateful to have met both of you, and wish you continued success in everything you do. I gratefully acknowledge all the funding agencies which made the contents of this thesis possible. Thank you to the Canadian Institutes of Health Research, the Heart & Stroke Foundation of Canada, the American Heart Association, Keystone Symposia Future of Science Fund, Research Manitoba, and the Bank of Montreal for the funding which allowed me to complete and share the results of my project. Many thanks are also extended to the administrative staff in the Department of Physiology & Pathophysiology. To the angels in the Department office, Gail McIndless, Judy Olfert, and Sharon McCartney, thank you for being endless wells of knowledge and wisdom, and for being so patient with all the lost paperwork, award deadlines, and committee meeting mix-ups. I would not have survived graduate studies without you! Last, but definitely not least, I am so thankful for the support provided by my dear friends and family, who have been endlessly understanding and generous cheerleaders throughout my graduate studies. A giant merci to my parents, Lise & Normand, who have unconditionally supported me my entire life. To Marc, Sylvie, Janelle, and Kim: thank you for always knowing how to make me laugh—there is no better cure for the dejection from a failed experiment than a reminder of how weird our pets are. And to my SMA family, Ashley, Mai, Chelsea, Alison, Lauren and Sabrina: despite not seeing you all as often as I would like, our quarterly “Meetings of the Minds” are a great reminder of how lucky I am to have such strong, independent women in my life. I am also grateful for my chosen family, Ray & Ann, and Debbie & Gregg, with whom I have been so fortunate to share my triumphs and failures. Finally, I must thank my incredibly loving and patient husband, Cameron, who saw me through all the highs and lows with unwavering positivity and constant encouragement. I am so lucky to have had my best friend by my side through this incredible journey.