Design and Evaluation of an In Vivo, Repeatable, and Real Time Reporter for Myofibroblast Cell Fate as a Surrogate for Cardiac Fibrotic Remodeling

Thesis (Master's)--University of Washington, 2018Heart disease is the leading cause of death in the United States accounting for 1 in every 4 deaths [“Heart Disease Facts”]. More than half of heart disease deaths are attributed to coronary heart disease which often leads to congestive heart failure costing the United States $30.7 billion annually [“Heart Disease Fact Sheet”]. Myofibroblasts lead coronary heart failure to heart disease through long term adverse fibrotic remodeling [Van Den Borne et al]. After myocardial infarction, fibroblasts transdifferentiate into activated myofibroblasts that orchestrate matrix remodeling through deposition of collagen, cytokine secretion, and contractility forming a fibrotic scar. While this is beneficial in acute wound healing, prolonged presence of the fibrotic scar interrupts proper heart function, increases cardiac workload, and encourages further fibrotic buildup resulting in heart failure. Research on elucidating the regulatory pathways involved in transdifferentiation is occurring slowly and is made difficult without proper cell specific reporting. Currently, methods to track myofibroblast activity are indirect and limited. Immunohistochemistry is the gold standard for measuring cardiac fibrosis in in vivo studies yet is limited by invasiveness, quantifiability, and inability for longitudinal study. MRI has recently been able to quantify and track fibrosis with high temporal and special resolution through protein targeted gadolinium contrast agents, however, this method is expensive and indirect [Caravan et al]. Here we perform literature and bioinformatic survey to investigate potential transcriptional activators in myofibroblast specific gene promoters and show initial in vitro support for a genetic luciferase reporter for myofibroblast cell fate driven by chimeric combinations of promoters from genes involved in carrying out fibrotic remodeling as a surrogate for cardiac fibrosis. The addition of double 9x tandem repeat enhancer sequences greatly boosted luciferase expression while maintaining reporter specificity to TGFb treatment and fibroblast cell type. Ultimately, a reporter of this kind will enable real time longitudinal study, reduce animal waste, and has potential applications in gene therapy