Defining murine cardiac fibroblast cellular states via fibroblast-vascular crosstalk and extracellular matrix remodeling

Abstract

The overarching focus of this thesis is to gain insight into novel categories of cardiac fibroblast cellular state by probing the biological underpinnings of how structure dictates function. Cardiac fibroblasts are responsible for scarring and disease progression in the heart, and they adapt both their morphology and roles depending on various external stimuli. The physiological and pathological interactions between fibroblasts in the heart and their environment are crucial to understanding cellular behavior and mechanisms of fibroblast-driven diseases that remodel the entire architecture of the heart over time. This work, resulting in novel insights to fibroblast structure-function relationships, required combinatorial approaches from cell biology, genetic engineering, three-dimensional imaging and image processing, and unbiased modeling. In this thesis work, we combined these techniques to study the relationship between cardiac fibroblasts, the extracellular matrix, and the vasculature of the murine heart. Within intact hearts, we meticulously assessed the three-dimensional morphology of fibroblasts in their native environment with a wide range of cell fate options in their decision-making processes. We then narrowed or controlled the cellular fate possibilities with injury, genetic knockdown or overexpression of activation pathways, and combined injury with genetic manipulation to encourage known cellular identities. The tools developed here allowed for visualization and quantification of previously unknown fibroblast association with the vasculature as well as spatially-dependent heterogeneity of fibroblast morphology. We also demonstrated the ability to unbiasedly categorize fibroblasts strictly based on morphology and distinguish specific genetic cellular states within that shape-based characterization. We investigated how various morphologically and spatially distinct fibroblast populations might impact basic physiology in 3D. These results demonstrate an important relationship between fibroblasts and the vasculature of the heart. Future work is needed to elucidate the exact mechanisms behind shifting fibroblast morphological states and their resultant specific roles in health and disease.