Cardiac fibroblasts represent an important connective tissue population in the heart with increasingly recognized roles in cardiac development, homeostasis, and disease. Heart failure, in particular, is debilitating cardiac disorder with hallmark features of extensive myocyte death, pathological remodeling, and fibrosis. In both pathological and age-related cardiac fibrotic states, resident cardiac fibroblasts could serve as a novel therapeutic target to reduce pathological remodeling or generate new functional cardiomyocytes. However, the detailed mechanistic understanding of how disease-driven phenotypic changes in cardiac fibroblasts influence their functional crosstalk with cardiomyocytes remains largely unknown. In addition, as an appealing therapeutic strategy, cardiac fibroblasts could be directly reprogrammed into functional cardiomyocytes. However, the direct reprogramming in two-dimensional (2D) culture remains ineffective and whether three-dimensional (3D) culture milieu could improve the reprogramming efficiency remains unexplored. Therefore, the primary goals of this dissertation have been to:​ 1) engineer a versatile 3D engineered co-culture system approximating environment of native cardiac tissue to systematically investigate age-dependent effects of cardiac fibroblasts on the function and molecular properties of surrounding cardiomyocytes, 2) identify subpopulations of pathologically activated cardiac fibroblasts in response to pressure overload and how they differentially affect cardiac function and fibrosis, and 3) determine whether the reprogramming efficiency of fibroblasts to cardiomyocytes can be enhanced in 3D engineered tissue environment.

To achieve these goals, we first developed a versatile and physiologically relevant hydrogel-based 3D cardiomimetic co-culture platform to systematically assess the direct cardiac fibroblast-induced effects on surrounding cardiomyocytes. By using this 3D co-culture system, we showed that the age of cardiac fibroblasts is a strong determinant of the structure, function, and molecular properties of co-cultured cardiomyocytes. In particular, adult, but not fetal, cardiac fibroblasts significantly deteriorated electrical and mechanical function of the co-cultured cardiomyocytes, as evidenced by slower action potential conduction, prolonged action potential duration, weaker contractions, higher tissue stiffness, and reduced calcium transient amplitude. This functional deficit was associated with structural and molecular signatures of pathological remodeling including fibroblast proliferation, interstitial collagen deposition, and upregulation of pro-fibrotic markers.

In response to cardiac insult, quiescent cardiac fibroblasts become pathologically activated myofibroblasts leading to dysregulated ECM deposition and eventually deterioration of cardiac function. Specifically targeting the pathologically activated myofibroblasts represent an appealing therapeutic goal to delay or reverse the progression of heart failure and pathological fibrosis. We thus set out to investigate whether there exist functional distinct subpopulations of cardiac fibroblasts in response to pressure overload. By utilizing a pressure-overload mouse model and flow cytometry-based cell sorting, we identified the previously uncharacterized Thy1neg (Thy1-/MEFSK4+/CD45-/CD31-) fibroblast population that displayed a more pathological activated phenotype and deteriorated cardiomyocyte function in 3D co-culture system. Additionally, in response to pressure overload, mice with global knockout of Thy1 developed more severe cardiac dysfunction and fibrosis compared to wild-type counterparts, further suggesting a functional role of Thy1 in cardiac pathogenesis.

Finally, we explored whether a tissue-engineered 3D hydrogel microenvironment would enhance direct reprogramming efficiency of fibroblasts into induced cardiomyocytes beyond what has been achievable in traditional 2D culture. We demonstrated that culturing cardiac fibroblasts reprogrammed by a cocktail of microRNAs (miR combo) within a tissue-engineered 3D environment dramatically enhanced the efficiency of reprogramming, which was associated with significant increases in the expression of several matrix metalloproteinases (MMPs). Pharmacological inhibition of MMPs blocked the enhanced reprogramming effects of the 3D environment suggesting a potential mechanism for this observation.

In summary, this dissertation studies established and utilized a physiologically relevant 3D co-culture platform for optimizing engineered cardiac tissue function, studying fibrotic heart disease, and improving fibroblast-reprogramming based cardiac therapeutics. Our studies provide the first evidence of critical roles of the age of supporting cells in engineering functional cardiac tissues, reveal a pathogenic subpopulation of cardiac fibroblasts characterized by lack of Thy1 expression, and demonstrate that 3D tissue-engineered environment can enhance the direct reprogramming of fibroblasts to cardiomyocytes via a MMP-dependent mechanism. These findings provide the foundation for the further development of novel fibroblast-targeted therapeutic strategies for myocardial infarction and pressure-overload induced heart failure.