Regenerating your Heart

In this thesis I study zebrafish heart regeneration to identify pro-regenerative genes that may have therapeutic benefits for ischemic heart failure patients. In chapter 1, I introduce zebrafish heart regeneration, how it compares to and differs from repair mechanisms in mammalian hearts, and what molecular mechanisms are known facilitate regeneration of the heart. In chapter 2, we identify Prrx1b as a crucial factor expressed in the zebrafish epicardium that ensures the balance between fibrosis and heart regeneration. We find Prrx1b restricts the amount of pro-fibrotic fibroblasts and stimulates cardiomyocyte proliferation by promoting Nrg1 expression in epicardial derived cells (EPDCs). In chapter 3, we perform a cross-species comparison between the border zones of zebrafish and mouse hearts and identify Hmga1 as driver of cardiomyocyte proliferation and regeneration. We find that Hmga1 reactivates developmentally silenced genes by reducing repressive H3K27me3 levels, thereby activating a pro-regenerative gene program. Notably, we find that AAV-mediated delivery of Hmga1 in the mouse heart post injury induces cardiomyocyte proliferation and functional improvement, positioning it as a potential therapeutic target for regenerative cardiac gene therapy. In chapter 4, we review the field of cardiac gene therapy for ischemic heart failure, highlighting the importance of robust preclinical models and focusing on promising therapeutic targets for the new generation of regenerative gene therapies. In chapter 5, we start the preclinical development of Hmga1 as a potential cardiac gene therapy by assessing optimized AAV-mediated delivery in rodent models. We find that optimized delivery of Hmga1 in mice achieves high cardiac transduction, with limited off target transduction and no unwanted proliferative effects. In addition, delayed administration of Hmga1 post injury is still able to achieve high transduction, increase cardiomyocyte proliferation and significantly reduce scar size. Finally, we find indications that Hmga1 acts through stimulating dedifferentiation of cardiomyocytes. In chapter 6, we test human cell-based ex vivo/in vitro models to compliment the in vivo preclinical evaluation of Hmga1 gene therapy. We assess AAV-mediated delivery of Hmga1 in human induced pluripotent derived cardiomyocytes (hIPSC-CM) derived spheroids, and in human adult live myocardial slices (LMS). We find that Hmga1 delivery stimulates proliferation in spheroids, but not in LMS. However, both require further optimization for use as reliable translational system. In chapter 7, the general discussion, I reflect on the results obtained in this thesis and place them in the broader context of zebrafish heart regeneration and cardiac gene therapy.