Mechanisms of myocardial regeneration mediated by Nkx2.5 in zebrafish

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PROJECT SUMMARY Due to the limited ability to mount a regenerative response in injured myocardium, cardiac dysfunction remains a key cause of morbidity and mortality. Coronary artery disease leads to myocardial infarction which results in fibrotic scarring, adverse remodeling, and heart failure. In contrast to adult mice and humans, neonatal mice and adult zebrafish retain the ability to undergo cardiomyocyte dedifferentiation and proliferation, ultimately stimulating cardiac regeneration following injury. Deciphering the fundamental transcriptional networks shared in development and regeneration will fuel our ability to awaken endogenous reparative mechanisms. In zebrafish, embryonic cardiac transcriptional factors necessary for cardiogenesis, such as Nkx2.5, are galvanized following injury to stimulate cardiomyocyte production. Our preliminary studies reveal that Fibulin 5, an extracellular matrix (ECM) glycoprotein functioning downstream of Nkx2.5, is upregulated in the injured heart. Moreover, we show that fibulin loss-of-function fish exhibit impaired regenerative capacity and crucial roles in migration and tissue elasticity. In this proposal, we will test the hypothesis that activation of Nkx2.5 is necessary to produce an ECM milieu comprise of cardinal pro-regenerative proteins such as Fibulin 5 that can be harnessed to augment cell- based therapies for patients. In Aim 1, we will elucidate the myriad mechanisms by which Nkx2.5 directs effectors to mediate deposition and remodeling of ECM elements employing mass spectrometry-based proteomic strategies. In Aim 2, we postulate that Fibulin 5 modulates TGF-β signaling to promote epicardial invasion and titrate tissue stiffness. We will dissect both the canonical and non-canonical TGF-β pathways to identify the key signal transducers downstream of Fibulin 5. Using time lapse imaging of cardiac slices, we will probe the intertwined movements of epicardial and myocardial cells in real time as they penetrate deep into the wound. We will benefit from atomic force microscopy to measure elastic modulus in regenerating myocardium to uncover the requirement of Fibulin 5 in preserving a malleable microenvironment to promote migration. In Aim 3, we will investigate the premise that Fibulin 5 is a vital component of the regenerative ECM with potential to enhance scar resolution by evaluating hiPSC-CMs mobility in vitro and host-graft integration in vivo. Together, we anticipate that our proposed work will launch a novel paradigm highlighting the importance of embryonic ECM components such as Fibulin 5 downstream of Nkx2-5 in promoting epicardial and cardiomyocyte migration and tissue elasticity to enhance therapeutic strategies for patients.