Human CRISPRa Cardiac Reprogramming: Model Characterization and Epigenetic Mechanisms

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PROJECT SUMMARY / ABSTRACT The preponderance of morbidity and mortality from cardiac disease results from ischemic damage to the myocardium. Because of the limited regenerative capacity of cardiomyocytes, ischemic damage results in permanent myocardial death followed by fibrosis and scar formation. Cardiac reprogramming is a promising new regenerative approach that has the potential to restore myocardium by converting resident fibroblasts into cardiomyocyte-like cells. In the decade of research that has followed the first report of cardiac reprogramming, the field has made significant strides in improving the efficiency of its methods and in understanding the signaling pathways, transcriptomic changes, and chromatin remodelers that are important for cardiac reprogramming. Our long-term goal is to help catalyze the application of cardiac reprogramming to human health by improving our techniques and by deepening our knowledge of the reprogramming process. A constant in cardiac reprogramming methods has been induction of reprogramming through forced expression of exogenous transcription factors, usually encoded by a retroviral vector. We have developed a new model of human cardiac reprogramming that uses CRISPR-based gene activation (CRISPRa) to promote MEF2C, GATA4, and TBX5 expression, and by comparing this model to our current methods, we can gain new insights into the basic biology of the reprogramming process. Aim 1 is to characterize the molecular, phenotypic, and functional features of our CRISPRa model. Sarcomeric structure and gap junction patterns will be assayed by immunofluorescence, and transcriptomic changes will be studied using RNA-seq. Calcium flux through a fluorescent reporter as well as observations of cell contraction will be measured as functional outcomes. Aim 2 is to study the epigenetic changes and mechanisms of reprogramming induced by CRISPRa. The epigenetic dynamics at MEF2C, GATA4, and TBX5 and other genes will be studied, and the epigenomic remodeling unique to CRISPRa reprogramming will be determined through ChIP-seq and ATAC-seq. The potential mechanisms that lead to unique epigenetic features will be explored through RNAi as well as conventional CRISPR editing. Overall, this study will provide key insights into a novel form of cardiac reprogramming, insights that will deepen our perspectives on cell identity, cell plasticity, and cell fate determination. Furthermore, by developing a CRISPRa- based model, we have opened a potential avenue for trial of cardiac reprogramming in humans. With further study, cardiac reprogramming can become safe and effective enough for human regenerative purposes. I will complete this work under the advisement of Dr. Li Qian, a world-renowned scientist with a passion for mentorship. My institution has ample resources for the work I have planned, and it offers many opportunities for learning and professional growth as detailed in my training plan. Upon completing this fellowship, I will be ready for the next phase of training in my path to becoming an independently funded physician scientist.