Investigating Mitogenic Mechanism and Therapeutic Efficacy of Adenosine Deaminase Knockout in Human and Murine Cardiomyocytes

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Abstract Mammalian cardiomyocytes (CMs) in embryonic and neonatal hearts are proliferative, which allows for heart regeneration to occur. This proliferative capacity of CMs is lost shortly after birth and in the adult hearts CMs are largely post-mitotic, renewing at very low rates of less than 1% per year. The inability of adult CMs to proliferate, along with the absence of resident stem cells capable of robust generation of new CMs, limit the ability of adult mammalian hearts to self-repair following injury such as myocardial infarction (MI). Preliminary results from my CRISPR knockout screen in cultured neonatal rat CMs show that knockout of the gene adenosine deaminase (ADA-KO) results in robust cell cycle activation in both primary rodent and pluripotent stem cell-derived human CMs in vitro. Follow-up RNA sequencing analysis suggests that ADA-KO alters CM metabolism. As metabolic alterations have been shown to underlie the pro-proliferative actions of other known cardiac mitogens, my proposed studies will determine the metabolic and molecular signaling mechanisms governing ADA-KO mediated proliferation in human CMs. Additionally, I will test the in vitro effects of ADA-KO on injured engineered cardiac tissues (ECTs) made using polyploid human CMs, which we have shown to be resistant to division similarly to polyploid CMs in vivo. Furthermore, in proof-of-concept in vivo studies, I will test therapeutic efficacy of ADA-KO in the setting of myocardial infarction by AAV delivery of ADA-KO sgRNAs to transgenic mice with CM-specific Cas9 expression, followed by studies of cardiac proliferation and function. When completed, these cross-species in vitro and in vivo studies will provide better understanding of the roles of ADA in cardiac metabolism and regeneration and will create a basis for the development of novel gene therapies for cardiac regeneration.