This research focuses on dissecting the molecular mechanism of cardiac regeneration in the animal model, zebrafish, upon a myocardial infarction like injury. Zebrafish are one of the few vertebrates that can fully regenerate their hearts after an injury in 30 days. This phenomenon is not seen in humans, who generate scar tissue after this injury with reduced circulatory efficiency. However, there is evidence that neonatal mice under 7 days old can regenerate their hearts, but this is lost upon adulthood. Determining this pathway is the first step to develop therapeutics in order to provide relief to people suffering from cardiac injuries. In this research, we used chemically ablated transgenic zebrafish to generate a 30% injury. We determined that upon an injury, both the Wnt pathway and the mTOR pathway are sequentially activated and upregulated to restart cardiac proliferation to regenerate the heart. Wnt pathway proteins like Axin and β-catenin are activated 3 days post injury and mTOR proteins like pS6 are activated gradually over 7 days post injury. The inhibition of the Wnt pathway using DKK showed a downregulation of the mTOR pathway and downregulation of cardiomyocyte proliferation. Inhibition of the mTOR pathway using Rapamycin also stopped cardiomyocyte proliferation from occurring. Mass spectrometry data showed a decrease in glutamine and an increase in leucine during the proliferative phase. Since leucine is one of the activators of the mTOR pathway, we see that the glutamine-leucine transporter is also upregulated post-injury. Thus, we show that heart regeneration in adult zebrafish occurs via cardiomyocyte proliferation by using the Wnt and mTOR pathways to upregulate cardiomyocyte proliferation upon injury.

The Polycomb Repressive Complex 2 (PRC2) is an important epigenetic remodeler in developmental transitions and cell fate determinations. PRC2 is responsible for the addition of H3K27me3 marks that repress developmental gene expression. The catalytic subunit of PRC2 is the methyltransferase (Enhancer of Zeste 2) EZH2 which binds to EED (Embryonic Ectoderm Development) to methylate H3K27 on gene promoter regions. To investigate the requirement of PRC2 in different developmental transitions, a computationally designed protein was utilized to inhibit EED-EZH2 interaction. The novel designed protein is named EED binder (EB) and competes over endogenous EZH2 on the EED binding cleft with 300 times greater affinity than endogenous EZH2. We cloned EB-GFP under heatshock inducible promoter and injected this construct to one cell zebrafish embryos to generate a germ line transmissible insertion. To study the requirement of PRC2 in early developing embryos (0-3dpf), we applied heatshock (HS) on EB-GFP positive and negative embryos. Western blot analysis revealed global downregulation of EZH2 and H3K27me3 in EB-GFP positive, but not control embryos. Additionally, Co-Immunoprecipitation experiments showed EB-GFP binding to EED. Finally, to test the requirement of PRC2 in caudal fin regeneration, adult (5 month old) EB-GFP positive and negative animals were fin-amputated and the regeneration growth rate was measured for 14 days. Our results show that EB-GFP positive fish were able to regenerate their fins faster, resulting in a large fin size compared to either negative or non-HS clutch mate. Overall, we have developed a computer designed inducible PRC2 inhibitory system to study PRC2 function in Zebrafish, at the whole animal level. In the future, we will utilize EB-GFP to explore PRC2 and other epigenetic modifiers that are required for tissue and organ regeneration before and after injury.