Reawakening cardiomyocyte proliferation from structural mediated quiescence

SUMMARY This project aims to examine the synergy between Hippo/Yap and Wnt/b-catenin signaling to induce cell cycle reentry and proliferation in adult mouse and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our team of experts in cardiac development, iPSC-based disease modeling, Yap and Wnt signaling, and cardiac regeneration is poised to address the overall hypothesis that Yap and Wnt signaling synergistically induce sarcomere disassembly and CM proliferation and their activities are enhanced by disruption of structural barriers such as cell-cell contact and cell-extracellular matrix (ECM) interactions. Based on compelling preliminary data from both Martin and Wu Labs, we show that induction of Yap signaling in the adult mouse heart drives cytoskeletal rearrangement and initiation of cell cycle activity after myocardial infarction (MI) while the treatment of hiPSC-CMs with CHIR, a small molecule activator of canonical Wnt/b-catenin signaling, induces their massive expansion that is enhanced by disrupting cell-cell contact. We propose to address our experimental hypotheses by completing three independent, yet interrelated Specific Aims: 1) Address the hypothesis that YAP and Wnt signaling synergize to breakdown sarcomeric assembly and cytoskeletal structure during cardiomyocyte proliferation. We will determine whether YAP and Wnt signaling synergistically regulate sarcomere gene expression and assembly by introducing the Yap5SA transgene and conditional b-catenin gain-of-function (GOF) alleles in the mouse heart to examine changes in the expression of sarcomeric genes and their myofibrillar assembly. In parallel, we will assess the effect of the Yap5SA transgene and CHIR treatment on hiPSC-CMs. We will also determine whether Wnt and YAP-induced reduction in sarcomere assembly enhances cell cycle gene expression and CM proliferation and maintains an active chromatin state in adult CMs by simultaneously inducing Yap5SA transgene and b-catenin signaling by b-catenin GOF allele in adult CM and in hiPSC-CMs beyond that achieved with each treatment alone. 2) Address the hypothesis that the disruption of cell-cell and cell-matrix interactions will enhance CM proliferation via gain in YAP and Wnt signaling. We will determine whether the disruption of cell-cell contacts will enhance CM proliferation via gain in YAP and Wnt signaling by knocking down cell contact protein N-Cadherin (N-Cad) while activating proliferation with Yap5SA and b-catenin GOF allele or with CHIR in mice and in hiPSC-CMs. We will also determine whether the disruption of cell-ECM interactions will enhance CM proliferation via gain in YAP and Wnt signaling. 3) Address the hypothesis that the induction of YAP and Wnt signaling specifically in the MI border zone will synergistically enhance CM proliferation. This translational aim will examine the synergy between Yap and Wnt signaling in the setting of MI using an infarct border zone-specific ANF enhancer to examine the synergy of Yap/b-catenin signaling in infarcted mice, stressed hiPSC-CMs, and infarcted pigs.