Traditionally the adult heart was viewed as having virtually no capacity for regeneration. However, work from multiple groups now supports a more dynamic model in which both animal and human hearts have some ' though limited ' endogenous regenerative potential. A deeper understanding of the heart's regenerative capacity and the underlying regulators is important for harnessing this capacity for therapy.We propose an innovative, collaborative approach to understand the regeneration of heart cells in vitro and in vivo using cutting-edge tools from synthetic biology. This proposal integrates disparate disciplines to answer two critical outstanding questions: what is the heart's regenerative capacity and what cellular population(s) is/are responsible? Moreover, we will test the hypothesis that the CITED4 transcriptional co-activator is sufficient to drive cardiomyogenesis.We propose a novel technology based on synthetic gene circuits called Cell Division Counters (CDCs) that can count cell divisions in single cells. This technology will quantify the extent of cardiomyogenesis in the adult heart, unambiguously identify the source and fate of new cardiomyocytes (CMs), and provide novel strategies for engineering cardiac regeneration. This work builds on the Lu lab's expertise in synthetic biology, the Rosenzweig lab's experience in cardiac biology, and their close collaboration.We will pursue three integrated Aims:(Aim 1) We will construct and validate CDCs for mammalian cells using recombinases that sequentially invert or excise specific stretches of DNA with each pulse of cell-phase-specific promoters. These DNA arrangements lead to a different DNA state for each event count and can be assayed through a variety of means.(Aim 2) We will deliver CDCs into cardiac cells in vitro, identify dividing versus non-dividing cell populations, and transcriptionally profile these populations to determine their lineages and key regulators. We will investigate the proliferation potential of c-kit-positive putative cardiac stem cells, adult CMs, and other cardiac cells.(Aim 3) We will deliver CDCs into mouse hearts in vivo to quantify cardiomyogenesis after infarction and in response to CITED4 transcriptional pathway perturbations. Proliferating cells will be sorted based on CDC state and assayed to determine their lineage, transcriptomic profiles, and key regulators, which will be validated to confirm their role in cardiac regeneration.
|Program type||Collaborative Sciences Award|
|Effective start/end date||07/01/2014 → 06/30/2017|