Project Summary Stem cell mediated cardiac repair is an exciting and controversial area of cardiovascular research that holds the potential to produce novel, revolutionary therapies for the treatment of heart disease. Extensive investigation to define cell types contributing to cardiac formation, homeostasis and regeneration has produced several candidates, including adult cardiac c-Kit+ expressing stem and progenitor cells that have even been employed in a Phase I clinical trial demonstrating beneficial outcome for many patients together with safety and feasibility of this therapeutic approach. Despite many promising studies including the SCIPIO clinical trial, the significance of the c-kit+ cell population in cardiac regeneration is persistently debated among many cardiovascular researchers and clinicians. Therefore, further investigation conducted with rigorous experimental design, methodology, and interpretation is essential to resolve current ambiguities and advance the field of cardiac regenerative therapy. In that spirit, this proposal determines the role of c-Kit+ cell participation in cardiac repair using a novel, inducible transgenic mouse model to genetically tag c-Kit expressing cells during development, following injury and in the aging heart. In contrast to “knock-in” approaches employed in many recent c-Kit lineage-tracing models that generate a hemizygous genotype for endogenous c-Kit, the strategy proposed here exploits overexpression of a validated exogenous c-Kit promoter construct to drive expression of the rtTA transactivator. This construct in combination with promoters regulated by the tetracycline responsive element (TRE) induces transcription of the TRE controlled reporter upon administration of doxycycline. c-Kit+ cells are tagged in a time-dependent manner transiently or permanently, depending upon the TRE controlled partner. Preliminary results show that c-Kit cells comprise a more diverse population than previously assumed, necessitating a more nuanced understanding of fundamental c-Kit biology and expression patterns. Specific Aims are: 1) c-Kit+ stem cells contribute to adult cardiac tissue during tissue homeostasis and repair as demonstrated using genetic models for lineage tracing in vivo, 2) c-Kit+ cells contribute to cardiac formation during development and 3) c-Kit+ cells contribute to homeostasis in the aging heart. The innovation of this proposal is in the approach used to tag and track c-Kit cells such that endogenous c-Kit biology remains intact while overexpression confers greater sensitivity and therefore more extensive labeling of the c-Kit cell population. The significance of these studies is to clarify existing unresolved controversies in the field by establishing c-Kit cell expression and participation in cardiac formation and repair. Collectively, these studies will contribute essential information defining the importance of c-Kit+ cardiac progenitor populations as reagents for cell based therapy in ...