Abstract 187: c-Kit Biology Revealed by Two Transgenic Reporter Models.

Abstract

Background: The biological significance of c-Kit as a marker of cardiac stem cells, and role(s) of c-Kit+ cells in myocardial development or in response to pathologic injury remain unresolved due to varied findings among investigators and experimental model systems. Alternative experimental models and approaches are needed to achieve a broader perspective of cardiac c-Kit biology that contextualizes discrepant published observations. Objectives: Tracking c-Kit expression using transgenesis overcomes limitations inherent to knock-in reporter models. Two novel, inducible transgenic c-Kit reporter models are presented in this study to further elaborate on myocardial c-Kit biology. Methods: A previously characterized mouse c-Kit promoter segment was engineered to generate a transgenic mouse in which rtTA transactivator is expressed in c-Kit+ cells (c-KitrtTA). c-KitrtTA crossed to Tet-Responsive-Element(TRE)-Histone2B-EGFP or TRE-Cre lines produces the CKH2B and CKCre double transgenic lines, which express doxycycline-inducible H2BEGFP or Cre proteins in c-Kit+ cells. The CKmTmG triple transgenic mouse, arising from CKCre crossed to the ROSAmTmG reporter line, utilizes doxycycline induced recombination to tag c-Kit+ cells irreversibly with membrane bound EGFP. Endogenous c-Kit and transgenic reporter expression was assessed in adult cardiac myocyte and nonmyocyte cells from these mice under resting and cellular stress conditions using immunohistochemistry and flow cytometry. Results: Coincidence of c-Kit and EGFP is observed in approximately 75% of freshly isolated nonmyocyte cells as detected by flow cytometry. A subpopulation of cardiomyocytes express H2BEGFP or mEGFP in the uninjured, doxycycline treated adult heart. H2BEGFP and c-Kit expression increase in myocytes in response to isoproterenol-induced pathologic stress in vivo and in vitro. Conclusion: These c-Kit transgenic reporter models provide sensitive, specific, inducible and persistent tracking of c-Kit promoter activation. Results presented here reveal an unexpected role for c-Kit expression in adult cardiomyocytes. Future studies will use both models to investigate c-Kit expression in all cell types during cardiac formation and repair.