Isolation of Embryonic Cardiomyocytes and Cell Proliferation Assay Using Genetically Engineered Reporter Mouse Model.

Abstract

Congenital heart disease (CHD) is often associated with myogenic defects. During heart development, cardiomyocyte growth requires essential cues from extrinsic factors such as insulin-like growth factor 2 (IGF-2). To determine whether and how growth factors account for embryonic cardiomyocyte proliferation, isolation followed by culturing of embryonic cardiomyocytes can be utilized as a useful tool for heart developmental studies. Current protocols for isolating cardiomyocytes from the heart do not include a cardiomyocyte-specific reporter to distinguish cardiomyocytes from other cell types. To optimize visualization of cardiomyocyte proliferation, our protocol utilizes a Tnnt2-promoter-driven H2B-GFP knock-in mouse model (TNNT2H2B-GFP/+) for in vitro visualization of nuclear-tagged cardiomyocyte-specific fluorescence. A cardiomyocyte-specific genetic reporter paired with an effective proliferation assay improves the reproducibility of mechanistic studies by increasing the accuracy of cell identification, proliferated cell counting, and cardiomyocyte tracking. Key features • This protocol refines previous methods of cardiomyocyte isolation to specifically target embryonic cardiomyocytes. • UsesH2B-GFP/+cardiomyocyte reporters as identified by Yan et al. (2016). • Traces cell proliferation with Phospho-Histone 3 (p-H3) assay. • Has applications in assessing the role of growth factors in cardiomyocyte proliferation.