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.