Protocol to identify small molecules promoting rat and mouse cardiomyocyte proliferation based on the FUCCI and MADM reporters

Discovery of small molecules promoting cardiomyocyte proliferation is important for heart regeneration and related heart disease. Here, we describe a protocol to isolate neonatal rat and mouse cardiomyocytes, infect cardiomyocytes with Tnnt2-mAG-hGeminin (1/110) or Tnnt2-Cre adenovirus, and identify small molecules that promote cardiomyocyte proliferation by high-content microscopy. This protocol can be modified to investigate other pro-proliferation factors in cardiomyocytes and other cell types. For complete details on the use and execution of this protocol, please refer to Du et al. (2022).¹