Abstract 156: Endogenous Cardiomyocyte Dedifferentiation and Cycling Revealed by Single-Cell Imaging and Single-Nucleus Transcriptomic Analysis

Although the mammalian heart had been recognized as a post-mitotic non-regenerative organ, recent evidence demonstrated a measurable cardiomyocyte (CM) renewal at low rates in normal hearts and augmented in post-injury myocardium. However, the resources and cellular processes of such a new CM formation, and the underlying molecular programs remained largely undetermined. While CMs in neonatal hearts or adult CMs in tissue culture can undergo (partial) dedifferentiation and proliferation, the roles of in vivo CM dedifferentiation and cycling were unclear. Here, we report a multi-reporter transgenic mouse models featuring efficient adult CM (ACM) genetic cell fate mapping and real-time CM dedifferentiation reporting. In this triple-transgenic model, upon tamoxifen-induced gene recombination, only ACMs switched from expressing red fluorescent protein (RFP) to green fluorescent protein (GFP), and non-CMs remained expressing RFP. The nuclear blue fluorescent protein (BFP) signal was expressed in normal mature ACMs but reduced in neonatal or cultured dedifferentiated ACMs. Hence, all 4 plausible modes of new CM formation can be visualized using this model. Using ImageStream for single-cell imaging analysis, we demonstrated that non-myocytes (including putative cardiac progenitor cells) contributed negligibly to new ACM formation at baseline or after myocardial infarction (MI). In contrast, there was a significant increase in dedifferentiated (e.g. GFP + BFP - ) CMs in post-MI hearts compared to the sham. Cell cycling activity was enhanced within the dedifferentiated CM population compared to normal ACMs (GFP + BFP + ). Furthermore, we applied massive whole-heart single-nucleus RNA-seq to dissect the transcriptomes of dedifferentiated CMs. We found that CMs in post-MI hearts identified with dedifferentiation reporter had significant down-regulations of genes in networks for hypertrophic growth, cardiac contractile functions and metabolism, but up-regulation in signaling pathways for cell survival, active cell cycle and proliferation. Therefore, cellular dedifferentiation may be an important prerequisite for endogenous CM cell cycling and proliferation and explain the measurable cardiac myogenesis seen after MI.