Background: Albeit the evident renewal of adult cardiomyocytes (CMs), studying cellular mechanisms of endogenous myogenesis in adult hearts is challenging due to the scarcity of new CM formation and the complexity of heart cell populations. We previously found that dedifferentiation in CMs renders them competent to proliferate in vitro. Nonetheless, its role in CM renewal in injured myocardium remained undetermined. OBJECTIVE: To investigate cellular mechanisms underlying endogenous CM renewal in adult hearts after myocardial infarction (MI).
Methods and Results: To enable high-throughput analysis including flow cytometry and cell imaging, we generated a CM nucleus-specific blue fluorescent protein (BFP) reporter mouse for real-time visualization of CM lineage. BFP signal also reflected the maturity of CMs. To further identify the source of endogenous CM renewal, we utilized an efficient CM lineage-tracking model by cross-breeding αMHC-MerCreMer mouse with dual-reporter mT/mG mouse (dubbed as RFPfl/GFP). The tri-transgenic RFPfl/GFP;BFP model generated from these multi-reporter mice enabled us to distinguish 4 plausible modes of CM renewal. We found over 99.5% adult CMs in tamoxifen-treated tri-transgenic mice expressed CM fate mapping reporter GFP, and nuclear and maturity reporter BFP, but became GFP+BFP- if CMs undergo dedifferentiation. Non-CMs including cardiac progenitor cells (CPCs), if any, would remain expressing RFP for their lack of gene recombination. In post-MI hearts, predominant CMs remained in GFP+, with minimal contribution from cardiac differentiation of CPCs that would have become RFP+BFP+. Furthermore, there was a significant increase in cycling dedifferentiated CMs (BrdU+GFP+BFP-) besides cycling but mature ones (BrdU+GFP+BFP+) in post-MI hearts, with more increase in the former than the latter, indicating dedifferentiation and proliferation of pre-existing CMs contribute to the endogenous cardiac myogenesis.
Conclusion: We developed a multi-reporter mouse model featuring efficient CM lineage tracking and maturity (vs dedifferentiation) reporting. Endogenous CM renewal is predominately due to the proliferation of pre-existing cardiomyocytes, with dedifferentiation as an intermediate state.