NORAD Overexpression Enhances hiPSC-CM Regenerative Potency via PUM2/MASTL Signaling

BACKGROUND: The engraftment of induced cells from human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for myocardial infarction therapy is critically hindered by their low cell-cycle activity and survival rates. In this study, we explored the role of the long noncoding RNA activated by DNA damage (NORAD) in enhancing the cell-cycle activity and engraftment of hiPSC-CMs, providing new insights into myocardial repair. METHODS: hiPSC-CMs overexpressing NORAD were generated via lentiviral transduction using a cardiac-specific promoter, whereas NORAD knockdown was achieved by small interfering RNA transfection. The effects of NORAD on cell-cycle activity, nuclear DNA content, nuclear number, maturation, and apoptosis in hiPSC-CMs were examined in vitro using Western blot, reverse transcription quantitative polymerase chain reaction, immunofluorescence, and flow cytometry. In a murine myocardial infarction model, hiPSC-CMs overexpressing NORAD were transplanted into the infarcted myocardium. Engraftment, cell-cycle activity, and infarct size were evaluated by immunofluorescence, and cardiac function was assessed by echocardiography. Furthermore, the molecular mechanisms underlying NORAD-mediated regulation of hiPSC-CM cell-cycle activity were investigated, including its role in exosome-mediated paracrine effects on host cardiomyocytes. RESULTS: NORAD overexpression significantly increased the percentages of Ki67-positive, phosphorylated histone 3-positive, Aurora B-positive, and EdU-positive cells. It also increased the proportion of diploid nuclei and mononucleated cells, induced a trend toward a less mature state, and reduced apoptosis in hiPSC-CMs. Transplantation of hiPSC-CMs overexpressing NORAD improved myocardial repair, with greater cell-cycle activity of engrafted cells and endogenous cardiomyocytes in the myocardial infarction model. Mechanistically, NORAD exerted its effects by sequestering PUM2, an RNA-binding protein, thereby alleviating its translational repression of MASTL, a key regulator of mitotic progression. Moreover, exosomes secreted by hiPSC-CMs overexpressing NORAD promoted the cell-cycle activity of recipient cardiomyocytes, suggesting a possible paracrine contribution to myocardial repair. CONCLUSIONS: NORAD overexpression promoted cell-cycle progression via the PUM2/MASTL mRNA axis. Moreover, exosomes derived from these cells stimulated endogenous cardiomyocyte cell-cycle activity, contributing to myocardial repair. These findings underscore the therapeutic potential of NORAD-modulated hiPSC-CMs for promoting myocardial repair.