Generation of ANP- and BNP-deficient human iPSC-derived cardiomyocytes reveals natriuretic peptide–dependent angiogenic paracrine effects

BACKGROUND: Cell therapy using pluripotent stem cell-derived cardiomyocytes (PSC-CMs) has emerged as a promising strategy for cardiac regeneration. Although transplanted PSC-CMs can contribute to remuscularization, accumulating evidence suggests that paracrine mechanisms also play an important role in functional recovery. Among potential paracrine factors, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are highly expressed in PSC-CMs; however, whether PSC-CM-derived ANP and BNP contribute to cardiac repair after transplantation remains to be determined. METHODS: In this study, we generated human induced pluripotent stem cell (hiPSC) lines deficient in ANP (NPPA knockout), BNP (NPPB knockout), or both peptides using CRISPR-Cas9 genome editing. These iPSCs were differentiated into cardiomyocytes, and their characteristics and paracrine effects were then examined in vitro using endothelial cell assays and co-culture with cardiac fibroblasts. RESULTS: ANP- and/or BNP-deficient hiPSC-derived cardiomyocytes (hiPSC-CMs) showed comparable cardiac differentiation efficiency, cell size, proliferative capacity, and susceptibility to apoptosis compared with wild-type hiPSC-CMs. In trans-well migration and Matrigel tube formation assays, conditioned medium from wild-type hiPSC-CMs significantly enhanced endothelial cell migration and tube formation compared with medium from double-knockout hiPSC-CMs lacking both ANP and BNP, suggesting that natriuretic peptides contribute to the pro-angiogenic effects of hiPSC-CMs in vitro. In contrast, co-culture with hiPSC-CMs attenuated TGF-β1-induced myofibroblast gene expression in cardiac fibroblasts regardless of natriuretic peptide deficiency, possibly reflecting minimal expression of the natriuretic peptide receptor NPRA in fibroblasts under in vitro conditions. CONCLUSIONS: These findings suggest that ANP and BNP are involved in the pro-angiogenic paracrine activity of hiPSC-CMs in vitro. This study provides genetically modified hiPSC-CM models that may serve as a platform for future in vivo transplantation studies to determine whether PSC-CM-derived natriuretic peptides contribute to cardiac repair after transplantation.