Disrupted N-Cadherin Expression Leads to Sarcomeric Disassembly and Cell Cycle Activation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: Induction of cardiomyocyte proliferation holds great promise for cardiac regenerative therapy. We showed previously that in the presence of Wnt signaling agonist, the removal of cell-cell contact between cardiomyocytes is the key to drive massive expansion of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the molecular mechanism regulating cell contact-mediated suppression of hiPSC-CM proliferation remains unknown. Hypothesis: We hypothesized that N-cadherin, the major adherens junction molecule expressed in cardiomyocytes, mediates contact inhibition of hiPSC-CM proliferation. Methods/Results: By qPCR and Western blotting, we showed comparable transcript and protein expression level of N-cadherin when hiPSC-CMs were cultured either densely (with cell contact) or sparsely (without cell contact), despite the observed N-cadherin localization at intercellular junction in densely cultured cells only. We disrupted N-cadherin expression with transient transfection of siRNA and found that the loss of N-cadherin expression resulted in 1.75-fold increase in Ki67 expression in densely but not sparsely cultured day 30 hiPSC-CM, suggesting that junction-localized N-cadherin may affect hiPSC-CM proliferation. Furthermore, we found that the loss of N-cadherin expression led to wide-spread sarcomeric disassembly. To examine whether sarcomeric disassembly alone is sufficient to stimulate cell cycle activity in densely cultured hiPSC-CMs, we knocked down α-actinin expression which led to sarcomere disruption and an increase in Ki67+ expression by 1.6-fold in day 30 hiPSC-CMs. Conclusions: Taken together, our results suggest that N-cadherin mediates the previously observed cell-cell contact inhibition of hiPSC-CM proliferation and the disruption of N-cadherin expression in densely cultured hiPSC-CMs leads to sarcomeric disassembly and hiPSC-CM proliferation. Furthermore, we showed that sarcomeric disassembly alone stimulates hiPSC-CM proliferation. These findings support the need for inducing cardiomyocyte dedifferentiation to achieve in situ cardiomyocyte proliferation for developing novel regenerative therapy to treat congenital and adult heart diseases in the future.