Disrupting methyl‐CpG‐binding protein 2 expression induces the transformation of induced pluripotent stem cell cardiomyocytes into pacemaker‐like cells by insulin gene enhancer binding protein 1

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Abstract
Background: The experiment will explore whether interfering with the expression of methyl‐CpG‐binding protein 2 (MecP2) can enhance the ability of insulin gene enhancer binding protein 1 (ISL1) to induce iPSC‐CMs to differentiate into pacemaker‐like cells.Methods: Differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs) can be induced via the regulation of the Wnt signaling pathway. Real‐time quantitative PCR (qPCR), western blotting, immunofluorescence staining, and patch‐clamp technique were used to analyze the ability of ISL1 to induce the transformation of iPSC‐CMs into pacemaker‐like cells. Calcium spark, patch‐clamp technique, and real‐time qPCR were used to verify whether disrupting the expression of MeCP2 enhanced this ability of ISL1 to induce the differentiation of iPSC‐CMs into pacemaker cells. Transplant pacemaker‐like cardiomyocytes into the myocardium of mice to observe whether the pacemaker cells can survive in the tissue for a long time.Results: RT‐qPCR and patch‐clamp analyses showed that overexpression of ISL1 induced the successful differentiation of iPSC‐CMs into pacemaker cells. ISL1‐overexpressing pacemaker‐like cells possessed typical characteristics of pacemaker morphology, including action potential and If inward current. Chromatin immunoprecipitation results showed that MeCP2 bound to the promoter region of HCN4. Following disruption of MeCP2 expression, the gene expression of sinoatrial node‐specific transcription factors, If inward current, and cardiac rhythm changes in iPSC‐CMs resembled those of sinoatrial node pacemaker cells. Therefore, ISL1 induced the differentiation of iPSC‐CMs into pacemaker‐like cells, and knockdown of MeCP2 increased this effect. Frozen section results showed that surviving pacemaker‐like cells could still be observed in myocardial tissue after 45 days.Conclusions: Experiments have found that interfering with the expression of MeCP2 can increase the ability of ISL1 to induce iPSC‐CM cells to differentiate into pacemaker‐like cells. And the pacemaker‐like cells obtained in this experiment can survive in myocardial tissue for a long time.
Funding Information
  • National Natural Science Foundation of China (81370305)
  • Jiangsu Provincial Department of Science and Technology (SKT20130102)

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