Promoting differentiation of human induced pluripotent stem cells into sinoatrial node-like cells through programmed regulation of AMPK signaling pathway

Background and aims The differentiation of human induced pluripotent stem cells (hiPSCs) into sinoatrial node-like cells (SANLCs) remains challenged by complex differentiation protocols and low efficiency. This study aims to reveal the role of AMPK signaling in the differentiation process of hiPSCs into SANLCs, providing new strategies for obtaining SANLCs in vitro. Methods Cells from the cardiac mesodermal stage, cardiac progenitor cell stage and cardiomyocyte stage during differentiation of hiPSCs to cardiomyocytes (hiPSC-CMs) were taken for transcriptome sequencing and analyses. PCR, immunostaining, Western blot, FACS and patch clamp techniques were employed for the analyses of differentiated hiPSC-CMs. Results KEGG analysis identified that the AMPK signaling pathway is significantly enriched with the expression of NKX2.5 (sinus node cell development-related transcription factor). The early activation and late inhibition of AMPK signaling were both effective in the upregulation of SANLCs markers. In addition, the combined manipulation of both stages further enhanced the differentiation efficiency reflected by higher SANLCs marker expression, which was also confirmed at the protein level by immunofluorescence, Western blot, flow cytometry analyses. SANLCs obtained from the differentiation with combined modulation of AMPK signaling displayed typical features of native pacemaker cells in the heart, including ion channel currents (IKAch, ICaT, ICaL, If), action potentials and robust autonomic responsiveness to both β-adrenergic and muscarinic stimulation. Conclusion Early activation and then inhibition of the AMPK signaling pathway during the differentiation process can promote hiPSCs differentiation to SANLCs, which may provide a novel strategy for obtaining SANLCs for studies on sinoatrial node diseases.