Background : Biological pacemakers derived from pluripotent stem cell (PSC) have been considered as a potential therapeutic surrogate for sick sinus syndrome. So it’s essential to develop high efficient strategies for enrichment of sinoatrial node-like cells (SANLC) as seed cells for biological pacemakers. It has reported that BMP, FGF and RA signaling pathways were involved specification of different cardiomyocyte subtypes, pacemaker, ventricle, and atria cells. Methods : During the differentiation process from human induced pluripotent stem cell (hiPSC) to cardiomyocyte through small molecule based temporal modulation of the Wnt signaling pathway, signaling of BMP, FGF and RA was manipulated at cardiac mesoderm stage. The methods of qRT-PCR, immunofluorescence, flow cytometry and whole cell patch clamp were used to identify the SANLC.Results : qRT-PCR results showed that manipulating each one of BMP, FGF and RA signaling was effective for the upregulation of SANLC markers. Moreover, combined modulation of such three pathways displayed the best efficiency for the expression of SANLC markers, which was further confirmed at protein level using immunofluorescence, flow cytometry. Finally, the electrophysiological characteristics of induced SANLC were verified by patch clamp method. Conclusion : An efficient transgene-independent differentiation protocol for generating SANLC from hPSC was developed, in which combined modulating BMP, FGF and RA signaling at cardiac mesoderm stage generates SANLC at high efficiency. It may serve as a potential approach for biological pacemaker construction.

Introduction GFA (Guan-Fu Base A) as one of the main active substances in the Chinese medicine Ranunculaceae Aconite, has been approved the effect of anti-atrial fibrillation via its atrial-selective Na channel-blocking action. It is recently undergoing phase IV clinical study. However, the molecular mechanism of Na 1.5 channel inhibition by GFA is largely unclear. Methods and Results Na 1.5 channel and its mutants were expressed in Xenopus oocytes and the currents were recorded with two-microelectrode voltage-clamp. GFA inhibited Na 1.5 currents in a concentration-dependent manner, with IC of 66.24 μM, 371.59 μM, and 381.08 μM for wild type (WT), Delta KPQ (∆KPQ) and R1623Q constructs, respectively. Both the mutations of ∆KPQ and R1623Q decreased inhibitory potency of GFA about 5~6-fold. N406K mutation significantly altered the inhibition effect of GFA. Even 1 mM GFA has almost no inhibitory effect on the mutant. For both the WT and mutant channels, GFA reduced the currents in concentration, voltage and time dependent manner. Conclusion: GFA is a potent blocker of Na 1.5 channel. N406, the aromatic residues in the transmembrane helical of DIS6, is most likely responsible for the high-affinity binding of GFA to Na 1.5 channel.

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Background: Biological pacemakers derived from pluripotent stem cell (PSC) have been considered as a potential therapeutic surrogate for sick sinus syndrome. So it’s essential to develop high efficient strategies for enrichment of sinoatrial node-like cells (SANLC) as seed cells for biological pacemakers. It has been reported that BMP, FGF and RA signaling pathways are involved in specification of different cardiomyocyte subtypes, pacemaker, ventricular, and atrial cells. We aimed to investigate whether combined modulation of BMP, FGF and RA signaling pathways could enrich the differentiation of SANLC from human pluripotent stem cell (hiPSC).Methods: During the differentiation process from human-induced pluripotent stem cell to cardiomyocyte through small molecule based temporal modulation of the Wnt signaling pathway, signaling of BMP, FGF and RA was manipulated at cardiac mesoderm stage. qRT-PCR, immunofluorescence, flow cytometry and whole cell patch clamp were used to identify the SANLC. Results: qRT-PCR results showed that manipulating each one of bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and retinoid acid (RA) signaling was effective for the upregulation of SANLC markers. Moreover, combined modulation of these three pathways displayed the best efficiency for the expression of SANLC markers, which was further confirmed at protein level using immunofluorescence and flow cytometry. Finally, the electrophysiological characteristics of upregulated SANLC were verified by patch clamp method.Conclusion: An efficient transgene-independent differentiation protocol for generating SANLC from hiPSC was developed, in which combined modulating BMP, FGF and RA signaling at cardiac mesoderm stage generates SANLC at high efficiency. This may serve as a potential approach for biological pacemaker construction.

Background: Biological pacemakers derived from pluripotent stem cell (PSC) have been considered as a potential therapeutic surrogate for sick sinus syndrome. So it’s essential to develop high efficient strategies for enrichment of sinoatrial node-like cells (SANLC) as seed cells for biological pacemakers. It has been reported that BMP, FGF and RA signaling pathways are involved in specification of different cardiomyocyte subtypes, pacemaker, ventricular, and atrial cells. Methods: During the differentiation process from human-induced pluripotent stem cell (hiPSC) to cardiomyocyte through small molecule based temporal modulation of the Wnt signaling pathway, signaling of BMP, FGF and RA was manipulated at cardiac mesoderm stage. qRT-PCR, immunofluorescence, flow cytometry and whole cell patch clamp were used to identify the SANLC. Results: qRT-PCR results showed that manipulating each one of bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and retinoid acid (RA) signaling was effective for the upregulation of SANLC markers. Moreover, combined modulation of these three pathways displayed the best efficiency for the expression of SANLC markers, which was further confirmed at protein level using immunofluorescence and flow cytometry. Finally, the electrophysiological characteristics of upregulated SANLC were verified by patch clamp method. Conclusion: An efficient transgene-independent differentiation protocol for generating SANLC from hiPSC was developed, in which combined modulating BMP, FGF and RA signaling at cardiac mesoderm stage generates SANLC at high efficiency. This may serve as a potential approach for biological pacemaker construction.