Cardiac biological pacing(BP)is one of the future directions for bradyarrhythmias intervention.Currently,cardiac pacemaker cells(PCs)used for cardiac BP are mainly derived from pluripotent stem cells(PSCs).However,the...Cardiac biological pacing(BP)is one of the future directions for bradyarrhythmias intervention.Currently,cardiac pacemaker cells(PCs)used for cardiac BP are mainly derived from pluripotent stem cells(PSCs).However,the production of high-quality cardiac PCs from PSCs remains a challenge.Here,we developed a cardiac PC differentiation strategy by adopting dual PC markers and simulating the developmental route of PCs.First,two PC markers,Shox2 and Hcn4,were selected to establish Shox2:EGFP;Hcn4:mCherry mouse PSC reporter line.Then,by stepwise guiding naïve PSCs to cardiac PCs following naïve to formative pluripotency transition and manipulating signaling pathways during cardiac PCs differentiation,we designed the FSK method that increased the yield of SHOX2^(+);HCN4^(+)cells with typical PC characteristics,which was 12 and 42 folds higher than that of the embryoid body(EB)and the monolayer M10 methods respectively.In addition,the in vitro cardiac PCs differentiation trajectory was mapped by single-cell RNA sequencing(scRNA-seq),which resembled in vivo PCs development,and ZFP503 was verified as a key regulator of cardiac PCs differentiation.These PSC-derived cardiac PCs have the potential to drive advances in cardiac BP technology,help with the understanding of PCs(patho)physiology,and benefit drug discovery for PC-related diseases as well.展开更多
Activation of the heart normally begins in the sinoatrial node(SAN).Electrical impulses spontaneously released by SAN pacemaker cells(SANPCs)trigger the contraction of the heart.However,the cellular nature of SANPCs r...Activation of the heart normally begins in the sinoatrial node(SAN).Electrical impulses spontaneously released by SAN pacemaker cells(SANPCs)trigger the contraction of the heart.However,the cellular nature of SANPCs remains controversial.Here,we report that SANPCs exhibit glutamatergic neuron-like properties.By comparing the single-cell transcriptome of SANPCs with that of cells from primary visual cortex in mouse,we found that SANPCs co-clustered with cortical neurons.Tissue and cellular imaging confirmed that SANPCs contained key elements of glutamatergic neurotransmitter system,expressing genes encoding glutamate synthesis pathway(G/s),ionotropic and metabotropic glutamate receptors(Grina,Gria3,Grm1 and Grm5)t and glutamate transporters(Slc17a7).SANPCs highly expressed cell markers of glutamatergic neurons(Snap25 and S/-c17a7)t whereas Gad1,a marker of GABAergic neurons,was negative.Functional studies revealed that inhibition of glutamate receptors or transporters reduced spontaneous pacing frequency of isolated SAN tissues and spontaneous Ca2+transients frequency in single SANPC.Collectively,our work suggests that SANPCs share dominant biological properties with glutamatergic neurons,and the glutamatergic neurotransmitter system may act as an intrinsic regulation module of heart rhythm,which provides a potential intervention target for pacemaker cell-associated arrhythmias.展开更多
基金National Natural Science Foundation of China(grant number 82088101 and 81930013 to Y.-H.C.,31871491 to J.Y.,82070338 and 82222008 to D.X.,82370396 to D.S.,82122007 to D.L.)National Key Research and Development Plan(grant number 2019YFA0801501 to Y.-H.C.)+4 种基金Program for the Research Unit of Origin and Regulation of Heart Rhythm,Chinese Academy of Medical Sciences(grant number 2019RU045 to Y.-H.C.)Top-Level Clinical Discipline Project of Shanghai Pudong(grant number PWYgf2021-01 to Y.-H.C.)Key Research Center Construction Project of Shanghai(grant number 2022ZZ01008 to Y.-H.C.)Shanghai Key Clinical Specialty Project(shslczdzk06202 to Y.-H.C.)National Key Clinical Specialty and Fundamental Research Funds for the Central Universities to Y.-H.C.
文摘Cardiac biological pacing(BP)is one of the future directions for bradyarrhythmias intervention.Currently,cardiac pacemaker cells(PCs)used for cardiac BP are mainly derived from pluripotent stem cells(PSCs).However,the production of high-quality cardiac PCs from PSCs remains a challenge.Here,we developed a cardiac PC differentiation strategy by adopting dual PC markers and simulating the developmental route of PCs.First,two PC markers,Shox2 and Hcn4,were selected to establish Shox2:EGFP;Hcn4:mCherry mouse PSC reporter line.Then,by stepwise guiding naïve PSCs to cardiac PCs following naïve to formative pluripotency transition and manipulating signaling pathways during cardiac PCs differentiation,we designed the FSK method that increased the yield of SHOX2^(+);HCN4^(+)cells with typical PC characteristics,which was 12 and 42 folds higher than that of the embryoid body(EB)and the monolayer M10 methods respectively.In addition,the in vitro cardiac PCs differentiation trajectory was mapped by single-cell RNA sequencing(scRNA-seq),which resembled in vivo PCs development,and ZFP503 was verified as a key regulator of cardiac PCs differentiation.These PSC-derived cardiac PCs have the potential to drive advances in cardiac BP technology,help with the understanding of PCs(patho)physiology,and benefit drug discovery for PC-related diseases as well.
基金The scRNA-seq data of mouse SANPCs in this study have been deposited in the NCBI Sequence Read Archive(accession number SRP192665)The single-cell expression matrix of primary visual CCs was downloaded from Gene Expression Omnibus as reported(GSE71585)Embryonic SAN and adjacent atrial cell data were obtained from Gene Expression Omnibus(GSE130461).
文摘Activation of the heart normally begins in the sinoatrial node(SAN).Electrical impulses spontaneously released by SAN pacemaker cells(SANPCs)trigger the contraction of the heart.However,the cellular nature of SANPCs remains controversial.Here,we report that SANPCs exhibit glutamatergic neuron-like properties.By comparing the single-cell transcriptome of SANPCs with that of cells from primary visual cortex in mouse,we found that SANPCs co-clustered with cortical neurons.Tissue and cellular imaging confirmed that SANPCs contained key elements of glutamatergic neurotransmitter system,expressing genes encoding glutamate synthesis pathway(G/s),ionotropic and metabotropic glutamate receptors(Grina,Gria3,Grm1 and Grm5)t and glutamate transporters(Slc17a7).SANPCs highly expressed cell markers of glutamatergic neurons(Snap25 and S/-c17a7)t whereas Gad1,a marker of GABAergic neurons,was negative.Functional studies revealed that inhibition of glutamate receptors or transporters reduced spontaneous pacing frequency of isolated SAN tissues and spontaneous Ca2+transients frequency in single SANPC.Collectively,our work suggests that SANPCs share dominant biological properties with glutamatergic neurons,and the glutamatergic neurotransmitter system may act as an intrinsic regulation module of heart rhythm,which provides a potential intervention target for pacemaker cell-associated arrhythmias.
