Unknown,Transcriptomics,Genomics,Proteomics

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Modulation of calcium activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker- like cells

ABSTRACT: Background: Ion channels are key determinants for the function of excitable cells but little is known about their role and involvement during cardiac development. Earlier work identified Ca2+-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here, we have investigated their impact on the differentiation of pluripotent cells towards the cardiac lineage. Methods and Results: We have applied the SKCa-activator EBIO on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa-activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation and diminished teratoma formation. Time-restricted SKCa-activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the EBIO-induced effects. Conclusions: SKCa-activation drives the fate of pluripotent cells towards the cardiac lineage and preferentially into pacemaker-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification. Untreated ES cells in three independent experiments: - Untreated control ES cells sample 1 (Con_1) - Untreated control ES cells sample 2 (Con_2) - Untreated control ES cells sample 3 (Con_3) EBIO-treated ES cells in three independent experiments: - EBIO-treated ES cells sample 1 (EBIO_1) - EBIO-treated ES cells sample 2 (EBIO_2) - EBIO-treated ES cells sample 3 (EBIO_3) Untreated differentiated ES cells in two independent experiments: - Untreated control differentiated ES cells sample 1 (Con_day5+10_1) - Untreated control differentiated ES cells sample 2 (Con_day5+10_2) EBIO-treated differentiated ES cells in two independent experiments: - EBIO-treated differentiated ES cells sample 1 (EBIO_day5+10_1) - EBIO-treated differentiated ES cells sample 2 (EBIO_day5+10_2)

ORGANISM(S): Mus musculus

SUBMITTER: Martin Zenke

PROVIDER: E-GEOD-18660 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells.

Kleger Alexander A Seufferlein Thomas T Malan Daniela D Tischendorf Michael M Storch Alexander A Wolheim Anne A Latz Stephan S Protze Stephanie S Porzner Marc M Proepper Christian C Brunner Cornelia C Katz Sarah-Fee SF Varma Pusapati Ganesh G Bullinger Lars L Franz Wolfgang-Michael WM Koehntop Ralf R Giehl Klaudia K Spyrantis Andreas A Wittekindt Oliver O Lin Qiong Q Zenke Martin M Fleischmann Bernd K BK Wartenberg Maria M Wobus Anna M AM Boeckers Tobias M TM Liebau Stefan S

Circulation 20101018 18

<h4>Background</h4>Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage.<h4>Methods and results</h4>We have applied the SKCa activator ...[more]

PMID: 20956206

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Project description:to study the effect of induced expression of TBX3 within the atrium of the heart Background: Treatment of congenital or acquired disorders of the sinus node or atrioventricular node requires insight into the molecular mechanisms for the development and homeostasis of these pacemaker tissues. In the developing heart, transcription factor TBX3 is required for pacemaker and conduction system development. Here, we explore the role of TBX3 in the adult heart and investigate whether TBX3 is able to reprogram terminally differentiated working cardiomyocytes into pacemaker cells. This would be an attractive approach in biological pacemaker formation. Methods and results: TBX3 expression was ectopically induced in cardiomyocytes of adult transgenic mice. Expression analysis revealed an efficient switch from the working myocardial expression profile to that of the pacemaker myocardium. This included suppression of genes encoding gap junction subunits (Cx40, Cx43), the cardiac Na+ channel (NaV1.5; INa) and inwardly rectifying K+ ion channels (Kir-genes; IK1). Concordantly, we observed conduction slowing in these hearts, and reductions in INa and IK1 in cardiomyocytes isolated from these hearts. The reduction in IK1 resulted in a more depolarized maximum diastolic potential, thus enabling spontaneous diastolic depolarization. Neither ectopic pacemaker activity nor pacemaker current, If, were observed. Lentiviral expression of TBX3 in ventricular cardiomyocytes resulted in conduction slowing and development of heterogeneous phenotypes, including depolarized and spontaneously active cardiomyocytes. Conclusions: TBX3 partially reprograms terminally differentiated working cardiomyocytes into pacemaker-like cells and induces important pacemaker properties. The ability of TBX3 to reduce intercellular coupling to overcome current-to-load mismatch and the ability to reduce IK1 density to enable diastolic depolarization, are very promising TBX3 characteristics for biological pacemaker formation strategies. 5 TBX3 expressing left atrial appendage samples (Tamoxifen-treated Myh6MCM;CTBX3 adult male mice) and 6 controls ((Tamoxifen-treated Myh6MCM adult male mice)

Project description:BACKGROUND: Single-cell RNA sequencing (scRNA-seq) is widely used in cancer research and organ development since its powerful ability to analyze cellular heterogeneity. However, its application in cardiomyocytes is rare mainly because the cardiomyocytes are too large and fragile to withstand traditional single-cell approaches. METHODS: Through designing the isolation procedure of neonatal mouse cardiac cells, we perform scRNA-seq with samples from 27 neonatal mice. Individual cell transcriptomes were subsequently clustered based on transcriptional profiles of highly variable genes. Then these clusters were used as the basis for between-cluster differential gene expression analyses and between-cluster interaction analyses. At last, we analyzed the intersection of these clusters with genetic and pharmacologic data. RESULTS: We provide detailed cellular atlases of the heart at single-cell resolution across four different stages after birth. We have luckily obtained tens of thousands of cardiomyocytes, to our knowledge, the most extensive reference framework so far. Moreover, we have discovered unexpected erythrocytes-like cardiomyocyte-terminal cardiomyocytes, comprising more than a third of all the cardiomyocytes. Only a few genes are highly expressed in these cardiomyocytes. They are highly differentiated cardiomyocytes that function as contraction pump. In addition, we have identified two cardiomyocyte-like conducting cells, lending support to the theory that the sinoatrial node pacemaker cells are specialized cardiomyocytes. Furthermore, we provide a substantial blueprint for comprehensive interactions between cardiomyocytes and other cardiac cells. We linked specific cell types to adverse drug reactions and congenital heart diseases and analyzed the roles of cell subtypes in adverse drug reactions and cardiac diseases. CONCLUSIONS: This mouse cardiac cell atlas improves our understanding of the heart and provides a valuable reference in response to varying physiological conditions and diseases.

Dataset Information

Modulation of calcium activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker- like cells

Publications

Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells.

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