XB-ART-50421Stem Cells April 1, 2015; 33 (4): 1113-29.
Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.
During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.
PubMed ID: 25524439
PMC ID: PMC6750130
Article link: Stem Cells
Species referenced: Xenopus laevis
Genes referenced: hcn4 isl1 mef2d myh6 nkx2-5 nodal nodal1 prkg1 tbx20 tbx5 tnni3
Article Images: [+] show captions
|Figure 4. Negative regulation of Isl1 by Nkx2–5 occurs during second heart field progenitor differentiation into cardiomyocytes in vivo. (A): Isl1‐Cre knock‐in mice (Isl1Cre/+) were crossed with mice carrying floxed Nkx2–5 alleles (Nkx2–5fl/fl) to generate tissue‐specific deletion of Nkx2–5. (B): Whole‐mount in situ analysis for expression of Nkx2–5 mRNA (dark blue) in Isl1‐Cre;Nkx2–5 mutants (Isl1Cre/+;Nkx2–5fl/fl, right panels) and somite‐matched littermate controls (Isl1+/+;Nkx2–5fl/fl, left panels) at ED9.5 (20–21 somite pairs). (C): Whole‐mount RNA in situ hybridization for Isl1 in ED9.0 embryos (16 somite pairs) from Isl1+/+;Nkx2–5fl/fl controls (left panels) and Isl1Cre/+;Nkx2–5fl/fl mutants (right panels). Arrows indicate persisting Isl1 expression in the forming ventricular and atrial regions of the mutant hearts. Images are representative of five embryos per genotype. (D): Representative images of transverse sections of control Isl1+/+;Nkx2–5fl/fl (left panels) and mutant Isl1Cre/+;Nkx2–5fl/fl (right panels) ED9.25 embryos (18–19 somite pairs) after immunofluorescence analysis of Isl1 (red) and cTnT (green) protein expression. Sections correspond to the position indicated by the lines drawn through the adjacent embryo view. Scale bars = 50 µm. Images are representative of three embryos per genotype. White bracket marks malformed RV/OFT in Nkx2–5 mutants. Abbreviations: A, atria; AVC, atrioventricular canal; LA, left atrium; LV, left ventricle; OFT, outflow tract; RV, right ventricle; SV, sinus venosus; V, ventricle.|
|Figure 6. Overexpression of Isl1 promotes expression of nodal lineage markers and represses ventricular program of differentiating myocytes. (A): Expression level of genes specific for nodal and working‐myocardium lineages was determined by qRT‐PCR in GFP and Isl1OE cardiomyocytes at days 14 and 21 in independent EB differentiation experiments (as indicated by numbers). All values are normalized to Gapdh and are relative to the average expression value measured in GFP cells from differentiations 1 and 2 at day 14. p values were calculated using the Mann‐Whitney test. Minimum (min) and maximum (max) values were taken as a reference for heatmap representation. (B, C): Immunofluorescence analysis of Mlc2a (green), Mlc2v (magenta), and F‐actin (red, visualized with Phalloidin) in 1‐month‐old GFP and Isl1OE cardiomyocytes. Scale bars = 25 µm (B). Quantification of Mlc2a+, Mlc2v+ and Mlc2a/Mlc2v‐double positive myocytes from GFP (top) and Isl1OE ESCs (bottom). Mean values ± SEM, n = 189 for GFP and n = 535 for Isl1OE from five to six independent experiments (C). (D, E): Immunofluorescence analysis of Mlc2a (green), Hcn4 (magenta), and F‐actin (red, visualized with Phalloidin) in 1‐month‐old GFP and Isl1OE cardiomyocytes. Scale bars = 25 µm (D). Quantification of Hcn4+ cells in the Mlc2a+ myocyte fraction from GPF and Isl1OE ESCs. Mean values ± SEM, n = 86 for GFP and n = 83 for Isl1OE from two to three independent experiments. Abbreviation: GFP, green fluorescent protein.|
|Figure 7. Isl1 overexpression affects subtype diversification of electrophysiologically functional cardiomyocytes. (A): Representative action potential (AP) traces and corresponding APD90/APD50 ratios (R) of 1‐month‐old ventricular‐, atrial‐, nodal‐like, and intermediate cardiomyocytes from GFP and Isl1OE ESCs, as determined with optical imaging using di8‐ANEPPS. (B): Quantification of 1‐month‐old ventricular‐, atrial‐, nodal‐like, and intermediate cardiomyocytes from GFP and Isl1OE ESCs based on the unique AP traits and the pharmacological response to ivabradine. Mean values ± SEM, n = 122 for GFP and n = 65 for Isl1OE from three independent experiments. (C): Percentage of reduction of early and total DD slope as well as AP frequency in 1‐month‐old nodal cardiomyocytes from GFP (black bars) and Isl1OE (red bars) ESCs after 3 µM Iva application. (D): Working model for the regulation of cardiomyocyte subtype specification by Isl1/Nkx2–5‐mediated mechanism. Abbreviations: DD, diastolic depolarization; GFP, green fluorescent protein; Iva, ivabradine.|
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