Direct Nkx2-5 Transcriptional Repression of Isl1 Controls Cardiomyocyte Subtype Identity

Abstract

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 1 precursors. Embryos deficient for Nkx2-5 in the Isl1 1 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.

Disruption of spatiotemporal hypoxic signaling causes congenital heart disease in mice

“…-cells in the FHF and particularly in the linear heart tube might be seen as cells that have already acquired a more mature cardiomyocyte fate, a conclusion that is also supported by the direct role of NKX2.5 in repression of progenitor genes (e.g., Isl1 and Fgf10) and the persistent signature of progenitor cell gene expression in the myocardium of Nkx2.5 mutants (19,21,22).…”

“…Repression of Isl1 expression might not rely solely on hypoxia responses, since inactivation of Nkx2.5 causes persistent expression of Isl1 in the forming heart tube (19). Furthermore, a recent finding demonstrated direct repression of Isl1 by NKX2.5 for proper development of the ventricular myocardium (21). However, we assume that hypoxia is the decisive initial step to silencing Isl1 expression in SHF CPCs migrating into the linear heart tube, which relieves suppression of Nkx2.5 by ISL1, subsequently enabling repression of Isl1 by NKX2.5.…”

“…TUNEL assays to monitor apoptosis were carried out using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's protocol. Wholemount in situ hybridizations for Isl1 mRNA and Nkx2.5 mRNA were performed as described previously (55) using dual DIG-labeled Isl1 or Nkx2.5 antisense probes synthesized from ISL1-and NKX2.5-cDNApCR-BluntII-TOPO clones (21).…”

The formation and function of the cardiac conduction system

“…In mouse, Isl1 is required for the proliferation and function of SAN cells, and the SAN-specific deletion of Isl1 results in embryonic lethality . Furthermore, Isl1-deficiency in mice leads to a downregulation of key regulators of SAN development, such as Tbx3, Shox2 and Bmp4, and ion channels for SAN function, including Hcn4, Hcn1 and Cacna1g Vedantham et al, 2015), whereas Isl1 overexpression in ESC-derived cardiomyocytes leads to upregulation of the SAN gene programme and downregulation of the chamber myocardium gene programme (Dorn et al, 2015). Isl1 is a target of Shox2 in the SAN and can rescue the bradycardia phenotype caused by Shox2 deficiency (Hoffmann et al, 2013).…”

Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue

“…We measured the spontaneous beating rate of cardiac constructs since automaticity is a key indicator of cardiac subtype and electrophysiological maturation. 18,42,43 In constructs with cardiomyocyte-only conditions in 1.25 mg/mL collagen, the average spontaneous beating rate is 1.2 Hz. Coculture conditions resulted in a slight decrease of spontaneous beating rate to *0.6 Hz in both 1.25 and 6 mg/mL collagen, although the decrease is not statistically significant (Fig.…”