Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system
Patrick Y. Jay, Brett S. Harris, Colin T. Maguire, Antje Buerger, Hiroko Wakimoto, Makoto Tanaka, Sabina Kupershmidt, Dan M. Roden, Thomas M. Schultheiss, Terrence X. O’Brien, Robert G. Gourdie, Charles I. Berul, Seigo Izumo
Patrick Y. Jay, Brett S. Harris, Colin T. Maguire, Antje Buerger, Hiroko Wakimoto, Makoto Tanaka, Sabina Kupershmidt, Dan M. Roden, Thomas M. Schultheiss, Terrence X. O’Brien, Robert G. Gourdie, Charles I. Berul, Seigo Izumo
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Article Cardiology

Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system

Abstract

Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause atrioventricular conduction defects in humans by unknown mechanisms. We show in KO mice that the number of cells in the cardiac conduction system is directly related to Nkx2-5 gene dosage. Null mutant embryos appear to lack the primordium of the atrioventricular node. In Nkx2-5 haploinsufficiency, the conduction system has half the normal number of cells. In addition, an entire population of connexin40–/connexin45+ cells is missing in the atrioventricular node of Nkx2-5 heterozygous KO mice. Specific functional defects associated with Nkx2-5 loss of function can be attributed to hypoplastic development of the relevant structures in the conduction system. Surprisingly, the cellular expression of connexin40, the major gap junction isoform of Purkinje fibers and a putative Nkx2-5 target, is unaffected, consistent with normal conduction times through the His-Purkinje system measured in vivo. Postnatal conduction defects in Nkx2-5 mutation may result at least in part from a defect in the genetic program that governs the recruitment or retention of embryonic cardiac myocytes in the conduction system.

Authors

Patrick Y. Jay, Brett S. Harris, Colin T. Maguire, Antje Buerger, Hiroko Wakimoto, Makoto Tanaka, Sabina Kupershmidt, Dan M. Roden, Thomas M. Schultheiss, Terrence X. O’Brien, Robert G. Gourdie, Charles I. Berul, Seigo Izumo

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Figure 1

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The absence of the AV node primordium in Nkx2-5–null mutant embryos. (A)...
The absence of the AV node primordium in Nkx2-5–null mutant embryos. (A) Schematic diagram of the Nkx2-5 genomic structure, targeting construct, and Nkx2-5neo allele and Northern blot analysis demonstrating a 50% reduction in Nkx2-5 mRNA in the Nkx2-5+/neo adult ventricular myocardium. (B) WT Nkx2-5 E9.5 embryos that carry the minK-lacZ gene show blue X-gal staining in the inner curvature of the AV canal (arrow). (C) Nkx2-5neo/neo E9.5 embryos show abnormal development of the heart tube and no minK-lacZ activity in the AV canal region where the AV node primordium is expected (arrow); staining is normal in the somites (arrowhead). In situ hybridization for minK mRNA and the corresponding brightfield images demonstrate expression throughout the myocardium of WT (D and E) and Nkx2-5neo/neo (F and G) embryos. All images are representative of three or more embryos or mice. Comparisons of minK-lacZ expression were made between animals with identical copy numbers of the reporter allele. B and C show minK-lacZ homozygotes; heterozygotes yield similar results. WT, Nkx2-5 WT; Het, Nkx2-5+/neo; KO, Nkx2-5neo/neo; H, HindIII; N, NotI; S, SpeI; e 1, exon; e 2, exon 2; L, loxP sequence; neo, pGK neomycin resistance cassette. Probe denotes fragments used for Southern blot analysis. Scale bars: 200 μm (B and C), 100 μm (D–G).