The HAND1 frameshift A126FS mutation does not cause hypoplastic left heart syndrome in mice

Abstract

Aims: To test if a human Hand1 frame shift mutation identified in human samples is causative of hypoplastic left heart syndrome (HLHS).

Methods and results: HLHS is a poorly understood single ventricle congenital heart defect that affects two to three infants in every 10 000 live births. The aetiologies of HLHS are largely unknown. The basic helix-loop-helix transcription factor HAND1 is required for normal heart development. Interrogation of HAND1 sequence from fixed HLHS tissues identified a somatic frame-shift mutation at Alanine 126 (NP_004812.1 p.Ala126Profs13X defined as Hand1A126fs). Hand1A126fs creates a truncated HAND1 protein that predictively functions as dominant negative. To determine if this mutation is causative of HLHS, we engineered a conditional Hand1A126fs mouse allele. Activation of this allele with Nkx2.5Cre results in E14.5 lethality accompanied by cardiac outflow tract and intraventricular septum abnormalities. Using αMHC-Cre or Mef2CAHF-Cre to activate Hand1A126fs results in reduced phenotype and limited viability. Left ventricles of Hand1A126FS mutant mice are not hypoplastic.

Conclusions: Somatically acquired Hand1A126FS mutation is not causative of HLHS. Hand1A126FS mutation does exhibit embryonic lethal cardiac defects that reflect a dominant negative function supporting the critical role of Hand1 in cardiogenesis.

Keywords: Cardiac development; Hand1; Hypoplastic left heart syndrome; Transcription; bHLH.

Figure 1

Hand1^A126FS localizes to the nucleus acting as a dominant negative. (A) Schematic of wild-type HAND1 and HAND1 A126FS and Dbox and Ebox luciferase reporters. (B) Luciferase assays from HEK293 lysates with the indicated plasmids (pIRESNeo), open bars; E12-pIRESNeo (E12), grey bars; Hand1-pIRESNeo (Hand1), black bars; Hand1^A126FS-pIRESNeo (Hand1FS), blue bars; Hand1-pIRESNeo + E12-pIRESNeo black diagonal-striped bars; Hand1FS pIRESNeo+ E12-pIRESNeo blue diagonal-striped bars; Hand1-pIRESNeo + Hand1FS-pIRESNeo, waved bars). *^,#P ≤ 0.05 by ANOVA compared with pIRESNeo and Hand1FS + E12, respectively. Error bars show standard error. (C) EMSA of in vitro expressed HAND1 (H1), Hand1 A126FS (H1^A126fs), and E12 in the indicated combinations. Unprogrammed reticulocyte lysates (UP) show non-specific complexes (ns). E12-programmed lysates reveal homodimer binding (red arrowhead) that is competed with specific competitor (SC) but not non-specific competitor (NS). Hand1-E12 heterodimers (black arrowhead). H1^A126fs-E12 heterodimers do not bind DNA (double asterisk). (D) HEK293s co-transfection of Hand1A^126fs-eGFP and E12-DsRed. DAPI (blue) marks nuclei. Both Hand1^A126fs-eGFP and E12-DsRed co-localize to the nucleus. Transfection and EMSA represent five experiments; only one EMSA is shown. Scale bar in (D), 10 μm.

Figure 2

Targeting strategy and expression validation of Hand1A^126FS/+ mice. (A) Schematic of the Hand1 locus and targeting construct. Hand1 targeting arms were utilized to insert a loxP element-flanked Neomyosin cassette (Flox STOP Neo). The 3′ targeting arm includes the A126fs mutation. Upon Cre-recombination and removal of the Stop-Flox cassette, activates the Hand1^A126fs allele. (B) Southern blot analysis shows targeting frequency exceeding 50%. (C) E10.5 wholemount Hand1 ISH in control (Hand1^SF-A126fs/+), recombined (Nkx2.5^Cre/+; Hand1^A126fs/+), and compound Hand1 heterozygotes (Nkx2.5^Cre/+; Hand1^A126fs/fx) single-copy mutants. I2, mandibular component of the first pharyngeal arch; oft, cardiac outflow tract; rv, right ventricle; lv, left ventricle; u, umbilicus. Scale bar 150 μm.

Figure 3

Nkx2.5^Cre/+; Hand1^A126FS/+ E12.5 mutants present with an elongation of the cardiac OFT. (A) Nkx2.5^+/+; Hand1^SF-A126FS/+ wholemount control compared with two Nkx2.5^Cre/+; Hand1A^126FS/+ mutants (F, K). Measure of cardiac OFT (white bars) shows that mutant OFTs extend farther into the forming RV, which appears smaller and shows some signs oedema (white arrowhead). Section analysis of the control shown in (A). (B–E) reveals expected structural development that includes a septated pulmonary trunk (pt) developing IVS and LV. Magnification in (E) shows a well-formed compact zone (black bar). Matched sections from Nkx2.5^+/+; Hand1A^126FS/+ mutants shown in (F) and (K). (G–O) A poorly formed IVS (asterisk), small RV, and a thinner compact zone (J, O). Size of the LV is indistinguishable between control and Nkx2.5^+/+; Hand1A^126FS/+ mutant hearts. Data represent an example of each genotype. Ten embryos per genotype were examined. ra, right atria; la, left atria; rv, right ventricle; lv, left ventricle; ivs, intraventricular septum; pt, pulmonary trunk. Scale bars 150 and 500 μm.

Figure 4

Nkx2.5^Cre/+; Hand1^A126FS/+ hearts display an increase in both cell autonomous and non-cell autonomous cell death. Control Hand1^SF-A126fs/+ (A, B) and Nkx2.5^Cre/+; Hand1^A126FS/+ mutants (C, D) were assayed for cell death using Lysotracker staining in wholemount at E10.5 and E11.5. Control hearts show little cell death. Increased cell death is observed in OFT, RV, and, LV of Nkx2.5^Cre/+; Hand1^A126FS/+ mutants (white arrows). (E–H) EdU wholemount proliferation analysis at E10.5 and E11.5. Results show no obvious differences in between control and mutant hearts. Data represent an example of each genotype. Six embryos per genotype were examined. ra, right atria; la, left atria; rv, right ventricle; lv, left ventricle. Scale bars 150 μm.

Figure 5

Wholemount ISH of Nkx2.5^Cre/+; Hand1^A126FS/+ mutant embryos reveals altered cardiac gene expression during heart formation. E11.5 Nkx2.5^+/+; Hand1^SF-A126FS/+ control (A, C, E, G) and Nkx2.5^Cre/+; Hand1^A126FS/+mutant (B, D, F, H) embryos hybridized to the indicated riboprobes. Wnt11 marks OFT tissue (white bars; A and B). Tbx5 is an LV chamber maker. Tbx5 expression appears more homogeneous in Nkx2.5^Cre/+; Hand1^A126FS/+mutants when compared with controls (white arrowhead; C and D). Dkk3 marks the IVS and ventricular chambers. Dkk3 expression appears more robust in Nkx2.5^Cre/+; Hand1^A126FS/+mutants (white arrowhead; E and F). Cited1 appears unchanged between control and Nkx2.5^Cre/+; Hand1^A126FS/+mutants (G and H). Data represent an example of each genotype. Eight embryos per genotype were examined. Scale bars 150 μm.

Figure 6

Section ISH in E10.5 and E12.5 control and Nkx2.5^Cre/+; Hand1^A126FS/+ mutants. Bmp10 (A–D) and Anf (E–H) expression marks trabeculae. No significant changes in expression are observed. Cited1 expression (I–L) expression is not altered. Cxcl12 cardiac expression is required for formation of intra-ventricular coronary arteries. At E10.5 (M, N) and E12.5 (O, P), Cxcl12 expression is upregulated (black arrowheads) compared with controls (M and O). Dkk3 (Q–T) shows expanded expression (black arrowheads) in Nkx2.5^Cre/+; Hand1^A126FS/+mutants (R, T) compared with control (Q, S). The compact zone markers Tbx20 (U–X) and Hey2 (Y–Ab) show altered expression in Nkx2.5^Cre/+; Hand1^A126FS/+mutants. Tbx20 is upregulated in Nkx2.5^Cre/+; Hand1^A126FS/+mutants (V, X black arrowheads). Hey2 expression is markedly down in Nkx2.5^Cre/+; Hand1^A126FS/+mutants (Z, Ab; asterisks) compared with Nkx2.5^+/+; Hand1^SF-A126FS/+ controls. Data represent an example of each genotype. Eight embryos per genotype were examined. Scale bars 200 μm.

Figure 7

Histological comparison and assessment of gene expression in E14.5 Nkx2.5^+/+; Hand1^SF-A126FS/+ control and Nkx2.5^Cre/+; Hand1A^126FS/+mutants. (A) H&E transverse Control hearts show a patent IVS and separate RV and LV with a well-established compact zone (black bar), RA, and LA. (B) Nkx2.5^Cre/+; Hand1^A126FS/+mutants consistently display VSDs (asterisk) and thin poorly developed compact zone (black bar). LV chamber size is unaffected. (C–F) E14.5 Bmp10 expression reveals a loss trabecular expression. Black boxes in (C, D) define the higher magnification images of (E) and (F). Lines through compact zone measures thickness. (G–J) Dkk3 expression is upregulated in E14.5 Nkx2.5^Cre/+; Hand1^A126FS/+mutants. Boxes in (G) and (H) define the higher magnification view shown in (I) and (J). Data represent an example of each genotype. Six embryos per genotype were examined. ra, right atria; la, left atria; rv, right ventricle; lv, left ventricle; ivs, intraventricular septum. Scale bars 500 and 100 μm.