1. GATA-Binding Factor 6 Contributes to Atrioventricular Node Development and FunctionCLINICAL PERSPECTIVE
by Fang Liu, Min Min Lu, Neil N. Patel, Kurt J. Schillinger, Tao Wang, and Vickas V. Patel
Circ Genom Precis Med
Volume 8(2):
April 21, 2015
Copyright © American Heart Association, Inc. All rights reserved.

2. Gata6 carboxyl zinc-finger deletion increases ventricular myocyte size.
Gata6 carboxyl zinc-finger deletion increases ventricular myocyte size. A to D, Bright field images of isolated ventricular myocytes from 2 mature Mlc2vCre-Gata6f/f mice (A and B) and Gata6f/f mice (C and D). Ventricular myocytes without the Gata6 carboxyl zinc-finger are significantly larger. Scale bar in A applies to B to D. E to H, Wheat germ agglutinin (WGA) staining of ventricular sections of mature Gata6f/f and mutant mice (E and F) and E16 Gata6f/f and mutant mice (G and H). Ventricular myocyte cross-sections are larger in the mutant ventricles at both ages. Scale bar in E applies to F to H. The plots show that mature ventricular myocyte area (I) and length (J) are increased in the Gata6 mutant hearts. Myocytes were isolated from 3 different mice of each genotype. The number of myocytes in each group is shown within the bars. K and L, The number of ventricular myocyte cross-sections in each μm2 region. The number of regions analyzed from 3 different hearts in each group is shown within the bars. *P<0.01. Cre-G6 indicates Mlc2vCre-Gata6f/f; and G6, Gata6f/f.
Fang Liu et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

3. Conduction defects in mature mice without the Gata6 carboxyl zinc-finger.
Conduction defects in mature mice without the Gata6 carboxyl zinc-finger. A and B, Surface ECG leads I, II, III, aVR, aVL, and aVF along with intracardiac recordings of the right atrial electrogram (RAE) and His-bundle electrogram (HBE) from an adult Mlc2vCre-Gata6f/f (A) and Gata6f/f (B) mouse. Note the PR-interval is longer (red dashed lines) in the mutant mouse, with no gross defects in overall QRS complex morphology or other intervals on the surface ECG leads. The Mlc2vCre-Gata6f/f mouse has a PR-interval of 57 vs 46 ms for the Gata6f/f mouse. Recordings from the HBE show atriohisian (AH) interval prolongation (solid blue lines) without functional Gata6. The Mlc2v-CreGata6f/f mouse has an AH interval of 42 vs 31 ms for the Gata6f/f mouse. a indicates atrial electrogram; P, P wave; T, T wave; and v, ventricular electrogram.
Fang Liu et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

4. Gata6 expression in the developing murine atrioventricular conduction system.
Gata6 expression in the developing murine atrioventricular conduction system. In situ hybridization with probes against Gata6-exon4, Tbx3, and Id2 mRNA in frontal sections from Gata6f/f mouse embryos are shown. At embryonic day (E) 12.5 with functional Gata6 (A), Gata6 expression overlaps with Tbx3 (D) and Id2 (G). At E14.5 Gata6 transcript expression (B) is weaker in the regions that overlap with Tbx3 (E) and Id2 (H). By E16.5 Gata6 expression (C) is lower in the regions where it overlaps with Tbx3 (F) and Id2 (I), but Gata6 is more robustly expressed in the Tbx3-negative ventricular myocardium. The white arrows point to regions of developing cardiac conduction system that are either marked or devoid of Gata6+ staining. The yellow arrowheads point to regions of positive signal within the developing atrioventricular bundle. The white arrowheads point to regions of positive signal within the developing bundle branches. Scale bar, 100 μm. Scale bar in A applies to all E12.5 images; scale bar in B applies to all E14.5 images; and scale bar in C applies to all E16.5 images.
Fang Liu et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

5. Effects of Gata6 carboxyl zinc-finger deletion in the developing mouse atrioventricular conduction system.
Effects of Gata6 carboxyl zinc-finger deletion in the developing mouse atrioventricular conduction system. In situ hybridization with probes against Gata6-exon4, Tbx3, and Id2 mRNA in frontal sections from Mlc2v-CreGata6f/f mouse embryos is shown. In the absence of Gata6 with a carboxyl zinc-finger, Gata6 expression at embryonic day (E) 12.5 (A) overlaps with that of Tbx3 (D) and Id2 (G), but the region stained by each of these transcripts is smaller compared with similar anatomic sections from hearts with intact Gata6. A reduced area stained by Gata6 (B), Tbx3 (E), and Id2 (H) persists at E14.5, as well at E16.5 (C, F, and I). The white arrows point to regions of the developing cardiac conduction system that are either marked or devoid of Gata6+ staining. The yellow arrowheads point to regions of positive signal within the developing atrioventricular bundle. The white arrowheads point to regions of positive signal within the developing bundle branches. Scale bar, 100 μm. Scale bar in A applies to all E12.5 images; scale bar in B applies to all E14.5 images; and scale bar in C applies to all E16.5 images.
Fang Liu et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

6. Gata6 carboxyl zinc-finger deletion reduces hyperpolarizing cyclic nucleotide–gated channel, subtype 4 (HCN4) expression in the atrioventricular node (AVN).
Gata6 carboxyl zinc-finger deletion reduces hyperpolarizing cyclic nucleotide–gated channel, subtype 4 (HCN4) expression in the atrioventricular node (AVN). Immunohistochemical staining of frontal sections from mature murine hearts against HCN4 (green) in control hearts (A–E), and Gata6 mutant hearts (F–J), shows HCN4 expression is significantly diminished within the AVN in the absence of Gata6 with a carboxyl zinc-finger (A and F). The dotted yellow line in F represents the region of TBX3+ staining in an adjacent section as shown in Figure II in the Data Supplement. In both the presence and absence of Gata6 with a carboxyl zinc-finger, HCN4-positive cells are expressed in the lower nodal region (LNR) and portions of the atrioventricular bundle (AVB; B–D and G–I). The expected lack of HCN4 staining in the bundle branches (BB) is seen in the control and mutant hearts (E and J). Blue arrowheads point to the LNR; white arrowheads point to HCN4+ staining in the AVB. Scale bar, 250 μm and applies to all panels.
Fang Liu et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

7. Gata6 carboxyl zinc-finger deletion does not affect connexin-40 (CX40) expression in the mouse cardiac conduction system.
Gata6 carboxyl zinc-finger deletion does not affect connexin-40 (CX40) expression in the mouse cardiac conduction system. Immunohistochemical staining of frontal sections from mature murine hearts against CX40 (red) in Gata6f/f hearts (A–E) and those without the Gata6 carboxyl zinc-finger (F–J) reveals the expected lack of CX40 staining in the atrioventricular node (AVN), despite the presence of Gata6 (A and F). In the presence and absence of Gata6 with a carboxyl zinc-finger, the expected expression of CX40 within the atrioventricular bundle (AVB) is observed (B–D and G–I). Deletion of the Gata6 carboxyl zinc-finger does not seem to affect the expression of CX40-positive cells within the bundle branches (E and J). The blue arrowheads point to the lower nodal region (LNR); the yellow arrowheads point to regions of CX40+ staining in the bundle branches (BB). Scale bar, 250 μm and applies to all panels.
Fang Liu et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

8. Gata6 carboxyl zinc-finger deletion reduces ID2 expression and cardiomyocyte cell-cycle exit in the developing atrioventricular bundle (AVB).
Gata6 carboxyl zinc-finger deletion reduces ID2 expression and cardiomyocyte cell-cycle exit in the developing atrioventricular bundle (AVB). Immunohistochemical staining in adult murine hearts against ID2 in Gata6f/f hearts (A and C) shows ID2 expression is higher in the proximal cardiac conduction system compared with the working myocardium. In the absence of Gata6 with a carboxyl zinc-finger (B and D), ID2 expression is reduced in the atrioventricular node (AVN) and AVB. E to K, Gata6 with a carboxyl zinc-finger is required for cell-cycle exit of cardiomyocytes in the developing AVB. Immunohistochemical staining of embryonic day 12.5 wild-type hearts (E–G) against proliferating nuclear cell antigen (PCNA; red) and TBX3 (green), as well as similarly staged Gata6 mutant hearts (H–J). The dashed yellow outline indicates the TBX3-expressing AVB. Note there are fewer PCNA/TBX3-positive cells in the AVB of Gata6f/f heart compared with the mutant heart. K, PCNA-positive nuclei in the crest of the septum that overlap with TBX3-expressing cells averaged from 5 hearts of each genotype. *P<0.01. Scale bar, 100 μm. Scale bar in A applies to A to D; scale bar in E applies to E to J.
Fang Liu et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

9. GATA6 transactivates Id2 and Ncx1.
GATA6 transactivates Id2 and Ncx1. A, VISTA comparison of the murine and human Id2 proximal promoter and exon 1, and (B) of the murine and human Ncx1 proximal promoter and exon 1. A and B, The x and y axes indicate sequence length (kb) and percent homology (≥75%, pink), respectively. GATA6 binding sites conserved in the mouse and human sequence are indicated by asterisks. C, Schematic of the Id2-LUC reporter containing the 5.2-kb proximal promoter upstream of firefly luciferase (LUC), and (D) the Ncx1-LUC reporter containing the 5.1-kb proximal promoter that is also upstream of firefly luciferase. E, Activation of the Id2-LUC reporter by GATA6. HL-1 cells were transiently transfected with Id2-LUC and 125 to 500 ng of expression plasmid encoding wild-type GATA6 or GATA6 lacking exon 4 (Gata6Δexon4). The reporter is activated by wild-type GATA6 but not by GATA6Δexon4. F, Activation of the Ncx1-LUC reporter by GATA6. HL-1 cells were similarly transiently transfected with Ncx1-LUC and 125 to 500 ng of expression plasmid encoding wild-type GATA6 or GATA6Δexon4. As with Id2-LUC, the Ncx1-LUC reporter was activated by the expression of wild-type GATA6 but not by GATA6Δexon4. The transient transfection studies in E and F show the mean change in report activity from ≥3 different experiments at each concentration of expression plasmid.
Fang Liu et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.