Evaluating the role of connexin43 in congenital heart disease: Screening for mutations in patients with outflow tract anomalies and the analysis of knock-in mouse models

Abstract

Background: GJA1 gene encodes a gap junction protein known as connexin 43 (Cx43). Cx43 is abundantly expressed in the ventricular myocardium and in cardiac neural crest cells. Cx43 is proposed to play an important role in human congenital heart disease, as GJA1 knock-out mice die neonatally from outflow tract obstruction. In addition, patients with visceroatrial heterotaxia or hypoplastic left heart syndrome were reported to have point mutations in GJA1 at residues that affect protein kinase phosphorylation and gating of the gap junction channel. However, as these clinical findings were not replicated in subsequent studies, the question remains about the contribution of GJA1 mutations in human congenital heart disease (CHD).

Materials and methods: We analyzed the GJA1 coding sequence in 300 patients with CHD from two clinical centers, focusing on outflow tract anomalies. This included 152 with Tetralogy of Fallot from over 200 patients exhibiting outflow tract anomalies, as well as other structural heart defects including atrioventricular septal defects and other valvar anomalies. Our sequencing analysis revealed only two silent nucleotide substitutions in 8 patients. To further assess the possible role of Cx43 in CHD, we also generated two knock-in mouse models with point mutations at serine residues subject to protein kinase C or casein kinase phosphorylation, sites that are known to regulate gating and trafficking of Cx43, respectively.

Results: Both heterozygous and homozygous knock-in mice were long term viable and did not exhibit overt CHD.

Conclusion: The combined clinical and knock-in mouse mutant studies indicate GJA1 mutation is not likely a major contributor to CHD, especially those involving outflow tract anomalies.

Keywords: Connexin43; congenital heart disease; gap junction; knock-in mouse model; outflow tract; phosphorylations.

Figure 1

Genomic deoxyribonucleic acid (DNA) amplification and targeting construct design (a) GJA1 genomic DNA amplification and sequencing. The location of primers used for polymerase chain reaction (PCR) amplification and sequencing of the coding region of GJA1 (black region) are indicated. Primers were designed to avoid amplification of the pseudogene.[26] (b) Gja1 knock-in construct design The knock-in vectors contained floxed PGK-Neo (S368A) or PGK-Neo-Stop (S330A/S328Y/S325A), a thymidine kinase cassette, and 5’/3’ homology arms (dashed lines). Mutation in the mouse Gja1 knock-in constructs (vertical arrow) consisted of S368A (AGC->GCT) or S330A:S328Y:S325A (TCC->GCA;TCC->TAC;AGC->GCA). Primers used for screening are described in Table 4.

Figure 2

Southern blot analysis of Cx43 KI mice. (a) On the top shown is the representation of the genomic region of Cx43 gene with positions of diagnostic restriction site. Below shown is the representation of Cx43 KI construct and position of diagnostic restriction site. Also shown is the position of primers that was used for genotyping the mice. (b) Southern blot of genomic DNA digested with either PstI (left panel) or XbaI (right panel) and hybridized with e 5’ and 3’ probe respectively. The position of the probe was shown on map of panel A. Pst I digested genomic DNA hybridized to with 5’ probe (left panel) should produce 8.0 Kb fragment for wildtype (lane 5) and 4.6 Kb fragment for KI mice (lane 1-4). Xba I digested genomic DNA hybridized to with 3’ probe (right panel) should produce 9.5 Kb fragment for wildtype (lane 5) and 8.7 Kb fragment for KI mice (lane 1-4).

Figure 3

Genotype distribution of Gja1 knock-in mice litters. Shown are the genotype distribution recovered in three litters of mice obtained from the mating of heterozygous Gja1 knock-in mice with the S330A:S328Y:S325A (CK1) mutation. The genotype obtained (blue) is not significantly different from the expected distribution (red).

Figure 4

Cx43 localization in heart tissue from wildtype, S368A and S235A/S328Y/S330A mice. Paraffin-embedded sections of mouse hearts were stained with Cx43 primary and secondary as indicated in the Materials and Methods. 4’,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei (bar = 500μm). The lower row of panels shows higher magnification views of the indicated portions from the panels above (bar = 60 μm)

Figure 5

Characterization of total Cx43 in mouse hearts. Western blots of whole heart lysates, isolated from two WT (Wild type-Cx43), CK1 (S325A/S328Y/S330A) or two PKC (S368A) hearts, simultaneously probed for total Cx43. Membranes were also probed with anti-vinculin antibody, as an internal loading control. Notice that Cx43 protein levels are decreased in CK1 and PKC mice compared with WT. Positions of the molecular weight markers are shown on the left