A Connexin 40 Mutation Associated with a Malignant Variant of Progressive Familial Heart Block Type-1

Genetic screening of PFHB1

Genomic screening by PCR and DNA sequencing was performed for GJA5 (Cx40), GJA1 (Cx43), GJC1 (Cx45), KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, KCNJ2, SCN1B, SCN4B, HCN4. (Primer information in online supplement.) All participating probands and family members gave written informed consent, in accordance with standards (Declaration of Helsinki) and local ethics committees.

Plasmid construction

A 1.1-kilobase Cx40-DNA fragment was subcloned into bicystronic plasmids pIRES2-EGFP and pIRES2-DsRED2. An EGFP or FLAG epitope was added at Cx40 C-terminal to generate EGFP- or FLAG-tagged Cx40. Site-directed mutagenesis (Q58L) was performed with QuikChange. Primer information and additional details in online supplement.

Cell culture and transfection

Constructs were introduced into connexin-deficient HeLa cells, or mouse neuroblastoma (N2A) cells using Lipofectamine as per manufacturer's protocol.

Electrophysiology

Gap junction currents were recorded from transiently-transfected N2A cell pairs using whole-cell double patch clamp techniques as previously described.^{18, 19} Further details in online supplement.

Immunocytochemistry and Western blotting

HeLa cells, transfected with pEGFPN1-Cx40-WT, pCMV-FLAG-Cx40-Q58L, or both, were stained with anti-FLAG M2 antibody and Alexa546-labeled secondary antibody. EGFP and Alexa546 fluorescence images were recorded by confocal microscopy. For western blotting, N2A cells were transiently transfected with 3μg Cx40 plasmids. Two days after transfection, cells were lysed, proteins extracted and separated by conventional methods. Further details in online supplement.

Statistical analysis

Results are presented as mean±SEM. Mann-Whitney rank-sum tests with Bonferroni's post-hoc correction were used in comparisons for which normality or equal variance assumptions were invalid. In other instances, differences between groups were assessed by one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc correction. Statistical significance was assumed for P < 0.05.

Genetic screening of PFHBI probands

We genetically screened 156 probands with clinical diagnosis of PFHBI. We identified 4 novel and 5 previously-reported mutations in SCN5A,^{13, 15} 3 mutations in SCN1B,¹⁴ and a novel germline heterozygous missense mutation in exon 2 of the Cx40 gene, GJA5 (Supplemental Table S1). Mutations were not found in connexin genes GJA1 (Cx43) or GJC1 (Cx45) or in other genes screened (KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, HCN4 or SCN4B). Of the novel SCN5A mutations, one caused a modification of the amplitude and voltage gating kinetics of the sodium current in heterologously expressing cells (supplemental Figure S1); three other mutant constructs failed to express functional channels, suggesting that patients carrying the mutation were functionally haploinsuficient for Nav1.5 (supplemental Figure S1). The GJA5 mutation (c. 173 A>T) caused an amino acid substitution (glutamine (Q) replaced by leucine (L)) at position 58 in Cx40 (Cx40-Q58L; Figures 1A-1B). The mutation was absent in 400 alleles from unaffected controls and in other 155 PFHBI probands. Screening of the entire gene panel (including SCN5A and SCN1B) revealed no other sequence modification in the DNA of this proband. Topological analysis placed amino acid 58 of Cx40 within the first extracellular loop (Figure 1C). The presence of glutamine in this position is highly conserved among GJA5 orthologs, and two other cardiac connexins, Cx43 and Cx45 (Figure 1D). The clinical and genotypic characteristics of proband and tested family members are described below.

Clinical phenotypes and genotype of the PFHBI pedigree with the GJA5 mutation

The proband, an 11-year-old male at time of death, was first referred for evaluation when he was 6 years-old because of ECG abnormalities. Although asymptomatic at that time, his ECG showed advanced atrioventricular (AV) block, complete left bundle branch block (LBBB) and left axis deviation (Figure 2A). Echocardiography and cardiac scintigraphy did not reveal signs of structural heart disease. He experienced an episode of syncope at age 9; implantation of a permanent pacemaker was recommended by the physician but not authorized by the legal guardian. The proband died suddenly two years later during exercise (running) and the family declined postmortem examination. The proband's younger sister shares the Cx40-Q58L mutation. She is asymptomatic with a QRS duration at the upper limit of normal, left axis deviation that has been progressive (Supplemental Table S2) and QRS notch. These findings are consistent with impaired intraventricular conduction (Figure 2B). The mother died suddenly at age 30, after delivering the second child. An ECG on record, obtained when she was 16 years old, was similar to that of the proband (compare Figure 2C with 2A). In addition, a ventricular tachycardia was recorded during the recovery phase of an exercise stress test (Supplemental Figure S2). DNA from the mother was not available for examination. Other family members, including proband's father, showed normal ECG. DNA analysis of proband's father and maternal grandparents revealed absence of the Cx40-Q58L mutation. Based on clinical data, and genotypic features of proband and sister, it is most likely that mutation Cx40-Q58L appeared de novo in the proband's mother. The data also indicate an early onset of PFHBI in this family, when compared to the natural history of the disease in most other cases.⁸ As an initial step to assess the functional implications of mutation Cx40-Q58L, modified constructs were transiently expressed in an exogenous system and evaluated for localization and function.

Electrophysiological properties of mutant Cx40-Q58L channels

Connexin-deficient neuroblastoma (N2A) cells were transiently transfected with cDNA for Cx40-WT or Cx40-Q58L; electrophysiological properties of homologous Cx40 channels were analyzed by conventional dual whole-cell patch clamp. Figure 3A shows representative junctional current traces elicited by a transjunctional voltage gradient of -60 mV. Average junctional conductance (Gj) decreased from 22.2±1.7 nS in Cx40-WT (n=14) to 0.56±0.34 nS in cells expressing the Cx40-Q58L mutant (n=14; p<0.001). The probability of functional coupling, calculated by dividing the number of electrically-coupled pairs by the number of pairs tested, was 100% and 57.1% for WT and Q58L, respectively.

Figure 3B depicts representative single channel recordings elicited by a transjunctional voltage of -60 mV in cell pairs expressing Cx40-WT or Cx40-Q58L. Unitary events for WT channels displayed current transitions corresponding to two conducting states (O₁ and O₂) of 43.3 pS and 119.5 pS, respectively. Figure 3C shows the event histograms for both cell types (Cx40-WT: N=3 cell pairs, n=303 events; Cx40-Q58L: N=3, n=416). The histogram for the Cx40-WT channels was best described by two Gaussian distributions centered at 136.2±2.3 pS and 53.1±5.3 pS. In contrast, the histogram for Cx40-Q58L channels was best described by a single Gaussian function, centered at 40.2±0.3 pS. Moreover, the length of time that a channel dwelled in the open state (dwell open time) was substantially longer for Cx40-Q58L than for WT channels (dwell open time for WT, 27.9±0.5 msec, N=4, n=186; for Q58L, 92.0±7.8 msec, N=3, n=163; Figure 3D). Of note, mutation Q58L had a strong dominant effect on formation of heterotypic functional gap junctions. Cells were transfected with either pIRES2-EGFP-Cx40WT or pIRES2-DsRED2-Cx40Q58L, and heterotypic pairs identified by fluorescence microscopy (an EGFP-expressing cell paired with a DsRED2-expressing cell). We recorded from 8 cell pairs and detected unitary current events in only two pairs. A total of 57 events were recorded, and average macroscopic junctional conductance was 0.04±0.03 nS. Collectively, the data demonstrated that mutation Q58L significantly affects the biophysical properties of Cx40 channels and the overall ability of Cx40 gap junctions to form a low-resistance pathway between cells.

Electrophysiological properties and gap junction plaque formation in cells co-expressing WT and Q58L proteins

In the clinical cases identified, mutation Q58L was detected only in one carrier allele. Therefore, we assessed the function of gap junctions in cells co-expressing WT and mutant proteins. N2A cells were co-transfected with cDNA for both GFP-tagged Cx40-WT and Cx40-Q58L (0.5 μg of pEGFPN1-Cx40-WT combined with 0.5 μg of pEGFPN1-Cx40- Q58L). Results were compared to those obtained when only one of the constructs (1 μg) was transfected. Cells expressing both constructs (WT/Q58L) showed intermediate conductance (15.4±3.7 nS, n=16) between WT (28.8±3.6 nS, n=16, p<0.001) and Q58L (0.28±0.11 nS, n=14, p<0.001; Figure 4A). These values were comparable to those obtained using the bicistronic pIRES2-EGFP constructs (WT: 22.2±1.7 nS, n=14; WT/Q58L: 13.0±2.4 nS, n=17; Q58L: 0.56±0.34 nS, n=14). The co-expression results were consistent with those obtained using pIRES plasmids that tagged the cells both green and red, if co-transfected (Supplemental Figure S1). The probability of finding functional coupling in co-transfected cells was 76.5%, which was intermediate between WT (100%) and Q58L (57.1%).

The characteristics of gap junction plaques observed in cells co-expressing WT and Q58L varied significantly between pairs (Figure 4B). Nearly half of transfected (fluorescence-positive) cells exhibited clear and discrete gap junction plaques (arrow (a)) whereas the rest of fluorescent-positive cells showed diffuse expression pattern and absence of well-defined plaques (arrow (b)). Fluorescence-positive and gap junction plaque-positive cells were counted in 10 different views for each group, and efficacy of gap junction plaque formation was statistically analyzed (Figure 4C) by calculating the ratio of cells with gap junction plaques to the number of fluorescence-positive cells. In the Cx40-WT group, almost all fluorescent-positive cells exhibited clear gap junction plaques (94.9±1.9%, n=940), whereas there was a more diffuse and homogenous pattern with only occasional plaque formation in the Cx40-Q58L group (6.6±0.7%, n=1318, p<0.001 compared with WT). In contrast, results varied widely in cells co-transfected with WT/Q58L; nearly half of fluorescence-positive cells exhibited gap junction plaques similar to those observed in cells transfected with the WT construct (48.2±2.4%, n=855, p<0.001), whereas the rest showed a diffuse expression pattern similar to that of Cx40-Q58L. To establish a better correlation between plaque formation and junctional conductance, both variables were measured concurrently in the same cell pair, for 39 N2A cell pairs where GFP-tagged plasmids of Cx40-WT and Cx40-Q58L were co-transfected. As shown in Figure 4D, about half of GFP-positive cell pairs showed a very small junctional conductance (<5 nS) and very few or negligible gap junction plaques (a). In the other half of cell pairs, small, dot-like junctional plaques correlated with intermediate junctional conductance (Gj) values (b), and there were clear, extensive gap junction plaques associated with Gj values larger than 25 nS (c). Overall, we found significant heterogeneity in the extent of electrical coupling, although the measurements of Gj correlated with the localization of proteins in transfected cells. These results indicate that the Q58L mutation significantly impairs the ability of cells to form gap junction plaques, though the effect is not purely dominant when both WT and mutant proteins are co-expressed.

Subcellular distribution of WT and Q58L Cx40 in transiently-transfected cells

To further analyze the subcellular distribution of Cx40-WT and Cx40-Q58L proteins, the C-terminal of Cx40-WT was tagged with GFP, whereas the C-terminal of Cx40-Q58L was FLAG-tagged. After transfection of N2A cells with the tagged constructs, the distribution of each protein was examined by confocal microscopy. As shown in Figure 5, green color indicates the position of GFP-tagged molecules, whereas red indicates the position of FLAG-tagged molecules. In cells transfected only with GFP-tagged Cx40-WT, fluorescence was consistently detected at sites of cell-cell apposition, following the pattern previously described for GFP-labeled gap junction plaques (Figure 5A). A similar distribution was found when cells were transfected with FLAG-tagged Cx40-WT (not shown). In contrast, most FLAG-tagged Cx40-Q58L signal was evenly distributed around the cell, in the vicinity of the plasma membrane (Figure 5B). Biotinylation experiments showed that the Q58L mutation did not prevent the Cx40 protein from inserting into the membrane and presenting a domain reachable form the extracellular space (Supplemental Figure S2). Micoscopy experiments in cells co-expressing GFP-tagged Cx40-WT and FLAG-tagged Cx40-Q58L proteins yielded results intermediate to those obtained when only one construct was expressed. Nearly half of cell pairs showed both proteins distributed homogenously at or near the cell membrane, without formation of well-defined gap junction plaques (Figure 5C). These images resembled those obtained when only Cx40-Q58L proteins were expressed (middle column of Figure 5B). In contrast, other cell pairs showed clustering of fluorescent signals within closely confined areas that appeared to be gap junction plaques (Figure 5D).

The experiments described above led us to speculate that the distribution and function of heteromeric connexons is determined by their mutant subunit content, whereby formation (or not) of plaques and channels are determined –at least in part- by the abundance of expression of one protein over the other. As an initial step to probe this hypothesis, we took advantage of the characteristics of the bicistronic plasmid pIRES, in which the expression rate of the upstream gene is several-fold greater than that of the downstream gene,²⁰ and explored the functional properties of heteromeric connexons. WT-Cx40 and GFP-tagged Q58L-Cx40 were subcloned into the pIRES vector, either alone or in combination, in the specific orientations shown in Figure 6A. Protein expression levels of WT-Cx40 and Q58L-Cx40 were determined by immunochemistry. In contrast to the data obtained when WT-Cx40 and GFP-tagged Q58L-Cx40 plasmids were co-transfected at a 1:1 ratio (lane 6), expression of heteromeric pIRES plasmids WT-IRES-Q58L-EGFP (lane 3) and Q58L-EGFP-IRES-WT (lane 4) resulted in uneven protein expression levels of WT (40 kDa) and Q58L-EGFP (67 kDa), depending upon their orientation in the pIRES vector. Based on these observations, we constructed a homomeric WT-Cx40 plasmid (WT-IRES-WT) and heteromeric plasmids of WT-Cx40 and Q58L-Cx40 with different orientations (WT-IRES-Q58L and Q58L-IRES-WT; Figure 6B). Junctional conductance of cell pairs expressing WT-IRES-Q58L (25.3±2.8 nS, n=17) was nearly indistinguishable from that of the homomeric plasmid, WT-IRES-WT (27.8±1.4 nS, n=17, NS). By contrast, the converse heteromeric construct Q58L-IRES-WT showed substantially reduced junctional conductance (0.29±0.12 nS, n=15; p<0.001), comparable with that of the homomeric Q58L (0.56 ± 0.34 nS, Figure 3A). These results suggest that the final electrophysiological properties of the heteromeric connexons are determined predominantly by the numbers of mutant subunits in each gap junction, rather than defined by a dominant-negative effect.