Connexin43 ablation in foetal atrial myocytes decreases electrical coupling, partner connexins, and sodium current

Abstract

Aims: Remodelling and regional gradients in expression of connexins (Cx) are thought to contribute to atrial electrical dysfunction and atrial fibrillation. We assessed the effect of interaction between Cx43, Cx40, and Cx45 on atrial cell-to-cell coupling and inward Na current (I(Na)) in engineered pairs of atrial myocytes derived from wild-type mice (Cx43(+/+)) and mice with genetic ablation of Cx43 (Cx43(-/-)).

Methods and results: Cell pairs were engineered by microcontact printing from atrial Cx43(+/+) and Cx43(-/-) murine myocytes (1 day before birth, 3-5 days in culture). Dual and single voltage clamp were used to measure intercellular electrical conductance, g(j), and its dependence on transjunctional voltage, V(j), single gap junction channel conductances, and I(Na). 3D reconstructions of Cx43, Cx40, and Cx45 immunosignals in gap junctions were made from confocal slices. Full genetic Cx43 ablation produced a decrease in immunosignals of Cx40 to 62 ± 10% (mean ± SE; n= 17) and Cx45 to 66 ± 8% (n= 16). G(j) decreased from 80 ± 9 nS (Cx43(+/+), n= 17) to 24 ± 2 nS (Cx43(-/-), n= 35). Single channel analysis showed a shift in the main peak of the channel histogram from 49 ± 1.7 nS (Cx43(+/+)) to 67 ± 1.8 nS (Cx43(-/-)) with a second minor peak appearing at 27 ± 1.5 pS. The dependence of g(j) on V(j) decreased with Cx43 ablation. Importantly, peak I(Na) decreased from -350 ± 44 pA/pF (Cx43(+/+)) to -154 ± 28 pA/pF (Cx43(-/-)).

Conclusions: The dependence of Cx40, Cx45, and I(Na) on Cx43 expression indicates a complex interaction between connexins and I(Na) in the atrial intercalated discs that is likely to be of relevance for arrhythmogenesis.

Figure 1

Engineered atrial cell pair. (A) Wide Field image of engineered murine atrial cell Cx43^+/+ pair overlaid with the immunofluorescence of N-Cadherin (green) and Cx40 (red). Note the sharply defined interface between the two myocytes (x/z plane). (B) 3D surface rendering of N-Cadherin (green) and Cx40 (red) immunofluorescence signals shown in the y/z plane. (C) 3D surface rendering of Cx40 immunofluorescence signal alone showing plaque-like arrangement of Cx40. Image deconvolution was applied to confocal signals before reconstruction.

Figure 2

Change in atrial intercellular electrical conductance, g_j,0 and voltage gating with germline Cx43 ablation. (A) Germline ablation of Cx43 decreased intercellular electrical conductance in atrial cell pairs from 80 ± 9 nS (n= 17) to 53 ± 7 nS (heterozygous deletion; n= 32) and to 23 ± 2 nS (homozygous deletion; n= 35). Asterisks denote statistical difference (P < 0.01). (B–C) Relative gap junctional conductance (g_j,ss/g_j,inst) in atrial cell pairs [Cx43^+/+: (B); Cx43^+/−: (C); Cx43^−/−: (D)]. With ablation of Cx43, the decrease in g_j at steady state with increasing transjunctional voltage, V_j, occurs at higher V_j, indicating dominance of Cx40 (for numerical analysis, see Table 1).

Figure 3

Change in atrial unitary single gap junction channel conductances, γ_j, with germline Cx43 ablation. (A, C, and D) Frequency histograms of single channel conductances, γ_j,main in Cx43^+/+ (A), Cx43^+/− (C), and Cx43^−/− (E) atrial pairs, showing main peaks (Gaussian fit) at 49 ± 1.7 pS (Cx43^+/+, n= 649) to 53 ± 1.4 pS (Cx43^+/−, n= 335), and 67 ± 1.8 pS (Cx43^−/−, n= 481) with a second minor peak appearing at 27.1 ± 1.5 pS. Bin width 5 pS. (B, D, and F) Examples of unitary currents recorded in atrial cell pairs. Vertical dotted line indicates switch of polarity of V_j.

Figure 4

Change in inward Na current I_Na with germline Cx43 ablation. (A) Original recordings of I_Na recorded in Cx43^+/+ (left), Cx43^+/− (middle), and Cx43^−/− (right) atrial myocytes. (B) Peak I_Na density in Cx43^+/+, Cx43^+/−, and Cx43^−/− atrial myocytes. Asterisk denotes statistical significance (P < 0.05).

Figure 5

Changes in Cx43, Cx40, and Cx45 immunosignals in atrial gap junctions with germline Cx43 ablation: Atrial Cx43^+/+ cell pair (A and B), Atrial Cx43^−/− cell pair (C and D). The top parts of A–D depict a shadow projection view on engineered atrial cell pairs with enhanced cytoplasmic background fluorescence (green) and nuclei (blue). The lower quadrangles of the panels show 3D surface renderings of the immunofluorescence signals in a semi-transparent mode in the z/y (sagittal) plane of the junctions. (A) and (C) show Cx43 immunofluorescence (red) and Cx45 immunofluorescence (green); (B) and (D) show Cx40 immunofluorescence (red) and Cx45 immunofluorescence (green). Scale bars: 10 μm.

Figure 6

Changes in Cx43, Cx40, and Cx45 immunosignals in atrial gap junctions with germline Cx43 ablation. The bars with an asterisk denote statistical significance among a given connexin (P < 0.05). The Cx43, Cx45, and Cx40 signals are expressed in relative volume, normalized to the respective wild-type. The numbers above the columns denote the number of cell pairs analysed.