Connexin Lateralization Contributes to Male Susceptibility to Atrial Fibrillation

Figures

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  Figure 1. Similar action potential configuration in left atrial myocytes of male and female mice. (A). Representative recordings of male (black) and female (red) action potentials recorded using perforated patch-clamp techniques at 4 Hz at 37 °C. Dotted line displays 0 mV. (B). Summary table of mean values of action potential parameters: maximum upstroke velocity (Vmax), action potential amplitude (APA), action potential duration at 20, 50 and 90% of repolarization (APD20, APD50 or APD90) and resting membrane potential (RMP). No statistical difference was observed between males and females.

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  Figure 2. No sex difference in Na+ and K+ currents in mouse left atrial myocytes. (A). Typical recordings of INa from male (black) and female (red) left atrial myocytes. The inset shows voltage protocol here and in panel D. (B). Mean IV curves of INa show comparable current density between male and female left atrial myocytes (at −35 mV: M: −20.3 ± 2.0 pA/pF, n = 11, N = 3; F: −19.1 ± 2.1 pA/pF, n = 13, N = 2; p = 0.70). (C). mRNA expression of the Scna5 gene, underlying the NaV1.5 channel, was not different between male and female left atrial tissues (M: 1.11 ± 0.07, n = 6; F: 1.00 ± 0.13, n = 6; p = 0.46). (D, left). Typical recordings of the total K+ current (Ipeak) from male (black) and female (red) left atrial myocytes. (D, Right). Mean IV curves of Ipeak show no sex difference in left atrial myocytes (at +30 mV: M: 20.5 ± 2.8 pA/pF, n = 13, N = 5; F: 21.4 ± 1.0 pA/pF, n = 26, N = 9, p = 0.78). Welch’s t-test was used due to unequal variances (p > 0.05 at all voltages). Dotted lines represent the zero current.

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  Figure 3. Gene expression and cellular distribution of Cx40 and Cx43. (A). No sex differences were observed in mRNA expression of Gja5 (left) and Gja1 (right) encoding for Cx40 and Cx43 (n = 6/group), respectively. (B). The percentage of Cx40 lateralization, defined as the percentage of positive signal present at the lateral membrane, is higher in male left atrial myocytes compared to females (M: 63 ± 4%, n = 22, N = 4; F: 45 ± 4%, n = 17, N = 3; ** p = 0.005). (C). Representative example of the Cx40 distribution (in green) in male and female atrial myocytes. (D). Scatter plot of Cx43 lateralization in male and female atrial myocytes (M: 66 ± 4%, n = 23, N = 4; F: 44 ± 4%, n = 12, N = 3; ** p = 0.002). (E). Representative examples of Cx43 distribution (in red). All scale bars represent 20 µm. ** p < 0.01.

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  Figure 4. Similar level of interstitial fibrosis in male and female atrial myocytes. (A). Typical collagen picrosirius red staining (stained red) of left atria of male and female mice, at a 4× and 20× magnification zoom. Scale bars represent 100 and 500 µm, at 4× and 20×, respectively. The scatter plots reveal no differences in atrial fibrous content between males and females (M: 4.9 ± 1.2%, n = 4; F: 5.3 ± 0.7%, n = 4; p = 0.79). (B). qPCR analysis shows that the mRNA expression level of genes involved in fibrosis pathways was similar between male and female atria (n = 4–6/group).

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  Figure 5. The left atria are heavier, and the atrial myocyte capacitances are greater in males. (A). Summary table of the numerical values of morphometric measurements of male and female mice. (B). The scatter plot displaying left atria weight normalized to tibia length (TL) shows a higher ratio in male than in female mice (M: 0.24 ± 0.01 mm/mg, n = 15; F: 0.16 ± 0.01 mm/mg, n = 16; **** p < 0.0001). (C). Representative examples of atrial myocytes isolated from male and female mice obtained from connexin immunofluorescence experiments, viewed under brightfield confocal microscopy. Scale bars represent 20 µm. (D). The cell capacitance of left atrial myocytes are higher in males than in females (M: 55.9 ± 1.0 pF, n = 149, N = 20; F: 44.8 ± 0.9 pF, n = 129, N = 17; **** p < 0.0001). (E). The length, width and cell area of left atrial myocytes are greater in males than in females (M: n = 55–68, N = 3; F: n = 40–67, N = 68) (** p < 0.01, *** p < 0.001). Cellular parameters in E were obtained from cells used for Ca2+ transient experiments performed for a separate study submitted to International Journal of Molecular Sciences (IJMS) (unpublish).

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  Figure 6. Orchiectomy reduces susceptibility of males to AF. (A). Typical recording of simultaneous surface ECG and intracardiac ECG (iECG) with induction of AF in response to burst pacing, followed by spontaneous recovery to NSR. The burst pacing duration has been cut for easier graphical illustration. (B). The bar graphs summarize data obtained on AF inducibility in intact and gonadectomized male and female mice in response to burst stimulation protocols (M: 65%, 13/20; ORC: 38%, 6/16; F: 42%, 10/24; OVX: 33%, 4/12). While ovariectomy has no effect on AF susceptibility in females, orchiectomy reduced AF susceptibility of males. The male and orchiectomized groups were compared using the one-sided chi square test (p = 0.050). Data for intact male (M) and female (F) presented in panel B represent a subset of data of a separate study submitted to International Journal of Molecular Sciences (IJMS) (unpublish).

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  Figure 7. Orchiectomy reduces connexin lateralization and cell size in males. (A,B). Left. Representative examples of the distribution of Cx40 (in green, panel (A)) and Cx43 (in red, panel (C)) in male and ORC atrial myocytes. Right. Lateralization of Cx40 (panel (A)) and Cx43 (panel (B)) in left atrial myocytes is reduced by orchiectomy (ORC) compared to control males. (C). Left. Typical images of left atrial myocytes from male and ORC mice. Right. Scatter plot of cell area of male and ORC left atrial myocytes from connexin immunofluorescence experiments. Scale bars represent 20 µm. * p < 0.05, ** p < 0.01, **** p < 0.0001.