Clinical observations have linked atrial fibrillation, the most common cardiac arrhythmia, with reduced electrical coupling of cardiac myocytes. During atrial fibrillation, spatial re-modelling of the gap junctional protein, connexin43 (Cx43), which is primarily responsible for electrical coupling at the intercalated discs, has been observed in human right atrial tissue (Kostin et al. 2002). In this study, we have shown expression of the Cx43 protein changes with age within the right atrium.
Guinea-pigs of 1 month and 38 months of age were humanely killed by anaesthetic overdose. The right atrium was dissected from the heart, then embedded in cryo-media and frozen until sectioned on a cryostat for immunocytochemistry, or punch biopsy samples (4 mm diameter) were frozen until Western blot. Sections of 14 µm were cut and labelled using the antibodies anti-Cx43 (Chemicon International, USA) plus anti-IgG conjugated to FITC (Dako, Denmark) and then imaged using confocal laser scanning microscopy. Data are presented as means ± S.E.M., and were analysed by ANOVA, followed by Student’s unpaired t test.
Intercalated disc area significantly increased from 33 ± 2.8 µm2 at 1 month to 83 ± 6.3 µm2 at 38 months. The dimensions of the intercalated disc significantly increased at the minor axis from 5.2 ± 0.2 µm at 1 month to 8.78 ± 0.3 µm at 38 months, and at the major axis from 7.8 ± 0.4 µm at 1 month to 12.3 ± 0.5 µm at 38 months (n = 30, P < 0.0001 in both cases). Within the intercalated discs, the area positive for Cx43 protein significantly decreased over 35 % with age (n = 30, P < 0.0001).
For Western blot, samples were ground in liquid nitrogen, prepared in protease inhibitors, separated by 10 % SDS-PAGE, transferred to nitrocellulose and processed with anti-Cx43 plus anti-IgG conjugated to HRP (Dako, Denmark). Bands of Cx43 protein at 43 kDa were detected by chemiluminescence. The band density of each sample was standardised to a Cx43 peptide control. Connexin43 expression of right atrium tissue significantly decreased 69 %, from 6.25 ± 1.2 µg at 1 month to 1.9 ± 0.5 µg at 38 months (P = 0.018, n = 5).
Therefore, we have shown a correlation between ageing and a decline in expression of Cx43 protein within the right atrium. This could explain the increased incidence of atrial arrhythmias in the elderly population.
This work was supported by the Wellcome Trust and British Heart Foundation.