In mammals, a decline in function of the pacemaker of the heart, the sinoatrial node (SAN), is associated with ageing and clinically exhibited as an increase in SAN conduction time and distance from the centre of the SAN to the crista terminalis, resulting in an increase in the velocity of conduction (Alings & Bouman, 1993). These clinical observations could be the result of a decrease in the density of gap junctions. In this study, we have shown expression of the principal cardiac gap junction protein, connexin43 (Cx43), changes with age within the SAN. Data were principally obtained by Western blot and compared with those from conduction maps and immunocytochemistry.

Guinea-pigs aged 1 day to 38 months were humanely killed by anaesthetic overdose. The SAN was removed from the intercaval region between the superior and inferior venae cavae of the right atrium of the heart. The preparation was maintained in oxygenated Tyrode solution at 37 °C and the extracellular electrode was moved at 0.5 or 1 mm intervals and the extracellular potential measured at each point to construct a map of conduction. From the centre of the SAN to the crista terminalis, the distance (1.2 ± 0.2 mm in the 1 day compared with 2.8 ± 0.4 mm in the 38 month animals; P = 0.005; n = 5) and conduction time (8.6 ± 0.6 ms in the 1 day compared with 17.5 ± 3.7 ms in the 38 month animals; P = 0.046; n = 5) increased with age. Sections of 14 µm were cut from SAN preparations and labelled using the antibodies anti-Cx43 (Chemicon International, USA) plus anti-IgG conjugated to FITC (Dako, Denmark). Immunofluorescence with confocal laser scanning microscopy demonstrated that anti-Cx43 labelling was absent from the centre of the SAN, increasing from 3.5 ± 0.6 mm² in the 1 month to 47.6 ± 2.0 mm² in the 38 month animals (P < 0.02; n = 5), demonstrating a 13-fold increase in area. However, there was no Cx43-negative area in the 1 day animals (n = 5). For Western blot, punch biopsy samples (4 mm diameter) were taken from the right atrial appendage and SAN centre from each animal. The paired samples were grouped by age, frozen and 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. For each sample the band density was standardised, and each data pair were expressed with the SAN as a proportion of the atrial, for each animal in all age groups. Anti-Cx43 labelled protein for 1 day (60.2 ± 2.2 %) and 1 month (56.3 ± 3.7 %) animals were not significantly different. However, anti-Cx43 labelling of 18 month animals was reduced to 15.1 ± 2.3 %, the 26 month animals to 9.2 ± 1.6 % and the 38 month animals to 5.2 ± 0.7 %, demonstrating an overall reduction of 45 % or 9-fold change (1 month to 38 months, P < 0.02; n = 5). Therefore, we have shown a correlation between ageing and the area absent of Cx43 protein expression in the centre of the SAN. This can explain the increase of the SAN conduction time with age. All mean data ± S.E.M. were analysed by Student’s unpaired t test.

This work was supported by the Medical Research Council and British Heart Foundation.

All procedures accord with current UK legislation.