Transverse (t)-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling and associated Ca2+ fluxes in mammalian cardiac myocytes. T-tubules were considered to be effectively absent from atrial myocytes. However, recent studies have demonstrated t-tubules in the atria of larger mammalian species (1-4). To date there have been no reports of the presence of t-tubules in pig atrial myocytes. The aim of the present study was to investigate the t-tubule network of pig atrial myocytes in comparison with ventricular cells.Animal procedures were approved by local ethics committee and conducted according to UK legislation. Atrial and ventricular tissue was obtained from 5-6 month old female Landrace White pigs (45-75 kg). Atrial cells were isolated by arterial perfusion with a collagenase-containing Tyrode's solution. Membranes of isolated cells were visualised using di-8-ANEPPS or AlexaFluor 488-conjugated wheat germ agglutinin (WGA). The spatio-temporal properties of Ca2+ transients were examined in field-stimulated (1 Hz) isolated cells loaded with fluo-4-AM (5 μM) and superfused with Tyrode's solution (pH 7.4, 22 °C). T-tubules were visualised in fixed, frozen tissue sections stained with AlexaFluor 488-conjugated WGA and AlexaFluor 633-conjugated phalloidin. Binary images were obtained by application of a threshold and t-tubule density (TTD) calculated as the percentage of bright pixels. A Gaussian function was fitted to the distribution of TTD. A distance mapping approach was used to calculate the percentage of pixels within 0.5 μm of t-tubule-like staining (%TT0.5) (2, 3). Data were analysed by Student's unpaired t-test or one or two-way ANOVA with Bonferroni post-tests. P<0.05 were considered significant.Isolated ventricular cells showed a well-developed t-tubule network whereas atrial myocytes did not. The spatio-temporal properties of the atrial Ca2+ transient were consistent with the absence of t-tubules. In tissue sections, TTD was significantly lower in atrial (2.69±0.378, n=33) compared to ventricular (7.52±0.379, n=32; P<0.0001) myocytes. However, t-tubules could be identified in 9 of 33 atrial cells. The probability distribution of TTD in atrial cells was consistent with the existence of a sub-population of cells with t-tubules (P<0.001). The %TT0.5 of atrial myocytes with t-tubules (24.0±2.50 %, n=9) was significantly greater than atrial myocytes without t-tubules (7.04±0.679 %, n=19; P<0.0001) but not different from ventricular cells (23.7±1.19 %, n=31).These data demonstrate heterogeneity in the extent of the t-tubule network in pig atrial myocytes, consistent with a previous report from the dog (4). Heterogeneity in the extent of the t-tubule network is likely to reflect heterogeneity in Ca transport and electrical activity within the atria and may thereby play a role in the origin of ectopic activity.
|Journal||Proceedings of The Physiological Society|
|Publication status||Published - 2014|