Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C40
Influence of connexin expression and co-expression levels on action potential propagation in the HL-1 cells
P. Dias1, T. Desplantez1, N. D. Ullrich2, K. Grikscheit1, N. J. Severs1, K. T. MacLeod1, E. Dupont3
1. National Heart and Lung Institute, Imperial College London, London, United Kingdom. 2. Department of Physiology, University of Bern, Bern, Switzerland. 3. Faculty of Health and Medical Science, University of Surrey, Guildford, United Kingdom.
In the myocardium gap junctions mediate the orderly spread of current flow from cell-to-cell on which the sequential contraction of the cardiac chambers depends on. In mammalian hearts gap junction proteins connexin43 (Cx43), Cx40 and Cx45 are co-expressed in distinctive combinations and relative quantities in functionally specialised subsets of cardiac myocytes. Numerous studies in heart failure have identified a loss of connexin expression as key contributors to arrhythmias alongside factors such as membrane excitability and tissue architecture. The functional consequences of connexin co-expression in modulating action potential propagation in vivo are poorly understood. To study the relative importance of voltage-gated ion channels and gap junctions in relation to propagation velocities, clones of the original HL-1 mouse atrial myocyte tumour line were used as an in vitro cell model. The values presented are means ± standard error of the mean, compared by an unpaired t test or ANOVA. Five clones were characterised for expression of myocytic markers, calcium handling proteins and connexins. Using microelectrode arrays, two of the clones, #2 and #6, displayed large differences in conduction velocities (#2: 4±1mm/s-1, n=6 #6: 41±2mm/s-1, n=13; p<0.001). Patch clamp recordings showed similar membrane current densities in both clones for sodium (#2: -77.5±6.0pA/pF; #6: -85.0±14.3pA/pF; p>0.05), L-type (#2: -0.89±0.07pA/pF; #6: -0.86±0.04pA/pF, p>0.05) and T-type (#2: -0.94±0.04pA/pF; #6: -0.89±0.03pA/pF, p>0.05) calcium channels. However, large differences were seen in the expression levels of Cx43, Cx40 and Cx45 between the two clones. RNA interference combined with microelectrode arrays was employed in clone 6 to establish the relative importance of each connexin in impulse propagation. Knockdown of Cx40, Cx43 or Cx45 resulted in a decrease in conduction velocity but the loss in conduction was connexin dependent (37±2mm/s-1 in control n=6; 28±2mm/s-1 in Cx43 knockdown, n=8; 24±2mm/s-1 in Cx40 knockdown n=10; 16±2mm/s-1 in Cx45 knockdown, n=3; p<0.05). Simultaneous knock down of two or more connexins caused a similar and not additive decrease in conduction velocities (21±2mm/s-1 in Cx43+Cx40 knockdown, n=3; 23±1mm/s-1 in Cx43+Cx45 knockdown, n=3; 22±1mm/s-1 in Cx40+Cx45 knockdown, n=3 and 23±2mm/s-1 in Cx43+Cx40+Cx45 knockdown, n=3; p<0.05). These results indicate that electrical coupling by gap junctions is the major determinant of conduction velocities, particularly Cx45 in the HL-1 clones. Further experiments on clone 6 are currently ongoing by dual cell patch clamp to assess the total gap-junctional conductance before and after RNA interference.
Where applicable, experiments conform with Society ethical requirements