Quantitative analysis of Na+-Ca2+ exchanger expression in guinea-pig heart

Abstract

In previous studies, regional variations in the expression of the Na+-Ca2+ exchanger (NCX) have been examined qualitatively in human heart using the C2C12 monoclonal antibody [Wang, J., Schwinger, R.H., Frank, K., Muller-Ehmsen, J., Martin-Vasallo, P., Pressley, T.A., Xiang, A., Erdmann, E. and McDonough, A.A. (1996) J. Clin. Invest. 98, 1650-1658]. Although NCX expression was found to be significantly lower in the atria compared to the septum, no significant differences were found between atrial and ventricular tissue. NCX has been located in the general sarcolemma and t-tubules of ventricular muscle and as t-tubules are sparse in atrial tissue compared to ventricular tissue, it is surprising that NCX expression was found to be similar in both atria and ventricles [Wang et al. (1996)]. To reinvestigate this, we have used SDS/PAGE and a quantitative Western blotting technique to determine the pattern of expression of NCX in guinea-pig heart in tissue samples from left atrium, right atrium, septum, left ventricle and fight ventricle. NCX protein expression was 17.5 ± 3.9 pmol·mg-1 of protein in the left atrium and 29.2 ± 6.1 pmol·mg-1 of protein in the fight atrium, which were both significantly lower (P < 0.05) than NCX expression in the septum, left ventricle and right ventricle (64.7 ± 15.2, 76.8 ± 19.5 and 69.4 ± 14.1 pmol·mg-1 of protein, respectively, n = 7). These differences in NCX expression may reflect variations in the cellular location of NCX protein in these regions. To study this, we used confocal immunofluorescence of single isolated myocytes to examine differences in the proportion of fluorescent staining on the general surface membrane compared with the interior of the cell (which presumably reflects a t-tubular location). We found that the general membrane staining was 79.0 ± 1.2% in cells from the atria which was significantly higher (P < 0.001) than that seen in cells from the septum, left ventricle and right ventricle, with 48.1 ± 1.1%, 48.2 ± 1.8% and 45.6 ± 1.3%, respectively (n = 20). These results illustrate a similar pattern of NCX expression in guinea-pig and human, with expression in atrial tissue significantly lower than in ventricular tissue. However, the cellular location of NCX differs regionally; in atrial tissue, the majority of the NCX protein is located in the general sarcolemma whereas in ventricular and septal tissue, approximately 50% of NCX protein is located within the cell (presumably at the level of the t-tubules).