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

Abstract

. (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 right ventricle. NCX protein expression was 17.5^3.9 pmol´mg 21 of protein in the left atrium and 29.2^6.1 pmol´mg 21 of protein in the right 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 21 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).Keywords: guinea-pig; heart; monoclonal antibody; physiology; sodium-calcium exchanger.In cardiac muscle, the Na 1 -Ca 21 exchanger (NCX) plays an important role in calcium homeostasis and hence in the control of cardiac contraction. The mAb C2C12 [1] has been used previously to detect the presence of the NCX in human heart and regional variations in the expression of the NCX have been studied qualitatively (expressed in arbitrary units of immunoblot signal intensity) in human heart using the C2C12 antibody [2]. Although NCX expression was found to be significantly lower in the atria compared to the septum, no significant differences were found between the atria and both ventricles [2]. The NCX has been located to the surface membrane and t-tubules in ventricular muscle [1,3]. However, in atrial muscle, t-tubules are sparse compared with ventricular muscle [4] and consequently it is ...

Comparison of ion channel distribution and expression in cardiomyocytes of canine pulmonary veins versus left atrium

“…The bands of interest (arrows) are seen at 160, 120 and 70 kDa. Similar NCX bands in cardiac tissue have been described in several previous studies [9,10]. McDonald et al [10] identified these three bands as containing amino acid residues 371-525 of NCX and quantified regional NCX expression by summing immunosignals from each band for a given region.…”

Myocardial Infarction Causes Increased Expression But Decreased Activity of the Myocardial Na+—Ca2+ Exchanger in the Rabbit

“…In all samples, an immunoreactive band at 120 kDa was evident, representing the mature NCX protein. Commonly, a weaker band was also observed at ≈ 70 kDa; this secondary band has been shown to be a proteolytic subfragment of the full‐length NCX (McDonald et al . 2000).…”

“…In all samples, an immunoreactive band at 120 kDa was evident, representing the mature NCX protein. Commonly, a weaker band was also observed at ~70 kDa; this secondary band has been shown to be a proteolytic subfragment of the full-length NCX (McDonald et al 2000). Analysis of band density was performed on both the 70 and 120 kDa signals and the two signals were summed to give the final NCX signal.…”

The Degree of t-System Remodeling Predicts Negative Force-Frequency Relationship and Prolonged Relaxation Time in Failing Human Myocardium

“…Probing for NCX1 yielded clear bands at 120kDa and 70kDa, as described in other studies using the same primary antibody. Because both bands have been shown to represent functional forms of NCX1, we included both in our analyses (McDonald et al, 2000). We found that the expression of Ca 2+ cycling proteins varied considerably and that the FFR did not correlate with NCX1 or SERCA2 (Figures 3B,C).…”