| Abstract: | Clinical characteristics: The clinical characteristics are shown in Table 1. There were 35 men and 19 women with a mean age of 67+-12 years (range 40 to 85). Patients with AF did not differ from control subjects with SR with respect to clinical parameters that were evaluated except the diameter of left atrium, which was larger in patients with AF. Ax6 gene expression in chronic AF The difference in transcription of Ax6 between the two groups was determined by comparing the Ax6/GAPDH mRNA ratio. As shown in Figure 1, the expression level of Ax6 mRNA was significantly decreased in both RA and LA of AF patients compared to the corresponding atrium of SR patients (AF vs SR; RA: 0.21 +- 0.11 vs 0.61 +- 0.32; LA: 0.20 +- 0.10 vs 0.62 +- 0.32; both p < 0.01). In addition, in either SR or AF group, the gene expression levels were equivalent between the right and left atrium. Ax6 protein expression in chronic AF The difference in Ax6 protein expression between the two groups was determined by comparing the Ax6/s-actin ratio in Western blotting. As shown in Figure 2, the expression levels of Ax6 protein in chronically fibrillating atria were nearly half compared to SR (AF vs SR; RA: 0.18 +- 0.10 vs 0.34 +- 0.16; LA: 0.17 +- 0.09 vs 0.37 +- 0.17; both p < 0.01). In addition, similar to the transcript, the protein expression levels between RA and LA in either AF or SR group were comparable. Immunohistochemistry Single-labeling experiments showed that Ax6 protein is more or less evenly distributed in each sample. Consistent with the Western blotting results, a marked difference existed between the SR and AF groups. In general, the labels of Ax6 were abundant in the SR group, regardless of the RA or LA (Figure 3, A and B), though variations existed between samples (Figures 4B, 5B, and 6B). In contrast, in the chronically fibrillating atria the labels of Ax6 were less compared to the SR group (Figure 3, C and D). However, similar to the SR group, variations in expression also existed between samples of the AF group (Figures 4F, 5F, and 6F); in some sections of the fibrillating atria it was even difficult to identify the staining of Ax6 (Figure 3C). Since the distribution of Ax6 appeared to surround the cell borders (Figure 3), we further studied the spatial relationship of Ax6 with NCX and L-type Ca2+ channel, both of which were known to exist in the sarcolemma, and ryanodine receptor, in the sarcoplasmic reticulum. For this purpose, randomly selected samples from the SR group and samples expressing Ax6 protein detectable in the single labeling experiment from the AF group were used. In Figure 4, double immunolocalization of Ax6 (shown in red) with NCX (in green) and in Figure 5 of Ax6 (in red) with L-type Ca2+ channel (in green) demonstrated extensive overlapping (in yellow) of the Ax6 labels with those of the L-type Ca2+ channel and the NCX, respectively. In contrast, the Ax6 labels do not overlap with those of ryanodine receptor-marked sarcoplasmic reticulum in the cytoplasmic compartment (Figure 6). Comparison of laboratory data and clinical parameters: A positive correlation was found between the mRNA ratio and the protein ratio of Ax6 in each atrium for all SR and AF patients (Figure 7, A and B). On the other hand, in the multivariate analysis, the protein level of Ax6 is not influenced by age, gender, history of diabetes, hypertension, severity of coronary artery disease, previous myocardial infarction, left ventricular ejection fraction, atrial filling pressure, and use of preoperative medication(s). |
