By Cx43 immuno-labeling of freshly isolated adult rabbit SANC, we confirmed that the average cell size (projected area) from the central area (Cx43-negative, 612.9 plus/minus 12.5 micrometers-squared, n=340) is indeed smaller than SANC from the peripheral area (Cx43-positive, 818.6 plus/minus 23.7 micrometers-squared, n=188, p<0.001), though the median values of the cell size are 571.0 and 747.5 micrometers-squared for Cx43-negative and Cx43-positive SANC, respectively, suggesting an uneven distribution for both cell originals. On top of that, the Cx43-negative SANC tend to have relatively longer perimeters, as their ratio of perimeter and projected area (0.350 plus/minus 0.005) is significantly bigger than the Cx43-positive SANC (0.300 plus/minus 0.006, p<0.001). Is there any functional relevance related to the cell origin? We did not detect any difference in AP firing rate (3.07 plus/minus 0.13 Hz, n=13 for Cx43-negative and 3.28 plus/minus 0.12 Hz, n=23 for Cx43-positive, respectively, p=0.26), though the cell size of Cx43-negative SANC (657.4 plus/minus 45.2 micrometers-squared) is smaller than Cx43-positive SANC (1020.0 plus/minus 61.0 micrometers-squared, p<0.001). In addition, there is no significant difference of AP amplitude, Maximum Diastolic Polarization or AP overshoot between Cx43-negative and Cx43-positive SANC, and no correlation was found between these amplitude-related parameters and cell size. On the other hand, we detected some substantial difference in AP characteristic parameters between Cx43-negative and Cx43-positive SANC. Cx43-negative SANC have a longer AP duration than Cx43-positive (APD50 = 93.0 plus/minus 4.0 vs. 78.0 plus/minus 2.9 ms, p<0.01; APD75 = 120.7 plus/minus 4.9 vs. 102.4 plus/minus 2.9, p<0.01), slower AP upstroke speed (dV/dtmax = 8.12 plus/minus 1.31 vs. 13.36 plus/minus 0.62 V/sec, p<0.001), but shorter linear diastolic depolarization (p<0.001). However, linear regression analysis failed to detect significant correlations between these kinetic parameters and cell size for both Cx43-negative and Cx43-positive SANC! Our data of AP-triggered global Ca2+ transient do not show significant difference of the major characteristic parameters between Cx43-negative (n=14) and Cx43-positive (n=20) SANC, though the cell size is different. In the same set of cells, spontaneous Local Ca2+ Releases (LCRs) are present beneath the cell membrane just prior to AP-induced global Ca2+ transient. No substantial difference is found in terms of LCR period, amplitude, size and duration between the two groups (n=128 or 206 for Cx43-negative or Cx43-positive SANC, respectively). Though the larger signal mass per LCR in Cx43-negative (p<0.001), when normalized by space and time, does not differ (p=0.85). Is there any difference between these two groups if the cells treated with acute beta-AR stimulation? With maximum concentration of ISO (1micromole/L), no significant difference was detected of the major characteristic parameters of APs between Cx43-negative (n=12) and Cx43-positive SANC (n=15). With the perfusion of 100nanomole/L ISO, there was no difference of the APs between Cx43-negative (n=12) and Cx43-positive (n=14). At the lower concentration of ISO (10nanomole/L) stimulation, similar results were obtained (n=12 or 15 for Cx43-negative or Cx43-positive SANC, respectively). To compare more precisely if there is any difference of the response to beta-AR stimulation between the two groups, we measured SANC beating rate without any dye or electrodes by tracking the cell edge, and performed self-control experiments with the perfusion of different concentrations of ISO (0, 0.1nanomole/L, 1nanomole/L, 10nanomole/L, 100nanomole/L, 1micromole/L and 10micromole/L). The dose-response curves were almost identical between Cx43-negative (n=10) and Cx43-positive (n=12) group, and the EC50 is 6.11nanomole/L. In addition, no difference was detected between the coefficience of variation of AP firing rate between Cx43-negative and Cx43-positive SANC at control and under ISO application. Our current results indicated that, even though the AP firing rate or Ca2+ transient cycle length or LCR period are similar, the cells from central or peripheral SAN area have some difference and the difference is not related to cell size. Is there any structural difference between the two cell types? Our immuno-labeling data suggested that the density of Sodium-Calcium exchanger along the cell membrane in Cx43-negative SANC (n=89) is relatively higher than in Cx43-positive SANC (n=148, p<0.01) and the staining pattern is similar. For Serca2 labeling, both the pattern and density are similar in Cx43-negative and Cx43-positive SANC. However, for phospholamban immune-labeling, the pattern differs between them, and the Cx43-nagative SANC (n=58) has a higher ratio of peri-nuclear to cytosolic immune-labeling than Cx43-positive SANC (n=50, p<0.001). More experiments to investigate the relationship of Ryanodine receptors and Cx43 are in the process by duo-immunolabeling. Our results indicate that although different in size, single isolated central and peripheral SANC, in the basal state or during beta-AR stimulation, do not differ from each other with respect to average spontaneous AP cycle length. Some differences are detected in the AP parameters, LCS size, protein expression and pattern. These differences merit further study.
