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Studies have confirmed that apamin sensitive SK channels express abundantly on cardiac myocytes and atrioventricular node cells, play an important role in human atrial repolarization, modulate human ventricular repolarization and participate in atrioventricular nodal function. So, the roles of SKchannels in heart electrical activities can not be ignored. In the present study, we investigate the expression and the function of apamin sensitive SK channels in sinoatrial node.
We isolate single sinoatrial node cells, the cells are identified by irregular cell morphology and immunofluorescent staining with HCN4, a specific ion channel that express only on sinoatrial node cells, and perform immunofluorescent staining with SK2, SK3 to observe the location of apamin sensitive SK channels in sinoatrial node under confocal laser scanning microscope. For the detection of the protein expression level, we carry out Western Blot. The functional roles of apamin sensitive SK channels are tested by observing the effect of apamin, a specific blocker of the channels, on the intrinsic heart rate and sinus node electrogram (SNE) under the condition of blocking autonomic nervous system by atropine and propranolol. We isolate rat working heart and perfuse with Tyrode's solution(contains 1.0uM propranolol and 100nM atropine to block autonomic nervous system) and record ECG, then add apamin (final concentration is 100nM) to Tyrode's and record ECG after 5min perfusion. The control group is perfused with Tyrode's, which does not contain apamin. The SNE is recorded from isolated sinoatrial node tissue as the same manner.
The single sinoatrial node cells isolated are spindle, winding or arc-shaped and HCN4 positive. The results of immunofluorescent staining indicate that SK2 and SK3 channels express abundantly on the membrane of sinoatrial node cells, and not in plasma and nuclear. Quantification of SK2 and SK3 protein in rat sinoatrial node by Western Blot shows consistent result with immunofluorescent staining. The relative SK2 and SK3 protein level normalized to the GAPDH is 0.95±0.12 and 0.79±0.13(n=6). After blocking SK channels by 100nM apamin, the intrinsic heart rate decrease significantly compare with before apamin (before apamin: 375±23 bpm vs 350±19 bpm, p＜0.05, n=7). The data of SNE demonstrates that apamin significantly reduce the sinus rhythm (before apamin: 360±18 bpm vs 331±15 bpm, p＜0.05, n=7).
SK2 and SK3 channels express abundantly on membrane of sinoatrial node cells, and play an important role in sinoatrial node fuction.