Inhibitory Effects of Cyclopiazonic Acid on the Pacemaker Current in Sinoatrial Nodal Cells.

Objective: The spontaneous action potential of isolated sinoatrial node (SAN) cells is regulated by a coupled-clock system of two clocks: the calcium clock and membrane clock. However, it remains unclear whether calcium clock inhibitors have a direct effect on the membrane clock. The purpose of this study was to investigate the direct effect of cyclopiazonic acid (CPA), a selective calcium clock inhibitor, on the function of the membrane clock of SAN cells.

Methods: at SAN cells were isolated by trypsinization and identified based on morphology and electrophysiology. I and HCN currents were recorded via patch clamp technique. The expression of the HCN channel protein was determined by Western blotting analysis.

Results: The diastolic depolarization rate of spontaneous action potentials and the current densities of I were reduced by exposure to 10 μM CPA. The inhibitory effect of CPA was concentration-dependent with an IC value of 16.3 μM and a Hill coefficient of 0.98. The effect of CPA on I current was also time-dependent, and the I current amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the I current was shifted to a negative potential, indicating that channel activation slowed down. Finally, the protein expression of HCN4 in HEK293 cells was markedly downregulated by CPA.

Conclusions: These results indicate that the direct inhibition effect of CPA on the I current in SAN cells is both concentration- and time-dependent. The underlying mechanisms may involve slowing down steady-state activation and the downregulation of pacemaker channel protein expression.