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

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.

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

The diastolic depolarization rate of spontaneous action potentials and the current densities of I_f were reduced by exposure to 10 μM CPA. The inhibitory effect of CPA was concentration-dependent with an IC₅₀ value of 16.3 μM and a Hill coefficient of 0.98. The effect of CPA on I_f current was also time-dependent, and the I_f current amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the I_f 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.

These results indicate that the direct inhibition effect of CPA on the I_f 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.