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-f 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-f were reduced by exposure to 10 mu M CPA. The inhibitory effect of CPA was concentration-dependent with an IC50 value of 16.3 mu M 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. Conclusions: 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 steadystate activation and the downregulation of pacemaker channel protein expression. (C) 2020 IBRO. Published by Elsevier Ltd. All rights reserved.