The Dynamic Clamp Induced Pacing in Single and Coupled Cells

The dynamic clamp is a method whereby an arbitrary current, a function of time and membrane potential, is applied to a preparation to create hybrid circuits of real and virtual cells.We tested the ability to add electronically two membrane currents, the inward rectifier current (IK1) and the pacemaker current (If), which contribute to myocyte pacemaker activity. Injection of computer-simulated If in addition to IK1 under current-clamp conditions into an isolated canine ventricle myocyte and/or HEK293 cell stably expressing SCN5a initiated pacing activity. The pacing rate and action-potential (AP) duration were modified by changing If and IK1 amplitudes.We also examined whether coupling of a source cell with If and IK1 to another cell creates a two-cell pacing unit. HEK293 cells were co-cultured either with HEK293/SCN5a cells or acutely isolated adult canine ventricular myocytes. Injecting If and IK1 currents into one cell of a pair generated sustained pacing in a coupled ventricular myocyte or HEK293/SCN5a cell once a critical level of electrical cell-cell coupling between the If+IK1 source cell and the neighboring cell was exceeded. Application of the gap-junction blocker carbenoxolone stopped pacing activity in the coupled myocyte and/or HEK293/SCN5a cell; washout of carbenoxolone restored activity. Hence, the currents from a If+IK1-injected cell delivered to a cardiac myocyte (or another cell type) via gap junctions can generate spontaneous APs allowing the cell pair to function as a pacemaker unit.Pacing activity also was investigated artificially by electrically connecting two separate single cells via a dual-cell dynamic clamp, permitting varying of coupling conductance while modulating If and/or IK1 within either or both of the two coupled cells.These results demonstrate that the dynamic clamp can be used to study the determinants of pacemaker activity.Supported by HL28958, GM88180, GM088181.