Dynamic clamp: a powerful tool in cardiac electrophysiology

Abstract

Dynamic clamp is a collection of closely related techniques that have been employed in cardiac electrophysiology to provide direct answers to numerous research questions regarding basic cellular mechanisms of action potential formation, action potential transfer and action potential synchronization in health and disease. Building on traditional current clamp, dynamic clamp was initially used to create virtual gap junctions between isolated myocytes. More recent applications include the embedding of a real pacemaking myocyte in a simulated network of atrial or ventricular cells and the insertion of virtual ion channels, either simulated in real time or simultaneously recorded from an expression system, into the membrane of an isolated myocyte. These applications have proven that dynamic clamp, which is characterized by the real-time evaluation and injection of simulated membrane current, is a powerful tool in cardiac electrophysiology. Here, each of the three different experimental configurations used in cardiac electrophysiology is reviewed. Also, directions are given for the implementation of dynamic clamp in the cardiac electrophysiology laboratory. With the growing interest in the application of dynamic clamp in cardiac electrophysiology, it is anticipated that dynamic clamp will also prove to be a powerful tool in basic research on biological pacemakers and in identification of specific ion channels as targets for drug development.

Figure 1. Block diagram of first dynamic clamp setup

Setup used to study the mutual synchronization of two small clusters (∼100 μm in diameter) of spontaneously active embryonic chick ventricular cells (Scott, 1979). An analog circuit is used to generate positive and negative command potentials proportional to the difference in membrane potential between the two clusters. These command potentials (V_N and −V_N) are sent to the main amplifiers to let them inject a ‘nexus current’ into each of the clusters such that these are effectively coupled by a nexus resistance R_N. The setup includes a ‘minicomputer’ (HP 2116A), but its role is limited to recording the membrane potential of each cluster. Redrawn from Fig 2–6 of Scott (1979).

Figure 2. Experimental design of dynamic clamp experiments in cardiac cellular electrophysiology

A, ‘coupling clamp’ configuration. The membrane potentials of two isolated myocytes (V_m,1 and V_m,2), both in current clamp mode, are sampled into a microcomputer (PC) and a coupling current (I_c) is computed, based on V_m,1 and V_m,2. Command potentials (V_cmd) are then sent to the patch-clamp amplifiers to inject this current into the myocytes as additional membrane current via the recording patch pipettes. The time step for updating input and output values is Δt. B, ‘model clamp’ configuration. The free-running membrane potential of a single isolated myocyte in current clamp mode (V_m) is sampled into a microcomputer (PC). An additional V_m-dependent membrane current (I_x) is computed and injected into the myocyte via the recording patch pipette. C, ‘dynamic action potential clamp’ configuration. The free-running membrane potential of a single isolated myocyte is recorded in current clamp mode and used to voltage clamp a HEK-293 cell, in which a specific ion current is expressed. This ion current (I_x) is fed back to the PC and, after on-line ‘correction’ (i.e. subtraction of endogenous background current of the HEK cell and appropriate scaling of the remaining current), injected into the myocyte as additional membrane current (I_x′).

Figure 3. Mutual synchronization of two adult pacemaker cells isolated from the rabbit sinoatrial node

The dynamic clamp technique of Fig. 2A was used to introduce an ohmic coupling conductance between the two cells, thus simulating gap junctional conductance. Membrane potential of the two cells (V_m, top) and coupling current flowing in the direction from cell 1 to cell 2 (I_c, bottom). Data from experiment 950803-2 of Verheijck et al. (1998). A, cells not coupled. B, cells coupled by 0.2 nS. C, cells coupled by 2 nS. D, cells coupled by 10 nS.

Figure 4. Effect of simulated ‘injury current’ on the human left ventricular action potential

The dynamic clamp technique of Fig. 2B was used to inject a time-varying current into an isolated human left ventricular myocyte. This current was computed as an ohmic current with a reversal potential of −40 mV and a conductance of 0, 10 or 20 nS, as indicated, and represents the ‘injury current’ that flows from normal myocardium to a depolarized ischaemic region. Data from Verkerk et al. (2000). A, superimposed action potentials of the myocyte. B, injected ‘injury current’. The myocyte was paced at 1 Hz.

Figure 5. Effect of a HERG channel mutation probed with dynamic action potential clamp

The native delayed rectifier potassium current (I_Kr) of an isolated rabbit left ventricular myocyte was blocked by E-4031 and replaced with either wild-type or R56Q mutant HERG current expressed in a HEK-293 cell using the dynamic clamp technique of Fig. 2C (Berecki et al. 2005). A, superimposed action potentials of the isolated myocyte, which was paced at 1 Hz. B, associated HERG current.