Action potential propagation failures in long-term recordings from embryonic stem cell-derived cardiomyocytes in tissue culture.

Three-dimensional cell aggregates (embryoid bodies, EBs) containing clusters of spontaneously beating cardiomyocytes were derived from permanent mouse embryonic stem (ES) cells. Extracellular recordings of the population action potentials of cardiomyocyte clusters were made using permanently mounted silver wire electrodes and microelectrode arrays integrated into the bottom of the culture dish. These techniques allowed long-term recordings (for up to several weeks) from individual EBs under cell culture conditions. The normal electrical activity consisted of regular spiking with a frequency of 0.5-5 Hz. However, most EBs (87%) spontaneously developed temporary or persistent complex activity patterns because of intermittent block of action potential propagation at narrow pathways connecting larger beating areas. Similar propagation blocks could also be reversibly induced in regularly spiking EBs by nimodipine (NDP). In addition to a slowing of pacemaker activity, NDP (20-200 nM) induced a stepwise decrease of the action potential frequency at the recording site. Perforated patch-clamp recordings from enzymatically isolated ES-cell-derived cardiomyocytes showed that similar activity patterns do not occur at the single-cell level. We suggest that this novel approach may provide a useful tool for in vitro studies of chronotropy and phenomena of propagation failure similar to AV block.

Role of ATP-dependent K(+) channels in the electrical excitability of early embryonic stem cell-derived cardiomyocytes.

Single, murine embryonic stem cell-derived early stage cardiomyocytes dissociated from embryoid bodies expressed two inward rectifier K(+) channels, I(K1) and the ATP dependent K(+) current. I(K1) exhibited low density in early stage cardiomyocytes, but increased significantly in late stage cells. In contrast, the ATP dependent K(+) current was expressed at similar densities in early and late stage cardiomyocytes. This current was found to be involved in the determination of the membrane potential, since glibenclamide depolarized early cardiomyocytes and exerted a positive chronotropic effect. Some cardiomyocytes displayed a bursting behavior of action potentials, characterized by alternating periods with and without action potentials. During the phases without action potentials, the membrane potential was hyperpolarized, indicating the involvement of K(+) channels in the generation of this bursting behavior. Extracellular recording techniques were applied to spontaneously contracting areas of whole embryoid bodies. In 20% of these bursting behavior similar to that seen in the single cells was observed. In regularly beating embryoid bodies, bursting could be induced by reduction of substrates from the extracellular medium as well as by superfusion with the positive chronotropic agents Bay K 8644 or isoproterenol. Perfusion with substrate-reduced medium induced bursting behavior after a short latency, isoproterenol and Bay K 8644 resulted in a positive chronotropic response followed by bursting behavior with longer latencies. The spontaneous bursting was blocked by glibenclamide. These experimental results suggest that intermittent activation of ATP dependent K(+) channels underlies the bursting behavior observed in single cardiomyocytes and in the whole embryoid body. Conditions of metabolic stress lead to the rhythmic suppression of action potential generation. Our data indicate that ATP dependent K(+) channels play a prominent role in the cellular excitability of early cardiomyocytes.