To provide further insights into the impulse propagation between cardiac myocytes, we performed multiparametric studies of excitation spread with cellular resolution in confluent monolayers of cultured cardiomyocytes (CM). Simultaneous paired intracellular recordings of action potentials in two individual
IntroductionThe cardiac conduction system, which enables fast and coordinated contraction of the heart, relies on specialized intercellular permeable junction channels, named gap junction [1,2]. Conduction abnormalities may occur either from changes in the action potential contour or from electrical uncoupling at gap junctions, or both [3,4]. However, the detailed electrophysiological understanding of this arrhythmic propensity is still unclear, because of the lack of experimental models allowing routine simultaneous monitoring of action potentials and of cell-cell electrical coupling. Experimental model are thus mandatory to get relevant insights into this kind of arrhythmogenesis. We confirmed here that cardiac muscle cells cultivated from newborn rat hearts display differentiated functional properties, and therefore represent a valuable cellular model of the myocardial excitability, as demonstrated earlier [5,6].To provide further insights into the impulse propagation between cardiac myocytes, we performed multiparametric studies of excitation spread in confluent monolayers of cultured cardiomyocytes (CM) using either simultaneous paired intracellular microelectrodes or microelectrode arrays (MEA). The section 2 describe the materials and methods used in our study, the section 3 shows some results and then the last section 4 is devoted to the conclusion and discussion.
2.Materials and methods
Cardiomyocyte culture preparationNeonatal ventricular myocytes were prepared from 1 to 4 days-old Wistar rats by trypsin-based enzymatic dispersion as described previously [7]. The cell suspension was preplated twice in Ham's F10 medium supplemented with fetal calf serum (FCS) and penicillin/streptomycin (100 U/ml) in order to increase cardiomyocyte proportion. Cardiomyocyte-rich cultures (> 90%) were seeded at a final density of 10 5 cells per cm 2 in supplemented Ham's F10 medium. Cultures were incubated in a humidified incubator (95% air, 5% CO 2 at 37 • C) and were used after 4-5 days of growth, a step at which confluent and spontaneously beating cell monolayers were obtained.
Paired intracellular recordingsFor intracellular electrophysiology, cardiomyocytes were plated onto 60 mm tissue culture dishes. CM cultures were mounted in a thermo-controlled (37 • C) bath on an inverted microscope as previously described [5]. Simultaneous paired intracellular recordings of action potentials in two individual CM spaced 100-300 µm apart were done using two independently driven conventional glass microelectrodes. The contractions were simultaneously recorded