Interactions of H2O2, EGTA and Patch Pipette Recording Methods in Feline Ventricular Myocytes

Free radical generating systems produce a sequence of changes in the action potentials of isolated cardiac myocytes. Efforts to understand the nature of the alterations in ionic currents have yielded mixed results. The use of patch-pipettes for voltage-clamp experiments and manipulation of pipette solution composition may contribute to the confusion. In this study, action potentials were recorded from feline ventricular myocytes during exposure to 100 μM H2O2. Action potential changes recorded with conventional, high resistance microelectrodes occurred in three stages as previously reported. The action potential changes recorded with patch-pipettes showed increases in action potential duration, notch and plateau potentials over time. These cells did not display after depolarizations or loss of excitability as seen with conventional microelectrodes. With the patch-pipette method, untreated control cells had time-dependent changes in the action potentials. When the pipette filling solution was modified by the addition of 5 mM EGTA, the H2O2-induced changes appeared reduced but not eliminated. The initial control values of action potential parameters before exposure to H2O2 varied with the presence or absence of EGTA. These data show both direct effects of EGTA and of time on action potential parameters independent of H2O2-exposure. These artifacts of the patch-pipette method and the composition of the pipette solutions affect the sequence of changes in the action potential during exposure to H2O2.