The resting membrane potential of hSC-CM in a syncytium is more hyperpolarised than that of isolated cells

Figures

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  Figure 1.. Schematic representation of the patch clamp technique and corresponding resistances. (a) Simplified overview of the patch clamp pipette attachment to the isolated cell (ruptured whole-cell configuration) is shown. Kir2.1 channels are represented in yellow and in blue the hemi-channels with no connection to other cells, leading to a high Rmem. A circuit diagram of the different resistances is shown on the right with Re, Rs, Ra and Rmem representing the electrode (pipette), seal, access and cell (or membrane) resistances, respectively. The leak current (I-leak), originating from the Rs, can counter the currents of the cell (I-cell). (b) Overview of patching a cell in a monolayer whereby cells are connected by gap junction channels (composed of two hemichannels from neighboring cells). The electrical and chemical connectivity between cells is indicated by the red arrows, which leads to a lower Rmem compared to isolated cells (panel a)

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  Figure 2.. Methodology of Ri calculation. (a) Circuit diagram of the resistances in the patched cell configuration. Electrode resistance (Re) stands in series with the patched cell, whereby the Rs stands in parallel with the Ra and the Rmem, which together form the input resistance Ri. (b) Ri can be calculated, in current clamp mode, from the difference in potential (ΔV) after a current injection (−0.05 nA injected), a representative current recording is shown on the left. Using Ohm’s law and taking into account the circuit represented in panel a, Ri can be calculated as shown on the right

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  Figure 3.. Representative and non-representative ventricular AP phenotype. (a) An example AP waveform of a cell with representative ventricular phenotype where the RMP is close to −80 mV and a fast upstroke velocity is observed. The insets at the bottom, on the left a magnification of the depolarization phase of the AP is shown with on the right the first differential of this depolarization phase that represents the dV/dtmax of the AP. (b) An example of a cell with a non-representative ventricular phenotype is represented, displaying a RMP more depolarized than −80 mV and a slow upstroke velocity. Magnification of the depolarization phase and the first differential of it are shown below. (c) Same cell as represented in panel b but now displaying the elicited AP upon a negative current injection (−0.05 nA). By the negative current injection the RMP was brought toward −80 mV. Subsequent induction of an AP leads to a fast upstroke velocity, which indicates that the cell is not a nodal type hSC-CM but a ventricular type cell with a more depolarized RMP

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  Figure 4.. Correlation between RMP, AP duration and upstroke velocity properties in monolayer and isolated hSC-CMs. (a) The RMP of monolayer cells (black triangles) and isolated cells (inverted gray triangles) are plotted against their respective APD90. It is observed that the RMP in monolayer cells is more hyperpolarised compared to the isolated cells and they have a lower variability in APD90 values. Each dot represents the mean of 5 AP´s recorded within one cell. (b) Plot shows the relation between maximum upstroke velocity (mV/ms), obtained by taking the maximum of the first differential of the depolarization phase of the AP, and APD90 (ms). (c) Maximum upstroke velocity is plotted against the respective RMP. Note that when the RMP is more depolarized than −60 mV no adequate upstroke velocity was obtained

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  Figure 5.. Seal and input resistance can modify RMP recordings from monolayer and isolated cells: (a) The seal resistance Rs of patched hSC-CMs in monolayer (black triangles) and isolated hSC-CMs (gray inverted triangles) is plotted against the RMP. Represented values are the mean of 5 APs recorded from the same cell. (b) RMP was plotted over their respective Ri which visualizes that for isolated cells the RMP is more depolarized when Ri is increased. (c) Plotting the ratio of Ri/Rs over RMP for monolayer and isolated cells clearly states that, mostly, when Rs/Ri ratio is >30 the RMP is more depolarized. Note that the Rs/Ri ratio is plotted as a log scale

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  Figure 6.. Effect of Cbx on the AP of hSC-CMs in monolayer. The RMP, peak, APD90 and maximal upstroke velocity of the AP from hSC-CMs in monolayer are represented for control condition (normal ECS) and upon 5 minutes wash-in of 100 µM or 200 µM Cbx. Note that these experiments were performed on a different batch of hSC-CMs, explaining the slight differences in values compared to the data in table 1, due to batch-to-batch variability in hSC-CMs

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  Figure 7.. Rs and Ri influence the reversal potential of the Kir2.1 channel. (a) Representative current traces of an adequate CHO cell that expresses sufficient Kir2.1. The red line indicates the zero level. The voltage pulse protocol to elicit the currents is shown below. On the right is the current density (pA/pF) plotted to their respective potentials, generating an IV plot, with a magnification of the positive current around −80 mV. Note that the current density was determined without performing a leak subtraction on the recording. (b) Example traces of an inadequate CHO cell, using the same voltage clamp protocol, expressing low levels of Kir2.1 with the red line indicating the zero level. On the right is the current density (pA/pF) plotted to their respective potentials with a magnification of the negative current until 0 mV