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

Abstract

Human-induced pluripotent stem cell (hiPSC) and stem cell (hSC) derived cardiomyocytes (CM) are gaining popularity as in vitro model for cardiology and pharmacology studies. A remaining flaw of these cells, as shown by single-cell electrophysiological characterization, is a more depolarized resting membrane potential (RMP) compared to native CM. Most reports attribute this to a lower expression of the Kir2.1 potassium channel that generates the I K1 current. However, most RMP recordings are obtained from isolated hSC/hiPSC-CMs whereas in a more native setting these cells are interconnected with neighboring cells by connexin-based gap junctions, forming a syncytium. Hereby, these cells are electrically connected and the total pool of I K1 increases. Therefore, the input resistance (Ri) of interconnected cells is lower than that of isolated cells. During patch clamp experiments pipettes need to be well attached or sealed to the cell, which is reflected in the seal resistance (Rs), because a nonspecific ionic current can leak through this pipette-cell contact or seal and balance out small currents within the cell such as I K1 . By recording the action potential of isolated hSC-CMs and that of hSC-CMs cultured in small monolayers, we show that the RMP of hSC-CMs in monolayer is approximately −20 mV more hyperpolarized compared to isolated cells. Accordingly, adding carbenoxolone, a connexin channel blocker, isolates the cell that is patch clamped from its neighboring cells of the monolayer and depolarizes the RMP. The presented data show that the recorded RMP of hSC-CMs in a syncytium is more negative than that determined from isolated hSC/hiPSC-CMs, most likely because the active pool of Kir2.1 channels increased.

Altered Electrical, Biomolecular, and Immunologic Phenotypes in a Novel Patient-Derived Stem Cell Model of Desmoglein-2 Mutant ARVC

“…Furthermore, these cells beat spontaneously, and because of their immaturity are more depolarized at rest than adult ventricular cells, which can affect the dynamics of voltage-gated ion channels and alter excitability [ 69 ]. On the other hand, hiPSC-CMs cultured in a syncytium such as ours may possess resting transmembrane potentials more similar to adult cardiomyocytes, as compared to single cell preparations [ 70 , 71 ]. We also note that our RT-qPCR studies are limited to three biological replicates for each group.…”

Human induced pluripotent stem cell-derived cardiomyocytes as an electrophysiological model: Opportunities and challenges—The Hamburg perspective

“…There are other advantages of sharp microelectrodes to measure AP. Proper cell-to-cell coupling may be critical for the generation of a correct diastolic potential in hiPSC-CM ( Van de Sande et al, 2021 ). From a more general perspective, variability of APD is much lower when measured in intact tissue than in isolated CM measurements, because of high inter-cell variation of ion channel activity ( Lachaud et al, 2022 ).…”

“…In fact, pharmacological blockers of I Kur have gained great interest since they were believed to effectively terminate AF without the risk of life-threatening ventricular proarrhythmic effects. However, the inefficacy of I Kur block to prolong atrial refractoriness in vitro ( Du et al, 2015 ; Lemoine et al, 2018 ; Van de Sande et al, 2021 ; Lachaud et al, 2022 ) may explain the lack of antiarrhythmic efficacy in AF in patients ( Shunmugam et al, 2018 ; Camm et al, 2019 ). Therefore, any pharmacological model where I Kur block increases APD 90 could be misleading ( Devalla et al, 2015 ).…”

Morpho-functional comparison of differentiation protocols to create iPSC-derived cardiomyocytes

“…After dissociation we saw that cells obtained with both protocols showed sufficient survival (>50% of single cells attached to the cover slips were round with smooth cell membrane). For AP characterization, we performed AP recordings on a non-dissociated monolayer, as these reflect a more physiologically relevant condition ( Van de Sande et al, 2021 ). We were able to obtain AP recordings for three of the differentiated cell lines (Control 2, BrS patient 1 and 2), however their quality impacted the number of recordings which could be analyzed ( ).…”

“…patches of monolayers). This way we mimicked better the physiological conditions, where cell–cell connections are intact and connecting proteins are structurally similar to that in cardiac tissue, as opposed to an isolated cell ( Van de Sande et al, 2021 ). We checked if the obtained AP characteristics of iPSC-CMs from protocol B fall within the normal ranges, based on the AP properties described for iPSC-CMs in seven previous literature reports, where differentiation protocols and patch-clamping conditions similar to our report were used ( Jeziorowska et al, 2017 ; Bett et al, 2013 ; Gibson et al, 2014 ; Meijer van Putten et al, 2015 ; Lopez-Redondo et al, 2016 ; Herron et al, 2016 ; Feaster et al, 2015 ).…”