Pharmacological Profile of the Sodium Current in Human Stem Cell-Derived Cardiomyocytes Compares to Heterologous Nav1.5+β1 Model

Sande, Dieter V. Van de; Kopljar, Ivan; Teisman, Ard; Gallacher, David J.; Snyders, Dirk J.; Lü, Hua; Labro, Alain J.

doi:10.3389/fphar.2019.01374

Cited by 8 publications

(12 citation statements)

References 54 publications

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“…In agreement, in monolayers the upstroke velocity of hSC-CMs was significantly faster (approximately a 10-fold, p-value <0.001) compared to isolated cells (Table 1). This correlates well with the reported sodium channel properties of these hSC-CMs whereby almost all sodium channels are inactivated at potentials above −60 mV [31]. A decrease of sodium channel availability is also reflected in peak potential, which in hSC-CM monolayer amounted to 50.4 ± 2.6 mV (n = 20) compared to only 34.7 ± 1.8 mV (n = 16) in isolated cells, consequently resulting in a larger mean AP amplitude in monolayer recordings (Table 1).…”

Section: Resultssupporting

confidence: 90%

“…Since the RMP influences sodium current availability at rest, it consequently influences peak potential, AP amplitude and upstroke velocity (mV/ms) [ 30 ]. A more depolarized RMP will cause more sodium channels to be inactivated, as the V 1/2 for inactivation is around −80 mV, such that fewer channels are available to open [ 31 ]. In agreement, in monolayers the upstroke velocity of hSC-CMs was significantly faster (approximately a 10-fold, p-value <0.001) compared to isolated cells ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…In hSC-CMs in monolayer the RMP seems hyperpolarized enough to induce a fast upstroke of the AP waveform and AP properties of hSC-CMs are more comparable to native human ventricular CMs than that of isolated cells supporting their usability in pharmacological screening assays. Combined with Nav1.5 current data from previous research, the RMP needs to be at least lower than −60 mV as otherwise most Nav1.5 channels will be inactivated and consequently not available for activation [ 30 , 31 ]. In our patch clamp recordings, we only observed a fast upstroke velocity when the RMP was more hyperpolarized than −60 mV.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…After particle analysis and dilation of the nuclear objects, the resulting regions of interest were used to measure the signal intensities in the cACT on the GFP channels within 50 µM around the nuclei. User-defined thresholds were set to classify cells as being positive for either marker, calibrated to commercial hSC-CMs ( Van de Sande et al, 2019 ) as a positive control and negative fibroblast cACT expression. Efficiency is presented as percentage of cells positive for cACT.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) offer an attractive platform for cardiovascular research. Patient-specific iPSC-CMs are very useful for studying disease development, and bear potential for disease diagnostics, prognosis evaluation and development of personalized treatment. Several monolayer-based serum-free protocols have been described for the differentiation of iPSCs into cardiomyocytes, but data on their performance are scarce. In this study, we evaluated two protocols that are based on temporal modulation of the Wnt/β-catenin pathway for iPSC-CM differentiation from four iPSC lines, including two control individuals and two patients carrying an SCN5A mutation. The SCN5A gene encodes the cardiac voltage-gated sodium channel (Nav1.5) and loss-of-function mutations can cause the cardiac arrhythmia Brugada syndrome. We performed molecular characterization of the obtained iPSC-CMs by immunostaining for cardiac specific markers and by expression analysis of selected cardiac structural and ionic channel protein-encoding genes with qPCR. We also investigated cell growth morphology, contractility and survival of the iPSC-CMs after dissociation. Finally, we performed electrophysiological characterization of the cells, focusing on the action potential (AP) and calcium transient (CT) characteristics using patch-clamping and optical imaging, respectively. Based on our comprehensive morpho-functional analysis, we concluded that both tested protocols result in a high percentage of contracting CMs. Moreover, they showed acceptable survival and cell quality after dissociation (>50% of cells with a smooth cell membrane, possible to seal during patch-clamping). Both protocols generated cells presenting with typical iPSC-CM AP and CT characteristics, although one protocol (that involves sequential addition of CHIR99021 and Wnt-C59) rendered iPSC-CMs, which were more accessible for patch-clamp and calcium transient experiments and showed an expression pattern of cardiac-specific markers more similar to this observed in human heart left ventricle samples.

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“…7 Despite the benefit of heterologous expression system, it does not represent full extent of the pharmacological profile of antiarrhythmic drugs. 8,9 In mammalian ventricular myocardium eugenol reduces the isometric force developed by the papillary muscles possibly due to the blockade of L-type Ca 2+ current (I Ca,L ). 10,11 Thus, in the present study, we investigated the antiarrhythmic properties of eugenol in an ex vivo model of ouabain-induced arrhythmia and its possible involvement of I Na .…”

Section: Introductionmentioning

confidence: 99%

Eugenol delays the onset of ouabain‐induced ventricular cardiac arrhythmias in guinea pigs

Teixeira‐Fonseca,

Santos‐Miranda,

Marques

et al. 2023

Basic Clin Pharma Tox

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Eugenol is an aromatic compound used in the manufacture of medicines, perfumes, cosmetics and as an anesthetic due to the ability of the drug to block the neuronal isoform of voltage‐gated Na+ channels (NaV). Some arrhythmias are associated with gain of function in the peak sodium current (INa) found in cardiomyocytes, and antiarrhythmic sodium channel blockers are commonly used in the clinical practice. This study sought to elucidate the potential mechanisms of eugenol’s protection in the arrhythmic model of ouabain‐induced arrhythmias in guinea pig heart. Ex vivo arrhythmias were induced using 50 μmol.l−1 of ouabain. The antiarrhythmic properties of eugenol were evaluated in the ex vivo heart preparation and isolated ventricular cardiomyocytes. The compound’s effects on cardiac sodium current and action potential using the patch‐clamp technique were evaluated. In all, eugenol decreased the ex vivo cardiac arrhythmias induced by ouabain. Furthermore, eugenol showed concentration dependent effect upon INa, left‐shifted the stationary inactivation curve and delayed the recovery from inactivation of the sodium current. All these aspects are considered to be antiarrhythmic. Our findings demonstrate that eugenol has antiarrhythmic activity, which may be partially explained by the ability of eugenol to change de biophysical properties of INa of cardiomyocytes.

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Pharmacological Profile of the Sodium Current in Human Stem Cell-Derived Cardiomyocytes Compares to Heterologous Nav1.5+β1 Model

Cited by 8 publications

References 54 publications

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

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

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

Eugenol delays the onset of ouabain‐induced ventricular cardiac arrhythmias in guinea pigs

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