Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of NaV1.5 sodium channels

Abstract

Loss-of-function mutations of the SCN5A gene encoding for the sodium channel α-subunit Na V 1.5 result in the autosomal dominant hereditary disease Brugada Syndrome (BrS) with a high risk of sudden cardiac death in the adult. We here engineered human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRISPR/Cas9 introduced BrS-mutation p.A735V-Na V 1.5 (g.2204C > T in exon 14 of SCN5A ) as a novel model independent of patient´s genetic background. Recent studies raised concern regarding the use of hiPSC-CMs for studying adult-onset hereditary diseases due to cells’ immature phenotype. To tackle this concern, long-term cultivation of hiPSC-CMs on a stiff matrix (27–42 days) was applied to promote maturation. Patch clamp recordings of A735V mutated hiPSC-CMs revealed a substantially reduced upstroke velocity and sodium current density, a prominent rightward shift of the steady state activation curve and decelerated recovery from inactivation as compared to isogenic hiPSC-CMs controls. These observations were substantiated by a comparative study on mutant A735V-Na V 1.5 channels heterologously expressed in HEK293T cells. In contrast to mutated hiPSC-CMs, a leftward shift of sodium channel inactivation was not observed in HEK293T, emphasizing the importance of investigating mechanisms of BrS in independent systems. Overall, our approach supports hiPSC-CMs’ relevance for investigating channelopathies in a dish.

iPSC-Cardiomyocyte Models of Brugada Syndrome—Achievements, Challenges and Future Perspectives

“…In most of the published iPSC-CM BrS models, no specific maturation strategies were used, just a slightly longer culture of at least 30 days before functional testing was performed (except for [ 55 ] with at least 19 days). Only de la Roche and colleagues used the cultivation of the iPSC-CMs on a stiff matrix to improve maturation [ 56 ]. Further purification of the cell culture with the selection of properly differentiated cardiomyocytes can be obtained with metabolic enrichment approaches, including simple glucose starvation, or substitution of glucose in culture media with lactic acid or fatty acids, to force the switch to a non-oxidative metabolism in the cell culture [ 57 , 58 , 59 ].…”

“…Most of the published functional reports from iPSC-CM models, focused on sodium channel genes such as SCN5A [ 56 , 66 , 67 , 68 , 69 , 70 , 71 ], SCN10A [ 72 ] or SCN1B [ 73 ]. In addition, pathogenic variants in other BrS-related genes, such as RRAD [ 74 ] or PKP2 [ 75 , 76 ] were modelled.…”

“…Until now, ten iPSC-CM models of BrS-related SCN5A variants have been published. Eight of them were generated from patients with a pure BrS phenotype [ 56 , 66 , 67 , 68 , 69 ] and two from patients with a mixed LQTS/BrS phenotype [ 70 , 71 ] and will be discussed as such in the following paragraphs.…”

Modeling Cardiac Disease Mechanisms Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Progress, Promises and Challenges

“…The possibility to couple the versatility of iPSCs together with CRISPR-Cas9 technology in a single impactful experiment is proved to have redrawn our approach to stem cells biology and biomedical research, allowing the establishment of a powerful approach to derive isogenic lines, meaning that diseased iPSCs and the control lines are genetically matched and they only differ in the disease-causing variant. In the past few years, the application of CRISPR-Cas9 technology in disease modeling has allowed the generation of isogenic iPSC-based disease models for several cardiomyopathies and channelopathies, including dilated cardiomyopathy (DCM) [ 24 ], Barth syndrome (BTHS) [ 25 ], long QT syndrome (LQTS) [ 26 ], Brugada syndrome (BS) [ 27 ], and left ventricular non-compaction (LVNC) [ 28 ]. However, as happens for the majority of technologies involving human cells and genome manipulation, CRISPR-Cas9 technology has also raised fundamental ethical concerns.…”

hiPSCs Derived Cardiac Cells for Drug and Toxicity Screening and Disease Modeling: What Micro- Electrode-Array Analyses Can Tell Us

“…Besides the generation of disease models from patient derived cells, new gene editing technologies find application: de la Roche et al generated a model for the Brugada syndrome carrying the A735V mutation in the SCN5A gene introduced by CRISPR/Cas9 in order to be independent of the patient’s genetic background. The generated CM showed electrophysiological alteration such as decreased upstroke velocity and sodium current density [126].…”