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Na_V1.5 knockout in iPSCs: a novel approach to study Na_V1.5 variants in a human cardiomyocyte environment.

ABSTRACT: Cardiomyocytes derived from patient-specific induced pluripotent stem cells (iPSC-CMs) successfully reproduce the mechanisms of several channelopathies. However, this approach involve cell reprogramming from somatic tissue biopsies or genomic editing in healthy iPSCs for every mutation found and to be investigated. We aim to knockout (KO) Na_V1.5, the cardiac sodium channel, in a healthy human iPSC line, characterize the model and then, use it to express variants of Na_V1.5. We develop a homozygous Na_V1.5 KO iPSC line able to differentiate into cardiomyocytes with CRISPR/Cas9 tool. The Na_V1.5 KO iPSC-CMs exhibited an organized contractile apparatus, spontaneous contractile activity, and electrophysiological recordings confirmed the major reduction in total Na⁺ currents. The action potentials (APs) exhibited a reduction in their amplitude and in their maximal rate of rise. Voltage optical mapping recordings revealed that the conduction velocity Ca²⁺ transient waves propagation velocities were slow. A wild-type (WT) Na_V1.5 channel expressed by transient transfection in the KO iPSC-CMs restored Na⁺ channel expression and AP properties. The expression of Na_V1.5/delQKP, a long QT type 3 (LQT3) variant, in the Na_V1.5 KO iPSC-CMs showed that dysfunctional Na⁺ channels exhibited a persistent Na⁺ current and caused prolonged AP duration that led to arrhythmic events, characteristics of LQT3.

SUBMITTER: Pierre M

PROVIDER: S-EPMC8387439 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RationaleThe cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined.ObjectiveTo characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria.Methods and resultsThrough a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A.ConclusionsWe describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.

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Na_V1.5 knockout in iPSCs: a novel approach to study Na_V1.5 variants in a human cardiomyocyte environment.

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Dataset Information

NaV1.5 knockout in iPSCs: a novel approach to study NaV1.5 variants in a human cardiomyocyte environment.

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OmicsDI is part of the ELIXIR infrastructure

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Na_V1.5 knockout in iPSCs: a novel approach to study Na_V1.5 variants in a human cardiomyocyte environment.