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. 2019 May 22;10:2267. doi: 10.1038/s41467-019-09929-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 9 — Correction of K219T mutation rescues Na⁺ current phenotype and SCN5A expression. a Gene editing strategy used for the correction of the mutation. b Representative electropherograms from DNA sequencing of exon 4 of the LMNA gene showing the correction of the alanine-to-cytosine transversion at position 656 (c.656A<C) in the generated isogenic K219T-corrected iPSC lines (the reverse sequence is shown in the figure; the T<C mutation is indicated by the arrow). The nucleotide mutation corresponds to the amino acid change p.K219T. The electropherogram from the parental K219T-LMNA iPSC line is shown on the top panel as reference. c Left panel: examples of Na⁺ current (I_Na) traces recorded in CNTR- (top), K219T-corrected (top) and K219T- (bottom) CMs (Scale bar, 2 ms, 50 pA/pF). Right panel: I-V curves constructed from average peak sodium current density as a function of voltage command measured in the three conditions described above (CNTR: n = 23; K219T-corrected: n = 14; K219T: n = 22), showing significant sodium current increases in K219T-corrected cells with respect to the parental mutant CMs. *adj.p < 0.05; **adj.p < 0.005 (two-way ANOVA). d I_Na density, measured at −30 mV, in K219T-corrected CMs (176.94 ± 61.05 pA/pF) relative to K219T-CMs (91.58 ± 30.69 pA/pF) and CNTR-CMs (156.12 ± 33.73 pA/pF) expressed as a percentage. e Voltage dependences: steady state activation (CNTR: n = 23; K219T-corrected: n = 14; K219T: n = 22)/inactivation (CNTR: n = 18; K219T-corrected: n = 12; K219T: n = 17) curves. f RT-qPCR showing that SCN5A gene expression is restored to control levels in CMs generated from K219T-corrected-iPSCs. Data are represented relative to CNTR-CMs and normalized to expression of the housekeeping genes 18S and HGPRT. g ChIP against Lamin A/C, showing loss of Lamin A/C binding at the SCN5A promoter region in K219T-corrected CMs vs mutant K219T-CMs. Levels of Lamin A/C binding are instead indistinguishable from those detected in CMs differentiated from control cells. All values are reported as means ± SD. *adj.p < 0.05; **adj.p < 0.005 (one-way ANOVA), unless otherwise specified