Figure 7. REM2 variant found in severely affected individuals drives significant increase in I_CaL, but is reverted back to WT levels with CRISPR/Cas9–mediated gene correction.

(A) Single-cell RT-PCR confirmation of REM2 expression and ventricular lineage in electrophysiology-verified ventricular patient iPSC-CMs (IV-3–derived cell shown here). MLC-2v, myosin light chain 2 ventricle (ventricular lineage marker); HCN4, hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (pacemaker lineage marker). (B) Immunohistochemical confirmation of REM2 expression in an iCell ventricular cardiomyocyte. Blue, nucleus; red, REM2. Scale bar: 50 μm. (C) Macroscopic L-type Ca²⁺ currents obtained from iCell ventricular cardiomyocytes transfected with either REM2 wild type or REM2 G96A (mimicking the SNP found in severely affected hERG R752W mutation–positive individuals, III-3 and IV-3). (D) Summary data of REM2 WT overexpression (2 μg cDNA) in iCell ventricular cardiomyocytes compared with REM2 G96A. REM2 WT and REM2 G96A are composed of 3 independent experiments (REM2 WT n = 12, REM2 G96A n = 11). Relative current for REM2 WT at 0 mV, –2.21 ± –0.34, compared with REM2 G96A, –3.60 ± –0.39. *P = 0.002 as determined by unpaired Student’s t test. (E) The effects of CRISPR/Cas9–mediated correction of the REM2 G96A mutant allele back to the wild type (from A96 to G96) on APD₉₀ (macroscopic action potential record shown). (F) Summary APD₉₀ and APD₅₀ data from IV-3 compared with CRISPR-corrected IV-3. (G) Macroscopic raw traces of L-type calcium current comparing IV-3 against CRISPR-corrected IV-3. (H) Summary L-type calcium current data comparing IV-3 against CRISPR-corrected IV-3. See Supplemental Table 8 for exact numbers of replicate measures (n) and statistical analysis. Three separate CRISPR/Cas9–corrected differentiations were characterized for data shown in E–H. *Statistical significance (P < 0.05) as determined by unpaired Student’s t test in F and H.