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. 2018 Dec 31;3(6):728–740. doi: 10.1016/j.jacbts.2018.08.008

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© 2018 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

MYH7 E848G Induces Systolic Dysfunction in Isogenic Line

(A) Schematic diagram of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9 system–mediated homozygous MYH7 knockout (KO) in a normal hiPSC line. (B) Sequence and chromatogram (homozygote) of the start region of MYH7 KO iPSC. (C) Representative immunofluorescent microscopic images reveal formation of sarcomeres despite the absence of MYH7 in MYH7 KO-CMs. (D) Immunoblot confirms MYH7 KO in MYH7 KO-CMs and approximately equal amounts of MYH7 protein 3 days after transduction with either 15 multiplicity of infections (MOIs) of Ad-MYH7 or 25 MOI of Ad-E848G. (E) Immunofluorescent microscopic images show incorporation of c-term flag-tagged MYH7 and MYH7 E848G into sarcomeres of MYH7 KO-CMs. (F, G) MYH7 KO-CMs treated with 25 MOI Ad-E848G demonstrate impaired fractional shortening compared with MYH7 KO-CMs treated with 50 MOI of green fluorescent protein–encoding adenovirus (Ad-GFP) or 15 MOI of Ad-MYH7 (Kruskal-Wallis test, p < 0.05; post hoc 2-tailed Mann-Whitney U test, p < 0.05 between Ad-GFP and Ad-E848G; post hoc 2-tailed Mann-Whitney U test, p < 0.05 between Ad-MYH7 and Ad-E848G). See Supplemental Video 3. GAPDH = glyceraldehyde-3-phosphate dehydrogenase; other abbreviations as in Figures 1 and 2.

graphic file with name grv3.jpg — MYH7 E848G Induces Systolic Dysfunction in Isogenic Line

(A) Schematic diagram of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9 system–mediated homozygous MYH7 knockout (KO) in a normal hiPSC line. (B) Sequence and chromatogram (homozygote) of the start region of MYH7 KO iPSC. (C) Representative immunofluorescent microscopic images reveal formation of sarcomeres despite the absence of MYH7 in MYH7 KO-CMs. (D) Immunoblot confirms MYH7 KO in MYH7 KO-CMs and approximately equal amounts of MYH7 protein 3 days after transduction with either 15 multiplicity of infections (MOIs) of Ad-MYH7 or 25 MOI of Ad-E848G. (E) Immunofluorescent microscopic images show incorporation of c-term flag-tagged MYH7 and MYH7 E848G into sarcomeres of MYH7 KO-CMs. (F, G) MYH7 KO-CMs treated with 25 MOI Ad-E848G demonstrate impaired fractional shortening compared with MYH7 KO-CMs treated with 50 MOI of green fluorescent protein–encoding adenovirus (Ad-GFP) or 15 MOI of Ad-MYH7 (Kruskal-Wallis test, p < 0.05; post hoc 2-tailed Mann-Whitney U test, p < 0.05 between Ad-GFP and Ad-E848G; post hoc 2-tailed Mann-Whitney U test, p < 0.05 between Ad-MYH7 and Ad-E848G). See Supplemental Video 3. GAPDH = glyceraldehyde-3-phosphate dehydrogenase; other abbreviations as in Figures 1 and 2.