Figure 5: MYH7 E848G Induces Systolic Dysfunction in Isogenic Line

<b>(A)</b> 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>(B)</b> Sequence and chromatogram (homozygote) of the start region of MYH7 KO iPSC. <b>(C)</b> Representative immunofluorescent microscopic images reveal formation of sarcomeres despite the absence of MYH7 in MYH7 KO-CMs. <b>(D)</b> 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. <b>(E)</b> Immunofluorescent microscopic images show incorporation of c-term flag-tagged MYH7 and MYH7 E848G into sarcomeres of MYH7 KO-CMs. <b>(F, G)</b> 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 &lt; 0.05; post hoc 2-tailed Mann-Whitney <i>U</i> test, p &lt; 0.05 between Ad-GFP and Ad-E848G; post hoc 2-tailed Mann-Whitney <i>U</i> test, p &lt; 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.