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.

<b>(A)</b> 规律间隔成簇短回文重复序列/CRISPR相关蛋白9（Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9）介导的正常人类诱导多能干细胞（human induced pluripotent stem cell，hiPSC）系中MYH7基因纯合敲除（knockout，KO）的示意图。
<b>(B)</b> MYH7敲除诱导多能干细胞起始区域的序列及色谱图（纯合子型）。
<b>(C)</b> 代表性免疫荧光显微图像显示，在MYH7敲除心肌细胞（MYH7 KO-CMs）中，即使缺乏MYH7蛋白，仍可形成肌节。
<b>(D)</b> 免疫印迹实验证实，MYH7敲除心肌细胞中成功实现MYH7敲除；且分别以15倍感染复数（multiplicity of infections，MOI）的Ad-MYH7或25倍感染复数的Ad-E848G转导3天后，各组MYH7蛋白表达量大致相当。
<b>(E)</b> 免疫荧光显微图像显示，C端FLAG标签标记的MYH7及MYH7 E848G可整合进入MYH7敲除心肌细胞的肌节中。
<b>(F, G)</b> 与分别感染50倍感染复数的绿色荧光蛋白编码腺病毒（Ad-GFP）或15倍感染复数的Ad-MYH7的MYH7敲除心肌细胞相比，感染25倍感染复数Ad-E848G的MYH7敲除心肌细胞的分数缩短率受损（克鲁斯卡尔-沃利斯检验，<i>p</i> < 0.05；事后双尾曼-惠特尼<i>U</i>检验：Ad-GFP组与Ad-E848G组间<i>p</i> < 0.05；Ad-MYH7组与Ad-E848G组间<i>p</i> < 0.05）。详见补充视频3。甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase，GAPDH）；其余缩写同图1和图2。