Liu Lian, Zhang Ran-Ran, Yang Qian, Wang Xu, Gui Yong-Hao
Department of Cardiology, Children's Hospital of Fudan University, Shanghai 201102, China.
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai 200032, China.
Sheng Li Xue Bao. 2017 Jun 25;69(3):267-275.
Cardiac troponin T (cTnT) serves as a structural protein of myocardial fiber, and participates in heart excitation-contraction coupling process. Here, we generated tnnt2a (cTnT-coding gene) deletion mutant zebrafish via CRISPR/Cas9 technique, and performed phenotypic analysis of the identified tnnt2a mutants. We observed that there was no significant difference between heterozygous mutant and wild type zebrafish, and the homozygous mutants displayed significant malformations in heart, including cardiac arrest, atrium and ventricle enlargement, pericardium effusion, and the individuals usually died before 7 day post fertilization (dpf). We further analyzed the expression alternations of heart sarcomere genes (tnnt2a, actc1a, tpm4a, myl7, vmhc) at transcriptional level in tnnt2a(Δ2) zebrafish by performing real time RT-PCR, and found that the RNA expression level of tnnt2a in tnnt2a zebrafish decreased constantly at each time point of developmental stages, and actc1a, tpm4a, myl7 and vmhc all showed higher expressions at early developmental stages and lower expressions at late developmental stages, in comparison with those of wild type zebrafish. Lastly, electron microscopy on cardiac tissues suggested that there were significant changes of the thick or thin filament structures in tnnt2a(Δ2) zebrafish, which was further confirmed by F-actin and Tpm4 immunofluorescence staining. The tnnt2a zebrafish generated by CRISPR/Cas9 bears the most common symptoms of patients with dilated cardiomyopathy, and therefore can be used as a tool to study TNNT2-deficiency related cardiomyopathy.
心肌肌钙蛋白T（cTnT）作为心肌纤维的一种结构蛋白，参与心脏兴奋-收缩偶联过程。在此，我们通过CRISPR/Cas9技术构建了tnnt2a（cTnT编码基因）缺失突变体斑马鱼，并对鉴定出的tnnt2a突变体进行了表型分析。我们观察到杂合突变体斑马鱼与野生型斑马鱼之间没有显著差异，而纯合突变体表现出明显的心脏畸形，包括心脏骤停、心房和心室扩大、心包积液，这些个体通常在受精后7天内死亡。我们通过实时RT-PCR进一步分析了tnnt2a(Δ2)斑马鱼心脏肌节基因（tnnt2a、actc1a、tpm4a、myl7、vmhc）在转录水平的表达变化，发现与野生型斑马鱼相比，tnnt2a斑马鱼中tnnt2a的RNA表达水平在发育阶段的每个时间点都持续下降，而actc1a、tpm4a、myl7和vmhc在发育早期均表现出较高的表达，在发育后期则表现出较低的表达。最后，对心脏组织的电子显微镜观察表明，tnnt2a(Δ2)斑马鱼的粗肌丝或细肌丝结构存在显著变化，F-肌动蛋白和Tpm4免疫荧光染色进一步证实了这一点。通过CRISPR/Cas9构建的tnnt2a斑马鱼具有扩张型心肌病患者最常见的症状，因此可作为研究TNNT2缺乏相关心肌病的工具。
