Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function
Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function
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DOI：
10.1161/circulationaha.119.039521
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发表时间：
2019-11-12
期刊：
影响因子：
37.8
通讯作者：
Murry, Charles E.
中科院分区：
文献类型：
--
作者：
Zaunbrecher, Rebecca J.;Abel, Ashley N.;Murry, Charles E.
背景：巨肌节蛋白肌联蛋白在心脏健康和疾病中都很重要。编码肌联蛋白（TTN）的基因突变是家族性扩张型心肌病的主要已知原因。TTN中这些突变的不均匀分布促使我们寻求对该基因及其在心肌细胞（CM）肌节形成和功能中编码的亚型的更完整的理解。研究方法：为了研究肌联蛋白在人CM中的功能，我们使用CRISPR/Cas9在人诱导多能干细胞中TTN基因的Z盘（TTN-Z（-/-））和A带（TTN-A（-/-））区域中产生纯合截短。用免疫染色、工程化心脏组织机械测量以及单细胞力和钙测量表征所得CM。结果如下：分化后，我们惊讶地发现，尽管上游突变较多，TTN-Z（-/-）-CM仍具有肌节并明显收缩，而TTN-A（-/-）-CM则没有。我们假设肌节的形成是由最近发现的肌联蛋白Cronos亚型的表达引起的，Cronos起始于TTN-Z（-/-）-CM的截短下游。使用定制的Cronos抗体，我们证明了这种亚型在人CM中表达并整合到肌原纤维中。TTN-Z（-/-）-CM仅表达Cronos肌联蛋白，但与表达全长和Cronos肌联蛋白的对照相比，这些细胞产生较低的收缩力，并且具有扰动的肌原纤维集束。Cronos肌联蛋白在人胎儿心脏组织中高度表达，并且当在人诱导多能干细胞衍生的CM中敲除时，尽管存在全长肌联蛋白，但这些细胞表现出收缩力降低和肌原纤维紊乱。结论：我们证明，Cronos肌联蛋白在发展中的人类CM中表达，并能够支持部分肌节形成的情况下，全长肌联蛋白。此外，Cronos肌联蛋白对于人诱导多能干细胞衍生的CM中的适当肌节功能是必需的。进一步的研究是必要的，以了解这种新的异构体的分子机制，以及它如何有助于人类心脏疾病。
Background: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin (TTN) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function. Methods: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z(-/-)) and A-band (TTN-A(-/-)) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements. Results: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z(-/-)-CMs had sarcomeres and visibly contracted, whereas TTN-A(-/-)-CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z(-/-)-CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z(-/-)-CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin. Conclusions: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.