Department of Bioengineering (R.J.Z., J.M., D.-H.K., M.R., C.E.M.), University of Washington, Seattle.
Center for Cardiovascular Biology (R.J.Z., A.N.A., K.B., A.L., P.A.F., L.P., H.R., X.Y., J.M., D.-H.K., N.J.S., M.R., C.E.M.), University of Washington, Seattle.
Circulation. 2019 Nov 12;140(20):1647-1660. doi: 10.1161/CIRCULATIONAHA.119.039521. Epub 2019 Oct 7.
The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin () are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function.
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 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.
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.
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.
巨大的肌节蛋白 titin 对于心脏健康和疾病都非常重要。编码 titin 的基因突变是家族性扩张型心肌病的主要已知原因。这些突变在基因中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同工型有更全面的了解。
为了研究 titin 在人类 CMs 中的功能，我们使用 CRISPR/Cas9 在人类诱导多能干细胞中生成 Z 盘（TTN-Z）和 A 带（TTN-A）区域的基因的纯合截断。通过免疫染色、工程化心脏组织力学测量以及单细胞力和钙测量对产生的 CMs 进行了表征。
分化后，我们惊讶地发现，尽管突变更上游，TTN-Z-CMs 仍具有肌节并可见收缩，而 TTN-A-CMs 则没有。我们假设肌节形成是由 titin 的一种最近发现的同工型 Cronos 引起的，该同工型在 TTN-Z-CMs 中的截断下游开始表达。使用定制的 Cronos 抗体，我们证明该同工型在人类 CMs 中表达并整合到肌原纤维中。TTN-Z-CMs 仅表达 Cronos titin，但与表达全长和 Cronos titin 的对照细胞相比，这些细胞产生的收缩力较低，并且肌原纤维束排列紊乱。Cronos titin 在人类胎儿心脏组织中高度表达，并且在人类诱导多能干细胞衍生的 CMs 中敲除时，尽管存在全长 titin，这些细胞仍表现出收缩力降低和肌原纤维排列紊乱。
我们证明 Cronos titin 在发育中的人类 CMs 中表达，并且能够在没有全长 titin 的情况下支持部分肌节形成。此外，Cronos titin 对于人类诱导多能干细胞衍生的 CMs 中的肌节功能正常是必需的。需要进一步研究以了解这种新型同工型的分子机制以及它如何导致人类心脏疾病。
