National Heart and Lung Institute, Imperial College London, Du Cane Road, London W12 0NN, UK.
Heart Science Centre, Magdi Yacoub Institute, Harefield Hospital, London UB9 6JH, UK.
Cardiovasc Res. 2022 Jan 7;118(1):241-253. doi: 10.1093/cvr/cvaa316.
Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN-truncating variants.
We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN-truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN-truncating variants.
Titin-truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN-truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation.
扩张型心肌病（DCM）与许多编码肌节蛋白的基因突变有关。肌联蛋白基因 TTN 的截断突变是最常见的。需要进行蛋白质组学和功能表征，以阐明疾病的起源和 TTN 截断变异的致病机制。
我们从携带截断 TTN 突变的 DCM 心脏中分离肌原纤维，并测量力的 Ca2+敏感性及其长度依赖性。还在去皮心肌细胞中同时测量力和三磷酸腺苷（ATP）消耗。使用蛋白激酶 A 和 λ 磷酸酶操纵肌钙蛋白 I（TnI）和肌球蛋白结合蛋白-C（MyBP-C）的磷酸化水平。采用 mRNA 测序来概述基因表达谱。我们发现携带 TTN 突变的肌原纤维的 Ca2+敏感性明显高于供体心脏的肌原纤维。在 DCM 肌原纤维中，TTN 截断变异的 Ca2+敏感性的长度依赖性缺失。DCM 组和供体组之间 TTN mRNA 的表达水平没有发现显著差异。TTN 外显子的使用和剪接也相似。然而，我们发现编码 Z 盘蛋白的基因下调，而在 DCM 患者中，心房特异性调节肌球蛋白轻链基因 MYL7 上调。
肌联蛋白截断突变导致肌原纤维的长度依赖性激活降低和弹性增加。在健康供体中，TnI 和 MyBP-C 的磷酸化水平对于 Ca2+敏感性的长度依赖性变化至关重要，但在 DCM 患者中，TTN 截断变异的磷酸化水平降低。Z 盘蛋白表达的降低可能解释了观察到的肌原纤维被动刚度和长度依赖性激活的降低。
