Single-molecule to sarcomere level investigation of human ventricular light chain 1 mutations that cause hypertrophic cardiomyopathy

在遗传性心脏病中，肥厚型心肌病（HCM）是人类收缩功能障碍和心力衰竭的最常见原因。心室β-心肌肌球蛋白II（β-CM）运动复合体（由β-肌球蛋白重链β-MyHC和轻链MLC 1v和MLC 2 v组成）中的单点突变占HCM病例的约40%。对于β-CM成分中的HCM突变，我们假设突变改变了个体运动特性，这是HCM表型发展的初始触发因素。因此，可以想象的是，了解肌球蛋白功能的主要变化的确切细节和选择性纠正突变型马达分子中的这些改变可能有助于预防症状性HCM表型的发展。人心室必需轻链MLC 1v中的点突变与引起严重形式的HCM有关。一些MLC 1v突变甚至与年轻时的心脏性猝死（SCD）和儿童期的HCM有关，从而突出了ELC在肌球蛋白功能中的关键作用。我们提出的研究的主要目标是了解导致患者HCM的原发性运动功能障碍。我们的目标是获得一个全面的了解改变电机特性的结果，一个新的错义突变MLC 1v（A57 D），和其他已知的突变（A57 G，E143 K，M149 V）与严重形式的HCM，包括SCD。我们所提出的工作的三个主要特征通过提供生理学相关的框架在人类心肌病研究中提供了显著的进展：1）使用完整的人源β-CM马达复合物或全酶，并通过用特定的突变ELC重建β-CM马达来产生突变马达。2)使用最先进的方法，如光学捕获和全内反射荧光（TIRF）显微镜，单分子功能分析的突变电机，促进电机的生化和机械功能的高时空分辨率，即使是细微的变化的识别。3)利用携带突变马达的人心肌肌原纤维测定肌节水平的收缩特性。通过在其他健康或野生型肌原纤维背景中重建突变的MLC 1v，可以鉴定纯粹的突变特异性效应。该实验设计有望揭示心肌细胞中肌原纤维水平变化的初始触发因素。这些研究将提供有关受损特征的精确细节、运动功能障碍的程度及其在不同分子组织尺度上的影响的信息。此外，这些突变使我们能够获得结构-功能关系的基本知识。重要的是，在电机的特定的生化或机械性能的改变可能阐明的潜在机制MLC 1v介导的触发HCM病理，从而铺平了道路，最终的治疗策略。

Among inherited cardiac diseases, hypertrophic cardiomyopathy (HCM) is the most frequent cause of contractile dysfunction and heart failure in humans. Single point mutations in the ventricular β-cardiac myosin II (β-CM) motor complex (composed of the β-myosin heavy chain, β-MyHC, and light chains, MLC1v and MLC2v) account for  40% of HCM cases. For HCM mutations in the β-CM components, we hypothesize that mutation alters the individual motor properties, which act as an initial trigger for the development of the HCM phenotype. Thus, it is conceivable that knowledge of the precise details of primary changes in myosin’s function and selective correction of these alterations in mutant motor molecules may help in preventing the development of the symptomatic HCM phenotype. Point mutations in the human ventricular essential light chain, MLC1v, are implicated in causing severe forms of HCM. Some of the MLC1v mutations are even linked to sudden cardiac death (SCD) at a young age and HCM in childhood, thus highlighting the critical role of ELC in myosin function. The main goal of our proposed research is to understand the primary motor dysfunction that led to the HCM in the patient. We aim to gain a comprehensive understanding of the altered motor characteristics as a consequence of a novel missense mutation in the MLC1v (A57D), and other known mutations (A57G, E143K, M149V) that are associated with serious forms of HCM, including SCD. Three main features of our proposed work offer a significant advance in human cardiomyopathy research by providing a physiologically relevant framework: 1) Use of the complete β-CM motor complex or holoenzyme of human origin and generation of mutant motors by reconstitution of the β-CM motor with specific mutant ELCs. 2) Single-molecule function analysis of the mutant motors using state-of-the-art methods, such as optical trapping and total internal reflection fluorescence (TIRF) microscopy, facilitating the identification of even subtle alterations in the motor’s biochemical and mechanical features with high spatiotemporal resolution. 3) Determining sarcomere-level contractile properties by employing human cardiac myofibrils bearing mutated motors. By reconstituting the mutated MLC1v in otherwise healthy or wild-type myofibril background, purely mutation-specific effects can be identified. This experimental design is expected to reveal the initial trigger for myofibril level changes in the cardiomyocytes. These studies will provide information on the precise details of impaired features, the extent of motor dysfunction, and their impact at different scales of molecular organization. Furthermore, these mutations allow us to gain basic knowledge of the structure-function relationships. Importantly, alteration in the motor’s specific biochemical or -mechanical properties may elucidate the underlying mechanism of MLC1v-mediated trigger for HCM pathology, thereby paving the way toward definitive therapeutic strategies.