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

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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.