Investigating Lmod2 cardiomyopathy using human iPSC-derived cardiomyocytes

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PROJECT SUMMARY/ABSTRACT Striated muscle cell contraction is dependent on the proper overlap of myosin (thick) filaments and actin (thin) filaments. Leiomodin (Lmod) and tropomodulin (Tmod) are proteins that bind to the pointed end of thin filaments in order to fine-tune their lengths. Mutations in these proteins have been shown to result in dysregulated thin filament lengths, sarcomere disassembly and the development of cardiomyopathies. Yet, the mechanism for how they contribute to this disease process is largely unknown. Recently, the first pathogenic mutation in Lmod2 was identified in a human. It was discovered that a newborn patient had a homozygous nonsense mutation in LMOD2 (c.1193G>A, p.Trp398*), which is predicted to result in a substantially truncated protein. Clinically, the patient presented with cardiac abnormalities at birth and received a heart transplant at 10 months of age. Explanted heart tissue confirmed the diagnosis of dilated cardiomyopathy. The main objective of this research proposal is to understand the consequences of this mutation on the structure and function of the heart, with the long-term goal of elucidating potential therapeutic options for Lmod2-mediated cardiac dysfunction. To do this, various experimental approaches will be utilized in vitro and in vivo to address the hypothesis that mutations in Lmod2 result in cardiac dysfunction and alterations in sarcomere structure, due to dysregulation of actin-thin filaments. In order to properly decipher the cardiac effects of this human nonsense mutation, two well-established model systems will be used: (1) human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the patient and (2) a novel CRISPR designed knock-in mouse model harbouring the same mutation as the patient. From these two systems, alterations in the expression, structure and functional properties of Lmod2 will be deduced through the following biochemical analyses and functional assays: First, the subcellular structure of the sarcomere will be analyzed and actin- thin filament lengths measured using immunocytochemistry from the patient's iPSC-CMs and CRISPR/Cas9 gene edited isogenic controls. Second, calcium and voltage sensitive fluorescent probes will provide information on intracellular calcium mobilization and changes in single cell electrical recordings, respectively. In addition RNA sequencing will give insight into the effects of the Lmod2 p.Trp398* mutation on sarcomeric transcriptome networks. Third, excised heart tissue from mutant mice will be used to study sarcomere architecture via immunohistochemistry and force/Ca2+ relationships via isolated single-fiber mechanics. Understanding how actin filament assembly is regulated is of broad interest since actin is the most abundant protein in many cell types and is involved in numerous essential cellular processes. The results obtained from this multidisciplinary project will likely decipher how a single mutation in Lmod2 can lead to human cardiomyopathy. It will also broaden our knowledge about actin filament structure and assembly dynamics, which are predicted to have implications beyond cardiac muscle.