Titin in Skeletal Muscle Health and Disease

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There is a rapidly increasing awareness of the importance of titin in causing neuromuscular disorders (titinopathies), two of which we focus on in this work, centronuclear myopathy (CNM) and hereditary myopathy with early respiratory failure (HMERF). Titinopathies frequently present early-onset muscle weakness and respiratory difficulty but understanding their underlying mechanisms is held back by our still limited understanding of the functional roles of titin in skeletal muscle. Titin comprises the third myofilament of muscle and spans along the sarcomere, from Z-disk to M-band. Titin’s I-band region functions as a molecular spring that generates passive stiffness with recent studies indicating that passive stiffness affects active force at sub- maximal activation levels. However, it is not known how important titin’s stiffness is to overall skeletal muscle health and if altered stiffness can cause a myopathy. That the importance might be high is suggested by our pilot studies that reveal deranged titin stiffness and reduced active tensions in titinopathy patients with CNM. Aims 1 and 2 address how altering titin-based stiffness affects both passive and active muscle properties and if it can be disease-causing. We will perform mechanical studies on biopsies from titinopathy patients (focus on CNM) as well as study two contrasting mouse models in which the stiffness of titin’s spring region is either increased (PEVK truncation) or reduced (inactivation of Rbm20). The working hypothesis is that altering titin’s spring region alters both passive muscle stiffness and active tension and that this causes myopathy. Aim 2 also studies a novel mouse model that mimics CNM with splice site mutations in titin’s spring region and we will examine the functional consequences at the RNA, protein, structural and functional levels. We also study the A-band segment of titin, specifically the C-zone. This zone has not been studied, it is clinically important as this is where a large number of disease-causing mutations are found. We investigate in Aim 3 a mutation in the C-zone exon 343 which causes HMERF, a myopathy with respiratory muscle involvement that can be fatal. Using a novel HMERF mouse that we made the mechanistic basis of the disease will be studied. Through excision of the mutated titin exon 343, the therapeutic potential of exon skipping for treating HMERF will be tested. With its basic science and translational goals and its in-depth and integrative approach, this application seeks to continue our track record of cutting-edge titin research. Powerful techniques and novel mouse models are in place, pilot data support the guiding hypotheses, and our research team is highly experienced. The proposal will greatly enhance insights in titin biology, titin’s role in muscle disease, and titin’s potential as a therapeutic target.