Titin-based stiffness regulation and mechanosensing in activated skeletal muscle.

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Titin is the third myofilament of skeletal muscle where it spans the I-band and A-band regions of the sarcomere. Multiple titin mutations have been described that result in debilitating myopathies, highlighting titin's importance in skeletal muscle and the need to understand all of titin's functions fully. Our current understanding of titin is largely based on studying passive skeletal muscle and assuming that no established properties change when skeletal muscle is activated. However, recent studies suggest that titin's I-band segment interacts with the thin filament in contracting or diseased muscle, altering titin's extensibility from that in passive muscle and impacting passive force and thick filament activation. Possible thin filament interaction sites are the PEVK element and the N2A of skeletal muscle, the latter is part of a recently discovered novel stiffness regulation mechanism that involves MARP1, a stress response protein. Using mouse models aims 1 and 2 focus on the roles of the N2A and PEVK elements in regulating titin stiffness in skeletal muscle, including the effects of upregulating MARP. We also study the role of titin in activating the thick filament in skeletal muscle. Important work in the myosin field has shown that muscle activation requires thin filament activation (as is well-known) and thick filament activation mechanisms (a more recent discovery). In relaxed skeletal muscle, myosin is either in the super-relaxed (SRX) state or the disordered-relaxed (DRX) state. The conversion of SRX to DRX turns thick filaments ON, promoting contraction. Several mechanisms have been proposed to regulate the ON state of the skeletal muscle thick filament, including a mechano-sensing mechanism that involves thick filament strain. We have previously obtained evidence that titin-based passive force strains the skeletal muscle thick filament. Aim three will test the hypothesis that this converts SRX to DRX myosin in skeletal muscle and switches the thick filament from OFF to ON. High-resolution ATP turnover assays have revealed that although the SRX state occurs in each of the A- band regions of skeletal muscle (the D-zone, C-zone, and P-zone), the C-zone has the highest level. In addition to titin, the C-zone contains MyBP-C. Aim 4 will study the importance of each in SRX. It will also address the effect of locally perturbing titin strain (by deleting single C-zone domains) on SRX in skeletal muscle. This work has high novelty and addresses fundamental questions that have clinical relevance. All required models and tools are available, an experienced team of collaborators is in place, and extensive pilot data support the guiding hypotheses of the proposed research. This proposal is a significant step towards our long-term goal, which is to gain a detailed understanding of the roles of titin in both passive and active skeletal muscle and contribute to our understanding of the mechanistic basis of skeletal muscle disease.