Renewal Proposal (HE 8530/3): Uncovering the molecular and structural role of titin and MyBP-C to skeletal and cardiac muscle performance.

• Applicant: Anthony Hessel, Ph.D.
• Subject Area: Anatomy and Physiology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 454867250

Muscle contraction theories were developed under the assumption that contraction force was entirely generated by the interaction of thick and thin filament proteins within muscle cells. Later, a third filament called titin was discovered that interacts directly with thick and thin filaments as a spring, stretching with muscle stretch and producing pulling forces against the other filaments. Titin-based forces regulate fundamental contraction properties that are highly regulated in vivo by mostly unknown mechanisms. During the previous DFG award period, we used the titin cleavage (TC) mouse line and the specialized small-angle X-ray diffraction technique at Argonne National Labs (USA) to directly measure changes to skeletal and cardiac muscle filaments and contraction before and immediately after the controlled cleavage of titin filaments in otherwise healthy muscle cells in vivo. We found that titin directly controls not only force production by re-orientation of motor proteins from an OFF to ON conformation, but also force transmission by increasing filament and sarcomere stiffness. We further found that cleaving away fast-isoform myosin-binding protein C (MyBP-C) reoriented motor proteins towards ON conformation, demonstrating that both titin and MyBP-C are critical myosin motor ON/OFF state controllers. In this renewal grant, we will conduct the most logical next experiments with two aims that lean heavily on the X-ray diffraction technique. The first aim is to continue our study of TC cardiac muscle to define the relationship between titin-based force and cardiac myofilament function. The second aim is to bolster our previous work with cleavable fast-isoform MyBP-C in skeletal muscle, and use novel MyBP-C mouse models to selectively cleave the slow isoform only, or both slow- and fast-isoform MyBP-Cs in skeletal muscle, which will allow for the characterization of the different functionalities of the isoforms. This is a critical characterization, as isoforms mix within a fiber and so could alter contraction properties. I hypothesize that an immediate reduction of titin-based force via targeted titin cleavage in cardiac muscle will reduce contraction force, with associated changes to thick and thin filament proteins similar to skeletal muscle. Furthermore, I hypothesize that fast- and slow-isoform MyBP-C both control myosin motor ON/OFF states, but with fast-isoform cleavage causing more contractile dysfunction than slow-isoform MyBP-C. Importantly, we will attempt to reverse functional changes prompted by the structural cleavage of sarcomere proteins by treatment with myosin modulators. My studies will be unique in reporting a purely titin-based effect on heart muscle function and a purely MyBP-C effect on skeletal muscle function and will be relevant to the study of prevalent titin-based myopathies, computerized muscle model algorithms, and bioinspired assistive devices for movement.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Wolfgang Linke