Single sarcomere and Structural Protein Mechanics Measurement System

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The way striated muscles contract and produce force, and so allow for human movement, is described by a theory developed in 1957 by Nobel Prize Winner AF Huxley. Huxley proposed that muscle contraction occurs through the sliding of two filamentous proteins called actin and myosin, and that this sliding is produced by small molecular motors called cross-bridges. This so-called cross-bridge theory of muscle contraction is described in textbooks of muscle physiology and is taught across the globe. This theory is excellent in describing the mechanics of contraction for muscles that either remain at a constant length (isometric contraction) or shorten (concentric contraction). However, the cross-bridge theory was never able to predict the mechanics of contraction when a muscle is stretched (a so-called eccentric contraction). Eccentric contractions occur during every movement, for example when we walk or run. In 2002, we discovered that there must be a "passive" structural element that also contributes to active force production. Using careful experiments at different structural levels, we identified that the force beyond that caused by actin and myosin comes from a structural protein called titin. However, despite this discovery 15 years ago, the molecular mechanism by which titin contributes to active force remains unknown, although ample theories have been proposed. The primary reason for this lack in progress has been because even though actin, myosin and cross-bridges can be seen using sophisticated imaging and diffraction techniques, titin does not provide a diffraction pattern, and thus cannot be seen using even the most sophisticated imaging techniques available today. Ten years ago, we proposed the theory that titin changes its inherent stiffness when a muscle is activated and does so by binding to the actin filament. Currently, this is the most accepted theory, but there is no direct experimental proof supporting the theory. In the current NSERC RTI application, we request the components for an equipment system that will allow us to probe our theory directly. In contrast to other equipment systems, the proposed system has unique features that allow for gaining unprecedented insights into the molecular interaction of actin, myosin, and titin: -It allows for mechanical testing of single sarcomeres (the smallest contractile unit of muscle) -It allows for controlled activation of any number of sarcomeres within a myofibril -It allows for measuring sarcomere mechanics with unprecedented accuracy -And it allows for visualizing titin-actin interactions in activated sarcomeres If our theory was proven correct with this new equipment system, it would fundamentally change how scientists view muscle contraction, it would produce a change in our textbooks and our teachings of muscle physiology: It would be the greatest paradigm shift in the field of muscle contraction and function since Huxley's 1957 proposal of the cross-bridge paradigm.