Grant: Research Tools and Instruments

Actin and myosin are two proteins that make up molecular motors that drive many functions in animals including cell division, ATP production, hearing, and most importantly, contractions in muscles. We have studied the molecular mechanisms of muscle contractions for the past 15 years and realized that the textbook version of muscle contraction cannot explain many experimentally observed phenomena. Specifically, when an active muscle is stretched, its force, energetics and stiffness are not explained well by the molecular motor comprised of actin and myosin. In 2002, we discovered that structural proteins, thought to play only passive roles, contribute to active force production in muscles. We identified, through molecular, sub-cellular and cellular experiments, that the sarcomeric protein titin plays a crucial role in active force production. We have preliminary evidence, that titin binds calcium upon muscle activation, thereby increasing its stiffness and force, and that titin binds to actin, thus shortening its free spring length, thereby increasing its resistance to stretch. If our theories are correct, then the proximal segments of titin are bound to actin upon muscle activation, and thus cannot be stretched in an active muscle like they are in a passive muscle.*In order to test these hypotheses and provide fundamentally new insight into the molecular mechanisms of muscle contraction, we request monies for the purchase of a unique single sarcomere and myofibril testing system that allows for simultaneous force measurements and fluorescent microscopy to measure structural changes in sarcomeric proteins during muscle contraction with nanometre and millisecond resolution.*The requested system will be assembled in house using a unique configuration, and will vastly expand our current possibilities by allowing for simultaneous measurement of muscle energetics, muscle mechanics, and the structural changes in single proteins, in situ, on the sarcomere level.*Trainees will be exposed to a variety of micro-, nano- and pico-mechanical techniques in cellular and molecular biomechanics that will be valuable beyond skeletal muscle research. Trainees will learn animal handling skills, and the harvesting of skeletal muscle cells and myofibrils. They will also learn state-of-the-art fluorescent and atomic force microscopy, fluorescent spectroscopy, and single sarcomere and myofibril physiological and mechanical testing. A crucial set of experiments will involve the measurement of stretching, unfolding and refolding of sub-segments of titin's I-band domain. Such measurements have not been performed to date and could change the fundamental textbook notion of how muscles contract and how they produce force. *In summary, the requested system is one of a kind, it will allow us to perform experiments that are currently not possible elsewhere, and will provide unique training opportunities for students in biomechanics and biomedical engineering.*