Grant: Research Tools and Instruments

The requested equipment consists of a mechanical testing system for single skeletal muscle myofibrils and single sarcomeres, combined with a cryo-preservation system that allows for fixing sarcomeres "instantaneously" at different contractile states for high resolution cryo-electron microscopy. This system will be unique in the world, and I am not aware of any scientist currently contemplating such a technically difficult approach that will require 6-12 months of training before it can be expertly and reliably used by students and other trainees. The system will be used to identify protein-protein interactions, protein movements relative to each other and protein unfolding and refolding in sarcomeric proteins for different contractile conditions including passive muscle, fully active muscle, fully active muscle following active stretching in the so-called force enhanced state and following active muscle shortening in the so-called force depressed state. The primary hypotheses we will be testing are: (i) titin binds to actin when muscle is activated thereby increasing its stiffness by a factor of about four, and (ii) titin binding to actin (if it indeed occurs) is regulated by movement of the structural proteins troponin and tropomyosin which exposes binding sites for titin on actin once cross-bridges have reached the strongly bound state. This research is important in our efforts to identify the details of the molecular mechanisms of muscle contraction. In today's muscle physiology textbooks, muscle contraction is exclusively described by the cross-bridge theory involving the contractile proteins actin and myosin. We demonstrated in 2002 that there are passive components that increase force when a muscle is activated and in 2008, we identified that this passive component contributing to active force production is the structural protein titin. However, the way titin achieves an increase in its force contribution in an active muscle (compared to passive) is not understood, although speculations are abundant. This research will be enabled with the new equipment system that will allow us to make fundamental contributions to the science of muscle physiology and mechanics. Our discovery of titin's involvement in active force production has already impacted the field in a way that today's thinking of muscle contraction is completely different than that of just a decade ago. The research proposed with the requested equipment system will again have a major impact in the field and will define research activities in the area of muscle contraction mechanisms for the next decade. The benefits to the research field in Canada are that major fundamental contributions in our understanding of the mechanisms underlying muscle contraction will be made, thus positioning research that has been made in Canada at the forefront of the field. It may also benefit Canada in the development of nanotechnology, and the advancement of high-resolution electron microscopy.