Probing an active role of titin in modulating actin-myosin interactions

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The goal of these experiments is to explore how titin filaments can modulate actin-myosin interaction, particularly in cardiac muscle. Titin is a multifunctional polypeptide which interacts at multiple sites with other sarcomeric proteins, such as myosin or actin. Wheras the 'passive' roles of titin in muscle contraction are well documented, hints at a more active involvement of titin are abundant but scattered and unsystematic. We wish to follow up and probe these hints in a more systematic way. To study the putative 'active' mechanical properties of titin, we will make use of unique methodology: the single myofibril preparation, combined with fluorophore-labelled anti-titin antibodies, provides an ideal tool to investigate the role of titin. We will perform force and stiffness measurements on single cardiac myofibrils to test whether the titin filament stiffens upon activation with Ca²+, being 'contractile' in a true sense. We will also determine the effect titin has on the active sarcomere length-tension curve of cardiac myofibrils, in particular on high stretch. Further, the in-vitro motility assay will be employed to test the hypothesis that titin has a specific effect on thin-filament sliding velocity. A variety of recombinant titin constructs will be used and the possibility studied that the effect of titin on actin sliding speed is influenced by [Ca²+]. If a specific effect of a construct is found, we will examine whether a comparable effect can also be detected in the single myofibril, to confirm a functional relevance of the observations in the highly ordered enviroment of the sarcomere.