The Role of Tropomyosin and Cardiac Myosin Binding Protein-C in Modulating Thin Filament Activity

Muscle contraction is a finely tuned mechanism involving cyclical interactions between actin and myosin, regulated by calcium through troponin and tropomyosin and modulated by myosin binding protein-C. Genetic mutations of the proteins involved in such complex mechanism can thus lead to potential life threatening diseases, such as Hypertrophic Cardiomyopathy (HCM). Although being mostly asymptomatic, HCM affects 1 in 500 people, ultimately leading to poor prognosis and sudden death, thought to occur through the impairment of relaxation during diastole. In this thesis I present the experiments conducted to improve our current understanding of the molecular mechanism behind HCM, specifically on the role of tropomyosin and myosin binding protein-C in modulating thin filament activation and relaxation. Using a single molecule approach, we first visualised fluorescent myosin binding to reconstituted thin filaments and examined their dynamics in the presence of the tropomyosin HCM causing E180G mutation, demonstrating a shift of the thin filament activation state towards the closed state, facilitating myosin binding at low calcium, and a reduction of the thin filament regulatory unit. We then looked at the dynamics of very highly concentrated clusters of myosin, showing how the sudden collapse of these active regions cannot be explained by normal relaxation mechanisms, thus suggesting an alternative mechanistic role for tropomyosin and how its mutations could lead to impaired relaxation in HCM. Finally, we turned our focus on N-terminal fragments of cardiac myosin binding protein-C (cMyBP-C) and study their role in thin filament activation, by looking at how they affect acto-myosin interactions. We found that only the presence of the whole cMyBP-C N-terminus was able to promote acto-myosin interactions at low Ca2+ or repressing them at high Ca2+. Moreover, by looking at the dynamics of the fragments, we were able to determine that cMyBP-C possesses a two steps binding mechanism to actin, leading us to define its mechanism by which it activates the thin filament