Dual-filament regulation of contraction in fast and slow human skeletal muscle fibres

Skeletal muscle weakness is a major feature of many muscle disorders, yet there are currently no effective treatments to restore muscle contractility. This lack of therapeutic options reflects a limited understanding of the molecular mechanisms that regulate contraction in human skeletal muscle. Traditionally, muscle contraction is controlled by calcium-dependent activation of the thin filament: following electrical stimulation, calcium ions released from the sarcoplasmic reticulum bind to troponin, triggering structural changes that enable actin filaments to interact with myosin motors on the thick filament, producing force and movement. However, recent evidence indicates that the thick filament itself plays a fundamental regulatory role in contraction. Calcium-independent mechanisms within the myosin filament control the transition of myosin motors between OFF and ON states, regulating force generation. So far, these newly identified mechanisms have been characterised only in “fast-contracting” fibres in animal models, whereas little is known about the two main fibre types of human skeletal muscle, the “slow-contracting” type-1 and the fast type-2A fibres. Our preliminary findings suggest that thick filament-based regulatory mechanisms operate differently in human muscle fibres compared to previously studied models. Disruption of these mechanisms is already implicated in sarcomere-based cardiomyopathies. Moreover, small molecules that modulate myosin motor ON/OFF transitions have recently been developed to treat those heart diseases, demonstrating the feasibility of pharmacologically restoring contractile function. Although these myosin modulators also target the slow myosin in skeletal muscle, their effects on skeletal muscle regulation, and their therapeutic potential in muscle disorders, remain largely unexplored. The overarching aim of this project is to define the fundamental regulatory mechanisms controlling human skeletal muscle contractility, in both health and disease, to enable rational therapeutic targeting of the contractile apparatus in skeletal muscle disorders. Our primary objective is to identify the mechanisms that regulate myosin filament activation and force generation in human slow (type-1) and fast (type-2A) fibres. To achieve this goal, we will combine complementary biophysical techniques, including small-angle X-ray scattering, fluorescence polarisation microscopy and advanced fibre mechanics, to study the regulatory structural changes in both actin and myosin filaments under near-physiological conditions. These approaches will be applied, for the first time, to isolated human muscle fibres obtained from biopsy samples of healthy donors, enabling us to dissect the distinct regulatory features of slow and fast muscle fibres. We will also investigate how the kinetics of contraction and relaxation are controlled by the structural dynamics of thick and thin filaments. Our secondary objective is to explore the therapeutic potential of small-molecule myosin modulators in skeletal muscle diseases. Using muscle tissue from patients with congenital myopathies as a proof-of-concept model, we will characterise disruptions in thick filament regulation and test the ability of myosin activators to restore contractile performance. This work will lay the foundation for extending pharmacological targeting of the myosin filament to a wider range of muscle disorders, including age-associated muscle weakness, and will guide the rational design of new therapies aimed at enhancing or suppressing skeletal muscle contractility as clinically required.