Toward a better understanding of the mechanisms by which myofilaments control the activation of striated muscle

My research activity during the PhD course was aimed at investigating in situ the function of the proteins involved in the regulation of contraction of striated (skeletal and cardiac) muscle. According to the most recent structural and mechanical evidence, in the sarcomere, the structural unit of striated muscle, all “three filaments”, the thin actin containing filament, the thick myosin containing filament and the cytoskeleton protein titin (the “third filament”) contribute to regulation of contraction. In the classical view thin filament is activated by Ca2+ binding to troponin, leading to tropomyosin displacement that exposes actin sites for interaction with myosin motors extending from the neighbouring thick filaments. Motor attachment to actin contributes to spreading of the activation along the thin filament, through a cooperative mechanism, not yet defined (Gordon et al. Physiol Rev 80:853, 2000). The evidence that the myosin motors in the resting state lie on the surface of the thick filament in an OFF state unable to bind actin and hydrolyse ATP (Woodhead et al. Nature 436:1196, 2005; Stewart et al. PNAS 107:430, 2010), made evident the need for a thick filament activation mechanism that allows the heads to move away from the surface of the thick filament to make them available for interaction with actin (ON state). Mechano-sensing in the thick filament has been shown to promote recruitment of the myosin motors from their OFF state in relation to the load during contraction (Linari et al. Nature 528:276, 2015; Reconditi et al. PNAS 114:3240, 2017), through a mechanism that implies stiffening of the cytoskeletal protein titin during muscle activation to allow the transmission of the stress on the sarcomere to the myosin motors even when they are detached (Squarci et al. PNAS 120:e2219346120, 2023). Deteriorations of thin or thick filament regulation related to mutations in sarcomeric proteins are the most common cause of myopathies. In this thesis I used sarcomere level mechanics either to investigate the mechanism of cooperativity in thin actin activation or to characterise the mechanical performance of the muscle of the medaka fish, a new animal model ideal for the investigation of the genotype-phenotype relation in human diseases related to mutations in sarcomeric proteins. The first aim was pursued applying microsecond-nanometre sarcomere mechanics to Ca2+-activated demembranated fibres of skeletal muscle of the rabbit to define protocols in which the force of the actin attached myosin motor can be selectively changed. In this way it was found that the cooperativity of thin filament activation increases with the force of the motor independent of the fraction of attached motors. The results, recently published in Communications Biology (Caremani, Marcello et al. Communications Biology 5:1266, 2022), introduce a dynamic component in the classical view of the steric-blocking model for thin filament activation. The second subject of my doctorate activity concerned the characterisation of the mechanical performance of the intact skeletal muscle of medaka fish, an animal model where it is possible to induce a specific mutation in titin gene (TTN), identified in patients with skeletal muscle diseases due to impairment of the thick filament regulation. The maximum tetanic force, the force-velocity relation and the power output showed comparable values as those reported for skeletal muscles of other animal species (frog and mouse), indicating the medaka fish as a suitable model to investigate the correlation between genotype and phenotype. The results of these experiments have been published in abstract form (Marcello et al. 49th EMC, Prague, Czech Republic, 22-26 September 2022) and are the object of a manuscript in preparation. In relation to this second aim, I spent six months in the laboratory of Prof. Bjarne Udd (Folkhälsan Research Center, Helsinki, Finland) to acquire the technical skills to identify genetic variants of titin in mutant animal models with respect to the wild type and to characterize the downstream effects at mRNA and/or protein levels. Using the CRISP-Cas9 technique, a medaka fish line with a specific mutation reported in human in TTN is under development. The existing animals models used to investigate titin physiopathology have been reviewed in collaboration with the research group in Helsinki (Marcello et al. J Cell Mol Med 26:5103, 2022).