Leader: Marco Linari (UNIFI); Other collaborator(s):
Using nanometer-microsecond technology for mechanical and structural studies either in vitro (single molecule and synthetic nanomachine mechanics) or in situ (sarcomere level mechanics and synchrotron-light X-ray diffraction) we aim to determine i) the effect of muscle ageing on the performance of contractile and regulatory proteins and their neurohormonal and metabolic regulation at different hierarchical levels of the skeletal and cardiac muscle, in particular determining how altered Ca2+ handling and metabolic stress in muscle ageing and sarcopenia affect muscle structure and function; ii) the action mechanism of small molecule effectors candidate as new therapeutic tools to improve muscle function and exercise resistance in aged muscle.
Brief description of the activities and of the intermediate results
Main policy, industrial and scientific implications
Brief description of the activities and of the intermediate results
In collaboration with Genethon (Evry, France), experiments were performed to define the mechanical performance of fast and slow muscles in wild type (WT) and FINmaj mutant mice. Specifically, the maximum isometric force T 0, the force velocity (T-V) relation, the unloaded shortening velocity and the maximum power output were determined. In homozygous FINmaj mice, the isometric force was reduced in both fast and slow muscles, the unloaded shortening velocity was reduced only in the slow muscle and the maximum power output was reduced in both fast and slow muscles.
Brief description of the activities and of the intermediate results
In a series of experiments the regional contribution of the myosin filament to force and its modulation in relation to the systolic performance were determined with X-ray diffraction from electrically stimulated trabeculae and papillary muscles of rat ventricle (temperature 27°C, sarcomere length 1.9-2.2 um). The force at the peak of the twitch was varied in the range 5-100 kPa under different protocols able to vary the number of attached motors (Caremani et al. 2016, PNAS 113:3675-3680). Thick filament activation starts from the periphery of the thick filament and spreads throughout the filament, in relation to titin activation (Squarci et al. 2023, PNAS 120:e2219346120), at relatively small systolic forces (≤1/2 the maximum force). At these low forces motors attachment occurs only in the central, Myosin Binding Protein-C containing zone, of the thick filament. At higher systolic forces motor attachment spreads toward the periphery of the thick filament, according to thin filament cooperative activation (McKillop and Geeves 1993, Biophys J 65: 693–701), operating at the sub-saturating level of intracellular Ca 2+ of the cardiac twitch. In this period, a manuscript has been prepared and it is now under the second revision to PNAS (Morotti et al. 2024, BiorXiv, doi: https://doi.org/10.1101/2024.05.11.593706).
Brief description of the activities and of the intermediate results
1) The performance of purified proteins re-assembled in a myosin-based synthetic nanomachine with a Dual Laser Optical Tweezers (DLOT) system has been measured. The system uniquely ensures the dynamic range in force and movement (Pertici et al. (2018). Nat Commun 9:3532). Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and shortening by cyclic ATP–driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform–dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle (Buonfiglio et al. (2024). Commun Biol 7:362). Data fitting with a stochastic model provides a self–consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin–attached motors and the rate of transition through the attachment–detachment cycle.
2) The suitability of the medaka fish (Oryzias latipes) as a model for investigating the molecular mechanisms of human myopathies caused by mutations of sarcomeric proteins is tested by combining structural analysis and sarcomere-level mechanics of the skeletal muscle of the transparent tail of medaka larva at stage 40. The mechanical performance of the contracting muscle of the wild-type larva has been defined at the level of the half-thick filament, where 300 myosin motors work in parallel as a collective motor, allowing a detailed comparison with the established performance of the skeletal muscle of different vertebrates (Marcello et al. (2024). Am J Physiol 326:C632). The mechanical performance of the medaka muscle, scaled at the level of the myosin- containing thick filament, together with its reduced genome duplication makes this model unique for investigations of the genotype/phenotype correlations in human myopathies.
3) Titin-related changes in filament structure able to switch ON the myosin motors have been investigated in combined mechanical and X-ray diffraction experiments. In heart muscle, switching ON of motors has been attributed to thick filament mechanosensing (Reconditi et al. (2017). PNAS 114:3240; Brunello et al. (2020). PNAS 117:8177), a mechanism originally demonstrated in skeletal muscle (Linari et al. 2015. Nature 528:276), which adapts the number of motors made available for interaction with actin to the load of the contraction. However, the molecular basis of mechanosensing as well as its hierarchical organization along the thick filament are not known. In this series of experiments the regional contribution of the myosin filament to force and its modulation in relation to the systolic performance are determined with X-ray diffraction from electrically stimulated trabeculae and papillary muscles of rat ventricle (temperature 27°C, sarcomere length 1.9-2.2 m). The force at the peak of the twitch is varied in the range 5-100 kPa under different protocols able to vary the number of attached motors (Caremani et al. (2016). PNAS 113:3675-3680). Thick filament activation starts from the periphery of the thick filament and spreads throughout the filament, in relation to titin activation (Squarci et al. (2023). PNAS 120:e2219346120), at relatively small systolic forces (≤1/2 the maximum force). At these low forces motors attachment occurs only in the central, Myosin Binding Protein-C containing zone, of the thick filament. At higher systolic forces motor attachment spreads toward the periphery of the thick filament, according to thin filament cooperative activation (McKillop and Geeves. (1993). Biophys J 65: 693–701), operating at the sub-saturating level of intracellular Ca 2+ of the cardiac twitch. Results of the research have been recently published (Morotti et al. (2024). PNAS 121:e2410893121).
Brief description of the activities and of the intermediate results
1) Creatine (Cr) is essential for cellular energy homeostasis, particularly in muscle and brain tissues. Creatine Transporter Deficiency (CTD), an X-linked disorder caused by mutations in the SLC6A8 gene, disrupts Cr transport, leading to intellectual disability, speech delay, autism, epilepsy, and various non-neurological symptoms. In addition to neurological alterations, Creatine Transporter knockout (CrT −/y ) mice exhibit severe muscle atrophy and functional impairments. We provided the first characterization of the skeletal muscle phenotype in CrT −/y mice, revealing profound ultrastructural abnormalities accompanied by reduced fibre cross-sectional area and muscle performance (Pertici et al. 2025. Cell Death and Diseases 16:99).
2) The molecular bases of the depression of the force by lowering the temperature below the physiological level has been investigated in heart (Ca 2+ -activated cardiac trabeculae from the rat ventricle). Results show that in the cardiac myosin, as in the skeletal muscle myosin, the force-generating transition is endothermic. The underlying large heat absorption indicates the interaction of extended hydrophobic surfaces within the myosin motor, like those suggested by the crystallographic model of the working stroke (Morotti et al. Int J Mol Sci 26:469).
3) The suitability of the medaka fish (Oryzias latipes) as a model for investigating the molecular mechanisms of human myopathies caused by mutations of sarcomeric proteins has been tested by combining structural analysis and sarcomere-level mechanics of the skeletal muscle of the transparent tail of medaka larva at stage 40 (Marcello et al. 2024. Am J Physiol 326:C632). In this period, in collaboration with Bjarne Udd (Folkhalsan Research Centre, Helsinki, Finland) and Vincenzo Nigro (TIgem Institute, Naples. Italy), a knockin line bearing the Hereditary Myopathy with Early Respiratory Failure (HMERF) patient missense mutation p.C31712R has been generated. In our lab a series of experiments to investigate how the mutation alters muscle performance is ongoing.
Scientific papers:
Abstract to national/international conferences:
2024
2023