Myogenesis – from forces to structure and treatment

Séminaire organisé par Frank Schnorrer du 29 août au 3 septembre 2022.

Participants

Andreas BAUSCH (Technische Universität München – Garching, ALLEMAGNE), Alexander BERSHADSKY (Mechanobiology Institute (MBI), National University of Singapore – Singapore, SINGAPOUR), Helen BLAU (Stanford University – Stanford, California, ÉTATS-UNIS), Elizabeth CHEN (University of Texas Southwestern Medical Center – DALLAS, ÉTATS-UNIS), Kristina DJINOVIC CARUGO (EMBL Grenoble – Grenoble, AUTRICHE), Benjamin FRIEDRICH (TU Dresden, Physics of Life – Dresden, ALLEMAGNE), Edgar GOMES (Instituto Medicina Molecular – LISBON, PORTUGAL), Anne HOUDUSSE (European Molecular Biology Laboratory – Paris, FRANCE), Nuno LUIS (IBDM – Institut de Biologie du Développement de Marseille – Marseille, FRANCE), Pascal MAIRE (Institut Cochin – Paris, FRANCE), Qiyan MAO (Institut de Biologie du Développement  – Marseille, FRANCE), Christophe MARCELLE (MeLis, UCBL – Lyon, FRANCE), Kevin Kit PARKER (Harvard University – Allston, MA, ÉTATS-UNIS), Norbert PERRIMON  (Brigham and Women’s Hospital/Harvard Medical School – Boston, ÉTATS-UNIS), Olivier POURQUIÉ (Brigham and Women’s Hospital/Harvard Medical School – Boston, ÉTATS-UNIS), Stefan RAUNSER (Max Planck Institute of Molecular Physiology – Dortmund, ALLEMAGNE), Alejandra RODRIGUEZ DE LA ROSA (Brigham and Women’s Hospital/Harvard Medical School – Boston, ÉTATS-UNIS), William ROMAN (Stanford University – Stanford, PORTUGAL), Frank SCHNORRER (Developmental Biology Institute Marseille – Marseille, FRANCE)

Résumé

Figure legend: (A) the musculoskeletal system is a biomechanical apparatus optimised to transmit the produced force into body motion. Contraction of the muscle fibers will change the position of the skeleton. (B) Sarcomeres have a stereotypic organisation and produce contractile forces by myosin sliding along parallel actin filaments that are mechanically linked to the myosin filaments by large titin molecules. Reproduced from Luis & Schnorrer Cells & Development 2021, https://pubmed.ncbi.nlm.nih.gov/34863916/)

Du 29 août au 3 septembre 2022, dix-neuf scientifiques, dont quatre juniors, se sont réunis dans les locaux de la Fondation des Treilles à Tourtour (Provence) pour participer à un atelier intitulé “Myogenèse – des forces à la structure et aux traitements”. Cet atelier organisé par Olivier Pourquié (Harvard University, Boston) et Frank Schnorrer (Aix-Marseille University, CNRS) a organisé un modèle de réunion interdisciplinaire ayant pour thème commun la myogenèse – comment les muscles sont construits et comment ils fonctionnent. Elle a réuni des biologistes du développement qui ont mis au point des technologies de cellules souches permettant de former des muscles in vitro, avec des biologistes cellulaires qui ont imagé en direct le développement musculaire in vivo et mesuré les forces mécaniques dans des sarcomères (unités contractiles de tous les muscles) en développement. L’apport des ingénieurs, qui ont pu développer des environnements mécaniques optimaux pour générer des micro-tissus musculaires et cardiaques in vitro, et des physiciens, qui étudient l’auto-assemblage de l’actomyosine in vitroet la modélisation in silico, a inspiré des discussions sur la manière dont les sarcomères peuvent s’assembler dans des environnements mécaniquement optimisés. Les spécialistes en biologie structurale, qui étudient la structure atomique des composants sarcomériques in vitro et in situ, ont permis d’avoir une vision sans précédent de la nanostructure du sarcomère et ont ainsi fourni des explications moléculaires pour diverses maladies musculaires causées par des variantes des composants sarcomériques. Enfin, les biologistes du développement qui étudient le maintien et la réparation à long terme des fibres musculaires ont discuté de la manière dont la réparation des muscles endommagés peut être stimulée. Ces discussions ont inspiré des perspectives sur la façon dont les dommages musculaires à un âge avancé pourraient être mieux traités à l’avenir. Le cadre informel et le petit nombre de participants interdisciplinaires ont suscité des discussions extrêmement animées et stimulé le développement de diverses collaborations inédites.

Abstract 

From August 29^th to September 3^rd 2022 nineteen scientists, including four junior scientists, met at the properties of the Fondation des Treilles in Tourtour (Provence) to participate at a workshop entitled “Myogenesis – from forces to structure and treatment”. This workshop organised by Olivier Pourquié (Harvard University, Boston) and Frank Schnorrer (Aix-Marseille University, CNRS) set a role model for an interdisciplinary meeting with the common theme of myogenesis – how muscles are built and how they function. It brought together developmental biologists that pioneered stem cell technologies allowing to make muscle in vitro, with cell biologists who imaged muscle development live in vivo and measured mechanical forces in developing sarcomeres, the contractile unit of all muscles. Input from engineers who were able to provide optimal mechanical environments to generate muscle and heart micro-tissues in vitro and physicists studying actomyosin self-assembly in vitro and modelling in silicoinspired discussions about how sarcomeres can assemble in mechanically optimised environments. Structural biologists, who investigate the atomic structure of sarcomeric components in vitro and in situ led to unprecedented insight into the nanostructure of the sarcomere and thus provided molecular explanations for various muscle diseases caused by variants in sarcomeric components. Finally, developmental biologists investigating the long-term maintenance and repair of muscle fibers discussed how repair of damaged muscles can be stimulated. These discussions have inspired perspectives about how muscle damage at old age could be better treated in the future. Together, the informal setting and the small number of interdisciplinary participants sparked extremely lively discussions and stimulated the development of various novel collaborations. 

Report

Muscles are one of the fundamental tissues common to all bilaterian animals, including humans. They produce the mechanical forces that enable animal movements. Importantly, many skeletal muscle fibers and in particular cardiomyocytes need to be maintained for the entire life of the animal. Thus, it is a major challenge for our society to improve the functional state of muscle at advanced age.

One unifying feature of mammalian skeletal and heart muscles as well as insect body muscles is the striated organisation of their contractile apparatus. Each muscle consists of large cells called muscle fibers, whose active forces are produced by periodic chains of contractile sarcomeres. Sarcomeres are highly ordered, pseudo-crystalline arrays of actin and myosin filaments, linked by gigantic titin springs that bear mechanical load (Figure 1).

            The Fondation des Treilles workshop entitled “Myogenesis – from forces to structure and treatment” (Aug 29 – Sept 3 2022) aimed to discuss latest discoveries about how functional muscles are made during development and how they can be maintained during the entire life of the animal. These broad goals were discussed in an exceptionally interdisciplinary setting by bringing physicists, who model or study actomyosin in vitro, and engineers, who build complex micro-environments, together with biologists that study muscle development or muscle regeneration in vitro and in vivo, bridging across model systems from human muscle in vitro to mouse, fish, chick and fly muscles in vivo.

            In the ‘Development and sarcomere function session’, Frank Schnorrer (Marseille) reported about quantification and manipulation of forces across titin and titin spring length in flies in vivo, and how titin spring length impacts sarcomere length. Elizabeth Chen (Dallas) discussed the actin-propelled invasive membrane protrusions that promote myoblast fusion, a process in which mononucleated myoblasts fuse to form multinucleated and contractile muscle fibers. She found that the actin regulators Arp2/3 work together with the fusion promotors Myomaker and Myomixer to regulate myoblast fusion during zebrafish muscle development. Qiyan Mao (Marseille) and Alejandra Rodriguez de la Rosa (Boston) reported on their joint efforts to produce human muscle fibers in vitro using induced-pluripotent stem cell technology to image human sarcomere formation in a dish. Interesting self-organisation patterns of muscle fibers were reported. Together, this session demonstrated how muscle in vivo and in vitro models synergise to better understand the cell and mechanobiology of muscle development.

            In the ‘Muscle Structure session’ Stefan Raunser (Dortmund) presented single particle and cryo electron tomography technologies that unravelled the atomic structure of actin filaments at unprecedented resolution, as well as the key parts of the sarcomere nanostructure in situ, including the Z-disc and the actin filaments decorated with Nebulin and Tropomyosin from isolated mouse muscle fibers.  Kristina Djinovic-Carugo (Vienna, Grenoble) provided structural insights into the myotilin:F-actin, a-Actinin:FATZ and a-Actinin:titin complexes at the Z-disc and discussed a possible phase separation mechanism of FATZ that interacts with a-Actinin as a potential basis of the first steps of Z-disc and sarcomere biogenesis starting from Z-bodies. Anne Houdusse (Paris) discussed how solving the atomic structure of muscle myosins allowed insights into how myosin forces can be manipulated with various specific myosin allosteric effectors that can reduce or increase the force produced in sarcomeres. This may allow the treatment of force-induced muscle diseases like Duchenne by reducing the force load. This session provided an impressive example how the molecular structure of the sarcomere can spark strategies to treat myopathies.

            In the ‘Communication and engineering session’, Norbert Perrimon (Boston) discussed strategies of inter-organ communication that regulate metabolites and lipid storage using the fly model, and a new CRISPR screening strategy to identify receptors for novel ligands. Kevin Kit Parker (Boston) reported the engineering of smart environments onto which cardiomyocytes self-organise to heart micro-tissues. This included nanofiber scaffolds with a helical alignment that achieve better organisation and contractility of the heart micro-tissues. Nuno Luis (Marseille) reported tight mechanical interactions between the growing sarcomeres and the maturing mitochondria in developing flight muscles of the fly. This included a transcriptional feedback mechanism from the squeezed mitochondria/sarcomeres back into the nucleus. Together, this session highlighted that sarcomeres and muscle cells communicate extensively with their microenvironment, inside and outside these cells, respectively.

            In the ‘Actomyosin dynamics and modelling’, Andreas Bausch (Munich) presented self-organised flows of actomyosin mixtures in test tubes and how assembling patterns of microtubules may be directed by actin filament scaffolds providing topological defects. This might be highly relevant for developing muscle fibers in vivo that display a very crowded environment. Alexander Bershadsky (Singapore) reported how forces control the maturation of focal adhesions and discussed mechanisms how nonmuscle myosin can organise into stacked 300 nm long periodic structures in nonmuscle cells. This is surprising as non-muscle cells are missing the titin protein. Benjamin Friedrich (Dresden) introduced a new theoretical model of sarcomere self-organisation from homogenous to a periodic pattern that accommodates titin as mechanical feedback from actin back to myosin. Together, this session highlighted to commonalities between actin and myosin in all cells and its adaptations by adding titin in muscle and heart.

            In the ‘Skeletal muscle in vitro’ session, Olivier Pourquie (Boston) reported how human stem cell technologies combined with 3-dimensional culture conditions can generate paraxial cells in vitro that not only show oscillating gene expression but also form correctly patterned ‘segmentoids’. As the oscillation frequencies in these cultures remain species specific, metabolic differences between human and mouse cells were suggested to set the pace. Edgar Gomes (Lisbon) presented mouse muscle primary cultures as a new genetic model to investigate the morphogenesis of the catalytic triad that couple neuronal signals with muscle contraction. Arp2/3 complex members are involved in its organisation. This session demonstrated particularly strongly that in vitro muscle models are essential to understand fundamental problems of in vivo muscle development.

            In the final session ‘Stem cells and repair’, Helen Blau (Stanford) demonstrated how boosting the production of prostaglandin E2 (PGE2) in old mouse muscle can rejuvenate the muscle tissue including its aged stem cells (satellite cells) and the aged mitochondria of old muscle fibers. This was achieved by blocking a PGE2 degradation enzyme – a strategy that may have therapeutic potential. William Roman (Stanford, Barcelona) reported how muscle fibers after eccentric exercise signal to repair small sarcomeric lesions without recruiting satellite cells: a close-by nucleus moves, attracted by a calcium signal, in a microtubule dependent manner to the lesion site and there induces transcription of repair genes coding for sarcomere components that then reassemble the damaged sarcomere. Christophe Marcelle (Lyon) used genetics in chick embryos by applying smart electroporation techniques to monitor Wnt-signalling activity in developing myoblasts: Wnt-signalling is needed for migration of myoblasts into the limb and for their proper dispersion, but not for their differentiation. Pascal Maire (Paris) studied the expression of the different myosin heavy chain isoforms (MyHC) in the mouse model in vivo. Different fast MyHCs are located close in the genome and are controlled by a myosin super enhancer that regulates the expression of the specific MyHC in each nucleus of the respective muscle fiber types during development, during repair and after denervation. Together, this session demonstrated that muscle repair can happen at all levels, the sarcomere, the stem cells or the mitochondria. Better understanding of these repair mechanisms will provide future avenues of treatment muscle diseases that appear at advanced age.

            This Les Treilles meeting on Myogenesis has been unique of its kind, as it was bridging classical developmental muscle models with new in vitro models and the physics of bioengineering and modelling as well as structural insights to better understand the development, the regeneration, the mechanobiology and the structure of the sarcomere, eventually until atomic resolution. Everybody left the meeting inspired with novel ideas and lists of future collaborations to explore.