Thèse Contribution des Noyaux et des Mitochondries Dérivées des Cellules Souches Musculaires Adultes à l'Homéostasie et la Plasticité de la Fibre Musculaire H/F Doctorat.Gouv.Fr

Établissement : Université Claude Bernard Lyon 1 École doctorale : EDISS - Interdisciplinaire Sciences-Santé Laboratoire de recherche : PGNM - PATHOPHYSIOLOGIE ET GENETIQUE DU NEURONE ET DU MUSCLE Direction de la thèse : Anna KNEPPERS Début de la thèse : 2026-11-09 Date limite de candidature : 2026-11-09T23:59:59 Le vieillissement de la population s'accompagne d'une augmentation des pathologies liées à l'âge, et notamment de la sarcopénie, qui est associée à une perte de la masse et de la fonction musculaires et entraîne une diminution de l'autonomie des personnes âgées. La compréhension des mécanismes cellulaires qui sous-tendent ce vieillissement musculaire offre des perspectives d'amélioration de la qualité de vie de ces personnes âgées et se place donc comme un enjeu santé publique. À l'échelle cellulaire, le vieillissement induit des altérations des fibres musculaires (i.e. myofibres, les cellules musculaires), qui présentent des défauts mitochondriaux et sécrètent des molécules ayant un effet délétère sur un autre type cellulaire crucial pour la maintenance du muscle strié squelettique et sa plasticité : les cellules souches musculaires. Ces cellules souches ont la capacité de fusionner avec la myofibre, et de lui transférer leur contenu, notamment leurs noyaux et leurs mitochondries. Dans une perspective de prévention des altérations de la myofibre liées au vieillissement, la compréhension du rôle de ces noyaux et mitochondries issus des cellules souches musculaires dans le fonctionnement de la myofibre est nécessaire.
Ce projet vise à comprendre la contribution des cellules souches musculaires à l'homéostasie et à la prévention des altérations musculaires associées au vieillissement des myofibres adultes. Il emploiera des technologies avancées permettant le traçage de ces noyaux et mitochondries dérivés des cellules souches musculaires afin de déterminer le rôle des molécules d'ARN messagers qui en sont issues dans les mécanismes de maintien de l'homéostasie cellulaire de la myofibre. Déterminer l'implication des cellules souches musculaires dans la prévention voire l'inversion des effets du vieillissement musculaire permettra ainsi d'ouvrir la voie à de nouvelles stratégies thérapeutiques. Context

Over the past two decades, humans experienced a marked increase in lifespan, which has led to a rapid aging of the human population and an increase in the prevalence of age-related diseases1. One of the most striking effects of advancing age is the progressive loss of skeletal muscle mass and function, termed sarcopenia2, which leads to a decreased quality of life and is a predictor of poor outcome in patients with other age-related diseases2.
Several cell types are impacted in sarcopenic muscle, but perhaps most relevant to functional defects are alterations in the functional units within skeletal muscle - the syncytial myofibers. Most notably, sarcopenic muscles display a fast-to-slow fiber type shift due to atrophy of fast glycolytic (type II) fibers3,4, and a loss of mitochondrial oxidative capacity4,5. The molecular processes that underlie this impaired cellular homeostasis are orchestrated by aging factors' categorized in 9 hallmarks: loss of proteostasis, mitochondrial dysfunction, genomic instability, telomere attrition, epigenetic alterations, deregulated nutrient-sensing, stem cell exhaustion, altered intercellular communication, and cellular senescence6. These hallmarks are strongly interrelated, but it has been posed that cellular senescence is the nexus linking the molecular causes of aging with the structural and functional effects of aging7.

Recent studies link cellular senescence to the development of sarcopenia8-10. Moreover, senescent cell removal from aged mice results in skeletal muscle rejuvenation' both in terms of mass and function8,10, emphasizing the detrimental impact of cellular senescence on skeletal muscle homeostasis. The rejuvenation effect of senescent cell removal on skeletal muscle can be partially attributed to beneficial effects on the skeletal muscle stem cells (MuSCs, or satellite cells), which support myofiber homeostasis through fusion. Indeed, given the post-mitotic nature of myofibers, MuSCs are essential for both myofiber cellular turnover during regeneration, and for the turnover of myofiber intracellular components during homeostasis and adaptation. Strong evidence suggests that the myriad of factors secreted by senescent cells, collectively referred to as the senescence-associated secretory phenotype (SASP)11-14, contribute to MuSC dysfunction with advancing age. Importantly, the dogma that only mitotic cell types can undergo senescence and produce SASP factors is now being questioned by a growing body of evidence for senescence in terminally differentiated cells, including aged myofibers11,14-17, and suggests that myofiber senescence can lead to both myofiber-intrinsic defects and impaired extrinsic support of myofiber homeostasis by MuSCs.
Interestingly, physical exercise reverses SASP factors18, restores the capacity of MuSC activation19,20, and induces MuSC-to-myofiber fusion (a.k.a. myonuclear accretion)21-23. Moreover, short-term whole body or myofiber-specific expression of the Yamanaka factors improved MuSC numbers and function24,25, and recapitulates exercise-induced methylome alterations26. Aside from the pleiotropic effects of exercise, this implies a direct contribution of MuSCs to skeletal muscle rejuvenation, that could have important therapeutic potential27-29. However, to date it remains unknown to which cellular processes MuSCs contribute in order to promote myofiber homeostasis and rejuvenation.

Strikingly, the molecular alterations seen in sarcopenic muscle - including the downregulation of ribosome and translation ontologies, encompassing genes encoding for mitochondrial ribosomal proteins30 - resemble a loss of scaling between cell size and mRNA and protein synthesis when DNA becomes rate-limiting31. Indeed, due to a loss of proteostasis, not only nuclear DNA (gDNA), but also mitochondrial DNA (mtDNA) may become rate-limiting in sarcopenic muscle, which could synergistically contribute to the progression of sarcopenia through the induction of myofiber senescence30-33. We speculate that the addition of MuSC-derived nuclei and mitochondria34, and their associated DNA, could break this vicious cycle of myofiber deterioration in sarcopenia by compensating for a loss of myofiber transcriptional capacity, by normalizing the transcriptional profile on a cellular level, and even by reversing the senescence signature of aged myonuclei and rejuvenating the aged myofiber mtDNA pool.

Working hypotheses and objectives

Broadly, myofiber size is influenced by its myonuclear number and their myonuclear domain, i.e., the cytoplasmic volume controlled by each nucleus35. Indeed, a MuSC-dependent increase in myonuclear number is required for sustained exercise-induced myofiber growth23, and age-related MuSC dysfunction is associated with a blunted response to exercise36. Moreover, type II fiber atrophy is linked with a type II fiber-specific loss of MuSCs and myonuclear number3. Short-term myofiber growth can occur in absence of myonuclear addition by plasticity of the myonuclear domain. However, a loss of scaling between nucleic acid and protein synthesis can lead to cytoplasm dilution and contributes to senescence31. Furthermore, the dogma that only mitotic cell types can undergo senescence is now being questioned by a growing body of evidence for senescence in terminally differentiated cells11,15,37. As such, we hypothesize that the addition of MuSC-derived nuclei could serve to compensate for a loss in myofiber transcriptional capacity, could normalize the transcriptional profile on the cellular level, and could even revert the senescence signature of aged myonuclei by improving scaling between nuclei acid and protein synthesis.
Another important characteristic of aging skeletal muscle is a loss of mitochondrial oxidative capacity4,5. Mitochondria are dynamic organelles endowed with their own DNA molecule (mtDNA) and replication and translation machinery. Thereby mitochondria contribute to the regulation of their own mass and function. In long-lived cells such as myofibers, this regulation is essential for mitochondrial quality maintenance38. However, aging is associated with the accumulation of mtDNA mutations, and an imbalanced rate and coordination of mitochondrial translation, that could lead to a vicious cycle of mitochondrial deterioration32. Interestingly, restoration of mitochondrial mass and function by mitochondrial transfer is an emerging therapeutic approach39. Mitochondrial transfer can occur through several physiological processes, including cell fusion. Yet, the potential of MuSCs to donate cellular components other than their nucleus is generally overlooked. A recent study demonstrated the existence of mitochondrial domains by conditional labelling of mitochondria under control of the MuSC-specific Pax7 promoter34. This shows a MuSC contribution to the myofiber mitochondrial pool, and suggests a regulatory role for MuSCs in the rate and coordination of myofiber mitochondrial translation and rejuvenation of the aged myofiber mtDNA pool.
Ultimately, answering the fundamental biological question to which cellular processes do MuSCs contribute in order to promote myofiber maintenance and rejuvenation', will allow for a better understanding of the contribution of MuSCs to skeletal muscle maintenance, and a better targeting of therapeutic approaches to counteract skeletal muscle aging.

We will for the first time directly address this through the following objectives:
1) Characterizing the transcriptional contribution of new' MuSC-derived nuclei and mitochondria to adult myofibers (Aim 1);
2) Unravelling the causal link between the transcriptional contribution of MuSCs to adult myofibers and the rate of MuSC fusion to myofibers (Aim 2);
3) Assessing the contribution of new' MuSC-derived nuclei and mitochondria to the reversal of senescence-associated transcriptional alterations upon skeletal muscle aging (Aim 3). Methodology
MuSC nucleus and mitochondria tracing: A major hurdle in tracing of MuSC-derived nuclei into myofibers is the propagation of nuclear fluorescent reporter proteins within the myofiber syncytium. To overcome this, the host lab has developed in vitro and in vivo tools for EdU-based nuclear tracing at the DNA level41, complementing their previous work assessing MuSC fate42. For snRNAseq applications, they used Pax7CreERT2;B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (TdTomato) mice to trace MuSC-derived nuclei at the mRNA level, through a specific bioinformatic strategy. For tracing of mitochondria in both imaging and ST applications, they use Pax7CreERT2;B6.Cg-Gt(ROSA)26Sortm1(CAG-EGFP)Brsy/J (MITO-Tag) mice. To this end, we will integrate the fluorescent signal (mitochondria) and MITO-Tag mRNA signal (nuclei).
Induction of MuSC contribution to myofibers: The rate of MuSC fusion to myofibers (i.e., myonuclear accretion) in unperturbed adult skeletal muscle is low. We benefit from this low basal rate to induce a semi-synchronous addition of new myonuclei upon 6 sessions of 80 submaximal isometric contractions invoked by non-damaging transcutaneous neuromuscular electrical stimulation (NMES) of mouse Plantar Flexor muscles, as characterized before40,41.