Role of the circadian clock in maintaining skeletal muscle homeostasis : Physiological and pathological implications

L’homéostasie du muscle squelettique est assurée par sa remarquable aptitude à réguler différents paramètres physiologiques tels que ses fonctions métaboliques ou sa masse. La régulation de la masse musculaire représente un enjeu important pour l’organisme car une dérégulation de cette dernière impacte le métabolisme énergétique global ainsi que d’autres paramètres tels que la locomotion. Ce tissu présente également une capacité importante de régénération suite à des blessures causées par des exercices intensifs ou des myopathies. La régénération du muscle squelettique nécessite une interaction spatio-temporelle précise entre les cellules satellites (SC) et les cellules immunitaires, qui fournissent le microenvironnement optimal pour la prolifération et la différenciation des SCs.Les rythmes circadiens, générés par notre horloge biologique, contrôlent diverses fonctions physiologiques telles que le métabolisme et l’immunité. Ce système permet aux organismes d'anticiper des changements environnementaux prévisibles tels que l’alternance jour/nuit. Au niveau métabolique, il coordonne la mise en place des voies nécessaires au stockage des nutriments ou au contraire à la dépense énergétique. Concernant l’immunité, le rôle majeur de l’horloge est de réguler la circulation et la fonction des différentes cellules immunitaires afin de préparer ce système durant les phases de la journée où le risque d’infection est le plus élevé. Dans ce contexte, nous nous sommes intéressés au rôle de l’horloge circadienne dans le contrôle de la masse du muscle squelettique mais également dans sa capacité régénérative. Le laboratoire a préalablement démontré un rôle majeur de Rev-erbα, un composant moléculaire de l’horloge circadienne, dans la fonction du muscle squelettique. En effet, ce récepteur nucléaire régule la capacité oxydative musculaire en contrôlant les processus de biogenèse mitochondriale et d’autophagie. Les résultats obtenus durant ma thèse mettent en évidence un rôle essentiel de Rev-erbα dans le contrôle de la masse musculaire. Précisément, une délétion globale de Rev-erbα chez la souris entraine une diminution de la masse musculaire associée à une augmentation de l’expression des gènes relatifs à l’atrophie musculaire. De façon intéressante, l’activation pharmacologique de ce récepteur permet de contrer l’atrophie musculaire induite par un traitement aux glucocorticoïdes.En parallèle, j’ai également mis en évidence le rôle de l’horloge biologique dans le processus de régénération musculaire. Nous avons montré que des perturbations environnementale et génétique de l'horloge ont un impact sur la régénération du muscle squelettique associé à une altération du recrutement des cellules immunitaires et principalement des cellules myéloïdes. En outre, l’utilisation de modèles génétiques d’altération de l’horloge dans les cellules myéloïdes a mis en évidence des défauts de régénération démontrant l’importance d’une horloge fonctionnelle au sein de cette population pour contrôler la réparation du muscle squelettique. Des analyses transcriptomiques nous ont permis de relier ce défaut de régénération à une expression perturbée de chimiokines, essentielles dans la communication entre les cellules immunitaires et les cellules satellites, pouvant être à l’origine du défaut de myogenèse observé dans nos modèles.Par ailleurs, nous nous sommes intéressés dans ce contexte de régénération musculaire à l’étude d’une population immunitaire nouvellement identifiée : les cellules lymphoïdes innées (ILCs). Ces cellules de l’immunité innée sont présentes essentiellement dans les tissus muqueux comme les poumons ou les intestins où elles assurent un rôle de sentinelle. Dans ce contexte, nous avons montré que les ILCs, et principalement les ILC2, étaient présentes dans le muscle squelettique après une blessure. De surcroît, nous avons mis en évidence que la délétion en ILC2 conduisait à un processus de régénération altéré.Skeletal muscle homeostasis is ensured by its remarkable ability to control many of its physiological parameters such as its metabolic function or its mass according to the needs of the organism. Muscle mass regulation is essential for global health since its deregulation not only impacts overall energy metabolism but also other parameters such as locomotion. This tissue has an important capacity to regenerate following injuries caused by intensive exercises or myopathies. Skeletal muscle regeneration requires a well-orchestrated spatio-temporal interaction between satellite cells (SCs) and immune cells, which provides the optimal microenvironment for SC proliferation and differentiation.Circadian rhythms, generated by our biological clock, control various physiological functions such as metabolism and immunity. This ancestral system is present in all organisms allowing them to anticipate and optimize physiological functions to predictable daily changes. The clock integrates signals related to energy state and, in turn, regulates many metabolic pathways gating them to the most relevant time of the day. Concerning immunity, the major role of the clock is to coordinate leucocyte circulation and function allowing the body to anticipate phases of the day with higher risk of infections. In this context, we are interested in the role of the circadian clock in the control of skeletal muscle mass but also in its regenerative capacity. The role of Rev-erbα, a key component of the biological clock, has already been demonstrated in this tissue by our laboratory. Indeed, this nuclear receptor regulates muscle oxidative capacity by controlling mitochondrial biogenesis and autophagy. My thesis results highlight that Rev-erbα is also essential in the regulation of muscle mass. Specifically, global deletion of Rev-erbα leads to muscle mass decrease associated with increased expression of genes related to muscle atrophy. Interestingly, pharmacological activation of this receptor prevents muscle atrophy induced by glucocorticoid treatment.During my thesis, I also highlighted the role of the circadian clock in the control of muscle regeneration process. We have shown that environmental and genetic clock disruption lead to defective skeletal muscle regeneration associated with an alteration of immune cells recruitment, mainly myeloid cells. Furthermore, regenerative process defects observed in our myeloid cells-specific genetic clock disruption models bring out the importance of a functional clock in these cells to control skeletal muscle repair. Transcriptomic analyses allowed us to associate this regeneration defect to disturbed expression of chemokines essential in the communication between immune cells and satellite cells, which could elicit myogenesis alteration.In the context of muscle regeneration, we also investigated the role of a newly identified immune population: innate lymphoid cells (ILCs). This innate immune cells are located essentially in mucosal tissues such as lung or intestine where they ensure a sentinel function. We have shown that ILCs, and mainly ILC2, are present in skeletal muscle after injury. Interestingly, we have demonstrated that ILC2 depletion results in impaired regenerative process

MuscleJ: a high-content analysis method to study skeletal muscle with a new Fiji tool

International audienceSkeletal muscle has the capacity to adapt to environmental changes and regenerate upon injury. To study these processes, most experimental methods use quantification of parameters obtained from images of immunostained skeletal muscle. Muscle cross-sectional area, fiber typing, localization of nuclei within the muscle fiber, the number of vessels, and fiber-associated stem cells are used to assess muscle physiology. Manual quantification of these parameters is time consuming and only poorly reproducible. While current state-of-the-art software tools are unable to analyze all these parameters simultaneously, we have developed MuscleJ, a new bioinformatics tool to do so

Hepatitis C virus alters the morphology and function of peroxisomes

International audienceDespite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells

Nuclear Receptor Subfamily 1 Group D Member 1 Regulates Circadian Activity Of NLRP3 Inflammasome to Reduce the Severity of Fulminant Hepatitis in Mice

International audienceBACKGROUND & AIMS:The innate immune system responds not only to bacterial signals, but also to non-infectious danger-associated molecular patterns that activate the NLRP3 inflammasome complex after tissue injury. Immune functions vary over the course of the day, but it is not clear whether these changes affect the activity of the NLRP3 inflammasome. We investigated whether the core clock component nuclear receptor subfamily 1 group D member 1 (NR1D1, also called Rev-erbα) regulates expression, activity of the NLRP3 inflammasome, and its signaling pathway.METHODS:We collected naïve peritoneal macrophages and plasma, at multiple times of day, from Nr1d1-/- mice and their Nr1d1+/+ littermates (controls) and analyzed expression of NLR family pyrin domain containing 3 (NLRP3), interleukin 1 beta (IL1B, in plasma), and IL18 (in plasma). We also collected bone marrow-derived primary macrophages from these mice. Levels of NR1D1 were knocked down with small hairpin RNAs in human primary macrophages. Bone marrow-derived primary macrophages from mice and human primary macrophages were incubated with lipopolysaccharide (LPS) to induce expression of NLRP3, IL1B and IL18; cells were incubated with LPS and ATP to activate the NLRP3 complex. We analyzed caspase 1 activity and cytokine secretion. NR1D1 was activated in primary mouse and human macrophages by incubation with SR9009; some of the cells were also incubated with an NLRP3 inhibitor or inhibitors of caspase 1. Nr1d1-/- mice and control mice were given intraperitoneal injections of LPS to induce peritoneal inflammation; plasma samples were isolated and levels of cytokines were measured. Nr1d1-/- mice, control mice, and control mice given injections of SR9009 were given LPS and galactosamine to induce fulminant hepatitis and MCC950 to specifically inhibit NLRP3; plasma was collected to measure cytokines and a marker of liver failure (ALAT); liver tissues were collected and analyzed by quantitative PCR, immunohistochemistry, and flow cytometry.RESULTS:In peritoneal macrophages, expression of NLRP3 and activation of its complex varied with time of day (circadian rhythm)-this regulation required NR1D1. Primary macrophages from Nr1d1-/- mice and human macrophages with knockdown of NR1D1 had altered expression patterns of NLRP3, compared to macrophages that expressed NR1D1, and altered patterns of IL1B and 1L18 production. Mice with disruption of Nr1d1 developed more-severe acute peritoneal inflammation and fulminant hepatitis than control mice. Incubation of macrophage with the NR1D1 activator SR9009 reduced expression of NLRP3 and secretion of cytokines. Mice given SR9009 developed less-severe liver failure and had longer survival times than mice given saline (control).CONCLUSIONS:In studies of Nr1d1-/- mice and human macrophages with pharmacologic activation of NR1D1, we found NR1D1 to regulate the timing of NLRP3 expression and production of inflammatory cytokines by macrophages. Activation of NR1D1 reduced the severity of peritoneal inflammation and fulminant hepatitis in mice

Glycogen Dynamics Drives Lipid Droplet Biogenesis during Brown Adipocyte Differentiation

International audienceBrowning induction or transplantation of brown adipose tissue (BAT) or brown/beige adipocytes derived from progenitor or induced pluripotent stem cells (iPSCs) can represent a powerful strategy to treat metabolic diseases. However, our poor understanding of the mechanisms that govern the differentiation and activation of brown adipocytes limits the development of such therapy. Various genetic factors controlling the differentiation of brown adipocytes have been identified, although most studies have been performed using in vitro cultured pre-adipocytes. We investigate here the differentiation of brown adipocytes from adipose progenitors in the mouse embryo. We demonstrate that the formation of multiple lipid droplets (LDs) is initiated within clusters of glycogen, which is degraded through glycophagy to provide the metabolic substrates essential for de novo lipogenesis and LD formation. Therefore, this study uncovers the role of glycogen in the generation of LDs

Endospanin-2 enhances skeletal muscle energy metabolism and running endurance capacity.

Metabolic stresses such as dietary energy restriction or physical activity exert beneficial metabolic effects. In the liver, endospanin-1 and endospanin-2 cooperatively modulate calorie restriction-mediated (CR-mediated) liver adaptations by controlling growth hormone sensitivity. Since we found CR to induce endospanin protein expression in skeletal muscle, we investigated their role in this tissue. In vivo and in vitro endospanin-2 triggers ERK phosphorylation in skeletal muscle through an autophagy-dependent pathway. Furthermore, endospanin-2, but not endospanin-1, overexpression decreases muscle mitochondrial ROS production, induces fast-to-slow fiber-type switch, increases skeletal muscle glycogen content, and improves glucose homeostasis, ultimately promoting running endurance capacity. In line, endospanin-2-/- mice display higher lipid peroxidation levels, increased mitochondrial ROS production under mitochondrial stress, decreased ERK phosphorylation, and reduced endurance capacity. In conclusion, our results identify endospanin-2 as a potentially novel player in skeletal muscle metabolism, plasticity, and function

Image_2_Hepatitis C virus alters the morphology and function of peroxisomes.TIF

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.</p

Image_5_Hepatitis C virus alters the morphology and function of peroxisomes.TIF

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.</p