Serotonin reuptake inhibitors improve muscle stem cell function and muscle regeneration in male mice.

Serotonin reuptake inhibitor antidepressants such as fluoxetine are widely used to treat mood disorders. The mechanisms of action include an increase in extracellular level of serotonin, neurogenesis, and growth of vessels in the brain. We investigated whether fluoxetine could have broader peripheral regenerative properties. Following prolonged administration of fluoxetine in male mice, we showed that fluoxetine increases the number of muscle stem cells and muscle angiogenesis, associated with positive changes in skeletal muscle function. Fluoxetine also improved skeletal muscle regeneration after single and multiples injuries with an increased muscle stem cells pool and vessel density associated with reduced fibrotic lesions and inflammation. Mice devoid of peripheral serotonin treated with fluoxetine did not exhibit beneficial effects during muscle regeneration. Specifically, pharmacological, and genetic inactivation of the 5-HT1B subtype serotonin receptor also abolished the enhanced regenerative process induced by fluoxetine. We highlight here a regenerative property of serotonin on skeletal muscle.

Intracellular activity and in vivo efficacy in a mouse model of septic arthritis of the novel pseudopeptide Pep16 against Staphylococcus aureus clinical isolates.

Objectives: Assessing the therapeutic potential of a novel antimicrobial pseudopeptide, Pep16, both in vitro and in vivo for the treatment of septic arthritis caused by Staphylococcus aureus. Methods: Seven clinical isolates of S. aureus (two MRSA and five MSSA) were studied. MICs of Pep16 and comparators (vancomycin, teicoplanin, daptomycin and levofloxacin) were determined through the broth microdilution method. The intracellular activity of Pep16 and levofloxacin was assessed in two models of infection using non-professional (osteoblasts MG-63) or professional (macrophages THP-1) phagocytic cells. A mouse model of septic arthritis was used to evaluate the in vivo efficacy of Pep16 and vancomycin. A preliminary pharmacokinetic (PK) analysis was performed by measuring plasma concentrations using LC-MS/MS following a single subcutaneous injection of Pep16 (10 mg/kg). Results: MICs of Pep16 were consistently at 8 mg/L for all clinical isolates of S. aureus (2- to 32-fold higher to those of comparators) while MBC/MIC ratios confirmed its bactericidal activity. Both Pep16 and levofloxacin (when used at 2 × MIC) significantly reduced the bacterial load of all tested isolates (two MSSA and two MRSA) within both osteoblasts and macrophages. In MSSA-infected mice, Pep16 demonstrated a significant (∼10-fold) reduction on bacterial loads in knee joints. PK analysis following a single subcutaneous administration of Pep16 revealed a gradual increase in plasma concentrations, reaching a peak of 5.6 mg/L at 12 h. Conclusions: Pep16 is a promising option for the treatment of septic arthritis due to S. aureus, particularly owing to its robust intracellular activity.

Synergistic Activity of Pep16, a Promising New Antibacterial Pseudopeptide against Multidrug-Resistant Organisms, in Combination with Colistin against Multidrug-Resistant Escherichia coli, In Vitro and in a Murine Peritonitis Model.

Colistin is a drug of last resort to treat extreme drug-resistant Enterobacterales, but is limited by dose-dependent toxicity and the emergence of resistance. A recently developed antimicrobial pseudopeptide, Pep16, which acts on the cell membrane, may be synergistic with colistin and limit the emergence of resistance. We investigated Pep16 activity against Escherichia coli with varying susceptibility to colistin, in vitro and in a murine peritonitis model. Two isogenic derivatives of E. coli CFT073 (susceptible and resistant to colistin) and 2 clinical isolates (susceptible (B119) and resistant to colistin (Af31)) were used. Pep16 activity, alone and in combination with colistin, was determined in vitro (checkerboard experiments, time-kill curves, and flow cytometry to investigate membrane permeability). Toxicity and pharmacokinetic analyses of subcutaneous Pep16 were performed in mice, followed by the investigation of 10 mg/kg Pep16 + 10 mg/kg colistin (mimicking human concentrations) in a murine peritonitis model. Pep16 alone was inactive (MICs = 32-64 mg/L; no bactericidal effect). A concentration-dependent bactericidal synergy of Pep16 with colistin was evidenced on all strains, confirmed by flow cytometry. In vivo, Pep16 alone was ineffective. When Pep16 and colistin were combined, a significant decrease in bacterial counts in the spleen was evidenced, and the combination prevented the emergence of colistin-resistant mutants, compared to colistin alone. Pep16 synergizes with colistin in vitro, and the combination is more effective than colistin alone in a murine peritonitis by reducing bacterial counts and the emergence of resistance. Pep16 may optimize colistin use, by decreasing the doses needed, while limiting the emergence of colistin-resistant mutants.

Using a multiomics approach to unravel a septic shock specific signature in skeletal muscle.

Sepsis is defined as a dysregulated host response to infection leading to organs failure. Among them, sepsis induces skeletal muscle (SM) alterations that contribute to acquired-weakness in critically ill patients. Proteomics and metabolomics could unravel biological mechanisms in sepsis-related organ dysfunction. Our objective was to characterize a distinctive signature of septic shock in human SM by using an integrative multi-omics approach. Muscle biopsies were obtained as part of a multicenter non-interventional prospective study. Study population included patients in septic shock (S group, with intra-abdominal source of sepsis) and two critically ill control populations: cardiogenic shock (C group) and brain dead (BD group). The proteins and metabolites were extracted and analyzed by High-Performance Liquid Chromatography-coupled to tandem Mass Spectrometry, respectively. Fifty patients were included, 19 for the S group (53% male, 64 ± 17 years, SAPS II 45 ± 14), 12 for the C group (75% male, 63 ± 4 years, SAPS II 43 ± 15), 19 for the BD group (63% male, 58 ± 10 years, SAPS II 58 ± 9). Biopsies were performed in median 3 days [interquartile range 1-4]) after intensive care unit admission. Respectively 31 patients and 40 patients were included in the proteomics and metabolomics analyses of 2264 proteins and 259 annotated metabolites. Enrichment analysis revealed that mitochondrial pathways were significantly decreased in the S group at protein level: oxidative phosphorylation (adjusted p = 0.008); branched chained amino acids degradation (adjusted p = 0.005); citrate cycle (adjusted p = 0.005); ketone body metabolism (adjusted p = 0.003) or fatty acid degradation (adjusted p = 0.008). Metabolic reprogramming was also suggested (i) by the differential abundance of the peroxisome proliferator-activated receptors signaling pathway (adjusted p = 0.007), and (ii) by the accumulation of fatty acids like octanedioic acid dimethyl or hydroxydecanoic. Increased polyamines and depletion of mitochondrial thioredoxin or mitochondrial peroxiredoxin indicated a high level of oxidative stress in the S group. Coordinated alterations in the proteomic and metabolomic profiles reveal a septic shock signature in SM, highlighting a global impairment of mitochondria-related metabolic pathways, the depletion of antioxidant capacities, and a metabolic shift towards lipid accumulation.ClinicalTrial registration: NCT02789995. Date of first registration 03/06/2016.

Defective angiogenesis in CXCL12 mutant mice impairs skeletal muscle regeneration.

BACKGROUND: During muscle regeneration, the chemokine CXCL12 (SDF-1) and the synthesis of some specific heparan sulfates (HS) have been shown to be critical. CXCL12 activity has been shown to be heavily influenced by its binding to extracellular glycosaminoglycans (GAG) by modulating its presentation to its receptors and by generating haptotactic gradients. Although CXCL12 has been implicated in several phases of tissue repair, the influence of GAG binding under HS influencing conditions such as acute tissue destruction remains understudied. METHODS: To investigate the role of the CXCL12/HS proteoglycan interactions in the pathophysiology of muscle regeneration, we performed two models of muscle injuries (notexin and freeze injury) in mutant CXCL12Gagtm/Gagtm mice, where the CXCL12 gene having been selectively mutated in critical binding sites of CXCL12 to interact with HS. Histological, cytometric, functional transcriptomic, and ultrastructure analysis focusing on the satellite cell behavior and the vessels were conducted on muscles before and after injuries. Unless specified, statistical analysis was performed with the Mann-Whitney test. RESULTS: We showed that despite normal histology of the resting muscle and normal muscle stem cell behavior in the mutant mice, endothelial cells displayed an increase in the angiogenic response in resting muscle despite the downregulated transcriptomic changes induced by the CXCL12 mutation. The regenerative capacity of the CXCL12-mutated mice was only delayed after a notexin injury, but a severe damage by freeze injury revealed a persistent defect in the muscle regeneration of CXCL12 mutant mice associated with vascular defect and fibroadipose deposition with persistent immune cell infiltration. CONCLUSION: The present study shows that CXCL12 is crucial for proper muscle regeneration. We highlight that this homing molecule could play an important role in drastic muscle injuries and that the regeneration defect could be due to an impairment of angiogenesis, associated with a long-lasting fibro-adipogenic scar.

Beneficial role of adipose-derived mesenchymal stem cells from microfragmented fat in a murine model of duchenne muscular dystrophy.

INTRODUCTION: No etiologic therapy is available for Duchenne muscular dystrophy (DMD), but mesenchymal stem cells were shown to be effective in preclinical models of DMD. The objective of this study is to investigate the effect of microfragmented fat extracted on a murine model of DMD. METHODS: Fat tissue was extracted from healthy human participants and injected IM into DMD mice. Histological analysis, cytokines, and force measurement were performed up to 4 weeks after injection. RESULTS: Duchenne muscular dystrophy mice injected with microfragmented fat exhibited an improved muscle phenotype (decreased necrosis and fibrosis), a decrease of inflammatory cytokines, and increased strength. DISCUSSION: Administration of microfragmented fat in key muscles may improve muscular phenotype in patients with DMD. Muscle Nerve, 2019.

A novel paradigm links mitochondrial dysfunction with muscle stem cell impairment in sepsis.

Sepsis is an acute systemic inflammatory response of the body to microbial infection and a life threatening condition associated with multiple organ failure. Survivors may display long-term disability with muscle weakness that remains poorly understood. Recent data suggest that long-term myopathy in sepsis survivors is due to failure of skeletal muscle stem cells (satellite cells) to regenerate the muscle. Satellite cells impairment in the acute phase of sepsis is linked to unusual mitochondrial dysfunctions, characterized by a dramatic reduction of the mitochondrial mass and hyperactivity of residual organelles. Survivors maintain the impairment of satellite cells, including alterations of the mitochondrial DNA (mtDNA), in the long-term. This condition can be rescued by treatment with mesenchymal stem cells (MSCs) that restore mtDNA alterations and mitochondrial function in satellite cells, and in fine their regenerative potential. Injection of MSCs in turn increases the force of isolated muscle fibers and of the whole animal, and improves the survival rate. These effects occur in the context of reduced inflammation markers that also raised during sepsis. Targeting muscle stem cells mitochondria, in a context of reduced inflammation, may represent a valuable strategy to reduce morbidity and long-term impairment of the muscle upon sepsis.

Muscle regeneration after sepsis.

Severe critical illness is often complicated by intensive care unit-acquired weakness (ICU-AW), which is associated with increased ICU and post-ICU mortality, delayed weaning from mechanical ventilation and long-term functional disability. Several mechanisms have been implicated in the pathophysiology of ICU-AW, but muscle regeneration has not been investigated to any extent in this context, even though its involvement is suggested by the protracted functional consequences of ICU-AW. Recent data suggest that muscle regeneration could be impaired after sepsis, and that mesenchymal stem cell treatment could improve the post-injury muscle recovery.

Comparative Study of Injury Models for Studying Muscle Regeneration in Mice.

BACKGROUND: A longstanding goal in regenerative medicine is to reconstitute functional tissues or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised. METHODS: We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®. RESULTS: We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models. CONCLUSIONS: Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.

Dormancy and quiescence of skeletal muscle stem cells.

The skeletal muscle of vertebrates has a huge regenerative capacity. When destroyed after different types of injury, this organ can regenerate very quickly (less than 20 days following myotoxin injection in the mouse) ad integrum and repeatedly. The cell responsible for this regeneration is the so-called satellite cell, the muscle stem cell that lies on top of the muscle fibre, a giant, multinucleated cell that contains the contractile material. When injected in the muscle, satellite cells can efficiently differentiate into contractile muscle fibres. The satellite cell shows great therapeutic potential; and its regenerative capacity has triggered particular interest in the field of muscular degeneration. In this review we will focus on one particular property of the satellite cell: its quiescence and dormancy. Indeed adult satellite cells are quiescent; they lie between the basal lamina and the basement membrane of the muscle fibre, ready to proliferate, and fuse in order to regenerate myofibers upon injury. It has recently been shown that a subpopulation of satellite cells is able to enter dormancy in human and mice cadavers. Dormancy is defined by a low metabolic state, low mobility, and a long lag before division when plated in vitro, compared to quiescent cells. This definition is also based on current knowledge about long-term hematopoietic stem cells, a subpopulation of stem cells that are described as dormant based on the same criteria (rare division and low metabolism when compared to progeny which are dividing more often). In the first part of this review, we will provide a description of satellite cells which addresses their quiescent state. We will then focus on the uneven distribution of satellite cells in the muscle and describe evidence that suggests that their dormancy differs from one muscle to the next and that one should be cautious when making generalisations regarding this cellular state. In a second part, we will discuss the transition between active dividing cells in developing animals to quiescence. This mechanism could be used or amplified in the switch from quiescence to dormancy. In a third part, we will review the signals and dynamics that actively maintain the satellite cell quiescent. The in-depth understanding of these mechanisms is key to describing how dormancy relies on quiescent state of the cells. In a fourth part, we will deal with dormancy per se: how dormant satellite cells can be obtained, their characteristics, their metabolic profile, and their molecular signature as compared to quiescent cells. Here, we will highlight one of the most important recent findings: that quiescence is a prerequisite for the entry of the satellite cell into dormancy. Since dormancy is a newly discovered phenomenon, we will review the mechanisms responsible for quiescence and activation, as these two cellular states are better known and key to understanding satellite cell dormancy. This will allow us to describe dormancy and its prerequisites.

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capacity.

The accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency are challenging issues for stem cell biology. Here we report the isolation of viable and functional skeletal myogenic cells from humans up to 17 days, and mice up to 14 days post mortem, much longer beyond previous reports. Muscle stem cells are enriched in post mortem tissue, suggesting a selective survival advantage compared with other cell types. Transplantation of mouse muscle and haematopoietic stem cells regenerates tissues robustly. Cellular quiescence contributes to this cell viability where cells adopt a reversible dormant state characterized by reduced metabolic activity, a prolonged lag phase before the first cell division, elevated levels of reactive oxygen species and a transcriptional status less primed for commitment. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and regenerative capacity. Thus, these cells provide a useful resource for studying stem cell biology.

A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division.

Satellite cells are adult skeletal muscle stem cells that are quiescent and constitute a poorly defined heterogeneous population. Using transgenic Tg:Pax7-nGFP mice, we show that Pax7-nGFP(Hi) cells are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFP(Lo) cells. Pax7-nGFP(Hi) can give rise to Pax7-nGFP(Lo) cells after serial transplantations. Proliferating Pax7-nGFP(Hi) cells exhibit lower metabolic activity, and the majority performs asymmetric DNA segregation during cell division, wherein daughter cells retaining template DNA strands express stem cell markers. Using chromosome orientation-fluorescence in situ hybridization, we demonstrate that all chromatids segregate asymmetrically, whereas Pax7-nGFP(Lo) cells perform random DNA segregation. Therefore, quiescent Pax7-nGFP(Hi) cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFP(Hi) cells generate distinct daughter cell fates by asymmetrically segregating template DNA strands to the stem cell. These findings provide major insights into the biology of stem cells that segregate DNA asymmetrically.

A critical requirement for notch signaling in maintenance of the quiescent skeletal muscle stem cell state.

Notch signaling plays a key role in virtually all tissues and organs in metazoans; however, limited examples are available for the regulatory role of this pathway in adult quiescent stem cells. We performed a temporal and ontological assessment of effectors of the Notch pathway that indicated highest activity in freshly isolated satellite cells and, unexpectedly, a sharp decline before the first mitosis, and subsequently in proliferating, satellite cell-derived myoblasts. Using genetic tools to conditionally abrogate canonical Notch signaling during homeostasis, we demonstrate that satellite cells differentiate spontaneously and contribute to myofibers, thereby resulting in a severe depletion of the stem cell pool. Furthermore, whereas loss of Rbpj function provokes some satellite cells to proliferate before fusing, strikingly, the majority of mutant cells terminally differentiate unusually from the quiescent state, without passing through S-phase. This study establishes Notch signaling pathway as the first regulator of cellular quiescence in adult muscle stem cells.

Numb promotes an increase in skeletal muscle progenitor cells in the embryonic somite.

Multiple cell types arise from cells in the dermomyotome of the somite that express Pax3 and Pax7, and myogenesis is regulated by Notch signaling. The asymmetric cell fate determinant Numb is thought to promote differentiation of skeletal muscle and other lineages by negatively regulating Notch signaling. We used transgenesis to overexpress Numb spatiotemporally in Pax3(+)/Pax7(+) somitic stem and progenitor cells in mouse embryos using a spatiotemporally regulated enhancer element from the Myf5 locus that can target muscle progenitor cells prior to cell commitment. Molecular analyses as well as examination of dermal and skeletal muscle cell fates in vivo show that although Numb is thought to be associated with muscle differentiation, unexpectedly the common stem/progenitor pool size for these lineages is increased in Numb-transgenic embryos. Prospective isolation of the relevant transgenic cells and analysis by quantitative reverse-transcription polymerase chain reaction demonstrated that, in this context, canonical Notch targets are not significantly downregulated. These findings were corroborated using a Notch reporter mouse during the formation of somites and prior to lineage segregation. Thus, we propose that Numb can regulate the self-renewal of dermal and muscle progenitors during a lineage progression.

Asymmetric cell divisions and asymmetric cell fates.

The regulation of self-renewal, cell diversity, and differentiation can occur by modulating symmetric and asymmetric cell divisions. Remarkably, asymmetric cell divisions can arise through multiple processes in which molecules in the cytoplasm and nucleus, as well as template "immortal" DNA strands, can segregate to one daughter cell during cell division. Explaining how these events direct distinct daughter cell fates is a major challenge to understanding how the organism is assembled and maintained for a lifetime. Numerous technical issues that are associated with assessing how distinct cell fates are executed in vivo have resulted in divergent interpretations of experimental findings. This review addresses some of these points and considers different developmental model systems that attempt to investigate how cell fate decisions are determined, as well as the molecules that guide these choices.

[Immortal DNA or epigenetic signature ?]. / ADN immortel ou signature épigénétique ?

During mitosis each daughter cell inherits a full copy of the maternal genomic material. DNA replication, however, is an imprecise process, thus errors can arise resulting in potentially deleterious mutations over extended rounds of cell division and these may lead to cancinogenesis. Over thirty years ago, J. Cairns proposed that a cell could avoid the accumulation of mutations arising from DNA replication if all template DNA strands are inherited in one daughter cell during cell division, thus giving rise to the notion of << immortal >> DNA strands. In this model the stem cells would retain the template DNA (older) strands. Proving or disproving this notion experimentally has been challenging. Further, it has recently become apparent that epigenetic regulation of gene expression plays a critical role in governing cell states, self-renewal and differentiation. In light of these data, can the phenomenon on template DNA strand segregation also reflect this epigenetic signature? In this review we explore these notions, discuss the evidence in support of this theory, the implications, and some of the mechanisms which could explain this phenomenon.