Dysfunction of programmed embryo senescence is linked to genetic developmental defects.

Developmental senescence is a form of programmed senescence that contributes to morphogenesis during embryonic development. We showed recently that the SIX1 homeoprotein, an essential regulator of organogenesis, is also a repressor of adult cellular senescence. Alterations in the SIX/EYA pathway are linked to the human branchio-oto-renal (BOR) syndrome, a rare congenital disorder associated with defects in the ears, kidneys and branchial arches. Here, we have used Six1-deficient mice, an animal model of the BOR syndrome, to investigate whether dysfunction of senescence underpins the developmental defects associated with SIX1 deficiency. We have focused on the developing inner ear, an organ with physiological developmental senescence that is severely affected in Six1-deficient mice and BOR patients. We show aberrant levels and distribution of senescence markers in Six1-deficient inner ears concomitant with defective morphogenesis of senescent structures. Transcriptomic analysis and ex vivo assays support a link between aberrant senescence and altered morphogenesis in this model, associated with deregulation of the TGFß/BMP pathway. Our results show that misregulation of embryo senescence may lead to genetic developmental disorders, significantly expanding the connection between senescence and disease.

Overlapping functions of SIX homeoproteins during embryonic myogenesis.

Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. Here, we provide a deep characterization of their role in distinct mouse developmental territories. We showed, at the hypaxial level, that the Six1:Six4 double knockout (dKO) somitic precursor cells adopt a smooth muscle fate and lose their myogenic identity. At the epaxial level, we demonstrated by the analysis of Six quadruple KO (qKO) embryos, that SIX are required for fetal myogenesis, and for the maintenance of PAX7+ progenitor cells, which differentiated prematurely and are lost by the end of fetal development in qKO embryos. Finally, we showed that Six1 and Six2 are required to establish craniofacial myogenesis by controlling the expression of Myf5. We have thus described an unknown role for SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades operating at the head level and in the genesis of myogenic stem cells.

SIX1 and SIX4 homeoproteins regulate PAX7+ progenitor cell properties during fetal epaxial myogenesis.

Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the sine oculis-related homeobox Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here, we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 knockout mice (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior muscle. Mutant stem cells contribute to hypotrophic myofibers that are not innervated but retain the ability to self-renew.

Population Pharmacokinetics of Vancomycin in Patients Receiving Hemodialysis in a Malian and a French Center and Simulation of the Optimal Loading Dose.

PURPOSE: Vancomycin dosing remains challenging in patients receiving intermittent hemodialysis, especially in developing countries, where access to therapeutic drug monitoring and model-based dose adjustment services is limited. The objectives of this study were to describe vancomycin population PK in patients receiving hemodialysis in a Malian and French center and examine the optimal loading dose of vancomycin in this setting. METHODS: Population pharmacokinetic analysis was conducted using Pmetrics in 31 Malian and 27 French hemodialysis patients, having a total of 309 vancomycin plasma concentrations. Structural and covariate analyses were based on goodness-of-fit criteria. The final model was used to perform simulations of the vancomycin loading dose, targeting a daily area under the concentration-time curve (AUC) of 400-600 mg.h/L or trough concentration of 15-20 mg/L at 48 hours. RESULTS: After 48 hours of therapy, 68% of Malian and 63% of French patients exhibited a daily AUC of <400. The final model was a 2-compartment model, with hemodialysis influencing vancomycin elimination and age influencing the vancomycin volume distribution. Younger Malian patients exhibited a lower distribution volume than French patients. Dosing simulation suggested that loading doses of 1500, 2000, and 2500 mg would be required to minimize underexposure in patients aged 30, 50, and 70 years, respectively. CONCLUSIONS: In this study, a low AUC was frequently observed in hemodialysis patients in Mali and France after a standard vancomycin loading dose. A larger dose is necessary to achieve the currently recommended AUC target. However, the proposed dosing algorithm requires further clinical evaluation.

Unexpected role of SIX1 variants in craniosynostosis: expanding the phenotype of SIX1-related disorders.

BACKGROUND: Pathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported. METHODS: We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1nLacZ/+ reporter mouse. RESULTS: From 1629 unrelated cases with craniosynostosis we identified seven different SIX1 variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of SIX1 loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme. CONCLUSION: Craniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis.

Myogenesis control by SIX transcriptional complexes.

SIX homeoproteins were first described in Drosophila, where they participate in the Pax-Six-Eya-Dach (PSED) network with eyeless, eyes absent and dachsund to drive synergistically eye development through genetic and biochemical interactions. The role of the PSED network and SIX proteins in muscle formation in vertebrates was subsequently identified. Evolutionary conserved interactions with EYA and DACH proteins underlie the activity of SIX transcriptional complexes (STC) both during embryogenesis and in adult myofibers. Six genes are expressed throughout muscle development, in embryonic and adult proliferating myogenic stem cells and in fetal and adult post-mitotic myofibers, where SIX proteins regulate the expression of various categories of genes. In vivo, SIX proteins control many steps of muscle development, acting through feedforward mechanisms: in the embryo for myogenic fate acquisition through the direct control of Myogenic Regulatory Factors; in adult myofibers for their contraction/relaxation and fatigability properties through the control of genes involved in metabolism, sarcomeric organization and calcium homeostasis. Furthermore, during development and in the adult, SIX homeoproteins participate in the genesis and the maintenance of myofibers diversity.

To Estimate or to Forecast? Lessons From a Comparative Analysis of Four Bayesian Fitting Methods Based on Nonparametric Models.

ABSTRACT: Using pharmacokinetic (PK) models and Bayesian methods in dosing software facilitates the analysis of individual PK data and precision dosing. Several Bayesian methods are available for computing Bayesian posterior distributions using nonparametric population models. The objective of this study was to compare the performance of the maximum a posteriori (MAP) model, multiple model (MM), interacting MM (IMM), and novel hybrid MM(HMM) in estimating past concentrations and predicting future concentrations during therapy. Amikacin and vancomycin PK data were analyzed in older hospitalized patients using 2 strategies. First, the entire data set of each patient was fitted using each of the 4 methods implemented in BestDose software. Then, the 4 methods were used in each therapeutic drug monitoring occasion to estimate the past concentrations available at this time and to predict the subsequent concentrations to be observed on the next occasion. The bias and precision of the model predictions were compared among the methods. A total of 406 amikacin concentrations from 96 patients and 718 vancomycin concentrations from 133 patients were available for analysis. Overall, significant differences were observed in the predictive performance of the 4 Bayesian methods. The IMM method showed the best fit to past concentration data of amikacin and vancomycin, whereas the MM method was the least precise. However, MM best predicted the future concentrations of amikacin. The MAP and HMM methods showed a similar predictive performance and seemed to be more appropriate for the prediction of future vancomycin concentrations than the other models were. The richness of the prior distribution may explain the discrepancies between the results of the 2 drugs. Although further research with other drugs and models is necessary to confirm our findings, these results challenge the widely accepted assumption in PK modeling that a better data fit indicates better forecasting of future observations.

Six1 is essential for differentiation and patterning of the mammalian auditory sensory epithelium.

The organ of Corti in the cochlea is a two-cell layered epithelium: one cell layer of mechanosensory hair cells that align into one row of inner and three rows of outer hair cells interdigitated with one cell layer of underlying supporting cells along the entire length of the cochlear spiral. These two types of epithelial cells are derived from common precursors in the four- to five-cell layered primordium and acquire functionally important shapes during terminal differentiation through the thinning process and convergent extension. Here, we have examined the role of Six1 in the establishment of the auditory sensory epithelium. Our data show that prior to terminal differentiation of the precursor cells, deletion of Six1 leads to formation of only a few hair cells and defective patterning of the sensory epithelium. Previous studies have suggested that downregulation of Sox2 expression in differentiating hair cells must occur after Atoh1 mRNA activation in order to allow Atoh1 protein accumulation due to antagonistic effects between Atoh1 and Sox2. Our analysis indicates that downregulation of Sox2 in the differentiating hair cells depends on Six1 activity. Furthermore, we found that Six1 is required for the maintenance of Fgf8 expression and dynamic distribution of N-cadherin and E-cadherin in the organ of Corti during differentiation. Together, our analyses uncover essential roles of Six1 in hair cell differentiation and formation of the organ of Corti in the mammalian cochlea.

[Injectable concentrated potassium concentrate, emblematic of the lack of safety of the drug use process in French hospitals]. / Le potassium injectable concentré, emblématique de l'insécurité médicamenteuse des hôpitaux français.

Despite its proven dangers, the ward stock drug distribution system predominates in French hospitals. This system allows 12 million injectable ampoules of concentrated potassium chloride to circulate uncontrolled each year. Such a situation is absurd for the following reasons : 1) injected by mistake, concentrated potassium kills within seconds ; 2) the true incidence of potassium-related fatalities and incidents is unknown ; 3) fatal intravenous injection of potassium produces no specific anatomical changes and subtle, if any, findings at autopsy ; 4) it is used for capital punishment by lethal injection in various countries ; and 5) healthcare worker serial killers benefit from the fact that potassium is not identifiable in post-mortem examinations and that investigations to find the murderer are complex and of uncertain outcome. Other medications classed as high-risk have similar characteristics to those of concentrated potassium solutions. Injectable potassium can therefore be regarded as emblematic of the lack of safety of the drug use process in French hospitals. The priority measure to protect patients from this deadly risk is to remove these drugs from uncontrolled ward stocks and to provide premixed potassium solutions. Evidence of the increased safety of the unit dose drug dispensing system should compel health policy makers to systematically implement it, thus bringing the drug use process into compliance with existing French and European regulations.

Le potassium injectable concentré, emblématique de l'insécurité médicamenteuse des hôpitaux français.

Despite its proven dangers, the ward stock drug distribution system predominates in French hospitals. This system allows 12 million injectable ampoules of concentrated potassium chloride to circulate uncontrolled each year. Such a situation is absurd for the following reasons : 1) injected by mistake, concentrated potassium kills within seconds ; 2) the true incidence of potassium-related fatalities and incidents is unknown ; 3) fatal intravenous injection of potassium produces no specific anatomical changes and subtle, if any, findings at autopsy ; 4) it is used for capital punishment by lethal injection in various countries ; and 5) healthcare worker serial killers benefit from the fact that potassium is not identifiable in post-mortem examinations and that investigations to find the murderer are complex and of uncertain outcome. Other medications classed as high-risk have similar characteristics to those of concentrated potassium solutions. Injectable potassium can therefore be regarded as emblematic of the lack of safety of the drug use process in French hospitals. The priority measure to protect patients from this deadly risk is to remove these drugs from uncontrolled ward stocks and to provide premixed potassium solutions. Evidence of the increased safety of the unit dose drug dispensing system should compel health policy makers to systematically implement it, thus bringing the drug use process into compliance with existing French and European regulations.

Engrailed controls epaxial-hypaxial muscle innervation and the establishment of vertebrate three-dimensional mobility.

Chordates are characterised by contractile muscle on either side of the body that promotes movement by side-to-side undulation. In the lineage leading to modern jawed vertebrates (crown group gnathostomes), this system was refined: body muscle became segregated into distinct dorsal (epaxial) and ventral (hypaxial) components that are separately innervated by the medial and hypaxial motors column, respectively, via the dorsal and ventral ramus of the spinal nerves. This allows full three-dimensional mobility, which in turn was a key factor in their evolutionary success. How the new gnathostome system is established during embryogenesis and how it may have evolved in the ancestors of modern vertebrates is not known. Vertebrate Engrailed genes have a peculiar expression pattern as they temporarily demarcate a central domain of the developing musculature at the epaxial-hypaxial boundary. Moreover, they are the only genes known with this particular expression pattern. The aim of this study was to investigate whether Engrailed genes control epaxial-hypaxial muscle development and innervation. Investigating chick, mouse and zebrafish as major gnathostome model organisms, we found that the Engrailed expression domain was associated with the establishment of the epaxial-hypaxial boundary of muscle in all three species. Moreover, the outgrowing epaxial and hypaxial nerves orientated themselves with respect to this Engrailed domain. In the chicken, loss and gain of Engrailed function changed epaxial-hypaxial somite patterning. Importantly, in all animals studied, loss and gain of Engrailed function severely disrupted the pathfinding of the spinal motor axons, suggesting that Engrailed plays an evolutionarily conserved role in the separate innervation of vertebrate epaxial-hypaxial muscle.

MyoD reprogramming requires Six1 and Six4 homeoproteins: genome-wide cis-regulatory module analysis.

Myogenic regulatory factors of the MyoD family have the ability to reprogram differentiated cells toward a myogenic fate. In this study, we demonstrate that Six1 or Six4 are required for the reprogramming by MyoD of mouse embryonic fibroblasts (MEFs). Using microarray experiments, we found 761 genes under the control of both Six and MyoD. Using MyoD ChIPseq data and a genome-wide search for Six1/4 MEF3 binding sites, we found significant co-localization of binding sites for MyoD and Six proteins on over a thousand mouse genomic DNA regions. The combination of both datasets yielded 82 genes which are synergistically activated by Six and MyoD, with 96 associated MyoD+MEF3 putative cis-regulatory modules (CRMs). Fourteen out of 19 of the CRMs that we tested demonstrated in Luciferase assays a synergistic action also observed for their cognate gene. We searched putative binding sites on these CRMs using available databases and de novo search of conserved motifs and demonstrated that the Six/MyoD synergistic activation takes place in a feedforward way. It involves the recruitment of these two families of transcription factors to their targets, together with partner transcription factors, encoded by genes that are themselves activated by Six and MyoD, including Mef2, Pbx-Meis and EBF.

Population pharmacokinetics of teicoplanin administered by subcutaneous or intravenous route and simulation of optimal loading dose regimen.

Objectives: To investigate the population pharmacokinetics of teicoplanin in patients treated by the subcutaneous (sc) and/or intravenous (iv) route. Patients and methods: Non-linear mixed-effects modelling described teicoplanin concentrations from 98 patients with infection caused by Gram-positive cocci. Monte Carlo simulations were performed to evaluate the probability of target attainment (PTA) of various dosage regimens. Results: Teicoplanin concentrations were best described by a two-compartment model with clearance predicted by estimated glomerular filtration rate. Estimated absorption rate constant (between-subject variability) was 0.039 h-1 (77%), clearance was 0.305 L/h (28%), central volume was 10.3 L (49%), inter-compartmental clearance was 4.42 L/h (66%) and peripheral volume was 97.4 L (51%). The sc route was associated with lower initial Cmin and AUC (day 3: loading phase) compared with the iv route. This difference appeared to vanish after 14 days, with comparable simulated PTAs based on the Cmin and AUC for all tested dosages (400, 600, 800 and 1000 mg every 12 h). However, a loading dose regimen with five administrations of either 400 or 600 mg was not sufficient to achieve the target Cmin (≥15 mg/L) for both routes. Also, PTAs for higher MIC (≥1.0 mg/L) were poor with all regimens for both routes. Conclusions: This is the first study examining the pharmacokinetic/pharmacodynamic implications of using the sc route for teicoplanin. Subcutaneous administration is associated with lower Cmin and AUC values after the loading phase compared with iv administration. Therefore, iv administration should be preferred in the first few days of therapy. This study also shows that loading doses of teicoplanin higher than currently recommended should be used to improve PTA.

Six homeoproteins and a Iinc-RNA at the fast MYH locus lock fast myofiber terminal phenotype.

Thousands of long intergenic non-coding RNAs (lincRNAs) are encoded by the mammalian genome. However, the function of most of these lincRNAs has not been identified in vivo. Here, we demonstrate a role for a novel lincRNA, linc-MYH, in adult fast-type myofiber specialization. Fast myosin heavy chain (MYH) genes and linc-MYH share a common enhancer, located in the fast MYH gene locus and regulated by Six1 homeoproteins. linc-MYH in nuclei of fast-type myofibers prevents slow-type and enhances fast-type gene expression. Functional fast-sarcomeric unit formation is achieved by the coordinate expression of fast MYHs and linc-MYH, under the control of a common Six-bound enhancer.

Mathematical modeling and systems pharmacology of tuberculosis: Isoniazid as a case study.

Tuberculosis (TB) treatment needs to be optimized as it is currently long and associated with increasing drug resistance. The antimycobacterial effect of isoniazid (INH) is characterized by a biphasic kill curve, whose causes are still debated. In this work, we developed a complete mathematical model describing the time-course of TB infection and its treatment by INH in human lung. This model was based on a pharmacokinetic model, a pharmacodynamic model and a pathophysiological model. It was used to simulate the antibacterial effect of INH during the first days of therapy. This full model adequately reproduced some qualitative and quantitative properties of the early bactericidal activity of INH observed in TB patients. The kill curves simulated with the model reproduced the biphasic killing effect of INH and the predicted declines in extracellular bacteria were comparable to clinical data. A sensitivity analysis provided interesting insights regarding the biphasic kill curve. The first phase appeared to be essentially driven by the drug effect. In the second phase, while drug pharmacology was the major determinant of the antibacterial effect, a slight influence of the dynamics of infected macrophages was also observed. This work permits to formulate hypotheses for optimizing the efficacy of TB drug candidates and confirms the utility of mathematical modeling to generate new assumptions for TB research.

Six homeoproteins directly activate Myod expression in the gene regulatory networks that control early myogenesis.

In mammals, several genetic pathways have been characterized that govern engagement of multipotent embryonic progenitors into the myogenic program through the control of the key myogenic regulatory gene Myod. Here we demonstrate the involvement of Six homeoproteins. We first targeted into a Pax3 allele a sequence encoding a negative form of Six4 that binds DNA but cannot interact with essential Eya co-factors. The resulting embryos present hypoplasic skeletal muscles and impaired Myod activation in the trunk in the absence of Myf5/Mrf4. At the axial level, we further show that Myod is still expressed in compound Six1/Six4:Pax3 but not in Six1/Six4:Myf5 triple mutant embryos, demonstrating that Six1/4 participates in the Pax3-Myod genetic pathway. Myod expression and head myogenesis is preserved in Six1/Six4:Myf5 triple mutant embryos, illustrating that upstream regulators of Myod in different embryonic territories are distinct. We show that Myod regulatory regions are directly controlled by Six proteins and that, in the absence of Six1 and Six4, Six2 can compensate.

Influence of renal function estimation on pharmacokinetic modeling of vancomycin in elderly patients.

Vancomycin is a renally excreted drug, and its body clearance correlates with creatinine clearance. However, the renal function estimation equation that best predicts vancomycin clearance has not been established yet. The objective of this study was to compare the abilities of different renal function estimation equations to describe vancomycin pharmacokinetics in elderly patients. The NPAG algorithm was used to perform population pharmacokinetic analysis of vancomycin concentrations in 78 elderly patients. Six pharmacokinetic models of vancomycin clearance were built, based on the following equations: Cockcroft-Gault (CG), Jelliffe (JEL), Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) (both in milliliters per minute per 1.73 m(2)), and modified MDRD and CKD-EPI equations (both in milliliters per minute). Goodness-of-fit and predictive performances of the six PK models were compared in a learning set (58 subjects) and a validation set (20 patients). Final analysis was performed to estimate population parameters in the entire population. In the learning step, the MDRD-based model best described the data, but the CG- and JEL-based models were the least biased. The mean weighted errors of prediction were significantly different between the six models (P = 0.0071). In the validation group, predictive performances were not significantly different. However, the use of a renal function estimation equation different from that used in the model building could significantly alter predictive performance. The final analysis showed important differences in parameter distributions and AUC estimation across the six models. This study shows that methods used to estimate renal function should not be considered interchangeable for pharmacokinetic modeling and model-based estimation of vancomycin concentrations in elderly patients.

AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle-specific AMPKα1α2 double-knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle exercise capacity, mitochondrial function, and contraction-stimulated glucose uptake. Exercise performance was significantly reduced in the mdKO mice, with a reduction in maximal force production and fatigue resistance. An increase in the proportion of myofibers with centralized nuclei was noted, as well as an elevated expression of interleukin 6 (IL-6) mRNA, possibly consistent with mild skeletal muscle injury. Notably, we found that AMPKα1 and AMPKα2 isoforms are dispensable for contraction-induced skeletal muscle glucose transport, except for male soleus muscle. However, the lack of skeletal muscle AMPK diminished maximal ADP-stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial substrate utilization but not baseline mitochondrial muscle content. Together, these results demonstrate that skeletal muscle AMPK has an unexpected role in the regulation of mitochondrial oxidative phosphorylation that contributes to the energy demands of the exercising muscle.-Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J. T., Pehmøller, C., Sanz, N., Sakakibara, I., Saint-Amand, E., Rimbaud, S., Maire, P., Marette, A., Ventura-Clapier, R., Ferry, A., Wojtaszewski, J. F. P., Foretz, M., Viollet, B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.

[New insights into adult muscle fiber-type diversity: involvement of Six homeoproteins]. / Diversité des fibres musculaires squelettiques, rôle des homéoprotéines Six.

The mechanisms by which fast/slow muscle fiber diversity emerges during mammalian development are poorly understood, although fast/slow motoneuron activity is known to be an important stimulus controlling fiber-type maintenance and transition in adults. The key role of innervation in the maintenance of the slow-phenotype of the mature adult fibers has been amply demonstrated through denervation and cross-innervation experiments, as well as by the use of various electrical stimulation paradigms. Several signaling pathways have been reported to link nerve stimulation (inducing intracellular calcium elevation) to the maintenance of the slow muscle fiber phenotype. Less is known about the transcription factors and signaling pathways responsible for the fast IIB-glycolytic phenotype. Six1 homeoprotein accumulates at higher levels in the nuclei of adult fast-type myofibers, and Six transcription complexes are far more active in fast/glycolytic fibers than in slow/oxidative fibers. Ectopic expression of Six proteins in the slow soleus muscle leads to a switch from the slow/oxidative phenotype to the fast/glycolytic phenotype, confirming the involvement of this transcriptional complex in the fast/glycolytic phenotype. We have further shown that Six homeoprotein-mutant mice exhibit an impaired capacity to generate fast-type myofibers during embryonic development, and that adult myofibers deprived of Six1 have a slower phenotype. We have also characterized the gene networks controlled by Six homeoproteins at various developmental stages and have shown that, in the adult myofiber, Six proteins control the activity of an enhancer at the fast myosin heavy chain cluster. We identified a three-element genetic partnership in which this enhancer under the control of the myogenic homeoprotein Six1, functions as a regulatory hub that controls the fast fiber phenotype. In this partnership, the enhancer positively controls the expression of both the adjacent fast myosin heavy chain (MYH) gene cluster and Linc-MYH. Linc-MYH is present only in adult fast-type skeletal myofibers, where it suppresses slow-type gene expression.

RhoA Is a Crucial Regulator of Myoblast Fusion.

Satellite cells (SCs) are adult muscle stem cells that are mobilized when muscle homeostasis is perturbed. Here we show that RhoA in SCs is indispensable to have correct muscle regeneration and hypertrophy. In particular, the absence of RhoA in SCs prevents a correct SC fusion both to other RhoA-deleted SCs (regeneration context) and to growing control myofibers (hypertrophy context). We demonstrated that RhoA is dispensable for SCs proliferation and differentiation; however, RhoA-deleted SCs have an inefficient movement even if their cytoskeleton assembly is not altered. Proliferative myoblast and differentiated myotubes without RhoA display a decreased expression of Chordin, suggesting a crosstalk between these genes for myoblast fusion regulation. These findings demonstrate the importance of RhoA in SC fusion regulation and its requirement to achieve an efficient skeletal muscle homeostasis restoration.