MuscleJ2: a rebuilding of MuscleJ with new features for high-content analysis of skeletal muscle immunofluorescence slides.

Histological analysis of skeletal muscle is of major interest for understanding its behavior in different pathophysiological conditions, such as the response to different environments or myopathies. In this context, many software programs have been developed to perform automated high-content analysis. We created MuscleJ, a macro that runs in ImageJ/Fiji on batches of images. MuscleJ is a multianalysis tool that initially allows the analysis of muscle fibers, capillaries, and satellite cells. Since its creation, it has been used in many studies, and we have further developed the software and added new features, which are presented in this article. We converted the macro into a Java-language plugin with an improved user interface. MuscleJ2 provides quantitative analysis of fibrosis, vascularization, and cell phenotype in whole muscle sections. It also performs analysis of the peri-myonuclei, the individual capillaries, and any staining in the muscle fibers, providing accurate quantification within regional sublocalizations of the fiber. A multicartography option allows users to visualize multiple results simultaneously. The plugin is freely available to the muscle science community.

[Nuclear receptors and circadian clock: Implications for inflammatory diseases]. / Récepteurs nucléaires et rythmes circadiens - Implications dans les maladies inflammatoires.

The biological clock is a set of evolutionarily conserved "clock proteins" that generate circadian rhythms in behavior and physiological processes. The clock programs these processes at specific times of the day, allowing the organism to optimize its functions by anticipating predictable daily changes such as day/night, hence sleep/wake or feeding/fasting cycles. Modern lifestyle, i.e., exposure to light at night, shift work and irregular eating patterns and sleep schedules desynchronize the clocks residing in each organ. This dissonance is associated with an increased risk of developing various diseases such as cancer, metabolic, cardiovascular and chronic inflammatory diseases.

Title: Récepteurs nucléaires et rythmes circadiens - Implications dans les maladies inflammatoires. Abstract: L'horloge circadienne programme l'ensemble des processus physiologiques, dont l'activité du système immunitaire, à des moments précis de la journée. Elle permet d'optimiser les fonctions de l'organisme en anticipant les changements quotidiens tels que les cycles jour/nuit. Nos habitudes de vie comme l'exposition à la lumière artificielle ou une prise alimentaire irrégulière désynchronisent cependant cette horloge et provoquent des maladies, par exemple inflammatoires. Au niveau moléculaire, elle consiste en un réseau de facteurs de transcription dont certains sont des récepteurs nucléaires, activables par des ligands. Une meilleure compréhension des rythmes biologiques et du rôle des récepteurs nucléaires de l'horloge circadienne permettrait d'ouvrir un champ thérapeutique nouveau. La chronothérapie qui consiste en l'administration d'un composé pharmacologique au moment de la journée le plus propice, permettrait, en ciblant ces récepteurs, d'optimiser l'efficacité du traitement et d'en réduire les possibles effets secondaires.

NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression.

The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions, such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impaired sarco/endoplasmic reticulum calcium ATPase-dependent (SERCA-dependent) SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracted the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from patients with Duchenne muscular dystrophy displayed lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorated SR calcium homeostasis and improved muscle structure and function in dystrophic mdx/Utr+/- mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic potential for mitigating myopathy.

The hepatocyte insulin receptor is required to program the liver clock and rhythmic gene expression.

Liver physiology is circadian and sensitive to feeding and insulin. Food intake regulates insulin secretion and is a dominant signal for the liver clock. However, how much insulin contributes to the effect of feeding on the liver clock and rhythmic gene expression remains to be investigated. Insulin action partly depends on changes in insulin receptor (IR)-dependent gene expression. Here, we use hepatocyte-restricted gene deletion of IR to evaluate its role in the regulation and oscillation of gene expression as well as in the programming of the circadian clock in the adult mouse liver. We find that, in the absence of IR, the rhythmicity of core-clock gene expression is altered in response to day-restricted feeding. This change in core-clock gene expression is associated with defective reprogramming of liver gene expression. Our data show that an intact hepatocyte insulin receptor is required to program the liver clock and associated rhythmic gene expression.

The Circadian Clock and Obesity.

The modern way of life has dramatically affected our biological rhythms. Circadian rhythms, which are generated by an endogenous circadian clock, are observed in a large number of physiological functions including metabolism. Proper peripheral clock synchronization by different signals including appropriate feeding/fasting cycles is essential to coordinate and temporally gate metabolic processes. In this chapter, we emphasize the importance of nutrient sensing by peripheral clocks and highlight the major role of peripheral and central clock communication to locally regulate metabolic processes and ensure optimal energy storage and expenditure. As a consequence, changes in eating behavior and/or bedtime, as occurs upon shift work and jet lag, have direct consequences on metabolism and participate in the increasing prevalence of obesity and associated metabolic diseases such as type 2 diabetes and non-alcoholic fatty liver disease. In this setting, time-restricted feeding has been suggested as an efficient approach to ameliorate metabolic parameters and control body weight.

Timed use of digoxin prevents heart ischemia-reperfusion injury through a REV-ERBα-UPS signaling pathway.

Myocardial ischemia-reperfusion injury (MIRI) induces life-threatening damages to the cardiac tissue and pharmacological means to achieve cardioprotection are sorely needed. MIRI severity varies along the day-night cycle and is molecularly linked to components of the cellular clock including the nuclear receptor REV-ERBα, a transcriptional repressor. Here we show that digoxin administration in mice is cardioprotective when timed to trigger REV-ERBα protein degradation. In cardiomyocytes, digoxin increases REV-ERBα ubiquitinylation and proteasomal degradation, which depend on REV-ERBα ability to bind its natural ligand, heme. Inhibition of the membrane-bound Src tyrosine-kinase partially alleviated digoxin-induced REV-ERBα degradation. In untreated cardiomyocytes, REV-ERBα proteolysis is controlled by known (HUWE1, FBXW7, SIAH2) or novel (CBL, UBE4B) E3 ubiquitin ligases and the proteasome subunit PSMB5. Only SIAH2 and PSMB5 contributed to digoxin-induced degradation of REV-ERBα. Thus, controlling REV-ERBα proteostasis through the ubiquitin-proteasome system is an appealing cardioprotective strategy. Our data support the timed use of clinically-approved cardiotonic steroids in prophylactic cardioprotection.

Nuclear Receptors and Clock Components in Cardiovascular Diseases.

Cardiovascular diseases (CVD) are still the first cause of death worldwide. Their main origin is the development of atherosclerotic plaque, which consists in the accumulation of lipids and inflammatory leucocytes within the vascular wall of large vessels. Beyond dyslipidemia, diabetes, obesity, hypertension and smoking, the alteration of circadian rhythms, in shift workers for instance, has recently been recognized as an additional risk factor. Accordingly, targeting a pro-atherogenic pathway at the right time window, namely chronotherapy, has proven its efficiency in reducing plaque progression without affecting healthy tissues in mice, thus providing the rationale of such an approach to treat CVD and to reduce drug side effects. Nuclear receptors are transcriptional factors involved in the control of many physiological processes. Among them, Rev-erbs and RORs control metabolic homeostasis, inflammatory processes and the biological clock. In this review, we discuss the opportunity to dampen atherosclerosis progression by targeting such ligand-activated core clock components in a (chrono-)therapeutic approach in order to treat CVD.

Nuclear Receptors in the Control of the NLRP3 Inflammasome Pathway.

The innate immune system is the first line of defense specialized in the clearing of invaders whether foreign elements like microbes or self-elements that accumulate abnormally including cellular debris. Inflammasomes are master regulators of the innate immune system, especially in macrophages, and are key sensors involved in maintaining cellular health in response to cytolytic pathogens or stress signals. Inflammasomes are cytoplasmic complexes typically composed of a sensor molecule such as NOD-Like Receptors (NLRs), an adaptor protein including ASC and an effector protein such as caspase 1. Upon stimulation, inflammasome complex components associate to promote the cleavage of the pro-caspase 1 into active caspase-1 and the subsequent activation of pro-inflammatory cytokines including IL-18 and IL-1ß. Deficiency or overactivation of such important sensors leads to critical diseases including Alzheimer diseases, chronic inflammatory diseases, cancers, acute liver diseases, and cardiometabolic diseases. Inflammasomes are tightly controlled by a two-step activation regulatory process consisting in a priming step, which activates the transcription of inflammasome components, and an activation step which leads to the inflammasome complex formation and the subsequent cleavage of pro-IL1 cytokines. Apart from the NF-κB pathway, nuclear receptors have recently been proposed as additional regulators of this pathway. This review will discuss the role of nuclear receptors in the control of the NLRP3 inflammasome and the putative beneficial effect of new modulators of inflammasomes in the treatment of inflammatory diseases including colitis, fulminant hepatitis, cardiac ischemia-reperfusion and brain diseases.

Circadian Control of Inflammasome Pathways: Implications for Circadian Medicine.

The innate immune system senses "non-self" molecules derived from pathogens (PAMPs) as well as endogenous damage-associated molecular patterns (DAMPs) and promotes sterile inflammation that is necessary for injury resolution, tissue repair/regeneration, and homeostasis. The NOD-, LRR- and pyrin domain containing protein 3 (NLRP3) is an innate immune signaling complex whose assembly and activation can be triggered by various signals ranging from microbial molecules to ATP or the abnormal accumulation of crystals, thus leading to IL-1ß and IL-18 maturation and secretion. Deregulation of the NLRP3 signaling cascade is associated with numerous inflammatory and metabolic diseases including rheumatoid arthritis, gout, atherosclerosis or type 2 diabetes. Interestingly, the circadian clock controls numerous inflammatory processes while clock disruption leads to or exacerbates inflammation. Recently, the biological clock was demonstrated to control NLRP3 expression and activation, thereby controlling IL-1ß and IL-18 secretion in diverse tissues and immune cells, particularly macrophages. Circadian oscillations of NLRP3 signaling is lost in models of clock disruption, contributing to the development of peritonitis, hepatitis, or colitis. Sterile inflammation is also an important driver of atherosclerosis, and targeting the production of IL-1ß has proven to be a promising approach for atherosclerosis management in humans. Interestingly, the extent of injury after fulminant hepatitis or myocardial infarction is time-of-day dependent under the control of the clock, and chronotherapy represents a promising approach for the management of pathologies involving deregulation of NLRP3 signaling.

Diet-Induced Obesity Alters the Circadian Expression of Clock Genes in Mouse Gustatory Papillae.

Diet-induced obesity (DIO) is associated with a defect of the orosensory detection of dietary lipids in rodents. This dysfunction is not anecdotic since it might worsen the negative effects of obesity by promoting the overconsumption of energy-dense foods. Previous studies have highlighted a progressive devaluation of reward value of lipid stimuli due to a desensitization of dopaminergic brain areas in DIO mice. Paradoxically, the putative deleterious impact of obesity on peripheral fat detection by the gustatory papillae remains poorly documented. Using a whole transcriptomic investigation of the circumvallate papillae (CVP), an analysis of CVP genes involved in fat taste transduction and signaling along the day, and two bottle choice tests, we have found that (i) CVP, known to house the most taste buds in the oral cavity, displays a genic circadian rhythm, (ii) DIO reduces the oscillation of key genes involved both in the circadian clock and lipid detection/signaling, and (iii) the gene invalidation of the clock gene Rev-Erbα does not significantly affect fat preference despite an oily solution intake slightly lower than littermate controls. Taken together these data bring the first demonstration that the gustatory function is under control of a peripheral clock in mammals, as already reported in fly and suggest that a disturbance of this rhythmicity might contribute to the lower fatty taste acuity found in obese mice.

Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver.

Liver injury triggers adaptive remodeling of the hepatic transcriptome for repair/regeneration. We demonstrate that this involves particularly profound transcriptomic alterations where acute induction of genes involved in handling of endoplasmic reticulum stress (ERS) is accompanied by partial hepatic dedifferentiation. Importantly, widespread hepatic gene downregulation could not simply be ascribed to cofactor squelching secondary to ERS gene induction, but rather involves a combination of active repressive mechanisms. ERS acts through inhibition of the liver-identity (LIVER-ID) transcription factor (TF) network, initiated by rapid LIVER-ID TF protein loss. In addition, induction of the transcriptional repressor NFIL3 further contributes to LIVER-ID gene repression. Alteration to the liver TF repertoire translates into compromised activity of regulatory regions characterized by the densest co-recruitment of LIVER-ID TFs and decommissioning of BRD4 super-enhancers driving hepatic identity. While transient repression of the hepatic molecular identity is an intrinsic part of liver repair, sustained disequilibrium between the ERS and LIVER-ID transcriptional programs is linked to liver dysfunction as shown using mouse models of acute liver injury and livers from deceased human septic patients.

Differential unfolded protein response in skeletal muscle from non-diabetic glucose tolerant or intolerant patients with obesity before and after bariatric surgery.

AIMS: Not all people with obesity become glucose intolerant, suggesting differential activation of cellular pathways. The unfolded protein response (UPR) may contribute to the development of insulin resistance in several organs, but its role in skeletal muscle remains debated. Therefore, we explored the UPR activation in muscle from non-diabetic glucose tolerant or intolerant patients with obesity and the impact of bariatric procedures. METHODS: Muscle biopsies from 22 normoglycemic (NG, blood glucose measured 120 min after an oral glucose tolerance test, G120 < 7.8 mM) and 22 glucose intolerant (GI, G120 between 7.8 and 11.1 mM) patients with obesity were used to measure UPR activation by RTqPCR and western blot. Then, UPR was studied in biopsies from 7 NG and 7 GI patients before and 1 year after bariatric surgery. RESULTS: Binding immunoglobulin protein (BIP) protein was ~ 40% higher in the GI compared to NG subjects. Contrastingly, expression of the UPR-related genes BIP, activating transcription factor 6 (ATF6) and unspliced X-box binding protein 1 (XBP1u) were significantly lower and C/EBP homologous protein (CHOP) tended to decrease (p = 0.08) in GI individuals. While BIP protein positively correlated with fasting blood glucose (r = 0.38, p = 0.01), ATF6 and CHOP were associated with G120 (r = - 0.38 and r = - 0.41, p < 0.05) and the Matsuda index (r = 0.37 and r = 0.38, p < 0.05). Bariatric surgery improved metabolic parameters, associated with higher CHOP expression in GI patients, while ATF6 tended to increase (p = 0.08). CONCLUSIONS: CHOP and ATF6 expression decreased in non-diabetic GI patients with obesity and was modified by bariatric surgery. These genes may contribute to glucose homeostasis in human skeletal muscle.

Glycogen Dynamics Drives Lipid Droplet Biogenesis during Brown Adipocyte Differentiation.

Browning 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.

The LPS/D-Galactosamine-Induced Fulminant Hepatitis Model to Assess the Role of Ligand-Activated Nuclear Receptors on the NLRP3 Inflammasome Pathway In Vivo.

The NLRP3 inflammasome is a cellular sensor of danger signals such as extracellular ATP or abnormally accumulating molecules like crystals. Activation of NLRP3 by such compounds triggers a sterile inflammatory response that may be involved in numerous pathologies including rheumatoid arthritis, atherosclerosis, diabetes, and Alzheimer's disease. A better understanding of the mechanisms that govern NLRP3 inflammasome activation is an important step toward the development of novel therapeutic strategies to dampen over-activation of the immune system. Recent findings demonstrate that ligand-activated nuclear receptors regulate the NLRP3 inflammasome pathway, thus representing possible therapeutic targets. It is therefore important to assess the potential of these putative targets in the regulation of the NLRP3 inflammasome activation in the most appropriate pathophysiological models. Fulminant hepatitis (FH) results from massive hepatocyte apoptosis, hemorrhagic necrosis, and inflammation. Low doses of LPS in combination with the specific hepatotoxic agent D-galactosamine (D-GalN) promote liver injury in mice and induce the production of inflammatory cytokines associated with increased NLRP3 protein and caspase 1 activity, thus recapitulating the clinical picture of FH in humans. We provide a simple method to examine the involvement of nuclear receptors in NLRP3-driven fulminant hepatitis, consisting in the induction of FH, in the isolation of liver macrophages, and in the extraction and analysis of RNA content.

Combinatorial regulation of hepatic cytoplasmic signaling and nuclear transcriptional events by the OGT/REV-ERBα complex.

The nuclear receptor REV-ERBα integrates the circadian clock with hepatic glucose and lipid metabolism by nucleating transcriptional comodulators at genomic regulatory regions. An interactomic approach identified O-GlcNAc transferase (OGT) as a REV-ERBα-interacting protein. By shielding cytoplasmic OGT from proteasomal degradation and favoring OGT activity in the nucleus, REV-ERBα cyclically increased O-GlcNAcylation of multiple cytoplasmic and nuclear proteins as a function of its rhythmically regulated expression, while REV-ERBα ligands mostly affected cytoplasmic OGT activity. We illustrate this finding by showing that REV-ERBα controls OGT-dependent activities of the cytoplasmic protein kinase AKT, an essential relay in insulin signaling, and of ten-of-eleven translocation (TET) enzymes in the nucleus. AKT phosphorylation was inversely correlated to REV-ERBα expression. REV-ERBα enhanced TET activity and DNA hydroxymethylated cytosine (5hmC) levels in the vicinity of REV-ERBα genomic binding sites. As an example, we show that the REV-ERBα/OGT complex modulates SREBP-1c gene expression throughout the fasting/feeding periods by first repressing AKT phosphorylation and by epigenomically priming the Srebf1 promoter for a further rapid response to insulin. Conclusion: REV-ERBα regulates cytoplasmic and nuclear OGT-controlled processes that integrate at the hepatic SREBF1 locus to control basal and insulin-induced expression of the temporally and nutritionally regulated lipogenic SREBP-1c transcript.

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

BACKGROUND: Skeletal 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. METHODS: Running on the popular open source Fiji software platform, MuscleJ simultaneously analyzes parameters from immunofluorescent staining, imaged by different acquisition systems in a completely automated manner. RESULTS: After segmentation of muscle fibers, up to three other channels can be analyzed simultaneously. Dialog boxes make MuscleJ easy-to-use for biologists. In addition, we have implemented color in situ cartographies of results, allowing the user to directly visualize results on reconstituted muscle sections. CONCLUSION: We report here that MuscleJ results were comparable to manual observations made by five experts. MuscleJ markedly enhances statistical analysis by allowing reliable comparison of skeletal muscle physiology-pathology results obtained from different laboratories using different acquisition systems. Providing fast robust multi-parameter analyses of skeletal muscle physiology-pathology, MuscleJ is available as a free tool for the skeletal muscle community.

Circadian misalignment induces fatty acid metabolism gene profiles and compromises insulin sensitivity in human skeletal muscle.

Circadian misalignment, such as in shift work, has been associated with obesity and type 2 diabetes. However, direct effects of circadian misalignment on skeletal muscle insulin sensitivity and the muscle molecular circadian clock have never been studied in humans. Here, we investigated insulin sensitivity and muscle metabolism in 14 healthy young lean men [age 22.4 ± 2.8 years; body mass index (BMI) 22.3 ± 2.1 kg/m2 (mean ± SD)] after a 3-d control protocol and a 3.5-d misalignment protocol induced by a 12-h rapid shift of the behavioral cycle. We show that short-term circadian misalignment results in a significant decrease in muscle insulin sensitivity due to a reduced skeletal muscle nonoxidative glucose disposal (rate of disappearance: 23.7 ± 2.4 vs. 18.4 ± 1.4 mg/kg per minute; control vs. misalignment; P = 0.024). Fasting glucose and free fatty acid levels as well as sleeping metabolic rate were higher during circadian misalignment. Molecular analysis of skeletal muscle biopsies revealed that the molecular circadian clock was not aligned to the inverted behavioral cycle, and transcriptome analysis revealed the human PPAR pathway as a key player in the disturbed energy metabolism upon circadian misalignment. Our findings may provide a mechanism underlying the increased risk of type 2 diabetes among shift workers.

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.