En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

Opposing effects of genetic variation in MTCH2 for obesity versus heart failure.

Genetic variation in genes regulating metabolism may be advantageous in some settings but not others. The non-failing adult heart relies heavily on fatty acids as a fuel substrate and source of ATP. In contrast, the failing heart favors glucose as a fuel source. A bootstrap analysis for genes with deviant allele frequencies in cardiomyopathy cases versus controls identified the MTCH2 gene as having unusual variation. MTCH2 encodes an outer mitochondrial membrane protein, and prior genome-wide studies associated MTCH2 variants with body mass index, consistent with its role in metabolism. We identified the referent allele of rs1064608 (p.Pro290) as being overrepresented in cardiomyopathy cases compared to controls, and linkage disequilibrium analysis associated this variant with the MTCH2 cis eQTL rs10838738 and lower MTCH2 expression. To evaluate MTCH2, we knocked down Mtch in Drosophila heart tubes which produced a dilated and poorly functioning heart tube, reduced adiposity and shortened life span. Cardiac Mtch mutants generated more lactate at baseline, and they displayed impaired oxygen consumption in the presence of glucose but not palmitate. Treatment of cardiac Mtch mutants with dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, reduced lactate and rescued lifespan. Deletion of MTCH2 in human cells similarly impaired oxygen consumption in the presence of glucose but not fatty acids. These data support a model in which MTCH2 reduction may be favorable when fatty acids are the major fuel source, favoring lean body mass. However, in settings like heart failure, where the heart shifts toward using more glucose, reduction of MTCH2 is maladaptive.

A fat- and sucrose-enriched diet causes metabolic alterations in mdx mice.

Duchenne muscular dystrophy (DMD), a progressive muscle disease caused by the absence of functional dystrophin protein, is associated with multiple cellular, physiological, and metabolic dysfunctions. As an added complication to the primary insult, obesity/insulin resistance (O/IR) is frequently reported in patients with DMD; however, how IR impacts disease severity is unknown. We hypothesized a high-fat, high-sucrose diet (HFHSD) would induce O/IR, exacerbate disease severity, and cause metabolic alterations in dystrophic mice. To test this hypothesis, we treated 7-wk-old mdx (disease model) and C57 mice with a control diet (CD) or an HFHSD for 15 wk. The HFHSD induced insulin resistance, glucose intolerance, and hyperglycemia in C57 and mdx mice. Of note, mdx mice on CD were also insulin resistant. In addition, visceral adipose tissue weights were increased with HFHSD in C57 and mdx mice though differed by genotype. Serum creatine kinase activity and histopathological analyses using Masson's trichrome staining in the diaphragm indicated muscle damage was driven by dystrophin deficiency but was not augmented by diet. In addition, markers of inflammatory signaling, mitochondrial abundance, and autophagy were impacted by disease but not diet. Despite this, in addition to disease signatures in CD-fed mice, metabolomic and lipidomic analyses demonstrated a HFHSD caused some common changes in C57 and mdx mice and some unique signatures of O/IR within the context of dystrophin deficiency. In total, these data revealed that in mdx mice, 15 wk of HFHSD did not overtly exacerbate muscle injury but further impaired the metabolic status of dystrophic muscle.

Spp1 (osteopontin) promotes TGFß processing in fibroblasts of dystrophin-deficient muscles through matrix metalloproteinases.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. Prior work has shown that DMD progression can vary, depending on the genetic makeup of the patient. Several modifier alleles have been identified including LTBP4 and SPP1. We previously showed that Spp1 exacerbates the DMD phenotype in the mdx mouse model by promoting fibrosis and by skewing macrophage polarization. Here, we studied the mechanisms involved in Spp1's promotion of fibrosis by using both isolated fibroblasts and genetically modified mice. We found that Spp1 upregulates collagen expression in mdx fibroblasts by enhancing TGFß signaling. Spp1's effects on TGFß signaling are through induction of MMP9 expression. MMP9 is a protease that can release active TGFß ligand from its latent complex. In support for activation of this pathway in our model, we showed that treatment of mdx fibroblasts with MMP9 inhibitor led to accumulation of the TGFß latent complex, decreased levels of active TGFß and reduced collagen expression. Correspondingly, we found reduced active TGFß in Spp1-/-mdxB10 and Mmp9-/-mdxB10 muscles in vivo. Taken together with previous observations of reduced fibrosis in both models, these data suggest that Spp1 acts upstream of TGFß to promote fibrosis in mdx muscles. We found that in the context of constitutively upregulated TGFß signaling (such as in the mdxD2 model), ablation of Spp1 has very little effect on fibrosis. Finally, we performed proof-of-concept studies showing that postnatal pharmacological inhibition of Spp1 reduces fibrosis and improves muscle function in mdx mice.

Dusp6 is a genetic modifier of growth through enhanced ERK activity.

Like other single-gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole-genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the dual-specificity phosphatase 6 (Dusp6) gene from the DBA/2 J (D2) mouse strain. DUSP6 is a broadly expressed dual-specificity phosphatase protein, which binds and dephosphorylates extracellular-signal-regulated kinase (ERK), leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DUSP6 and ERK resulting in increased ERK phosphorylation and ERK activity. In dystrophic muscle, DUSP6 Met62Ile is strongly upregulated to counteract its reduced activity. We found that myoblasts from the D2 background were insensitive to a specific small molecule inhibitor of DUSP6, while myoblasts expressing the canonical DUSP6 displayed enhanced proliferation after exposure to DUSP6 inhibition. These data identify DUSP6 as an important regulator of ERK activity in the setting of muscle growth and muscular dystrophy.

Guide Cells Support Muscle Regeneration and Affect Neuro-Muscular Junction Organization.

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor ß (TGFß) pathway, osteopontin encoded by the SPP1 gene and latent TGFß binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFß and TGFß-associated pathways. We identified that increased TGFß resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFß and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.

Outside in: The matrix as a modifier of muscular dystrophy.

Muscular dystrophies are genetic conditions leading to muscle degeneration and often, impaired regeneration. Duchenne Muscular Dystrophy is a prototypical form of muscular dystrophy, and like other forms of genetically inherited muscle diseases, pathological progression is variable. Variability in muscular dystrophy can arise from differences in the manner in which the primary mutation impacts the affected protein's function; however, clinical heterogeneity also derives from secondary mutations in other genes that can enhance or reduce pathogenic features of disease. These genes, called genetic modifiers, regulate the pathophysiological context of dystrophic degeneration and regeneration. Understanding the mechanistic links between genetic modifiers and dystrophic progression sheds light on pathologic remodeling, and provides novel avenues to therapeutically intervene to reduce muscle degeneration. Based on targeted genetic approaches and unbiased genomewide screens, several modifiers have been identified for muscular dystrophy, including extracellular agonists of signaling cascades. This review will focus on identification and possible mechanisms of recently identified modifiers for muscular dystrophy, including osteopontin, latent TGFß binding protein 4 (LTBP4) and Jagged1. Moreover, we will review the investigational approaches that aim to target modifier pathways and thereby counteract dystrophic muscle wasting.

Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy.

The muscular dystrophies are genetically diverse. Shared pathological features among muscular dystrophies include breakdown, or loss of muscle, and accompanying fibrotic replacement. Novel strategies are needed to enhance muscle repair and function and to slow this pathological remodeling. Glucocorticoid steroids, like prednisone, are known to delay loss of ambulation in patients with Duchenne muscular dystrophy but are accompanied by prominent adverse effects. However, less is known about the effects of steroid administration in other types of muscular dystrophies, including limb-girdle muscular dystrophies (LGMDs). LGMD 2B is caused by loss of dysferlin, a membrane repair protein, and LGMD 2C is caused by loss of the dystrophin-associated protein, γ-sarcoglycan. Herein, we assessed the efficacy of steroid dosing on sarcolemmal repair, muscle function, histopathology, and the regenerative capacity of primary muscle cells. We found that in murine models of LGMD 2B and 2C, daily prednisone dosing reduced muscle damage and fibroinflammatory infiltration. However, daily prednisone dosing also correlated with increased muscle adipogenesis and atrophic remodeling. Conversely, intermittent dosing of prednisone, provided once weekly, enhanced muscle repair and did not induce atrophy or adipogenesis, and was associated with improved muscle function. These data indicate that dosing frequency of glucocorticoid steroids affects muscle remodeling in non-Duchenne muscular dystrophies, suggesting a positive outcome associated with intermittent steroid dosing in LGMD 2B and 2C muscle.

Myomir dysregulation and reactive oxygen species in aged human satellite cells.

Satellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells.

Fate choice of post-natal mesoderm progenitors: skeletal versus cardiac muscle plasticity.

Regenerative medicine for skeletal and cardiac muscles still constitutes a fascinating and ambitious frontier. In this perspective, understanding the possibilities of intrinsic cell plasticity, present in post-natal muscles, is vital to define and improve novel therapeutic strategies for acute and chronic diseases. In addition, many somatic stem cells are now crossing the boundaries of basic/translational research to enter the first clinical trials. However, it is still an open question whether a lineage switch between skeletal and cardiac adult myogenesis is possible. Therefore, this review focuses on resident somatic stem cells of post-natal skeletal and cardiac muscles and their plastic potential toward the two lineages. Furthermore, examples of myogenic lineage switch in adult stem cells are also reported and discussed.

Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo.

Mice deficient in α-sarcoglycan (Sgca-null mice) develop progressive muscular dystrophy and serve as a model for human limb girdle muscular dystrophy type 2D. Sgca-null mice suffer a more severe myopathy than that of mdx mice, the model for Duchenne muscular dystrophy. This is the opposite of what is observed in humans and the reason for this is unknown. In an attempt to understand the cellular basis of this severe muscular dystrophy, we isolated clonal populations of myogenic progenitor cells (MPCs), the resident postnatal muscle progenitors of dystrophic and wild-type mice. MPCs from Sgca-null mice generated much smaller clones than MPCs from wild-type or mdx dystrophic mice. Impaired proliferation of Sgca-null myogenic precursors was confirmed by single fiber analysis and this difference correlated with Sgca expression during MPC proliferation. In the absence of dystrophin and associated proteins, which are only expressed after differentiation, SGCA complexes with and stabilizes FGFR1. Deficiency of Sgca leads to an absence of FGFR1 expression at the membrane and impaired MPC proliferation in response to bFGF. The low proliferation rate of Sgca-null MPCs was rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted into dystrophic muscle, Sgca-null MPCs exhibited reduced engraftment. The reduced proliferative ability of Sgca-null MPCs explains, at least in part, the severity of this muscular dystrophy and also why wild-type donor progenitor cells engraft efficiently and consequently ameliorate disease.

The human genetic variant rs6190 unveils Foxc1 and Arid5a as novel pro-metabolic targets of the glucocorticoid receptor in muscle.

Genetic variations in the glucocorticoid receptor (GR) gene NR3C1 can impact metabolism. The single nucleotide polymorphism (SNP) rs6190 (p.R23K) has been associated in humans with enhanced metabolic health, but the SNP mechanism of action remains completely unknown. We generated a transgenic knock-in mice genocopying this polymorphism to elucidate how the mutant GR impacts metabolism. Compared to non-mutant littermates, mutant mice showed increased muscle insulin sensitivity and strength on regular chow and high-fat diet, blunting the diet-induced adverse effects on weight gain and exercise intolerance. Overlay of RNA-seq and ChIP-seq profiling in skeletal muscle revealed increased transactivation of Foxc1 and Arid5A genes by the mutant GR. Using adeno-associated viruses for in vivo overexpression in muscle, we found that Foxc1 was sufficient to transcriptionally activate the insulin response pathway genes Insr and Irs1. In parallel, Arid5a was sufficient to transcriptionally repress the lipid uptake genes Cd36 and Fabp4, reducing muscle triacylglycerol accumulation. Collectively, our findings identify a muscle-autonomous epigenetic mechanism of action for the rs6190 SNP effect on metabolic homeostasis, while leveraging a human nuclear receptor coding variant to unveil Foxc1 and Arid5a as novel epigenetic regulators of muscle metabolism.

Intermittent glucocorticoid treatment improves muscle metabolism via the PGC1α/Lipin1 axis in an aging-related sarcopenia model.

Sarcopenia burdens the older population through loss of muscle energy and mass, yet treatments to functionally rescue both parameters are lacking. The glucocorticoid prednisone remodels muscle metabolism on the basis of frequency of intake, but its mechanisms in sarcopenia are unknown. We found that once-weekly intermittent prednisone administration rescued muscle quality in aged 24-month-old mice to a level comparable to that seen in young 4-month-old mice. We discovered an age- and sex-independent glucocorticoid receptor transactivation program in muscle encompassing peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α) and its cofactor Lipin1. Treatment coordinately improved mitochondrial abundance through isoform 1 and muscle mass through isoform 4 of the myocyte-specific PGC1α, which was required for the treatment-driven increase in carbon shuttling from glucose oxidation to amino acid biogenesis. We also probed myocyte-specific Lipin1 as a nonredundant factor coaxing PGC1α upregulation to the stimulation of both oxidative and anabolic effects. Our study unveils an aging-resistant druggable program in myocytes for the coordinated rescue of energy and mass in sarcopenia.

Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis.

Skeletal muscle is dynamically controlled by the balance of protein synthesis and degradation. Here we discover an unexpected function for the transcriptional repressor B cell lymphoma 6 (BCL6) in muscle proteostasis and strength in mice. Skeletal muscle-specific Bcl6 ablation in utero or in adult mice results in over 30% decreased muscle mass and force production due to reduced protein synthesis and increased autophagy, while it promotes a shift to a slower myosin heavy chain fibre profile. Ribosome profiling reveals reduced overall translation efficiency in Bcl6-ablated muscles. Mechanistically, tandem chromatin immunoprecipitation, transcriptomic and translational analyses identify direct BCL6 repression of eukaryotic translation initiation factor 4E-binding protein 1 (Eif4ebp1) and activation of insulin-like growth factor 1 (Igf1) and androgen receptor (Ar). Together, these results uncover a bifunctional role for BCL6 in the transcriptional and translational control of muscle proteostasis.

Effects of Glucocorticoids in Murine Models of Duchenne and Limb-Girdle Muscular Dystrophy.

In vivo testing of glucocorticoid steroids in dystrophic mice offers important insights in benefits and risks of those drugs in the pathological context of muscular dystrophy. Frequency of dosing changes the spectrum of glucocorticoid effects on muscle and metabolic homeostasis. Here, we describe a combination of non-invasive and invasive methods to quantitatively discriminate the specific effects of intermittent (once-weekly) versus mainstay (once-daily) regimens on muscle fibrosis, muscle function, and metabolic homeostasis in murine models of Duchenne and limb-girdle muscular dystrophies.

Comprehensive Analyses of Muscle Function, Lean and Muscle Mass, and Myofiber Typing in Mice.

Skeletal muscle disorders commonly affect the function and integrity of muscles. Novel interventions bring new potential to rescue or alleviate the symptoms associated with these disorders. In vivo and in vitro testing in mouse models allows quantitative evaluation of the degree of muscle dysfunction, and therefore, the level of potential rescue/restoration by the target intervention. Several resources and methods are available to assess muscle function and lean and muscle mass, as well as myofiber typing as separate concepts; however, a technical resource unifying these methods is missing. Here, we provide detailed procedures for analyzing muscle function, lean and muscle mass, and myofiber typing in a comprehensive technical resource paper. Graphical abstract.

Microbiota dysbiosis influences immune system and muscle pathophysiology of dystrophin-deficient mice.

Duchenne muscular dystrophy (DMD) is a progressive severe muscle-wasting disease caused by mutations in DMD, encoding dystrophin, that leads to loss of muscle function with cardiac/respiratory failure and premature death. Since dystrophic muscles are sensed by infiltrating inflammatory cells and gut microbial communities can cause immune dysregulation and metabolic syndrome, we sought to investigate whether intestinal bacteria support the muscle immune response in mdx dystrophic murine model. We highlighted a strong correlation between DMD disease features and the relative abundance of Prevotella. Furthermore, the absence of gut microbes through the generation of mdx germ-free animal model, as well as modulation of the microbial community structure by antibiotic treatment, influenced muscle immunity and fibrosis. Intestinal colonization of mdx mice with eubiotic microbiota was sufficient to reduce inflammation and improve muscle pathology and function. This work identifies a potential role for the gut microbiota in the pathogenesis of DMD.

Glucocorticoid intermittence coordinates rescue of energy and mass in aging-related sarcopenia through the myocyte-autonomous PGC1alpha-Lipin1 transactivation.

Sarcopenia burdens the elderly population through loss of muscle energy and mass, yet treatments to functionally rescue both parameters are missing. The glucocorticoid prednisone remodels muscle metabolism based on frequency of intake, but its mechanisms in sarcopenia are unknown. We found that once-weekly intermittent prednisone rescued muscle quality in aged 24-month-old mice to levels comparable to young 4-month-old mice. We discovered an age- and sex-independent glucocorticoid receptor transactivation program in muscle encompassing PGC1alpha and its co-factor Lipin1. Treatment coordinately improved mitochondrial abundance through isoform 1 and muscle mass through isoform 4 of the myocyte-specific PGC1alpha, which was required for the treatment-driven increase in carbon shuttling from glucose oxidation to amino acid biogenesis. We also probed the myocyte-specific Lipin1 as non-redundant factor coaxing PGC1alpha upregulation to the stimulation of both oxidative and anabolic capacities. Our study unveils an aging-resistant druggable program in myocytes to coordinately rescue energy and mass in sarcopenia.

Light-phase prednisone promotes glucose oxidation in heart through novel transactivation targets of cardiomyocyte-specific GR and KLF15.

Circadian time of intake determines the cardioprotective outcome of glucocorticoids in normal and infarcted hearts. The cardiomyocyte-specific glucocorticoid receptor (GR) is genetically required to preserve normal heart function in the long-term. The GR co-factor KLF15 is a pleiotropic regulator of cardiac metabolism. However, the cardiomyocyte-autonomous metabolic targets of the GR-KLF15 concerted epigenetic action remain undefined. Here we report that circadian time of intake determines the activation of a transcriptional and functional glucose oxidation program in heart by the glucocorticoid prednisone with comparable magnitude between sexes. We overlayed transcriptomics, epigenomics and cardiomyocyte-specific inducible ablation of either GR or KLF15. Downstream of a light-phase prednisone stimulation in mice, we found that both factors are non-redundantly required in heart to transactivate the adiponectin receptor expression (Adipor1) and promote insulin-stimulated glucose uptake, as well as transactivate the mitochondrial pyruvate complex expression (Mpc1/2) and promote pyruvate oxidation. We then challenged this time-specific drug effect in obese diabetic db/db mice, where the heart shows insulin resistance and defective glucose oxidation. Opposite to dark-phase dosing, light-phase prednisone rescued glucose oxidation in db/db cardiomyocytes and diastolic function in db/db hearts towards control-like levels with sex-independent magnitude of effect. In summary, our study identifies novel cardiomyocyte-autonomous metabolic targets of the GR-KLF15 concerted program mediating the time-specific cardioprotective effects of glucocorticoids on cardiomyocyte glucose utilization.