A microbiome-dependent gut-brain pathway regulates motivation for exercise.

Exercise exerts a wide range of beneficial effects for healthy physiology1. However, the mechanisms regulating an individual's motivation to engage in physical activity remain incompletely understood. An important factor stimulating the engagement in both competitive and recreational exercise is the motivating pleasure derived from prolonged physical activity, which is triggered by exercise-induced neurochemical changes in the brain. Here, we report on the discovery of a gut-brain connection in mice that enhances exercise performance by augmenting dopamine signalling during physical activity. We find that microbiome-dependent production of endocannabinoid metabolites in the gut stimulates the activity of TRPV1-expressing sensory neurons and thereby elevates dopamine levels in the ventral striatum during exercise. Stimulation of this pathway improves running performance, whereas microbiome depletion, peripheral endocannabinoid receptor inhibition, ablation of spinal afferent neurons or dopamine blockade abrogate exercise capacity. These findings indicate that the rewarding properties of exercise are influenced by gut-derived interoceptive circuits and provide a microbiome-dependent explanation for interindividual variability in exercise performance. Our study also suggests that interoceptomimetic molecules that stimulate the transmission of gut-derived signals to the brain may enhance the motivation for exercise.

Tibetan PHD2, an allele with loss-of-function properties.

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.

Mitochondrial ultrastructural adaptations in fast muscles of mice lacking IL15RA.

The pro-inflammatory cytokine interleukin-15 (IL15) and its receptor α (IL15RA) participate in the regulation of musculoskeletal function and metabolism. Deletion of the Il15ra gene in mice increases spontaneous activity, improves fatigue resistance in the glycolytic extensor digitorum longus (EDL) and protects from diet-induced obesity. In humans, IL15RA single-nucleotide polymorphisms (SNPs) have been linked to muscle strength, metabolism and performance in elite endurance athletes. Taken together, these features suggest a possible role for IL15RA in muscle mitochondrial structure and function. Here, we have investigated the consequences of loss of IL15RA on skeletal muscle fiber-type properties and mitochondrial ultrastructure. Immunostaining of the EDL for myosin heavy chain (MyHC) isoforms revealed no significant changes in fiber type. Electron microscopy (EM) analysis of the EDL indicated an overall higher mitochondria content, and increased cristae density in subsarcolemmal and A-band mitochondrial subpopulations. The higher cristae density in Il15ra-/- mitochondria was associated with higher OPA1 and cardiolipin levels. Overall, these data extend our understanding of the role of IL15RA signaling in muscle oxidative metabolism and adaptation to exercise.

Elongated mitochondrial constrictions and fission in muscle fatigue.

Mitochondria respond to stress and undergo fusion and fission at variable rates, depending on cell status. To understand mitochondrial behavior during muscle fatigue, we investigated mitochondrial ultrastructure and expression levels of a fission- and stress-related protein in fast-twitch muscle fibers of mice subjected to fatigue testing. Mice were subjected to running at increasing speed until exhaustion at 45 min-1 h. In further experiments, high-intensity muscle stimulation through the sciatic nerve simulated the forced treadmill exercise. We detected a rare phenotype characterized by elongated mitochondrial constrictions (EMCs) connecting two separate segments of the original organelles. EMCs are rare in resting muscles and their frequency increases, albeit still at low levels, in stimulated muscles. The constrictions are accompanied by elevated phosphorylation of Drp1 (Dnm1l) at Ser 616, indicating an increased translocation of Drp1 to the mitochondrial membrane. This is indicative of a mitochondrial stress response, perhaps leading to or facilitating a long-lasting fission event. A close apposition of sarcoplasmic reticulum (SR) to the constricted areas, detected using both transmission and scanning electron microscopy, is highly suggestive of SR involvement in inducing mitochondrial constrictions.

The nuclear receptor Rev-erbα controls circadian thermogenic plasticity.

Circadian oscillation of body temperature is a basic, evolutionarily conserved feature of mammalian biology. In addition, homeostatic pathways allow organisms to protect their core temperatures in response to cold exposure. However, the mechanism responsible for coordinating daily body temperature rhythm and adaptability to environmental challenges is unknown. Here we show that the nuclear receptor Rev-erbα (also known as Nr1d1), a powerful transcriptional repressor, links circadian and thermogenic networks through the regulation of brown adipose tissue (BAT) function. Mice exposed to cold fare considerably better at 05:00 (Zeitgeber time 22) when Rev-erbα is barely expressed than at 17:00 (Zeitgeber time 10) when Rev-erbα is abundant. Deletion of Rev-erbα markedly improves cold tolerance at 17:00, indicating that overcoming Rev-erbα-dependent repression is a fundamental feature of the thermogenic response to cold. Physiological induction of uncoupling protein 1 (Ucp1) by cold temperatures is preceded by rapid downregulation of Rev-erbα in BAT. Rev-erbα represses Ucp1 in a brown-adipose-cell-autonomous manner and BAT Ucp1 levels are high in Rev-erbα-null mice, even at thermoneutrality. Genetic loss of Rev-erbα also abolishes normal rhythms of body temperature and BAT activity. Thus, Rev-erbα acts as a thermogenic focal point required for establishing and maintaining body temperature rhythm in a manner that is adaptable to environmental demands.

IL-15Rα is a determinant of muscle fuel utilization, and its loss protects against obesity.

IL-15Rα is the widely expressed primary binding partner for IL-15. Because of the wide distribution in nonlymphoid tissues like skeletal muscle, adipose, or liver, IL-15/IL-15Rα take part in physiological and metabolic processes not directly related to immunity. In fast muscle, lack of IL-15Rα promotes an oxidative switch, with increased mitochondrial biogenesis and fatigue resistance. These effects are predicted to reproduce some of the benefits of exercise and, therefore, improve energy homeostasis. However, the direct effects of IL-15Rα on metabolism and obesity are currently unknown. We report that mice lacking IL-15Rα (IL-15Rα(-/-)) are resistant to diet-induced obesity (DIO). High-fat diet-fed IL-15Rα(-/-) mice have less body and liver fat accumulation than controls. The leaner phenotype is associated with increased energy expenditure and enhanced fatty acid oxidation by muscle mitochondria. Despite being protected against DIO, IL-15Rα(-/-) are hyperglycemic and insulin-resistant. These findings identify novel roles for IL-15Rα in metabolism and obesity.

Erythrocytosis and pulmonary hypertension in a mouse model of human HIF2A gain of function mutation.

The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation.

A knock-in mouse model of human PHD2 gene-associated erythrocytosis establishes a haploinsufficiency mechanism.

The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2(P294R/+) mice display a degree of erythrocytosis equivalent to that seen in Phd2(+/-) mice. The Phd2(P294R/+)-associated erythrocytosis is reversed in a Hif2a(+/-), but not a Hif1a(+/-) background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2.

COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to human collagen VI congenital muscular dystrophy.

Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a Col6a3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The Col6a3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy.

Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in dystrophin and the subsequent disruption of the dystrophin-associated protein complex (DAPC). Utrophin is a dystrophin homolog expressed at high levels in developing muscle that is an attractive target for DMD therapy. Here we show that the extracellular matrix protein biglycan regulates utrophin expression in immature muscle and that recombinant human biglycan (rhBGN) increases utrophin expression in cultured myotubes. Systemically delivered rhBGN up-regulates utrophin at the sarcolemma and reduces muscle pathology in the mdx mouse model of DMD. RhBGN treatment also improves muscle function as judged by reduced susceptibility to eccentric contraction-induced injury. Utrophin is required for the rhBGN therapeutic effect. Several lines of evidence indicate that biglycan acts by recruiting utrophin protein to the muscle membrane. RhBGN is well tolerated in animals dosed for as long as 3 months. We propose that rhBGN could be a therapy for DMD.

Pharmacology of myopia and potential role for intrinsic retinal circadian rhythms.

Despite the high prevalence and public health impact of refractive errors, the mechanisms responsible for ametropias are poorly understood. Much evidence now supports the concept that the retina is central to the mechanism(s) regulating emmetropization and underlying refractive errors. Using a variety of pharmacologic methods and well-defined experimental eye growth models in laboratory animals, many retinal neurotransmitters and neuromodulators have been implicated in this process. Nonetheless, an accepted framework for understanding the molecular and/or cellular pathways that govern postnatal eye development is lacking. Here, we review two extensively studied signaling pathways whose general roles in refractive development are supported by both experimental and clinical data: acetylcholine signaling through muscarinic and/or nicotinic acetylcholine receptors and retinal dopamine pharmacology. The muscarinic acetylcholine receptor antagonist atropine was first studied as an anti-myopia drug some two centuries ago, and much subsequent work has continued to connect muscarinic receptors to eye growth regulation. Recent research implicates a potential role of nicotinic acetylcholine receptors; and the refractive effects in population surveys of passive exposure to cigarette smoke, of which nicotine is a constituent, support clinical relevance. Reviewed here, many puzzling results inhibit formulating a mechanistic framework that explains acetylcholine's role in refractive development. How cholinergic receptor mechanisms might be used to develop acceptable approaches to normalize refractive development remains a challenge. Retinal dopamine signaling not only has a putative role in refractive development, its upregulation by light comprises an important component of the retinal clock network and contributes to the regulation of retinal circadian physiology. During postnatal development, the ocular dimensions undergo circadian and/or diurnal fluctuations in magnitude; these rhythms shift in eyes developing experimental ametropia. Long-standing clinical ideas about myopia in particular have postulated a role for ambient lighting, although molecular or cellular mechanisms for these speculations have remained obscure. Experimental myopia induced by the wearing of a concave spectacle lens alters the retinal expression of a significant proportion of intrinsic circadian clock genes, as well as genes encoding a melatonin receptor and the photopigment melanopsin. Together this evidence suggests a hypothesis that the retinal clock and intrinsic retinal circadian rhythms may be fundamental to the mechanism(s) regulating refractive development, and that disruptions in circadian signals may produce refractive errors. Here we review the potential role of biological rhythms in refractive development. While much future research is needed, this hypothesis could unify many of the disparate clinical and laboratory observations addressing the pathogenesis of refractive errors.

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

AKT controls protein synthesis and oxidative metabolism via combined mTORC1 and FOXO1 signalling to govern muscle physiology.

BACKGROUND: Skeletomuscular diseases result in significant muscle loss and decreased performance, paralleled by a loss in mitochondrial and oxidative capacity. Insulin and insulin-like growth factor-1 (IGF-1) are two potent anabolic hormones that activate a host of signalling intermediates including the serine/threonine kinase AKT to influence skeletal muscle physiology. Defective AKT signalling is associated with muscle pathology, including cachexia, sarcopenia, and disuse; however, the mechanistic underpinnings remain unresolved. METHODS: To elucidate the role of AKT signalling in muscle mass and physiology, we generated both congenital and inducible mouse models of skeletal muscle-specific AKT deficiency. To understand the downstream mechanisms mediating AKT's effects on muscle biology, we generated mice lacking AKT1/2 and FOXO1 (M-AKTFOXO1TKO and M-indAKTFOXO1TKO) to inhibit downstream FOXO1 signalling, AKT1/2 and TSC1 (M-AKTTSCTKO and M-indAKTTSCTKO) to activate mTORC1, and AKT1/2, FOXO1, and TSC1 (M-QKO and M-indQKO) to simultaneously activate mTORC1 and inhibit FOXO1 in AKT-deficient skeletal muscle. Muscle proteostasis and physiology were assessed using multiple assays including metabolic labelling, mitochondrial function, fibre typing, ex vivo physiology, and exercise performance. RESULTS: Here, we show that genetic ablation of skeletal muscle AKT signalling resulted in decreased muscle mass and a loss of oxidative metabolism and muscle performance. Specifically, deletion of muscle AKT activity during development or in adult mice resulted in a significant reduction in muscle growth by 30-40% (P  < 0.0001; n = 12-20) and 15% (P < 0.01 and P < 0.0001; n = 20-30), respectively. Interestingly, this reduction in muscle mass was primarily due to an ~40% reduction in protein synthesis in both M-AKTDKO and M-indAKTDKO muscles (P < 0.05 and P < 0.01; n = 12-20) without significant changes in proteolysis or autophagy. Moreover, a significant reduction in oxidative capacity was observed in both M-AKTDKO (P < 0.05, P < 0.01 and P < 0.001; n = 5-12) and M-indAKTDKO (P < 0.05 and P < 0.01; n = 4). Mechanistically, activation and inhibition of mTORC1/FOXO1, respectively, but neither alone, were sufficient to restore protein synthesis, muscle oxidative capacity, and muscle function in the absence of AKT in vivo. In a mouse model of disuse-induced muscle loss, simultaneous activation of mTORC1 and inhibition of FOXO1 preserved muscle mass following immobilization (~5-10% reduction in casted M-indFOXO1TSCDKO muscles vs. ~30-40% casted M-indControl muscles, P < 0.05 and P < 0.0001; n = 8-16). CONCLUSIONS: Collectively, this study provides novel insights into the AKT-dependent mechanisms that underlie muscle protein homeostasis, function, and metabolism in both normal physiology and disuse-induced muscle wasting.

Persistent NF-κB activation in muscle stem cells induces proliferation-independent telomere shortening.

During the repeated cycles of damage and repair in many muscle disorders, including Duchenne muscular dystrophy (DMD), the muscle stem cell (MuSC) pool becomes less efficient at responding to and repairing damage. The underlying mechanism of such stem cell dysfunction is not fully known. Here, we demonstrate that the distinct early telomere shortening of diseased MuSCs in both mice and young DMD patients is associated with aberrant NF-κB activation. We find that prolonged NF-κB activation in MuSCs in chronic injuries leads to shortened telomeres and Ku80 dysregulation and results in severe skeletal muscle defects. Our studies provide evidence of a role for NF-κB in regulating stem-cell-specific telomere length, independently of cell replication, and could be a congruent mechanism that is applicable to additional tissues and/or diseases characterized by systemic chronic inflammation.

Distinctive patterns of microRNA expression in extraocular muscles.

The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other muscles. We and others have shown that EOMs have a unique transcriptome and proteome. Here we investigated the expression pattern of microRNAs (miRNAs), as they may play a role in generating the unique EOM allotype. We isolated RNA and screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. Seventy-four miRNAs were found to be differentially regulated (P value <0.05) of which 31 (14 upregulated, 17 downregulated) were differentially regulated at signal strength >500. Muscle-specific miRNAs miR-206 and miR-499 were upregulated and miR-1, miR-133a, and miR-133b were downregulated in EOM. Quantitative PCR (qPCR) analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using cotransfection of precursor miRNAs with reporter constructs containing the 3'-untranslated region of predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular makeup of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne muscular dystrophy and may assist in development of therapeutic strategies.

Pretreatment with a soluble activin type IIB receptor/Fc fusion protein improves hypoxia-induced muscle dysfunction.

Hypoxia, or reduced oxygen, occurs in a variety of clinical and environmental situations. Hypoxic exposure is associated with decreased muscle mass and a concomitant reduction in exercise capacity, although the exact mechanisms are not completely understood. The activin type IIB receptor (ActRIIB) is a receptor for transforming growth factor-beta (TGFbeta) superfamily members that are involved in the negative regulation of lean tissue mass. Given that hypoxia has negative effects on muscle mass and function and that modulation of the ActRIIB has been shown to increase muscle mass, we tested the hypothesis that pharmacological targeting of the ActRIIB for 2 wk would attenuate the loss of muscle mass and function in mice after exposure to normobaric hypoxia. ActRIIB modulation was achieved using a soluble activin receptor/Fc fusion protein (sActRIIB) in mice housed in a hypoxic chamber for 1 or 2 wk. Hypoxia induced a reduction in body weight in PBS- and sActRIIB-treated mice, although sActRIIB-treated mice remained larger throughout the hypoxic exposure. The absolute forces generated by extensor digitorum longus muscles were also significantly greater in sActRIIB- than PBS-treated mice and were more resistant to eccentric contraction-induced force drop after eccentric lengthening contractions. In summary, sActRIIB pretreatment attenuated hypoxia-induced muscle dysfunction. These data suggest that targeting the ActRIIB is an effective strategy to counter hypoxia-induced muscle dysfunction and to preacclimatize to hypoxia in clinical or high-altitude settings.

Superior calcium homeostasis of extraocular muscles.

Extraocular muscles (EOMs) are a unique group of skeletal muscles with unusual physiological properties such as being able to undergo rapid twitch contractions over extended periods and escape damage in the presence of excess intracellular calcium (Ca(2+)) in Duchenne's muscular dystrophy (DMD). Enhanced Ca(2+) buffering has been proposed as a contributory mechanism to explain these properties; however, the mechanisms are not well understood. We investigated mechanisms modulating Ca(2+) levels in EOM and tibialis anterior (TA) limb muscles. Using Fura-2 based ratiometric Ca(2+) imaging of primary myotubes we found that EOM myotubes reduced elevated Ca(2+) ˜2-fold faster than TA myotubes, demonstrating more efficient Ca(2+) buffering. Quantitative PCR (qPCR) and western blotting revealed higher expression of key components of the Ca(2+) regulation system in EOM, such as the cardiac/slow isoforms sarcoplasmic Ca(2+)-ATPase 2 (Serca2) and calsequestrin 2 (Casq2). Interestingly EOM expressed monomeric rather than multimeric forms of phospholamban (Pln), which was phosphorylated at threonine 17 (Thr17) but not at the serine 16 (Ser16) residue. EOM Pln remained monomeric and unphosphorylated at Ser16 despite protein kinase A (PKA) treatment, suggesting differential signalling and modulation cascades involving Pln-mediated Ca(2+) regulation in EOM. Increased expression of Ca(2+)/SR mRNA, proteins, differential post-translational modification of Pln and superior Ca(2+) buffering is consistent with the improved ability of EOM to handle elevated intracellular Ca(2+) levels. These characteristics provide mechanistic insight for the potential role of superior Ca(2+) buffering in the unusual physiology of EOM and their sparing in DMD.

Ets-2 repressor factor silences extrasynaptic utrophin by N-box mediated repression in skeletal muscle.

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPalpha/beta to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest "repressing repressors" as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.

PMO-based let-7c site blocking oligonucleotide (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne muscular dystrophy (DMD).

Upregulation of utrophin, a dystrophin related protein, is considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a set of miRNAs, among which let-7c is evolutionarily highly conserved. We designed PMO-based SBOs complementary to the let-7c binding site in UTRN 3'UTR, with the goal of inhibiting let-7c interaction with UTRN mRNA and thus upregulating utrophin. We used the C2C12UTRN5'luc3' reporter cell line in which the 5'- and 3'-UTRs of human UTRN sequences flank luciferase, for reporter assays and the C2C12 cell line for utrophin western blots, to independently evaluate the site blocking efficiency of a series of let-7c PMOs in vitro. Treatment of one-month old mdx mice with the most effective let-7c PMO (i.e. S56) resulted in ca. two-fold higher utrophin protein expression in skeletal muscles and the improvement in dystrophic pathophysiology in mdx mice, in vivo. In summary, we show that PMO-based let-7c SBO has potential applicability for upregulating utrophin expression as a therapeutic approach for DMD.

Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells.

Utrophin upregulation is considered a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). A number of microRNAs (miRNAs) post-transcriptionally regulate utrophin expression by binding their cognate sites in the 3' UTR. Previously we have shown that miRNA: UTRN repression can be alleviated using miRNA let-7c site blocking oligonucleotides (SBOs) to achieve utrophin upregulation and functional improvement in mdx mice. Here, we used CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c, miR-196b) clustered in a 500 bp inhibitory miRNA target region (IMTR) within the UTRN 3' UTR, for achieving higher expression of endogenous utrophin. Deleting the UTRN IMTR in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. 2-fold higher levels of utrophin protein. Differentiation of the UTRN edited DMD-hiPSCs (UTRNΔIMTR) by MyoD overexpression resulted in increased sarcolemmal α-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based UTRN genome editing offers a novel utrophin upregulation therapeutic strategy applicable to all DMD patients, irrespective of the dystrophin mutation status.