TRIM7 inhibits enterovirus replication and promotes emergence of a viral variant with increased pathogenicity.

To control viral infection, vertebrates rely on both inducible interferon responses and less well-characterized cell-intrinsic responses composed of "at the ready" antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell-intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7 but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor.

A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.

Functional micropeptides can be concealed within RNAs that appear to be noncoding. We discovered a conserved micropeptide, which we named myoregulin (MLN), encoded by a skeletal muscle-specific RNA annotated as a putative long noncoding RNA. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), which inhibit SERCA, the membrane pump that controls muscle relaxation by regulating Ca(2+) uptake into the sarcoplasmic reticulum (SR). MLN interacts directly with SERCA and impedes Ca(2+) uptake into the SR. In contrast to PLN and SLN, which are expressed in cardiac and slow skeletal muscle in mice, MLN is robustly expressed in all skeletal muscle. Genetic deletion of MLN in mice enhances Ca(2+) handling in skeletal muscle and improves exercise performance. These findings identify MLN as an important regulator of skeletal muscle physiology and highlight the possibility that additional micropeptides are encoded in the many RNAs currently annotated as noncoding.

A myocardin-adjacent lncRNA balances SRF-dependent gene transcription in the heart.

Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.

Myogenin and class II HDACs control neurogenic muscle atrophy by inducing E3 ubiquitin ligases.

Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.

Myofiber-type-dependent 'boulder' or 'multitudinous pebble' formations across distinct amylopectinoses.

At least five enzymes including three E3 ubiquitin ligases are dedicated to glycogen's spherical structure. Absence of any reverts glycogen to a structure resembling amylopectin of the plant kingdom. This amylopectinosis (polyglucosan body formation) causes fatal neurological diseases including adult polyglucosan body disease (APBD) due to glycogen branching enzyme deficiency, Lafora disease (LD) due to deficiencies of the laforin glycogen phosphatase or the malin E3 ubiquitin ligase and type 1 polyglucosan body myopathy (PGBM1) due to RBCK1 E3 ubiquitin ligase deficiency. Little is known about these enzymes' functions in glycogen structuring. Toward understanding these functions, we undertake a comparative murine study of the amylopectinoses of APBD, LD and PGBM1. We discover that in skeletal muscle, polyglucosan bodies form as two main types, small and multitudinous ('pebbles') or giant and single ('boulders'), and that this is primarily determined by the myofiber types in which they form, 'pebbles' in glycolytic and 'boulders' in oxidative fibers. This pattern recapitulates what is known in the brain in LD, innumerable dust-like in astrocytes and single giant sized in neurons. We also show that oxidative myofibers are relatively protected against amylopectinosis, in part through highly increased glycogen branching enzyme expression. We present evidence of polyglucosan body size-dependent cell necrosis. We show that sex influences amylopectinosis in genotype, brain region and myofiber-type-specific fashion. RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), the only known cellular machinery for head-to-tail linear ubiquitination critical to numerous cellular pathways. We show that the amylopectinosis of RBCK1 deficiency is not due to loss of linear ubiquitination, and that another function of RBCK1 or LUBAC must exist and operate in the shaping of glycogen. This work opens multiple new avenues toward understanding the structural determinants of the mammalian carbohydrate reservoir critical to neurologic and neuromuscular function and disease.

Ectopic PLAG1 induces muscular dystrophy in the mouse.

Even though various genetic mutations have been identified in muscular dystrophies (MD), there is still a need to understand the biology of MD in the absence of known mutations. Here we reported a new mouse model of MD driven by ectopic expression of PLAG1. This gene encodes a developmentally regulated transcription factor known to be expressed in developing skeletal muscle, and implicated as an oncogene in certain cancers including rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma composed of myoblast-like cells. By breeding loxP-STOP-loxP-PLAG1 (LSL-PLAG1) mice into the MCK-Cre line, we achieved ectopic PLAG1 expression in cardiac and skeletal muscle. The Cre/PLAG1 mice died before 6 weeks of age with evidence of cardiomyopathy significantly limiting left ventricle fractional shortening. Histology of skeletal muscle revealed dystrophic features, including myofiber necrosis, fiber size variation, frequent centralized nuclei, fatty infiltration, and fibrosis, all of which mimic human MD pathology. QRT-PCR and Western blot revealed modestly decreased Dmd mRNA and dystrophin protein in the dystrophic muscle, and immunofluorescence staining showed decreased dystrophin along the cell membrane. Repression of Dmd by ectopic PLAG1 was confirmed in dystrophic skeletal muscle and various cell culture models. In vitro studies showed that excess IGF2 expression, a transcriptional target of PLAG1, phenocopied PLAG1-mediated down-regulation of dystrophin. In summary, we developed a new mouse model of a lethal MD due to ectopic expression of PLAG1 in heart and skeletal muscle. Our data support the potential contribution of excess IGF2 in this model. Further studying these mice may provide new insights into the pathogenesis of MD and perhaps lead to new treatment strategies.

Activation of Autophagic Flux Blunts Cardiac Ischemia/Reperfusion Injury.

[Figure: see text].

Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development.

HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes. Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality. Hand2 expression is tightly regulated by upstream enhancers that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications. Here we show that transcription of a Hand2-associated long non-coding RNA, which we named upperhand (Uph), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome, the Uph-Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment.

Mechanistic basis of neonatal heart regeneration revealed by transcriptome and histone modification profiling.

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.

Development of a Chronic Canine Ovariohysterectomy Model to Evaluate Vaginal Cuff Healing Using Two Closure Systems.

Background and Purpose. This study established a suitable animal model of ovariohysterectomy; characterized the course and pattern of vaginal healing after ovariohysterectomy; and compared healing obtained after closure of the vaginal cuff with a novel cuff-closure device (Zip-stitch® clips) and VICRYL® sutures. Research Design and Study Sample. This prospective, randomized, controlled, blinded animal study was conducted in 27 mongrel hounds according to an IACUC-approved protocol. Each animal underwent ovariohysterectomy followed by vaginal cuff closure with Zip-stitch or VICRYL. At two or six weeks, animals were sacrificed for gross and histological analysis. Data Collection. The primary endpoint was the difference in the fraction of vaginal cuff healed six weeks after application of the closure device. Secondary endpoints included histopathologic cellular and tissue responses, including inflammation, necrosis, infection, and vascular and muscle changes. Results. In the test group, there were two distinct locations where fibrotic or granular tissue fusion between the anterior and posterior vaginal walls was observed: in tissue "captured" by a clip or in tissue around the clip. The fraction of the vaginal cuff healed was similar in animals treated with Zip-stitch clips and those treated with sutures at six weeks (68±10% vs 67±18%; P=.148, test for non-inferiority) after surgery. The test article performed similarly or better than the control article in terms of the intensity or extent of the secondary endpoints. Conclusions. Subject to further confirmation, this study supports Zip-stitch clips as a method to maintain immediate post-operative approximation of the vaginal cuff leading to healing but did not achieve statistical significance in its primary endpoint.

The G protein-coupled receptor Gpr161 regulates forelimb formation, limb patterning and skeletal morphogenesis in a primary cilium-dependent manner.

The role of basal suppression of the sonic hedgehog (Shh) pathway and its interaction with Indian hedgehog (Ihh) signaling during limb/skeletal morphogenesis is not well understood. The orphan G protein-coupled receptor Gpr161 localizes to primary cilia and functions as a negative regulator of Shh signaling by promoting Gli transcriptional repressor versus activator formation. Here, we show that forelimb buds are not formed in Gpr161 knockout mouse embryos despite establishment of prospective limb fields. Limb-specific deletion of Gpr161 resulted in prematurely expanded Shh signaling and ectopic Shh-dependent patterning defects resulting in polysyndactyly. In addition, endochondral bone formation in forearms, including formation of both trabecular bone and bone collar was prevented. Endochondral bone formation defects resulted from accumulation of proliferating round/periarticular-like chondrocytes, lack of differentiation into columnar chondrocytes, and corresponding absence of Ihh signaling. Gpr161 deficiency in craniofacial mesenchyme also prevented intramembranous bone formation in calvarium. Defects in limb patterning, endochondral and intramembranous skeletal morphogenesis were suppressed in the absence of cilia. Overall, Gpr161 promotes forelimb formation, regulates limb patterning, prevents periarticular chondrocyte proliferation and drives osteoblastogenesis in intramembranous bones in a cilium-dependent manner.

Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart.

Although the adult mammalian heart is incapable of meaningful functional recovery following substantial cardiomyocyte loss, it is now clear that modest cardiomyocyte turnover occurs in adult mouse and human hearts, mediated primarily by proliferation of pre-existing cardiomyocytes. However, fate mapping of these cycling cardiomyocytes has not been possible thus far owing to the lack of identifiable genetic markers. In several organs, stem or progenitor cells reside in relatively hypoxic microenvironments where the stabilization of the hypoxia-inducible factor 1 alpha (Hif-1α) subunit is critical for their maintenance and function. Here we report fate mapping of hypoxic cells and their progenies by generating a transgenic mouse expressing a chimaeric protein in which the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 recombinase. In mice bearing the creERT2-ODD transgene driven by either the ubiquitous CAG promoter or the cardiomyocyte-specific α myosin heavy chain promoter, we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart. These results indicate that hypoxia signalling is an important hallmark of cycling cardiomyocytes, and suggest that hypoxia fate mapping can be a powerful tool for identifying cycling cells in adult mammals.

Correction of Three Prominent Mutations in Mouse and Human Models of Duchenne Muscular Dystrophy by Single-Cut Genome Editing.

Duchenne muscular dystrophy (DMD), one of the most common neuromuscular disorders of children, is caused by the absence of dystrophin protein in striated muscle. Deletions of exons 43, 45, and 52 represent mutational "hotspot" regions in the dystrophin gene. We created three new DMD mouse models harboring deletions of exons 43, 45, and 52 to represent common DMD mutations. To optimize CRISPR-Cas9 genome editing using the single-cut strategy, we identified single guide RNAs (sgRNAs) capable of restoring dystrophin expression by inducing exon skipping and reframing. Intramuscular delivery of AAV9 encoding SpCas9 and selected sgRNAs efficiently restored dystrophin expression in these new mouse models, offering a platform for future studies of dystrophin gene correction therapies. To validate the therapeutic potential of this approach, we identified sgRNAs capable of restoring dystrophin expression by the single-cut strategy in cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) with each of these hotspot deletion mutations. We found that the potential effectiveness of individual sgRNAs in correction of DMD mutations cannot be predicted a priori, highlighting the importance of sgRNA design and testing as a prelude for applying gene editing as a therapeutic strategy for DMD.

Fusogenic micropeptide Myomixer is essential for satellite cell fusion and muscle regeneration.

Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.

Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities.

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

High-Phosphate Diet Induces Exercise Intolerance and Impairs Fatty Acid Metabolism in Mice.

BACKGROUND: Inorganic phosphate (Pi) is used extensively as a preservative and a flavor enhancer in the Western diet. Physical inactivity, a common feature of Western societies, is associated with increased cardiovascular morbidity and mortality. It is unknown whether dietary Pi excess contributes to exercise intolerance and physical inactivity. METHODS: To determine an association between Pi excess and physical activity in humans, we assessed the relationship between serum Pi and actigraphy-determined physical activity level, as well as left ventricular function by cardiac magnetic resonance imaging, in DHS-2 (Dallas Heart Study phase 2) participants after adjusting for relevant variables. To determine direct effects of dietary Pi on exercise capacity, oxygen uptake, serum nonesterified fatty acid, and glucose were measured during exercise treadmill test in C57/BL6 mice fed either a high-Pi (2%) or normal-Pi (0.6%) diet for 12 weeks. To determine the direct effect of Pi on muscle metabolism and expression of genes involved in fatty acid metabolism, additional studies in differentiated C2C12 myotubes were conducted after subjecting to media containing 1 to 3 mmol/L Pi (pH 7.0) to simulate in vivo phosphate conditions. RESULTS: In participants of the DHS-2 (n=1603), higher serum Pi was independently associated with reduced time spent in moderate to vigorous physical activity ( P=0.01) and increased sedentary time ( P=0.004). There was no association between serum Pi and left ventricular ejection fraction or volumes. In animal studies, compared with the control diet, consumption of high-Pi diet for 12 weeks did not alter body weight or left ventricular function but reduced maximal oxygen uptake, treadmill duration, spontaneous locomotor activity, fat oxidation, and fatty acid levels and led to downregulation of genes involved in fatty acid synthesis, release, and oxidation, including Fabp4, Hsl, Fasn, and Pparγ, in muscle. Similar results were recapitulated in vitro by incubating C2C12 myotubes with high-Pi media. CONCLUSIONS: Our data demonstrate a detrimental effect of dietary Pi excess on skeletal muscle fatty acid metabolism and exercise capacity that is independent of obesity and cardiac contractile function. Dietary Pi may represent a novel and modifiable target to reduce physical inactivity associated with the Western diet.

Adiponectin protects against development of metabolic disturbances in a PCOS mouse model.

Adiponectin, together with adipocyte size, is the strongest factor associated with insulin resistance in women with polycystic ovary syndrome (PCOS). This study investigates the causal relationship between adiponectin levels and metabolic and reproductive functions in PCOS. Prepubertal mice overexpressing adiponectin from adipose tissue (APNtg), adiponectin knockouts (APNko), and their wild-type (WT) littermate mice were continuously exposed to placebo or dihydrotestosterone (DHT) to induce PCOS-like traits. As expected, DHT exposure led to reproductive dysfunction, as judged by continuous anestrus, smaller ovaries with a decreased number of corpus luteum, and an increased number of cystic/atretic follicles. A two-way between-groups analysis showed that there was a significant main effect for DHT exposure, but not for genotype, indicating adiponectin does not influence follicle development. Adiponectin had, however, some protective effects on ovarian function. Similar to in many women with PCOS, DHT exposure led to reduced adiponectin levels, larger adipocyte size, and reduced insulin sensitivity in WTs. APNtg mice remained metabolically healthy despite DHT exposure, while APNko-DHT mice were even more insulin resistant than their DHT-exposed littermate WTs. DHT exposure also reduced the mRNA expression of genes involved in metabolic pathways in gonadal adipose tissue of WT and APNko, but this effect of DHT was not observed in APNtg mice. Moreover, APNtg-DHT mice displayed increased pancreatic mRNA levels of insulin receptors, Pdx1 and Igf1R, suggesting adiponectin stimulates beta cell viability/hyperplasia in the context of PCOS. In conclusion, adiponectin improves metabolic health but has only minor effects on reproductive functions in this PCOS-like mouse model.

Myomaker is a membrane activator of myoblast fusion and muscle formation.

Fusion of myoblasts is essential for the formation of multi-nucleated muscle fibres. However, the identity of muscle-specific proteins that directly govern this fusion process in mammals has remained elusive. Here we identify a muscle-specific membrane protein, named myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is downregulated thereafter. Overexpression of myomaker in myoblasts markedly enhances fusion, and genetic disruption of myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibres. Remarkably, forced expression of myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacological perturbation of the actin cytoskeleton abolishes the activity of myomaker, consistent with previous studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein that controls mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.

Requirement of MEF2A, C, and D for skeletal muscle regeneration.

Regeneration of adult skeletal muscle following injury occurs through the activation of satellite cells, an injury-sensitive muscle stem cell population that proliferates, differentiates, and fuses with injured myofibers. Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors play essential roles in muscle differentiation during embryogenesis, but their potential contributions to adult muscle regeneration have not been systematically explored. To investigate the potential involvement of MEF2 factors in muscle regeneration, we conditionally deleted the Mef2a, c, and d genes, singly and in combination, within satellite cells in mice, using tamoxifen-inducible Cre recombinase under control of the satellite cell-specific Pax7 promoter. We show that deletion of individual Mef2 genes has no effect on muscle regeneration in response to cardiotoxin injury. However, combined deletion of the Mef2a, c, and d genes results in a blockade to regeneration. Satellite cell-derived myoblasts lacking MEF2A, C, and D proliferate normally in culture, but cannot differentiate. The absence of MEF2A, C, and D in satellite cells is associated with aberrant expression of a broad collection of known and unique protein-coding and long noncoding RNA genes. These findings reveal essential and redundant roles of MEF2A, C, and D in satellite cell differentiation and identify a MEF2-dependent transcriptome associated with skeletal muscle regeneration.