Muscle Cell Differentiation Research Articles

Adaptive changes in the DNA damage response during skeletal muscle cell differentiation

DNA double-strand breaks (DSBs) trigger specialized cellular mechanisms that collectively form the DNA damage response (DDR). In proliferating cells, the DDR serves the function of mending DNA breaks and satisfying the cell-cycle checkpoints. Distinct goals exist in differentiated cells that are postmitotic and do not face cell-cycle checkpoints. Nonetheless, the distinctive requirements and mechanistic details of the DDR in differentiated cells are still poorly understood. In this study, we set an in vitro differentiation model of human skeletal muscle myoblasts into multinucleated myotubes that allowed monitoring DDR dynamics during cell differentiation. Our results demonstrate that myotubes have a prolonged DDR, which is nonetheless competent to repair DSBs and render them significantly more resistant to cell death than their progenitors. Using live-cell microscopy and single-molecule kinetic measurements of transcriptional activity, we observed that myotubes respond to DNA damage by rapidly and transiently suppressing global gene expression and rewiring the epigenetic landscape of the damaged nucleus. Our findings provide novel insights into the DDR dynamics during cellular differentiation and shed light on the strategy employed by human skeletal muscle to preserve the integrity of the genetic information and sustain long-term organ function after DNA damage.

G6PD Orchestrates Genome-Wide DNA Methylation and Gene Expression in the Vascular Wall.

Recent advances have revealed the importance of epigenetic modifications to gene regulation and transcriptional activity. DNA methylation, a determinant of genetic imprinting and the de novo silencing of genes genome-wide, is known to be controlled by DNA methyltransferases (DNMT) and demethylases (TET) under disease conditions. However, the mechanism(s)/factor(s) influencing the expression and activity of epigenetic writers and erasers, and thus DNA methylation, in healthy vascular tissue is incompletely understood. Based on our recent studies, we hypothesized that glucose-6-phosphate dehydrogenase (G6PD) is a modifier of DNMT and TET expression and activity and an enabler of gene expression. In the aorta of CRISPR-edited rats with the Mediterranean G6PD variant, we determined DNA methylation by whole-genome bisulfite sequencing, gene expression by RNA sequencing, and large artery stiffness by echocardiography. Here, we documented higher expression of Dnmt1, Dnmt3a, Tet2, and Tet3 in aortas from Mediterranean G6PDS188F variant (a loss-of-function single nucleotide polymorphism) rats than their wild-type littermates. Concomitantly, we identified 17,618 differentially methylated loci genome-wide (5787 hypermethylated loci, including down-regulated genes encoding inflammation- and vasoconstriction-causing proteins, and 11,827 hypomethylated loci, including up-regulated genes encoding smooth muscle cell differentiation- and fatty acid metabolism-promoting proteins) in aortas from G6PDS188F as compared to wild-type rats. Our results demonstrated that nitric oxide, which is generated in a G6PD-derived NADPH-dependent manner, increases TET and decreases DNMT activity. Further, we observed less large artery (aorta) stiffness in G6PDS188F as compared to wild-type rats. These results establish a noncanonical function of the wild-type G6PD and G6PDS188F variant in the regulation of DNA methylation and gene expression in healthy vascular tissue and reveal that the G6PDS188F variant contributes to reducing large artery stiffness.

BACH1 regulates the differentiation of vascular smooth muscle cells from human embryonic stem cells via CARM1-mediated methylation of H3R17

The role of BACH1 in the process of vascular smooth muscle cell (VSMC) differentiation from human embryonic stem cells (hESCs) remains unknown. Here, we find that the loss of BACH1 in hESCs attenuates the expression of VSMC marker genes, whereas overexpression of BACH1 after mesoderm induction increases the expression of VSMC markers during invitro hESC-VSMC differentiation. Mechanistically, BACH1 binds directly to coactivator-associated arginine methyltransferase 1 (CARM1) during invitro hESC-VSMC differentiation, and this interaction is mediated by the BACH1 bZIP domain. BACH1 recruits CARM1 to VSMC marker gene promoters and promotes VSMC marker expression by increasing H3R17me2 modification, thus facilitating invitro VSMC differentiation from hESCs after the mesoderm induction. The increased expression of VSMC marker genes by BACH1 overexpression is partially abolished by inhibition of CARM1 or the H3R17me2 inhibitor TBBD in hESC-derived cells. These findings highlight the critical role of BACH1 in hESC differentiation into VSMCs by CARM1-mediated methylation of H3R17.

Abstract 15295: Myocardin Condensation Drives Cell Lineage Specification During Cardiogenesis

During cardiovascular development, cells make switch-like decisions to activate the expression of new gene programs leading to lineage specification. The mechanisms underlying these decisive choices remain unclear. Myocardin is a cardiomyocyte and smooth muscle cell specific transcriptional coactivator, which is necessary and important for activating cardiovascular genes. We show that Myocardin activates lineage-specific gene programs by concentration-dependent and switch-like formation of nuclear condensates. By modeling the natural changes in Myocardin concentration during development, coupled with quantitative fluorescence microscopy, single-cell resolution reporter assays, and cellular differentiation assays, we demonstrate that condensate formation is directly linked to transcriptional activation and cardiovascular lineage specification. During cardiomyocyte and smooth muscle cell differentiation, the formation of Myocardin condensates precedes activation of cell identity genes and these condensates are present at sites of cell identity gene transcription. Myocardin condensates form, activate gene expression, and specify cell state at critical concentration thresholds, dependent upon the C-terminal disordered region of Myocardin. Disrupting condensate formation by manipulating the sequence of this region abolishes gene activation, which can be rescued by replacing this region with condensate-forming disordered regions from other unrelated proteins. Our work demonstrates that Myocardin condensation at critical concentrations enables switch-like changes in gene expression programs, providing new insights into cardiomyocyte and smooth muscle cell lineage specification during cardiovascular development.

Improved culture procedure for bovine muscle satellite cells for cultured meat

Many researchers and companies around the world are reported to have developed cultured meat, but their specific techniques have rarely been disclosed. Thus, the purpose of this study is to provide an improved procedure for cultured meat. There are four major steps in this cultured meat production: muscle cell isolation, proliferation, differentiation, and validation. The improved isolation enabled the efficient removal of unnecessary cells and tissues compared to previous procedures. In addition, proper use of basal media can improve the proliferation efficiency by about 2-fold. During the differentiation process, improved procedure was performed by using 10 % horse serum-containing media after 3 days of initial differentiation for myotube induction. This method demonstrated significantly enhanced myotube formation, up to 2.6-fold increase in area and up to 1.9-fold increase in fusion index compared to the previous method. This study provides a simple, improved procedure to enable more effective cultured meat production compared to previous procedures and is expected to help produce inexpensive and safe cultured meat.

A newly identified lncRNA lnc000100 regulates proliferation and differentiation of cattle skeletal muscle cells

ABSTRACT Cattle skeletal muscle development is a complex and highly coordinated biological process mediated by a series of myogenic regulators, which plays a critical role in beef yield and quality. Long non-coding RNAs (lncRNAs) have been shown to regulate skeletal muscle development. However, the molecular mechanism by which lncRNAs regulate skeletal muscle development is largely unknown. We performed transcriptome analysis of muscle tissues of adult and embryo Angus cattle to investigate the mechanism by which lncRNA regulates skeletal muscle development between adult and embryo cattle. A total of 37,115 candidate lncRNAs were detected, and a total of 1,998 lncRNAs were differentially expressed between the muscle tissue libraries of adult and embryo cattle, including 1,229 up-regulated lncRNAs and 769 down-regulated lncRNAs (adult cattle were the control group). We verified the expression of 7 differentially expressed lncRNAs by quantitative real-time PCR (RT-qPCR), and analysed the tissue expression profile of lnc000100, which is down-regulated in the longest dorsal muscle during foetal life and which is highly specifically expressed in muscle tissue. We found that the interference of lnc000100 significantly inhibited cell proliferation and promoted cell differentiation. Lnc000100 was located in the nucleus by RNA-FISH. Our research provides certain resources for the analysis of lncRNA regulating cattle skeletal muscle development, and may also provide new insights for improving beef production and breed selection.

Histopathological Evaluation of Pulmonary Thromboendarterectomy Specimens of Chronic Thromboembolic Pulmonary Hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a condition with a poor prognosis in which the pulmonary arteries are occluded by organized thrombi. P ulmonary thromboendarterectomy (PEA) is an effective treatment for CTEPH; however, the literature on its histopathological examination is lacking. This study aimed to investigate the histopathological findings and protein and gene expression in PEA specimens, establish an optimal histopathological evaluation method, and clarify the mechanisms of thrombus organization and disease progression in CTEPH. In total, 50 patients with CTEPH who underwent PEA were analyzed. The patients were categorized according to their clinical data into two groups: good and poor postoperative courses. The relationship between their histopathological findings and the clinical course was examined. Immunohistochemical studies confirmed the expression of oxidants, antioxidants, and s mooth muscle cell (SMC) differentiation markers and their changes during the progression of thrombus organization. The mRNA expression analysis of 102 samples from 27 cases included oxidants, antioxidants, and vasoconstrictor endothelin-1. In the PEA specimens, colander-like lesions (aggregations of recanalized blood vessels with well-differentiated SMCs) were significantly more common in the good postoperative course group than in the poor postoperative course group; analysis of proteins and genes proposed that oxidative and antioxidant mechanisms were involved. In the colander-like lesions, there was an increase in endothelin-1 mRNA and protein expression of endothelin receptor A. Colander-like lesions in PEA specimens must be identified. Additionally, SMC differentiation in recanalized vessels and the expression of vasoconstrictors and their receptors may contribute to the progression of CTEPH.

Disturbed flow regulates protein disulfide isomerase A1 expression via microRNA-204 and controls vascular smooth muscle cell differentiation

Disturbed flow regulates protein disulfide isomerase A1 expression via microRNA-204 and controls vascular smooth muscle cell differentiation

193 Creatine Monohydrate on In Vitro Cultivated Bovine Cells Leading to Guanidinoacetic Acid Supplementation In Vivo

Abstract Guanidinoacetic acid (GAA) is an amino acid-like compound that acts as a precursor to creatine (CRE) when either made in the body or digested. Creatine is directly used by skeletal muscle tissue as an energy source with the potential to act as a growth-promoting agent. The study objectives were to evaluate the effects of various CRE concentrations on bovine satellite cells for the rate of proliferation and myogenic differentiation. Satellite cells (SC) were isolated from the Longissimus muscle of 3-month-old Holstein bulls (n = 5). Cells were seeded at a density of 1 × 104 cells/mL on 96-well plates and incubated with 0 (CON), 2.5, 5, 7.5, and 10 mM of CRE. The absorbance rate was measured at 0, 24, 48, 72, and 96 h of incubation. Cells for gene expression were plated on 6-well plates at a density of 1 × 105 in 10% FBS/DMEM. Following 24 h of adhesion, SC were incubated with 5 and 7.5mM CRE for 48 h. After which, cells were harvested after 48 h for Pax3, Pax7, and Myf5 mRNA gene expression analysis. To identify the effects of CRE during myogenic differentiation, SCs were induced myogenic differentiation. Upon reaching approximately 90% confluency, the growth medium was replaced by differentiation media (2% horse serum/ DMEM) containing 0 (CON), 5, and 7.5 mM CRE. A two-way ANOVA and Tukey's HSD test were conducted regarding cell proliferation. A one-way ANOVA and Tukey's HSD test were performed for gene expression. Interaction of treatment against time was noted (P < 0.01) in Con, 2.5, 5, 7.5, and 10 mM CRE. Cell proliferation rate was influenced by CRE treatment (P < 0.01). At 72 h of incubation, all CRE treatments increased cell proliferation rate compared with Con (P < 0.05). At 96 h of incubation, cells treated with 2.5, 5, and 7.5 mM of CRE increased proliferation compared with Con (P < 0.05). At 96 h, CRE treatment 2.5, 5, and 7.5 mM increased proliferation rate compared with 10mM CRE treated cells (P < 0.05). No treatment effect was found in the mRNA gene expression of Pax7, Pax3, and Myf5 (P > 0.05). Cells incubated with CRE showed no change in mRNA gene expression of IGF-1 and Myogenin (P > 0.05), and a tendency was noted for S6KB1 increase expression under treatment (P = 0.0643). S6KB1 is a protein kinase that, through mTOR pathway expression, leads to an increase in cell growth, protein synthesis, and cell proliferation. Results suggest that the treatment of BSCs with CRE impacted proliferation, CRE at 5mM vs. 7.5 mM concentrations treatment of differentiated muscle cells led to a trend in altering S6KB1 gene expression.

Comparison of muscle structure and transcriptome analysis reveals the mechanism of growth variation in Yangtze sturgeon (Acipenser dabryanus)

Sturgeons display a slow growth rate and delayed initial sexual maturity, which extends the captive breeding and conservation processes. To date, few studies have revealed the molecular mechanism underlying the growth difference of sturgeons. In the present study, juvenile Yangtze sturgeons (Acipenser dabryanus) with different growth rates were selected from the same cultured pool for muscle structure, enzyme activity and comparative transcriptome analysis. Histological assays showed that the fiber diameter of the fast-growing group was significantly larger than that of the slow-growing group. The activities of four glycometabolism related enzymes, including two key rate-limiting enzymes hexokinase and pyruvate kinase, were significantly higher in fast-growing group. RNA-Seq of muscle tissues identified 7408 differentially expressed genes (DEGs), which consisted of 3637 upregulated genes and 3771 downregulated genes, in the fast-growing group vs. the slow-growing group. In the brain, a total of 12,213 unigenes were differentially expressed (6050 upregulated unigenes and 6163 downregulated unigenes) between the two groups. The expression levels of IGF1 and IGF1-binding proteins (IGFBPs) in muscle were significantly higher in the fast-growing group and might be promising candidate biomarkers for measuring growth performance in Yangtze sturgeon. Two myogenic regulatory factors (myog and myf6) were significantly upregulated, which indicated that the growth variation in juvenile Yangtze sturgeon might be partly due to the difference in the differentiation of muscle cells. In addition, pathway enrichment analysis revealed that the glycolysis and cell cycle pathways were significantly enhanced in the fast-growing groups, suggesting that they might also play important roles in the improved growth performance of A. dabryanus. Furthermore, the WGCNA as well as hub gene network analyses supported that cell cycle and gluconeogenesis might be involved in the differential growth performance of A. dabryanus. The results of this study may contribute to our understanding of the underlying molecular mechanism of growth variation in fish.

RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis

Alternative splicing is a prevalent gene-regulatory mechanism, with over 95% of multi-exon human genes estimated to be alternatively spliced. Here, we describe a tissue-specific, developmentally regulated, highly conserved, and disease-associated alternative splicing event in exon 7 of the eyes absent homolog 3 (Eya3) gene. We discovered that EYA3 expression is vital to the proliferation and differentiation of myoblasts. Genome-wide transcriptomic analysis and mass spectrometry-based proteomic studies identified SIX homeobox 4 (SIX4) and zinc finger and BTB-domain containing 1 (ZBTB1), as major transcription factors that interact with EYA3 to dictate gene expression. EYA3 isoforms differentially regulate transcription, indicating that splicing aids in temporal control of gene expression during muscle cell differentiation. Finally, we identified RNA-binding fox-1 homolog 2 (RBFOX2) as the main regulator of EYA3 splicing. Together, our findings illustrate the interplay between alternative splicing and transcription during myogenesis.

Gene Expression Analyses in Models of Rosiglitazone-Induced Physiological and Pathological Mineralization Identify Novel Targets to Improve Bone and Vascular Health.

Clinical studies revealed detrimental skeletal and vascular effects of the insulin sensitizer rosiglitazone. We have shown earlier that rosiglitazone accelerates osteoblast differentiation from human mesenchymal stem cells (hMSC) at the expense of increased oxidative stress and cell death. In calcifying human vascular cells, rosiglitazone stimulates pathological mineralization, an effect diminished by the antioxidant resveratrol. Here, we aimed to elucidate transcriptional networks underlying the rosiglitazone-enhanced mineralization phenotype. We performed genome-wide transcriptional profiling of osteogenic hMSCs treated with rosiglitazone for short-term periods of 1 up to 48 h during the first two days of differentiation, a phase that we show is sufficient for rosiglitazone stimulation of mineralization. Microarray-based mRNA expression analysis revealed 190 probes that were differently expressed in at least one condition compared to vehicle-treated control. This rosiglitazone gene signature contained well-known primary PPAR targets and was also endogenously regulated during osteogenic hMSC differentiation and osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) into calcifying vascular cells (CVCs). Comparative analysis revealed rosiglitazone targets that were commonly enriched in osteoblasts and CVCs or specifically enriched in either osteoblasts or CVCs. Finally, we compared expression patterns of CVC-specific genes with patient expression data from carotid plaque versus intact adjacent tissue, and identified five rosiglitazone targets to be differentially regulated in CVCs and carotid plaque but not osteoblasts when compared to their non-mineralizing counterparts. These targets, i.e., PDK4, SDC4, SPRY4, TCF4 and DACT1, may specifically control extracellular matrix mineralization in vascular cells, and hence provide target candidates for further investigations to improve vascular health.

Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives.

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.

Hypocalcemia in combination with hyperphosphatemia impairs muscle cell differentiation in vitro

PurposeHypoparathyroidism is a rare endocrine disorder characterized by low or absent secretion of parathyroid hormone (PTH), which leads to decreased calcium and increased phosphorus levels in the serum. The diagnosis of hypoparathyroidism is based on the identification of the aforementioned biochemical abnormalities, which may be accompanied by clinical manifestations. Symptoms of hypoparathyroidism, primarily attributed to hypocalcemia, include muscle cramps or spasms, facial, leg, and foot pain, seizures, and tingling in the lips or fingers. The treatment of hypoparathyroidism depends on the severity of symptoms and the underlying pathology. Over the long term, calcium supplements, active vitamin D analogs, and thiazide diuretics may be needed. In fact, in patient cohorts in which optimal disease control still remains elusive, replacement therapy with recombinant parathyroid hormone analogs may be contemplated. Despite the predominantly neuromuscular symptoms of hypoparathyroidism, further effects of parathyroid hormone deficiency at the muscle cell level remain poorly understood. Thus, the aim of our study was to evaluate the effects of hypocalcemia in combination with hyperphosphatemia on muscle cells differentiation in vitro.MethodsC2C12 cells, an in vitro model of muscle cells, were differentiated for 2 or 6 days in the presence of hypocalcemia (CaCl2 0.9 mmol/l) and moderate (PO4 1.4 mmol/l) or severe (PO4 2.9 mmol/l) hyperphosphatemia, or combinations of both conditions. Cell differentiation and expression of genes linked to muscle differentiation were evaluated.ResultsThe combination of hypocalcemia with hyperphosphatemia induced a significant reduction (50%) in differentiation marker levels, such as MyoD (protein 1 for myoblast determination) and myogenin on the 1st day of differentiation, and MHC (myosin heavy chains) after 6 days of differentiation compared to control. Furthermore, this condition induced a statistically significant reduction of insulin-like growth factor-1 (IGF-1) mRNA expression and inhibition of IGF signaling and decrease in ERK phosphorylation compared to control cells.ConclusionsOur results showed that a condition of hypocalcemia with hyperphosphatemia induced an alteration of muscle cell differentiation in vitro. In particular, we observed the reduction of myogenic differentiation markers, IGF-1 signaling pathway, and ERK phosphorylation in differentiated skeletal myoblasts. These data suggest that this altered extracellular condition might contribute to the mechanisms causing persistence of symptoms in patients affected by hypoparathyroidism.

Icariin attenuates the calcification of vascular smooth muscle cells through ERα - p38MAPK pathway.

To investigate the relationship between icariin and the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and the signal pathway involved. We applied a universally accepted calcification model of VSMCs induced by β glycerophosphate. Then the VSMCs calcification was observed by treatment with icariin and/or inhibitors of estrogen receptors (ERs) and p38-mitogen-activated protein kinase (MAPK) signaling. Icariin inhibited osteoblastic differentiation and mineralization of VSMCs due to decreased ALP activity and Runx2 expression. Further study demonstrated that icariin exerted this suppression effect through activating p38-MAPK but not extracellular-regulated kinase, JNK or Akt. An inhibitor of p38-MAPK partially reversed the inhibitory effects of icariin on osteoblastic differentiation. Interestingly, treatment of VSMCs with an ER antagonist ICI182780 and a selective ERα receptor antagonist PPT attenuated icariin-mediated inhibition effect of VSMCs calcification, associated with suppression of p38-MAPK phosphorylation. Icariin inhibited the osteoblastic differentiation of VSMCs, and that the inhibitory effects were mediated by p38-MAPK pathways through ERα.

Apabetalone, a Clinical-Stage, Selective BET Inhibitor, Opposes DUX4 Target Gene Expression in Primary Human FSHD Muscle Cells.

Facioscapulohumeral dystrophy (FSHD) is a muscle disease caused by inappropriate expression of the double homeobox 4 (DUX4) gene in skeletal muscle, and its downstream activation of pro-apoptotic transcriptional programs. Inhibitors of DUX4 expression have the potential to treat FSHD. Apabetalone is a clinical-stage bromodomain and extra-terminal (BET) inhibitor, selective for the second bromodomain on BET proteins. Using primary human skeletal muscle cells from FSHD type 1 patients, we evaluated apabetalone for its ability to counter DUX4's deleterious effects and compared it with the pan-BET inhibitor JQ1, and the p38 MAPK inhibitor-and DUX4 transcriptional repressor-losmapimod. We applied RNA-sequencing and bioinformatic analysis to detect treatment-associated impacts on the transcriptome of these cells. Apabetalone inhibited the expression of DUX4 downstream markers, reversing hallmarks of FSHD gene expression in differentiated muscle cells. JQ1, but not apabetalone, was found to induce apoptosis. While both BET inhibitors modestly impacted differentiation marker expression, they did not affect myotube fusion. Losmapimod also reduced expression of DUX4 target genes but differed in its impact on FSHD-associated pathways. These findings demonstrate that apabetalone inhibits DUX4 target gene expression and reverses transcriptional programs that contribute to FSHD pathology, making this drug a promising candidate therapeutic for FSHD.

Nuclear Smooth Muscle α-actin Participates in Vascular Smooth Muscle Cell Differentiation.

Missense variants throughout ACTA2, encoding smooth muscle α-actin (αSMA), predispose to adult-onset thoracic aortic disease, but variants disrupting arginine 179 (R179) lead to Smooth Muscle Dysfunction Syndrome (SMDS) characterized by diverse childhood-onset vascular diseases. Here we show that αSMA localizes to the nucleus in wildtype (WT) smooth muscle cells (SMCs), enriches in the nucleus with SMC differentiation, and associates with chromatin remodeling complexes and SMC contractile gene promotors. The ACTA2 p.R179 αSMA variant shows decreased nuclear localization. Primary SMCs from Acta2 SMC-R179C/+ mice are less differentiated than WT SMCs in vitro and in vivo and have global changes in chromatin accessibility. Induced pluripotent stem cells from patients with ACTA2 p.R179 variants fail to fully differentiate from neuroectodermal progenitor cells to SMCs, and single-cell transcriptomic analyses of an ACTA2 p.R179H patient's aortic tissue show increased SMC plasticity. Thus, nuclear αSMA participates in SMC differentiation, and loss of this nuclear activity occurs with ACTA2 p.R179 pathogenic variants.

MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular disorders and has no cure. Due to an unknown molecular mechanism, FSHD displays overlapping manifestations with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FSHD is caused by aberrant gain of expression of the transcription factor double homeobox 4 (DUX4), which triggers a pro-apoptotic transcriptional program resulting in inhibition of myogenic differentiation and muscle wasting. Regulation of DUX4 activity is poorly known. We identify Matrin 3 (MATR3), whose mutation causes ALS and dominant distal myopathy, as a cellular factor controlling DUX4 expression and activity. MATR3 binds to the DUX4 DNA-binding domain and blocks DUX4-mediated gene expression, rescuing cell viability and myogenic differentiation of FSHD muscle cells, without affecting healthy muscle cells. Finally, we characterize a shorter MATR3 fragment that is necessary and sufficient to directly block DUX4-induced toxicity to the same extent as the full-length protein. Collectively, our data suggest MATR3 as a candidate for developing a treatment for FSHD.

Upregulated LncRNA H19 Sponges MiR-106a-5p and Contributes to Aldosterone-Induced Vascular Calcification via Activating the Runx2-Dependent Pathway.

Excess aldosterone is implicated in vascular calcification (VC), but the mechanism by which aldosterone-MR (mineralocorticoid receptor) complex promotes VC is unclear. Emerging evidence indicates that long-noncoding RNA H19 (H19) plays a critical role in VC. We examined whether aldosterone-induced osteogenic differentiation of vascular smooth muscle cells (VSMCs) through H19 epigenetic modification of Runx2 (runt-related transcription factor-2) in a MR-dependent manner. We induced in vivo rat model of chronic kidney disease using a high adenine and phosphate diet to explore the relationship among aldosterone, MR, H19, and VC. We also cultured human aortic VSMCs to explore the roles of H19 in aldosterone-MR complex-induced osteogenic differentiation and calcification of VSMCs. H19 and Runx2 were significantly increased in aldosterone-induced VSMC osteogenic differentiation and VC, both in vitro and in vivo, which were significantly blocked by the MR antagonist spironolactone. Mechanistically, our findings reveal that the aldosterone-activated MR bound to H19 promoter and increased its transcriptional activity, as determined by chromatin immunoprecipitation, electrophoretic mobility shift assay, and luciferase reporter assay. Silencing H19 increased microRNA-106a-5p (miR-106a-5p) expression, which subsequently inhibited aldosterone-induced Runx2 expression at the posttranscriptional level. Importantly, we observed a direct interaction between H19 and miR-106a-5p, and downregulation of miR-106a-5p efficiently reversed the suppression of Runx2 induced by H19 silencing. Our study clarifies a novel mechanism by which upregulation of H19 contributes to aldosterone-MR complex-promoted Runx2-dependent VSMC osteogenic differentiation and VC through sponging miR-106a-5p. These findings highlight a potential therapeutic target for aldosterone-induced VC.

Comparative analysis of the myoglobin gene in whales and humans reveals evolutionary changes in regulatory elements and expression levels.

Cetacea and other diving mammals have undergone numerous adaptations to their aquatic environment, among them high levels of the oxygen-carrying intracellular hemoprotein myoglobin in skeletal muscles. Hypotheses regarding the mechanisms leading to these high myoglobin levels often invoke the induction of gene expression by exercise, hypoxia, and other physiological gene regulatory pathways. Here we explore an alternative hypothesis: that cetacean myoglobin genes have evolved high levels of transcription driven by the intrinsic developmental mechanisms that drive muscle cell differentiation. We have used luciferase assays in differentiated C2C12 cells to test this hypothesis. Contrary to our hypothesis, we find that the myoglobin gene from the minke whale, Balaenoptera acutorostrata, shows a low level of expression, only about 8% that of humans. This low expression level is broadly shared among cetaceans and artiodactylans. Previous work on regulation of the human gene has identified a core muscle-specific enhancer comprised of two regions, the "AT element" and a C-rich sequence 5' of the AT element termed the "CCAC-box". Analysis of the minke whale gene supports the importance of the AT element, but the minke whale CCAC-box ortholog has little effect. Instead, critical positive input has been identified in a G-rich region 3' of the AT element. Also, a conserved E-box in exon 1 positively affects expression, despite having been assigned a repressive role in the human gene. Last, a novel region 5' of the core enhancer has been identified, which we hypothesize may function as a boundary element. These results illustrate regulatory flexibility during evolution. We discuss the possibility that low transcription levels are actually beneficial, and that evolution of the myoglobin protein toward enhanced stability is a critical factor in the accumulation of high myoglobin levels in adult cetacean muscle tissue.