MEF2C mRNA Research Articles

Stoichiometry of long noncoding RNA interactions with other RNAs: Insights from OIP5-AS1.

Long noncoding (lnc)RNAs modulate gene expression programs in a range of developmental processes in different organs. In skeletal muscle, lncRNAs have been implicated in myogenesis, the process whereby muscle precursor cells form muscle fibers during embryonic development and regenerate muscle fibers in the adult. Here, we discuss OIP5-AS1, a lncRNA that is highly expressed in skeletal muscle and is capable of coordinating protein expression programs during myogenesis. Given that several myogenic functions of OIP5-AS1 involve interactions with MEF2C mRNA and with the microRNA miR-7, it was critical to carefully evaluate the precise levels of OIP5-AS1 during myogenesis. We discuss the approaches used to examine lncRNA copy number using OIP5-AS1 as an example, focusing on quantification by quantitative PCR analysis with reference to nucleic acids of known abundance, by droplet digital (dd)PCR measurement, and by microscopic visualization of individual lncRNAs in cells. We discuss considerations of RNA stoichiometry in light of developmental processes in which lncRNAs are implicated. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

METTL3 promotes microglial inflammation via MEF2C in spinal cord injury.

Spinal cord injury (SCI) is a significant contributor to disability in contemporary society, resulting in substantial psychological and economic burdens for patients and their family. Microglia-mediated inflammation is an important factor affecting the nerve repair of SCI patients. N6-methyladenosine (m6A) is a prevalent epigenetic modification in mammals, which shows a strong association with inflammation. However, the mechanism of m6A modification regulating microglia-mediated inflammation is still unclear. Here, we observed that METTL3, a m6A methylase, was increased in SCI mice and lipopolysaccharide (LPS)-exposed BV2 cells. Knockdown of METTL3 inhibited the increased expression of iNOS and IL-1β induced by LPS in vitro. Subsequently, MEF2C, myocyte-specific enhancer factor 2C, was decreased in SCI mice and LPS-exposed BV2 cells. Knockdown of MEF2C promoted the expression of iNOS and IL-1β. Sequence analysis showed that there were multiple highly confident m6A modification sites on the MEF2C mRNA. METTL3 antibody could pull down a higher level of MEF2C mRNA than the IgG in RNA binding protein immunoprecipitation assay. Knockdown of METTL3 promoted MEF2C protein expression and MEF2C mRNA expression, accompanied by a reduced m6A modification level on the MEF2C mRNA. Knockdown of MEF2C inhibited the anti-inflammatory effect of METTL3 siRNA. Our results suggest that METTL3 promotes microglia inflammation via regulating MEF2C mRNA m6A modification induced by SCI and LPS treatment.

The sodium borate relieves the hypertrophic damage induced during pregnancy, it improves contractibility, reduces oxidative stress and stimulates cell proliferation

IntroductionFetal and postnatal hypertrophy develop in response to such different exposures or illnesses the mother suffers during gestation as anti-infectious and physical agents, obesity, hypertension, diabetes, and even advanced maternal age. This gives rise to high comorbidities in the newborn; therefore, looking for alternatives that contribute to cardiac homeostasis is quite necessary to inhibit the overgrowth of myocytes. Boron-derivative compounds could play a key role in exerting a repairing effect on chronic cardiac damage induced during gestation. MethodologyThe cardiotoxic effect of 6.4, 12 and 100 mg/kg of sodium tetraborate administered by oral delivery route to healthy pregnant mice was assessed. After that, the use of the chemical compound was tested in the treatment of pregnant mice previously subjected to isoproterenol (fetal hypertrophy model) on the fifth day post coitus. Prior to the sacrifice of the pups of mice an electrocardiography (ECG) was done. Morphological and histological changes of heart were assessed in newborn pups. As a damage marker, the concentration of p38 nitrogen-activated protein kinases were evaluated by using Western Blot and the levels of malondialdehyde (MDA) as well as glutathione antioxidants (GSH) and glutathione peroxidase (GPx) were tested by spectrometry. Moreover, the mRNA expression for early response genes (c-jun, c-fos y c-myc), late response (GATA-4, Mef2c, NFAT) and heart damage (ANP and BNP) was measured by qPCR real time. ResultsThe supply of 6,4 and 12 mg/kg-sodium tetraborate favored ventricular remodeling with histological alterations. By comparison, 100 mg/kg of sodium tetraborate administered during the fetal stage did not alter neither the cardiac morphology of six-week old pups nor the p38/P-p38MAPK ratio remained the same and no oxidative stress was observed.When pregnant females treated with isoproterenol were treated with 100 mg/kg sodium tetraborate during the fetal stage, an improvement in contractility was detected in the pups with an actual reduction in myocardial fibrosis and oxidative stress, but cardiac mass increased. In addition, the expression levels of c-jun, c-myc, GATA-4, MEF2c and ANP mRNA declined in comparison with CTR. However, the hypertrophic damage mechanism was sustained by c-fos, NFAT and BNP expressions. ConclusionsThe set of results achieved suggests that high concentrations of sodium tetraborate have no cardiotoxic effects. Furthermore, sodium tetraborate mitigates hypertrophy induced during pregnancy, thereby improving contractibility, reducing oxidative stress and stimulating cell proliferation. Therefore, sodium tetraborate could be an excellent prophylactic treatment administered by delivery oral route during pregnancy when there is a risk of developing fetal left ventricular hypertrophy (LVH).

MEF2C Expression Is Regulated by the Post-transcriptional Activation of the METTL3-m6A-YTHDF1 Axis in Myoblast Differentiation.

N6-methyladenosine (m6A) plays an essential role in regulating gene expression. However, the effect of m6A on skeletal myoblast differentiation and the underlying mechanisms are still unclear. Here, we ascertained mRNA m6A methylation exhibited declined changes during bovine skeletal myoblast differentiation, and both MEF2C mRNA expression and m6A levels were significantly increased during myoblast differentiation. We found that MEF2C with mutated m6A sites significantly inhibited myoblast differentiation compared with wild-type MEF2C. METTL3 promoted MEF2C protein expression through posttranscriptional modification in an m6A-YTHDF1-dependent manner. Moreover, MEF2C promoted the expression of METTL3 by binding to its promoter. These results revealed that there is a positive feedback loop between these molecules in myoblast differentiation. Our study provided new insights into skeletal muscle differentiation and fusion, which may provide an RNA methylation-based approach for molecular genetics and breeding in livestock as well as for the treatment of muscle-related diseases.

The Cell Adhesion Molecule L1 Interacts with Methyl CpG Binding Protein 2 via Its Intracellular Domain.

Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1’s intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1−55). Proximity ligation assay indicates that metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1) and ɣ-secretase, are involved in the generation of L1−55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1−55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.

Identification of Potential circRNA-microRNA-mRNA Regulatory Network in Skeletal Muscle.

Circular RNAs (circRNAs) are a newly discovered family of regulatory RNAs generated through backsplicing. Genome-wide profiling of circRNAs found that circRNAs are ubiquitously expressed and regulate gene expression by acting as a sponge for RNA-binding proteins (RBPs) and microRNAs (miRNAs). To identify circRNAs expressed in mouse skeletal muscle, we performed high-throughput RNA-sequencing of circRNA-enriched gastrocnemius muscle RNA samples, which identified more than 1,200 circRNAs. In addition, we have identified more than 14,000 and 15,000 circRNAs in aging human skeletal muscle tissue and satellite cells, respectively. A subset of abundant circRNAs was analyzed by RT-PCR, Sanger sequencing, and RNase R digestion assays to validate their expression in mouse skeletal muscle tissues. Analysis of the circRNA-miRNA-mRNA regulatory network revealed that conserved circNfix might associate with miR-204-5p, a suppressor of myocyte enhancer factor 2c (Mef2c) expression. To support the hypothesis that circNfix might regulate myogenesis by controlling Mef2c expression, silencing circNfix moderately reduced Mef2c mRNA expression and inhibited C2C12 differentiation. We propose that circNfix promotes MEF2C expression during muscle cell differentiation in part by acting as a sponge for miR-204-5p.

Substitution of fish meal with krill meal in rice field eel (Monopterus albus) diets: Effects on growth, immunity, muscle textural quality, and expression of myogenic regulation factors

An 8-week feeding experiment was conducted to evaluate the effects of replacing fish meal (FM) with krill meal (KM) on the growth, immunity, muscle textural quality, and expression of myogenic regulation factors of Monopterus albus (initial body weight: 23.00 ± 0.10 g). Five experimental diets consisted of different proportions of FM (0, 25, 50, 75, and 100 g/kg) were substituted with KM, corresponding to dietary inclusion levels of 0 (KM0), 28.8 g/kg (KM29), 57.6 g/kg (KM58), 86.4 g/kg (KM86), and 115.2 g/kg (KM115). The results showed that: (1) the weight gain rate (WGR) of the fish in the KM58, KM86, and KM115 groups were significantly higher than those of the KM0 group, whereas the opposite result was observed for the feed conversion ratio. (2) Serum complement 3 content and superoxide dismutase activity in the KM58 group and alkaline phosphatase activity in the KM29, KM58, and KM115 groups were significantly higher than those in the KM0 group. However, there were no significant differences in serum immunoglobulin M, complement 4, malondialdehyde content, and catalase activity among all groups. (3) The highest levels of hardness, cohesiveness, springiness, chewiness, gumminess, and adhesiveness of muscle tissues of M. albus fed with the KM58 diet were significantly higher than those of the other treatments. (4) No remarkable difference was detected in MEF2A, MEF2D, MyoG, and Myf5 mRNA expression levels in the muscle tissues among the dietary treatments. However, MyoD2, Myh6, MyoD1, MRF4, MEF2B, and MEF2C mRNA expression levels were significantly upregulated by KM replacement. In conclusion, these results suggested that dietary inclusion levels of 57.6–115.2 g/kg KM improved the growth performance, immune function, muscle textural quality, and expression of myogenic regulation factors of M. albus. Based on the WGR, the optimal level of KM was 103.08 g/kg.

Interaction of OIP5-AS1 with MEF2C mRNA promotes myogenic gene expression.

Long noncoding (lnc)RNAs potently regulate gene expression programs in physiology and disease. Here, we describe a key function for lncRNA OIP5-AS1 in myogenesis, the process whereby myoblasts differentiate into myotubes during muscle development and muscle regeneration after injury. In human myoblasts, OIP5-AS1 levels increased robustly early in myogenesis, and its loss attenuated myogenic differentiation and potently reduced the levels of the myogenic transcription factor MEF2C. This effect relied upon the partial complementarity of OIP5-AS1 with MEF2C mRNA and the presence of HuR, an RNA-binding protein (RBP) with affinity for both transcripts. Remarkably, HuR binding to MEF2C mRNA, which stabilized MEF2C mRNA and increased MEF2C abundance, was lost after OIP5-AS1 silencing, suggesting that OIP5-AS1 might serve as a scaffold to enhance HuR binding to MEF2C mRNA, in turn increasing MEF2C production. These results highlight a mechanism whereby a lncRNA promotes myogenesis by enhancing the interaction of an RBP and a myogenic mRNA.

Significance of MEF2C and RUNX3 Regulation for Endochondral Differentiation of Human Mesenchymal Progenitor Cells

Guiding progenitor cell development between chondral versus endochondral pathways is still an unachieved task of cartilage neogenesis, and human mesenchymal progenitor cell (MPC) chondrogenesis is considered as a valuable model to better understand hypertrophic development of chondrocytes. Transcription factors Runx2, Runx3, and Mef2c play prominent roles for chondrocyte hypertrophy during mouse development, but little is known on the importance of these key fate-determining factors for endochondral development of human MPCs. The aim of this study was to unravel the regulation of RUNX2, RUNX3, and MEF2C during MPC chondrogenesis, the pathways driving their expression, and the downstream hypertrophic targets affected by their regulation. RUNX2, RUNX3, and MEF2C gene expression was differentially regulated during chondrogenesis of MPCs, but remained low and unregulated when non-hypertrophic articular chondrocytes were differentiated under the same conditions. RUNX3 and MEF2C mRNA and protein levels rose in parallel to hypertrophic marker upregulation, but surprisingly, RUNX2 gene expression changed only by trend and RUNX2 protein remained undetectable. While RUNX3 expression was driven by TGF-β and BMP signaling, MEF2C responded to WNT-, BMP-, and Hedgehog-pathway inhibition. MEF2C but not RUNX3 levels correlated significantly with COL10A1, IHH, and IBSP gene expression when hypertrophy was attenuated. IBSP was a downstream target of RUNX3 and MEF2C but not RUNX2 in SAOS-2 cells, underlining the capacity of RUNX3 and MEF2C to stimulate osteogenic marker expression in human cells. Conclusively, RUNX3 and MEF2C appeared more important than RUNX2 for human endochondral MPC chondrogenesis. Pathways altering the speed of chondrogenesis (FGF, TGF-β, BMP) affected RUNX2 or RUNX3, while pathways changing hypertrophy (WNT, PTHrP/HH) regulated mainly MEF2C. Taken together, reduction of MEF2C levels is a new goal to shift human cartilage neogenesis toward the chondral pathway.

MiR-199a-3p inhibition facilitates cardiomyocyte differentiation of embryonic stem cell through promotion of MEF2C.

MicroRNAs (miRNAs) is a small molecule (19-25 nucleotide) noncoding RNA that inhibits the expression of target messenger RNA (mRNA) at the posttranscriptional level as an endogenous regulator. There is an increasing evidence that miR-199a-3p has a significant effect on the development of multiple tumors. However, the specific roles of miR-199a-3p in myocardial differentiation of embryonic stem cell still need to be investigated. Method of the hanging drop was used to build the model of cardiomyocyte differentiation of stem cell and beating rate of embryoid bodies (EBs) was calculated. The levels of intracellular MEF2C, a-MHC, GATA4, Nkx2.5, and cTnT mRNA were measured by real-time quantitative polymerase chain reaction, while the expressions of miR-199a-3p were detected simultaneously. Protein levels of MEF2C, a-MHC, GATA4, Nkx2.5, and cTnT were quantified by western blot analysis. Immunoreactivities of MEF2C and cTnT were analyzed by immunofluorescence. The interaction between miR-199a-3p and its predicted target (3'-untranslated region of MEF2C mRNA) was verified by luciferase assay. MiR-199a-3p levels increased during cardiogenesis. MiR-199a-3p inhibitor increased the beating rate of EBs and promoted expressions of cardiac-specific markers (GATA4, Nkx2.5, cTnT, and a-MHC). Notably, miR-199a-3p inhibition brought upregulation of MEF2C, which is the target of miR-199a-3p that we predicted and verified experimentally. In addition, MEF2C siRNA decreased miR-199a-3p inhibitor promoted EBs beating and attenuated miR-199a-3p inhibitor-induced cTnT and MEF2C expressions. The results above showed that MEF2C was involved in the process of promoting the differentiation of stem cells into cardiac myocytes by miR-199a-3p inhibitors.

Increased Expression of miR-1 in Fast-Twitch Skeletal Muscle in Response to Resistance Exercise

Background: The response of microRNA (miR-1), upstream regulators (Myogenic Differentiation 1 (MyoD1) and Myocyte Enhancer Factor 2C (Mef2C)), and a downstream target (Histone Deacetylase 4 (Hdac4)) to an acute bout of Resistance Exercise (RE) in Extensor Digitorum Longus (EDL) and soleus muscles has remained elusive. Objectives: In this experimental study, we investigated the effect of an acute bout of RE on the expression of miR-1, MyoD1, Mef2C, and Hdac4 genes in the slow and fast-twitch muscle of rats. Methods: The current study was conducted at Tarbiat Modares University, Tehran, Iran, in 2017. Fifteen male Wistar rats were randomly divided into two groups, control (n = 5) and RE (n = 10). The RE protocol consisted of four sets of five repetitions of climbing a ladder with weights attached to the tails of rats. The soleus and EDL muscles of rats were collected at 3 h (n = 5) and 6 h (n = 5) post-RE. The real-time polymerase chain reaction was used to measure the levels of miR-1, MyoD1, Mef2C, and Hdac4 mRNA expression. Results: The miR-1 expression in EDL muscle was significantly lower at 3 h and 6 h post-exercise in the RE group than in the control group (P < 0.01). The miR-1 expression in soleus muscle was significantly lower at 3 h post-exercise in the RE group than in the control group (P < 0.007) but it was significantly higher at 6 h post-exercise (P < 0.036). The expression of MyoD1, Mef2C, and Hdac4 genes in the EDL muscle was higher in the RE group than in the control group (P < 0.01). The expression of these genes in response to RE had more fluctuations in soleus muscle. Conclusions: It can be concluded that miR-1 expression in extensor digitorum longus and soleus muscles of rats responds to resistance exercise in different manners and this coincides with a change in upstream regulators and downstream target.

OR20-6 Ketones and Sepsis-Induced Muscle Weakness: Signal or Fuel for Protection?

Background: Septic patients often develop muscle weakness, contributing to ICU morbidity and mortality. We recently observed that obese septic mice and patients do not develop such weakness, explained by increased lipolysis and hepatic conversion of fatty acids into the ketone bodies. Furthermore, in lean septic mice, supplementing nutrition with 3-hydroxybutyrate (3HB) reproduced the protection against muscle weakness seen with obesity. The aim of the current study was to investigate underlying mechanisms of the 3HB-protection. Methods: We performed a mouse study (n=49) in a centrally-catheterized fluid-resuscitated model of prolonged (5 days) abdominal sepsis (cecal ligation and puncture). Lean septic mice received standard parenteral nutrition (PN) supplemented with either D,L-3HB (5 mg/kg/day) or isocaloric glucose (gluc). Healthy pair-fed mice served as controls. The use of 3HB as energy substrate in the muscle was assessed by quantifiying protein, triglyceride and glycogen content and markers of muscle ketolysis, proteolysis and fatty acid oxidation (FAO). Signaling function of 3HB was assessed by markers of muscle autophagy activation, inflammation and regeneration. Results: Although 3HB infusion abated sepsis-induced upregulation of ubiquitin-proteasome system marker Fxbo32 (p=0.002), loss of muscle mass was not prevented (p=0.8). Sepsis reduced triglyceride content (p≤0.01) equally in PN+gluc and PN+3HB mice (p=0.9), but did not affect markers of muscle FAO (Cd36, Cpt1b, Acadl, Hadha mRNA) in PN+gluc and PN+3HB mice (p≥0.2). Muscle glycogen content was unaltered by sepsis (p=0.1), and the illness-induced decrease in plasma glucose concentrations at day 5 (p≤0.004) was similar in PN+gluc and PN+3HB mice (p=1). Gene expression of the ketolysis enzyme Oxct1 was lower in PN+3HB compared to PN+gluc septic mice and healthy controls (p≤0.05). Also, ketone body transporter Mct1 mRNA was elevated by sepsis (p≤0.01) but attenuated by 3HB (p=0.05) compared to glucose. Sepsis-induced elevation of inflammation markers (Tnfα, IL-1β, Nlrp3 mRNA levels; p≥0.4) and autophagy (Atg5, Atg7 mRNA; p≥0.4; LC3 II/I, p-ULK1/ULK1 protein; p≥0.4) were not countered by 3HB infusion compared to glucose. However, PN+3HB septic mice did show enhanced markers of muscle regeneration (Myod1, Myf5 and Myog mRNA; p≤0.04) compared to PN+gluc septic mice and healthy controls. Also, sepsis-induced elevation of expression of Hdac4, an inhibitor of myoblast differentiation stimulator Mef2c, was reduced by 3HB (p=0.03) and resulted in increased Mef2c mRNA levels (p=0.01) compared to glucose. Conclusion: In conclusion, the observed protection against sepsis-induced muscle weakness with 3HB infusion coincided with enhanced markers of muscle regeneration, but not with changes in substrate handling. Supplemented 3HB during sepsis thus acted as a signaling molecule rather than as an energy source.

Paediatric patients with acute leukaemia and KMT2A (MLL) rearrangement show a distinctive expression pattern of histone deacetylases.

Histone deacetylase inhibitors (HDACi) had emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. There was a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles identified specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with KMT2A (MLL) rearrangement, with high HDAC9 and MEF2D expression, regardless of age, KMT2A partner and lineage. Moreover, we observed an adverse prognostic value of HDAC9 overexpression, regardless of KMT2A rearrangement. Our results provide useful knowledge on the complex picture of HDAC expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.

Retinal expression and localization of Mef2c support its important role in photoreceptor gene expression

Photoreceptor-specific gene expression is controlled by a hierarchical network of transcription factors, including the master regulators cone-rod homeobox (Crx) and neural retina leucine zipper (Nrl). Myocyte-enhancer factor 2c (Mef2c) is an ubiquitously expressed transcription factor with important functions in the cardiovascular system. Here, we performed a detailed analysis of Mef2c expression, localization and function in the retina to further elucidate its potential role for photoreceptor gene regulation. We showed that murine retinal Mef2c mRNA expression was high at birth and peaked at late postnatal developmental stages. Using immunohistochemistry and Western blot, Mef2c protein was detected in the outer nuclear layer of adult mouse and human retinas and localized to the nucleus of 661W photoreceptor-like cells. Mef2c knock-down in 661W cells reduced the expression of arrestin 3 (Arr3) and medium-wave-sensitive cone opsin (Opn1mw) but increased transcript levels of mitogen-activated protein kinase 15 (Mapk15) and phosphodiesterase 6h (Pde6h). In conclusion, Mef2c is highly expressed in the retina where it modulates photoreceptor-specific gene expression.

Maternal dietary linoleic acid supplementation promotes muscle fibre type transformation in suckling piglets.

As meat quality is basically dependent on muscle fibre characteristics, it is important to know how muscle fibres are regulated and transformed. This study aimed to investigate the effect of maternal dietary supplementation on muscle fibre types using 3% saturated fatty acid (palmitic acid, PA) or 3% unsaturated fatty acid (linoleic acid, LA) from 80days of gestation to the weaning of offspring (25days post-natal). The results indicated that higher mRNA levels of MyHCI type genes were found in the soleus muscles of piglets that suckled from LA-supplemented sows than from PA-supplemented sows. In addition, LA treatment increased the gene expression of the type I muscle fibre marker troponin I (p<0.01), suggesting that LA promoted muscle fibre type transformation to type I fibres. Moreover, PGC-1α (p<0.01) and MEF2c (p<0.05) mRNA levels were higher in the piglets from the LA treatment group than in those from the PA treatment group. Furthermore, LA supplementation also significantly increased AMP-activated protein kinase (AMPK) mRNA levels (p<0.05), which is an upstream regulator of PGC-1α. Collectively, these findings demonstrated that maternal dietary LA supplementation promoted muscle fibre transformation to type I fibre and that this process may be mediated through an AMPK-dependent pathway.

Crucial Role of Elovl6 in Chondrocyte Growth and Differentiation during Growth Plate Development in Mice.

ELOVL family member 6, elongation of very long chain fatty acids (Elovl6) is a microsomal enzyme, which regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 has been shown to be associated with various pathophysiologies including insulin resistance, atherosclerosis, and non-alcoholic steatohepatitis. To investigate a potential role of Elovl6 during bone development, we here examined a skeletal phenotype of Elovl6 knockout (Elovl6-/-) mice. The Elovl6-/- skeleton was smaller than that of controls, but exhibited no obvious patterning defects. Histological analysis revealed a reduced length of proliferating and an elongated length of hypertrophic chondrocyte layer, and decreased trabecular bone in Elovl6-/- mice compared with controls. These results were presumably due to a modest decrease in chondrocyte proliferation and accelerated differentiation of cells of the chondrocyte lineage. Consistent with the increased length of the hypertrophic chondrocyte layer in Elovl6-/- mice, Collagen10α1 was identified as one of the most affected genes by ablation of Elovl6 in chondrocytes. Furthermore, this elevated expression of Collagen10α1 of Elovl6-null chondrocytes was likely associated with increased levels of Foxa2/a3 and Mef2c mRNA expression. Relative increases in protein levels of nuclear Foxa2 and cytoplasmic histone deacethylase 4/5/7 were also observed in Elovl6 knockdown cells of the chondrocyte lineage. Collectively, our data suggest that Elovl6 plays a critical role for proper development of embryonic growth plate.

MEF2D and MEF2C pathways disruption in sporadic and familial ALS patients

Amyotrophic lateral sclerosis (ALS) is a progressive neuro-muscular disease characterized by motor neuron loss. MEF2D and MEF2C are members of the myocyte enhancer factor 2 family (MEF2), a group of transcription factors playing crucial roles both in muscle and in neural development and maintenance; for this reason, a possible involvement of MEF2 in ALS context has been investigated. Since the transcriptional activity of each tissue specific MEF2 isoform is conserved in different cell types, we chose to assess our parameters in an easily accessible and widely used experimental tool such as peripheral blood mononuclear cells (PBMCs) obtained from 30 sporadic ALS patients (sALS), 9 ALS patients with mutations in SOD1 gene (SOD1+) and 30 healthy controls. Gene expression analysis showed a significant up-regulation of MEF2D and MEF2C mRNA levels in both sporadic and SOD1+ ALS patients. Although protein levels were unchanged, a different pattern of distribution for MEF2D and MEF2C proteins was evidenced by immunohistochemistry in patients. A significant down-regulation of MEF2 downstream targets BDNF, KLF6 and RUFY3 was reported in both sALS and SOD1+ ALS patients, consistent with an altered MEF2 transcriptional activity. Furthermore, the potential regulatory effect of histone deacetylase 4 and 5 (HDAC4 and HDAC5) on MEF2D and MEF2C activity was also investigated. We found that MEF2D and HDAC4 colocalize in PBMC nuclei, while HDAC5 was localized in the cytoplasm. However, the unchanged HDACs localization and protein levels between sALS and controls seem to exclude their involvement in MEF2 altered function. In conclusion, our results show a systemic alteration of MEF2D and MEF2C pathways in both sporadic and SOD1+ ALS patients, underlying a possible common feature between the sporadic and the familial form of disease. Although further analyses in other neuromuscular diseases are needed to determine the specificity of changes in these pathways to ALS, measuring MEF2 alterations in accessible biofluids may be useful as biomarkers for disease diagnosis and progression.

Effects of hyperacetylation of H3K9ac mediated by histone acetylases on the overexpression of MEF2C induced by alcohol in the hearts of fetal mice during pregnancy

Objective To investigate the effects of H3K9ac hyperacetylation mediated by histone acetylases on the overexpression of MEF2C in the hearts of fetal mice exposed to alcohol during pregnancy, and provide new intervention targets for prevention and treatment of cardiac dysplasia caused by alcohol exposure. Methods C57BL/6 mice were divided into 5 groups randomly, blank control group, dimethylsulfoxide (DMSO) group, alcohol group, alcohol+ anacardic acid group and anacardic acid group, and then the hearts of fetal mice were collected to be analyzed.Chromatin immunoprecipitation and Western blot were used in assaying the binding of histone acetylases and the level of H3K9ac to the promoter of MEF2C in the hearts of fetal mice.The mRNA expression of MEF2C was tested by adopting real-time PCR. Results The level of H3K9ac in the promoter of MEF2C in the hearts of fetal mice exposed to alcohol was higher than that in the hearts of fetal mice exposed to saline (1.30±0.19 vs 0.45±0.01), there was statistically significant difference (P<0.05), while the binding of E1A-binding protein (p300), p300/cyclic adenosine monophosphate response element binding protein-associated factor (PCAF) and steroid receptor coactivator-1 (SRC1) to the promoter of MEF2C were abnormally elevated in the hearts of fetal mice treated by alcohol (1.68±0.08 vs 0.82±0.08, 1.08±0.05 vs 0.42±0.02, 1.18±0.05 vs 0.39±0.08), and there were statistically significant differences (all P<0.05). The expression of MEF2C mRNA in alcohol group was higher than that in blank control group (1.36±0.12 vs 0.29±0.03), there was statistically significant difference(P<0.05). However, a pan-acetylases inhibitor, anacardic acid, could decrease significantly the binding of p300 and PCAF to the promoter of MEF2C (1.52±0.05 vs 0.63±0.09, 1.13±0.04 vs 0.45±0.04), and correct abnormal hyperacetylation of H3K9ac induced by alcohol (1.58±0.08 vs 0.67±0.05), and down-regulate the over-expression of MEF2C in the hearts of fetal mice exposed to alcohol (1.36±0.12 vs 0.41±0.05), and there were statistically significant differences (all P<0.05). Conclusions Hyperacetylation of H3K9ac mediated by p300 and PCAF may be a key regulatory factor in the over-expression of cardiac nuclear transcription factor MEF2C in the hearts of fetal mice exposed to alcohol during pregnancy.Anacardic acid can significantly attenuate the level of H3K9ac through inhibiting the binding of p300 and PCAF to the promoter of MEF2C, and down-regulate the over-expression of cardiac nuclear transcription factor MEF2C in the hearts of fetal mice. Key words: Histone acetylases; Transcription factor; Alcohol; Acetylation; Overexpression

Abstract 14356: Blood Tests That Improve the Accuracy of a Brugada Syndrome Diagnosis

Objectives: To discover the role of altered gene expression regulation in Brugada Syndrome (BrS) and to find biomarkers for BrS diagnosis. Methods: Twenty-five control patients (Control), 25 BrS patients without SCN5A mutation (SCN5A(-)) and 20 BrS patients with SCN5A mutation (SCN5A(+)) were included in this study. Specified gene expression of white blood cells (WBC) were measured by RT-qPCR using TaqMan® Gene Expression assay. Results: MEF2C and MESP1 are the two major cardiac specific transcription factors expressed in WBC. The mRNA expression levels of SCN5A, MEF2C and HuR, one of mRNA stabilizers, were decreased in the SCN5A (+) group (P=0.047, 0.02, 0.000 vs. control group, respectively). The mRNA expression of MESP1 in WBCs was significantly lower in both SCN5A(-) (P=0.012 vs. control) and SCN5A(+) (P=0.000 vs. control) groups. There was no difference between the two BrS groups in MESP1 expression (P=0.215). The area under the Receiver Operating Characteristics (ROC) analysis curve for prediction of BrS using MESP1 levels was 0.775 (95% CI 0.668, 0.882, asymptotic Sig.=0.000). At the optimal cutoff, the corresponding maximum sensitivity and specificity were 0.62 (95% CI: 0.47, 0.76) and 0.88 (0.69, 0.97), respectively. The diagnostic odds ratio (DOR) of MESP1 for BrS diagnosis was 11.96 (95% CI: 5.79, 24.73). The assessment of the mRNA levels in blood SCN5A, MEF2C and HuR were useful for predicting BrS patients with an SCN5A mutation. The area under the ROC analysis curve for prediction of BrS with an SCN5A mutation using SCN5A, MEF2C and HuR mRNA levels in WBCs was 0.847 (95% CI 0.752, 0.942, asymptotic Sig.=0.000), 0.685 (95% CI 0.542, 0.828, asymptotic Sig.=0.016) and 0.777 (95% CI 0.652, 0.902, asymptotic Sig.=0.000), respectively. At the optimal cutoff, the DOR of SCN5A, MEF2C and HuR for SCN5A(+) BrS diagnosis was 17.5 (95% CI: 8.06, 37.86), 4.9 (95% CI: 2.61, 9.17) and 23.5 (95% CI: 9.39, 58.80), respectively. Conclusions: Our results suggest that assessment of circulating MESP1 may be used as a biomarker for BrS diagnosis while decreased SCN5A, MEF2C and HuR mRNA in WBCs is associated with BrS patients with an SCN5A mutation. Our results also suggest that decreased expression of SCN5A, MEF2C, MESP1, and HuR may be pathophysiologically related to BrS.

Chronic binge alcohol (CBA) decreases miR‐206 expression in the skeletal muscle of Simian Immunodeficiency Virus (SIV)‐infected macaques

Studies using a non-human primate model of SIV infection have demonstrated that myoblasts isolated from CBA/SIV macaques have altered myogenic gene expression and decreased myoblast differentiation potential, suggesting impaired skeletal muscle (SKM) regeneration as a mechanism contributing to loss of SKM at end stage SIV. We hypothesized that the altered SKM milieu in CBA/SIV macaques dysregulates SKM specific microRNAs that regulate gene transcription necessary for myogenesis. Histone deacetylase 4 (HDAC4) negatively regulates myogenesis by associating with myocyte enhancer factor 2 (MEF2) and is a target of miR-206. CBA or isocaloric sucrose (SUC) were administered daily via a gastric catheter to male rhesus macaques beginning 3 mo. prior to SIV infection and continued throughout the duration of study. At ten weeks post-SIV infection, daily administration of antiretroviral therapy (ART; tenofovir and emtricitabine) was initiated in a subset of both SUC/SIV and CBA/SIV groups and continued for the duration of the study. SKM was obtained at necropsy (~11 mo. post-SIV) from SUC/SIV/ART± and CBA/SIV/ART± macaques. SKM of CBA/SIV macaques showed significantly decreased expression of miR-206 and MEF2C mRNA compared to SKM of SUC/SIV irrespective of ART. In contrast, SKM of CBA/SIV macaques showed increased mRNA expression of HDAC4 compared to SUC/SIV irrespective of ART. These results suggest that decreased miR-206 in CBA/SIV macaques favors upregulation of HDAC4, which represses MEF2C potentially contributing to impaired myogenesis. Future studies will verify the functional implications of the altered myomiR profile in myoblasts and its contribution to impaired SKM regeneration in CBA/SIV macaques.