Gene Expression In Muscle Cells Research Articles

Identification of immune-related signatures and pathogenesis differences between thoracic aortic aneurysm patients with bicuspid versus tricuspid valves via weighted gene co-expression network analysis.

Thoracic aortic aneurysm (TAA) occurs due to pathological aortal dilation, and both individuals with normal tricuspid aortic valves (TAV) or abnormal bicuspid aortic valves (BAV), the latter being a congenital condition, are at risk. However, some differences are present between TAA/BAV and TAA/TAV with respect to their pathophysiological processes and molecular mechanisms, but their exact nature is still mostly unknown. Therefore, it is necessary to elucidate TAA developmental differences among BAV vs. TAV patients. Publically-available gene expression datasets, aortic tissue derived from TAA/BAV and TAA/TAV individuals, were analyzed by weighted gene co-expression network analysis (WGCNA) to identify gene modules associated with those conditions. Gene Ontology (GO) enrichment analysis was performed on those modules to identify the enriched genes within those modules, which were verified by Gene Set Variation Analysis (GSVA) on a dataset derived from aortic smooth muscle cell gene expression between TAA/TAV and TAV/BAV patients. Immune cell infiltration patterns were then analyzed by CIBERSORT, and a protein-protein interaction (PPI) network was constructed based on WGCNA and enrichment analysis results to identify hub genes, followed by validation via stepwise regression analysis. Three signatures most strongly associated with TAA/TAV were confirmed by receiver operating characteristic (ROC) and decision curve analyses (DCA) between prior-established training and testing gene sets. WGCNA delineated 2 gene modules being associated with TAA/TAV vs. TAA/BAV; both were enriched for immune-associated genes, such as those relating to immune responses, etc., under enrichment analysis. TAA/TAV and TAA/BAV tissues also had differing infiltrating immune cell proportions, particularly with respect to dendritic, mast and CD4 memory T cells. Identified three signatures, CD86, integrin beta 2 (ITGB2) and alpha M (ITGAM), as yielding the strongest associations with TAA/TAV onset, which was verified by areas under the curve (AUC) at levels approximating 0.8 or above under ROC analysis, indicating their predictive value for TAA/TAV onset. However, we did not examine possible confounding variables, so there are many alternative explanations for this association. TAA/TAV pathogenesis was found to be more associated with immune-related gene expression compared to TAA/BAV, and the identification of three strongly-associated genes could facilitate their usage as future biomarkers for diagnosing the likelihood of TAA/TAV onset vs. TAA/BAV, as well as for developing future treatments.

Repressive epigenetic mechanisms, such as the H3K27me3 histone modification, were predicted to affect muscle gene expression and its mineral content in Nelore cattle

Epigenetic repression has been linked to the regulation of different cell states. In this study, we focus on the influence of this repression, mainly by H3K27me3, over gene expression in muscle cells, which may affect mineral content, a phenotype that is relevant to muscle function and beef quality. Based on the inverse relationship between H3K27me3 and gene expression (i.e., epigenetic repression) and on contrasting sample groups, we computationally predicted regulatory genes that affect muscle mineral content. To this end, we applied the TRIAGE predictive method followed by a rank product analysis. This methodology can predict regulatory genes that might be affected by repressive epigenetic regulation related to mineral concentration. Annotation of orthologous genes, between human and bovine, enabled our investigation of gene expression in the Longissimus thoracis muscle of Bos indicus cattle. The animals under study had a contrasting mineral content in their muscle cells. We identified candidate regulatory genes influenced by repressive epigenetic mechanisms, linking histone modification to mineral content in beef samples. The discovered candidate genes take part in multiple biological pathways, i.e., impulse transmission, cell signalling, immunological, and developmental pathways. Some of these genes were previously associated with mineral content or regulatory mechanisms. Our findings indicate that epigenetic repression can partially explain the gene expression profiles observed in muscle samples with contrasting mineral content through the candidate regulators here identified.

A Human atlas of smooth muscle cell gene expression; insights from the FANTOM consortium CAGE dataset

Abstract Background Under normal physiological conditions, smooth muscle cells (SMC's) have a role in both vessel contraction and generation of the extra-cellular matrix. It has now become apparent that SMCs throughout the vasculature play a key role in several cardiovascular conditions including hypertension, atherosclerosis, vasculopathy and myocardial infarction. However, how SMC gene expression compares in different vascular and non-vascular anatomical regions remains unknown. Purpose To create a human SMC gene expression map using data from the FANTOM consortium Cap-analysis gene expression (CAGE) dataset, a primary database of almost 600 human primary cell samples. Methods The gene level expression data was obtained by aggregating expression levels of all the promoters belonging to same gene as defined by the FANTOM CAGE dataset. The anatomical regions (n: 13) included the following vascular sites: aortic, brachiocephalic, carotid, coronary, internal thoracic & pulmonary arteries. Non-vascular regions included colon, oesophagus, prostate, uterine, trachea and bronchial. Weighted correlation network analysis (WGCNA) was used as an unsupervised approach to identify clustering of gene sets. Results Hierarchical clustering (Figure 1) demonstrates a distinct clustering of vascular SMC gene sets (aortic, carotid, coronary, internal thoracic, pulmonary & subclavian arteries). The same pattern of vascular SMC clustering was also seen in WGCNA (Figure 2). Gene ontology enrichment of vascular SMCs that clustered in WGCNA identified common gene sets relating to cytokine and chemokine activity. KEGG pathway analysis highlighted several pathways linked to inflammation (TNF, IL17 & NF-κB signalling) as well as pathways linked to atherosclerosis and lipid metabolism. Conclusion Vascular SMCs distinctly cluster away from non-vascular SMC types in humans and the separation is primarily driven by gene sets relating to inflammation, lipid metabolism & atherosclerosis. Mapping SMC gene expression at various anatomical regions in both healthy and diseased states may give us better insight into the role of SMCs in disease progression and could identify novel mechanistic targets for future therapy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Medial Collagen Type and Quantity Influence Mechanical Properties of Aneurysm Wall in Bicuspid Aortic Valve Patients

Collagen has an essential role in aortic biomechanics, and collagen remodeling has been associated with the development and progression of aortic aneurysm. However, the exact mechanisms behind collagen remodeling and the biomechanical implications are not well understood. This study presents an investigation of the relationship between collagen remodeling in the aortic wall and biomechanics, by means of collagen assays, smooth muscle cell gene expression, and mechanical testing on human aortic specimens collected from patients with bicuspid aortic valve. Collagen assay analysis was employed to determine collagen-I and total collagen content; quantitative real-time PCR was used to determine amountsCOL1A1andCOL3A1expression in the tissue. These parameters were compared with the local biomechanical properties determined from biaxial and uniaxial tensile testing. Collagen-I content was found to relate to improved mechanical properties, while total collagen content did not exhibit a relationship with biomechanics.COL1A1andCOL3A1expression were found to relate to the collagen-I content of the tissue, but not the total collagen content or biomechanical performance. Relationships between variables appeared to be dependent on the collagen content in specific layers of the aortic wall. The effect of age is also noted, as total collagen content and biomechanics were found to have significant associations with increasing age, while collagen-I content and collagen gene expression did not exhibit any correlation. Varying relationships were observed when looking at younger versus older patients. Findings highlight the importance of type and location in determining the influence of collagen on aortic biomechanics, as well as the role of gene expression in the onset and progression of collagen remodeling in aortic aneurysm.

RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts.

SUMMARYRBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression in muscle cells, contributing to heart disease. Here, we employ both 3′-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. RBFOX2-mediated APA modulates mRNA levels and/or isoform expression of a collection of genes, including contractile and mitochondrial genes. Depletion of RBFOX2 adversely affects mitochondrial health in myoblasts, correlating with disrupted APA of mitochondrial gene Slc25a4. Mechanistically, RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site, enforcing the use of the proximal polyadenylation site. In sum, our results unveil a role for RBFOX2 in fine-tuning expression of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

MicroRNAs in Skeletal Muscle and Hints on Their Potential Role in Muscle Wasting During Cancer Cachexia.

Cancer-associated cachexia is a heterogeneous, multifactorial syndrome characterized by systemic inflammation, unintentional weight loss, and profound alteration in body composition. The main feature of cancer cachexia is represented by the loss of skeletal muscle tissue, which may or may not be accompanied by significant adipose tissue wasting. Such phenotypic alteration occurs as the result of concomitant increased myofibril breakdown and reduced muscle protein synthesis, actively contributing to fatigue, worsening of quality of life, and refractoriness to chemotherapy. According to the classical view, this condition is primarily triggered by interactions between specific tumor-induced pro-inflammatory cytokines and their cognate receptors expressed on the myocyte membrane. This causes a shift in gene expression of muscle cells, eventually leading to a pronounced catabolic condition and cell death. More recent studies, however, have shown the involvement of regulatory non-coding RNAs in the outbreak of cancer cachexia. In particular, the role exerted by microRNAs is being widely addressed, and several mechanistic studies are in progress. In this review, we discuss the most recent findings concerning the role of microRNAs in triggering or exacerbating muscle wasting in cancer cachexia, while mentioning about possible roles played by long non-coding RNAs and ADAR-mediated miRNA modifications.

MicroRNA-499-5p regulates skeletal myofiber specification via NFATc1/MEF2C pathway and Thrap1/MEF2C axis

AimsThis study aimed to investigate the role of microRNA-499-5p (miR-499-5p) in the regulation of skeletal myofiber specification and its underlying mechanisms. Main methodsMouse C2C12 cells were used in this study. Cyclosporin A and siRNA targeting Thrap1 (si-Thrap1) were used to inhibit NFATc1/MEF2C pathway and knockdown Thrap1, respectively. The expressions of miR-499-5p and genes were evaluated by real-time quantitative PCR and western blot analysis. Key findingsOverexpression of miR-499-5p promoted oxidative fiber gene expression and repressed glycolytic fiber gene expression, affecting several factors associated with fiber specification including NFATc1/MEF2C pathway, PGC-1α, FoxO1 and Wnt5a. Inhibition of NFATc1/MEF2C pathway partly reduced the effect of miR-499-5p overexpression on muscle fiber gene expression. MiR-499-5p targeted Thrap1 in proliferating and differentiating C2C12 cells. Knockdown of Thrap1 showed a parallel function with miR-499-5p overexpression on muscle fiber gene expression and NFATc1/MEF2C pathway, accompanied by an increase of miR-499-5p level. The effects of miR-499-5p inhibitor on muscle fiber type specific gene expression and NFATc1/MEF2C pathway were effectively reversed by Thrap1 knockdown. SignificanceMiR-499-5p regulated skeletal myofiber specification and affected several factors associated with fiber specification. MiR-499-5p regulated muscle gene expression partly through NFATc1/MEF2C pathway. We also showed a clue that miR-499-5p regulates skeletal muscle fiber specification in C2C12 cells through targeting Thrap1, thereby, promoting NFATc1/MEF2C pathway and then triggering a series of oxidative muscle fiber gene expression.

Characterization of the Regulation and Function of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle

Skeletal muscle atrophy is a debilitating medical condition that can arise due to aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of neurogenic atrophy, mouse gastrocnemius muscle was isolated 3 and 14 days after sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Zinc Finger Protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) analysis showed that Zfp593 is more highly expressed in proliferating myoblasts compared to differentiated myotubes, while Western blot analysis confirmed that Zfp593 is expressed at the protein level in proliferating and differentiated mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, fragments of the proximal promoter region located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Furthermore, analysis of the promoter region of Zfp593 revealed four conserved E‐box elements in the proximal regulatory region, which are known myogenic regulatory factor (MRF) binding sites. Therefore, in order to determine how these elements may regulate the transcription of Zfp593, the E‐Boxes where either mutated or deleted and the resulting mutant reporter constructs were then transfected into C2C12 mouse myoblasts in combination with ectopic expression of MRFs. Additionally, it is predicted that the Zfp593 protein contains a zinc finger domain and is believed to function as a transcription factor. Therefore, to assess if Zfp593 is localized to the nucleus, we fused Zfp593 with green fluorescent protein (GFP), transfected the Zfp593‐GFP expression plasmid into C2C12 mouse myoblasts cells, visualized by confocal microscopy, and confirmed that Zfp593 does localize to the nucleus. Finally, the Zfp593 transcript possess a particularly long 3′‐untranslated region (UTR) region, so we cloned several fragments of the UTR, fused them with a reporter gene, and found that the UTR region has a negative regulatory effect on reportor gene expression in muscle cells. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that Zfp593, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cyclic Stretch Effects on Adipose-Derived Stem Cell Stiffness, Morphology and Smooth Muscle Cell Gene Expression.

Recent investigations consider adipose-derived stem cells (ASCs) as a promising source of stem cells for clinical therapies. To obtain functional cells with enhanced cytoskeleton and aligned structure, mechanical stimuli are utilized during differentiation of stem cells to the target cells. Since function of muscle cells is associated with cytoskeleton, enhanced structure is especially essential for these cells when employed in tissue engineering. In this study by utilizing a custom-made device, effects of uniaxial tension (1Hz, 10% stretch) on cytoskeleton, cell alignment, cell elastic properties, and expression of smooth muscle cell (SMC) genes in ASCs are investigated. Due to proper availability of ASCs, results can be employed in cardiovascular engineering when production of functional SMCs in arterial reconstruction is required. Results demonstrated that cells were oriented after 24 hours of cyclic stretch with aligned pseudo-podia. Staining of actin filaments confirmed enhanced polymerization and alignment of stress fibers. Such phenomenon resulted in stiffening of cell body which was quantified by atomic force microscopy (AFM). Expression of SM α-actin and SM22 α-actin as SMC associated genes were increased after cyclic stretch while GAPDH was considered as internal control gene. Finally, it was concluded that application of cyclic stretch on ASCs assists differentiation to SMC and enhances functionality of cells.

Exendin-4 increases oxygen consumption and thermogenic gene expression in muscle cells.

Glucagon-like peptide-1 (GLP1) has many anti-diabetic actions and also increases energy expenditure in vivo As skeletal muscle is a major organ controlling energy metabolism, we investigated whether GLP1 can affect energy metabolism in muscle. We found that treatment of differentiated C2C12 cells with exendin-4 (Ex-4), a GLP1 receptor agonist, reduced oleate:palmitate-induced lipid accumulation and triglyceride content compared with cells without Ex-4 treatment. When we examined the oxygen consumption rate (OCR), not only the basal OCR but also the OCR induced by oleate:palmitate addition was significantly increased in Ex-4-treated differentiated C2C12 cells, and this was inhibited by exendin-9, a GLP1 receptor antagonist. The expression of uncoupling protein 1 (UCP1), β3-adrenergic receptor, peroxisome proliferator-activator receptor a (PPARa) and farnesoid X receptor mRNA was significantly upregulated in Ex-4-treated differentiated C2C12 cells, and the upregulation of these mRNA was abolished by treatment with adenylate cyclase inhibitor (2'5'-dideoxyadenosine) or PKA inhibitor (H-89). As well, intramuscular injection of Ex-4 into diet-induced obese mice significantly increased the expression of UCP1, PPARa and p-AMPK in muscle. We suggest that exposure to GLP1 increases energy expenditure in muscle through the upregulation of fat oxidation and thermogenic gene expression, which may contribute to reducing obesity and insulin resistance.

Influence of Pulsing Electromagnetic Field Therapy on Gene Expression in Muscle Cells, Peripheral Circulation, and Metabolic Factors in Aging Adults

This study tested the acute effects of pulsating electromagnetic field (PEMF) therapy session on gene expression in muscle cells, and the changes in cardiovascular and metabolic function in a group of older adults after 12 weeks of treatment. Each therapy session involved 15 minutes of exposure to low frequency pulsating asymmetrical waveforms emitted by the Impulser™ Pro mattress. The treatment was provided five days per week for a total of 60 sessions. Pre and post session muscle biopsies provided tissue for analyses of selected genes. Blood pressure and arterial stiffness was determined for cardiovascular function while cholesterol and glucose levels were evaluated using serum samples. Skin temperature was measured using temperature probes. Fifty-five older men and women (mean age 59.75) completed the entire treatment while six subjects in this group consented to muscle biopsies. The results indicate no change in mRNA content of Ku70 during the single PEMF session however mRNA levels of IL-6 and c-Fos increased. The chronic effects of PEMF treatment include significant reductions in blood pressure, and increases in skin temperature. These findings suggest that due to no change in Ku70 levels, a gene associated with DNA damage, an acute bout of PEMF did not cause acute molecular damage. The increase in IL-6, a gene associated with expression of a glucose transporter, might have an influence on glucose metabolism. The increase in c- Fos, a gene linked to growth factors in skeletal muscle, might influence lose in muscle size associated with aging. The increase in skin temperature is difficult to interpret since it might be linked to either heightened metabolism or improvements in peripheral circulation. More studies are needed to determine if these changes impact the health of aging adults. Keywords: Pulsating electromagnetic field (PEMF) therapy, metabolic function, skin temperature.

Generation of an efficient artificial promoter of bovine skeletal muscle α-actin gene (ACTA1) through addition of cis-acting element.

The promoter of skeletal muscle α-actin gene (ACTA1) is highly muscle specific. The core of the bovine ACTA1 promoter extends from +29 to -233, about 262 base pairs (bp), which is sufficient to activate transcription in bovine muscle satellite cells. In this study, analysis by PCR site-specific mutagenesis showed that the cis-acting element SRE (serum response element binding factor) was processed as a transcriptional activator. In order to enhance the bovine ACTA1 promoter's activity, we used a strategy to modify it. We cloned a fragment containing three SREs from the promoter of ACTA1, and then one or two clones were linked upstream of the core promoter (262 bp) of ACTA1. One and two clones increased the activity of the ACTA1 promoter 3-fold and 10-fold, respectively, and maintained muscle tissue specificity. The modified promoter with two clones could increase the level of ACTA1 mRNA and protein 4-fold and 1.1-fold, respectively. Immunofluorescence results showed that green fluorescence of ACTA1 increased. Additionally, the number of total muscle microfilaments increased. These genetically engineered promoters might be useful for regulating gene expression in muscle cells and improving muscle mass in livestock.

Single muscle fiber gene expression with run taper.

This study evaluated gene expression changes in gastrocnemius slow-twitch myosin heavy chain I (MHC I) and fast-twitch (MHC IIa) muscle fibers of collegiate cross-country runners (n = 6, 20±1 y, VO2max = 70±1 ml•kg−1•min−1) during two distinct training phases. In a controlled environment, runners performed identical 8 kilometer runs (30∶18±0∶30 min:s, 89±1% HRmax) while in heavy training (∼72 km/wk) and following a 3 wk taper. Training volume during the taper leading into peak competition was reduced ∼50% which resulted in improved race times and greater cross-section and improved function of MHC IIa fibers. Single muscle fibers were isolated from pre and 4 hour post run biopsies in heavily trained and tapered states to examine the dynamic acute exercise response of the growth-related genes Fibroblast growth factor-inducible 14 (FN14), Myostatin (MSTN), Heat shock protein 72 (HSP72), Muscle ring-finger protein-1 (MURF1), Myogenic factor 6 (MRF4), and Insulin-like growth factor 1 (IGF1) via qPCR. FN14 increased 4.3-fold in MHC IIa fibers with exercise in the tapered state (P<0.05). MSTN was suppressed with exercise in both fiber types and training states (P<0.05) while MURF1 and HSP72 responded to running in MHC IIa and I fibers, respectively, regardless of training state (P<0.05). Robust induction of FN14 (previously shown to strongly correlate with hypertrophy) and greater overall transcriptional flexibility with exercise in the tapered state provides an initial molecular basis for fast-twitch muscle fiber performance gains previously observed after taper in competitive endurance athletes.

DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscle.

Facioscapulohumeral dystrophy (FSHD) is caused by decreased epigenetic repression of the D4Z4 macrosatellite array and recent studies have shown that this results in the expression of low levels of the DUX4 mRNA in skeletal muscle. Several other mechanisms have been suggested for FSHD pathophysiology and it remains unknown whether DUX4 expression can account for most of the molecular changes seen in FSHD. Since DUX4 is a transcription factor, we used RNA-seq to measure gene expression in muscle cells transduced with DUX4, and in muscle cells and biopsies from control and FSHD individuals. We show that DUX4 target gene expression is the major molecular signature in FSHD muscle together with a gene expression signature consistent with an immune cell infiltration. In addition, one unaffected individual without a known FSHD-causing mutation showed the expression of DUX4 target genes. This individual has a sibling with FSHD and also without a known FSHD-causing mutation, suggesting the presence of an unidentified modifier locus for DUX4 expression and FSHD. These findings demonstrate that the expression of DUX4 accounts for the majority of the gene expression changes in FSHD skeletal muscle together with an immune cell infiltration.

Effect of metformin and lipid emulsion on the circadian gene expression in muscle cells

The circadian clock influences nearly all aspects of metabolism. However, little is known regarding the effect of the energy status on circadian rhythms. Our aim was to test the effect of two opposing energy situations, metformin and lipid emulsion (LE), on clock and metabolic circadian expression in differentiated C2C12 myotubes. Metformin treatment led to depleted ATP levels accompanied by elevated NADH levels, whereas LE treatment led to increased ATP and NAD+ levels. Nevertheless, both LE and metformin treatments activated the AMP-activated protein kinase (AMPK) pathway. In contrast, the effect on circadian rhythms was completely different. LE led to disrupted clock and metabolic gene expression, whereas metformin led to mainly high-amplitude shifted rhythms. Combination of metformin and LE led to an antagonistic effect on circadian gene expression. Although metformin and LE have an opposing effect on circadian gene expression and on the cellular energy status, they both lead to AMPK activation.

Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells

BackgroundA sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown.MethodsYeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression.ResultsWe found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells.ConclusionsBased on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy.

Differential Expression of Myogenic Regulatory Factor Genes in the Skeletal Muscles of TambaquiColossoma macropomum(Cuvier 1818) from Amazonian Black and Clear Water

Hypothesizing that the Amazonian water system differences would affect the expression of muscle growth-related genes in juvenile tambaqui Colossoma macropomum (Cuvier 1818), this study aimed to analyze the morphometric data and expression of myogenic regulatory factors (MRFs) in the white and red muscle from tambaqui obtained from clear and black Amazonian water systems. All of the MRF transcript levels (myod, myf5, myogenin, and mrf4) were significantly lower in the red muscle from black water fish in comparison to clear water fish. However, in white muscle, only the myod transcript level was significantly decreased in the black water tambaqui. The changes in MRFs gene expression in muscle fibers of tambaqui from black water system provide relevant information about the environmental influence as that of water systems on gene expression of muscle growth related genes in the C. macropomum. Our results showed that the physical and chemical water characteristics change the expression of genes that promote muscle growth, and these results may be also widely applicable to future projects that aim to enhance muscle growth in fish that are of substantial interest to the aquaculture.

A Novel RhoA/ROCK-CPI-17-MEF2C Signaling Pathway Regulates Vascular Smooth Muscle Cell Gene Expression

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17.

The transcription factor Sp1 plays a crucial role in dok-7 gene expression

Dok-7 is a cytoplasmic activator of the muscle-specific receptor tyrosine kinase MuSK, and these two proteins are essential for neuromuscular junction (NMJ) formation. Mutations of the human DOK7 gene underlie a limb-girdle type of congenital myasthenic syndrome, a group of disorders characterized by NMJ synaptopathy. Because MuSK governs postsynaptic specialization of NMJs and controls where the NMJ forms in the skeletal muscle, it is crucial to appropriately regulate when and where Dok-7 is expressed to activate MuSK. However, the mechanisms underlying expression of the dok-7 gene remain unclear. Here, we show that two Sp1 consensus sequences in the mouse dok-7 5′-flanking region are necessary for dok-7 gene expression in muscle cells. We further demonstrate that the transcription factor Sp1 activates dok-7 gene expression through interaction with these two Sp1 sites. Taken together, these results indicate that Sp1 plays a crucial role in the regulation of the dok-7 gene.

CGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation

Although the regulation of smooth muscle cell (SMC) gene expression by cGMP-dependent protein kinase (PKG) is now recognized, the mechanisms underlying these effects are not fully understood. In this study, we report that PKG-I stimulates myocardin/serum response factor (SRF)-dependent gene expression in vascular SMCs. The expression of PKG in PKG-deficient cells enhanced myocardin-induced SM22 promoter activity in a concentration-dependent fashion. However, neither SRF nor myocardin expression was affected. To investigate alternative mechanisms, we examined whether PKG affects the phosphorylation of E26-like protein-1 (Elk-1), a SRF/myocardin transcription antagonist. The activation of PKG caused an increase in a higher molecular mass form of phospho-Elk-1 that was determined to be small ubiquitin-related modifier (sumo)ylated Elk-1. PKG increased Elk-1 sumoylation twofold compared with the PKG-deficient cells, and Elk-1 sumoylation was reduced using dominant-negative sumo-conjugating enzyme, DN-Ubc9, confirming PKG-dependent sumoylation of phospho-Elk-1 in vascular SMCs. In addition, PKG stimulated Elk-1 sumoylation in COS-7 cells overexpressing Elk-1, sumo-1, and PKG-I. The increased expression of PKG in vascular SMCs inhibited Elk-1 binding to SMC-specific promoters, SM22 and smooth muscle myosin heavy chain, as measured by EMSA and chromatin immunoprecipitation assay, and PKG suppressed the Elk-1 inhibition of SM22 reporter gene expression. Taken together, these data suggest that PKG-I decreases Elk-1 activity by sumo modification of Elk-1, thereby increasing myocardin-SRF activity on SMC-specific gene expression.