We previously reported that excess retinoic acid (RA) resulted in hypoplastic and derangement of myofilaments in embryonic tongue by inhibiting myogenic proliferation and differentiation through ...CamKIID pathway. Our further studies revealed that the expression of a series of miRNAs was altered by RA administration in embryonic tongue as well as in C2C12 cells. Thus, if excess RA impairs myogenic proliferation and differentiation through miRNAs is taken into account. In present study, miR-27b-3p was found up-regulated in RA-treated C2C12 cells as in embryonic tongue, and predicted to target the 3′UTR of α-dystrobrevin (DTNA). Luciferase reporter assays confirmed the direct interaction between miR-27b-3p and the 3′UTR of DTNA. MiR-27b-3p mimics recapitulated the RA repression on DTNA expression, C2C12 proliferation and differentiation, while the miR-27b-3p inhibitor circumvented these defects resulting from excess RA. As expected, the effects of siDTNA on C2C12 were coincided with those by RA treatment or miR-27b-3p mimics. Therefore, these findings indicated that excess RA inhibited the myoblast proliferation and differentiation by up-regulating miR-27b-3p to target DTNA, which implied a new mechanism in myogenic hypoplasia.
•A mechanism that RA results in tongue deformity by disrupting the myogenesis.•A non-muscle specific miR mediating the RA suppression on tongue myogenesis.•A target gene of non-muscle specific miR involved in RA induced tongue deformity.
Metal‐regulatory transcription factor 1 (MTF1) is a conserved metal‐binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements. MTF1 ...responds to both metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. To examine the role for MTF1 in cell differentiation, we use multiple experimental strategies including gene knockdown (KD) mediated by small hairpin RNA and clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9), immunofluorescence, chromatin immunopreciptation sequencing, subcellular fractionation, and atomic absorbance spectroscopy and report a previously unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, both MTF1 expression and nuclear localization increased. Mtf1 KD impaired differentiation, whereas addition of nontoxic concentrations of Cu+‐enhanced MTF1 expression and promoted myogenesis. Furthermore, we observed that Cu+ binds stoichiometrically to a C terminus tetra‐cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes, and Cu addition stimulated this binding. Of note, MTF1 formed a complex with myogenic differentiation (MYOD)1, the master transcriptional regulator of the myogenic lineage, at myogenic promoters. These findings uncover unexpected mechanisms by which Cu and MTF1 regulate gene expression during myoblast differentiation.—Tavera‐Montañez, C., Hainer, S. J., Cangussu, D., Gordon, S. J. V., Xiao, Y., Reyes‐Gutierrez, P., Imbalzano, A. N., Navea, J. G., Fazzio, T. G., Padilla‐Benavides, T. The classic metal‐sensing transcription factor MTF1 promotes myogenesis in response to copper. FASEB J. 33, 14556‐14574 (2019). www.fasebj.org
Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well ...characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.
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•CircRNAs are conserved, abundant, and regulated in myogenesis•High-throughput phenotypic screening reveals functional circRNAs•Circ-ZNF609 regulates myoblast proliferation•Circ-ZNF609 can be translated
Legnini et al. identified circ-ZNF609, a circular RNA expressed in murine and human myoblasts, which controls myoblast proliferation. Circ-ZNF609 contains an open reading frame and is translated into a protein in a splicing-dependent/cap-independent manner. Circ-ZNF609 translation can be modulated by stress conditions.
The cell-cell/cell-matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in ...vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.
► CDA-II inhibits myogenic differentiation in a dose-dependent manner. ► CDA-II repressed expression of muscle transcription factors and structural proteins. ► CDA-II inhibited proliferation and ...migration of C2C12 myoblasts.
CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.
The vasculature plays an indispensible role in organ development and maintenance of tissue homeostasis, such that disturbances to it impact greatly on developmental and postnatal health. Although ...cell turnover in healthy blood vessels is low, it increases considerably under pathological conditions. The principle sources for this phenomenon have long been considered to be the recruitment of cells from the peripheral circulation and the re-entry of mature cells in the vessel wall back into cell cycle. However, recent discoveries have also uncovered the presence of a range of multipotent and lineage-restricted progenitor cells in the mural layers of postnatal blood vessels, possessing high proliferative capacity and potential to generate endothelial, smooth muscle, hematopoietic or mesenchymal cell progeny. In particular, the tunica adventitia has emerged as a progenitor-rich compartment with niche-like characteristics that support and regulate vascular wall progenitor cells. Preliminary data indicate the involvement of some of these vascular wall progenitor cells in vascular disease states, adding weight to the notion that the adventitia is integral to vascular wall pathogenesis, and raising potential implications for clinical therapies. This review discusses the current body of evidence for the existence of vascular wall progenitor cell subpopulations from development to adulthood and addresses the gains made and significant challenges that lie ahead in trying to accurately delineate their identities, origins, regulatory pathways, and relevance to normal vascular structure and function, as well as disease.
Recent trends in scaffold design have focused on materials that can provide appropriate guidance cues for particular cell types to modulate cell behavior. In this study highly aligned and ...electrically conductive nanofibers that can simultaneously provide topographical and electrical cues for cells were developed. Thereafter their potential to serve as functional scaffolds for skeletal muscle tissue engineering was investigated. Well-ordered nanofibers, composed of polyaniline (PANi) and poly(ε-caprolactone) (PCL), were electrospun by introducing an external magnetic field in the collector region. Incorporation of PANi into PCL fibers significantly increased the electrical conductivity from a non-detectable level for the pure PCL fibers to 63.6±6.6mScm⁻¹ for the fibers containing 3wt.% PANi (PCL/PANi-3). To investigate the synergistic effects of topographical and electrical cues using the electrospun scaffolds on skeletal myoblast differentiation, mouse C2C12 myoblasts were cultured on random PCL (R-PCL), aligned PCL (A-PCL), random PCL/PANi-3 (R-PCL/PANi) and aligned PCL/PANi-3 (A-PCL/PANi) nanofibers. Our results showed that the aligned nanofibers (A-PCL and A-PCL/PANi) could guide myoblast orientation and promote myotube formation (i.e. approximately 40% and 80% increases in myotube numbers) compared with R-PCL scaffolds. In addition, electrically conductive A-PCL/PANi nanofibers further enhanced myotube maturation (i.e. approximately 30% and 23% or 15% and 18% increases in the fusion and maturation indices) compared with non-conductive A-PCL scaffolds or R-PCL/PANi. These results demonstrated that a combined effect of both guidance cues was more effective than an individual cue, suggesting a potential use of A-PCL/PANi nanofibers for skeletal muscle regeneration.
Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance and its plasticity are only partially understood. In this ...study we identify musclin—a peptide with high homology to natriuretic peptides (NP)—as an exercise-responsive myokine that acts to enhance exercise capacity in mice. We use human primary myoblast culture and in vivo murine models to establish that the activity-related production of musclin is driven by Ca2+-dependent activation of Akt1 and the release of musclin-encoding gene (Ostn) transcription from forkhead box O1 transcription factor inhibition. Disruption ofOstnand elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with recombinant musclin. Reduced exercise capacity in mice with disrupted musclin signaling is associated with a trend toward lower levels of plasma atrial NP (ANP) and significantly smaller levels of cyclic guanosine monophosphate (cGMP) and peroxisome proliferator-activated receptor gamma coactivator 1-α in skeletal muscles after exposure to exercise. Furthermore, in agreement with the established musclin ability to interact with NP clearance receptors, but not with NP guanyl cyclase-coupled signaling receptors, we demonstrate that musclin enhances cGMP production in cultured myoblasts only when applied together with ANP. Elimination of the activity-related musclin-dependent boost of ANP/cGMP signaling results in significantly lower maximum aerobic capacity, mitochondrial protein content, respiratory complex protein expression, and succinate dehydrogenase activity in skeletal muscles. Together, these data indicate that musclin enhances physical endurance by promoting mitochondrial biogenesis.
Strain and physical trauma to mechanically active cells, such as skeletal muscle myofibers, injures their plasma membranes, and mitochondrial function is required for their repair. We found that ...mitochondrial function was also needed for plasma membrane repair in myoblasts as well as nonmuscle cells, which depended on mitochondrial uptake of calcium through the mitochondrial calcium uniporter (MCU). Calcium uptake transiently increased the mitochondrial production of reactive oxygen species (ROS), which locally activated the guanosine triphosphatase (GTPase) RhoA, triggering F-actin accumulation at the site of injury and facilitating membrane repair. Blocking mitochondrial calcium uptake or ROS production prevented injury-triggered RhoA activation, actin polymerization, and plasma membrane repair. This repair mechanism was shared between myoblasts, nonmuscle cells, and mature skeletal myofibers. Quenching mitochondrial ROS in myofibers during eccentric exercise ex vivo caused increased damage to myofibers, resulting in a greater loss of muscle force. These results suggest a physiological role for mitochondria in plasma membrane repair in injured cells, a role that highlights a beneficial effect of ROS.
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