AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro.

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy.

BACKGROUND: The repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported. RESULTS: RGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to 'adherens junsctions' and 'extracellular-cell adhesion', while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation. CONCLUSIONS: These results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

Microbial composition of a hydropower cooling water system reveals thermophilic bacteria with a possible role in primary biofilm formation.

Microfouling, ie biofilm formation on surfaces, can have an economic impact and requires costly maintenance in water-powered energy generation systems. In this study, the microbiota of a cooling system (filter and heat exchanger) in the Irapé hydroelectric power plant in Brazil was examined. The goal was to identify bacteria that could be targeted to more efficiently reduce biofilm formation. Two sampling campaigns were made corresponding to two well-defined seasons of the Brazilian Cerrado biome: the dry (campaign 1) and the wet (campaign 2). Microfouling communities varied considerably over time in samples obtained at different times after the last clearance of the heat exchanger. The thermophilic bacteria Meiothermus, Thermomonas and Symbiobacterium were exclusive and abundant in the microfouling of the heat exchanger in campaign 2, while methanotrophs and iron-reducing bacteria were abundant only in filter sediments. These findings could help to guide strategies for ecofriendly measures to reduce biofilm fouling in hydroelectric power plants, minimizing environmental and economic losses.

In vitro analysis of the influence of mineralized and EDTA-demineralized allogenous bone on the viability and differentiation of osteoblasts and dental pulp stem cells.

Grafting based on both autogenous and allogenous human bone is widely used to replace areas of critical loss to induce bone regeneration. Allogenous bones have the advantage of unlimited availability from tissue banks. However, their integration into the remaining bone is limited because they lack osteoinduction and osteogenic properties. Here, we propose to induce the demineralization of the allografts to improve these properties by exposing the organic components. Allografts fragments were demineralized in 10% EDTA at pH 7.2 solution. The influence of the EDTA-DAB and MAB fragments was evaluated with respect to the adhesion, growth and differentiation of MC3'T3-E1 osteoblasts, primary osteoblasts and dental pulp stem cells (DPSC). Histomorphological analyses showed that EDTA-demineralized fragments (EDTA-DAB) maintained a bone architecture and porosity similar to those of the mineralized (MAB) samples. BMP4, osteopontin, and collagen III were also preserved. All the cell types adhered, grew and colonized both the MAB and EDTA-DAB biomaterials after 7, 14 and 21 days. However, the osteoblastic cell lines showed higher viability indexes when they were cultivated on the EDTA-DAB fragments, while the MAB fragments induced higher DPSC viability. The improved osteoinductive potential of the EDTA-DAB bone was confirmed by alkaline phosphatase activity and calcium deposition analyses. This work provides guidance for the choice of the most appropriate allograft to be used in tissue bioengineering and for the transport of specific cell lineages to the surgical site.

Didelphis albiventris: an overview of unprecedented transcriptome sequencing of the white-eared opossum.

BACKGROUND: The white-eared opossum (Didelphis albiventris) is widely distributed throughout Brazil and South America. It has been used as an animal model for studying different scientific questions ranging from the restoration of degraded green areas to medical aspects of Chagas disease, leishmaniasis and resistance against snake venom. As a marsupial, D. albiventris can also contribute to the understanding of the molecular mechanisms that govern the different stages of organogenesis. Opossum joeys are born after only 13 days, and the final stages of organogenesis occur when the neonates are inside the pouch, depending on lactation. As neither the genome of this opossum species nor its transcriptome has been completely sequenced, the use of D. albiventris as an animal model is limited. In this work, we sequenced the D. albiventris transcriptome by RNA-seq to obtain the first catalogue of differentially expressed (DE) genes and gene ontology (GO) annotations during the neonatal stages of marsupial development. RESULTS: The D. albiventris transcriptome was obtained from whole neonates harvested at birth (P0), at 5 days of age (P5) and at 10 days of age (P10). The de novo assembly of these transcripts generated 85,338 transcripts. Approximately 30% of these transcripts could be mapped against the amino acid sequences of M. domestica, the evolutionarily closest relative of D. albiventris to be sequenced thus far. Among the expressed transcripts, 2077 were found to be DE between P0 and P5, 13,780 between P0 and P10, and 1453 between P5 and P10. The enriched GO terms were mainly related to the immune system, blood tissue development and differentiation, vision, hearing, digestion, the CNS and limb development. CONCLUSIONS: The elucidation of opossum transcriptomes provides an out-group for better understanding the distinct characteristics associated with the evolution of mammalian species. This study provides the first transcriptome sequences and catalogue of genes for a marsupial species at different neonatal stages, allowing the study of the mechanisms involved in organogenesis.

Effects of strontium ranelate treatment on osteoblasts cultivated onto scaffolds of trabeculae bovine bone.

Blocks of Bovine bone have shown promising results as implantable scaffolds to promote bone regeneration. Strontium ranelate (SrR) is both an antiresorptive and an anabolic drug that has been indicated for oral administration to treat osteoporosis. Few studies, however, have investigated the local effects of SrR and its use in association with biomaterials thus far. In this work, we investigated SrR effects in cultures of primary osteoblasts (PO, from Wistar rats calvaria) and immortalized osteoblasts (IO, from MC3T3-E1 cell line) cultivated as a monolayer or in association with scaffolds of bovine bone in mineralized (MBB) and demineralized (DBB) forms. The optimum dose to induce SrR effects on cell viability was established as 0.1 mM. Our results suggested that the local administration of SrR is biocompatible and non-cytotoxic. In addition, SrR appeared to accelerate primary osteoblast cell differentiation by enhancing alkaline phosphatase activity, the expression of osteogenic differentiation markers, the synthesis of the organic matrix, and a decrease of Ca2+ ions in mineralized nodules. DBB was found to be a better scaffold material to promote PO and IO cell proliferation. Exposing the proteins of the demineralized bone matrix might improve scaffold osteoconductive properties. Our results indicated the importance of further investigation of the administration of SrR at sites of bone repair. The association of SrR and bone grafts suggests the possibility of using SrR as a co-adjuvant for bone tissue bioengineering and in bone regeneration therapies.

Repulsive Guidance Molecules a, b and c Are Skeletal Muscle Proteins, and Repulsive Guidance Molecule a Promotes Cellular Hypertrophy and Is Necessary for Myotube Fusion.

Repulsive guidance molecules (RGMs) compose a family of glycosylphosphatidylinositol (GPI)-anchored axon guidance molecules and perform several functions during neural development. New evidence has suggested possible new roles for these axon guidance molecules during skeletal muscle development, which has not been investigated thus far. In the present study, we show that RGMa, RGMb and RGMc are all induced during skeletal muscle differentiation in vitro. Immunolocalization performed on adult skeletal muscle cells revealed that RGMa, RGMb and RGMc are sarcolemmal proteins. Additionally, RGMa was found to be a sarcoplasmic protein with a surprisingly striated pattern. RGMa colocalization with known sarcoplasmic proteins suggested that this axon guidance molecule is a skeletal muscle sarcoplasmic protein. Western blot analysis revealed two RGMa fragments of 60 and 33 kDa, respectively, in adult skeletal muscle samples. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa overexpression produced hypertrophic cells, whereas RGMa knockdown resulted in the opposite phenotype. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types. Our results are the first to show an axon guidance molecule as a skeletal muscle sarcoplasmic protein and to include RGMa in a system that regulates skeletal muscle cell size and differentiation.

Derivation of sperm from xenografted testis cells and tissues of the peccary (Tayassu tajacu).

Because the collared peccary (Tayassu tajacu) has a peculiar Leydig cell cytoarchitecture, this species represents a unique mammalian model for investigating testis function. Taking advantage of the well-established and very useful testis xenograft technique, in the present study, testis tissue and testis cell suspensions from immature collared peccaries (n=4; 3 months old) were xenografted in SCID mice (n=48) and evaluated at 2, 4, 6, and 8 months after grafting. Complete spermatogenesis was observed at 6 and 8 months after testis tissue xenografting. However, probably due to de novo testis morphogenesis and low androgen secretion, functionally evaluated by the seminal vesicle weight, a delay in spermatogenesis progression was observed in the testis cell suspension xenografts, with the production of fertile sperm only at 8 months after grafting. Importantly, demonstrating that the peculiar testicular cytoarchitecture of the collared peccary is intrinsically programmed, the unique Leydig cell arrangement observed in this species was re-established after de novo testis morphogenesis. The sperm collected from the xenografts resulted in diploid embryos that expressed the paternally imprinted gene NNAT after ICSI. The present study is the first to demonstrate complete spermatogenesis with the production of fertile sperm from testis cell suspension xenografts in a wild mammalian species. Therefore, due to its unique testicular cytoarchitecture, xenograft techniques, particularly testis cell suspensions, may represent a new and very promising approach to evaluate testis morphogenesis and to investigate spermatogonial stem cell physiology and niche in the collared peccary.

Ovarian Puncture Triggers an Inflammatory Response that did not Affect Late Folliculogenesis, Ovulation Rate, and Fertility.

Ovarian puncture has been widely used in assisted reproduction, but there are still gaps about its effects on ovarian morphophysiology, as well as the relationship between inflammation caused by this procedure and the follicular growth and fertility. The aim of this study was to investigate the effects of ovarian puncture on folliculogenesis and fertility. Mice (n = 24) were divided into two groups: (1) SHAM-both ovaries were exposed and repositioned and (2) Punctured-ovaries were exposed, punctured, and repositioned. After 96 h of surgery, ovaries were collected for morphofunctional analysis. New females were used for the superovulation (n = 10) and fertility assays (n = 10). Increased volumetric density of inflammatory cells-p = 0.0005, p = 0.0013; hemorrhagic foci-p < 0.0001; and inflammatory exudate-p < 0.0001 could be noticed on the punctured group, compared to SHAM. The percentage of primordial follicles was lower on the punctured ovaries (p = 0.00294). Ovarian puncture has also induced an increase in the proliferation of granulosa cells of primary (p = 0.0321) and antral follicles (p = 0.0395), and an increased apoptotic index of antral follicles (p = 0.0100). There was no influence on expression of some genes related to inflammation, collagen deposition and folliculogenesis progression. The reproductive aspects (oocyte retrieval and number of fetuses per female) were not altered (p > 0.05). Taken together, our findings strongly suggest that ovarian puncture results in a local inflammation that affects follicular growth and atresia. However, it does not affect female fertility, which strengthens the safety of this procedure.

Diversity and distribution of iron-oxidising bacteria belonging to Gallionellaceae in different sites of a hydroelectric power plant.

Iron (Fe) is the fourth most abundant element on the planet, and iron-oxidising bacteria (FeOB) play an important role in the biogeochemical cycle of this metal in nature. FeOB stands out as Fe oxidisers in microaerophilic environments, and new members of this group have been increasingly discussed in the literature, even though their isolation can still be challenging. Among these bacteria is the Gallionellaceae family, mainly composed of neutrophilic FeOB, highlighting Gallionella ferruginea, and nitrite-oxidiser genera. In the previous metagenomic study of the biofilm and sediments of the cooling system from the Irapé hydroelectric power plant (HPP-Irapé), 5% of the total bacteria sequences were related to Gallionellaceae, being 99% unclassified at genus level. Thus, in the present study, a phylogenetic tree based on this family was constructed, in order to search for shared and unique Gallionellaceae signatures in a deep phylogenetic level affiliation and correlated them with geomorphologic characteristics. The results revealed that Gallionella and Ferrigenium were ubiquitous reflecting their ability to adapt to various locations in the power plant. The cave was considered a hotspot for neutrophilic FeOB since it harboured most of the Gallionellaceae diversity. Microscopic biosignatures were detected only in the CS1 sample, which presented abundance of the stalk-forming Ferriphaselus and of the sheath-forming Crenothrix. Further studies are required to provide more detailed insights on Gallionellaceae distribution and diversity patterns in hydroelectric power plants, particularly its biotechnological potential in this industry.

Naproxen administration affects murine late folliculogenesis, reduces granulosa cell proliferation and the number of ovulated oocytes.

Naproxen reduces the production of prostaglandins via inhibition of the cyclooxygenase. Studies have shown that its administration in women can be related to failed ovulation. Therefore, preclinical investigations must be performed in order to investigate its effects in experimental models. Thus, the aim of this study was to evaluate the effects of naproxen on murine folliculogenesis, ovulation, and female fertility. Female C57BL/6 mice (n = 128 - 6 weeks old) were divided into Control, low (10 mg/kg), and high naproxen (50 mg/kg) groups, who were treated for 8 days and directed to morphofunctional analyses. Follicular quantification showed a reduced percentage of antral follicles in naproxen-treated animals. These treated animals also showed smaller oocytes included in secondary and antral follicles, and the diameter of secondary and antral follicles was also reduced. A reduction in the percentage of Ki67-positive granulosa cells was observed in treated animals that also showed down-regulation of Igf1r compared to control. After an ovarian stimulation protocol, naproxen-treated animals showed a reduction in the percentage of secondary and antral follicles, a reduced number of ovulated oocytes and, corpora lutea, and an increased number of failed ovulations. Finally, naproxen-treated animals also showed a reduction in mating index and pregnancy rate. Our findings suggested that, in mice, naproxen administration (eight days treatment) negatively affects molecular and morphological aspects related to late folliculogenesis, ovulation, and fertility.

Corrigendum: Random cellulose acetate nanofibers: a breakthrough for cultivated meat production.

[This corrects the article DOI: 10.3389/fnut.2023.1297926.].

RGMa and RGMb expression pattern during chicken development suggest unexpected roles for these repulsive guidance molecules in notochord formation, somitogenesis, and myogenesis.

BACKGROUND: Repulsive guidance molecules (RGM) are high-affinity ligands for the Netrin receptor Neogenin, and they are crucial for nervous system development including neural tube closure; neuronal and neural crest cell differentiation and axon guidance. Recent studies implicated RGM molecules in bone morphogenetic protein signaling, which regulates a variety of developmental processes. Moreover, a role for RGMc in iron metabolism has been established. This suggests that RGM molecules may play important roles in non-neural tissues. RESULTS: To explore which tissues and processed may be regulated by RGM molecules, we systematically investigated the expression of RGMa and RGMb, the only RGM molecules currently known for avians, in the chicken embryo. CONCLUSIONS: Our study suggests so far unknown roles of RGM molecules in notochord, somite and skeletal muscle development.

Drastic Loss of Antral Follicles Due to Gene Expression Dysregulation Occurs on the First Day After Subcutaneous Ovarian Transplantation.

Ovarian cryopreservation is an alternative for the preservation of fertility, and the subcutaneous transplantation site is considered one of the most promising. Studies evaluating the follicular growth and its relationship with gene expression and vascular perfusion are essential for improving this technique and its clinical application. Thus, the aim of this study was to evaluate the effect of subcutaneous autotransplantation and vitrification on follicular growth and atresia and their relationship with vascular perfusion and gene expression. Therefore, female mice were ovariectomized, and the ovaries were divided in two experimental groups (1) vitrified (treatment, n = 97) and (2) not vitrified (control, n = 97) and subsequently were transplanted. Then grafts, from both groups, were recovered after 1, 12, or 23 days (D1, D12, D23) and subjected to follicular quantification, morphometry, and qPCR. Non-transplanted ovaries (D0) were also used. The estrous cycle and vascular perfusion were monitored throughout the experiment. On D9, 100% of the animals had reestablished their estrous cycles (p > 0.05). Blood perfusion at the transplant site was similar for both treatments (p > 0.05), with greater perfusion at the site of vitrified transplants only on D1 (p < 0.05). A drastic reduction in the number of antral follicles and an increased number of atretic follicles were observed on D1 (p < 0.0001), associated with upregulation of Casp3, Fshr, and Igf1r; and downregulation of Bax, Acvr1, Egfr, and Lhcgr (p < 0.05). Our findings indicate that the first day after subcutaneous transplantation is a critical period for follicular survival, with intense follicular atresia independent of Bax upregulation.

Bioactive cellulose acetate nanofiber loaded with annatto support skeletal muscle cell attachment and proliferation.

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212 nm vs. 284 ± 130 nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

Acute Exposure to Two Biocides Causes Morphological and Molecular Changes in the Gill Ciliary Epithelium of the Invasive Golden Mussel Limnoperna fortunei (Dunker, 1857).

Limnoperna fortunei, the golden mussel, is a bivalve mollusk considered an invader in South America. This species is responsible for ecological and economic damages due to its voluminous fouling capability. Chemical biocides such as MXD-100™ and sodium dichloroisocyanurate (NaDCC) are often used to control L. fortunei infestations in hydraulic systems. Thus, we proposed to investigate the effects of different periods (24, 48 and 72 h) of exposure to MXD-100™ (0.56 mg L-1) and NaDCC (1.5 mg L-1) on the gills of L. fortunei through morphological and molecular analyses. NaDCC promoted progressive morphological changes during the analyzed periods and only an upregulation of SOD and HSP70 expression during the first 24 h of exposure. MXD-100™ led to severe morphological changes from the first period of exposure, in addition to an upregulation of SOD, CAT, HSP70 and CYP expression during the first 24 h. In contrast, MXD-100™ led to a downregulation of CAT transcription between 24 and 48 h. In static conditions, NaDCC causes lethal damage after 72 h of exposure, and that exposure needs to be continuous to achieve the control of the species. Meanwhile, the MXD-100™ treatment presented several effects during the first 24 h, showing acute toxicity in a shorter period of time.

Random cellulose acetate nanofibers: a breakthrough for cultivated meat production.

Overcoming the challenge of creating thick, tissue-resembling muscle constructs is paramount in the field of cultivated meat production. This study investigates the remarkable potential of random cellulose acetate nanofibers (CAN) as a transformative scaffold for muscle tissue engineering (MTE), specifically in the context of cultivated meat applications. Through a comparative analysis between random and aligned CAN, utilizing C2C12 and H9c2 myoblasts, we unveil the unparalleled capabilities of random CAN in facilitating muscle differentiation, independent of differentiation media, by exploiting the YAP/TAZ-related mechanotransduction pathway. In addition, we have successfully developed a novel process for stacking cell-loaded CAN sheets, enabling the production of a three-dimensional meat product. C2C12 and H9c2 loaded CAN sheets were stacked (up to four layers) to form a ~300-400 µm thick tissue 2 cm in length, organized in a mesh of uniaxial aligned cells. To further demonstrate the effectiveness of this methodology for cultivated meat purposes, we have generated thick and viable constructs using chicken muscle satellite cells (cSCs) and random CAN. This groundbreaking discovery offers a cost-effective and biomimetic solution for cultivating and differentiating muscle cells, forging a crucial link between tissue engineering and the pursuit of sustainable and affordable cultivated meat production.

Ultrastructure of the gill ciliary epithelium of Limnoperna fortunei (Dunker 1857), the invasive golden mussel.

BACKGROUND: Limnoperna fortunei is a freshwater bivalve mollusc originally from southern Asia that invaded South America in the 1990's. Due to its highly efficient water pumping and filtering, and its capacity to form strong adhesions to a variety of substrates by byssus thread, this invasive species has been able to adapt to several environments across South America, causing significant ecological and economic damages. By gaining a deeper understanding of the biological and ecological aspects of L. fortunei we will be able to establish more effective strategies to manage its invasion. The gills of the mollusc are key structures responsible for several biological functions, including respiration and feeding. In this work, we characterized the ultrastructure of L. fortunei gills and its ciliary epithelium using light microscopy, transmission and scanning electron microscopies. This is the first report of the morphology of the epithelial cells and cilia of the gill of L. fortunei visualized in high resolution. RESULTS: The analysis showed highly organized and abundant ciliary structures (lateral cilia, laterofrontal cirri and frontal cilia) on the entire length of the branchial epithelium. Mitochondria, smooth endoplasmic reticulum and glycogen granules were abundantly found in the epithelial cells of the gills, demonstrating the energy-demanding function of these structures. Neutral mucopolysaccharides (low viscosity mucus) were observed on the frontal surface of the gill filaments and acid mucopolysaccharides (high viscosity mucus) were observed to be spread out, mainly on the lateral tract. Spherical vesicles, possibly containing mucus, could also be observed in these cells. These findings demonstrate the importance of the mucociliary processes in particle capture and selection. CONCLUSIONS: Our data suggest that the mechanism used by this mollusc for particle capture and selection could contribute to a better understanding of key aspects of invasion and also in the establishment of more efficient and economically viable strategies of population control.

RGMa can induce skeletal muscle cell hyperplasia via association with neogenin signalling pathway.

Although originally discovered inducing important biological functions in the nervous system, repulsive guidance molecule a (RGMa) has now been identified as a player in many other processes and diseases, including in myogenesis. RGMa is known to be expressed in skeletal muscle cells, from somites to the adult. Functional in vitro studies have revealed that RGMa overexpression could promote skeletal muscle cell hypertrophy and hyperplasia, as higher efficiency in cell fusion was observed. Here, we extend the potential role of RGMa during C2C12 cell differentiation in vitro. Our results showed that RGMa administrated as a recombinant protein during late stages of C2C12 myogenic differentiation could induce myoblast cell fusion and the downregulation of different myogenic markers, while its administration at early stages induced the expression of myogenic markers with no detectable morphological effects. We also found that RGMa effects on skeletal muscle hyperplasia are performed via neogenin receptor, possibly as part of a complex with other proteins. Additionally, we observed that RGMa-neogenin is not playing a role as an inhibitor of the BMP signalling in skeletal muscle cells. This work contributes to placing RGMa as a component of the mechanisms that determine skeletal cell fusion via neogenin receptor.

SAP30 Gene Is a Probable Regulator of Muscle Hypertrophy in Chickens.

Animals with muscle hypertrophy phenotype are targeted by the broiler industry to increase the meat production and the quality of the final product. Studies characterizing the molecular machinery involved with these processes, such as quantitative trait loci studies, have been carried out identifying several candidate genes related to this trait; however, validation studies of these candidate genes in cell culture is scarce. The aim of this study was to evaluate SAP30 as a candidate gene for muscle development and to validate its function in cell culture in vitro. The SAP30 gene was downregulated in C2C12 muscle cell culture using siRNA technology to evaluate its impact on morphometric traits and gene expression by RNA-seq analysis. Modulation of SAP30 expression increased C2C12 myotube area, indicating a role in muscle hypertrophy. RNA-seq analysis identified several upregulated genes annotated in muscle development in treated cells (SAP30-knockdown), corroborating the role of SAP30 gene in muscle development regulation. Here, we provide experimental evidence of the involvement of SAP30 gene as a regulator of muscle cell hypertrophy.