Myogenic Proteins Research Articles

EFFECT OF EXTREMELY LOW-FREQUENCY ELECTROMAGNETIC FIELDS ON PROMOTING MYOGENESIS IN THE EARLY PROCESS OF DENERVATED MUSCLEATROPHY

Objective: To investigate the effects of extremely low-frequency electromagnetic fields (ELF-EMF) on myogenesis during the early stage of denervated muscle atrophy in Sprague-Dawley (SD) rats. Methods: a total of 36 female SD rats were fed in cages and divided into three groups: the normal control group, the muscle atrophy group and the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group. The rats in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group received a magnetic field intervention for 2[Formula: see text]h per day. The posterior muscle groups of the lower limbs of rats were sampled on the first, second, third and fourth weeks for evaluating the underlying changes. The harvested tissues were stained with hematoxylin–eosin (HE) dye to observe the morphology of the muscle atrophy and to measure the muscle fiber cross-sectional area ratio. A reverse transcription polymerase chain reaction was used to detect the expression of Myf5 mRNA in each group. The myogenesis-related proteins in the harvested muscle tissue were detected by western blot assay. Results: (1) HE staining showed that with the extension of denervation time, the structural disorder of muscle fibers was aggravated, and in the second week of the denervated muscle atrophy, muscle fiber cross-sectional area tended to increase in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group. Overall muscle atrophy was inevitable. (2) Results of RT-PCR showed that the expression of Myf5 mRNA in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group was significantly high in the second week of denervation, and the difference was statistically significant compared with the muscle atrophy group (*[Formula: see text]). (3) Western blot analysis showed that the expression of MYF5 proteins in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group was significantly higher than that in the muscle atrophy group (**[Formula: see text]) in the second week of denervation. Further, the expression of MYOG protein in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group was significantly higher than that in the muscle atrophy group (****[Formula: see text]). Conclusions: 10[Formula: see text]Hz/1.5[Formula: see text]mT ELF-EMF can significantly promote myogenesis during early denervated muscle atrophy in SD rats.

Three-dimensional pore structure of the decellularized parsley scaffold regulates myogenic differentiation for cell cultured meat.

Decellularized plant scaffolds have been used to develop edible scaffolds for cell cultured meat because of their natural structures similar to that of mammalian tissues. However, their diverse three-dimensional (3D) porous structures may lead to differences in myogenic differentiation of skeletal muscle cells. In this study, parsley plant tissues were decellularized and modified by type A gelatin and transglutaminase while retaining, respectively, longitudinal fibrous and transverse honeycomb pore structures. The effects of the structure of the decellularized parsley scaffold on the proliferation and myogenic differentiation of C2C12 cells were investigated and the quality of cell cultured meat was evaluated. The results showed that fibrous pore structure guided cells to be arranged in parallel, whereas honeycomb pore structure connected cells in a circular pattern. After induced differentiation, the fibrous scaffolds were more inclined to form multinucleated myotubes with higher expression of myogenic genes and proteins, and the final cell-based meat contained higher total protein content. Decellularized plant scaffolds with fibrous pore structure were more suitable for myogenic differentiation of C2C12 cells, providing support to the development of edible scaffolds for cultured meat. PRACTICAL APPLICATION: This study investigated the different three-dimensional (3D) pore structure of parsley parenchyma to gain insight into how the 3D pore structure of decellularized plant scaffolds regulates myogenic differentiation, which is expected to address the unstable myogenic differentiation of skeletal muscle cells on decellularized plant scaffolds in cell culture meat production.

Developmental ability of Hanwoo muscle satellite cells under culture conditions mimicking the in vivo environment

Cultivated meat refers to edible meat obtained by proliferating cells without killing livestock in a laboratory. The selection of donor animals is a crucial factor for efficient cell culture production. Hanwoo is a native Korean taurine cattle breed raised as livestock in Korea since before 2000 B.C. Cells isolated from Hanwoo, which has little genetic diversity, are expected to be advantageous in cell culture because of the existence of fewer individual differences. However, cells collected from Hanwoo are in a state where efficient culture conditions have not been established. Therefore, in this study, we investigated the effects of mimicking an in vivo environment on the proliferation and differentiation of Hanwoo muscle satellite cells. The culture conditions consisted of CON (37°C/20% O2), T1 (37°C/2% O2), T2 (39°C/20% O2), and T3 (39°C/2% O2). Cell numbers decreased and expression levels of PAX7 and MYF5 decreased at a temperature of 39°C (p < 0.05). Conversely, 2% oxygen increased the number of cells and expression levels of PAX7 and MYF5 (p < 0.05). A temperature of 39°C inhibited the proliferation of Hanwoo muscle satellite cells by reducing the expression of PAX7 and MYF5 (p < 0.05). Conversely, 2% oxygen promoted the proliferation of Hanwoo muscle satellite cells by enhancing the expression of PAX7 and MYF5 (p < 0.05). During differentiation, a temperature of 39°C improved the myotube area and fusion index (p < 0.05). The RT-qPCR and Western blotting results revealed that a culture temperature of 39°C increased expression levels of the MYH2 gene and DES and MYOG proteins (p < 0.05). Additionally, an interactive condition increased expression levels of MYOD1, DES, and MYOG genes (p < 0.05). These results indicated that a temperature of 39°C promoted the differentiation of Hanwoo muscle satellite cells by increasing DES and MYOG protein expression. Thus, the production of cultivated meat using Hanwoo muscle satellite cells is expected to be efficient under 2% oxygen for proliferation and 39°C for differentiation.

Influence of Frequent Corticosteroid Local Injections on the Expression of Genes and Proteins Related to Fatty Infiltration, Muscle Atrophy, Inflammation, and Fibrosis in Patients With Chronic Rotator Cuff Tears: A Pilot Study.

The effect of local corticosteroid (CS) injections on rotator cuff muscles remains poorly defined, despite the significance of muscle quality as a crucial prognostic factor for patients with rotator cuff tears (RCTs). To compare alterations in gene and protein expression patterns in the rotator cuff muscles of patients with RCTs who received frequent joint CS injections with alterations in those without a history of CS injections. Controlled laboratory study. A total of 24 rotator cuff muscle samples with medium-sized tears from 12 patients with a frequent joint CS injection history (steroid group; 7 men and 5 women who had received ≥5 injections with at least 1 within the previous 3 months; mean age, 63.0 ± 7.2 years) and 12 age- and sex-matched control patients without a history of CS injections (no-steroid group) were acquired. Alterations in the expression of genes and proteins associated with adipogenesis, myogenesis, inflammation, and muscle fibrosis were compared between the groups using quantitative reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Statistical analysis included comparison of group means using the Mann-Whitney U test, chi-square test, or Fisher exact test and logistic regression for multivariate analysis. In the steroid group, the mRNA expression levels of adipogenic CCAAT/enhancer-binding protein alpha (C/EBPα; P = .008) and muscle atrophy-related genes (atrogin; P = .019) were significantly higher, and those of myogenic differentiation 1 (MyoD; P = .035), inflammatory interleukin 6 (IL-6; P = .035), and high mobility group box 1 (P = .003) were significantly lower compared with the no-steroid group. In addition, MyoD (P = .041) and IL-6 (P = .026) expression were decreased in the steroid versus no-steroid group. Immunohistochemistry revealed increased expression of C/EBPα and atrogin and decreased expression of MyoD and IL-6 in the steroid versus no-steroid group. Patients with RCTs and a history of frequent CS injections exhibited an upregulation of adipogenic and muscle atrophy-related genes and proteins within the rotator cuff muscles and a downregulation in the expression of myogenic and inflammatory genes and proteins in the same muscles. These altered gene and protein expressions by frequent local CS injections may cause poor outcomes in patients with RCTs.

Running improves muscle mass by activating autophagic flux and inhibiting ubiquitination degradation in mdx mice

BackgroundExercise therapy can improve muscle mass, strengthen muscle and cardiorespiratory function, and may be an excellent adjunctive treatment option for Duchenne muscular dystrophy. MethodsThis article investigates the effects of 10 weeks of treadmill training on skeletal muscle in control and mdx mice. Hematoxylin and eosin (H&E) staining was used to detect the morphometry of skeletal muscle; the grip strength test, suspension test, and rotarod test were used to detect limb muscle strength of mice, and Aurora Scientific Instruments were used to detect in vivo Muscle Stimulation Measuring Maximum Force of pre-fatigue and post-fatigue. The expression levels of myogenic proteins, ubiquitination markers, autophagy pathway proteins, and the proportion of different muscle fiber types were detected. ResultsThe experimental results show that running exercise can significantly improve the muscle mass of mdx mice, promote muscle strength, endurance, and anti-fatigue ability, reverse the pathological state of skeletal muscle destruction in mdx mice, and promote muscle regeneration. WB experiments showed that running inhibited the ubiquitination and degradation of muscle protein in mdx mice, inhibited AKT activation, decreased phosphorylated FoxO1 and FoxO3a, and restored the suppressed autophagic flux. Running enhances muscle strength and endurance by comprehensively promoting the expression of Myh1/2/4/7 fast and slow muscle fibers in mdx mice. ConclusionsRunning can inhibit the degradation of muscle protein in mdx mice, and promote the reuse and accumulation of proteins, thereby slowing down muscle loss. Running improves skeletal muscle mass by activating autophagic flux and inhibiting ubiquitination degradation in mdx mice.

In-ovo feeding with creatine monohydrate: implications for chicken energy reserves and breast muscle development during the pre-post hatching period.

The most dynamic period throughout the lifespan of broiler chickens is the pre-post-hatching period, entailing profound effects on their energy status, survival rate, body weight, and muscle growth. Given the significance of this pivotal period, we evaluated the effect of in-ovo feeding (IOF) with creatine monohydrate on late-term embryos' and hatchlings' energy reserves and post-hatch breast muscle development. The results demonstrate that IOF with creatine elevates the levels of high-energy-value molecules (creatine and glycogen) in the liver, breast muscle and yolk sac tissues 48h post IOF, on embryonic day 19 (p < 0.03). Despite this evidence, using a novel automated image analysis tool on day 14 post-hatch, we found a significantly higher number of myofibers with lower diameter and area in the IOF creatine group compared to the control and IOF NaCl groups (p < 0.004). Gene expression analysis, at hatch, revealed that IOF creatine group had significantly higher expression levels of myogenin (MYOG) and insulin-like growth factor 1 (IGF1), related to differentiation of myogenic cells (p < 0.01), and lower expression of myogenic differentiation protein 1 (MyoD), related to their proliferation (p < 0.04). These results imply a possible effect of IOF with creatine on breast muscle development through differential expression of genes involved in myogenic proliferation and differentiation. The findings provide valuable insights into the potential of pre-hatch enrichment with creatine in modulating post-hatch muscle growth and development.

BIO101 stimulates myoblast differentiation and improves muscle function in adult and old mice.

Muscle aging is associated with a consistent decrease in the ability of muscle tissue to regenerate following intrinsic muscle degradation, injury or overuse. Age-related imbalance of protein synthesis and degradation, mainly regulated by AKT/mTOR pathway, leads to progressive loss of muscle mass. Maintenance of anabolic and regenerative capacities of skeletal muscles may be regarded as a therapeutic option for sarcopenia and other muscle wasting diseases. Our previous studies have demonstrated that BIO101, a pharmaceutical grade 20-hydroxyecdysone, increases protein synthesis through the activation of MAS receptor involved in the protective arm of renin-angiotensin-aldosterone system. The purpose of the present study was to assess the anabolic and pro-differentiating properties of BIO101 on C2C12 muscle cells in vitro and to investigate its effects on adult and old mice models in vivo. The effects of BIO101 on C2C12 differentiation were assessed using myogenic transcription factors and protein expression of major kinases of AKT/mTOR pathway by Western blot. The in vivo effects of BIO101 have been investigated in BIO101 orally-treated (50mg/kg/day) adult mice (3months) for 28days. To demonstrate potential beneficial effect of BIO101 treatment in a sarcopenic mouse model, we use orally treated 22-month-old C57Bl6/J mice, for 14weeks with vehicle or BIO101. Mice body and muscle weight were recorded. Physical performances were assessed using running capacity and muscle contractility tests. Anabolic properties of BIO101 were confirmed by the rapid activation of AKT/mTOR, leading to an increase of C2C12 myotubes diameters (+26%, P<0.001). Pro-differentiating effects of BIO101 on C2C12 myoblasts were revealed by increased expression of muscle-specific differentiation transcription factors (MyoD, myogenin), resulting in increased fusion index and number of nuclei per myotube (+39% and +53%, respectively, at day 6). These effects of BIO101 were like those of angiotensin (1-7) and were abolished with the use of A779, a MAS receptor specific antagonist. Chronic BIO101 oral treatment induced AKT/mTOR activation and anabolic effects accompanied with improved physical performances in adult and old animals (maximal running distance and maximal running velocity). Our data suggest beneficial anabolic and pro-differentiating effects of BIO101 rendering BIO101 a potent drug candidate for treating sarcopenia and possibly other muscle wasting disorders.

Moracin E and M isolated from Morus alba Linné induced the skeletal muscle cell proliferation via PI3K-Akt-mTOR signaling pathway

Twigs of Morus alba have been used in traditional medicine to treat muscle-related symptoms such as aches, numbness, and stiffness. Despite its clinical use in traditional medicine, its active compounds and mode of action have not yet been investigated. Therefore, we aimed to isolate the compounds from the twigs of M. alba and deduce active compounds, key gene targets, and mechanism of action against sarcopenia using network pharmacology analysis. Using various isolation techniques and spectroscopic methods, 43 phytochemicals, including 3 new flavonoids, were isolated and performed network pharmacology analysis. According to the computational-assistant analysis, 28 compounds, 9 genes, and the PI3K-Akt-mTOR signaling pathway were deduced as expected active compounds (EAC), key targets, and the main signaling pathway. To verify the predicted results, the cell proliferation activities of the EAC were evaluated. Especially, moracin E and M significantly increased by 130% (p < 0.001) and 57% (p < 0.05), respectively, which have more than 2- and 1.5-fold stronger effects compared to the control. Furthermore, both increased the expression level of proteins involved in the PI3K-Akt-mTOR signaling pathway and myogenic proteins, including myogenin and MyoD. This study demonstrated that moracin E and M exhibit cell proliferative effects on skeletal muscle cells through the PI3K-Akt-mTOR signaling pathway.

Culture Method and Regulation Mechanisms of Stages of C2C12 Cell Line Myogenesis

INTRODUCTION: C2C12 is an immortalized mouse myoblast cell line actively used as in vitro experimental models to study skeletal muscle metabolism in biomedical research. C2C12 cells can differentiate into myocytes under appropriate culture conditions. It is important to understand the specifics of each stage of myogenesis and its regulatory mechanisms for the targeted exposure and development of medical drugs, taking into account the mechanisms of pathogenesis of various diseases.&#x0D; AIM: To describe the method of culturing С2С12 cell line and to study regulatory mechanisms of С2С12 cell line myogenesis.&#x0D; MATERIALS AND METHODS: The study was conducted on C2C12 cell line. Differentiation of myoblasts was induced in the nutrient medium containing 2% horse serum. The differentiation stages were evaluated by studying the cells on the 1st, 4th and 7th days. Cells before differentiation were used as a comparison group. At each differentiation stage, the myoblast fusion index (IM) was evaluated with preliminary staining of the cells by Romanowsky–Giemsa method. The cell differentiation mechanism was evaluated by the level of myosin, a-actin, myogenic differentiation protein (MyoD), myogenin (MyoG) using Western blot method.&#x0D; RESULTS: On the 1st day of C2C12 cells culturing, formation of myotubes was observed (IM = 0.15 ± 0.05), on the 4th day — fusion of myoblasts with the formation of binucleated cells accompanied by an increase in the amount of MyoD and α-actin protein (IM = 0.44 ± 0.14). By the 7th day of differentiation, the fusion of cells increased with the formation of myotubes containing more than two nuclei; the content of MyoD and α-actin did not differ from the control, and the amount of MyoG and myosin increased (IM = 0.77 ± 0.04).&#x0D; CONCLUSION: The described method of culturing C2C12 cell line in Dulbecco’s Modified Eagle’s Medium with a high glucose content (4500 mg/l), containing 2% horse serum, L-glutamine (4 mМ), 100 Un/ml and 100 µg/ml of penicillin and streptomycin, is suitable for the formation of muscle cells, where MyoD and MyoG participate in the regulation of the increase in the amount of specific muscle proteins — α-actin and myosin.

Effect of 20-hydroxyecdysone and its metabolites in the absence or presence of IGF-1 on regulation of skeletal muscle cell growth

Purpose: To investigate the effect of 20-hydroxyecdysone (20E) and its metabolites and their synergistic effect with IGF-1 on regulation of skeletal muscle cell growth.&#x0D; Methods: Mouse skeletal muscle cell line (C2C12) was solely treated with 20E and its metabolites (14- deoxy- 20-hydroxyecdysone, poststerone, and 14-deoxypoststerone) at doses of 0.1, 1, and 10 µM or co-treated with IGF-1 (10 ng/ml). Cell viability and proliferative capacity were evaluated using MTT and BrdU incorporation assays, respectively. Myogenic differentiation proteins [embryonic myosin heavy chain (EbMHC) and MHC], androgen receptor (AR), and IGF-1 receptor (IGF-1R) protein expression were investigated using immunocytochemistry.&#x0D; Results: Treatments of 20E and its metabolites had no toxicity on skeletal muscle cells or induced AR/IGF-1R expression. In addition, solely treatment of 20E and its metabolites or co-treatment with IGF-1 had no significant effect on cell proliferation and myogenic differentiation capacity. In contrast, IGF-1 treatment alone significantly increased EbMHC expression (p&lt;0.0001), MHC expression (p&lt;0.05), and myotube number (p&lt;0.05).&#x0D; Conclusion: These results indicate that 20E and its metabolites have no direct or synergistic effect with IGF-1 on skeletal muscle cell growth. Nevertheless, the pharmacological effects of 20E on skeletal muscle mass and strength in vivo that raises its therapeutic potential may associate with its indirect action.

A synergistic blend of Garcinia mangostana fruit rind and Cinnamomum tamala leaf extracts enhances myogenic differentiation and mitochondrial biogenesis in vitro and muscle growth and strength in mice.

A proprietary combination of Garcinia mangostana fruit rind and Cinnamomum tamala leaf extracts (LI80020F4, CinDura®) improved the physical performance and muscle strength of resistance-trained adult males. This study assessed the underlying mechanisms of the ergogenic potential of LI80020F4 in in vitro and in vivo models. The individual extracts and their combination (LI80020F4) were assessed for nitrite production in EAhy926 human endothelial cells. Subsequent experiments evaluated the effect of LI80020F4 in myotube formation in C2C12 mouse myoblasts, expression of mammalian target of rapamycin (mTOR) signaling proteins, myogenic factors, and mitochondrial functions in L6 rat myoblasts.Moreover, adult male ICR mice were randomly assigned (n = 15) into vehicle control (G1), exercise alone (G2), oxymetholone-16 mg/kg body weight (bw) (G3), and 75 (G4)-, 150 (G5)-, or 300 (G6) mg/kg bw of LI80020F4, orally gavaged for 28 days. G1 and G2 mice received 0.5% carboxymethylcellulose sodium. Following completion, muscle strength and physical performance were assessed on forelimb grip strength and forced swimming test (FST), respectively. Gastrocnemius (GA), tibialis anterior (TA) muscle weights, muscle fiber cross-sectional area (CSA), levels of muscle, and serum protein markers were also determined. LI80020F4 increased nitrite production in EAhy926 cells in a dose-dependent manner. LI80020F4 induced C2C12 myotube formation, increased mitochondrial biogenesis, upregulated the expressions of activated mTOR and other mitochondria and myogenic proteins, and mitigated H2O2-induced mitochondrial membrane depolarization in the myoblast cells. In the animal study, 75, 150, and 300 mg/kg bw LI80020F4 doses significantly (P < 0.05) increased the animals' forelimb grip strength. Mid- and high-dose groups showed increased swimming time, increased muscle weight, CSA, muscle growth-related, and mitochondrial protein expressions in the GA muscles. LI80020F4 increases nitric oxide production in the endothelial cells, mitochondrial biogenesis and function, upregulates skeletal muscle growth-related protein expressions and reduces oxidative stress; together, it explains the basis of the ergogenic potential of LI80020F4.

Klotho regulates the myogenic response of muscle to mechanical loading and exercise.

What is the central question of this study? Does the hormone Klotho affect the myogenic response of muscle cells to mechanical loading or exercise? What is the main finding and its importance? Klotho prevents direct, mechanical activation of genes that regulate muscle differentiation, including genes that encode the myogenic regulatory factor myogenin and proteins in the canonical Wnt signalling pathway. Similarly, elevated levels of klotho expression in vivo prevent the exercise-induced increase in myogenin-expressing cells and reduce exercise-induced activation of the Wnt pathway. These findings demonstrate a new mechanism through which the responses of muscle to the mechanical environment are regulated. Muscle growth is influenced by changes in the mechanical environment that affect the expression of genes that regulate myogenesis. We tested whether the hormone Klotho could influence the response of muscle to mechanical loading. Applying mechanical loads to myoblasts in vitro increased RNA encoding transcription factors that are expressed in activated myoblasts (Myod) and in myogenic cells that have initiated terminal differentiation (Myog). However, application of Klotho to myoblasts prevented the loading-induced activation of Myog without affecting loading-induced activation of Myod. This indicates that elevated Klotho inhibits mechanically-induced differentiation of myogenic cells. Elevated Klotho also reduced the transcription of genes encoding proteins involved in the canonical Wnt pathway or their target genes (Wnt9a, Wnt10a, Ccnd1). Because the canonical Wnt pathway promotes differentiation of myogenic cells, these findings indicate that Klotho inhibits the differentiation of myogenic cells experiencing mechanical loading. We then tested whether these effects of Klotho occurred in muscles of mice experiencing high-intensity interval training (HIIT) by comparing wild-type mice and klotho transgenic mice. The expression of a klotho transgene combined with HIIT synergized to tremendously elevate numbers of Pax7+ satellite cells and activated MyoD+ cells. However, transgene expression prevented the increase in myogenin+ cells caused by HIIT in wild-type mice. Furthermore, transgene expression diminished the HIIT-induced activation of the canonical Wnt pathway in Pax7+ satellite cells. Collectively, these findings show that Klotho inhibits loading- or exercise-induced activation of muscle differentiation and indicate a new mechanism through which the responses of muscle to the mechanical environment are regulated.

Establishment and characterization of a skeletal myoblast cell line of grass carp (Ctenopharyngodon idellus).

Skeletal muscle myoblastic cell lines can provide a valuable new in vitro model for the exploration of the mechanisms that control skeletal muscle development and its associated molecular regulation. In this study, the skeletal muscle tissues of grass carp were digested with trypsin and collagenase I to obtain the primary myoblast cell culture. Myoblast cells were obtained by differential adherence purification and further analyzed by cryopreservation and resuscitation, chromosome analysis, immunohistochemistry, and immunofluorescence. A continuous grass carp myoblast cell line (named CIM) was established from grass carp (Ctenopharyngodon idellus) muscle and has been subcultured > 100 passages in a year and more. The CIM cells revived at 79.78-95.06% viability after 1-6 months of cryopreservation, and shared a population doubling time of 27.24 h. The number of modal chromosomes of CIM cells was 48, and the mitochondrial 12S rRNA sequence of the CIM cell line shared 99% identity with those of grass carp registered in GenBank. No microorganisms (bacteria, fungi, or mycoplasma) were detected during the whole study. The cell type of CIM cells was proven to be myoblast by immunohistochemistry of specific myogenic protein markers, including CD34, desmin, MyoD, and MyHC, as well as relative expression of key genes. And the myogenic rate and fusion index of this cell line after 10 days of induced differentiation were 8.96 ~ 9.42% and 3-24%, respectively. The telomerase activity and transfection efficiency of CIM cell line were 0.027 IU/mgprot and 23 ~ 24%, respectively. These results suggest that a myoblast cell line named CIM with normal biological function has been successfully established, which may provide a valuable tool for related in vitro studies.

Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder (Paralichthys olivaceus): Isolation, Culture Conditions, and Myogenic Protein Expression.

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl2 and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.

Comparative evaluation of soybean meal vs. extruded soybean meal as a replacer for fishmeal in diets of olive flounder (Paralichthys olivaceus): Effects on growth performance and muscle quality

A 56-day feeding trial was performed to compare the impacts of replacement of dietary fishmeal by soybean meal (SM) or extruded soybean meal (ESM) on the growth performance and muscle quality of olive flounder (Paralichthys olivaceus) (initial weight: 6.32 ± 0.01 g). Nine isonitrogenous and isolipidic diets were prepared by substituting 0% (FM, control), 12% (SM12/ESM12), 24% (SM24/ESM24), 36% (SM36/ESM36) and 48% (SM48/ESM48) of fishmeal with SM or ESM respectively. Results indicated that contents of anti-nutritional factors (trypsin inhibitors, glycinin and β-conglycinin) in ESM were significantly lower than those in SM. The weight gain rate, specific growth rate, feed efficiency ratio, protein efficiency ratio, activities of intestinal trypsin and lipase, as well as expressions of myogenic regulatory factors (myf5, myod and mrf4) and protein synthesis-related genes (akt, pi3k, tor and s6) in fish muscle decreased linearly and quadratically with increasing replacement levels of dietary fishmeal by SM or ESM. Expressions of mstn and protein degradation-related genes (foxo1, murf-1, mafbx, capn2 and ctsl) in muscle showed a trend opposite to growth parameters responding to dietary SM or ESM levels. Significant differences in all the above parameters were observed in the SM36, SM48 and ESM48 groups, accompanied by the declines in contents of most muscle free amino acids. The increase of dietary fishmeal replacement level by SM linearly and quadratically decreased muscle hardness, water holding capacity (WHC), pH and total collagen content. Markedly lower values were found in the SM36 and SM48 groups. However, ESM could replace up to 36% of dietary fishmeal without significantly impacts on above muscle quality parameters. Additionally, reduced myofiber density and sarcomere length were observed in the SM36, SM48, ESM36 and ESM48 groups. However, at the same substitution level, dietary ESM relieved the effects of SM on myofiber morphology to some extent. In conclusion, according to broken-line regression on SGR, the optimal replacing level of fishmeal by SM and ESM for olive flounder was 22.07% and 34.39% respectively. The improved replacement level of fishmeal by ESM, in terms of fish growth, muscle hardness and WHC, may be due to the fact that dietary ESM alleviated the adverse impacts of SM on digestive enzyme activity, muscle growth-related genes expression, muscle pH, collagen content and myofiber morphology of fish.

Human adipose-derived mesenchymal stem cells laden in gellan gum spongy-like hydrogels for volumetric muscle loss treatment

Background: volumetric muscle loss (VML) is a traumatic massive loss of muscular tissue which frequently leads to amputation, limb loss, or lifetime disability. The current medical intervention is limited to autologous tissue transfer, which usually leads to non-functional tissue recovery. Tissue engineering holds a huge promise for functional recovery. Methods: in this work, we evaluated the potential of human adipose-derived mesenchymal stem cells (hASCs) pre-cultured in gellan gum based spongy-like hydrogels (SLHs). Results: in vitro, hASCs were spreading, proliferating, and releasing growth factors and cytokines (i.e. fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor 1, interleukin-6 (IL-6), IL-8, IL-10, vascular endothelial growth factor) important for muscular regeneration. After implantation into a volumetric muscle loss (VML) mouse model, implants were degrading overtime, entirely integrating into the host between 4 and 8 weeks. In both SLH and SLH + hASCs defects, infiltrated cells were observed inside constructs associated with matrix deposition. Also, minimal collagen deposition was marginally observed around the constructs along both time-points. Neovascularization (CD31+ vessels) and neoinnervation (β-III tubulin+ bundles) were significantly detected in the SLH + hASCs group, in relation to the SHAM (empty lesion). A higher density of α-SA+ and MYH7+ cells were found in the injury site among all different experimental groups, at both time-points, in relation to the SHAM. The levels of α-SA, MyoD1, and myosin heavy chain proteins were moderately increased in the SLH + hASCs group after 4 weeks, and in the hASCs group after 8 weeks, in relation to the SHAM. Conclusions: taken together, defects treated with hASCs-laden SLH promoted angiogenesis, neoinnervation, and the expression of myogenic proteins.

Comparative muscle transcriptome of Mali and Hampshire breeds of pigs: a preliminary study

Muscle development is an important priority of pig breeding programs. There is a considerable variation in muscularity between the breeds, but the regulation mechanisms of genes underlying myogenesis are still unclear. Transcriptome data from two breeds of pigs with divergent muscularity (Mali and Hampshire) were integrated with histology, immunofluorescence and meat yield to identify differences in myogenesis during the early growth phase. The muscle transcriptomics analysis revealed 17,721 common, 1413 and 1115 unique transcripts to Hampshire and Mali, respectively. This study identified 908 differentially expressed genes (p < 0.05; log2FC > ±1) in the muscle samples, of which 550 were upregulated and 358 were downregulated in Hampshire pigs, indicating differences in physiological process related to muscle function and development. Expression of genes related to myoblast fusion (MYMK), skeletal muscle satellite cell proliferation (ANGPT1, CDON) and growth factors (HGF, IGF1, IGF2) were higher in Hampshire than Mali, even though transcript levels of several other myogenesis-related genes (MYF6, MYOG, MSTN) were similar. The number of fibers per fascicle and the expression of myogenic marker proteins (MYOD1, MYOG and PAX7) were more in Hampshire as compared to Mali breed of pig, supporting results of transcriptome studies. The results suggest that differences in muscularity between breeds could be related to the regulation of myoblast fusion and myogenic activities. The present study will help to identify genes that could be explored for their utility in the selection of animals with different muscularities.

Programmable scaffolds with aligned porous structures for cell cultured meat

Porous scaffolds for cell cultured meat are currently limited in the food-grade material requirements, the cell adhesion, proliferation, and differentiation capacities, and the ignored appearance design. We proposed programmable scaffolds specially tailored for cell cultured meat. The scaffold with aligned porous structures was fabricated with the ice-templated directional freeze-drying of the food-grade collagen hydrogel. Due to the abundant tripeptide presence and well-aligned porous structures, the scaffold could not only provide sites for cell adhesion and proliferation, but also promote the oriented growth and differentiation of cells. The up-regulation of myogenic related genes, synthesis of myogenic related proteins and formation of matured myotubes furtherly proved the differentiation of cells on aligned scaffold. These characteristics would facilitate the traditional meat characteristics simulation of cell cultured meat in term of texture and microstructure. Meanwhile, patterned scaffolds were achievable as well with the help of mold-assisted ice templating, which would improve the people’s interest, recognition, and acceptance of the tailored cell cultured meat. These characteristics indicate great application prospects of the proposed programmable scaffolds in cell cultured meat.

Tea polyphenols coated sodium alginate-gelatin 3D edible scaffold for cultured meat

This study aimed to use edible scaffolds as a platform for animal stem cell expansion, thus constructing block-shaped cell culture meat. The tea polyphenols (TP)-coated 3D scaffolds were constructed of sodium alginate (SA) and gelatin (Gel) with good biocompatibility and mechanical support. Initially, the physicochemical properties and mechanical properties of SA-Gel-TP scaffolds were measured, and the biocompatibility of the scaffolds was evaluated by C2C12 cells. SEM results showed that the scaffold had a porous laminar structure with TP particles attached to the surface, while FT-IR results also demonstrated the encapsulation of TP coating on the scaffold. In addition, the porosity of all scaffolds was higher than 40% and the degradation rate during the incubation cycle was less than 40% and the S2-G1-TP0.1–3 h scaffold has excellent cell adhesion and extension. Subsequently, we inoculated rabbit skeletal muscle myoblasts (RbSkMC) on the scaffold and induced differentiation. The results showed good adhesion and extension behavior of RbSkMC on S2-G1-TP0.1–3 h scaffolds with high expression of myogenic differentiation proteins and genes, and SEM results confirmed the formation of myotubes. Additionally, the adhesion rate of cells on scaffolds with TP coating was 1.5 times higher than that on scaffolds without coating, which significantly improved the cell proliferation rate and the morphology of cells with extension on the scaffolds. Furthermore, rabbit-derived cultured meat had similar appearance and textural characteristics to fresh meat. These conclusions indicate the high potential of the scaffolds with TP coating as a platform for the production of cultured meat products.

Differential histological features and myogenic protein levels in distinct muscles of d-sarcoglycan null muscular dystrophy mouse model.

Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of β-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.