Myoblast Cells Research Articles

Abstract 15451: Regulations of Nrf2 by Nf-κB in Ischemic Heart Failure

Introduction: Oxidative stress is an important pathophysiological factor in the development and progression of ischemic heart failure (IHF). Nuclear factor E2-related factor 2 (Nrf2) is a major regulator of the protective antioxidant response and has been found to decrease by 50% in IHF. However, the mechanism of Nrf2 downregulation remains incompletely understood. NF-kB is a critical transcription factor that influences many cellular processes and is activated in IHF with increased reactive oxygen species (ROS). We hypothesize that activation of NF-kB may underlie the decreased Nrf2 activity in IHF. Methods and Results: To induce IHF, we performed left anterior descending coronary artery (LAD) ligation surgery in Sprague Dawley rats and FVB/NJ mice. We found a significant reduction in cardiac function after LAD surgery in ischemia/reperfusion (I/R) mice relative to sham-operated mice using echocardiography. Tissue sample analyses reveal a crucial role of NF-kB activation in the regulation of Nrf2 in IHF. Additionally, after 2 weeks of I/R, we found that rats treated with Nrf2 overexpressed lentivirus demonstrated greater pressure development than rats with vehicle treatment. Using rat cardiac myoblast cells (H9C2) and western blot analysis, we tested the expression of cytosolic and nuclear Nrf2 and p65 (a component of NF-kB) following time- and dose-dependent treatments of H2O2. We found that p65 activation inhibited Nrf2 activity, while inhibitions of p65 nuclear translocation prevented the decrease in Nrf2 nuclear activities. We then generated a knockout of p65 in H9C2 cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9 technology to test NF-κB regulation on the Nrf2 activities, which further strengthened our previous findings. Conclusions: Taken together, our data support the critical role of NF-kB activation in the regulation of Nrf2 in IHF, providing new insights into possible molecular targets for the treatment of IHF through the regulation of the protective antioxidant response.

Pharmacological Evaluation of Anti-Diabetic Activity of Myricetin in Different Cell Lines

The aim of this research work was to perform pharmacological evaluation of Myricetin by using different cell lines. To check the short-term cytotoxic effect of Myricetin in HepG2 and L6 cells, a colorimetric based MTT assay was performed also the glucose utilization in both HepG2 and L6 cells was estimated in order to measure the in vitro anti-diabetic activity of Myricetin. For Lipid accumulation T3-L1 preadipocytes cells are used. For measurement of nitric oxide (NO) the RAW-264.7 cells are used. The alpha-amylase assay and alpha-glucosidase inhibition assay was performed to check if our drug is having effect in term of inhibiting α-amylase and α-glucosidase respectively. Western blot analysis was performed to check the expressions of iNOS protein in LPS treated RAW 264.7 cells before and after the exposure of Myricetin. The cytotoxicity data suggested that Myricetin displayed low level of cytotoxicity in HepG2 cells and in L6 myoblast cell lines. For glucose utilization experiment, it discovered that glucose uptake level was notably increase in HepG2 cells and in L6 myoblast cell lines with the increasing concentration of Myricetin. Myricetin increase lipid accumulation in 3T3-L1 preadipocytes and also inhibits nitric oxide production in RAW 264.7 cell lines. Research was found that Myricetin had no significant effect in order to alter the expression of alpha-amylase and alpha-glucosidase. Our research concludes that Myricetin can be used as a potent anti-diabetic agent.

Comprehensive metabolite and biological profile of “Sulmona Red Garlic” ecotype’s aerial bulbils

“Sulmona Red Garlic” is a well-known Italian traditional product. Bulbs, used for culinary purposes, have been largely investigated for their medicinal properties whereas aerial bulbils are usually removed as waste material. Here, for the first time, chemical composition and biological properties of the hydroalcoholic extract from aerial bulbils were investigated. Complementary information on metabolite composition were obtained using both NMR based untargeted and HPLC-DAD targeted methodologies. The NMR analysis revealed the presence of sugars, organic acids, amino acids, organosulphur compounds (methiin, alliin, allicin and cycloalliin), and other secondary metabolites. In particular, methiin and alliin were identified for the first time in the NMR spectra of aerial bulbil garlic extracts. Polyphenol content was determined by HPLC-DAD analysis: catechin, chlorogenic acid, and gallic acid turned out to be the most abundant phenolics.Hydroalcoholic extract blocked cell proliferation of colon cancer cell line HCT116 with an IC50 of 352.07 µg/mL, while it was non-toxic to myoblast cell line C2C12. In addition, it caused seedling germination reduction of two edible and herbaceous dicotyledon species, namely Cichorium intybus and C. endivia. Moreover, the same extract reduced the gene expression of TNF-α (tumor necrosis factor), HIF1-α (hypoxia-inducible factor), VEGFA (vascular endothelial growth factor), and transient receptor potential (TRP) M8 (TRPM8) indicating the ability to contrast cancer development through the angiogenic pathway. Final, in silico experiments were also carried out supporting the biological effects of organosulphur compounds, particularly alliin, which may directly interact with TRPM8.The results here reported suggest the potential use of garlic aerial bulbils often considered a waste product as a source in phytotherapeutic remedies.

Roles of Fatty Acid Chain Length and Enzyme-Oriented Drug Controlled Release from pH-Triggering Self-Assembled Fatty Acid Conjugated Quetiapine Nanosuspensions.

Quetiapine (QTP) is a first-line antipsychotic drug, but its therapeutic druggability and patient adherence were limited due to high oral dose strength, low bioavailability and physicochemical/biopharmaceutical issues. To investigate the roles of fatty acid chain length and enzyme-oriented QTP controlled release from pH-triggering self-assembled fatty acid conjugated QTP nanosuspensions (NSPs). QTP was conjugated with different chain length fatty acids (C10-decanoic acid, C14-myristic acid, C18-stearic acid) to obtain QTP-fatty acid conjugates (QFCs: QD, QM, QS) by exploiting 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/4-dimethylaminopyridine (EDC/DMAP) conjugation chemistry. Then, the solubility, partitioning coefficient (log P), cell viability and cleavage kinetics of QFCs were evaluated. The pH-triggering self-assembled behaviors of QFCs to form QTP-fatty acid NSPs (QDN, QMN, QSN) by varying pH, QFC concentration and proton-to-QTP ratios were characterized. The morphological images, critical micelle concentration (CMC), physicochemical properties and enzyme-oriented QTP controlled release of NSPs were examined. Three QFCs were synthesized with different chain length fatty acids from QTP after desalting fumarate from QTP fumarate. The pH, QFC concentration and proton-to-quetiapine molar ratio could influence physicochemical properties and nanonization behaviors of QFCs. All three QFCs showed no effect on the viability of myoblast cells. The pH-triggering self-assembly of amphiphilic QFCs to form nanoparticles (NPs) occurred as the amine moiety of QTP was readily ionized in a strongly acidic environment (pH 1.2). Interestingly, the longer the fatty acid chain length, the lower water solubility, the higher log P (lipophilicity) and the smaller NP particle size were observed. The conversion rate of QFCs to liberate QTP by esterase in human plasma and liver S9 fractions was also inversely proportional to the fatty acid carbon chain length. Interestingly, the freeze-dried QMN showed the esterase-oriented controlled release of QTP over one month, unlike the initial burst release of QDN or the slowly delayed release pattern of QSN. A new pH-triggering self-assembled nanonization platform was developed using different chain length fatty acid conjugated QTP in low pH environment. By varying fatty acid chain length, the enzyme-oriented QTP controlled release dosage form was challenged to enhance the therapeutic effectiveness of QTP.

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.

Elucidating the Pharmacological Properties of Zingiber officinale Roscoe (Ginger) on Muscle Ageing by Untargeted Metabolomic Profiling of Human Myoblasts.

(1) Background: Muscle loss is associated with frailty and a reduction in physical strength and performance, which is caused by increased oxidative stress. Ginger (Zingiber officinale Roscoe) is a potential herb that can be used to reduce the level of oxidative stress. This study aimed to determine the effect of ginger on the expression of metabolites and their metabolic pathways in the myoblast cells to elucidate the mechanism involved and its pharmacological properties in promoting myoblast differentiation. (2) Methods: The myoblast cells were cultured into three stages (young, pre-senescent and senescent). At each stage, the myoblasts were treated with different concentrations of ginger extract. Then, metabolomic analysis was performed using liquid chromatography-tandem mass spectrometry (LCMS/MS). (3) Results: Nine metabolites were decreased in both the pre-senescent and senescent control groups as compared to the young control group. For the young ginger-treated group, 8-shogaol and valine were upregulated, whereas adipic acid and bis (4-ethyl benzylidene) sorbitol were decreased. In the pre-senescent ginger-treated group, the niacinamide was upregulated, while carnitine and creatine were downregulated. Ginger treatment in the senescent group caused a significant upregulation in 8-shogaol, octadecanamide and uracil. (4) Conclusions: Ginger extract has the potential as a pharmacological agent to reduce muscle loss in skeletal muscle by triggering changes in some metabolites and their pathways that could promote muscle regeneration in ageing.

Vitisin A-13-O-β-D-glucoside and Vitisin A from Iris lactea inhibit lipogenesis and promote lipolysis via the PKA/HSL pathway during adipogenic transdifferentiation of C2C12 cells

Oligostilbenes are a group of natural products derived from the polymerization of stilbene monomers. Despite the demonstration of their activities in regulating lipid metabolism, the function of oligostilbenes in the adipogenic transdifferentiation of multipotent myoblast cells remains unknown. Hence, the five oligostilbenes from Iris lactea were tested for their regulatory effects on adipogenic transdifferentiation of C2C12 myoblast cells. As a result, it was shown that Vitisin A-13-O-β-D-glucoside (VitAOG), Vitisin A (VitA) and Hopeaphenol (Hop) can greatly inhibit the adipogenic transdifferentiation of C2C12 cells by reducing lipid accumulation and downregulating the expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein beta (C/EBPβ) and fatty acid binding proteins 4 (FABP4). In contrast, Vitisin D (VitD) and Isohopeaphenol (Isohop) promote adipogenic transdifferentiation of C2C12 cells by increasing lipid accumulation and upregulating the expression of adipogenesis and lipogenesis markers. Further research found that the lipolytic protein levels of adipocyte triglyceride lipase (ATGL) and phosphorylation of hormone-sensitive lipase (HSL) were elevated by VitAOG and VitA. Additionally, VitAOG and VitA maintain lipid homeostasis by improving mitochondrial function. Taken together, our study reveals an effect of oligostilbenes on lipid metabolism in C2C12 cells, and VitAOG and VitA can be regarded as potential candidates for the treatment of obesity and other disorders of lipid metabolism.

NCAPG Regulates Myogenesis in Sheep, and SNPs Located in Its Putative Promoter Region Are Associated with Growth and Development Traits.

Previously, NCAPG was identified as a candidate gene associated with sheep growth traits. This study aimed to investigate the direct role of NCAPG in regulating myogenesis in embryonic myoblast cells and to investigate the association between single-nucleotide polymorphisms (SNPs) in its promoter region and sheep growth traits. The function of NCAPG in myoblast proliferation and differentiation was detected after small interfering RNAs (siRNAs) knocked down the expression of NCAPG. Cell proliferation was detected using CCK-8 assay, EdU proliferation assay, and flow cytometry cell cycle analysis. Cell differentiation was detected via cell immunofluorescence and the quantification of myogenic regulatory factors (MRFs). SNPs in the promoter region were detected using Sanger sequencing and genotyped using the improved multiplex ligation detection reaction (iMLDR®) technique. As a result, a notable decrease (p < 0.01) in the percentage of EdU-positive cells in the siRNA-694-treated group was observed. A significant decrease (p < 0.01) in cell viability after treatment with siRNA-694 for 48 h and 72 h was detected using the CCK-8 method. The quantity of S-phase cells in the siRNA-694 treatment group was significantly decreased (p < 0.01). After interfering with NCAPG in myoblasts during induced differentiation, the relative expression levels of MRFs were markedly (p < 0.05 or p < 0.01) reduced compared with the control group on days 5-7. The myoblast differentiation in the siRNA-694 treatment group was obviously suppressed compared with the control group. SNP1, SNP2, SNP3, and SNP4 were significantly (p < 0.05) associated with all traits except body weight measured at birth and one month of age. SNP5 was significantly (p < 0.05) associated with body weight, body height, and body length in six-month-old sheep. In conclusion, interfering with NCAPG can inhibit the proliferation and differentiation of ovine embryonic myoblasts. SNPs in its promoter region can serve as potential useful markers for selecting sheep growth traits.

Trichinella spiralis-Secreted Products Promote Collagen Capsule Formation through TGF-β1/Smad3 Pathway.

Trichinella spiralis (T. spiralis) muscle larvae colonize in the host's skeletal muscle cells, which are surrounded by collagen capsules. The mechanism underlying muscle stage larva-induced collagen capsule formation remains unknown. To clarify the mechanism, a T. spiralis muscular-infected mouse model was established by a single lateral tail vein injection with 20,000 T. spiralis newborn larvae (NBL). The infected mice were treated with or without SB525334 (TGF-β1 receptor type I inhibitor). Diaphragms were obtained post-infection, and the expression levels of the TGF-β1/Smad3 pathway-related genes and collagen genes (type IV and VI) were observed during the process of collagen capsule formation. The changes in myoblasts under stimulation of the excretory-secretory (ES) products of NBL with or without SB525334 were further investigated. Results showed that the expression levels of type IV collagen gene, type VI collagen gene, Tgfb1, and Smad3 were significantly increased in infected mice muscle cells. The expression levels of all the above genes were enhanced by the products of NBL in myoblast cells. These changes were reversed by co-treatment with SB525334 in vivo and in vitro. In conclusion, the TGF-β1/Smad3 pathway can be activated by T. spiralis infection in muscle cells. The activated TGF-β1/Smad3 pathway can stimulate the secretion of collagens by myocytes and plays a promoting role in the process of collagen capsule formation. The research has the limitation that the protein identification of the products of NBL has yet to be performed. Therefore, the specific components in the T. spiralis ES products that induce collagen synthesis should be further investigated.

Dielectrophoretic Movement of Cell Passing Between Surface Electrodes in Flow Channel

Abstract In this study, cell behavior in a microchannel was tracked for the application of dielectrophoresis to biological cell sorting. A pair of titanium surface electrodes was fabricated inside the microchannel by photolithographic techniques: a triangular electrode with a tip angle of 0.26 rad and a rectangular electrode. A periodic alternating current of square wave with a period of 1 μs was introduced between the electrodes to induce an asymmetric electric field perpendicular to the mainstream direction. The behavior of mouse myoblasts (C2C12: mouse myoblast cell line) was measured in vitro while the suspension was flowing. The relationship between cell shift motion near the electrode and cell shape on the two-dimensional projection plane was investigated. Experimental results showed that cell movement in the direction perpendicular to the mainstream increased with geometries away from the circle in the two-dimensional projection plane. This method can be applied to sort cells according to their degree of shape deviation from a sphere. The dielectrophoretic effect can be applied to sort cells not only by cell size but also by cell deformation.

Myogenesis Effects of RGX365 to Improve Skeletal Muscle Atrophy.

Age-related skeletal muscle atrophy and weakness not only reduce the quality of life of those afflicted, but also worsen the prognosis of underlying diseases. We evaluated the effect of RGX365, a protopanaxatriol-type rare ginsenoside mixture, on improving skeletal muscle atrophy. We investigated the myogenic effect of RGX365 on mouse myoblast cells (C2C12) and dexamethasone (10 µM)-induced atrophy of differentiated C2C12. RGX365-treated myotube diameters and myosin heavy chain (MyHC) expression levels were analyzed using immunofluorescence. We evaluated the myogenic effects of RGX365 in aging sarcopenic mice. RGX365 increased myoblast differentiation and MyHC expression, and attenuated the muscle atrophy-inducing F-box (Atrogin-1) and muscle RING finger 1 (MuRF1) expression. Notably, one month of oral administration of RGX365 to 23-month-old sarcopenic mice improved muscle fiber size and the expression of skeletal muscle regeneration-associated molecules. In conclusion, rare ginsenosides, agonists of steroid receptors, can ameliorate skeletal muscle atrophy during long-term administration.

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.

A Convergent fabrication of silk fibroin nanoparticles on quercetin loaded metal-organic frameworks for promising nanocarrier of myocardial infraction

The biomacromolecule silk fibroin (SF) may be constructed to promote biomimetic nucleation and nanostructures of inorganic nanomaterials, offering it a promising candidate for use in various biomimetic applications. We combined SF-NPs and ZIF-8-NPs to fabricate new drug vehicles that effectively release the drug. SF nanoparticles (SF-NPs) were assembled into quercetin (QCT), a myocardial drug added to fabricate QSF-NPs. By acting as a template for the ZIF-8 nucleation onto the surface, the QSF-NPs fabricated core-shell-structured nanocomposites (named QSF@Z-NCs) with ZIF-8 as the core-shell and the QSF-NPs. The biocompatibility analysis using the MTT assay revealed that the developed QCT, SF-NPs, and QSF@Z-NCs are not harmful to cardiac myoblast (H9C2) cells. The in vivo model demonstrated that H9C2 cells encouraged cardiomyocyte fibre regeneration in myocardial infarction rats. We fabricated a brand-new technique using H9C2 cells and QSF@Z-NCs that might encourage the healing processes in myocardial ischemia cells. This study's results demonstrate that it successfully treats myocardial injury.

Organometallic N‐acylhydrazones of 5‐nitrofuran and 5‐nitrothiophene based: Synthesis, electrochemical, antiparasitic evaluation, and computational study

In searching for new therapeutic agents for treating American trypanosomiasis and Human African trypanosomiasis, four nitroheterocyclic acylhydrazones of general formulae [R1‐CH=N‐NH‐C(O)‐(5‐C4H2X)] (where R1 = ferrocenyl or cyrhetrenyl, and X = O or S) have been synthesized and characterized by spectroscopic techniques. Comparative studies of their stability by 1H‐NMR and UV–Vis experiments were reported. Single‐crystal X‐ray diffraction confirmed the molecular structures of NF‐1 and NT‐2. Their X‐ray crystal structures reveal that both adopt an E‐configuration on the C=N moiety. Regarding the ‐NH‐C(O)‐ bond, the structure of NF‐1 confirmed a trans conformation, while NT‐2 exhibited a cis‐amide conformation. The cyclic voltammetry and electron spin resonance (ESR) experiments were conducted to study the electrochemical behavior of N‐acylhydrazones. The antiparasitic activities of compounds against Trypanosoma cruzi (epimastigotes) and Trypanosoma brucei (trypomastigotes) revealed that cyrhetrenyl complexes were more effective than their ferrocenyl analogs. The cyrhetrenyl derivative NT‐2 (EC50 = 2.25 μM) showed activity against T. brucei comparable to the standard drug nifurtimox (Nfx, EC50 = 3.56 μM). The ferrocenyl compound NT‐1 (&gt;200 μM) was at least two times less cytotoxic than the Nfx (88.7 μM) against the L6 rat skeletal myoblast cell line and exhibited a selectivity like Nfx toward T. brucei. Density functional theory (DFT) calculations were utilized as an approximation to explain the impact of organometallic and heterocyclic rings on antiparasitic activities. This study supported the experimental results, confirming that the cyrhetrenyl fragment in N‐acylhydrazone derivatives plays a significant role in the antitrypanosomal activity, which can be attributed to an increase in positive charge on the metal.

B-175 Phytochemicals in K. pinnata and the Antioxidant Activity of Combined K. pinnata and Metformin Preparations on Normal and Diabetic Skeletal Muscle Cells

Abstract Background Diabetes Mellitus is associated with oxidative stress, which may play a central role in developing diabetic complications. With the rise in healthcare costs, more people globally are choosing to complement their pharmacological regimen with dietary supplements from natural products. In this study, the compound composition of K. pinnata and the effects of combined preparations of K. pinnata and metformin on antioxidant activity in human skeletal muscle myoblasts (HSMM) and human diabetic skeletal muscle myoblasts (DHSMM) were investigated. Methods K. pinnata leaves were crushed and extracted with hot water, then gravity filtered and freeze-dried. A 1 mL aliquot of the Kalanchoe pinnata extract solution was utilized to determine the chemical composition of the herbal extract. Liquid chromatography-mass spectrometry (LC-MS) was run in negative ion mode, with the K. pinnata extract analyzed in triplicate. A computer program further analyzed the LC-MS data to determine the chemical components in K. pinnata aqueous extract. A database algorithm within Compound Discoverer was used to identify the compounds in K. pinnata aqueous extract. For subsequent cell culture experiments, the K. pinnata leaf extract was weighed and combined with aqueous metformin solutions to prepare various combinatorial treatments. Human skeletal muscle myoblast cells and diabetic human skeletal muscle myoblast cells (Lonza Inc, Walkersville, MD) were cultured and incubated at 37 °C with 5% CO2. A CCK-8 cytotoxicity assay was performed to determine cellular viability, while trypan blue was used to assess the cellular proliferation of in-vitro cells. The effects of combinatorial preparations of K. pinnata and metformin on antioxidant indices were determined in HSMM, DHSMM, and HSMM H2O2 stress-induced cells. Results Liquid chromatography-mass spectrometry (LC-MS) showed that K. pinnata contains biologically active flavanols. The main compounds identified in locally grown K. pinnata were quercetin, kaempferol, apigenin, epigallocatechin gallate (EGCG), and avicularin. Antioxidant results indicated that a combinatorial preparation of K. pinnata with metformin might modulate antioxidant responses by increasing the enzymatic activity of superoxide dismutase and increasing levels of reduced glutathione. A combination of 50 μM and 150 μg/mL of metformin and K. pinnata resulted in a significant increase in reduced glutathione levels in non-diabetic and diabetic human skeletal muscle myoblasts and H2O2 stress-induced human skeletal muscle myoblasts. Additionally, a K. pinnata treatment (400 µg/mL) alone significantly increased catalase (CAT) activity for non-diabetic and diabetic human skeletal muscle myoblasts and an H2O2 stress-induced human skeletal muscle myoblast cell line while significantly lowering malondialdehyde (MDA) levels. The antioxidant activity of the treatment options was more effective at promoting cell viability after 24 h of treatment vs 72 h. Conclusion These treatment options show promise, especially after 24 h for treating oxidative stress-mediated pathophysiological complications associated with type II diabetes.

Highly selective, sensitive and biocompatible rhodamine-based isomers for Al3+ detection: A comparative study

Selective detection of a metal ion with high selectivity is of great importance to understand its existence and its role in many chemical and biological processes. We report here the synthesis, characterization and Al3+ sensing properties of two rhodamine-based isomers, (E)-2-((2-(allyloxy)benzylidene)amino)ethyl)-3′,6′-bis(ethylamine)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-one (L-2-oxy) and (E)-2-((4-(allyloxy)benzylidene)amino)ethyl)-3′,6′-bis(ethylamine)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-one (L-4-oxy). L-2-oxyand L-4-oxy show pink coloration, significant enhancement in absorbance at 530 nm and fluorescence intensity at 553 nm in the presence of Al3+ among several cations. Quantum yield and lifetime of the probes increase in the presence of Al3+. LOD values have been determined as low as ∼1.0 nM for both the isomers. DFT study suggests that the cation induces opening of spirolactam ring resulting in the changes of the rhodamine dyes. Additional reason could be Chelation Enhanced Fluorescence (CHEF) effect due to the subsequent chelation of the metal ion. Between two isomers, L-2-oxy displays better sensing ability towards Al3+ in terms of fluorescence enhancement, limit of detection, lifetime enhancement. Both the probes have been utilized in cell imaging studies using rat skeletal myoblast cell line (L6 cell line).

Cardiolipin deficiency leads to the destabilization of mitochondrial magnesium channel MRS2 in Barth syndrome.

Barth syndrome (BTHS) is a debilitating X-linked cardio-skeletal myopathy caused by loss-of-function mutations in TAFAZZIN, a cardiolipin (CL)-remodeling enzyme required for the maintenance of normal levels of CL species in mitochondrial membranes. At present, how perturbations in CL abundance and composition lead to many debilitating clinical presentations in BTHS patients have not been fully elucidated. Inspired by our recent findings that CL is essential for optimal mitochondrial calcium uptake, we measured the levels of other biologically important metal ions in BTHS mitochondria and found that in addition to calcium, magnesium levels are significantly reduced. Consistent with this observation, we report a decreased abundance of the mitochondrial magnesium influx channel MRS2 in multiple models of BTHS including yeast, murine myoblast, and BTHS patient cells and cardiac tissue. Mechanistically, we attribute reduced steady-state levels of MRS2 to its increased turnover in CL-deficient BTHS models. By expressing Mrs2 in well-characterized yeast mutants of the phospholipid biosynthetic pathways, we demonstrate a specific requirement of CL for Mrs2 abundance and assembly. Finally, we provide in vitro evidence for the direct binding of CL with human MRS2. Together, our study has identified a critical requirement of CL for MRS2 stability and suggests perturbation of mitochondrial magnesium homeostasis as a novel contributing factor to BTHS pathology.

Anti-Staphylococcal Activity of Ligilactobacillus animalis SWLA-1 and Its Supernatant against Multidrug-Resistant Staphylococcus pseudintermedius in Novel Rat Model of Acute Osteomyelitis.

Osteomyelitis caused by staphylococcal infection is a serious complication of orthopedic surgery. Staphylococcus pseudintermedius is the main causative agent of osteomyelitis in veterinary medicine. Methicillin-resistant S. pseudintermedius (MRSP) has been reported in companion animals, especially dogs. Multidrug-resistant S. pseudintermedius is an emerging pathogen and has acquired antibiotic resistance against various commercial antimicrobial agents. New antimicrobial compounds are urgently needed to address antibiotic resistance, and the development of novel agents has become an international research hotspot in recent decades. Antimicrobial compounds derived from probiotics, such as bacteriocins, are promising alternatives to classical antibiotics. In this study, the antibacterial activities of Ligilactobacillus animalis SWLA-1 and its concentrated cell-free supernatant (CCFS) were evaluated in vitro and in vivo. The CCFS of this bacterium showed no toxicity against osteoblast and myoblast cells in vitro, while significantly inhibiting the multidrug-resistant S. pseudintermedius KUVM1701GC strain in a newly established rat model. The CCFS significantly inhibited multidrug-resistant staphylococci both in vitro and in vivo. This suggests that CCFS derived from L. animalis SWLA-1 has potential as an alternative to classic antibiotics for staphylococcal infections in dogs.

Hyperglycemia may induce a decrease in myoblast viability: A Preliminary Study

Link of Video Abstract: https://youtu.be/6j-cheXQzQE Background: Hyperglycemia plays a significant role in developing insulin resistance, leading to a continuous decline in metabolism. Uncontrolled long-term hyperglycemia has deleterious effects on muscles, leading to a decline in muscle mass, impaired capillarization, and muscle atrophy. We identified the effect of hyperglycemia on reducing myoblast cell viability through observation using the MTT assay. Methods: Primary culture of myoblast cells was performed using rat's muscle tissue which was further induced by hyperglycemia by adding D-glucose with concentrations of 10 mM, 25 mM, and 50 mM in the plating media. Cell viability was observed using the MTT assay by identifying the absorbance using an ELISA reader with λ=550-600 nm (595 nm) Results: There was a significant decrease in cell viability (p=0.000) due to administering various glucose concentrations in the plating media. There is a negative correlation between glucose concentration and myoblast cell viability (P-value = 0.000; t = -8.284; r = -0.926) Conclusion: Elevated blood sugar levels result in a significant reduction in the viability of myoblast cells, as indicated by the MTT assay. The MTT assay proved invaluable for precisely evaluating myoblast cell viability in the context of hyperglycemia.

Dipyridamole activates adenosine A2B receptor and AMPK/cAMP signaling and promotes myogenic differentiation of myoblastic C2C12 cells.

Introduction: Sarcopenia is defined as a loss of muscle mass and strength. ATP homeostasis is crucial during myogenesis. We determined how the purinergic system modulates myogenesis using dipyridamole (blocks adenosine taken up by the cells) and tenofovir (inhibits ATP release) in a myoblast cell line. Methods: C2C12 cells were differentiated in the presence/absence of tenofovir/dipyridamole, with/without the A2B selective inhibitor PSB-603. Extra-/intracellular nucleotides were examined via HPLC. The expression of muscle differentiation proteins (Pax7, Mif5, MyoD, MyoG, and MHC), PKA/CREB, adenosine receptors (A1, A2A, A2B, and A3), ATP-channel pannexin-1 and the P2X7 receptor was analyzed via WB and RT-PCR. cAMP and AMPK activation was measured. Results: Tenofovir increased intracellular ATP and reduced extracellular adenosine, decreasing Pax7 expression and increasing MHC expression prematurely. Dipyridamole increased intracellular AMP and extracellular adenosine, counteracting the premature myogenesis promoted by tenofovir. All adenosine receptors were expressed during differentiation with dipyridamole, increasing A2B expression. Tenofovir maintained inactive AMPK and decreased cAMP levels, as well as PKAα and pCREB expression, which were recovered with dipyridamole. Discussion: Adenosine and ATP act as mediators in muscle myogenesis. The blockade of ATP release by tenofovir promotes premature myogenesis, with dipyridamole counteracting the premature differentiation promoted by tenofovir via the adenosine A2B receptor and cAMP/AMPK pathways. Therefore, dipyridamole might be of interest as a therapeutic approach in sarcopenia.