Sugammadex affects GH/GHR’s signaling transduction on muscle cells by regulating the membrane-localized GHR level

Objectives Sugammadex (also known as bridion) is a modified γ-cyclodextrin, which is a reversal agent for the neuromuscular block. Growth hormone (GH) has an important biological effect on muscle, regulating muscle growth and development. In the current work, we explored the effect of Sugammadex on GH’s bioactivities. Methods Confocal laser scanning microscope (CLSM), flow cytometry, indirect immunofluorescence, Western-blot, and IP-WB were used to explore the effect of Sugammadex on GH’s bioactivities. Results We found that Sugammadex reduced the activity of GH on muscle cells, which down-regulated GH/GHR-mediated intracellular signaling pathway, such as Janus kinase 2 (JAK2) and signal transducers and activators of transcription 5 (STAT5). We further study the potential biological mechanism by which Sugammadex down-regulated GH/GHR-mediated signaling pathway, a series of related experiments were conducted, and found that Sugammadex may inhibit the proliferation of C2C12 cell via regulating the membrane-localized GHR, which may be the underlying mechanism by which Sugammadex suppressed GHR-induced signaling transduction. This work has laid the theoretical and experimental basis for further exploring the relationship between Sugammadex and GH’s activity. Conclusions In conclusion, this study laid a foundation for further study on the relationship between Sugammadex and GH’s activity.

MicroRNA-202 Induces Myoblast to Myocyte Differentiation through Targeting Rock-1

The expression patterns of microRNAs (small non-coding RNAs) are altered in many biological processes such as myogenesis. In this study, we aimed to investigate the impact of predicted miR-202, its target genes Akt2 and Rock-1 as a potential regulator of myoblast in the myocyte differentiation process using the C2C12 cell line. After confirmation of the differentiation process induced by 3% horse serum, the expression level of miRNA and its targets were evaluated. In the following, a luciferase assay was conducted to approve the effect of miRNA on its target. Our results indicated that miR-202 and Akt2 were significantly up-regulated during differentiation, while Rock-1 was downregulated. Co-transfection of miRNA with psiCHECK2-Rock-1 significantly presented that Rock-1 was directly targeted by miR-202. On the contrary, miR-202 has failed to enforce its inhibitory effect on Akt2 expression. In particular, miR-202 seems to be a regulator of muscle differentiation pathway thought targeting Rock-1.

KRAS Affects Adipogenic Differentiation by Regulating Autophagy and MAPK Activation in 3T3-L1 and C2C12 Cells

Kirsten rat sarcoma 2 viral oncogene homolog (Kras) is a proto-oncogene that encodes the small GTPase transductor protein KRAS, which has previously been found to promote cytokine secretion, cell survival, and chemotaxis. However, its effects on preadipocyte differentiation and lipid accumulation are unclear. In this study, the effects of KRAS inhibition on proliferation, autophagy, and adipogenic differentiation as well as its potential mechanisms were analyzed in the 3T3-L1 and C2C12 cell lines. The results showed that KRAS was localized mainly in the nuclei of 3T3-L1 and C2C12 cells. Inhibition of KRAS altered mammalian target of rapamycin (Mtor), proliferating cell nuclear antigen (Pcna), Myc, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein beta (C/ebp-β), diacylglycerol O-acyltransferase 1 (Dgat1), and stearoyl-coenzyme A desaturase 1 (Scd1) expression, thereby reducing cell proliferation capacity while inducing autophagy, enhancing differentiation of 3T3-L1 and C2C12 cells into mature adipocytes, and increasing adipogenesis and the capacity to store lipids. Moreover, during differentiation, KRAS inhibition reduced the levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), p38, and phosphatidylinositol 3 kinase (PI3K) activation. These results show that KRAS has unique regulatory effects on cell proliferation, autophagy, adipogenic differentiation, and lipid accumulation.

Oyster Hydrolysates Attenuate Muscle Atrophy via Regulating Protein Turnover and Mitochondria Biogenesis in C2C12 Cell and Immobilized Mice

Sarcopenia, also known as skeletal muscle atrophy, is characterized by significant loss of muscle mass and strength. Oyster (Crassostrea gigas) hydrolysates have anti-cancer, antioxidant, and anti-inflammation properties. However, the anti-sarcopenic effect of oyster hydrolysates remains uninvestigated. Therefore, we prepared two different oyster hydrolysates, namely TGPN and PNY. This study aimed to determine the anti-muscle atrophy efficacy and molecular mechanisms of TGPN and PNY on both C2C12 cell lines and mice. In vitro, the TGPN and PNY recovered the dexamethasone-induced reduction in the myotube diameters. In vivo, TGPN and PNY administration not only improved grip strength and exercise endurance, but also attenuated the loss of muscle mass and muscle fiber cross-sectional area. Mechanistically, TGPN and PNY increased the expression of protein synthesis-related protein levels via phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of the rapamycin pathway, and reduced the expression of protein degradation-related protein levels via the PI3K/Akt/forkhead box O pathway. Also, TGPN and PNY stimulated NAD-dependent deacetylase sirtuin-1(SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1,2, mitochondrial transcription factor A, along with mitochondrial DNA content via SIRT1/PGC-1α signaling. These findings suggest oyster hydrolysates could be used as a valuable natural material that inhibits skeletal muscle atrophy via regulating protein turnover and mitochondrial biogenesis.

Septin-7 is indispensable for proper skeletal muscle architecture and function

Today septins are considered as the fourth component of the cytoskeleton with the Septin-7 isoform playing a critical role in the formation of higher order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin-7 conditional knock-down mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers the organization and localization of septin filaments is revealed, and an ontogenesis-dependent expression of Septin-7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knock-out of Septin-7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin-7 is essential in proper myotube differentiation. These and the transient increase in Septin-7 expression following muscle injury demonstrate its vital contribution to muscle regeneration and development.

IUGR with catch-up growth programs impaired insulin sensitivity through LRP6/IRS-1 in male rats

Intrauterine growth restriction combined with postnatal accelerated growth (CG-IUGR) could lead to long-term detrimental metabolic outcomes characterized by insulin resistance. As an indispensable co-receptor of Wnt signaling, LRP6 plays a critical role in the susceptibility of metabolic disorders. However, whether LRP6 is involved in the metabolic programing is still unknown. We hypothesized that CG-IUGR programed impaired insulin sensitivity through the impaired LRP6-mediated Wnt signaling in skeletal muscle. A CG-IUGR rat model was employed. The transcriptional and translational alterations of the components of the Wnt and the insulin signaling in the skeletal muscle of the male CG-IUGR rats were determined. The role of LRP6 on the insulin signaling was evaluated by shRNA knockdown or Wnt3a stimulation of LRP6. Compared with controls, the male CG-IUGR rats showed an insulin-resistant phenotype, with impaired insulin signaling and decreased expression of LRP6/β-catenin in skeletal muscle. LRP6 knocked-down lead to reduced expression of the IR-β/IRS-1 in C2C12 cell line, while Wnt3a-mediated LRP6 expression increased the expression of IRS-1 and IGF-1R but not IR-β in the primary muscle cells of male CG-IUGR rats. The impaired LRP6/β-catenin/IGF-1R/IRS-1 signaling is probably one of the critical mechanisms underlying the programed impaired insulin sensitivity in male CG-IUGR.

Mori Ramulus Suppresses Hydrogen Peroxide-Induced Oxidative Damage in Murine Myoblast C2C12 Cells through Activation of AMPK

Mori Ramulus, the dried twigs of Morus alba L., has been attracting attention for its potent antioxidant activity, but its role in muscle cells has not yet been elucidated. The purpose of this study was to evaluate the protective effect of aqueous extracts of Mori Ramulus (AEMR) against oxidative stress caused by hydrogen peroxide (H2O2) in C2C12 mouse myoblasts, and in dexamethasone (DEX)-induced muscle atrophied models. Our results showed that AEMR rescued H2O2-induced cell viability loss and the collapse of the mitochondria membrane potential. AEMR was also able to activate AMP-activated protein kinase (AMPK) in H2O2-treated C2C12 cells, whereas compound C, a pharmacological inhibitor of AMPK, blocked the protective effects of AEMR. In addition, H2O2-triggered DNA damage was markedly attenuated in the presence of AEMR, which was associated with the inhibition of reactive oxygen species (ROS) generation. Further studies showed that AEMR inhibited cytochrome c release from mitochondria into the cytoplasm, and Bcl-2 suppression and Bax activation induced by H2O2. Furthermore, AEMR diminished H2O2-induced activation of caspase-3, which was associated with the ability of AEMR to block the degradation of poly (ADP-ribose) polymerase, thereby attenuating H2O2-induced apoptosis. However, compound C greatly abolished the protective effect of AEMR against H2O2-induced C2C12 cell apoptosis, including the restoration of mitochondrial dysfunction. Taken together, these results demonstrate that AEMR could protect C2C12 myoblasts from oxidative damage by maintaining mitochondrial function while eliminating ROS, at least with activation of the AMPK signaling pathway. In addition, oral administration of AEMR alleviated gastrocnemius and soleus muscle loss in DEX-induced muscle atrophied rats. Our findings support that AEMR might be a promising therapeutic candidate for treating oxidative stress-mediated myoblast injury and muscle atrophy.

Effects of Geniposide and Geniposidic Acid on Fluoxetine-Induced Muscle Atrophy in C2C12 Cells

Fluoxetine, an antidepressant known as a selective 5-hydroxytryptamine reuptake inhibitor (SSRI), can cause side effects such as muscle atrophy with long-term use, but the mechanism is not fully understood. Geniposide (GPS) and geniposidic acid (GPSA), the main components of Gardenia jasminoides fruit, have been shown to have biological activity in disease prevention, but their role in preventing FXT-related side effects such as muscle atrophy remains unclear. The process of muscle atrophy is a complex physiological mechanism involving the balance of protein synthesis and catabolism. In this study, we hypothesized that FXT may suppress hypertrophy signaling and activate the atrophy mechanisms, resulting in proteolysis and reduced protein synthesis, while geniposide (GPS) and geniposide acid (GPSA) may be beneficial in improving muscle weakness caused by FXT. The C2C12 cell model was used to examine the expression of hypertrophy signaling (PI3K, Akt, and mTOR) and protein break signals (FOXO, MuRF-1, and MyHC). Our data indicated that FXT inhibited MyHC and promoted MuRF-1 protein expression by downregulating the signaling pathways of p-ERK1/2, p-Akt, p-mTOR, and p-FOXO, resulting in a decrease in differentiation and myotube formation in C2C12 muscle cells, which further resulted in muscle atrophy. However, GPS and GPSA can positively regulate the atrophy mechanism induced by FXT in muscle cells, thereby ameliorating the imbalance in muscle synthesis. In conclusion, GPS and GPSA have the potential to attenuate the muscle loss caused by long-term FXT administration, diseases, or the aging process.

Srpk3 Decrease Associated with Alpha-Synuclein Increase in Muscles of MPTP-Induced Parkinson’s Disease Mice

Parkinson’s disease (PD) is characterized by a loss of dopaminergic cells in the substantia nigra, and its histopathological features include the presence of fibrillar aggregates of α-synuclein (α-syn), which are called Lewy bodies and Lewy neurites. Lewy pathology has been identified not only in the brain but also in various tissues, including muscles. This study aimed to investigate the link between serine/arginine-rich protein specific kinase 3 (srpk3) and α-syn in muscles in PD. We conducted experiments on the quadriceps femoris of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and the C2C12 cell line after treatment with 1-methyl-4-phenylpyridinium (MPP+) and srpk3 short interfering RNA (siRNA). Compared to the control group, the MPTP group showed significantly reduced expression of srpk3, but increased expression of α-syn. In MPP+-treated C2C12 cells, srpk3 expression gradually decreased and α-syn expression increased with the increasing MPP+ concentration. Moreover, experiments in C2C12 cells using srpk3 siRNA showed increased expressions of α-syn and phosphorylated α-syn. Our results showed that srpk3 expression could be altered by MPTP intoxication in muscles, and this change may be related to changes in α-syn expression. Furthermore, this study could contribute to advancement of research on the mechanism by which srpk3 plays a role in PD.