scholarly journals A Low-Therapeutic Dose of Lithium Inhibits GSK3 and Enhances Myoblast Fusion in C2C12 Cells

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1340 ◽  
Author(s):  
Kurgan ◽  
Whitley ◽  
Maddalena ◽  
Moradi ◽  
Stoikos ◽  
...  
Keyword(s):  
Bipolar Disorder ◽  
Therapeutic Dose ◽  
Specific Activity ◽  
Glycogen Synthase ◽  
Myogenic Differentiation ◽  
Myoblast Fusion ◽  
C2c12 Cells ◽  
Glycogen Synthase Kinase 3 ◽  
C2c12 Myotubes ◽  
Control Myotubes

: Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/β-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3β) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5–1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3β (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2–2.5 fold, p < 0.05), a result associated with an increase in total β-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (−86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions.

scholarly journals Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion

2019 ◽  
Vol 317 (5) ◽  
pp. C1025-C1033 ◽  
Author(s):  
Val A. Fajardo ◽  
Colton J. F. Watson ◽  
Kirsten N. Bott ◽  
Fereshteh Moradi ◽  
Lucas A. Maddalena ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Myogenic Differentiation ◽  
Vastus Lateralis ◽  
Critical Role ◽  
Myoblast Fusion ◽  
C2c12 Cells ◽  
C2c12 Myotubes ◽  
Mammalian Skeletal Muscle ◽  
Mrna Levels ◽  
Calcineurin Signaling

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

Mania and Depression: What's New?

2017 ◽  
Vol 41 (S1) ◽  
pp. S15-S15
Author(s):  
A. Fagiolini ◽  
G. Amodeo
Keyword(s):  
Bipolar Disorder ◽  
Atypical Antipsychotics ◽  
Unmet Needs ◽  
Glycogen Synthase ◽  
Treatment Strategies ◽  
Opiate Receptor ◽  
Glycogen Synthase Kinase 3 ◽  
Sustained Effects ◽  
Precise Understanding ◽  
Cellular Apoptosis

Despite the high burden of bipolar disorder and the noticeable progress in its treatment, the disorder still goes frequently misdiagnosed, unrecognized, or not optimally treated. To date, no medication has been specifically developed on the basis of a precise understanding of the pathophysiology of the disorder, or based on the unique characteristics of several subtypes of bipolar disorder or on the medication mechanism of action. Lithium remains on of the gold standard treatments for bipolar disorder. Its mood-stabilizing properties are thought to occur via specific cellular signaling pathways, such as inhibition of glycogen synthase kinase 3, which is considered to regulate cellular apoptosis. Divalproex, carbamazepine and several atypical antipsychotics are also approved for bipolar disease Evidence also suggests that antipsychotics show the ability to treat and prevent mania and/or depression but are often burdened by side effects such as sedation, hortostatic hypotension and weight gain. Hence, while it is clear that there still are several unmet needs especially for what pertains tolerability, efficacy for specific subtypes, and predictability. Novel and more effective treatments are needed and researchers are currently engaging in targeted drug development for bipolar illness, aimed at improving pharmacological strategies with marked and sustained effects. A variety of newer medications are being tested. Some of these drugs target pathways that are similar to those targeted by lithium, while others focus on newer targets, such as opiate receptor and N-methyl-D-aspartate (NMDA) receptors. Newer and older treatment strategies for bipolar disorder will be presented and critically reviewed.Disclosure of interestAndrea Fagiolini is/has been a consultant and/or a speaker and/or has received research grants from Allergan, Angelini, Astra Zeneca, Boehringer Ingelheim, Pfizer, Eli Lilly, Ferrer, Janssen, Lundbeck, Novartis, Otsuka, Roche.

scholarly journals Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation

2020 ◽  
Vol 72 (3) ◽  
pp. 379-391
Author(s):  
Ana Stancic ◽  
Ivana Drvenica ◽  
Branko Bugarski ◽  
Vesna Ilic ◽  
Diana Bugarski
Keyword(s):  
Cell Therapy ◽  
Experimental Model ◽  
Myogenic Differentiation ◽  
Calf Serum ◽  
C2c12 Cells ◽  
C2c12 Myotubes ◽  
Heme Oxygenase 1 ◽  
C2c12 Myoblast ◽  
Free Hemoglobin ◽  
Functional Characteristics

Functional characteristics of satellite cells (SCs) that act as myogenesis initiators and have emerged as a promising target for cell therapy, are dependent on their microenvironment. The aim of this study was to investigate the effect of cell-free hemoglobin, as a part of the microenvironment of SCs, on their functional characteristics. The C2C12 cell line served as the experimental model of SCs; hemoglobin isolated from porcine (PHb) and bovine (BHb) slaughterhouse blood served as the experimental model for extracellular hemoglobin. The proliferation rate of C2C12 cells was assessed by the MTT test, migration capacity by the scratch assay, and myogenic differentiation capacity by histochemical staining and RT-PCR analysis of the expression of genes specific for myogenic lineage. The effect of hemoglobin on the proliferation and migration of C2C12 cells was dependent on its concentration and the animal species it was isolated from, but the effect of BHb was more prominent. Both PHb and BHb decreased the expression levels of myogenin and muscle specific creatine kinase at a 10 ?M concentration. While PHb had no effect on the morphometric parameters of C2C12 myotubes, BHb modified the area and length of C2C12 myotubes cultivated in DMEM/2% horse serum and DMEM/10% fetal calf serum. While PHb and BHb had no effect on heme oxygenase 1 (Hmox1) expression, they stimulated the expression of hypoxia-inducible factor 1-alpha (Hif1?) at a concentration of 10 ?M. The mainly inhibitory effect of cell-free hemoglobin on myogenic differentiation suggests that it could be a relevant factor in the outcome of cell therapy of muscle injury.

287 PORCINE ADIPOSE-DERIVED STEM CELLS IN CO-CULTURE FUSE ACTIVELY WITH MOUSE MYOTUBES AND EXPRESS MYOGENIC MARKERS

2013 ◽  
Vol 25 (1) ◽  
pp. 291
Author(s):  
S. Jain ◽  
D. J. Milner ◽  
M. Bionaz ◽  
J. A. Cameron ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Myogenic Differentiation ◽  
Primary Source ◽  
Cell Types ◽  
C2c12 Cells ◽  
Immunofluorescent Staining ◽  
C2c12 Myotubes ◽  
Adipose Derived Stem Cells ◽  
Myogenic Markers

Mesenchymal stem cells (MSC) have been shown to be useful in regenerative medicine with their capability to give rise to various different cell types, including osteoblasts, adipocytes, chondrocytes, muscle cells, and neurons. Among MSC, bone marrow-derived stem cells (BMSC) are considered the primary source. Recently, many studies have shown the regenerative capabilities of adipose-derived stem cells (ASC). The ASC, with their greater abundance and ease of harvest, provide clear advantages over BMSC. We have previously demonstrated the myogenic differentiation of porcine ASC when co-cultured with differentiating C2C12 myoblasts in a myogenic differentiation-promoting medium. In this study, we sought to examine the myogenic potential of porcine ASC when co-cultured with fully differentiated murine myotubes. For the present study, we used porcine ASC isolated from the back fat of a transgenic gree fluorescent protein (GFP)-expressing pig at passage 3. The ASC were added to mouse C2C12 myotube cultures that had been induced towards myogenesis for 72 h. As controls, we co-cultured, in the same conditions, GFP-expressing endothelial cells (ENDO) from the aorta of the same pig. Additionally, we cultured ASC, ENDO, and C2C12 cells alone in myogenic-differentiation medium. Cultures were harvested at 12, 24, and 48 h after addition of porcine cells to myotube cultures for measurement of mouse- or porcine-specific myogenic markers by quantitative RT-PCR and immunohistochemistry. We were able to observe fusion of ASC GFP-expressing cells with pre-formed mouse myotubes by detection of myotubes expressing GFP. Additionally, immunofluorescent staining of co-cultures with an antibody specific for porcine nuclear Lamin A demonstrated the presence of ASC nuclei incorporated into myotubes. We observed large increases in gene expression of porcine-specific myogenin (MYOG; >900-fold) and desmin (DES; 8-fold). Unexpectedly, ENDO in co-culture with myotubes also had increased expression of DES (4-fold) and MYOG (400-fold), possibly indicating their de-differentiation and adaptation to a myogenic phenotype. In addition, expression of mouse-specific DES and MYOG were boosted in C2C12 myotubes when co-cultured for 48 h compared with C2C12 alone, suggesting enhanced myogenesis or prolonged survival of myotubes in co-culture. Cultures of ASC and ENDO alone did not display increased expression of myogenic markers. These results provide support for the use of ASC for muscle regeneration strategies, as in the case of damaged muscles and muscular dystrophy. In addition, compared with our previous observations, where ASC were co-cultured with undifferentiated C2C12, the ASC co-cultured with myotubes appeared to have an enhanced fusion and expression of myogenic markers. Finally, the capacity of ENDO to fuse and actively expressed muscle-specific genes deserves further investigation.

scholarly journals Creatine enhances differentiation of myogenic C2C12 cells by activating both p38 and Akt/PKB pathways

2007 ◽  
Vol 293 (4) ◽  
pp. C1263-C1271 ◽  
Author(s):  
Louise Deldicque ◽  
Daniel Theisen ◽  
Luc Bertrand ◽  
Peter Hespel ◽  
Louis Hue ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glycogen Synthase ◽  
Troponin T ◽  
Mammalian Target Of Rapamycin ◽  
C2c12 Cells ◽  
Glycogen Synthase Kinase 3 ◽  
Myocyte Enhancer Factor 2 ◽  
Chain Type ◽  
Dependent Mechanism ◽  
Enhancer Factor

In myogenic C2C12 cells, 5 mM creatine increased the incorporation of labeled [35S]methionine into sarcoplasmic (+20%, P < 0.05) and myofibrillar proteins (+50%, P < 0.01). Creatine also promoted the fusion of myoblasts assessed by an increased number of nuclei incorporated within myotubes (+40%, P < 0.001). Expression of myosin heavy chain type II (+1,300%, P < 0.001), troponin T (+65%, P < 0.01), and titin (+40%, P < 0.05) was enhanced by creatine. Mannitol, taurine, and β-alanine did not mimic the effect of creatine, ruling out an osmolarity-dependent mechanism. The addition of rapamycin, the inhibitor of mammalian target of rapamycin/70-kDa ribosomal S6 protein kinase (mTOR/p70s6k) pathway, and SB 202190, the inhibitor of p38, completely blocked differentiation in control cells, and creatine did not reverse this inhibition, suggesting that the mTOR/p70s6k and p38 pathways could be potentially involved in the effect induced by creatine on differentiation. Creatine upregulated phosphorylation of protein kinase B (Akt/PKB; +60%, P < 0.001), glycogen synthase kinase-3 (+70%, P < 0.001), and p70s6k (+50%, P < 0.001). Creatine also affected the phosphorylation state of p38 (−50% at 24 h and +70% at 96 h, P < 0.05) as well as the nuclear content of its downstream targets myocyte enhancer factor-2 (−55% at 48 h and +170% at 96 h, P < 0.05) and MyoD (+60%, P < 0.01). In conclusion, this study points out the involvement of the p38 and the Akt/PKB-p70s6k pathways in the enhanced differentiation induced by creatine in C2C12 cells.

scholarly journals The Present State of Lithium for the Prevention of Dementia Related to Alzheimer’s Dementia in Clinical and Epidemiological Studies: A Critical Review

2021 ◽  
Vol 18 (15) ◽  
pp. 7756
Author(s):  
Nobuyoshi Ishii ◽  
Takeshi Terao ◽  
Hirofumi Hirakawa
Keyword(s):  
Bipolar Disorder ◽  
Present State ◽  
Critical Review ◽  
Clinical Studies ◽  
Clinical Evidence ◽  
Glycogen Synthase ◽  
Epidemiological Studies ◽  
Glycogen Synthase Kinase 3 ◽  
Phosphorylated Tau ◽  
Dementia Prevention

Despite the unavailability of essential anti-dementia drugs, lithium may inhibit glycogen synthase kinase-3 (GSK-3) and decrease beta-amyloid and hyper-phosphorylated tau. In this review, we hypothesized that trace to standard levels of lithium (i.e., corresponding to the therapeutic levels for bipolar disorder) may be effective for dementia prevention. Excluding three insufficient level studies, we obtained two and one excellent clinical studies on standard and trace lithium levels, respectively, all of which supported the effects of lithium for dementia prevention. In addition, we identified good clinical and epidemiological studies (four each) on standard lithium levels, of which six studies supported the effects of lithium. Moreover, of three good epidemiological studies on trace lithium levels, two supported the aforementioned effects of lithium. The number of studies were substantially small, particularly those on trace lithium levels. Moreover, studies on standard lithium levels were insufficient to establish the efficacy of lithium for dementia prevention. This necessitates accumulating good or excellent clinical evidence for the effects of trace to standard lithium levels on dementia prevention.

Sign in / Sign up

Export Citation Format

Share Document