C2C12 Mouse Muscle Cells Research Articles

Characterization of TSSK6 Activating Co‐chaperone (TSACC) in Skeletal Muscle

Skeletal muscle atrophy is a physiological condition that is caused by a variety of conditions, including immobilization, denervation, spinal cord injury and aging and results in decreased muscle size and strength. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated gastrocnemius muscle from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that TSSK6 Activating Co‐chaperone (TSACC) is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that TSACC is expressed in muscle, the predicted TSACC cDNA was cloned from cultured muscle cells, as was a novel TSACC splice variant that contains an additional exon. Quantitative PCR (qPCR) was then used to assess TSACC expression levels of the full‐length and novel splice variant in proliferating and differentiated muscle cells. The results demonstrate that full‐length TSACC expression levels are relatively low in proliferating myoblasts but show significantly elevated expression in differentiated myotubes, whereas the novel splice variant is expressed relatively evenly in proliferating and differentiated muscle cells. In addition, characterization of the transcriptional regulation of TSACC was assessed by fusing fragments of the proximal promoter located immediately upstream of the start of transcription with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Interestingly, there are several predicted E‐box elements in the proximal promoter region of the TSACC gene further suggesting that TSACC may be regulated by muscle‐specific transcription factors. Finally, to determine a possible role for TSACC in skeletal muscle, sub‐cellular localization was determined by fusing the cDNA with GFP and expressing the fusion protein in cultured muscle cells. The discovery that TSACC is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle atrophy and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of the Regulation and Function of Family with Sequence Similarity 83D (Fam83d) in Skeletal Muscle

Skeletal muscle atrophy is a serious condition that can arise due to aging, cancer, corticosteroid exposure, and denervation. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated mouse gastrocnemius muscle from mice at 3 and 14 days following sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Fam83d is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) analysis revealed that Fam83d is more highly expressed in proliferating myoblasts compared to differentiated myotubes. Characterization of the transcriptional regulation of Fam83d was analyzed by cloning fragments of the proximal promoter region located immediately upstream from the start of transcription and fusing them with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Analysis of the promoter of Fam83d revealed conserved E‐box elements in the proximal regulatory region, which are known myogenic regulatory factor (MRF) binding sites. To assess where Fam83d is localized in the cell, we fused Fam83d with green fluorescent protein (GFP), transfected the Fam83d‐GFP expression plasmid into C2C12 mouse myoblasts cells, visualized by confocal microscopy, and observed that Fam83d is localized exclusively to the cytoplasm. It has recently been determined that Fam83d contains a putative phospholipase domain, is mis‐regulated in several different cancer types, and may modulate MAP Kinase signaling. Therefore, Fam83d was ectopically expressed in cultured muscle cells and markers of muscle cell differentiation and MAP Kinase signaling were analyzed. Fam83d overexpression resulted in significantly repressed levels of myosin heavy chain and myogenin expression, while levels of phosphorylated ERK were dramatically repressed. The discovery that Fam83d is expressed in skeletal muscle combined with the observation that Fam83d, a potential modulator of MAP Kinase signaling, is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and Characterization of TNF Receptor Associated Factor 3 Interacting Protein 3 (Traf3ip3) in Skeletal Muscle

Skeletal muscle atrophy is a physiological condition that results in decreased muscle size and strength and occurs in response to immobilization, denervation, spinal cord injury and aging. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated gastrocnemius muscle from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed that TNF Receptor Associated Factor 3 Interacting Protein 3 (Traf3ip3) is expressed in skeletal muscle and is induced in response to denervation. To confirm that Traf3ip3 is expressed in muscle cells, we cloned the Traf3ip3 cDNA from cultured muscle cells and performed quantitative PCR (qPCR) to assess expression levels in both proliferating and differentiated muscle cells. The qPCR results demonstrated that Traf3ip3 expression levels are relatively low in proliferating myoblasts but show dramatically elevated expression in differentiated myotubes. To characterize the transcriptional regulation of Traf3ip3, fragments of the proximal promoter located immediately upstream of the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in differential effects on reporter gene activity. The discovery that Traf3ip3 is expressed in muscle combined with the observation that this gene is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cost‐effective synthesis of polyethylene glycol diacrylate‐acrylic acid (PEGDA‐AA), a tissue engineering hydrogel ideal for C2C12 mouse muscle cells

Current research in tissue engineering focuses on growing and functionalizing cells in vitro to assemble viable constructs that restore, maintain, or improve damaged tissues or whole organs. Specific to the field of tissue engineering, hydrogels have proven successful in the development of multiple cell types with the goal of one day serving as grafts that will replace large areas of lost or damaged tissue. This assembly depends on the presence of a scaffold, or three‐dimensional structure that guides the growth of new tissue, and hydrogel polymers in particular have been known to be an excellent building block for scaffold formation. This research focused on the synthesis and characterization of the hydrogel polyethylene glycol diacrylate‐acrylic acid (PEGDA‐AA), a hydrophilic material that provides a viable environment for cell maturation because of its favorable characteristics for the development and maturation of mouse C2C12 muscle cells. Using modified procedures adapted from Browe et al. (2016) to create a cost‐effective way of synthesizing this precursor, PEGDA was successfully synthesized and characterized through 13C and 1H‐NMR spectroscopy. Current research focuses on analyzing the effects of the hydrogel on the growth and development of C2C12 cells with the long‐term goal of establishing an efficient model for human tissue engineering.Support or Funding InformationHampden‐Sydney College Office of Undergraduate ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low Citrate Synthase Activity Is Associated with Glucose Intolerance and Lipotoxicity.

Citrate synthase (CS) is a key mitochondrial enzyme. The aim of this study was to test the hypothesis that low CS activity impairs the metabolic health of mice fed a high fat diet (HFD) and promotes palmitate-induced lipotoxicity in muscle cells. C57BL/6J (B6) mice and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A), a strain which carries the A/J allele of CS on the B6 strain background, were fed HFD (45% kcal from fat) for 12 weeks. C2C12 mouse muscle cells were used to investigate effects of CS knockdown on cell viability and signalling after incubation in 0.8 mM palmitate. CS activity, but not that of β-hydroxyacyl-coenzyme-A dehydrogenase was lower in the gastrocnemius muscle and heart of B6.A mice compared to B6 mice (P < 0.001). During HFD feeding, glucose tolerance of mice decreased progressively and to a greater extent in B6.A females compared to B6 females, with males showing a similar trend. Body weight and fat gain did not differ between B6.A and B6 mice. After an 18 h incubation in 0.8 mM palmitate C2C12 muscle cells with ∼50% shRNA mediated reduction in CS activity showed lower (P < 0.001) viability and increased (P < 0.001) levels of cleaved caspase-3 compared to the scramble shRNA treated C2C12 cells. A/J strain variant of CS is associated with low enzyme activity and impaired metabolic health. This could be due to impaired lipid metabolism in muscle cells.

Identification and Characterization of Calcium Binding and Coiled Coil Domain 1 (Calcoco1) in Skeletal Muscle

Skeletal muscle atrophy results in reduced muscle size and strength and is caused by a range of physiological conditions, including aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of atrophy, skeletal muscle was isolated from mice following 3 days and 14 days of denervation. The gene expression profile of the denervated muscle tissue was analyzed by microarray and compared to control muscle in order to identify novel atrophy‐induced genes. The microarray revealed that Calcium Binding and Coiled Coil Domain 1 (Calcoco1) is expressed in skeletal muscle and is induced in response to denervation. To confirm that Calcoco1 is expressed in muscle, we successfully cloned the cDNA of Calcoco1 from cultured muscle cells, demonstrating that this gene is transcriptionally active. In addition, quantitative PCR (qPCR) was used to assess Calcoco1 expression in both proliferating and differentiated muscle cells and the results demonstrated that Calcoco1 expression levels are moderate in proliferating myoblasts, but show significant elevation in differentiated myotubes. Furthermore, Western Blot analysis of protein homogenates isolated from muscle cells confirmed that Calcoco1 is expressed in both proliferating myoblasts and differentiated myotubes. In order to better characterize the transcriptional regulation of Calcoco1, fragments of the proximal promoter located immediately upstream of the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Furthermore, bioinformatic analysis of the promoter region of Calcoco1 revealed a conserved canonical E‐box element, which is a known myogenic regulatory factor (MRF) binding site, further suggesting that Calcoco1 may be regulated by muscle specific transcription factors. Finally, the Calcoco1 protein is predicted to have both a calcium binding domain and a coiled‐coil domain, suggesting that it may function in protein‐protein interactions and/or as a putative transcription factor. Therefore, in order to determine the sub‐cellular localization of Calcoco1 in muscle cells, the Calcoco1 cDNA was fused with the green fluorescent protein (GFP), expressed in muscle cells, and visualized by confocal microscopy revealing that Calcoco1 is localized exclusively to the cytoplasm in unchallenged myoblast cells. The discovery that Calcoco1 is expressed in skeletal muscle combined with the observation that this gene is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle atrophy and may eventually lead to the identification of new therapeutic targets for the treatment and prevention of muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and Characterization of TSSK6 Activating Co‐chaperone (TSACC) in Skeletal Muscle

Skeletal muscle atrophy is a physiological condition that is caused by a variety of conditions, including immobilization, denervation, spinal cord injury and aging and results in decreased muscle size and strength. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that TSSK6 Activating Co‐chaperone (TSACC) is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that TSACC is expressed in muscle cells, we cloned the predicted TSACC cDNA from muscle cells, as well as a novel TSACC splice variant that contains an additional exon. Furthermore, quantitative PCR (qPCR) was used to assess TSACC expression levels in both proliferating and differentiated muscle cells and the results demonstrate that TSACC expression levels are relatively low in proliferating myoblasts, but show significantly elevated expression in differentiated myotubes. In order to characterize the transcriptional regulation of TSACC, fragments of the proximal promoter located immediately upstream of the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Interestingly, there are several predicted MyoD binding elements in the proximal promoter region of the TSACC gene further suggesting that TSACC may be regulated by muscle specific transcription factors. The discovery that TSACC is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle atrophy and may eventually lead to the identification of new therapeutic targets for the treatment and prevention of muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of the Regulation and Function of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle

Skeletal muscle atrophy is a debilitating medical condition that can arise due to aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of neurogenic atrophy, mouse gastrocnemius muscle was isolated 3 and 14 days after sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Zinc Finger Protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) analysis showed that Zfp593 is more highly expressed in proliferating myoblasts compared to differentiated myotubes, while Western blot analysis confirmed that Zfp593 is expressed at the protein level in proliferating and differentiated mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, fragments of the proximal promoter region located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Furthermore, analysis of the promoter region of Zfp593 revealed four conserved E‐box elements in the proximal regulatory region, which are known myogenic regulatory factor (MRF) binding sites. Therefore, in order to determine how these elements may regulate the transcription of Zfp593, the E‐Boxes where either mutated or deleted and the resulting mutant reporter constructs were then transfected into C2C12 mouse myoblasts in combination with ectopic expression of MRFs. Additionally, it is predicted that the Zfp593 protein contains a zinc finger domain and is believed to function as a transcription factor. Therefore, to assess if Zfp593 is localized to the nucleus, we fused Zfp593 with green fluorescent protein (GFP), transfected the Zfp593‐GFP expression plasmid into C2C12 mouse myoblasts cells, visualized by confocal microscopy, and confirmed that Zfp593 does localize to the nucleus. Finally, the Zfp593 transcript possess a particularly long 3′‐untranslated region (UTR) region, so we cloned several fragments of the UTR, fused them with a reporter gene, and found that the UTR region has a negative regulatory effect on reportor gene expression in muscle cells. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that Zfp593, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and Characterization of Mago Homology B (Magohb) in Skeletal Muscle

Skeletal muscle atrophy is a physiological condition that occurs due to immobilization, denervation, spinal cord transection and aging. Muscle wasting causes significant loss of muscle size, a measurable reduction is strength, and decreased mobility. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Mago Homology B (Magohb), an exon junction core complex component involved in splicing and mRNA surveillance, is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that Magohb is expressed in muscle cells, quantitative PCR (qPCR) was used to assess Magohb expression levels in both proliferating and differentiated muscle cells and the results demonstrate that Magohb is expressed in both proliferating myoblasts and differentiated myotubes. In order to characterize the transcriptional regulation of Magohb, fragments of the proximal promoter located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Furthermore, bioinformatic analysis of the promoter region of Magohb revealed a conserved canonical E‐box element, which is a known myogenic regulatory factor (MRF) binding site, further suggesting that Magohb may be regulated by muscle specific transcription factors. Finally, since Magohb is believed to play a role in exon‐exon junction identification and may play a role in mRNA splicing, we fused Magohb to GFP, transfected mouse myoblasts with the Magohb‐GFP expression plasmid, imaged the cells using confocal microscopy, and confirmed that Magohb is predominately localized to the nucleus. The discovery that Magohb is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of mRNA processing, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Cortisol on Endoplasmic Reticulum-stress, Apoptosis, and Autophagy in Mouse Muscle C2C12 Cells

Effects of Cortisol on Endoplasmic Reticulum-stress, Apoptosis, and Autophagy in Mouse Muscle C2C12 Cells

Cytotoxicity of Malondialdehyde and Cytoprotective Effects of Taurine via Oxidative Stress and PGC-1α Signal Pathway in C2C12 Cells

One of the end-products of ROS-induced peroxidation, malondialdehyde (MDA), induces the cross-links in proteins, which leads to perturbation of the physiological functions of cells and contributes to abnormal biological regulation and various disorders. Taurine (2-aminoethanesulfonic acid, Tau) aids in adjusting normal physiological functions to confer stress resistance. The protective effects of Tau against MDA stress in vitro or in vivo were reported previously. In this study, we had investigated the protective effects of taurine on viability, oxidative stress levels and mitochondrial biogenesis in mouse muscle C2C12 cells undergoing MDA induced stress. We show that the treatment with 100 μM MDA leads to increase in cell oxidative stress levels, inhibition of mitochondrial biogenesis and the reduction of the cell survival rates. The pretreatment with 0.1 μM taurine reduced MDA-induced death rate via inhibition of oxidative stress, restoration of mitochondrial functions of the mitochondrial membrane potential (MMP) and ATP production. In MDA stress, the pre-treatment with 0.1 μM taurine leads to upregulation of the factors of mitochondrial biogenesis. These observations suggest that the cytoprotective effects of taurine may be due to an induction of mitochondrial biogenesis.

Evodiamine promotes differentiation and inhibits proliferation of C2C12 muscle cells.

Evodiamine is a botanical alkaloid compound extracted from Tetradium plants. Previous studies have reported that evodiamine (Evo) treatment can reduce food uptake and improve insulin resistance in animals . The skeletal muscle comprises about 40% of the body mass of adults and has a vital role in regulating whole body glucose metabolism and energy metabolism. However, the effect of Evo on skeletal muscle is unclear. The main aim of the present study was to investigate the effect of Evo on the differentiation and proliferation of the mouse C2C12 muscle cell line. The results demonstrated that Evo promoted the expression of myogenic marker genes (Myogenin and muscle myosin heavy chain) and increased myoblast differentiation, potentially via activation of the Wnt/β‑catenin pathway. Furthermore, Evo increased mRNA expression of p21, reduced mRNA expression of CyclinB, CyclinD and CyclinE and reduced the percentage of proliferating cells. Also, phosphorylation of ERK1/2 was decreased by Evo treatment during cell proliferation. In conclusion, these findings indicated that Evo has marked effects on skeletal muscle development.

Characterization of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle

Skeletal muscle atrophy is a debilitating medical condition which can arise due to aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that zinc finger protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Furthermore, Western blot analysis confirmed that Zfp593 is expressed at the protein level in C2C12 mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, fragments of the proximal promoter located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with MyoD1 or myogenin expression plasmids, resulting in a differential effect on reporter activity. Furthermore, analysis of the promoter region of Zfp593 has revealed four conserved E‐box elements, which are known myogenic regulatory factor (MRF) binding sites. Zfp593 is predicted to possess a zinc finger domain and is believed to function as a transcription factor. Furthermore, two predicted putative nuclear localization signal sequences have been identified in the Zfp593 protein sequence, further suggesting that this protein is localized to the nucleus. Therefore, in order to determine the sub‐cellular localization of Zfp593 in muscle cells, the Zfp593 cDNA was isolated from C2C12 cells, cloned into a green fluorescent protein (GFP) expression plasmid and transfected into proliferating C2C12 cells. Transfected cells were visualized by confocal microscopy revealing that Zfp593 is localized to the nucleus of proliferating myoblasts. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.Support or Funding InformationThe work was supported by University of North Florida Transformational Learning grants to D.W.

Muscle Protein Signaling in C2C12 Cells Is Stimulated to Similar Degrees by Diverse Commercial Food Protein Sources and Experimental Soy Protein Hydrolysates.

Dietary protein stimulates muscle protein synthesis and is essential for muscle health. We developed a screening assay using C2C12 mouse muscle cells to assess the relative abilities of diverse commercial protein sources and experimental soy protein hydrolysates (ESH), after simulated gut digestion (SGD), to activate the mechanistic target of rapamycin complex I (mTORC1) muscle protein synthesis signaling pathway (p70S6K(Thr389) phosphorylation). Activation of mTORC1 was expressed as a percentage of a maximal insulin response. The bioactivities of proteins grouped by source including fish (81.3 ± 10.6%), soy (66.2 ± 4.7%), dairy (61.8 ± 4.3%), beef (53.7 ± 8.6%), egg (52.3 ± 10.6%), soy whey (43.4 ± 8.6%), and pea (31.4 ± 10.6%) were not significantly different from each other. Bioactivity for ESH ranged from 28.0 ± 7.5 to 98.2 ± 6.6%. The results indicate that both the protein source and processing conditions are key determinants for mTORC1 activation. Regression analyses demonstrated that neither leucine nor total branched-chain amino acid content of proteins is the sole predictor of mTORC1 activity and that additional factors are necessary.

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys(520) (mouse Cys(533)). In addition, an HIF-1α Cys(520) serine mutant is resistant to 2-AAPA-induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys(520) promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles.

358. AAV-MitoTimer to Asses Mitochondrial Turnover

Mitochondrial turnover is a key marker in the process of cellular senescence. In order to track mitochondrial turnover rates in various cell lines, an indicator is necessary to identify the formation of new mitochondria along with the degradation of old mitochondria. This measure is critical for the study of disease processes in which there is an up or down regulation of mitochondrial turnover rates. One dynamic tool to measure this balance is the pTRE-Tight-MitoTimer plasmid. The MitoTimer transgene combines the red fluorescent protein, dsRed, and a human cytochrome c oxidase subunit VIII (Cox 8) mitochondrial localization sequence to traffic the protein to the mitochondria where the color shifts from green to red over time (approximately 24 hours). Our goal is to use this as a tool to assess the increase or decrease in mitochondrial turnover as a result of mutations that cause mitochondrial dysfunction. We successfully developed two plasmids through the ligation of the MitoTimer transgene into a double stranded AAV-ITR containing plasmid with a 1) Desmin promoter pds-AAV-Des-MitoTimer to restrict expression to striated muscle and 2) CMV promoter pds-AAV-CMV-MitoTimer to enable ubiquitous expression among many cell types. Once cloned, these novel plasmids were then transfected into an immortalized C2C12 mouse muscle cell line to confirm successful generation of functional plasmids. An immediate application for these novel plasmids in our laboratory is the characterization of mitochondrial turnover rates in Barth Syndrome (BTHS) patient derived cells. BTHS is an X-linked mitochondrial disease that results from mutations of the gene TAZ, which encodes tafazzin. This gene serves to orchestrate the production of mature cardiolipin (CL) within the mitochondrial membrane. Mutations in the TAZ gene result in altered CL maturation, with implications on mitochondrial function. Thus far, mitochondrial turnover rates have not been carefully assessed in this disorder. As a preliminary experiment using human patient derived BTHS fibroblasts (harboring an exon 10: 748G &gt T nonsense mutation), pds-AAV-CMV-MitoTimer was successfully transfected and displayed variation in green and red fluorescence ratios as compared to a healthy control cell line. This demonstration of the impact of BTHS on mitochondrial turnover dynamics supports future use of these plasmids in BTHS induced pluripotent stem cells (iPSCs) that have been differentiated into cardiomyocytes or skeletal myotubes - the two cell-types most affected in BTHS. This will enable future comparisons of mitochondrial turnover in clinically relevant BTHS cells harboring a variety of different mutations and provide another metric by which to measure the success of pre-clinical therapeutics for BTHS. We are preparing an AAV Quad-mutant serotype for in vitro testing, and other serotypes will be used for in vivo models of mitochondrial dysfunction. AAV-MitoTimer will continue to be used as a measure of mitochondrial turnover, and provide an effective characterization method for metabolic diseases.

Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice.

Metformin is the main drug of choice for treating type 2 diabetes, yet the therapeutic regimens and side effects of the compound are all undesirable and can lead to reduced compliance. The aim of this study was to elucidate the mechanism of action of two novel compounds which improved glucose handling and weight gain in mice on a high-fat diet. Wildtype C57Bl/6 male mice were fed on a high-fat diet and treated with novel, anti-diabetic compounds. Both compounds restored the glucose handling ability of these mice. At a cellular level, these compounds achieve this by inhibiting complex I activity in mitochondria, leading to AMP-activated protein kinase activation and subsequent increased glucose uptake by the cells, as measured in the mouse C2C12 muscle cell line. Based on the inhibition of NADH dehydrogenase (IC50 27µmolL(-1)), one of these compounds is close to a thousand fold more potent than metformin. There are no indications of off target effects. The compounds have the potential to have a greater anti-diabetic effect at a lower dose than metformin and may represent a new anti-diabetic compound class. The mechanism of action appears not to be as an insulin sensitizer but rather as an insulin substitute.

Isolation, expression analysis and characterization of NEFA-interacting nuclear protein 30 and RING finger and SPRY domain containing 1 in skeletal muscle

Muscle atrophy results from a range of physiological conditions, including immobilization, spinal cord damage, inflammation and aging. In this study we describe two genes, NEFA-interacting nuclear protein 30 (Nip30) and RING Finger and SPRY domain containing 1 (Rspry1), which have not previously been characterized or shown to be expressed in skeletal muscle. Furthermore, Nip30 and Rspry1 were transcriptionally induced in response to neurogenic muscle wasting in mice and were also found to be expressed endogenously at the RNA and protein level in C2C12 mouse muscle cells. Interestingly, during analysis of Nip30 and Rspry1 it was observed that these genes share a 230 base pair common regulatory region that contains several putative transcription regulatory elements. In order to assess the transcriptional activity of the Nip30 and Rspry1 regulatory regions, a fragment of the promoter of each gene was cloned, fused to a reporter gene, and transfected into cells. The Nip30 and Rspry1 reporters were both found to have significant transcriptional activity in cultured cells. Furthermore, the Nip30-Rspry1 common regulatory region contains a conserved E-box enhancer, which is an element bound by myogenic regulatory factors that function in the regulation of muscle-specific gene expression. Therefore, in order to determine if the predicted E-box was functional, Nip30 and Rspry1 reporters were transfected into cells ectopically expressing the myogenic regulatory factor, MyoD1, resulting in significant induction of both reporter genes. In addition, mutation of the conserved E-box element eliminated MyoD1 activation of the Nip30 and Rspry1 reporters. Finally, GFP-tagged Nip30 was found to localize to the nucleus, while GFP-tagged Rspry1 was found to localize to the cytoplasm of muscle cells.

Transcriptional Activation of Zing Finger Protein 593 (Zfp593) During Neurogenic Skeletal Muscle Atrophy

Skeletal muscle atrophy is a serious medical condition resulting from an array of physiological conditions such as aging, corticosteroid use, cancer and loss of innervation. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase up-regulated under most atrophy conditions, is believed to play a role in targeting proteins for degradation during muscle wasting. However, data represented in this study indicates that MuRF1 may also function as a regulator of atrophy-induced gene expression. In response to neurogenic atrophy, zinc finger protein 593 (Zfp593) is strongly induced in wild-type mice, while up-regulation of Zfp593 is significantly blunted in MuRF1-null mice. To further study the transcriptional regulation of Zfp593, the promoter region was cloned and fused to a reporter gene. The reporter plasmid was then transfected into C2C12 mouse muscle cells alone or in combination with a MuRF1 expression plasmid. The results of these assays demonstrate that the Zfp593 reporter gene has significant activity in muscle cells, while reporter activity is repressed in response to MuRF1 ectopic expression. Analysis of the promoter region of Zfp593 revealed two conserved E-box elements, which are known myogenic regulatory factor (MRF) binding sites. To determine if MRF proteins modulate the transcriptional activity of Zfp593, MyoD1 or myogenin expression plasmids were co-transfected with the Zfp593 reporter into C2C12 cells resulting in a modest effect on reporter activity. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.

Transcriptional Activation of Neuromuscular Junction Genes is Altered in Muscle RING Finger 1 (MuRF1) Null Mice

Skeletal muscle atrophy occurs as a result of numerous conditions, including disruption of the neuromuscular junction (NMJ), which is the integration point between muscle cells and the neurons that regulate their activity. MuRF1 is an E3 ubiquitin ligase that is activated under must atrophy conditions and is believed to target substrate proteins for degradation during muscle wasting. However, data presented here suggests that MuRF1 may also regulate the transcriptional activity of a subset of genes during neurogenic atrophy, including MuSK and the nicotinic acetylcholine receptor (nAChR) subunit genes. The MuSK and nAChR proteins are essential for proper NMJ function and are rapidly and robustly activated in the muscle of wild-type mice following neuron damage. Interestingly, MuSK and the nAChR genes show blunted activation in MuRF1-null mice in response to denervation. To further investigate a potential transcriptional regulatory role for MuRF1, the promoter regions of MuSK and the nAChR subunit genes were cloned and fused to a reporter gene. These reporter constructs were then transfected into C2C12 mouse muscle cells alone or in combination with expression plasmids for MuRF1 and/or the myogenic regulatory factors (MRF) MyoD1 or myogenin. The results from reporter assays support previous findings demonstrating that the MRFs significantly up-regulate MuSK and nAChR subunit gene expression. Furthermore, when MuRF1 is co-transfected with MyoD1 or myogenin, the MRF-mediated MuSK and nAChR subunit reporter gene induction is significantly repressed. While the mechanism by which MuRF1 represses MRF-mediated gene activation remains unknown, the data presented here provides evidence that MuRF1 may modulate the transcriptional activity of skeletal muscle genes.