bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of UPRER Does Not Impair Signaling to Mitochondrial Biogenesis During Chronic Contractile Activity
Authors: Oliveira Ashley N , Memme Jonathan M , Hood David 
Published: November 2023

Cellular stresses that perturb the protein folding environment can be detrimental to cell health and may provoke apoptosis. In order to combat these stresses, the cell is equipped with compartment‐specific mechanisms to help regain homeostasis: the mitochondrial (UPRmt) and endoplasmic reticulum (UPRER) unfolded protein responses. Acute exercise is capable of eliciting the UPR, however the role of the UPR in chronic exercise‐induced adaptations is still unclear. Exercise training can be modeled by direct stimulation (6V, 10Hz; 3hrs/day) of the peroneal nerve in Sprague Dawley rats to induce chronic contractile activity (CCA) for 7 days. We can model endurance training in one hindlimb, while the other serves as an internal control. Previously, we have shown that UPRER signaling is upregulated during the early phases of CCA, as shown by 1.5–4 fold increases in BiP, CHOP and XBP1 spliced mRNA. We then sought to investigate the role of the UPRER in mitochondrial adaptations by inhibiting UPR signaling with tauroursodeoxycholic acid (TUDCA). TUDCA treatment during 7 days of CCA attenuated CHOP activation by 63%, suggesting effective impairment of UPRER signaling. Despite this, mitochondrial biogenesis (MB) was not reduced by TUDCA treatment during CCA, evidenced by comparable 22–24% increases in COX activity, a measure of mitochondrial content, after 7 days of CCA. Since the vast majority of proteins that are required for MB are nuclear‐encoded and require import into the organelle, this could imply an important role for protein import in UPRmt function. Tim23, a major translocase of the inner membrane, increased by an average of 23% as early as 1 day of CCA, suggesting a greater potential for the transport of nuclear‐transcribed proteins into the matrix. If protein import into the matrix is increased, this could be accompanied by a greater protein folding/handling capacity. Using this CCA model we can further investigate the role of protein import and its relationship with the UPRmt in potentially mediating mitochondrial adaptations to chronic exercise.Support or Funding InformationSupported by NSERC.
ROLE OF CENTRAL TRPV4 IN THE ACTIVATION OF HEAT LOSS RESPONSES IN WISTAR RATS
Authors: Bicego Kenia C , Scarpellini Carolina da S. , Almeida Maria Camila , Gargaglioni Luciane H. 
Published: November 2023

In vitro results and studies with knockout mice for TRPV4 have suggested that this receptor is activated by warm temperatures. Recently, our group has described that peripheral TRPV4, activated by ambient temperatures (Ta) between 26 and 30oC, stimulates warmth‐defence responses in non‐genetically modified rats; however no data was available about the role of hypothalamic TRPV4 in thermoregulation. Then, the aim of this study was to verify if central TRPV4 in the medial preoptic area (MPA) plays a role in the heat loss activation in rats. To this end, body temperature (Tc), metabolic rate (VO2) and heat loss index (HLI) were measured at Ta of 21 and 28°C after TRPV4 in MPA were antagonized by HC‐067047 (0.5, 5 and 25ng/50nL) . The antagonism of central TRPV4 caused an increase in the Tb of animals exposed to 28°C by approximately 1°C (5ng/50nL; p< 0.001), but those animals exposed to 21°C had their Tb increased only by ~0.4°C (5 and 25ng/50nL; p< 0.001). At 28°C, VO2 (p= 0.012), but not HLI, was also increased by the antagonist. Our results suggest, for the first time, that central TRPV4 plays a role in autonomic heat loss responses in rats. Moreover, this action appears to be dependent on peripheral activation, i.e., when peripheral TRPV4 are not activated, the central ones have a less evident role in the stimulation of heat loss.Support or Funding InformationFAPESP 2011/19131‐2 and 2015/04849‐6
Molecular mechanisms behind the accumulation of lipid that occurs after skeletal muscle injury
Authors: Gumucio Jonathan , Rittman Danielle , McDonagh Brian , Mendias Christopher 
Published: November 2023

Myosteatosis is the accumulation of lipid that occurs after skeletal muscle injury or in certain chronic neuromuscular or metabolic diseases. Muscle fiber atrophy and fibrosis often accompany myosteatosis. The amount of lipid accumulation is negatively correlated with skeletal muscle functional capacity, but the causes of myosteatosis are unknown. To gain a greater understanding of the ontogeny of myosteatosis, we induced a severe injury to the rotator cuff musculature in rats, isolated tissues 10, 30 or 60 days after tear, and used a combination of RNA sequencing and shotgun lipidomics to identify global changes in gene expression and lipid content in injured muscles. The RNA sequencing data and shotgun lipidomics results were then analyzed by Ingenuity Pathway Analysis (IPA, Qiagen) software to determine the biochemical pathways involved in myosteatosis. Samples were also prepared for electron microscopy and single muscle fiber contractility. Following muscle injury, there was a time dependent increase in total lipid content, largely due to a dramatic rise in triacylglyceride species. Interestingly, genes related to lipid synthesis (DGAT1, DGAT2) and lipid breakdown (LPL, HSL) were largely downregulated over time. IPA of RNASeq data predicted increased mitochondrial dysfunction and oxidative stress, and decreased lipid oxidation to be highly associated with myosteatosis at all time points. Genes related to lipid utilization and mitochondrial function (CPT1/2, SDH, COXIV) were also decreased with time. From electron micrographs, there was increased subsarcolemmal mitochondria at all time points after injury and dramatic sarcomere streaming at 30d and 60d compared to controls. Additionally, there was an increase in carbonylated proteins at 60d. At 30 and 60d, IPA predicted increased inflammatory pathways in chronically torn rotator cuff muscles compared to controls, and there was an overall increase in inflammatory gene markers (IL6, IL1b, COX1) at all time points. These changes corresponded to a time dependent decrease in muscle fiber specific force production, or muscle fiber maximum isometric force normalized to cross sectional area. Combined, these data suggest that the accumulation of lipid that occurs after muscle injury is likely due to a decrease in lipid utilization and breakdown rather than an increase in lipid synthesis. Sustained mitochondrial dysfunction and oxidative stress may result in the increased lipid infiltration and atrophy of muscle fibers over time. While further investigation is necessary, this study provided important insight into the development of myosteatosis and identifying potential therapeutic targets to improve muscle regeneration.Support or Funding InformationThis work was supported by NIH grants (R01AR063649 and F31AR065931)
Inhibition of Toll‐like Receptor 4 does not prevent palmitate‐induced endoplasmic reticulum stress in cultured skeletal muscle cells
Authors: Perry Ben Douglas , Rahnert Jill A , Zheng Bin , Price Russ 
Published: November 2023

Chronically elevated circulating saturated fatty acids (SFA), as seen in Type 2 Diabetes Mellitus, impairs insulin sensitivity, protein synthesis, and upregulates proteolytic pathways in skeletal muscle. The SFA‐induced impairment of muscle protein synthesis is due, in part, to increased endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response. However, the upstream receptor(s) that initiate ER stress in response to SFA in skeletal muscle are not well delineated. This study investigated the role of TLR4, which is activated by SFA and induces pro‐inflammatory signaling, on markers of ER stress and protein synthesis signaling in cultured mouse muscle cells (C2C12). C2C12 myotubes were treated with either vehicle, a TLR4‐specific ligand (lipopolysaccharides; 100 ng/mL), palmitate (SFA; 500 μM), or a combination of palmitate and TLR4‐specific inhibitor (TAK‐242, 1 μM). After 24 hours, cells were harvested and analyzed for markers of ER stress and protein synthesis using RT PCR and western blotting. Palmitate treatment substantially increased the levels of CHOP, spliced XBP1, and ATF 4 mRNAs as well as the protein expression of p‐PERK/PERK and p‐eIF2α/eIF2α. The palmitate/TLR4 inhibitor treatment unexpectedly elevated both CHOP mRNA and protein expression of p‐eIF2α/eIF2α to levels greater than palmitate alone. Compared to control, the p‐p70S6k/p70S6k ratio was reduced to a similar extent with both palmitate and palmitate/TLR4 inhibitor treatments. These data suggest that in conditions involving lipotoxicity, activation of TLR4 in skeletal muscle is not responsible for palmitate‐induced ER stress. Instead, activation of TLR4 may induce signaling which is protective during heightened ER stress in muscle.Support or Funding InformationSupported by NIH RO1 DK95610, NIH T32 DK007656 and VA Merit I01BX001456
Eutherian BetaM, a Muscle‐Specific Integral Protein of Inner Nuclear Membrane, is Implicated in Regulation of MyoD Expression
Authors: Modyanov Nikolai , Pestov Nikolay , Ahmad Nisar 
Published: November 2023

Vertebrate X‐chromosome ATP1B4 genes represent a rare instance of the orthologous gene co‐option that radically changed functions of encoded BetaM proteins during evolution. In lower vertebrates, BetaM is a subunit of Na,K‐ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through radical changes in the structure of its N‐terminal domain gained entirely new properties as a skeletal and atrial cardiac muscle‐specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. BetaM expression is strictly temporally regulated being the highest in late fetal and early postnatal myocytes. Here we show that expression of exogenous BetaM in cultured muscle cells up‐regulates expression of endogenous MyoD, a master regulator of myogenesis, both at transcriptional and translational levels. We determined that eutherian BetaM associates with nuclear transcriptional co‐regulator Ski‐interacting protein (SKIP) and both are identified by ChIP assays as components of MyoD transcriptional complex in vivo in rat neonatal skeletal muscle. Presence of BetaM was detected in the DRR but not in PRR and CE regions of the MyoD promoter. According to the results of Electrophoretic Mobility Shift Assays (EMSA) BetaM‐containing transcriptional complexes are bound to specific E‐box4 and CArG elements of the DRR region. Deletion of either E‐box4 or CArG or both in DRR luciferase reporter abolished its activation by BetaM, These findings demonstrate that BetaM is a novel eutherian‐specific transcriptional regulator which is involved in the regulation of MyoD expression during muscle development. Moreover, structural and functional alterations acquired through ATP1B4 gene co‐option and a unique pattern of expression argue that eutherian‐specific BetaM functions are essential, even might be necessary for survival of placental mammals in natural conditions, and that they provide evolutionary advantage.Support or Funding InformationSupported by Department of Physiology & Pharmacology, University of Toledo College of Medicine.
Intramuscular NF‐κβ Signaling in Response to Resistance Exercise and Recovery in Untrained Males
Authors: Townsend Jeremy , Stout Jeffrey , Jajtner Adam , Church David , Beyer Kyle , Oliveira Leonardo , Fukuda David , Hoffman Jay 
Published: November 2023

The nuclear factor kappa B (NF‐κβ) signaling cascade is responsible for mediating stress‐activated catabolic effects of TNF‐α downstream of the TNF receptor. NF‐κβ transcription factor remains inhibited in the cytosol of the muscle and can be stimulated for translocation and transcription by a variety of external stimuli, most notably by pro‐inflammatory cytokines and oxidative stress. Nevertheless, currently there is a gap in the literature with regard to the time course effect of NF‐κβ signaling following acute resistance exercise in humans.PURPOSETo observe the effects of an acute lower‐body resistance exercise protocol and subsequent recovery on intramuscular NF‐κβ signaling.METHODSTwenty‐eight untrained males (22.3 ± 3.2 y, 1.7 ± 0.1 m, 81.1 ± 15.5 kg) were assigned to either a control (CON; n=11) or exercise group (EX; n=17) and completed a lower‐body resistance exercise protocol consisting of the back squat, leg press, and leg extension exercise. Skeletal muscle microbiopsies were obtained from the vastus lateralis pre‐exercise (PRE), 1‐hour (1HR), 5‐hour (5HR), and 48‐hours (48HR) post‐resistance exercise. Multiplex signaling assay kits (EMD Millipore, Billerica, MA, USA) were used to quantify the total protein (TNFR1, c‐Myc) or phosphorylation status of proteins (IKKa/b, NF‐κβ, IkB) specific to apoptotic signaling pathways using MAGPIX® (Luminex, Austin, TX, USA). Mann Whitney U analysis was used to determine the effects of the exercise bout on intramuscular signaling at each timepoint. Additionally, change scores were analyzed by magnitude based inferences to determine a mechanistic interpretation.RESULTSMann‐Whitney U analysis revealed that resistance exercise increased total c‐Myc at 1H (p=0.001) and tended to increase with exercise (p=0.053) at 5H. A trend for an increase in IkB phosphorylation was observed at 5H (p=0.053). There were no other significant differences observed. Magnitude based inferences revealed a “Very Likely” increase in total c‐Myc from PRE‐5H and a “Likely” increase in IkB phosphorylation from PRE‐5H.CONCLUSIONIt appears that c‐Myc transcription factor and phosphorylation of IkB are elevated following acute intense resistance exercise in untrained males. These data may indicate that NF‐κβ signaling plays a role in skeletal muscle regeneration following resistance exercise in young untrained males.
Methylsulfonylmethane (MSM) Treatment Enhances C2C12 Wound Closure and Protects Cells from Oxidative Stress
Authors: Touchberry Chad D. , Von Schulze Alex , Amat‐Fernandez Clara , Lee Harold , Chow Ying , Wetmore Lori A 
Published: November 2023

MSM is known to provide anti‐inflammatory and anti‐oxidant effects in both animals and humans. Because both exercise and chronic disease have the potential to induce inflammation and oxidative stress in skeletal muscle, MSM may be a novel therapy to counter the negative consequences of these stressors on skeletal muscle function. Recent evidence has shown that MSM supplementation in human subjects can increase serum MSM concentrations to 2–3 mM. These serum concentrations of MSM have been associated with reduced muscle pain and reduced markers of muscle damage (creatine kinase) following damaging exercise. Therefore, MSM may protect muscle against damage or favorably influence the adaptive capacity of skeletal muscle under stress. However, it is not known if MSM induces this protection by directly acting on skeletal muscle. Therefore, our objective was to determine if MSM may provide cytoprotection from oxidative stress, or enhance the recovery processes in C2C12 myoblasts. We found that treating C2C12 myoblasts with increasing concentrations of MSM (0–200 mM) for 24, 48 and 72 hours decreased cell metabolism (via Alamar Blue) at high concentrations (100 & 200 mM); however this decline was not associated with increased cell death (via Trypan Blue). Flow cytometry analysis showed MSM treatment (200 mM) trapped myoblasts in G1 phase (+51%) and reduced the number of cells moving into S phase (DNA synthesis; −38%) or G2+M phase(mitosis; −43%) of cell division. MSM had no effect on C2C12 differentiation (d1, d4, d6). High concentrations of MSM (100 & 200 mM) were able to reduce cell death (−36% & −89% respectively) induced by oxidative stress (tert‐butyl hydrogen peroxide; 20 uM). Because heat shock proteins (HSPs) are known to be cytoprotective, we determined if increasing concentrations of MSM induced HSP expression; however, we found no changes in HSP70 and HSP27 expression. Lastly, lower concentrations (12.5 mM) of MSM enhanced wound closure (+35%) from a scratch assay 24 following treatment, while higher concentrations (200 mM) were associated with an impaired closure rate (−51%). While more applied research is needed, these findings suggest that MSM may directly alter the skeletal muscle response to stress and injury. Therefore, MSM supplementation may serve as an ergogenic aide to protect skeletal muscle from injury.Support or Funding InformationFunded by Bergstrom Nutrition to CDT
A Role for Titin During Doublet Potentiation of Fast and Slow Twitch Muscles
Authors: Monroy Jenna A , Bailey Elizabeth , Nishikawa Kiisa 
Published: November 2023

Doublet potentiation is the increase in muscle force as a result of the addition of a single pulse, or doublet, at the beginning of a low‐frequency stimulus train. The force output is higher in amplitude, generated at a faster rate, persists throughout activation. However, current muscle models fail to accurately predict muscle force during doublet potentiation. Recent work suggests that the elastic protein, titin, may play a role. In this study, we investigated the role of titin in doublet potentiation by using the muscular dystrophy with myositis (mdm) mouse, which is characterized by a deletion in the N2A region of the titin gene. Previous research on the winding filament hypothesis suggests that the N2A region of titin binds to the thin filaments upon activation, which increases titin stiffness and total muscle force. We hypothesized that the absence of N2A‐thin filament binding in mdm muscles reduces doublet potentiation. Using a servomotor force lever, we measured doublet potentiation in the soleus and extensor digitorum longus (EDL) muscles of wildtype and mdm mice at different muscle lengths. Potentiation was 20% lower in mdm than in wildtype soleus at all lengths (p = 0.04). In contrast to soleus, there was no difference in potentiation between wildtype and mdm EDL muscles (p = 0.7). In addition, potentiation was greater at shorter lengths than on the descending limb of the force‐length relationship in both soleus and EDL wildtype muscles. Mdm muscles did not show length dependence. Results from soleus muscles are consistent with the hypothesis that titin contributes to the increase in muscle force during doublet potentiation and that the lack of titin‐actin binding in mdm soleus muscles reduces doublet potentiation of muscle force. Differences in myosin isoforms, calcium flux, or titin isoforms, alone or in combination, may contribute to the observed differences between soleus and EDL in doublet potentiation.Support or Funding InformationThis work was funded by the Anderson Endowment at Denison University and NSF IOS‐1456868.
Peroxisomes in skeletal muscle protect against lipid‐induced insulin resistance
Authors: Noland Robert C. , Worsham Emily A. , Simon Jacob , Fuller Scott E. , Baes Myriam , Ghosh Sujoy , Mynatt Randall L. 
Published: November 2023

Excess intramuscular lipids are thought to play a causal role in the pathogenesis of insulin resistance; thus, strategies aimed at reducing this lipid burden offer therapeutic potential. Peroxisomes provide an intriguing option to meet this goal as they have the ability to process a broad spectrum of lipid species. As such, the purpose of this study was to determine if skeletal muscle peroxisomes protect against lipid‐induced insulin resistance. To test this hypothesis, we developed a muscle‐specific peroxisome‐deficient mouse model by deleting Pex5 in skeletal muscle (Pex5m−/−) and weaned them onto a moderate fat (25%) diet to which they had ad libitum access for 20 weeks. Pex5m−/− mice had similar body weight and composition when compared to Pex5fl/fl littermate controls, but exhibited impaired glucose tolerance. This impairment in whole body glucose homeostasis was associated with a 25–30% decrease in mitochondrial function in Pex5m−/− mice, suggesting that peroxisomes helped protect against mitochondrial dysfunction in skeletal muscle. To further test this potential link between peroxisomes and mitochondria in skeletal muscle we analyzed peroxisomal adaptations in a murine model where mitochondrial fatty acid oxidation was limited via muscle‐specific deletion of the rate‐limiting enzyme of mitochondrial lipid entry, Cpt1b (Cpt1bm−/−). As expected, the limitation in mitochondrial fatty acid entry in Cpt1bm−/−mice resulted in a compensatory upregulation of peroxisomes which helped maintain adequate lipid oxidation. These adaptations were associated with robust improvements in glucose tolerance in Cpt1bm−/−mice. To test the importance of peroxisomal adaptations in mediating these improvements, we developed a Pex5:Cpt1bm−/−mouse model to prevent peroxisomal compensation in the Cpt1bm−/− strain. Results from these studies revealed that skeletal muscle fatty acid oxidation was severely compromised in Pex5:Cpt1bm−/−and that abrogation of peroxisomal function prevented the improvement in glucose tolerance that was incurred by muscle‐specific Cpt1b deletion alone. Overall these results suggest that peroxisomes in skeletal muscle play an important role in maintaining whole body glucose homeostasis and that this is likely at least partially attributed to the fact that peroxisomes provide an alternate route for lipid disposal which limits mitochondrial lipid overload.Support or Funding InformationThis work utilized PBRC core facilities (Genomics, Transgenic and Animal Phenotyping, and Animal Metabolism and Behavior) that are supported in part by COBRE (NIH 8 P20‐GM103528) and NORC (NIH 2P30‐DK072476) center grants from the National Institutes of Health. This research was supported by NIH grants R01DK103860 (R.C.N.), R01DK089641 (R.L.M.), and a pilot and feasibility grant from NORC 2P30‐DK072476 (R.C.N.). Also, S.E.F. is supported by T32 fellowship NIH T32 AT004094.
Assessment Of Different Trophoblast Cell Lines As In Vitro Models For Placental Development
Authors: Daoud Georges , Barrak Joanna , Abou‐Kheir Wassim 
Published: November 2023

BackgroundThe placenta is a temporary organ of pregnancy that is involved in insuring optimal growth and development of the fetus. Since the 1980s, there has been a growing interest in the isolation of villous trophoblasts from human placenta for primary culture. Although very interesting, isolated primary trophoblasts have the disadvantage of being extremely difficult to maintain in culture which motivated investigators to generate villous and extravillous trophoblastic cell lines. Nevertheless, when aspiring to extrapolate the results they obtain in the trophoblastic cell lines to the trophoblasts in the placenta, the scientific community must be cautious as to whether these cell lines are truly representative of the physiologic setting.AimOur study raises questions regarding the validity of using the choriocarcinoma cell lines (BeWo, JEG‐3, JAR) vs. the extravillous cell line (HTR‐8/SVneo) as in vitro model systems for human trophoblast studies.MethodsImmunofluorescence staining was used to investigate the expression of CK7, e‐cadherin and vimentin in BeWo, JEG‐3, JAR and HTR‐8/SVneo cell lines. RT‐PCR allowed the measurement of mRNA levels of CK7, epCAM and vimentin in BeWo, JEG‐3, JAR and HTR‐8/SVneo while western blots assessed the protein expression of CK7, e‐cadherin and vimentin in BeWo, JEG‐3, JAR and HTR‐8/SVneo.ResultsBeWo, JEG‐3 and JAR cell lines all expressed CK7, e‐cadherin and epCAM and did not express vimentin while HTR‐8/SVneo cell line showed lower expression of CK7 and e‐cadherin and expressed vimentin. Additionally, two populations were observed in HTR‐8/SVneo cell line: a CK7+/Vimentin‐ population vs. CK7‐/Vimentin+ population.ConclusionOur study indicated that even though BeWo, JEG‐3 and JAR cell lines have proved to be epithelial trophoblastic cell lines, HTR‐8/SVneo has been disqualified in that regard and results based on this cell line should be controlled in primary trophoblast models.Support or Funding InformationMedical Practice plan (MPP), American University of Beirut Medical Center, Beirut, Lebanon