bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cytokines IL‐6 and LIF can differentially regulate myotube protein synthesis
Authors: GAO SONG , Carson James A. 
Published: November 2023

IL‐6 and LIF are members of the IL‐6 family of cytokines that have recognized paradoxical regulatory roles in skeletal muscle mass, but there are gaps in our understanding of the cellular mechanisms that contribute to this regulatory dichotomy. The IL‐6 family of cytokines can induce several upstream regulators of mTORC1, including PI3K/Akt, ERK1/2 and AMPK; these pathways have the ability to regulate muscle mTORC1 signaling and protein synthesis. However, mTORC1 signaling regulation of by the IL‐6 family of cytokines remains poorly understood. Therefore, we investigated the effects of IL‐6 and LIF dose and duration on mTORC1 signaling and protein synthesis in myotubes. C2C12 myotubes were treated with varying doses of IL‐6 or LIF for either a short (1–4h) or long duration (24h). Signaling through the gp130 receptor and downstream effectors, including PI3K/Akt, STAT3 and AMPK were investigated by specific siRNA or pharmaceutical inhibitors administration. The Akt‐mTORC1 signaling axis and protein synthesis was induced by short term IL‐6 or LIF administration, which was blocked by siRNA knock‐down of gp130 receptor or pharmaceutical inhibition of Akt signaling. Interestingly, inhibition of mTOR or STAT3 signaling decreased basal myotube protein synthesis, but did not block the protein synthesis induction by short term IL‐6 or LIF administration. Long term IL‐6 or LIF administration suppressed Akt‐mTORC1 signaling and protein synthesis, which coincided with increased AMPK phosphorylation. However, inhibition of AMPK signaling by siRNA knock‐down did not rescue the protein synthesis suppression by long term IL‐6 or LIF treatment. These results demonstrate that IL‐6 family of cytokines have the capacity to either activate or suppress myotube mTORC1 signaling and protein synthesis, and the ultimate effect depends on the degree of the cytokine exposure.Support or Funding InformationR01 CA121249 to J. A. Carson from the National Cancer Institute, P20 RR‐017698 to J. A. Carson from National Center for Research Resources Grant
Characterizing the Role of Muscle‐Specific RING Finger 1 (MuRF1) as a Regulator of Atrophy‐Induced Gene Expression
Authors: Kakareka Karina , Waddell David 
Published: November 2023

Skeletal muscle wasting occurs as a consequence of numerous physiological conditions, including denervation, immobilization, and aging. The E3 ubiquitin ligases, MuRF1 and MAFbx, are induced under nearly all atrophy conditions and are believed to play a key role in protein degradation. Preliminary data presented here provides intriguing evidence that MuRF1 may also act as a transcriptional modulator of muscle atrophy‐induced gene expression. The transcriptional regulation of MuRF1 and MAFbx were analyzed using a reporter gene system and exhibited repression in C2C12 cells overexpressing MuRF1. Furthermore, ectopic expression of the myogenic regulatory factors (MRFs), MyoD and myogenin, caused significant activation of the MuRF1 and MAFbx reporter gene constructs, while co‐overexpression of MuRF1 with MRFs resulted in reversal of MRF induction of reporter gene activity. Ectopic expression of a catalytically dead MuRF1 RING mutant failed to reverse MRF activation of the reporters, suggesting that ubiquitin ligase activity may be important for MuRF1 transcriptional regulation. In order to further investigate a potential mechanism for MuRF1 modulation of MRF activity, Western blot analysis was performed to analyze MRF protein expression levels in C2C12 cells overexpressing MuRF1 or MuRF1 RING mutant. Cells with ectopic MuRF1 or MuRF1 RING mutant showed repressed levels of myogenin. Additionally, cells overexpressing MuRF1 and MuRF1 RING mutant treated with MG132 showed a modest rescue of myogenin, but myogenin protein levels were still reduced. Finally, chromatin immunoprecipitation (ChIP) was performed in order to analyze MRF occupancy of the MuRF1 promoter. As expected, MRF overexpression resulted in increased MuRF1 promoter immunoprecipitation. Interestingly, co‐overexpression of MuRF1 with MRFs resulted in a reversal of promoter occupancy. These findings suggest that MuRF1 may regulate MRF transcriptional activity by interfering with promoter binding, which provides additional insight into the potential mechanism by which MuRF1 may act to transcriptionally regulate atrophy‐induced gene expression.Support or Funding InformationThis work was supported by University of North Florida Transformational Learning Opportunity Grants to D.W.
Deletion of Kvβ2 Alters Murine Skeletal Muscle Morphology and Phenotype
Authors: Tipparaju Srinivas M , Tur Jared , Chapalamadugu Kalyan C 
Published: November 2023

IntroductionMammalian skeletal muscle expresses various potassium channels that regulate myogenesis. Recent evidence points to the role of Kv1.5 in cell cycle progression in myoblasts as pharmacological inhibition of this channel in L6E9 cells inhibited G0/G1 transition. Additionally, skeletal muscle also expresses voltage‐gated potassium channel beta subunits 1–3 (Kvβ, 2, 3) of aldo‐keto reductases (AK6) family, although their role in skeletal myogenesis is not yet known. Given that Kvβ subunits binds to and regulate members of Kv1 family including Kv1.5, it is plausible that Kvβ subunits play a significant role in skeletal myogenesis.Hypothesiswe hypothesized that Kvβ2 modulates skeletal myogenesis.MethodsAge matched, 8–12 week old, wild type (Wt) and Kvβ2 knockout mice (KO) mice were used in this study. Transcript expression analysis of key transcription factors, muscle growth regulators and contractile genes was conducted utilizing real time quantitative PCR (qPCR). Myofiber size was estimated in biceps femoris muscle cross‐sections using Hematoxylin and eosin staining. Protein expression was assessed by Western blots in lysates isolated from Biceps femoris muscle.ResultsBody weight of KO mice recorded at 6, 11 or 16 weeks of age, was significantly (p < 0.05) less than that of age matched Wt mice. Body weight normalized to tibia length in 16 week old KO mice was nearly 30% less than that of age matched Wt mice. Biceps femoris muscle cross‐sections in KO mice revealed significantly smaller myofiber area when compared to that of Wt mice (p < 0.05). Myosin heavy chain mRNA expression profiles were also significantly different between the KO and Wt mice. when compared with that of Wt mice, gastrocnemius and biceps femoris of KO mice showed marginal (p < 0.1) to significantly (p < 0.05) higher expression of myosin heavy chain IIA and IIX isoforms, while myosin heavy chain I mRNA expression was not different between the groups. Soleus muscle of KO mice showed significantly higher myosin heavy chain I expression, while the expression of myosin heavy chain 2B was profoundly (p < 0.05) decreased. Interestingly, the expression of major myogenic regulatory factors was unchanged between the two genotypes, with the exception that MyoD expression showed a consistently decreasing trend in all three muscles of KO mice when compared with that of Wt. In C2C12 cells, Kvβ2 mRNA expression declined steadily during the course of differentiation reaching to approximately 10% of that observed before differentiation (p < 0.05).ConclusionOur data suggest that Kvβ2 subunit is essential for skeletal myogenesis. Kvβ2 appear to effect muscle fitness by altering myosin heavy chain composition, suggesting a significant role of Kvβ subunits in skeletal muscle size and phenotype determination.
Does Vehicle Matter on Skeletal Muscle Regeneration Study?
Authors: Srikuea Ratchakrit , Suhatcho Kanokwan 
Published: November 2023

Although lipid‐soluble and water‐soluble vehicles were routinely used in research investigation and clinical application, however, whether these vehicles have the effect on skeletal muscle regeneration after intramuscular injection are currently unseeing. Therefore, this study was designed to investigate the effects of intramuscular injections of lipid‐soluble vehicles [olive oil, 40% dimethyl sulfoxide (DMSO), and 40% propylene glycol (PG)] and water‐soluble vehicles [0.9% NaCl, phosphate‐buffered saline (PBS), 0.1% ethanol, and distilled water] on skeletal muscle regenerative capacity. Tibialis anterior muscle of young adult C57BL/6 mice was injured by 1.2% BaCl2 solution to induce skeletal muscle regeneration. Histological analysis on regenerating muscle fiber morphology was evaluated using hematoxylin and eosin staining. Expression of proteins that regulate skeletal muscle regeneration including skeletal muscle stem cell differentiation (myogenin) and regenerative muscle fiber formation (EbMHC) were investigated using immunohistochemical analysis. The results revealed that regenerating muscle fiber morphology, myogenin positive nuclei, and EbMHC protein expression were not significantly changed in almost tested vehicles compared to no vehicle administration. Nevertheless, presence of degenerative muscle fibers paralleled with increased number of myogenin positive nuclei and EbMHC protein expression were evident after intramuscular injections of 40% DMSO and 40% PG. In contrast, this deteriorate effect did not observe after intramuscular injections of 0.9% NaCl and distilled water which were the solutions that used for 40% DMSO and 40% PG preparation in this study. Moreover, the effects of 40% DMSO and 40% PG was comparable with 2% trypsin (degenerative substance) administration. These results suggest that intramuscular injections of 40% DMSO and 40% PG into regenerating muscle could lead to the recurrent of muscle injury during skeletal muscle regeneration. Hence, the applications of DMSO and PG as vehicle for intramuscular injection could potentially affect the experimental results that obtain during skeletal muscle regeneration study.Support or Funding InformationThis research project is supported by Mahidol University.
Regulation Of Histone 3 Acetylation By Intermittent Hypoxia
Authors: Nanduri Jayasri , Wang Ning , Semenza Gregg L , Prabhakar Nanduri R 
Published: November 2023

Acetylation of nucleosome core histone proteins determines the structure of chromatin, which regulates the accessibility of DNA sequences to the transcriptional machinery. In general, histone hyperacetylation activates gene transcription and histone hypoacetylation leads to gene silencing. Intermittent hypoxia (IH), a hallmark feature of sleep apnea, induces transcriptional activation of several genes. Whether histone acetylation contributes to IH‐induced gene transcription is not known. The goal of the present study was to determine the effects of IH on histone acetylation and assess the underlying mechanisms. Experiments were performed on rat pheochromocytoma (PC12) cells exposed to alternating cycles of 1.5% O2 for 30 sec followed by 20% O2 for 5 min. Exposure of cells to 60 cycles of IH increased global acetylation of histone 3 (H3) but not histone 4 (H4) in a stimulus‐dependent and reversible manner compared to cell exposed to normoxia (20% O2). Specific lysine residues K9, K14, K18 and K27 contributed to increased H3 acetylation by IH. Histone deacetylase (HDAC) inhibitors mimicked IH‐increased H3 acetylation under normoxic conditions. IH reduced HDAC3 and HDAC5 protein expression. Pharmacological blockade of NADPH oxidase with triazolo pyrimidine (VAS‐2870), a specific Nox inhibitor inhibited the increased H3 acetylation and decreased HDAC expression that were induced by IH. These results suggest that ROS generated by NADPH oxidase triggers IH‐induced HDAC downregulation, leading to increased H3 acetylation.Support or Funding InformationSupported by NIH‐PO1‐HL90554.
Chronic Intermittent Hypoxia Promotes Multiple Myeloma Development in Black/6 Mice
Authors: Kowkuntla Sandeep , Xu Hongwei , Tomasson Michael H , Bates Melissa 
Published: November 2023

Multiple myeloma (MM) is an incurable malignancy of bone marrow plasma B cells and is associated with obesity. Decreased levels of the lipokine adiponectin are associated with disease risk in obese patients with MM, but the mechanisms by which obesity contributes to MM remain unclear. Obesity is also associated with sleep apnea and chronic intermittent hypoxia (CIH) and MM cells develop aggressive features when exposed to hypoxia, so we hypothesized that CIH would facilitate disease progression in a mouse model of MM. 5TGM1 cells are a murine MM cell line derived from KaLwRij strain that fail to engraft efficiently in Black6 strain mice. We injected 5TGM1 MM cells into two cohorts of Black/6 mice, one control and one exposed to intermittent hypoxia for one week prior to injection and another three weeks after injection for a total of four weeks. None of the control mice developed disease after two months, but two of five (40%) CIH mice developed MM disease characterized by hind limb paralysis and bone marrow infiltration with GFP‐positive 5TGM1 cells. These results suggest that CIH may contribute to the development of MM. Further studies are warranted.Support or Funding InformationUniversity of Iowa Start‐Up Fund
Remodeling of neuronal properties in the preBötzinger Complex (preBötC) causes destabilized rhythm generation following chronic intermittent hypoxia (CIH)
Authors: Garcia Alfredo , Dashevskiy Tatiana , Ramirez Jan‐Marino 
Published: November 2023

CIH is a cardinal trait of conditions such as apneas of prematurity and sleep apnea. We have previously reported that CIH increases the irregularity of rhythm generation from the preBötC which leads to failed transmission to hypoglossal motor pool. This ongoing study aims to further understand how CIH affects preBötC neurons responsible for premotor generation of the inspiratory rhythm. Electrophysiological studies were conducted in the preBötC of isolated brainstem slices prepared from mice either exposed to room air or to CIH (10 days). Intracellular recordings from synaptically isolated preBötC neurons reveals that CIH exposure causes an increased rheobase and increased threshold for action potential generation. Moreover, in network, preBötC neurons generated fewer action potentials and tended to fire later during the network burst. Computational modeling of the respiratory network suggests that changes in intrinsic properties of preBötC neurons may alter their firing phenotypes (i.e., tonic vs. silent vs. burster). These may lead to relative changes in network composition that destabilizes rhythm generation. We conclude that intrinsic reconfiguration at the neuronal level is necessary for preserving network rhythm generation when experiencing recurrent perturbation such as CIH.Support or Funding InformationR01 HL126523; P01 HL090554
Response to Sustained Handgrip Exercise and Post‐Exercise Circulatory Arrest in Renal Denervation Responders and Non‐Responders
Authors: Hearon Christopher M , Howden Erin J , Verhees Myrthe , Lawley Justin S , East Cara , Stickford Abigail , Fu Qi , Levine Benjamin D 
Published: November 2023

PURPOSEHypertension is characterized by an exaggerated hemodynamic response to sympathoexcitatory stimuli such as exercise and metaboreflex activation. Renal Denervation (RDN) lowers blood pressure in some individuals with resistant hypertension, however the effect of RDN on reflex control of blood pressure remains unclear.METHODSA subset of subjects from the simplicity HTN‐3 study who underwent RDN (n=11) were classified as responders (R; n=7, 6M:1F, 61±10 years) or non‐responders (NR; n=4, 3M:1F, 57±4 years) based on a target decrease in office blood pressure of 20mmHg observed on at least 3 occasions post‐RDN (~27 months). Heart rate (HR), mean arterial blood pressure (MAP; plethysmography), and muscle sympathetic nervous system activity (MSNA, microneurography) were measured during supine rest, sustained handgrip exercise to fatigue (HGE; 40% maximal voluntary contraction), and post‐exercise circulatory arrest (PECA; 2 mins). Maximal changes (Δ) in HR, MAP, and MSNA (frequency: burst/min and total MSNA: AU/min) were calculated from baseline in response to HGE and PECA, and comparisons made using unpaired t‐tests and reported as mean ± SEM.RESULTSDuring supine rest, R had lower MAP (R: 97±7; NR: 118±8 mmHg, P<0.05), and similar HR (R: 70±4; NR: 75±9, P=0.26) and MSNA (R: 41.8±4; NR: 41±3 burst/min, P=0.50) compared to NR. During HGE, R had a similar rise in bursts frequency and a larger rise in total MSNA (ΔMSNA: R: 2897±825; NR: 382±573 AU/min, P<0.05) compared to NR. Despite this increase, the HR and BP response to HGE was similar between groups (ΔHR: R: 17±4; NR: 11±3 beats/min; ΔMAP: R: 33±8; NR: 27±7 mmHg, both P>0.05). During PECA, MSNA tended to remain elevated (ΔMSNA: R: 2345±687; NR: 775±493 AU/min, P=0.10) and HR was higher (ΔHR: R: 9±3; NR: −1±2 beats/min, P=0.02) in R compared to NR, while MAP was not different between groups (ΔMAP: R: 25±5; NR: 15±6 mmHg, P=0.21).CONCLUSIONR had a greater rise in MSNA in response to the same sympathoexcitatory stimuli (HGE, PECA) compared to NR. Despite this larger increase in MSNA, the HR and BP response to HGE and PECA was similar between groups. Thus, RDN may impact both central integration, and end organ responsiveness to sympathoexcitatory stimuli.
Leptin Increases Blood Pressure Acting via Transient Receptor Potential (TRP) Channels in the Carotid Body
Authors: Shin Mi Kyung , Hwang Min Woo , Yeung Bonnie Ho‐yee , Shirahata Machiko , Tang Winnie Wan‐yee , Sham James S.K. , Polotsky Vsevolod Y 
Published: November 2023

RationaleObesity leads to cardiovascular morbidity and mortality acting via multiple mechanisms including hypertension and obstructive sleep apnea. Obesity leads to high levels of adipokine leptin. Increased leptin levels have been previously reported in sleep apnea and implicated in increased sympathetic activity and the pathogenesis of hypertension. However, mechanisms of the effects of leptin on blood pressure are unclear. The carotid bodies (CB) express leptin receptor (LepR), but the mechanisms and consequences of leptin action in CB are unknown. We have previously demonstrated that leptin enhances carotid sinus nerve activity in response to hypoxia and this effect is abolished by non‐selective blockers of transient receptor potential (TRP) channels (Shirahata et al., 2015). We hypothesized that high levels of circulating leptin signal via LepR in CB leading to hypertension and that leptin's effect is mediated via TRP channels.Methods1) Male C57BL/6J mice (n=6) were implanted with telemetry in the left femoral artery for blood pressure monitoring at baseline, during leptin infusion (120ug/day for 3 days via a SC pump) before and after CB denervation; 2) LepR deficient db/db mice were implanted with telemetry; blood pressure measurements were performed followed by infection with adenovirus carrying the LepR gene (Ad‐LepR, n=5) or luciferase (control, Ad‐Luc, n=5) followed by BP measurements; 3) Expression of different TRP channels was measured in CB of wildype C57BL/6J, leptin deficient ob/ob mice and LepR deficient db/db mice; 4) TRP channel expression was measured in db/db mice infected with Ad‐LepR vs Ad‐Luc; 5) blood pressure monitoring was performed in male C57BL/6J mice (n= 9) at baseline, during leptin infusion with or without Trpm7 inhibitor, FTY720 (10mg/kg, sc, n=3) for 24 hrs.Results1) In mice with intact CB, leptin increased mean arterial pressure by 13 mm Hg during the day and by 16 mm Hg at night (p = 0.003 for the effect of leptin). CB denervation completely abolished leptin‐induced hypertension (p < 0.001). 2) wildtype C57BL/6J mice showed significantly higher CB expression of Trpm7 compared to ob/ob and db/db mice; 3) LepR expression in CB of db/db mice dramatically increased Trpm7 expression; 4) Trpm7 inhibitor FTY720 abolished leptin‐induced hypertension.ConclusionsLeptin increases blood pressure by acting via LepR and downstream Trpm7 channels in the CB.Support or Funding InformationHL133100, HL128970
High salt promotes NADPH oxidase‐dependent formation and activation of dendritic cells
Authors: Barbaro Natalia , Foss Jason David , Montaniel Kim Ramil , Chen Wei , Harrison David Glenn , Kirabo Annet 
Published: November 2023

It has become apparent that sodium can accumulate in the interstitium and promote inflammation through poorly defined mechanisms. We have previously shown that increased NADPH oxidase‐ dependent oxidative stress in dendritic cells (DCs) leads to formation of isolevuglandin (IsoLG)‐ modified proteins which act as neo‐antigens to activate T cells. We hypothesized that increasing sodium chloride (NaCl) in excess of the physiological interstitial space concentrations leads to monocyte differentiation into DCs in an NADPH oxidase‐dependent manner and formation of immunogenic IsoLGs. We exposed murine bone marrow cells to normal physiological NaCl (NS: 150 mM/L), elevated NaCl concentrations (HS: 190 mM/L), or HS plus gp91dstat, an inhibitor of the NADPH oxidase assembly. Compared to NS, HS significantly increased the number cells expressing the DC marker CD11c (NS: 14.4 ± 7.0 vs HS: 52 ± 11.7 % p<0.001), and this was prevented by co‐treatment with gp91dstat (39.8 ± 16.0 p<0.05 vs HS). In additional experiments, we found that exposure of monocytes isolated from human volunteers to HS, but not mannitol, caused a 2‐fold increase in formation of IsoLG‐modified proteins. This was associated with an increase in activation marker CD86 (NS: 466 ± 192 vs HS: 596 ± 324 MFI p<0.05) and production of inflammatory cytokines IL‐6, IL‐β and TNF‐α. Interestingly, these cells expressed surface markers indicative of transformation to activated DCs, as evidenced by their acquisition of surface marker CD83. In additional studies, we found that monocytes exposed to high salt for 7 days acquire a DC like morphology. Moreover, using flow cytometry, we confirmed that high salt exposure causes these cells to lose the monocyte marker CD14 (NS: 41.1 ± 15.4 vs HS: 19.9 ± 6.4 MFI; p<0.05), and gain the DC marker CD209 (NS: 24.2 ± 1.0 vs HS: 49.3 ± 0.7 MFI; p<0.001). Co‐treatment with gp91dstat and scavenging of IsoLGs during high salt exposure prevented conversion of monocytes into DCs. High salt dramatically increased mRNA expression of GM‐CSF (NS: 758 ± 440.8 vs HS: 5476 ± 2268 MFI; p<0.05), IL‐4 NS: 1868 ± 560.6 vs HS: 4867 ± 1152 MFI; p<0.05) and Flt3 (NS: 2526 ± 636.8 vs HS: 10014 ± 2370 MFI; p<0.05), which are known to mediate monocyte conversion to DCs. Thus, we have defined a novel pathway whereby high NaCl concentrations lead to transformation of monocytes to DCs due to increased formation of immunogenic isoketals. These observations provide insight into how elevated sodium environments lead to an inflammatory state.Support or Funding InformationThis work was supported by the American Heart Association grants 16POST29090001 and 14SFRN20420046, and National Institutes of Health grants 1K01HL13049701, 1R01HL12586501, and 5R01HL03900626