bluuebunny/crossref_metadata_2025_split huggingface dataset:
Estradiol treatment and exercise improve hepatic mitochondrial outcomes in mice following ovariectomy
Authors: Fuller Kelly N.Z. , Houchen Claire , Choi Michael , Allen Julie A. , McCoin Colin S. , Morris E. Matthew , Von Schulze Alex T. , Thyfault John P. 
Published: June 2021

We recently reported that female mice have increased hepatic mitochondrial respiratory capacity, and differential responses to exercise compared to male mice. Here we sought to determine the role of estrogen in the enhanced hepatic mitochondrial function found in female mice. Female C57BL mice (n= 4–6 per group) were randomly assigned to sham surgery (Sham), ovariectomy (OVX), or OVX plus estradiol replacement therapy from ~8 to 13 weeks of age. Half of the mice in each group were assigned to sedentary (SED) or voluntary wheel running (VWR). All mice had ad libitum access to high‐fat diet (HFD). We assessed isolated hepatic mitochondrial respiratory capacity using the Oroboros O2k with both pyruvate and palmityl‐carnitine as substrates. VWR reduced basal respiratory capacity of palmityl‐carnitine across all treatment groups. The OVX+Est group displayed increased State 3 respiratory capacity with pyruvate. Interestingly, in the absence of estrogen (OVX), both VWR and exogenous estradiol treatment independently improve the coupling control ratio (Basal/State 3: representative of coupling efficiency) and leak control ratio (Basal/Uncoupled: indicative of free ETS capacity) with pyruvate. We further assessed liver whole homogenate electron transport chain (ETC) protein content and found independent and combined effects of estradiol and exercise that were complex‐specific. The OVX group had a unique compensatory effect of increased Complex I protein with VWR, that was diminished with estradiol add‐back. Further investigation of treatment effects for complexes I‐IV, suggest that statistical differences are driven by the OVX. Interestingly, the sedentary OVX+Est group presented similarly to the Sham VWR group. In conclusion, these data suggest that ovariectomy leads to reduced hepatic mitochondrial respiratory control, and that estradiol replacement and modest exercise can aid in rescuing this phenotype.Support or Funding InformationThis work was funded by VA Merit Review: 1I01BX002567‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hindlimb Denervation Alters the Regulation of Autophagy and Mitophagy
Authors: Triolo Matthew , Hood David A 
Published: June 2021

During chronic muscle disuse, fibre atrophy occurs concomitantly with reductions in mitochondrial content and function. The reduction in organelle content is due, in part, to depressed mitochondrial biogenesis and elevations in intracellular degradation processes. The regulation of mitochondrial biogenesis has been studied extensively. However, the processes that underlie the degradation of mitochondria have yet to be fully elucidated. The breakdown of the organelle is due to a selective form of autophagy, termed mitophagy. When a mitochondrion becomes dysfunctional it dissociates from the mitochondrial network within the muscle and is subsequently degraded by lysosomes. The objective of this work is to better understand the processes of autophagy and mitophagy in the context of muscle disuse. Accordingly, we employed a hindlimb denervation protocol in which we unilaterally sectioned the peroneal nerve of one hindlimb, using the contralateral limb as a control in Sprague‐Dawley rats. Protein measurements of autophagy and mitochondrial content markers were made at 1, 3 and 7 days post‐denervation. We observed significant 25–30% (p<0.05) reductions in extensor digitorum longus (EDL) and tibialis anterior (TA) muscle mass by 7 days post‐denervation. Significant elevations in the autophagy proteins Tfeb, Beclin1 and ATG‐7 were also measured 7 days post denervation, while mitochondrial protein COX subunit‐IV and COX enzyme activity were reduced by 25% and 40%, respectively. However, SS and IMF mitochondrial respiratory capacity was diminished, accompanied by enhanced reactive oxygen species production, as early as 1–3 days following denervation. To investigate the early changes in autophagy and mitophagy flux that may explain the alterations in mitochondrial content and function, a subgroup of animals was treated with colchicine (4mg/kg/day) for 2 days to inhibit autophagic breakdown in response to 1 and 3 days of denervation. LC3‐II autophagy flux was increased as early as 1 and 3 days post‐denervation, but diminished by 7 days, in part as a result of a denervation‐induced increase in LC3‐II expression. Mitophagy flux was increased in parallel in SS mitochondria, and by 3 days in the IMF subfraction. These changes appeared to coincide with the declines in mitochondrial function, suggesting that although mitophagy is elevated, it is not sufficient to eliminate all of the dysfunctional organelles. Furthermore, the immediate elevations in autophagy/mitophagy flux at 1 and 3 days occurred prior to the upregulation of autophagy and lysosomal markers at 7 days post‐denervation, suggesting that the intrinsic activity of the autophagosomal breakdown pathway is sufficient in the early time course, but is subsequently upregulated to meet the demands of prolonged denervation.Support or Funding InformationSupported by NSERC, CanadaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myotube Morphology and Protein Metabolism are Negatively Regulated by Chemotherapy Drugs
Authors: Mora Stephen , Adegoke Olasunkanmi John 
Published: June 2021

Cachexia, a condition prevalent in many cancer patients, is characterized by weight loss and fatigue resulting from decreases in muscle mass and function. Severity of cachexia negatively correlates with treatment outcomes, drug toxicity and survival. Tumor burden and cancer related‐malnutrition have been implicated in the development of the condition. Other studies have suggested a causative link between chemotherapy treatment and cachexia. To understand the mechanisms of effects of these drugs on cachexia, we investigated the effects of a common chemotherapy drug cocktail on myotube morphology, myofibrillar protein abundance, and mTORC1 signalling. On day 4 of differentiation, myotubes were treated with vehicle or a chemotherapy drug cocktail (a mixture of cisplatin (20 μg/mL), leucovorin (10 μg/mL), and 5 fluorouracil (50 μg/mL)). Compared to myotubes treated with vehicle, those treated with the drug cocktail showed dysmorphic shape and shrinkage. Drug treatment also induced a 50% decrease in the abundance of myosin heavy chain (n = 5 independent experiments, P = 0.0001), 80% reduction in troponin and tropomyosin (n=3–4, P < 0.0003) by day 6 of differentiation. To explore the reasons for the low abundance of these myofibrillar proteins, we examined treatment effects on mTORC1 (mammalian/mechanistic target of rapamycin complex 1), a signaling complex whose activity is vital for muscle anabolism. Myotubes treated with the drug cocktail showed 75% reduction in the phosphorylation of mTORC1 activator AKT (n = 3, P = 0.0061), and ≥70% reduction in phosphorylation status of mTORC1 substrates ribosomal protein S6 and its kinase, S6K1 (n = 3, P < 0.03). Drug treatment also led to 50–75% decreases in markers of mitochondria content cytochrome C oxidase (COX IV) and pyruvate dehydrogenase (P < 0.05). Because of the link between AKT activation and regulation of both protein synthesis and proteolysis, these data suggest that chemotherapy drugs decrease the abundance of myofibrillar proteins likely through the modulation of pathways that regulate protein synthesis and degradation, and mitochondrial content. To improve treatment outcomes and quality of life of patients, it is critical to identify interventions that can limit the negative effects of these drugs on muscle protein status and mitochondrial content.Support or Funding InformationNatural Science and Engineering Research Council of CanadaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diaphragm Fiber Morphology of Ts65Dn Mice: A Model of Down Syndrome
Authors: Pascolla Jessica I. , Recceno Candace N. , Liang Chen , Greco Jacob , DeRuisseau Lara R. , DeRuisseau Keith C. 
Published: June 2021

Down Syndrome (DS) is a developmental disability that results from triplication of chromosome 21. Roughly 40% of people with DS experience sleep apnea. Consistent with this finding, the Ts65Dn mouse model of DS was previously shown to display hypoxemia. Our interests center on the involvement of the nervous system and diaphragm in the etiology of hypoxemia in the Ts65Dn mouse. We hypothesized that Ts65Dn mice would exhibit a greater number of apneas and proportion of diaphragm fast twitch fibers, which may be associated with hypoxia in these mice. Male Ts65Dn mice and colony controls (WT) (n=8/group) were studied at 3 months of age using barometric plethysmography to determine the occurrence of apneas (mean±SD) over a thirty‐minute evaluation. At ten months of age the spinal cord and diaphragm tissues were harvested from mice under isoflurane anesthesia. Ts65Dn mice displayed more apneas (36.3±28.0) than WT (7.1±9.1) mice at 3 months of age (p<0.05). Diaphragm fiber type distributions and areas were determined from a subset of mice (n=2/group). Compared to WT controls, diaphragm muscles from Ts65Dn mice displayed smaller Type I/IIa and Type IIx fibers (18.8% and 5.6% smaller, respectively) and larger Type IIb fibers (17.7% larger). The distribution of Type I/IIa fibers was similar in Ts65Dn mice compared to WT (64.1% vs. 58.8%) and distribution of Type IIb fibers was less than WT (4.7% vs. 8.2%). The distribution of Type IIx fibers was similar between the groups (31.1% DS; 33.0% WT). Statistical analyses were not performed on diaphragm morphological comparisons due to the small sample size. Our preliminary analyses indicate the Type IIb fibers, in particular, of Ts65Dn diaphragms mice may be larger and less prevalent compared with control diaphragms. Our work continues to expand upon these findings by including a larger sample size to achieve appropriate statistical power and incorporating phrenic motoneuron counts in spinal cord sections.Support or Funding InformationSupported by NIH 1R15HD076379‐01A1, 1R15HD076379‐01A1S1, and 1R15HD076379‐01A1S2This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Limited Fatty Acid Oxidation (FAO) in CPT2 Knockout Myocytes Associates with Insulin Resistance and Cell Stress: possible role of acylcarnitine lipotoxicity
Authors: Blackburn Michael L. , Ono‐Moore Kikumi D. , Hoppel Charles L. , Ellis Jessica M. , Adams Sean H. 
Published: June 2021

Lipotoxicity (the accumulation of fat‐derived metabolites that activate cell stress pathways) has been implicated in muscle insulin resistance. We have proposed that lipotoxicity, and in particular accumulation of long‐chain acylcarnitines (LCACs), also contributes to the episodic myopathy and rhabdomyolysis that is evident in persons with inherited disorders of long‐chain FAO. Many studies in the arena of LCAC bioactivity have employed exogenous application of LCACs to cells; few studies have modified endogenously‐produced LCACs to determine myocyte outcomes. Thus, conclusions about the (patho)physiological relevance of LCAC‐associated lipotoxicity have been difficult to determine. Herein, we used CRISPR‐Cas9 technology to knock out carnitine palmitoyltransferase‐2 (CPT2) in murine C2C12 myotubes: as expected, this maneuver led to almost complete inhibition of FAO, plus significant cellular and media accumulation of palmitoylcarnitine in palmitate‐treated cells. In CPT2 KO myotubes, treatment with 500 μM palmitate led to ~2‐fold higher production of IL‐6 (a myocyte cell stress marker) and a ~1.5 to 2‐fold increased release of adenylate kinase (cell death and permeability marker) at 3 and 6 hr post‐treatment (when compared to palmitate‐treated wildtype control cells; n=4/treatment). Furthermore, CPT2 KO myotubes displayed higher sensitivity to palmitate‐induced insulin resistance: the % inhibition in terms of insulin‐induced p‐Akt abundance was 60%, 80%, and 95% in CPT2 KO cells (at 200, 500, and 750 μM palmitate, respectively), vs. 38%, 53%, and 67% in controls (p<0.01, n=8/treatment). The results indicate that limitation of long‐chain FAO by loss of CPT2 in muscle cells elicits cell stress and insulin resistance, consistent with a working model in which lipotoxicity and accumulation of LCAC trigger these outcomes. Further studies are needed to determine if endogenously‐produced LCACs contribute to the phenotypes observed in CPT2 KO myotubes, or if LCAC‐independent mechanisms are involved. It remains to be established whether these phenomena underlie the cardiac and skeletal muscle myopathies typical of inherited disorders of FAO, or contribute to the insulin resistance accompanying type 2 diabetes.Support or Funding InformationThis work was supported by USDA‐ARS Project 6026‐51000‐010‐05S.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased Fiber Excitability Does Not Contribute to Post‐Tetanic Potentiation in Mice
Authors: LE GENGYUN , Baumann Cory W , Warren Gordon L , Lowe Dawn A 
Published: June 2021

PurposePost‐tetanic potentiation (PTP) is the enhancement of twitch force following tetanic stimulation and is an important component when studying skeletal muscle physiology and plasticity. Mechanistically, it is plausible that PTP could be associated with an increase in fiber excitability and/or any event following sarcoplasmic reticulum (SR) calcium release (i.e., at the contractile apparatus). Therefore, the goal of this study was to determine if increased fiber excitability contributes to PTP.MethodsAdult female C57BL/6J mice were chronically implanted with stimulating electrodes on the left common peroneal nerve and electromyographic (EMG) electrodes on the left tibialis anterior (TA) muscle. Following surgical recovery (at least 2 weeks after EMG electrode implantation), in vivo PTP of the anterior crural muscles was performed with simultaneous M‐wave recording from the TA muscle. To measure PTP, baseline twitch torque production was measured using a 0.1‐ms electrical stimulus pulse. Ten seconds later, muscles were stimulated using 0.1‐ms pulses at 100 Hz for 1 s to produce a prolonged, isometric tetanic contraction. Starting 2 s later, two post‐tetanic twitches were elicited with 2 s between the two. Changes in twitch torque (i.e., PTP) and M‐wave amplitude were calculated as percent increases in twitch torque and the corresponding M‐wave from baseline to the highest values following tetanic stimulation. All data were analyzed using paired t‐tests and are reported as means ± SE.ResultsTwitch torque at baseline was 0.45 ± 0.07 mN·m and represented the unpotentiated twitch. Following the tetanic stimulation, twitch torque increased by 34.0 ± 7.3% to 0.60 ± 0.07 mN·m (p=0.01). However, no change was detected in twitch M‐wave amplitude from before to after the tetanic stimulation (0.37 ± 0.03 to 0.39 ± 0.04 mV, p=0.48).ConclusionOn the basis of the disproportionate increase in twitch torque (34%) compared to the corresponding M‐wave amplitude increase (5%) after tetanic stimulation (p=0.02), we suggest that the PTP was not due to an increase in fiber excitability. Instead, it is more likely that PTP was attributable to an enhancement at or distal to SR calcium release such as phosphorylation of myosin regulatory light chain, which is consistent with previously reported mechanisms underlying PTP.Support or Funding InformationSupported by NIH grants R01‐AG031743 and T32‐AR007612, and Metropolitan State University Professional Development Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CRISPR/Cas9‐mediated Genome Editing in Patient‐Derived iPSC‐Cardiomyocytes Recapitulates an MYH6‐R443P Phenotype in a HLHS Family
Authors: Kim Min‐Su , Lough John , Geurts Aron , Mitchell Michael , Tomita‐Mitchell Aoy 
Published: June 2021

Hypoplastic Left Heart Syndrome (HLHS) is a clinically and anatomically severe form of Congenital Heart Disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. We previously demonstrated that rare variants in the α‐myosin heavy chain (MYH6) gene are significantly enriched in HLHS (p<1×10−5, observed in 10.5% or 20/190 HLHS subjects). Analysis of clinical outcomes showed that survival without cardiac transplant was reduced in HLHS subjects with MYH6 variants (p=6×10−3) as compared to those without MYH6 variants. Transcriptome and protein expression analyses from cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β‐myosin heavy chain (MYH7) gene in subjects with MYH6 variants. Visualizing α actinin in atria showed intrinsic disrupted sarcomere structure in HLHS patients with MYH6 variants.We observed that patient‐specific induced pluripotent stem cells (iPSCs) were defective in cardiomyogenic differentiation in vitro. Additionally, sarcomere structure in cardiomyocytes derived from iPSCs produced from a family trio (unaffected mother‐affected father‐patient) carrying a variant in MYH6 (MYH6‐R443P) was dysmorphic.We introduced the MYH6‐R443P variant into iPSCs derived from the unaffected parent using CRISPR/cas9 technology and observed that the MYH6‐R443P phenotype in HLHS‐iPSCs was recapitulated; specifically, gene‐edited MYH6‐R443P+/− and MYH6‐R443P−/− iPSCs displayed defective cardiomyogenic differentiation, and sarcomere structure in these cardiomyocytes was dysmorphic. We conclude that the etiology of MYH6‐associated HLHS may be informed utilizing iPSCs.Support or Funding InformationThe Advancing a Healthier Wisconsin Endowment (AHW)Wolfe Family FoundationGreat Milwaukee FoundationChildren's Research InstituteLittle Heart for LifeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Auto‐antibodies Targeting Components of Sarcolemma Repair Represent a Pathogenic Mechanism in Idiopathic Immune Myopathies
Authors: McElhanon Kevin E. , Capati Ana , Young Nicholas , Hampton Jeffrey , Paleo Brian J. , Beck Eric X , Sahenk Zarife , Aggarwal Rohit , Oddis Chester V. , Jarjour Wael N. , Weisleder Noah 
Published: June 2021

Idiopathic immune myopathies (IIM) represent a group of disorders causing chronic inflammation and significant damage to skeletal muscle due to an unchecked autoimmune response. The main challenge slowing study of IIM is an understanding of the pathogenic mechanism responsible for initiation and progression of skeletal muscle inflammation. Two mouse models of IIM have recently provided new insights into the pathogenesis of the disease. The synaptotagmin VII null (Syt VII−/−) mouse is characterized by defects in membrane resealing and presents with a mild form myositis at 2 months of age. A more robust model of IIM combines knock‐out of Syt VII with a FoxP3 mutation resulting in a mouse with impaired membrane resealing and regulatory T‐cell deficiency. Adoptive transfer of lymphocyte preparations isolated from this double mutant mouse model to recombination‐activating gene 1 (RAG‐1) null mice results in severe skeletal muscle inflammation. Plasma membrane resealing is a highly conserved mechanism allowing an injured cell to survive following a disruption to the lipid bilayer that compromises barrier function. Given the importance of plasma membrane barrier function in preventing exposure of intracellular antigens to the immune system, specifically in an immune‐compromised environment, it is possible that compromised sarcolemma resealing could drive pathogenesis of IIM. We show, for the first time, that a deficiency in T‐regulatory cells is not sufficient to induce sarcolemma fragility, however, purified antibodies against critical proteins facilitating the sarcolemma repair process are sufficient to reduce membrane integrity. We also demonstrate that sarcolemma integrity is reduced in distal skeletal muscle in the absence of inflammation in our novel murine model of IIM. We have established by direct ELISA that auto‐antibodies against multiple proteins involved in sarcolemma repair are elevated in IIM patient sera and find that exogenous delivery of IIM positive patient serum can compromise sarcolemma resealing in healthy skeletal muscle. These findings represent a novel mechanism that drives the progression of IIM when decreased sarcolemma integrity induces a vicious cycle of antigen presentation that directly contributes to the pathophysiology of idiopathic immune myopathies.Support or Funding InformationNIH, National Institute of Arthritis and Musculoskeletal and Skin Diseases Award Number F31AR071745This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adaptations of Bone and Bone Vasculature to Muscular Stretch Training
Authors: Barsoum Mina‐Michael , Hotta Kazuki , Delp Michael , Ghosh Payal , Behnke Bradley , Churchill Alexandra , Boyd Brandon , Muller‐Delp Judy Marie 
Published: June 2021

The magnitude of bone formation and remodeling is linked to both the magnitude of strain placed on the bone and the perfusion of bone. We have previously reported an increase in bone perfusion and bone density that occurs in the femur of old rats with moderate aerobic exercise training. The purpose of this study was to determine the acute and chronic effects of static muscle stretching on bone blood flow and to determine whether chronic stretching affects bone remodeling. Old male Fischer 344 rats were randomized to either a naïve or stretch‐trained group. Static stretching of ankle flexor muscles was achieved by placement of a dorsflexion splint on the left ankle for 30 min/day, 5d/wk for 4wks. The right hindlimb served as a contralateral control (unstretched) limb. Bone blood flow was assessed during and after an acute bout of stretching in naïve rats, and at rest and during exercise in stretch‐trained rats. Following 4 weeks of daily muscle stretching, micro‐CT analysis was used to determine the volume and density of bone and the bone vasculature in the tibia. In naïve rats, placement of the splint increased blood flow to the proximal tibial metaphasis. After 4 weeks of daily stretching, blood flow to the proximal tibial metaphysis was higher in the chronically stretched limb during exercise. Chronic stretching also increased tibial bone weight, and increased total bone volume in both the proximal and distal tibial metaphyses. In trabecular bone immediately below the proximal tibial growth plate, the total volume of bone was increased, but the bone density was not changed. Total intravascular volume also increased in the region of the bone below the growth plate. These data suggest that static muscle stretching increases loading of bone and bone blood flow, leading to bone remodeling and growth which is matched by an increase in bone intravascular volume.Support or Funding InformationNIH R15AG055029This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.