Mouse Quadriceps Research Articles

Targeting the transferrin receptor to transport antisense oligonucleotides across the mammalian blood-brain barrier.

Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.

The role of interleukin-10 in mitigating endoplasmic reticulum stress in aged mice through exercise.

Although unfolded protein response (UPR) is essential for cellular protection, its prolonged activation may induce apoptosis, compromising cellular longevity. The aging process increases the endoplasmic reticulum (ER) stress in skeletal muscle. However, whether combined exercise can prevent age-induced ER stress in skeletal muscle remains unknown. Evidence suggests that ER stress may increase inflammation by counteracting the positive effects of interleukin-10 (IL-10), whereas its administration in cells inhibits ER stress and apoptosis. This study verified the effects of aging and combined exercise on physical performance, ER stress markers, and inflammation in the quadriceps of mice. Moreover, we verified the effects of IL-10 on ER stress markers. C57BL/6 mice were distributed into young (Y, 6 mo old), old sedentary (OS, sedentary, 24 mo old), and old trained group (OT, submitted to short-term combined exercise, 24 mo old). To clarify the role of IL-10 in UPR pathways, knockout mice lacking IL-10 were used. The OS mice presented worse physical performance and higher ER stress-related proteins, such as C/EBP homologous protein (CHOP) and phospho-eukaryotic translation initiation factor 2 alpha (p-eIF2α/eIF2α). The exercise protocol increased muscle strength and IL-10 protein levels in OT while inducing the downregulation of CHOP protein levels compared with OS. Furthermore, mice lacking IL-10 increased BiP, CHOP, and p-eIF2α/eIF2α protein levels, indicating this cytokine can regulate the ER stress response in skeletal muscle. Bioinformatics analysis showed that endurance and resistance training downregulated DNA damage inducible transcript 3 (DDIT3) and XBP1 gene expression in the vastus lateralis of older people, reinforcing our findings. Thus, combined exercise is a potential therapeutic intervention for promoting adjustments in ER stress markers in aged skeletal muscle.NEW & NOTEWORTHY Aging elevates endoplasmic reticulum (ER) stress in skeletal muscle, potentially heightening inflammation by opposing interleukin-10 (IL-10) effects. This study found that short-term combined exercise boosted strength and IL-10 protein levels while reducing CHOP protein levels in older mice. In addition, IL-10-deficient mice exhibited increased ER stress markers, highlighting IL-10's role in regulating ER stress in skeletal muscle. Consequently, combined exercise emerges as a therapeutic intervention to elevate IL-10 and adjust ER stress markers in aging.

Competing endogenous RNA networks were associated with fat accumulation in skeletal muscle of aged male mice

Muscle aging contributed to morbidity and mortality in the elderly adults by leading to severe outcomes such as frailty, falls and fractures. Post-transcriptional regulation especially competing endogenous RNA (ceRNA) mechanism may modulate the process of skeletal muscle aging. RNA-seq was performed in quadriceps of 6-month-old (adult) and 22-month-old (aged) male mice to identify differentially expressed ncRNAs and mRNAs and further construct ceRNA networks. Decreased quadriceps-body weight ratio and muscle fiber cross-sectional area as well as histological characteristics of aging were observed in the aged mice. Besides, there were higher expressions of atrogin-1 and MuRF-1 and lower expression of Myog, Myf4 and Myod1 in the quadriceps of aged mice relative to that of adult mice. The expression of 85 lncRNAs, 52 circRNAs, 10 miRNAs and 277 mRNAs were significantly dysregulated in quadriceps between the two groups, among which two ceRNA networks lncRNA 2700081O15Rik/circRNA_0000820-miR-673–3p-Tmem120b were constructed. Level of triglycerides and expression of PPARγ, C/EBPα, FASN and leptin were elevated and the expression of adiponectin was reduced in quadriceps of aged mice compared with that of adult mice. LncRNA 2700081O15Rik/circRNA_0000820-miR-673–3p-Tmem120b were possibly associated with the adipogenesis and fat accumulation in skeletal muscle of age male mice.

Mitochondrial Transplantation's Role in Rodent Skeletal Muscle Bioenergetics: Recharging the Engine of Aging.

Mitochondria are the 'powerhouses of cells' and progressive mitochondrial dysfunction is a hallmark of aging in skeletal muscle. Although different forms of exercise modality appear to be beneficial to attenuate aging-induced mitochondrial dysfunction, it presupposes that the individual has a requisite level of mobility. Moreover, non-exercise alternatives (i.e., nutraceuticals or pharmacological agents) to improve skeletal muscle bioenergetics require time to be effective in the target tissue and have another limitation in that they act systemically and not locally where needed. Mitochondrial transplantation represents a novel directed therapy designed to enhance energy production of tissues impacted by defective mitochondria. To date, no studies have used mitochondrial transplantation as an intervention to attenuate aging-induced skeletal muscle mitochondrial dysfunction. The purpose of this investigation, therefore, was to determine whether mitochondrial transplantation can enhance skeletal muscle bioenergetics in an aging rodent model. We hypothesized that mitochondrial transplantation would result in sustained skeletal muscle bioenergetics leading to improved functional capacity. Fifteen female mice (24 months old) were randomized into two groups (placebo or mitochondrial transplantation). Isolated mitochondria from a donor mouse of the same sex and age were transplanted into the hindlimb muscles of recipient mice (quadriceps femoris, tibialis anterior, and gastrocnemius complex). The results indicated significant increases (ranging between ~36% and ~65%) in basal cytochrome c oxidase and citrate synthase activity as well as ATP levels in mice receiving mitochondrial transplantation relative to the placebo. Moreover, there were significant increases (approx. two-fold) in protein expression of mitochondrial markers in both glycolytic and oxidative muscles. These enhancements in the muscle translated to significant improvements in exercise tolerance. This study provides initial evidence showing how mitochondrial transplantation can promote skeletal muscle bioenergetics in an aging rodent model.

Combining electromyographic and electrical impedance data sets through machine learning: A study in D2-mdx and wild-type mice.

Needle impedance-electromyography (iEMG) assesses the active and passive electrical properties of muscles concurrently by using a novel needle with six electrodes, two for EMG and four for electrical impedance myography (EIM). Here, we assessed an approach for combining multifrequency EMG and EIM data via machine learning (ML) to discriminate D2-mdx muscular dystrophy and wild-type (WT) mouse skeletal muscle. iEMG data were obtained from quadriceps of D2-mdx mice, a muscular dystrophy model, and WT animals. EIM data were collected with the animals under deep anesthesia and EMG data collected under light anesthesia, allowing for limited spontaneous movement. Fourier transformation was performed on the EMG data to provide power spectra that were sampled across the frequency range using three different approaches. Random forest-based, nested ML was applied to the EIM and EMG data sets separately and then together to assess healthy versus disease category classification using a nested cross-validation procedure. Data from 20 D2-mdx and 20 WT limbs were analyzed. EIM data fared better than EMG data in differentiating healthy from disease mice with 93.1% versus 75.6% accuracy, respectively. Combining EIM and EMG data sets yielded similar performance as EIM data alone with 92.2% accuracy. We have demonstrated an ML-based approach for combining EIM and EMG data obtained with an iEMG needle. While EIM-EMG in combination fared no better than EIM alone with this data set, the approach used here demonstrates a novel method of combining the two techniques to characterize the full electrical properties of skeletal muscle.

Human milk extracellular vesicles enhance muscle growth and physical performance of immature mice associating with Akt/mTOR/p70s6k signaling pathway

Extracellular vesicles (EVs) play an important role in human and bovine milk composition. According to excellent published studies, it also exerts various functions in the gut, bone, or immune system. However, the effects of milk-derived EVs on skeletal muscle growth and performance have yet to be fully explored. Firstly, the current study examined the amino acids profile in human milk EVs (HME) and bovine milk EVs (BME) using targeted metabolomics. Secondly, HME and BME were injected in the quadriceps of mice for four weeks (1 time/3 days). Then, related muscle performance, muscle growth markers/pathways, and amino acids profile were detected or measured by grip strength analysis, rotarod performance testing, Jenner-Giemsa/H&E staining, Western blotting, and targeted metabolomics, respectively. Finally, HME and BME were co-cultured with C2C12 cells to detect the above-related indexes and further testify relative phenomena. Our findings mainly demonstrated that HME and BME significantly increase the diameter of C2C12 myotubes. HME treatment demonstrates higher exercise performance and muscle fiber densities than BME treatment. Besides, after KEGG and correlation analyses with biological function after HME and BME treatment, results showed L-Ornithine acts as a “notable marker” after HME treatment to affect mouse skeletal muscle growth or functions. Otherwise, L-Ornithine also significantly positively correlates with the activation of the AKT/mTOR pathway and myogenic regulatory factors (MRFs) and can also be observed in muscle and C2C12 cells after HME treatment. Overall, our study not only provides a novel result for the amino acid composition of HME and BME, but the current study also indicates the advantage of human milk on skeletal muscle growth and performance.

Traumatic muscle injury promotes aberrant fibro-adipogenic progenitor cell population, leading to fibrosis and impaired muscle stem cell function

Skeletal muscle has an exceptional regenerative capacity that relies on the temporal activation and regulation of immune cells, fibro-adipogenic progenitors (FAPs), and muscle stem cells (MuSCs). However, following traumatic injury to skeletal muscle, such as in extensive muscle deficits caused by injury or surgery, also known as volumetric muscle loss (VML), this coordinated regenerative response is diminished, leading to fibrosis, inflammation, and chronic functional deficiencies. The goal of this study was to compare the temporal response and phenotype of key cell populations, including macrophages, FAPs, and MuSCs following critical and sub-critical VML injuries in the mouse quadriceps. We hypothesize that traumatic injury, specifically VML, drives cell phenotypic variability amongst these cell populations. Unilateral VML injuries were used to quantify cell type distribution via flow cytometry at 1, 3, and 7 days post-VML (pVML, n=4 per injury size and time point). At day 7 pVML, anti-inflammatory M2 macrophages, FAPs, and MuSCs were all present in significantly elevated numbers per tissue volume in critical (3-mm) injuries compared to sub-critical (2-mm) injuries (p<0.05). Using multi-dimensional flow cytometry analysis, a distinct sub-population of FAPs was identified in critically sized VML injuries which had larger cell content and highly expressed the β1-integrin cell-surface marker. FAPs from quadriceps at day 7 pVML (VML-FAPs) were purified using fluorescence-activated cell sorting and cultured in vitro. VML-FAPs exhibited increased proliferation, increased cell area, and showed elevated levels of β1-integrin expression. In co-culture experiments, VML-FAPs were also shown to significantly decrease the myogenic capacity of primary MuSCs by quantification of myotube formation (p<0.05). When cultured with the anti-inflammatory cytokine TGF-β1, VML-FAPs more readily differentiated down a fibroblastic lineage. Collectively, these results indicate that following traumatic injury, namely VML, there is an aberrant shift in the FAPs cell population, possibly driven by the accumulation of M2 macrophages, towards a lineage which is persistent, pro-fibrotic, and anti-myogenic. The identification of this shift in VML-FAPs will be key to the development of pro-regenerative therapeutics for VML, as success of these VML-FAP interventions will rely on the ability to downregulate the fibrotic lineage of FAPs while maintaining their traditional pro-myogenic qualities during regeneration. Furthermore, this study also provokes investigation of differing FAP phenotypes in other traumatic muscle injuries to identify possible trends or incongruencies that may exist. Funding: This work was supported by the National Institute of Health under award number R01AG072309, R01AR071708, and Department of Defense under award number W81XWH-20-1-0336. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Depressed Protein Synthesis and Anabolic Signaling Potentiate ACL Tear–Resultant Quadriceps Atrophy

Background: Anterior cruciate ligament (ACL) tear (ACLT) leads to protracted quadriceps muscle atrophy. Protein turnover largely dictates muscle size and is highly responsive to injury and loading. Regulation of quadriceps molecular protein synthetic machinery after ACLT has largely been unexplored, limiting development of targeted therapies. Purpose: To define the effect of ACLT on (1) the activation of protein synthetic and catabolic signaling within quadriceps biopsy specimens from human participants and (2) the time course of alterations to protein synthesis and its molecular regulation in a mouse ACL injury model. Study Design: Descriptive laboratory study. Methods: Muscle biopsy specimens were obtained from the ACL-injured and noninjured vastus lateralis of young adult humans after an overnight fast (N = 21; mean ± SD, 19 ± 5 years). Mice had their limbs assigned to ACLT or control, and whole quadriceps were collected 6 hours or 1, 3, or 7 days after injury with puromycin injected before tissue collection for assessment of relative protein synthesis. Muscle fiber size and expression and phosphorylation of protein anabolic and catabolic signaling proteins were assessed at the protein and transcript levels (RNA sequencing). Results: Human quadriceps showed reduced phosphorylation of ribosomal protein S6 (–41%) in the ACL-injured limb (P = .008), in addition to elevated phosphorylation of eukaryotic initiation factor 2α (+98%; P = .006), indicative of depressed protein anabolic signaling in the injured limb. No differences in E3 ubiquitin ligase expression were noted. Protein synthesis was lower at 1 day (P = .01 vs control limb) and 3 days (P = .002 vs control limb) after ACLT in mice. Pathway analyses revealed shared molecular alterations between human and mouse quadriceps after ACLT. Conclusion: (1) Global protein synthesis and anabolic signaling deficits occur in the quadriceps in response to ACL injury, without notable changes in measured markers of muscle protein catabolism. (2) Importantly, these deficits occur before the onset of significant atrophy, underscoring the need for early intervention. Clinical Relevance: These findings suggest that blunted protein anabolism as opposed to increased catabolism likely mediates quadriceps atrophy after ACL injury. Thus, future interventions should aim to restore muscle protein anabolism rapidly after ACLT.

RF03 | PMON302 A Unique Estrogen-Sensitive Fibroblast Population Drives Abdominal Muscle Fibrosis

Abstract Weakening of the lower abdominal wall muscle via fibrosis causes visceral contents to bulge out, forming inguinal hernias. Humanized aromatase (Aromhum) transgenic male mice develop this weakness in the lower abdominal muscle (LAM) tissue, characterized by extensive fibrosis, muscle atrophy, and scrotal hernias. This mouse model incorporates the human version of the aromatase gene allowing for excessive estradiol (E2) production in skeletal muscle tissue. We have previously shown that the LAM fibroblasts are sensitive to local E2 production due to high levels of estrogen receptor alpha (ERα, Esr1)1. Here, we explore the origins of estrogen-driven muscle fibrosis in the LAM tissue of Aromhum mice at a single-cell level. Wild-type (WT) and Aromhum LAM tissues were processed via single-cell RNA sequencing (n = 3) and analyzed using Seurat. We observed a total of 22 UMAP clusters, including six fibroblast-like clusters. Five of these clusters express the fibro-adipogenic progenitor (FAP) marker Pdgfrα, and two of them are exclusively found in Aromhum LAM tissues. One Aromhum-enriched cluster highly expresses Esr1 and its downstream estrogen-responsive genes such as Pgr and Greb1. Pseudo-time analysis suggested that this cluster differentiates into a pathogenic fibroblast cluster characterized by high expression pro-inflammatory (Il33, Il6, Ccl8, C4b) and fibrosis-associated (Mmp3, Cthrc1, Saa3, Ptx3) genes. We further validated our findings via flow cytometry, immunocytochemistry, and western blots in LAM, upper abdominal muscle tissues, and quadriceps of both WT and Aromhum mice. We show that protein levels of Pdgfrα, ERα, Mmp3, C4b are greater in the fibroblasts freshly isolated from LAM tissues of Aromhum mice compared to WT mice. Moreover, Aromhum LAM tissue cell cycle analysis reveals that ∼15% of all ERα+ cells are in the G2-S phase compared with <1% of ERα- cells. Overall, we provide an insight into the cell types, genes, and proteins that are dysregulated in the LAM tissues from Aromhum mice with hernias. Inguinal hernias are found in ∼25% of the elderly male population2. Using an inguinal hernia mouse model that mimics the hernia pathophysiology of elderly men with estrogen excess and testosterone deficiency, we defined a specific population of estrogen-sensitive fibroblasts responsible for fibrosis and weakening of lower abdominal skeletal muscle tissue associated with hernias. We provide an insight into the specific disease-causing fibroblast populations and molecular targets that may be the basis for future therapeutics to prevent or treat inguinal hernias.

Downregulation of Sparc-like protein 1 during cisplatin-induced inhibition of myogenic differentiation of C2C12 myoblasts

Patients with cancer often experience muscle atrophy, which worsens their prognosis. Decreased muscle regenerative capacity plays an important role in the complex processes involved in muscle atrophy. Administration of cisplatin, a cancer chemotherapeutic agent, has been implicated as a cause of muscle atrophy. In this study, we examined whether cisplatin affects the differentiation of myoblasts into myotubes. We treated C2C12 myoblasts with a differentiation medium containing cisplatin and its vehicle during for 8 days and observed the changes in the expression of myosin heavy chain (MyHC) and myogenin in the myoblasts. Cisplatin was injected in mice for 4 consecutive days; on Day 5, the mice quadriceps muscles were sampled and examined. The expression of MyHCs increased and that of myogenin decreased after cisplatin treatment. The secretion of acidic cysteine-rich proteins (e.g., Sparc proteins) reportedly promotes C2C12 myoblast differentiation. Therefore, we investigated the Sparc family gene expression during myogenesis in C2C12 myoblasts after cisplatin treatment. Of all the genes investigated, Sparc-like protein 1 (Sparcl1) expression was significantly suppressed by cisplatin on Days 4–8. Simultaneous treatment with recombinant mouse Sparcl1 almost inhibited the cisplatin-induced suppression of total MyHC and myogenin protein levels. Moreover, Sparcl1 expression decreased in the skeletal muscles of mice, leading to cisplatin-induced muscle atrophy. Our results suggest that cisplatin-induced myogenesis suppression causes muscle atrophy and inhibits the expression of Sparcl1, which promotes C2C12 cell differentiation during myogenesis.

IFIT2-depleted metastatic oral squamous cell carcinoma cells induce muscle atrophy and cancer cachexia in mice.

BackgroundInterferon‐induced protein with tetratricopeptide repeat 2 (IFIT2) is a reported metastasis suppressor in oral squamous cell carcinoma (OSCC). Metastases and cachexia may coexist. The effect of cancer metastasis on cancer cachexia is largely unknown. We aimed to address this gap in knowledge by characterizing the cachectic phenotype of an IFIT2‐depleted metastatic OSCC mouse model.MethodsGenetically engineered and xenograft tumour models were used to explore the effect of IFIT2‐depleted metastatic OSCC on cancer cachexia. Muscle and organ weight changes, tumour burden, inflammatory cytokine profiles, body composition, food intake, serum albumin and C‐reactive protein (CRP) levels, and survival were assessed. The activation of the IL6/p38 pathway in atrophied muscle was measured.ResultsIFIT2‐depleted metastatic tumours caused marked body weight loss (−18.2% vs. initial body weight, P < 0.001) and a poor survival rate (P < 0.01). Skeletal muscles were markedly smaller in IFIT2‐depleted metastatic tumour‐bearing mice (quadriceps: −28.7%, gastrocnemius: −29.4%, and tibialis: −24.3%, all P < 0.001). Tumour‐derived circulating granulocyte‐macrophage colony‐stimulating factor (+772.2‐fold, P < 0.05), GROα (+1283.7‐fold, P < 0.05), IL6 (+245.8‐fold, P < 0.001), IL8 (+616.9‐fold, P < 0.001), IL18 (+24‐fold, P < 0.05), IP10 (+18.8‐fold, P < 0.001), CCL2 (+439.2‐fold, P < 0.001), CCL22 (+9.1‐fold, P < 0.01) and tumour necrosis factor α (+196.8‐fold, P < 0.05) were elevated in IFIT2‐depleted metastatic tumour‐bearing mice. Murine granulocyte colony‐stimulating factor (+61.4‐fold, P < 0.001) and IL6 (+110.9‐fold, P < 0.01) levels were significantly increased in IFIT2‐depleted metastatic tumour‐bearing mice. Serum CRP level (+82.1%, P < 0.05) was significantly increased in cachectic shIFIT2 mice. Serum albumin level (−26.7%, P < 0.01) was significantly decreased in cachectic shIFIT2 mice. An assessment of body composition revealed decreased fat (−81%, P < 0.001) and lean tissue (−21.7%, P < 0.01), which was consistent with the reduced food intake (−19.3%, P < 0.05). Muscle loss was accompanied by a smaller muscle cross‐sectional area (−23.3%, P < 0.05). Muscle atrophy of cachectic IFIT2‐depleted metastatic tumour‐bearing mice (i.v.‐shIFIT2 group) was associated with elevated IL6 (+2.7‐fold, P < 0.05), phospho‐p38 (+2.8‐fold, P < 0.05), and atrogin‐1 levels (+2.3‐fold, P < 0.05) in the skeletal muscle. Neutralization of IL6 rescued shIFIT2 conditioned medium‐induced myotube atrophy (+24.6%, P < 0.01).ConclusionsOur results suggest that the development of shIFIT2 metastatic OSCC lesions promotes IL6 production and is accompanied by the loss of fat and lean tissue, anorexia, and muscle atrophy. This model is appropriate for the study of OSCC cachexia, especially in linking metastasis with cachexia.

Determination of Taurine in Quadriceps Femoris by High Performance Liquid Chromatography with Precolumn Derivatization

A method for determination of taurine in mouse quadriceps femoris by high performance liquid chromatography with precolumn derivatization ophthalaldehyde (OPA) was established. The sample was extracted and treated with canon exchange resin. Results showed that taurine in quadriceps femoris was separated and quantified on C18 reversed phase column by high performance liquid chromatographic (HPLC) after derivatization with OPA in 3min, using mixture of methanol and phosphate （V/V＝1:1，pH＝4.9） as mobile phase, rate of flow is 0.6mL/min, detecting at 340nm by UV-detector, L-Glutamine as internal standard. The result showed that the linear ranger of taurine was 6.25-187.7ng/mL, correlation coefficient R2=0.9994, the recoveries were 91.8%-101.8%, RSD=3.2% (n=6). The retention time of taurine is 7.32 min. The concentration of taurine in mouse quadriceps femoris is 3.18mg/g. The protein and amino acid were separated by sample pre-treatment. The method is of good separation effect, simple, reliable, and can be used to analyze the taurine concentration in mammal tissue.

Effect of cigarette smoke extract on mitochondrial division in mouse quadriceps femoris cells

BackgroundTo observe the effect of cigarette smoke extract (CSE) on mitochondrial division in mouse quadriceps femoris cells and to explore the potential molecular mechanism of skeletal muscle dysfunction (SMD) in patients with chronic obstructive pulmonary disease (COPD).MethodsQuadriceps femoris were cultured, passaged, and stimulated with different concentrations of CSE. We divided cells into four groups (Control, 2.5%, 5%, 10%). The growth of cells, the expression of Dynamin related protein 1 (Drp-1), and apoptosis were observed and evaluated by fluorescence microscopy, RT-PCR, Western blot, and flow cytometry.ResultsThe longer the intervention time, the more obvious the decrease in cell number. In the 5% and 10% groups, the cells became round with gaps. Under an inverted fluorescence microscope, the green fluorescence of cells in 5% and 10% stained with Mito-Tracker Green was significantly less than that of the Control and 2.5%. Red fluorescence was reduced and the green fluorescence was increased in the 5% and 10% stained with JC-1. Flow cytometry analysis showed that reactive oxygen species (ROS) and apoptosis were increased in the CSE intervention groups. In the Control, 2.5%, 5%, and 10%, the levels of ROS were 0.052±0.015, 0.170±0.030, 5.340±0.500, and 24.400±1.900, respectively. The apoptotic rates (%) were 0.270±0.009, 2.650±0.060, 11.850±0.020, and 31.820±1.260, respectively. The relative expression levels were, 0.900±0.093, 1.141±0.099, 1.361±0.034, 2.155±0.092 for DNM1L mRNA, and 0.509±0.008, 0.569±0.028, 0.792±0.048, 0.940±0.062 for Drp-1. There were significant differences in the apoptotic rate, and Drp-1 expression between 5% and 10% compared with the Control and 2.5% (P<0.05).ConclusionsCSE may enhance mitochondrial division of quadriceps femoris cells by up-regulating the expression of Drp-1, affecting cellular energy metabolism and promoting quadriceps femoris apoptosis, ultimately leading to the occurrence and development of skeletal muscle dysfunction in COPD.

Branched-chain amino acid metabolism is regulated by ERR\u03b1 in primary human myotubes and is further impaired by glucose loading in type 2 diabetes

Aims/hypothesisIncreased levels of branched-chain amino acids (BCAAs) are associated with type 2 diabetes pathogenesis. However, most metabolomic studies are limited to an analysis of plasma metabolites under fasting conditions, rather than the dynamic shift in response to a metabolic challenge. Moreover, metabolomic profiles of peripheral tissues involved in glucose homeostasis are scarce and the transcriptomic regulation of genes involved in BCAA catabolism is partially unknown. This study aimed to identify differences in circulating and skeletal muscle BCAA levels in response to an OGTT in individuals with normal glucose tolerance (NGT) or type 2 diabetes. Additionally, transcription factors involved in the regulation of the BCAA gene set were identified.MethodsPlasma and vastus lateralis muscle biopsies were obtained from individuals with NGT or type 2 diabetes before and after an OGTT. Plasma and quadriceps muscles were harvested from skeletal muscle-specific Ppargc1a knockout and transgenic mice. BCAA-related metabolites and genes were assessed by LC-MS/MS and quantitative RT-PCR, respectively. Small interfering RNA and adenovirus-mediated overexpression techniques were used in primary human skeletal muscle cells to study the role of PPARGC1A and ESRRA in the expression of the BCAA gene set. Radiolabelled leucine was used to analyse the impact of oestrogen-related receptor α (ERRα) knockdown on leucine oxidation.ResultsImpairments in BCAA catabolism in people with type 2 diabetes under fasting conditions were exacerbated after a glucose load. Branched-chain keto acids were reduced 37–56% after an OGTT in the NGT group, whereas no changes were detected in individuals with type 2 diabetes. These changes were concomitant with a stronger correlation with glucose homeostasis biomarkers and downregulated expression of branched-chain amino acid transaminase 2, branched-chain keto acid dehydrogenase complex subunits and 69% of downstream BCAA-related genes in skeletal muscle. In primary human myotubes overexpressing peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, encoded by PPARGC1A), 61% of the analysed BCAA genes were upregulated, while 67% were downregulated in the quadriceps of skeletal muscle-specific Ppargc1a knockout mice. ESRRA (encoding ERRα) silencing completely abrogated the PGC-1α-induced upregulation of BCAA-related genes in primary human myotubes.Conclusions/interpretationMetabolic inflexibility in type 2 diabetes impacts BCAA homeostasis and attenuates the decrease in circulating and skeletal muscle BCAA-related metabolites after a glucose challenge. Transcriptional regulation of BCAA genes in primary human myotubes via PGC-1α is ERRα-dependent.Graphical abstract

Muscle atrophy induced by overexpression of ALAS2 is related to muscle mitochondrial dysfunction

BackgroundALAS2 (delta-aminolevulinate synthase 2) is one of the two isoenzymes catalyzing the synthesis of delta-aminolevulinic acid (ALA), which is the first precursor of heme synthesis. ALAS2-overexpressing transgenic mice (Tg mice) showed syndrome of porphyria, a series of diseases related to the heme anabolism deficiency. Tg mice showed an obvious decrease in muscle size. Muscle atrophy results from a decrease in protein synthesis and an increase in protein degradation, which ultimately leads to a decrease in myofiber size due to loss of contractile proteins, organelles, nuclei, and cytoplasm.MethodsThe forelimb muscle grip strength of age-matched ALAS-2 transgenic mice (Tg mice) and wild-type mice (WT mice) were measured with an automated grip strength meter. The activities of serum LDH and CK-MB were measured by Modular DPP. The histology of skeletal muscle (quadriceps femoris and gastrocnemius) was observed by hematoxylin and eosin (HE) staining, immunohistochemistry, and transmission electron microscope. Real-time PCR was used to detect mtDNA content and UCP3 mRNA expression. Evans blue dye staining was used to detect the membrane damage of the muscle fiber. Single skeletal muscle fiber diameter was measured by single-fiber analyses. Muscle adenosine triphosphate (ATP) levels were detected by a luminometric assay with an ATP assay kit.ResultsCompared with WT mice, the strength of forelimb muscle and mass of gastrocnemius were decreased in Tg mice. The activities of serum CK-MB and LDH, the number of central nuclei fibers, and Evans blue positive fibers were more than those in WT mice, while the diameter of single fibers was smaller, which were associated with suppressed expression levels of MHC, myoD1, dystrophin, atrogin1, and MuRF1. Re-expression of eMyHC was only showed in the quadriceps of Tg mice, but not in WT mice. Muscle mitochondria in Tg mice showed dysfunction with descented ATP production and mtDNA content, downregulated UCP3 mRNA expression, and swelling of mitochondria.ConclusionALAS2 overexpressing-transgenic mice (Tg mice) showed muscle dystrophy, which was associated with decreased atrogin-1 and MuRF-1, and closely related to mitochondrial dysfunction.

Gene-Metabolite Network Linked to Inhibited Bioenergetics in Association With Spaceflight-Induced Loss of Male Mouse Quadriceps Muscle.

ABSTRACTProlonged residence of mice in spaceflight is a scientifically robust and ethically ratified model of muscle atrophy caused by continued unloading. Under the Rodent Research Program of the National Aeronautics and Space Administration (NASA), we assayed the large‐scale mRNA and metabolomic perturbations in the quadriceps of C57BL/6j male mice that lived in spaceflight (FLT) or on the ground (control or CTR) for approximately 4 weeks. The wet weights of the quadriceps were significantly reduced in FLT mice. Next‐generation sequencing and untargeted mass spectroscopic assays interrogated the gene‐metabolite landscape of the quadriceps. A majority of top‐ranked differentially suppressed genes in FLT encoded proteins from the myosin or troponin families, suggesting sarcomere alterations in space. Significantly enriched gene‐metabolite networks were found linked to sarcomeric integrity, immune fitness, and oxidative stress response; all inhibited in space as per in silico prediction. A significant loss of mitochondrial DNA copy numbers in FLT mice underlined the energy deprivation associated with spaceflight‐induced stress. This hypothesis was reinforced by the transcriptomic sequencing–metabolomics integrative analysis that showed inhibited networks related to protein, lipid, and carbohydrate metabolism, and adenosine triphosphate (ATP) synthesis and hydrolysis. Finally, we discovered important upstream regulators, which could be targeted for next‐generation therapeutic intervention for chronic disuse of the musculoskeletal system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Single-cell transcriptional profiles in human skeletal muscle

Skeletal muscle is a heterogeneous tissue comprised of muscle fiber and mononuclear cell types that, in addition to movement, influences immunity, metabolism and cognition. We investigated the gene expression patterns of skeletal muscle cells using RNA-seq of subtype-pooled single human muscle fibers and single cell RNA-seq of mononuclear cells from human vastus lateralis, mouse quadriceps, and mouse diaphragm. We identified 11 human skeletal muscle mononuclear cell types, including two fibro-adipogenic progenitor (FAP) cell subtypes. The human FBN1+ FAP cell subtype is novel and a corresponding FBN1+ FAP cell type was also found in single cell RNA-seq analysis in mouse. Transcriptome exercise studies using bulk tissue analysis do not resolve changes in individual cell-type proportion or gene expression. The cell-type gene signatures provide the means to use computational methods to identify cell-type level changes in bulk studies. As an example, we analyzed public transcriptome data from an exercise training study and revealed significant changes in specific mononuclear cell-type proportions related to age, sex, acute exercise and training. Our single-cell expression map of skeletal muscle cell types will further the understanding of the diverse effects of exercise and the pathophysiology of muscle disease.

Activin A-Induced Cachectic Wasting Is Attenuated by Systemic Delivery of Its Cognate Propeptide in Male Mice.

In cancer, elevated activin levels promote cachectic wasting of muscle, irrespective of tumor progression. In excess, activins A and B use the myostatin signaling pathway in muscle, triggering a decrease in protein synthesis and an increase in protein degradation, which ultimately leads to atrophy. Recently, we demonstrated that local delivery of engineered activin and myostatin propeptides (natural inhibitors of these growth factors) could induce profound muscle hypertrophy in healthy mice. Additionally, the expression of these propeptides effectively attenuated localized muscle wasting in models of dystrophy and cancer cachexia. In this study, we examined whether a systemically administered recombinant propeptide could reverse activin A-induced cachectic wasting in mice. Chinese hamster ovary cells stably expressing activin A were transplanted into the quadriceps of nude mice and caused an 85-fold increase in circulating activin A levels within 12 days. Elevated activin A induced a rapid reduction in body mass (-16%) and lean mass (-10%). In agreement with previous findings, we demonstrated that adeno-associated virus-mediated delivery of activin propeptide to the tibialis anterior muscle blocked activin-induced wasting. In addition, despite massively elevated levels of activin A in this model, systemic delivery of the propeptide significantly reduced activin-induced changes in lean and body mass. Specifically, recombinant propeptide reversed activin-induced wasting of skeletal muscle, heart, liver, and kidneys. This is the first study to demonstrate that systemic administration of recombinant propeptide therapy effectively attenuates tumor-derived activin A insult in multiple tissues.

Phosphorylation of CRY1 Serine 71 Alters Voluntary Activity but Not Circadian Rhythms In Vivo.

Circadian clocks allow organisms to anticipate repetitive changes in their environment such as food availability, temperature, and predation. While they most clearly manifest at the behavioral level, driving sleep-wake cycles, for example, they also provide critical temporal regulation at the level of individual tissues. Circadian clocks within organs act to ensure that each tissue is functioning in a coordinated manner to anticipate the needs of the organism as a whole but also allow for adaptation of organs to their local environment. One critical aspect of this environment is energy availability, which is communicated at the cellular level via changes in metabolites such as ATP, calcium, and NADH. AMP-activated protein kinase (AMPK) is both sensitive to fluctuations in secondary metabolites and capable of resetting the circadian clock via destabilization of the core clock components CRY and PER. Phosphorylation of serine 71 of CRY1 by AMPK destabilizes CRY1 by decreasing its interaction with binding partner PER2, thus enabling greater association with the SCF complex substrate adaptor FBXL3. Here, we describe a transgenic mouse harboring germline mutation of CRY1 serine 71 to alanine. Unexpectedly, this mutation does not affect the steady-state level of CRY1 protein in mouse livers or quadriceps. We also did not detect changes in either behavioral or molecular circadian rhythms, but female Cry1S71A mice exhibit decreased voluntary locomotor activity compared with wild-type littermates. Together, these findings suggest that phosphorylation of CRY1 serine 71 is not required for the regulation of circadian rhythms under normal physiological conditions. However, it may be involved in responding to metabolic challenges or in other aspects of physiology that contribute to voluntary activity levels.

Determination of a Critical Size Threshold for Volumetric Muscle Loss in the Mouse Quadriceps.

The goal of this study was to determine the threshold for a critically sized, nonhealing muscle defect by characterizing key components in the balance between fibrosis and regeneration as a function of injury size in the mouse quadriceps. There is currently limited understanding of what leads to a critically sized muscle defect and which muscle regenerative components are functionally impaired. With the substantial increase in preclinical VML models as testbeds for tissue engineering therapeutics, defining the critical threshold for VML injuries will be instrumental in characterizing therapeutic efficacy and potential for subsequent translation.