Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Mice.

Chronic kidney disease (CKD) is associated with various pathologic changes, including elevations in serum phosphate levels (hyperphosphatemia), vascular calcification, and skeletal muscle atrophy. Elevated phosphate can damage vascular smooth muscle cells and cause vascular calcification. Here, we determined whether high phosphate can also affect skeletal muscle cells and whether hyperphosphatemia, in the context of CKD or by itself, is associated with skeletal muscle atrophy. As models of hyperphosphatemia with CKD, we studied mice receiving an adenine-rich diet for 14 weeks and mice with deletion of Collagen 4a3 (Col4a3-/-). As models of hyperphosphatemia without CKD, we analyzed mice receiving a high-phosphate diet for three and six months as well as a genetic model for klotho deficiency (kl/kl). We found that adenine, Col4a3-/-, and kl/kl mice have reduced skeletal muscle mass and function and develop atrophy. Mice on a high-phosphate diet for six months also had lower skeletal muscle mass and function but no significant signs of atrophy, indicating less severe damage compared with the other three models. To determine the potential direct actions of phosphate on skeletal muscle, we cultured primary mouse myotubes in high phosphate concentrations, and we detected the induction of atrophy. We conclude that in experimental mouse models, hyperphosphatemia is sufficient to induce skeletal muscle atrophy and that, among various other factors, elevated phosphate levels might contribute to skeletal muscle injury in CKD.

The androgen receptor in mesenchymal progenitors regulates skeletal muscle mass via Igf1 expression in male mice.

Androgens exert their effects primarily by binding to the androgen receptor (AR), a ligand-dependent nuclear receptor. While androgens have anabolic effects on skeletal muscle, previous studies reported that AR functions in myofibers to regulate skeletal muscle quality, rather than skeletal muscle mass. Therefore, the anabolic effects of androgens are exerted via nonmyofiber cells. In this context, the cellular and molecular mechanisms of AR in mesenchymal progenitors, which play a crucial role in maintaining skeletal muscle homeostasis, remain largely unknown. In this study, we demonstrated expression of AR in mesenchymal progenitors and found that targeted AR ablation in mesenchymal progenitors reduced limb muscle mass in mature adult, but not young or aged, male mice, although fatty infiltration of muscle was not affected. The absence of AR in mesenchymal progenitors led to remarkable perineal muscle hypotrophy, regardless of age, due to abnormal regulation of transcripts associated with cell death and extracellular matrix organization. Additionally, we revealed that AR in mesenchymal progenitors regulates the expression of insulin-like growth factor 1 (Igf1) and that IGF1 administration prevents perineal muscle atrophy in a paracrine manner. These findings indicate that the anabolic effects of androgens regulate skeletal muscle mass via, at least in part, AR signaling in mesenchymal progenitors.

Lactobacillus delbrueckii alleviates lipopolysaccharide-induced muscle inflammation and atrophy in weaned piglets associated with inhibition of endoplasmic reticulum stress and protein degradation.

Pro-inflammatory cytokines in muscle play a pivotal role in physiological responses and in the pathophysiology of inflammatory disease and muscle atrophy. Lactobacillus delbrueckii (LD), as a kind of probiotics, has inhibitory effects on pro-inflammatory cytokines associated with various inflammatory diseases. This study was conducted to explore the effect of dietary LD on the lipopolysaccharide (LPS)-induced muscle inflammation and atrophy in piglets and to elucidate the underlying mechanism. A total of 36 weaned piglets (Duroc × Landrace × Large Yorkshire) were allotted into three groups with six replicates (pens) of two piglets: (1) Nonchallenged control; (2) LPS-challenged (LPS); (3) 0.2% LD diet and LPS-challenged (LD+LPS). On d 29, the piglets were injected intraperitoneally with LPS or sterilized saline, respectively. All piglets were slaughtered at 4 h after LPS or saline injection, the blood and muscle samples were collected for further analysis. Our results showed that dietary supplementation of LD significantly attenuated LPS-induced production of pro-inflammatory cytokines IL-6 and TNF-α in both serum and muscle of the piglets. Concomitantly, pretreating the piglets with LD also clearly inhibited LPS-induced nuclear translocation of NF-κB p65 subunits in the muscle, which correlated with the anti-inflammatory effects of LD on the muscle of piglets. Meanwhile, LPS-induced muscle atrophy, indicated by a higher expression of muscle atrophy F-box, muscle RING finger protein (MuRF1), forkhead box O 1, and autophagy-related protein 5 (ATG5) at the transcriptional level, whereas pretreatment with LD led to inhibition of these upregulations, particularly genes for MuRF1 and ATG5. Moreover, LPS-induced mRNA expression of endoplasmic reticulum stress markers, such as eukaryotic translational initiation factor 2α (eIF-2α) was suppressed by pretreatment with LD, which was accompanied by a decrease in the protein expression levels of IRE1α and GRP78. Additionally, LD significantly prevented muscle cell apoptotic death induced by LPS. Taken together, our data indicate that the anti-inflammatory effect of LD supply on muscle atrophy of piglets could be likely regulated by inhibiting the secretion of pro-inflammatory cytokines through the inactivation of the ER stress/NF-κB singling pathway, along with the reduction in protein degradation.

Accelerometer-derived movement features as predictive biomarkers for muscle atrophy in neurocritical care: a prospective cohort study.

BACKGROUND: Physical inactivity and subsequent muscle atrophy are highly prevalent in neurocritical care and are recognized as key mechanisms underlying intensive care unit acquired weakness (ICUAW). The lack of quantifiable biomarkers for inactivity complicates the assessment of its relative importance compared to other conditions under the syndromic diagnosis of ICUAW. We hypothesize that active movement, as opposed to passive movement without active patient participation, can serve as a valid proxy for activity and may help predict muscle atrophy. To test this hypothesis, we utilized non-invasive, body-fixed accelerometers to compute measures of active movement and subsequently developed a machine learning model to predict muscle atrophy. METHODS: This study was conducted as a single-center, prospective, observational cohort study as part of the MINCE registry (metabolism and nutrition in neurointensive care, DRKS-ID: DRKS00031472). Atrophy of rectus femoris muscle (RFM) relative to baseline (day 0) was evaluated at days 3, 7 and 10 after intensive care unit (ICU) admission and served as the dependent variable in a generalized linear mixed model with Least Absolute Shrinkage and Selection Operator regularization and nested-cross validation. RESULTS: Out of 407 patients screened, 53 patients (age: 59.2 years (SD 15.9), 31 (58.5%) male) with a total of 91 available accelerometer datasets were enrolled. RFM thickness changed - 19.5% (SD 12.0) by day 10. Out of 12 demographic, clinical, nutritional and accelerometer-derived variables, baseline RFM muscle mass (beta - 5.1, 95% CI - 7.9 to - 3.8) and proportion of active movement (% activity) (beta 1.6, 95% CI 0.1 to 4.9) were selected as significant predictors of muscle atrophy. Including movement features into the prediction model substantially improved performance on an unseen test data set (including movement features: R2 = 79%; excluding movement features: R2 = 55%). CONCLUSION: Active movement, as measured with thigh-fixed accelerometers, is a key risk factor for muscle atrophy in neurocritical care patients. Quantifiable biomarkers reflecting the level of activity can support more precise phenotyping of ICUAW and may direct tailored interventions to support activity in the ICU. Studies addressing the external validity of these findings beyond the neurointensive care unit are warranted. TRIAL REGISTRATION: DRKS00031472, retrospectively registered on 13.03.2023.

A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single-nucleus resolution.

Cancer cachexia is a prevalent and often fatal wasting condition that cannot be fully reversed with nutritional interventions. Muscle atrophy is a central component of the syndrome, but the mechanisms whereby cancer leads to skeletal muscle atrophy are not well understood. We performed single-nucleus multi-omics on skeletal muscles from a mouse model of cancer cachexia and profiled the molecular changes in cachexic muscle. Our results revealed the activation of a denervation-dependent gene program that upregulates the transcription factor myogenin. Further studies showed that a myogenin-myostatin pathway promotes muscle atrophy in response to cancer cachexia. Short hairpin RNA inhibition of myogenin or inhibition of myostatin through overexpression of its endogenous inhibitor follistatin prevented cancer cachexia-induced muscle atrophy in mice. Our findings uncover a molecular basis of muscle atrophy associated with cancer cachexia and highlight potential therapeutic targets for this disorder.

Anti-muscle atrophy effect of fermented Tenebrio molitor larvae extract by modulating the PI3K-Akt-mTOR/FoxO3α pathway in mice treated with dexamethasone.

This study investigated the anti-sarcopenic effect of fermented Tenebrio molitor larvae (mealworms) extract (FME) in both dexamethasone (DEX)-treated C2C12 cells and mice. FME (100 µg/mL) increased the diameter of myotubes and inhibited the gene and protein expression of atrogin-1 compared to DEX- or non-fermented mealworms extract (ME)-treated C2C12 cells. Male C57BL/6N mice were divided into five groups: Normal Control (NC), DEX (10 mg/kg, intraperitoneal), and three groups of DEX+FME (100, 200, or 500 mg FME/kg/day, oral) for two weeks. FME at doses of 200 and 500 mg/kg effectively improved grip strength when compared to the DEX group. Histological analysis of the quadriceps muscle showed a larger muscle fiber size in the DEX+FME groups compared to DEX group. FME (200 and 500 mg/kg) significantly increased cross-sectional area of the muscle fiber compared to DEX group. FME (500 mg/kg) significantly decreased the ubiquitin, atrogin-1 and MuRF-1 protein levels, and increased levels of MHC and MyoG in DEX-treated mice. The puromycin labeling assay revealed that FME increased protein synthesis in DEX-induced muscle atrophy. The FME treatment demonstrated significant upregulation in phosphorylation levels, including mTOR, FoxO3α, Akt, and PI3K compared to DEX group. In conclusion, FME inhibited the increase in proteins associated with muscle atrophy, including, atrogin-1 and MuRF-1, by regulating the PI3K-Akt-FoxO3α pathway. FME improved the PI3K-Akt-mTOR signaling pathway, which was reduced by DEX. This study suggests that FME has the potential for use in sarcopenia therapy, possibly serving as a natural agent that counteracts the negative effects of DEX on muscle tissue.

Muscle-derived IL-1ß regulates EcSOD expression via the NBR1-p62-Nrf2 pathway in muscle during cancer cachexia.

Oxidative stress contributes to the loss of skeletal muscle mass and function in cancer cachexia. However, this outcome may be mitigated by an improved endogenous antioxidant defence system. Here, using the well-established oxidative stress-inducing muscle atrophy model of Lewis lung carcinoma (LLC) in 13-week-old male C57BL/6J mice, we demonstrate that extracellular superoxide dismutase (EcSOD) levels increase in the cachexia-prone extensor digitorum longus muscle. LLC transplantation significantly increased interleukin-1ß (IL-1ß) expression and release from extensor digitorum longus muscle fibres. Moreover, IL-1ß treatment of C2C12 myotubes increased NBR1, p62 phosphorylation at Ser351, Nrf2 nuclear translocation and EcSOD protein expression. Additional studies in vivo indicated that intramuscular IL-1ß injection is sufficient to stimulate EcSOD expression, which is prevented by muscle-specific knockout of p62 and Nrf2 (i.e. in p62 skmKO and Nrf2 skmKO mice, respectively). Finally, since an increase in circulating IL-1ß may lead to unwanted outcomes, we demonstrate that targeting this pathway at p62 is sufficient to drive muscle EcSOD expression in an Nrf2-dependent manner. In summary, cancer cachexia increases EcSOD expression in extensor digitorum longus muscle via muscle-derived IL-1ß-induced upregulation of p62 phosphorylation and Nrf2 activation. These findings provide further mechanistic evidence for the therapeutic potential of p62 and Nrf2 to mitigate cancer cachexia-induced muscle atrophy. KEY POINTS: Oxidative stress plays an important role in muscle atrophy during cancer cachexia. EcSOD, which mitigates muscle loss during oxidative stress, is upregulated in 13-week-old male C57BL/6J mice of extensor digitorum longus muscles during cancer cachexia. Using mouse and cellular models, we demonstrate that cancer cachexia promotes muscle EcSOD protein expression via muscle-derived IL-1ß-dependent stimulation of the NBR1-p62-Nrf2 signalling pathway. These results provide further evidence for the potential therapeutic targeting of the NBR1-p62-Nrf2 signalling pathway downstream of IL-1ß to mitigate cancer cachexia-induced muscle atrophy.

Chikungunya and Mayaro Viruses Induce Chronic Skeletal Muscle Atrophy Triggered by Pro-Inflammatory and Oxidative Response.

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle-articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, nuclear magnetic resonance imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced inflammation promote a rapid loss of muscle mass and reduction in fiber cross-sectional area by upregulation of muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1 expression, both key regulators of SkM fibers atrophy. Despite a reduction in inflammation and clearance of infectious viral particles, SkM atrophy persists until 30 days post-infection. The genomic CHIKV and MAYV RNAs were still detected in SkM in the late phase, along with the upregulation of chemokines and anti-inflammatory cytokine expression. In agreement with the involvement of inflammatory mediators on induced atrophy, the neutralization of TNF and a reduction in oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogen expression and atrophic fibers while increasing weight gain in treated mice. These data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphavirus pathogenesis and with a description of potentially new targets of therapeutic intervention.

Vigeo Promotes Myotube Differentiation and Protects Dexamethasone-Induced Skeletal Muscle Atrophy via Regulating the Protein Degradation, AKT/mTOR, and AMPK/Sirt-1/PGC1α Signaling Pathway In Vitro and In Vivo.

Sarcopenia, a condition caused by an imbalance between muscle growth and loss, can severely affect the quality of life of elderly patients with metabolic, inflammatory, and cancer diseases. Vigeo, a nuruk-fermented extract of three plants (Eleutherococcus senticosus Maxim (ESM), Achyranthes japonica (Miq.) Nakai (AJN), and Atractylodes japonica Koidzumi (AJK)) has been reported to have anti-osteoporotic effects. However, evidence of the effects of Vigeo on muscle atrophy is not available. Here, in the in vivo model of dexamethasone (Dex)-induced muscle atrophy, Vigeo treatment significantly reversed Dex-induced decreases in calf muscle volume, gastrocnemius (GA) muscle weight, and histological cross-section area. In addition, in mRNA and protein analyses isolated from GA muscle, we observed that Vigeo significantly protected against Dex-induced mouse muscle atrophy by inhibiting protein degradation regulated by atrogin and MuRF-1. Moreover, we demonstrated that Vigeo significantly promoted C2C12 cell line differentiation, as evidenced by the increased width and length of myotubes, and the increased number of fused myotubes with three or more nuclei. Vigeo alleviated the formation of myotubes compared to the control group. Vigeo also significantly increased the mRNA and protein expression of myosin heavy chain (MyHC), MyoD, and myogenin compared to that in the control. Vigeo treatment significantly reduced the mRNA and protein expression of muscle degradation markers atrogin-1 and muscle RING Finger 1 (MuRF-1) in the C2C12 cell line in vitro. Vigeo also activated the AMP-activated protein kinase (AMPK)/silent information regulator 1 (Sirt-1)/peroxisome proliferator-activated receptor-γ co-activator-1α (PGC1α) mitochondrial biogenesis pathway and the Akt/mTOR protein synthesis signaling pathway in Dex-induced myotube atrophy. These findings suggest that Vigeo may have protective effects against Dex-induced muscle atrophy. Therefore, we propose Vigeo as a supplement or potential therapeutic agent to prevent or treat sarcopenia accompanied by muscle atrophy and degeneration.

Association of serum iron metabolism with muscle mass and frailty in older adults: A cross-sectional study of community-dwelling older adults.

This study aimed to explore the correlation between serum ferritin and additional biomarkers associated with iron metabolism, as well as their connection to muscle atrophy and frailty in the community-dwelling middle-aged and elderly population. The study included 110 middle-aged and elderly participants. Participants were categorized into an iron accumulation group (31 cases) and a normal iron group (79 cases) based on the standard ferritin values for men and women. Based on the criteria of the Asian Working Group on Muscular Dystrophy, participants were classified into a sarcopenia group (31 cases) and a non-sarcopenia group (79 cases). Using the Fried frailty syndrome criteria, participants were categorized into non-frailty (7 cases), pre-frailty (50 cases), and frailty (53 cases) groups. We employed multiple linear regression, binary logistic regression, partial correlation analysis, and ordinal logistic regression to assess the associations between iron metabolism indices and the presence of muscle atrophy and frailty. Compared with the normal iron group, the iron overload group had significantly higher ferritin, weight loss, fatigue, slow gait, and frailty scores (P < .05). Among the 3 models we set, ferritin was not significantly correlated with muscle mass in models 1 and 3 (P > .05), ferritin was positively correlated with muscle mass in model 2 (Pmodel2 = .048), but Transferrin saturation was positively correlated with muscle mass in all 3 models (Pmodel1 = .047, Pmodel2 = .026, Pmodel3 = .024). Ferritin, body mass index and iron overload were the influencing factors of sarcopenia (Pferritin = .027, PBMI < .001, Piron overload = .028). Ferritin was positively correlated with weight loss, fatigue, slow gait, frailty score, and frailty grade (P < .05). Age, gender and ferritin were the influencing factors of frailty classification (P < .05). Disrupted iron metabolism can lead to decreased muscle mass and function among the middle-aged and elderly, increasing frailty risk. It's crucial to prioritize community-based frailty screening and prevention, focusing on iron utilization as well as storage, since accelerating the body's iron metabolism cycle might influence muscle health more significantly than iron reserves.

Muscle degeneration in aging Drosophila flies: the role of mechanical stress.

Muscle wasting is a universal hallmark of aging which is displayed by a wide range of organisms, although the causes and mechanisms of this phenomenon are not fully understood. We used Drosophila to characterize the phenomenon of spontaneous muscle fiber degeneration (SMFD) during aging. We found that SMFD occurs across diverse types of somatic muscles, progresses with chronological age, and positively correlates with functional muscle decline. Data from vital dyes and morphological markers imply that degenerative fibers most likely die by necrosis. Mechanistically, SMFD is driven by the damage resulting from muscle contractions, and the nervous system may play a significant role in this process. Our quantitative model of SMFD assessment can be useful in identifying and validating novel genetic factors that influence aging-related muscle wasting.

Effects of Quality of Integrity on Clinical and Structural Outcomes of Rotator Cuff Repair: A Retrospective Cohort Study of 504 Cases.

BACKGROUND: The clinical implications of structural integrity have been a subject of long debate. The oversimplified binary categorization of structural integrity into either healing or retear, along with faulty preoperative baselines for comparison, may contribute to the controversy. PURPOSE: To determine how the quality of structural integrity in a repaired cuff tendon affects both clinical and structural outcomes by dividing the patients into groups based on integrity and using the immediate postoperative baseline (time zero). STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A total of 504 patients with a full-thickness rotator cuff tear who underwent arthroscopic rotator cuff repair and were followed up for at least a year with magnetic resonance imaging (MRI) were included. The quality of structural integrity was graded using the Sugaya classification. To evaluate clinical outcomes, pain, range of motion, strength, functional scores, and overall satisfaction and function were used for within- and between-group analyses at the last follow-up. For the assessment of structural outcomes, the Goutallier classification for fatty infiltration (FI) and the tangent sign, occupation ratio, and normalized cross-sectional area for muscle atrophy (MA) were used. The baselines for these structural measurements were both the preoperative and the time-zero MRI scans. RESULTS: The mean clinical follow-up period was 31.8 ± 27.5 months, and the MRI follow-up period was 10.9 ± 5.3 months. There were 178 (35.3%), 228 (45.2%), 58 (11.5%), 14 (2.8%), and 26 (5.2%) shoulders with Sugaya grades 1, 2, 3, 4, and 5, respectively. Regardless of structural integrity, all clinical outcomes at a mean follow-up of 31.8 months after repair significantly improved compared with those before repair. Only in shoulders with Sugaya grade 1 did the FI of the supraspinatus muscle improve significantly from baseline. FI of the infraspinatus muscle did not change significantly in those with grades 1 and 2 but worsened in those with grades 3 and 5. MA measured using the occupation ratio improved significantly in shoulders with Sugaya grades 1 and 2 but declined in those with grade 5. CONCLUSION: This study established a correlation between improved structural integrity of the repaired cuff tendon and enhanced structural outcomes in rotator cuff muscles. Furthermore, the findings revealed that both FI and MA could be reversed in patients exhibiting high-quality structural integrity. However, these structural improvements were not mirrored in the clinical outcomes.

Proteomic Profiling of Hindlimb Skeletal Muscle Disuse in a Murine Model of Sepsis.

CONTEXT: Sepsis leads to multiple organ dysfunction and negatively impacts patient outcomes. Skeletal muscle disuse is a significant comorbidity in septic patients during their ICU stay due to prolonged immobilization. HYPOTHESIS: Combination of sepsis and muscle disuse will promote a unique proteomic signature in skeletal muscle in comparison to disuse and sepsis separately. METHODS AND MODELS: Following cecal ligation and puncture (CLP) or Sham surgeries, mice were subjected to hindlimb suspension (HLS) or maintained normal ambulation (NA). Tibialis anterior muscles from 24 C57BL6/J male mice were harvested for proteomic analysis. Proteomic profiles were assessed using nano-liquid chromatography with tandem mass spectrometry, followed by data analysis including Partial Least Squares Discriminant Analysis (PLS-DA), to compare the differential protein expression across groups. RESULTS: A total of 2876 differentially expressed proteins were identified, with marked differences between groups. In mice subjected to CLP and HLS combined, there was a distinctive proteomic signature characterized by a significant decrease in the expression of proteins involved in mitochondrial function and muscle metabolism, alongside a marked increase in proteins related to muscle degradation pathways. The PLS-DA demonstrated a clear separation among experimental groups, highlighting the unique profile of the CLP/HLS group. This suggests an important interaction between sepsis-induced inflammation and disuse atrophy mechanisms in sepsis-induced myopathy. INTERPRETATIONS AND CONCLUSIONS: Our findings reveal a complex proteomic landscape in skeletal muscle exposed to sepsis and disuse, consistent with an exacerbation of muscle protein degradation under these combined stressors. The identified proteins and their roles in cellular stress responses and muscle pathology provide potential targets for intervention to mitigate muscle dysfunction in septic conditions, highlighting the importance of addressing both sepsis and disuse concurrently in clinical and experimental settings.

Brain of miyoshi myopathy/dysferlinopathy patients presents with structural and metabolic anomalies.

Miyoshi myopathy/dysferlinopathy (MMD) is a rare muscle disease caused by DYSF gene mutations. Apart from skeletal muscles, DYSF is also expressed in the brain. However, the impact of MMD-causing DYSF variants on brain structure and function remains unexplored. To investigate this, we utilized magnetic resonance (MR) modalities (MR volumetry and 31P MR spectroscopy) in a family with seven children, four of whom have the illness. The MMD siblings showed distinct differences from healthy controls: (1) a significant (p < 0.001) right-sided volume asymmetry (+ 232 mm3) of the inferior lateral ventricles; and (2) a significant (p < 0.001) decrease in [Mg2+], along with a modified energy metabolism profile and altered membrane turnover in the hippocampus and motor and premotor cortices. The patients' [Mg2+], energy metabolism, and membrane turnover measures returned to those of healthy relatives after a month of 400 mg/day magnesium supplementation. This work is the first to describe anatomical and functional abnormalities characteristic of neurodegeneration in the MMD brain. Therefore, we call for further examination of brain functions in larger cohorts of MMD patients and testing of magnesium supplementation, which has proven to be an effective corrective approach in our study.

Impact of combined intermittent fasting and high-intensity interval training on apoptosis and atrophy signaling in rat fast- and slow-twitch muscles.

This study aimed to evaluate the influence of combined intermittent fasting (IF) and high-intensity interval training (HIIT) on morphology, caspase-independent apoptosis signaling pathway, and myostatin expression in soleus and gastrocnemius (white portion) muscles from healthy rats. Sixty-day-old male Wistar rats (n = 60) were divided into four groups: control (C), IF, high-intensity-interval training (T), and high-intensity-interval training and intermittent fasting (T-IF). The C and T groups received ad libitum chow daily; IF and T-IF received the same standard chow every other day. Animals from T and T-IF underwent a HIIT protocol five times a week for 12 weeks. IF reduced gastrocnemius mass and increased pro-apoptotic proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in soleus and cleaved-to-non-cleaved PARP-1 ratio and myostatin expression in gastrocnemius white portion. HIIT increased AIF and apoptosis repressor with caspase recruitment domain expression in soleus and cleaved-to-total PARP-1 ratio in gastrocnemius muscle white portion. The combination of IF and HIIT reduced fiber cross-sectional area in both muscles, increased EndoG and AIF expression, and decreased cleaved-to-non-cleaved PARP-1 ratio in gastrocnemius muscle white portion. Muscle responses to IF and HIIT are directly impacted by the muscle fiber type composition and are modulated, at least in part, by myostatin and caspase-independent apoptosis signaling.

Apolipoprotein E knockout, but not cholesteryl ester transfer protein (CETP)-associated high-density lipoprotein cholesterol (HDL-C) lowering, exacerbates muscle wasting in dysferlin-null mice.

BACKGROUND: Dysferlin-deficient limb-girdle muscular dystrophy type 2B (Dysf) mice are notorious for their mild phenotype. Raising plasma total cholesterol (CHOL) via apolipoprotein E (ApoE) knockout (KO) drastically exacerbates muscle wasting in Dysf mice. However, dysferlinopathic patients have abnormally reduced plasma high-density lipoprotein cholesterol (HDL-C) levels. The current study aimed to determine whether HDL-C lowering can exacerbate the mild phenotype of dysferlin-null mice. METHODS: Human cholesteryl ester transfer protein (CETP), a plasma lipid transfer protein not found in mice that reduces HDL-C, and/or its optimal adapter protein human apolipoprotein B (ApoB), were overexpressed in Dysf mice. Mice received a 2% cholesterol diet from 2 months of age and characterized through ambulatory and hanging functional tests, plasma analyses, and muscle histology. RESULTS: CETP/ApoB expression in Dysf mice caused reduced HDL-C (54.5%) and elevated ratio of CHOL/HDL-C (181.3%) compared to control Dysf mice in plasma, but without raising CHOL. Compared to the severe muscle pathology found in high CHOL Dysf/ApoE double knockout mice, Dysf/CETP/ApoB mice did not show significant changes in ambulation, hanging capacity, increases in damaged area, collagen deposition, or decreases in cross-sectional area and healthy myofibre coverage. CONCLUSIONS: CETP/ApoB over-expression in Dysf mice decreases HDL-C without increasing CHOL or exacerbating muscle pathology. High CHOL or nonHDL-C caused by ApoE KO, rather than low HDL-C, likely lead to rodent muscular dystrophy phenotype humanization.

Zfp697 is an RNA-binding protein that regulates skeletal muscle inflammation and remodeling.

Skeletal muscle atrophy is a morbidity and mortality risk factor that happens with disuse, chronic disease, and aging. The tissue remodeling that happens during recovery from atrophy or injury involves changes in different cell types such as muscle fibers, and satellite and immune cells. Here, we show that the previously uncharacterized gene and protein Zfp697 is a damage-induced regulator of muscle remodeling. Zfp697/ZNF697 expression is transiently elevated during recovery from muscle atrophy or injury in mice and humans. Sustained Zfp697 expression in mouse muscle leads to a gene expression signature of chemokine secretion, immune cell recruitment, and extracellular matrix remodeling. Notably, although Zfp697 is expressed in several cell types in skeletal muscle, myofiber-specific Zfp697 genetic ablation in mice is sufficient to hinder the inflammatory and regenerative response to muscle injury, compromising functional recovery. We show that Zfp697 is an essential mediator of the interferon gamma response in muscle cells and that it functions primarily as an RNA-interacting protein, with a very high number of miRNA targets. This work identifies Zfp697 as an integrator of cell-cell communication necessary for tissue remodeling and regeneration.

The role of ZEB1 in mediating the protective effects of metformin on skeletal muscle atrophy.

Metformin is an important antidiabetic drug that has the potential to reduce skeletal muscle atrophy and promote the differentiation of muscle cells. However, the exact molecular mechanism underlying these functions remains unclear. Previous studies revealed that the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), which participates in tumor progression, inhibits muscle atrophy. Therefore, we hypothesized that the protective effect of metformin might be related to ZEB1. We investigated the positive effect of metformin on IL-1ß-induced skeletal muscle atrophy by regulating ZEB1 in vitro and in vivo. Compared with the normal cell differentiation group, the metformin-treated group presented increased myotube diameters and reduced expression levels of atrophy-marker proteins. Moreover, muscle cell differentiation was hindered, when we artificially interfered with ZEB1 expression in mouse skeletal myoblast (C2C12) cells via ZEB1-specific small interfering RNA (si-ZEB1). In response to inflammatory stimulation, metformin treatment increased the expression levels of ZEB1 and three differentiation proteins, MHC, MyoD, and myogenin, whereas si-ZEB1 partially counteracted these effects. Moreover, marked atrophy was induced in a mouse model via the administration of lipopolysaccharide (LPS) to the skeletal muscles of the lower limbs. Over a 4-week period of intragastric administration, metformin treatment ameliorated muscle atrophy and increased the expression levels of ZEB1. Metformin treatment partially alleviated muscle atrophy and stimulated differentiation. Overall, our findings may provide a better understanding of the mechanism underlying the effects of metformin treatment on skeletal muscle atrophy and suggest the potential of metformin as a therapeutic drug.

Chlorogenic acid ameliorates muscle wasting by upregulating mRNA expressions of calcineurin and PGC-1α in diabetic rat model.

INTRODUCTION: Muscle health in diabetes mellitus (DM) is often neglected, which leads to muscle wasting. Increased reactive oxygen species in DM could decrease antioxidant enzymes such as superoxide dismutase-1 (SOD-1) and -2 (SOD-2) and inhibit calcineurin (CN) and PGC-1α signalling pathways. Chlorogenic acid (CGA) is known as a potent antioxidant and activators of CN and PGC-1α. This study aimed to determine the effect of CGA on mRNA expressions of SOD-1, SOD-2, CN and PGC-1α in inhibiting the progression of DM to muscle wasting. MATERIALS AND METHODS: This study was conducted at Department of Anatomy, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada starting on July 20th, 2020. A total of 24 male Wistar rats were randomly divided into six groups (four rats per group), i.e., control, DM 1.5 months (DM1.5), and DM 2 months (DM2); and DM groups treated with CGA in three different doses, namely CGA1 (12.5 mg/kg BW), CGA2 (25 mg/kg BW), and CGA3 (50 mg/kg BW). Control group was only injected with normal saline, while diabetic model was induced by intraperitoneal injection of streptozotocin. Blood glucose levels were measured twice (one week after diabetic induction and before termination). The soleus muscle tissue was harvested to analyse the mRNA expressions of SOD-1, SOD- 2, CN and PGC-1α using RT-PCR. In addition, the tissue samples were stained with immunohistochemistry for CN and haematoxylin-eosin (HE) for morphologic analysis under light microscopy. RESULTS: The mRNA expressions of SOD-1 and SOD-2 in the CGA1 group were relatively higher compared to the DM2 groups. The mRNA expression of CN in the CGA1 group was significantly higher compared to the DM2 group (p = 0.008). The mRNA expression of PGC-1α in the CGA1 group was significantly higher compared to the DM2 group (p = 0.025). Immunohistochemical staining showed that CNimmunopositive expression in the CGA1 group was more evident compared to the other groups. Haematoxylin-eosin staining showed that muscle tissue morphology in the CGA1 group was similar to that in the control group. CONCLUSION: Chlorogenic acid at a dose of 12.5 mg/kg BW shows lower blood glucose level, good skeletal muscle tissue morphology and higher mRNA expressions of SOD-1, SOD-2, CN and PGC-1α compared to the DM groups.

Epigenetics of Skeletal Muscle Atrophy.

Skeletal muscle atrophy, characterized by diminished muscle strength and mass, arises from various causes, including malnutrition, aging, nerve damage, and disease-related secondary atrophy. Aging markedly escalates the prevalence of sarcopenia. Concurrently, the incidence of muscle atrophy significantly rises among patients with chronic ailments such as heart failure, diabetes, and chronic obstructive pulmonary disease (COPD). Epigenetics plays a pivotal role in skeletal muscle atrophy. Aging elevates methylation levels in the promoter regions of specific genes within muscle tissues. This aberrant methylation is similarly observed in conditions like diabetes, neurological disorders, and cardiovascular diseases. This study aims to explore the relationship between epigenetics and skeletal muscle atrophy, thereby enhancing the understanding of its pathogenesis and uncovering novel therapeutic strategies.