MicroRNA 322 Aggravates Dexamethasone-Induced Muscle Atrophy by Targeting IGF1R and INSR

Dexamethasone (Dex) has been widely used as a potent anti-inflammatory, antishock, and immunosuppressive agent. However, high dose or long-term use of Dex is accompanied by side effects including skeletal muscle atrophy, whose underlying mechanisms remain incompletely understood. A number of microRNAs (miRNAs) have been shown to play key roles in skeletal muscle atrophy. Previous studies showed significantly increased miR-322 expression in Dex-treated C2C12 myotubes. In our study, the glucocorticoid receptor (GR) was required for Dex to increase miR-322 expression in C2C12 myotubes. miR-322 mimic or miR-322 inhibitor was used for regulating the expression of miR-322. Insulin-like growth factor 1 receptor (IGF1R) and insulin receptor (INSR) were identified as target genes of miR-322 using luciferase reporter assays and played key roles in Dex-induced muscle atrophy. miR-322 overexpression promoted atrophy in Dex-treated C2C12 myotubes and the gastrocnemius muscles of mice. Conversely, miR-322 inhibition showed the opposite effects. These data suggested that miR-322 contributes to Dex-induced muscle atrophy via targeting of IGF1R and INSR. Furthermore, miR-322 might be a potential target to counter Dex-induced muscle atrophy. miR-322 inhibition might also represent a therapeutic approach for Dex-induced muscle atrophy.

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Trimetazidine attenuates dexamethasone-induced muscle atrophy via inhibiting NLRP3/GSDMD pathway-mediated pyroptosis

Cell Death Discovery ◽

10.1038/s41420-021-00648-0 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Li Wang ◽

Xin-Feng Jiao ◽

Cheng Wu ◽

Xiao-Qing Li ◽

Hui-Xian Sun ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Muscle Performance ◽

Skeletal Muscle Atrophy ◽

C2c12 Myotubes ◽

High Dose ◽

Protective Effects ◽

Akt Inhibitor ◽

Caspase 1

AbstractSkeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can improve skeletal muscle performance both in humans and mice. We here showed that dexamethasone-induced atrophy, as evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression, and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and gasdermin-D (GSDMD). Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine treatment ameliorated dexamethasone-induced muscle pyroptosis and atrophy both in vivo and in vitro. Activation of NLRP3 using LPS and ATP not only increased the cleavage and activation of Caspase-1 and GSDMD, but also increased the expression levels of atrophy markers MuRF1 and Atrogin-1 in trimetazidine-treated C2C12 myotubes. Mechanically, dexamethasone inhibited the phosphorylation of PI3K/AKT/FoxO3a, which could be attenuated by trimetazidine. Conversely, co-treatment with a PI3K/AKT inhibitor, picropodophyllin, remarkably increased the expression of NLRP3 and reversed the protective effects of trimetazidine against dexamethasone-induced C2C12 myotube pyroptosis and atrophy. Taken together, our study suggests that NLRP3/GSDMD-mediated pyroptosis might be a novel mechanism for dexamethasone-induced skeletal muscle atrophy. Trimetazidine might be developed as a potential therapeutic agent for the treatment of dexamethasone-induced muscle atrophy.

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Trimetazidine Attenuates Dexamethasone-Induced Muscle Atrophy via Inhibiting NLRP3/GSDMD Pathway-Mediated Pyroptosis

10.20944/preprints202012.0643.v1 ◽

2020 ◽

Author(s):

Li Wang ◽

Ming-Qing He ◽

Xi-Yu Shen ◽

Kang-Zhen Zhang ◽

Can Zhao ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Ring Finger ◽

Muscle Performance ◽

Skeletal Muscle Atrophy ◽

C2c12 Myotubes ◽

High Dose ◽

Therapeutic Compound

Skeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can also improve skeletal muscle performance both in human and mice. We here showed that dexamethasone induced atrophy, evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression , and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLRP3, Caspase-1 and GSDMD. Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine administration ameliorated dexamethasone-induced muscle atrophy both in vivo and in vitro. Moreover, trimetazidine improved exercise tolerance, as evidenced by increased running distance and running time, as well as increased skeletal muscle mass in dexamethasone-treated mice. Mechanically, trimetazidine could reverse dexamethasone-induced activation of pyroptosis both in C2C12 myotubes and in mice. Taken together, our present study demonstrated that NLRP3/GSDMD pathway-mediated pyroptosis was involved in dexamethasone-induced skeletal muscle atrophy. Trimetazidine could partially alleviate dexamethasone-induced skeletal muscle atrophy, and increase the diameter of C2C12 myotubes via inhibiting pyroptosis. Thus, trimetazidine might be a potential therapeutic compound for the prevention of muscle atrophy in glucocorticoid-treated patients.

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miR-182 attenuates atrophy-related gene expression by targeting FoxO3 in skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00395.2013 ◽

2014 ◽

Vol 307 (4) ◽

pp. C314-C319 ◽

Cited By ~ 56

Author(s):

Matthew B. Hudson ◽

Jill A. Rahnert ◽

Bin Zheng ◽

Myra E. Woodworth-Hobbs ◽

Harold A. Franch ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Cultured Cells ◽

Cathepsin L ◽

Immunoblot Analysis ◽

Skeletal Muscle Atrophy ◽

Luciferase Reporter ◽

C2c12 Myotubes ◽

Ubiquitin Proteasome ◽

The Media

Skeletal muscle atrophy occurs in response to a variety of conditions including chronic kidney disease, diabetes, cancer, and elevated glucocorticoids. MicroRNAs (miR) may play a role in the wasting process. Activation of the forkhead box O3 (FoxO3) transcription factor causes skeletal muscle atrophy in patients, animals, and cultured cells by increasing the expression of components of the ubiquitin-proteasome and autophagy-lysosome proteolytic systems. To identify microRNAs that potentially modulate the atrophy process, an in silico target analysis was performed and miR-182 was predicted to target FoxO3 mRNA. Using a combination of immunoblot analysis, quantitative real-time RT-PCR, and FoxO3 3′-UTR luciferase reporter genes, miR-182 was confirmed to regulate FoxO3 expression in C2C12 myotubes. Transfection of miR-182 into muscle cells decreased FoxO3 mRNA 30% and FoxO3 protein 67% ( P < 0.05) and also prevented a glucocorticoid-induced upregulation of multiple FoxO3 gene targets including MAFbx/atrogin-1, autophagy-related protein 12 (ATG12), cathepsin L, and microtubule-associated protein light chain 3 (LC3). Treatment of C2C12 myotubes with dexamethasone (Dex) (1 μM, 6 h) to induce muscle atrophy decreased miR-182 expression by 63% ( P < 0.05). Similarly, miR-182 was decreased 44% ( P < 0.05) in the gastrocnemius muscle of rats injected with streptozotocin to induce diabetes compared with controls. Finally, miR-182 was present in exosomes isolated from the media of C2C12 myotubes and Dex increased its abundance. These data identify miR-182 as an important regulator of FoxO3 expression that participates in the control of atrophy-inducing genes during catabolic diseases.

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A cell-autonomous role for the glucocorticoid receptor in skeletal muscle atrophy induced by systemic glucocorticoid exposure

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00512.2011 ◽

2012 ◽

Vol 302 (10) ◽

pp. E1210-E1220 ◽

Cited By ~ 57

Author(s):

Monica L. Watson ◽

Leslie M. Baehr ◽

Holger M. Reichardt ◽

Jan P. Tuckermann ◽

Sue C. Bodine ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucocorticoid Receptor ◽

Muscle Atrophy ◽

Target Genes ◽

Prolonged Exposure ◽

Skeletal Muscle Atrophy ◽

High Dose ◽

Nerve Lesion ◽

Nutritional Deprivation

Glucocorticoids (GCs) are important regulators of skeletal muscle mass, and prolonged exposure will induce significant muscle atrophy. To better understand the mechanism of skeletal muscle atrophy induced by elevated GC levels, we examined three different models: exogenous synthetic GC treatment [dexamethasone (DEX)], nutritional deprivation, and denervation. Specifically, we tested the direct contribution of the glucocorticoid receptor (GR) in skeletal muscle atrophy by creating a muscle-specific GR-knockout mouse line (MGRe3KO) using Cre-lox technology. In MGRe3KO mice, we found that the GR is essential for muscle atrophy in response to high-dose DEX treatment. In addition, DEX regulation of multiple genes, including two important atrophy markers, MuRF1 and MAFbx, is eliminated completely in the MGRe3KO mice. In a condition where endogenous GCs are elevated, such as nutritional deprivation, induction of MuRF1 and MAFbx was inhibited, but not completely blocked, in MGRe3KO mice. In response to sciatic nerve lesion and hindlimb muscle denervation, muscle atrophy and upregulation of MuRF1 and MAFbx occurred to the same extent in both wild-type and MGRe3KO mice, indicating that a functional GR is not required to induce atrophy under these conditions. Therefore, we demonstrate conclusively that the GR is an important mediator of skeletal muscle atrophy and associated gene expression in response to exogenous synthetic GCs in vivo and that the MGRe3KO mouse is a useful model for studying the role of the GR and its target genes in multiple skeletal muscle atrophy models.

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Protective Effect of Pyropia yezoensis Peptide on Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes

Marine Drugs ◽

10.3390/md17050284 ◽

2019 ◽

Vol 17 (5) ◽

pp. 284 ◽

Cited By ~ 4

Author(s):

Min-Kyeong Lee ◽

Jeong-Wook Choi ◽

Youn Hee Choi ◽

Taek-Jeong Nam

Keyword(s):

Skeletal Muscle ◽

Growth Factor ◽

Signaling Pathway ◽

Muscle Atrophy ◽

Skeletal Muscle Atrophy ◽

C2c12 Myotubes ◽

Protective Effects ◽

Factor I ◽

Igf I ◽

Foxo Signaling Pathway

Dexamethasone (DEX), a synthetic glucocorticoid, causes skeletal muscle atrophy. This study examined the protective effects of Pyropia yezoensis peptide (PYP15) against DEX-induced myotube atrophy and its association with insulin-like growth factor-I (IGF-I) and the Akt/mammalian target of rapamycin (mTOR)-forkhead box O (FoxO) signaling pathway. To elucidate the molecular mechanisms underlying the effects of PYP15 on DEX-induced myotube atrophy, C2C12 myotubes were treated for 24 h with 100 μM DEX in the presence or absence of 500 ng/mL PYP15. Cell viability assays revealed no PYP15 toxicity in C2C12 myotubes. PYP15 activated the insulin-like growth factor-I receptor (IGF-IR) and Akt-mTORC1 signaling pathway in DEX-induced myotube atrophy. In addition, PYP15 markedly downregulated the nuclear translocation of transcription factors FoxO1 and FoxO3a, and inhibited 20S proteasome activity. Furthermore, PYP15 inhibited the autophagy-lysosomal pathway in DEX-stimulated myotube atrophy. Our findings suggest that PYP15 treatment protected against myotube atrophy by regulating IGF-I and the Akt-mTORC1-FoxO signaling pathway in skeletal muscle. Therefore, PYP15 treatment appears to exert protective effects against skeletal muscle atrophy.

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Myoferlin Regulates Wnt/β-Catenin Signaling-Mediated Skeletal Muscle Development by Stabilizing Dishevelled-2 Against Autophagy

International Journal of Molecular Sciences ◽

10.3390/ijms20205130 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5130 ◽

Cited By ~ 1

Author(s):

Shunshun Han ◽

Can Cui ◽

Haorong He ◽

Xiaoxu Shen ◽

Yuqi Chen ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Muscle Development ◽

Skeletal Muscle Atrophy ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

Skeletal Muscle Development ◽

Muscle Tissues ◽

Underlying Mechanisms ◽

Canonical Wnt

Myoferlin (MyoF), which is a calcium/phospholipid-binding protein expressed in cardiac and muscle tissues, belongs to the ferlin family. While MyoF promotes myoblast differentiation, the underlying mechanisms remain poorly understood. Here, we found that MyoF not only promotes C2C12 myoblast differentiation, but also inhibits muscle atrophy and autophagy. In the present study, we found that myoblasts fail to develop into mature myotubes due to defective differentiation in the absence of MyoF. Meanwhile, MyoF regulates the expression of atrophy-related genes (Atrogin-1 and MuRF1) to rescue muscle atrophy. Furthermore, MyoF interacts with Dishevelled-2 (Dvl-2) to activate canonical Wnt signaling. MyoF facilitates Dvl-2 ubiquitination resistance by reducing LC3-labeled Dvl-2 levels and antagonizing the autophagy system. In conclusion, we found that MyoF plays an important role in myoblast differentiation during skeletal muscle atrophy. At the molecular level, MyoF protects Dvl-2 against autophagy-mediated degradation, thus promoting activation of the Wnt/β-catenin signaling pathway. Together, our findings suggest that MyoF, through stabilizing Dvl-2 and preventing autophagy, regulates Wnt/β-catenin signaling-mediated skeletal muscle development.

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UBE2L3, a Partner of MuRF1/TRIM63, Is Involved in the Degradation of Myofibrillar Actin and Myosin

Cells ◽

10.3390/cells10081974 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1974

Author(s):

Dulce Peris-Moreno ◽

Mélodie Malige ◽

Agnès Claustre ◽

Andrea Armani ◽

Cécile Coudy-Gandilhon ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Ubiquitin Proteasome System ◽

Contractile Proteins ◽

Skeletal Muscle Atrophy ◽

C2c12 Myotubes ◽

Skeletal Muscle Wasting ◽

Life Prognosis ◽

Ubiquitin Conjugating Enzymes ◽

Alpha Actin

The ubiquitin proteasome system (UPS) is the main player of skeletal muscle wasting, a common characteristic of many diseases (cancer, etc.) that negatively impacts treatment and life prognosis. Within the UPS, the E3 ligase MuRF1/TRIM63 targets for degradation several myofibrillar proteins, including the main contractile proteins alpha-actin and myosin heavy chain (MHC). We previously identified five E2 ubiquitin-conjugating enzymes interacting with MuRF1, including UBE2L3/UbcH7, that exhibited a high affinity for MuRF1 (KD = 50 nM). Here, we report a main effect of UBE2L3 on alpha-actin and MHC degradation in catabolic C2C12 myotubes. Consistently UBE2L3 knockdown in Tibialis anterior induced hypertrophy in dexamethasone (Dex)-treated mice, whereas overexpression worsened the muscle atrophy of Dex-treated mice. Using combined interactomic approaches, we also characterized the interactions between MuRF1 and its substrates alpha-actin and MHC and found that MuRF1 preferentially binds to filamentous F-actin (KD = 46.7 nM) over monomeric G-actin (KD = 450 nM). By contrast with actin that did not alter MuRF1–UBE2L3 affinity, binding of MHC to MuRF1 (KD = 8 nM) impeded UBE2L3 binding, suggesting that differential interactions prevail with MuRF1 depending on both the substrate and the E2. Our data suggest that UBE2L3 regulates contractile proteins levels and skeletal muscle atrophy.

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Decreased miR-497-5p Suppresses IL-6 Induced Atrophy in Muscle Cells

Cells ◽

10.3390/cells10123527 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3527

Author(s):

Paula P. Freire ◽

Sarah S. Cury ◽

Letícia O. Lopes ◽

Geysson J. Fernandez ◽

Jianming Liu ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Muscle Wasting ◽

Target Genes ◽

Cellular Proliferation ◽

Skeletal Muscle Regeneration ◽

C2c12 Myotubes ◽

Published Data ◽

Compensatory Mechanism ◽

Control Of Gene Expression

Interleukin-6 (IL-6) is a pro-inflammatory cytokine associated with skeletal muscle wasting in cancer cachexia. The control of gene expression by microRNAs (miRNAs) in muscle wasting involves the regulation of thousands of target transcripts. However, the miRNA-target networks associated with IL6-induced muscle atrophy remain to be characterized. Here, we show that IL-6 promotes the atrophy of C2C12 myotubes and changes the expression of 20 miRNAs (5 up-regulated and 15 down-regulated). Gene Ontology analysis of predicted miRNAs targets revealed post-transcriptional regulation of genes involved in cell differentiation, apoptosis, migration, and catabolic processes. Next, we performed a meta-analysis of miRNA-published data that identified miR-497-5p, a down-regulated miRNAs induced by IL-6, also down-regulated in other muscle-wasting conditions. We used miR-497-5p mimics and inhibitors to explore the function of miR-497-5p in C2C12 myoblasts and myotubes. We found that miR-497-5p can regulate the expression of the cell cycle genes CcnD2 and CcnE1 without affecting the rate of myoblast cellular proliferation. Notably, miR-497-5p mimics induced myotube atrophy and reduced Insr expression. Treatment with miR-497-5p inhibitors did not change the diameter of the myotubes but increased the expression of its target genes Insr and Igf1r. These genes are known to regulate skeletal muscle regeneration and hypertrophy via insulin-like growth factor pathway and were up-regulated in cachectic muscle samples. Our miRNA-regulated network analysis revealed a potential role for miR-497-5p during IL6-induced muscle cell atrophy and suggests that miR-497-5p is likely involved in a compensatory mechanism of muscle atrophy in response to IL-6.

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miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export

AJP Cell Physiology ◽

10.1152/ajpcell.00266.2013 ◽

2014 ◽

Vol 306 (6) ◽

pp. C551-C558 ◽

Cited By ~ 74

Author(s):

Matthew B. Hudson ◽

Myra E. Woodworth-Hobbs ◽

Bin Zheng ◽

Jill A. Rahnert ◽

Mitsi A. Blount ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Ring Finger ◽

Skeletal Muscle Atrophy ◽

C2c12 Myotubes ◽

Activated T Cells ◽

Streptozotocin Induced Diabetes ◽

The Media ◽

The Relationship ◽

Deficient Mice

Skeletal muscle atrophy is prevalent in chronic diseases, and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and muscle RING-finger protein-1 (MuRF1) in muscle. It also was reported to be regulated by cytoplasmic nuclear factor of activated T cells 3 (NFATc3) in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/NFAT signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with dexamethasone (Dex) for 48 h also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 h of Dex administration, suggesting a link between Cn signaling and miR-23a. The level of miR-23a was lower in primary myotubes from mice lacking the α- or β-isoform of the CnA catalytic subunit than wild-type mice. Dex did not further suppress miR-23a in myotubes from Cn-deficient mice. Overexpression of CnAβ in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes, and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions downregulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.

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