Acute Heat Exposure Alters Autophagy Signaling in C2C12 Myotubes.
ABSTRACT: Autophagy is a major intracellular degradation process that is essential for the clearance of unnecessary proteins/organelles and the maintenance of cellular homeostasis. The inhibition of autophagy results in cellular consequences associated with many skeletal muscle pathologies, and therapies designed to elevate autophagic activity may provide protection from such pathologies. Acute exposure to low levels of heat has therapeutic effects; however, the impact of heat on skeletal muscle autophagy remains unclear. In the present study, C2C12 myotubes were maintained at 37°C thermoneutral (TN) or heated at 40°C heat treatment (HT) for 1 h. Myotubes were harvested immediately after heating, or returned to 37°C for recovery of 2 or 24 h. HT resulted in an elevation in pAMPK (T172), Beclin-1, and LC3 II, a marker for autophagosome formation, but no change in p62. In the context of autophagy inhibition with Bafilomycin A1, HT resulted in lower LC3 II compared to TN. The applied heat load induced the heat shock response, as evidenced by immediate upregulation of HSF1 and Hsp70. Hsp70 continued to increase during recovery, whereas pHsp27 was downregulated acutely in response to HT, but retuned to TN levels by 2 h of recovery. HT also reduced the phosphorylation of the MAP-kinases p38 and JNK. These findings suggest that an acute, short bout of mild heat may be beneficial to skeletal muscle by increasing AMPK activity, markers of autophagasome formation, and the heat shock response.
Project description:Hyperammonemia and sarcopenia (loss of skeletal muscle) are consistent abnormalities in cirrhosis and portosystemic shunting. We have shown that muscle ubiquitin-proteasome components are not increased with hyperammonemia despite sarcopenia. This suggests that an alternative mechanism of proteolysis contributes to sarcopenia in cirrhosis. We hypothesized that autophagy could be this alternative pathway since we observed increases in classic autophagy markers, increased LC3 lipidation, beclin-1 expression, and p62 degradation in immunoblots of skeletal muscle protein in cirrhotic patients. We observed similar changes in these autophagy markers in the portacaval anastamosis (PCA) rat model. To determine the mechanistic relationship between hyperammonemia and autophagy, we exposed murine C(2)C(12) myotubes to ammonium acetate. Significant increases in LC3 lipidation, beclin-1 expression, and p62 degradation occurred by 1 h, whereas autophagy gene expression (LC3, Atg5, Atg7, beclin-1) increased at 24 h. C(2)C(12) cells stably expressing GFP-LC3 or GFP-mCherry-LC3 constructs showed increased formation of mature autophagosomes supported by electron microscopic studies. Hyperammonemia also increased autophagic flux in mice, as quantified by an in vivo autophagometer. Because hyperammonemia induces nitration of proteins in astrocytes, we quantified global muscle protein nitration in cirrhotic patients, in the PCA rat, and in C(2)C(12) cells treated with ammonium acetate. Increased protein nitration was observed in all of these systems. Furthermore, colocalization of nitrated proteins with GFP-LC3-positive puncta in hyperammonemic C(2)C(12) cells suggested that autophagy is involved in degradation of nitrated proteins. These observations show that increased skeletal muscle autophagy in cirrhosis is mediated by hyperammonemia and may contribute to sarcopenia of cirrhosis.
Project description:Low estrogen levels may predispose women to increased bodyweight and dyslipidemia. Previous studies from our laboratory suggest an involvement of depressed heat shock response (HSR) in this scenario because estrogen potently stimulates HSR. As heat treatment induces the expression of the anti-inflammatory heat shock proteins of the 70-kDa family (HSP70) and its accompanying HSR, we aimed to investigate whether chronic heat treatment promotes beneficial effects on biometric, lipid profile, oxidative stress, and HSR in ovariectomized rats. Wistar adult female rats (n?=?32) were divided into four groups: control (C, n?=?7), ovariectomized (OVX, n?=?9), heat-treated (HT, n?=?9), and heat-treated ovariectomized rats (OVX+HT, n?=?7). HT and OVX+HT rats were anesthetized and submitted to heat treatment (once a week for 12 weeks) in a water bath (41 °C) to increase rats' rectal temperature up to 41 °C for 15 min, while C and OVX animals were submitted to a 36 °C water bath. HT attenuated the weight gain induced by OVX and increased HDL cholesterol and triglyceride serum levels. Also, OVX rats showed increased total cholesterol and LDL cholesterol levels that were not influenced by HT. Interestingly, it was found that an overall trend for HT to decrease tissue catalase and superoxide dismutase antioxidant activities was paralleled by a decrease in malondialdehyde levels (indicative of lower lipoperoxidation), especially in the skeletal muscle. Surprisingly, OVX was not able to depress intracellular HSP70 expression in the skeletal muscle, as expected, and this remained unchanged with HT. However, chronic HT did enhance intracellular HSP70 contents in white adipose tissue of OVX animals. As both glucose and insulin tolerance tests were not affected by OVX, which was not modified by HT, we suppose that estrogen absence alone is not sufficient to determine a state of insulin resistance associated with low intramuscular HSP70 content.
Project description:Chemerin is a novel adipocyte-derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin-mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3-I to LC3-II and higher expression levels of Beclin1 and autophagy-related protein-5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine-like receptor 1 or the use of mitochondria-targeting antioxidant Mito-TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin-stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3? (FoxO3?). Chemerin-induced mitochondrial autophagy likely through an AKT-FoxO3?-dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.
Project description:Satellite cells are crucial for skeletal muscle regeneration, but the molecular mechanisms regulating satellite cells are not entirely understood. Here, we show that the immunoglobulin superfamily containing leucine-rich repeat (Islr), a newly identified marker for mesenchymal stem cells, stabilizes canonical Wnt signaling and promote skeletal muscle regeneration. Loss of Islr delays skeletal muscle regeneration in adult mice. In the absence of Islr, myoblasts fail to develop into mature myotubes due to defective differentiation. Islr interacts with Dishevelled-2 (Dvl2) to activate canonical Wnt signaling, consequently regulating the myogenic factor myogenin (MyoG). Furthermore, Islr stabilizes Dvl2 by reducing the level of LC3-labeled Dvl2 and preventing cells from undergoing autophagy. Together, our findings identify Islr as an important regulator for skeletal muscle regeneration.
Project description:TGF-beta1 has been shown to induce autophagy in certain cells but whether and how this action is exerted in muscle and whether this activity relates to TGF-beta1 control of muscle cell differentiation remains unknown. Here, we show that expression of the autophagy-promoting protein phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) progressively declines during L6 and C2C12 skeletal muscle cell differentiation. PED/PEA-15 underwent rapid induction upon TGF-beta1 exposure of L6 and C2C12 myoblasts, accompanied by impaired differentiation into mature myotubes. TGF-beta1 also induced autophagy in the L6 and C2C12 cells through a PP2A/FoxO1-mediated mechanism. Both the TGF-beta1 effect on differentiation and that on autophagy were blocked by specific PED/PEA-15 ShRNAs. Myoblasts stably overexpressing PED/PEA-15 did not differentiate and showed markedly enhanced autophagy. In these same cells, the autophagy inhibitor 3-methyladenine rescued TGF-beta1 effect on both autophagy and myogenesis, indicating that PED/PEA-15 mediates TGF-beta1 effects in muscle. Muscles from transgenic mice overexpressing PED/PEA-15 featured a significant number of atrophic fibers, accompanied by increased light chain 3 (LC3)II to LC3I ratio and reduced PP2A/FoxO1 phosphorylation. Interestingly, these mice showed significantly impaired locomotor activity compared with their non-transgenic littermates. TGF-beta1 causes transcriptional upregulation of the autophagy-promoting gene PED/PEA-15, which in turn is capable to induce atrophic responses in skeletal muscle in vivo.
Project description:C/EBP? is a key mediator of cancer-induced skeletal muscle wasting. However, the signaling mechanisms that activate C/EBP? in the cancer milieu are poorly defined. Here, we report cancer-induced muscle wasting requires the transcriptional cofactor p300, which is critical for the activation of C/EBP?. Conditioned media from diverse types of tumor cells as well as recombinant HSP70 and HSP90 provoked rapid acetylation of C/EBP? in myotubes, particularly at its Lys39 residue. Overexpression of C/EBP? with mutated Lys39 impaired Lewis lung carcinoma (LLC)-induced activation of the C/EBP?-dependent catabolic response, which included upregulation of E3 ligases UBR2 and atrogin1/MAFbx, increased LC3-II, and loss of muscle proteins both in myotubes and mouse muscle. Silencing p300 in myotubes or overexpressing a dominant negative p300 mutant lacking acetyltransferase activity in mouse muscle attenuated LLC tumor-induced muscle catabolism. Administration of pharmacologic p300 inhibitor C646, but not PCAF/GCN5 inhibitor CPTH6, spared LLC tumor-bearing mice from muscle wasting. Furthermore, mice with muscle-specific p300 knockout were resistant to LLC tumor-induced muscle wasting. These data suggest that p300 is a key mediator of LLC tumor-induced muscle wasting whose acetyltransferase activity may be targeted for therapeutic benefit in this disease. SIGNIFICANCE: These findings demonstrate that tumor-induced muscle wasting in mice is abrogated by knockout, mutation of Lys39 or Asp1399, and pharmacologic inhibition of p300.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1331/F1.large.jpg.
Project description:Muscle LIM protein (MLP) is a microtubule-associated protein expressed in cardiac and muscle tissues that belongs to the cysteine-rich protein (CSRP/CRP) family. MLP has a central role during muscle development and for architectural maintenance of muscle cells. However, muscle cells rely on autophagy during differentiation and for structural maintenance. To study the role of MLP in autophagy, we have used C2C12 mouse myoblasts silenced or overexpressing MLP. Our results show that MLP contributes to the correct autophagosome formation and flux by interacting with LC3 as demonstrated by co-immunoprecipitation and PLA assay. In fact, MLP silencing results in decreased LC3-II staining and absent degradation of long-lived proteins. Moreover, MLP silencing impaired myoblasts differentiation as measured by decreased expression of MyoD1, MyoG1 and myosin heavy chain. Ultrastructural analysis revealed the presence of large empty autophagosomes in myoblasts and multimembranous structures in myotubes from MLP-silenced clones. Impaired autophagy in MLP-silenced cells resulted in increased susceptibility to apoptotic cell death. In fact, treatment of MLP-silenced C2C12 myoblasts and myotubes with staurosporine resulted in increased caspase-3 and PARP cleavage as well as increased percentage of cell death. In conclusion, we propose that MLP regulates autophagy during muscle cell differentiation or maintenance through a mechanism involving MLP/LC3-II interaction and correct autophagosome formation.
Project description:Skeletal muscle apoptosis and autophagy are catabolic processes that contribute to muscle atrophy during aging, disease, and following muscle injury. In this article, we present data on skeletal muscle apoptosis, autophagy, and morphology in C57BL/6 mice following doxorubicin administration. More specifically, time-course data on caspase-3, caspase-8, caspase-9, calpain, and cathepsin activity are presented, along with data on ATG7, p62, LC3-I, and LC3-II protein expression. Data on skeletal muscle reactive oxygen species (ROS) production, muscle morphology, as well as body and muscle weights are also presented.
Project description:Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.
Project description:We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ? 0.7), but poor reliability for three individual Western blots (interclass correlation, ? 0.4) (Table 3).