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The E3 ubiquitin ligase TRIM62 and inflammation-induced skeletal muscle atrophy.

Schmidt F, Kny M, Zhu X, Wollersheim T, Persicke K, Langhans C, Lodka D, Kleber C, Weber-Carstens S, Fielitz J - Crit Care (2014)

Bottom Line: Trim62 was expressed in myocytes, and its overexpression activated the atrophy-inducing activator protein 1 signal transduction pathway.Knockdown of Trim62 by small interfering RNA inhibited lipopolysaccharide-induced interleukin 6 expression.It could play a role in the pathogenesis of ICUAW by activating and maintaining inflammation in myocytes.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: ICU-acquired weakness (ICUAW) complicates the disease course of critically ill patients. Inflammation and acute-phase response occur directly within myocytes and contribute to ICUAW. We observed that tripartite motif-containing 62 (TRIM62), an E3 ubiquitin ligase and modifier of inflammation, is increased in the skeletal muscle of ICUAW patients. We investigated the regulation and function of muscular TRIM62 in critical illness.

Methods: Twenty-six critically ill patients with Sequential Organ Failure Assessment scores ≥8 underwent two skeletal muscle biopsies from the vastus lateralis at median days 5 and 15 in the ICU. Four patients undergoing elective orthopedic surgery served as controls. TRIM62 expression and protein content were analyzed in these biopsies. The kinetics of Trim62, Atrogin1 and MuRF1 expression were determined in the gastrocnemius/plantaris and tibialis anterior muscles from mouse models of inflammation-, denervation- and starvation-induced muscle atrophy to differentiate between these contributors to ICUAW. Cultured myocytes were used for mechanistic analyses.

Results: TRIM62 expression and protein content were increased early and remained elevated in muscles from critically ill patients. In all three animal models, muscular Trim62 expression was early and continuously increased. Trim62 was expressed in myocytes, and its overexpression activated the atrophy-inducing activator protein 1 signal transduction pathway. Knockdown of Trim62 by small interfering RNA inhibited lipopolysaccharide-induced interleukin 6 expression.

Conclusions: TRIM62 is activated in the muscles of critically ill patients. It could play a role in the pathogenesis of ICUAW by activating and maintaining inflammation in myocytes.

Trial registration: Current Controlled Trials ID: ISRCTN77569430 (registered 13 February 2008).

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Related in: MedlinePlus

Trim62 is expressed in myocytes. (A) Immunocytochemistry of differentiated C2C12 myotubes was performed using an anti-Trim62 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 50 μm. (B) C2C12 myoblasts were transfected with expression plasmids encoding Trim62-FLAG (top panel), Trim62 myc/His-6 (bottom panel) or an empty vector control plasmid. Immunofluorescence analysis was performed using anti-FLAG and anti-Myc antibodies, respectively, as well as an Alexa Fluor 488–coupled secondary antibody. Nuclei were stained with DAPI (blue). Scale bar, 25 μm.
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Fig6: Trim62 is expressed in myocytes. (A) Immunocytochemistry of differentiated C2C12 myotubes was performed using an anti-Trim62 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 50 μm. (B) C2C12 myoblasts were transfected with expression plasmids encoding Trim62-FLAG (top panel), Trim62 myc/His-6 (bottom panel) or an empty vector control plasmid. Immunofluorescence analysis was performed using anti-FLAG and anti-Myc antibodies, respectively, as well as an Alexa Fluor 488–coupled secondary antibody. Nuclei were stained with DAPI (blue). Scale bar, 25 μm.

Mentions: Immunocytochemistry was performed to elucidate whether endogenous Trim62 protein was contained in myocytes. As expected, differentiated C2C12 myotubes contained Trim62 endogenously. Trim62 was exclusively localized in the cytoplasm of these cells (Figure 6A). The localization of Trim62 in the cytoplasm was confirmed when we transfected expression plasmids encoding Trim62 with either an amino-terminal FLAG tag or a carboxy-terminal myc/His-6 tag in C2C12 myoblasts (Figure 6B). These data indicate that Trim62 is contained in myocytes, where it is localized mainly in the cytoplasm.Figure 6


The E3 ubiquitin ligase TRIM62 and inflammation-induced skeletal muscle atrophy.

Schmidt F, Kny M, Zhu X, Wollersheim T, Persicke K, Langhans C, Lodka D, Kleber C, Weber-Carstens S, Fielitz J - Crit Care (2014)

Trim62 is expressed in myocytes. (A) Immunocytochemistry of differentiated C2C12 myotubes was performed using an anti-Trim62 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 50 μm. (B) C2C12 myoblasts were transfected with expression plasmids encoding Trim62-FLAG (top panel), Trim62 myc/His-6 (bottom panel) or an empty vector control plasmid. Immunofluorescence analysis was performed using anti-FLAG and anti-Myc antibodies, respectively, as well as an Alexa Fluor 488–coupled secondary antibody. Nuclei were stained with DAPI (blue). Scale bar, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4231194&req=5

Fig6: Trim62 is expressed in myocytes. (A) Immunocytochemistry of differentiated C2C12 myotubes was performed using an anti-Trim62 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 50 μm. (B) C2C12 myoblasts were transfected with expression plasmids encoding Trim62-FLAG (top panel), Trim62 myc/His-6 (bottom panel) or an empty vector control plasmid. Immunofluorescence analysis was performed using anti-FLAG and anti-Myc antibodies, respectively, as well as an Alexa Fluor 488–coupled secondary antibody. Nuclei were stained with DAPI (blue). Scale bar, 25 μm.
Mentions: Immunocytochemistry was performed to elucidate whether endogenous Trim62 protein was contained in myocytes. As expected, differentiated C2C12 myotubes contained Trim62 endogenously. Trim62 was exclusively localized in the cytoplasm of these cells (Figure 6A). The localization of Trim62 in the cytoplasm was confirmed when we transfected expression plasmids encoding Trim62 with either an amino-terminal FLAG tag or a carboxy-terminal myc/His-6 tag in C2C12 myoblasts (Figure 6B). These data indicate that Trim62 is contained in myocytes, where it is localized mainly in the cytoplasm.Figure 6

Bottom Line: Trim62 was expressed in myocytes, and its overexpression activated the atrophy-inducing activator protein 1 signal transduction pathway.Knockdown of Trim62 by small interfering RNA inhibited lipopolysaccharide-induced interleukin 6 expression.It could play a role in the pathogenesis of ICUAW by activating and maintaining inflammation in myocytes.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: ICU-acquired weakness (ICUAW) complicates the disease course of critically ill patients. Inflammation and acute-phase response occur directly within myocytes and contribute to ICUAW. We observed that tripartite motif-containing 62 (TRIM62), an E3 ubiquitin ligase and modifier of inflammation, is increased in the skeletal muscle of ICUAW patients. We investigated the regulation and function of muscular TRIM62 in critical illness.

Methods: Twenty-six critically ill patients with Sequential Organ Failure Assessment scores ≥8 underwent two skeletal muscle biopsies from the vastus lateralis at median days 5 and 15 in the ICU. Four patients undergoing elective orthopedic surgery served as controls. TRIM62 expression and protein content were analyzed in these biopsies. The kinetics of Trim62, Atrogin1 and MuRF1 expression were determined in the gastrocnemius/plantaris and tibialis anterior muscles from mouse models of inflammation-, denervation- and starvation-induced muscle atrophy to differentiate between these contributors to ICUAW. Cultured myocytes were used for mechanistic analyses.

Results: TRIM62 expression and protein content were increased early and remained elevated in muscles from critically ill patients. In all three animal models, muscular Trim62 expression was early and continuously increased. Trim62 was expressed in myocytes, and its overexpression activated the atrophy-inducing activator protein 1 signal transduction pathway. Knockdown of Trim62 by small interfering RNA inhibited lipopolysaccharide-induced interleukin 6 expression.

Conclusions: TRIM62 is activated in the muscles of critically ill patients. It could play a role in the pathogenesis of ICUAW by activating and maintaining inflammation in myocytes.

Trial registration: Current Controlled Trials ID: ISRCTN77569430 (registered 13 February 2008).

Show MeSH
Related in: MedlinePlus