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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

Inflammation leads to skeletal muscle atrophyin vivo. Six- to eight-week-old male C57BL/6 N mice were subjected to sham operations (sham, n =5) or cecal ligation and puncture (CLP) surgery (n =5), as indicated, to induce polymicrobial sepsis. (A) Weights of the gastrocnemius/plantaris and tibialis anterior muscles were determined after 24 hours, 48 hours, 72 hours or 96 hours and normalized to tibia length. Data are presented as mean ± SEM. ***P <0.001, *P <0.05. Quantitative RT-PCR analyses of Atrogin1(B) and MuRF1(C) expression, and immunoblotting of proteins using anti-MuRF1 antibody (D), in gastrocnemius/plantaris and tibialis anterior muscles at 24 hours, 48 hours, 72 hours or 96 hours after surgery, as indicated. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expression and protein content were used as reference values, and data shown are relative changes. Data are presented as mean ± SEM. ***P <0.001, **P <0.01, *P <0.05. MuRF1, Muscle RING (really interesting new gene) finger–containing protein 1.
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Fig2: Inflammation leads to skeletal muscle atrophyin vivo. Six- to eight-week-old male C57BL/6 N mice were subjected to sham operations (sham, n =5) or cecal ligation and puncture (CLP) surgery (n =5), as indicated, to induce polymicrobial sepsis. (A) Weights of the gastrocnemius/plantaris and tibialis anterior muscles were determined after 24 hours, 48 hours, 72 hours or 96 hours and normalized to tibia length. Data are presented as mean ± SEM. ***P <0.001, *P <0.05. Quantitative RT-PCR analyses of Atrogin1(B) and MuRF1(C) expression, and immunoblotting of proteins using anti-MuRF1 antibody (D), in gastrocnemius/plantaris and tibialis anterior muscles at 24 hours, 48 hours, 72 hours or 96 hours after surgery, as indicated. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expression and protein content were used as reference values, and data shown are relative changes. Data are presented as mean ± SEM. ***P <0.001, **P <0.01, *P <0.05. MuRF1, Muscle RING (really interesting new gene) finger–containing protein 1.

Mentions: Inflammation-induced muscle atrophy was induced by the CLP method of polymicrobial sepsis as previously described [13,25]. Recently, we reported that increased expression of Il-6 and Saa1 in muscles of CLP mice might contribute to inflammation-induced atrophy [13,25]. In the present study, general inflammation was confirmed by an early and persistent increase in gene expression of the inflammatory cytokines Il-6 and tumor necrosis factor α (Tnf-α), as well as of the acute-phase protein Saa1, in the liver of CLP mice (Additional file 3: Figure S2). Septic mice showed a significant decrease in body weight as soon as 24 hours after surgery. CLP mice continued their weight loss, which reached its maximum after 96 hours at a reduction of 19% of body weight (P <0.05) (Additional file 4: Figure S3A). A significant decrease in muscle weights was found after 72 hours and 96 hours of sepsis, indicative of muscular atrophy (Figure 2A). Using qRT-PCR, we found a significant increase in Atrogin1 and MuRF1 expression after 24 hours of sepsis in gastrocnemius/plantaris and tibialis anterior muscles. Atrogin1 and MuRF1 expression followed a time course with early induction and a subsequent decrease (Figures 2B and 2C). Whereas MuRF1 expression was highest at the 24-hour time point, the induction of Atrogin1 peaked at 48 hours of sepsis (Figures 2B and 2C). Immunoblot analysis confirmed that muscular MuRF1 protein content went in parallel with its expression, showing an early induction and a subsequent decrease in gastrocnemius/plantaris and tibialis anterior muscles of CLP mice (Figure 2D).Figure 2


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)

Inflammation leads to skeletal muscle atrophyin vivo. Six- to eight-week-old male C57BL/6 N mice were subjected to sham operations (sham, n =5) or cecal ligation and puncture (CLP) surgery (n =5), as indicated, to induce polymicrobial sepsis. (A) Weights of the gastrocnemius/plantaris and tibialis anterior muscles were determined after 24 hours, 48 hours, 72 hours or 96 hours and normalized to tibia length. Data are presented as mean ± SEM. ***P <0.001, *P <0.05. Quantitative RT-PCR analyses of Atrogin1(B) and MuRF1(C) expression, and immunoblotting of proteins using anti-MuRF1 antibody (D), in gastrocnemius/plantaris and tibialis anterior muscles at 24 hours, 48 hours, 72 hours or 96 hours after surgery, as indicated. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expression and protein content were used as reference values, and data shown are relative changes. Data are presented as mean ± SEM. ***P <0.001, **P <0.01, *P <0.05. MuRF1, Muscle RING (really interesting new gene) finger–containing protein 1.
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Related In: Results  -  Collection

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Fig2: Inflammation leads to skeletal muscle atrophyin vivo. Six- to eight-week-old male C57BL/6 N mice were subjected to sham operations (sham, n =5) or cecal ligation and puncture (CLP) surgery (n =5), as indicated, to induce polymicrobial sepsis. (A) Weights of the gastrocnemius/plantaris and tibialis anterior muscles were determined after 24 hours, 48 hours, 72 hours or 96 hours and normalized to tibia length. Data are presented as mean ± SEM. ***P <0.001, *P <0.05. Quantitative RT-PCR analyses of Atrogin1(B) and MuRF1(C) expression, and immunoblotting of proteins using anti-MuRF1 antibody (D), in gastrocnemius/plantaris and tibialis anterior muscles at 24 hours, 48 hours, 72 hours or 96 hours after surgery, as indicated. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expression and protein content were used as reference values, and data shown are relative changes. Data are presented as mean ± SEM. ***P <0.001, **P <0.01, *P <0.05. MuRF1, Muscle RING (really interesting new gene) finger–containing protein 1.
Mentions: Inflammation-induced muscle atrophy was induced by the CLP method of polymicrobial sepsis as previously described [13,25]. Recently, we reported that increased expression of Il-6 and Saa1 in muscles of CLP mice might contribute to inflammation-induced atrophy [13,25]. In the present study, general inflammation was confirmed by an early and persistent increase in gene expression of the inflammatory cytokines Il-6 and tumor necrosis factor α (Tnf-α), as well as of the acute-phase protein Saa1, in the liver of CLP mice (Additional file 3: Figure S2). Septic mice showed a significant decrease in body weight as soon as 24 hours after surgery. CLP mice continued their weight loss, which reached its maximum after 96 hours at a reduction of 19% of body weight (P <0.05) (Additional file 4: Figure S3A). A significant decrease in muscle weights was found after 72 hours and 96 hours of sepsis, indicative of muscular atrophy (Figure 2A). Using qRT-PCR, we found a significant increase in Atrogin1 and MuRF1 expression after 24 hours of sepsis in gastrocnemius/plantaris and tibialis anterior muscles. Atrogin1 and MuRF1 expression followed a time course with early induction and a subsequent decrease (Figures 2B and 2C). Whereas MuRF1 expression was highest at the 24-hour time point, the induction of Atrogin1 peaked at 48 hours of sepsis (Figures 2B and 2C). Immunoblot analysis confirmed that muscular MuRF1 protein content went in parallel with its expression, showing an early induction and a subsequent decrease in gastrocnemius/plantaris and tibialis anterior muscles of CLP mice (Figure 2D).Figure 2

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