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Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2.

Witt CC, Witt SH, Lerche S, Labeit D, Back W, Labeit S - EMBO J. (2007)

Bottom Line: The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues.Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis.Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany.

ABSTRACT
The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.

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Elevated muscle protein synthesis after inactivation of MuRF1 and MuRF2. (A) The translation elongation factor EEF1G (interacting with MuRF1 and MuRF2; see Figure 3), its binding protein elF3a subunit INT6, and the translation-promoting p70S6 kinase are upregulated in dKO myocardium. (B) Immunohistochemistry with phospho-specific antibodies demonstrated the upregulation of the activated forms of p70S6K and its substrate S6 (phospho-p70S6K and phospho-S6, respectively) in dKO myocardium, suggesting activation of the AKT/mTOR pathway. Scale bar, 20 μm. (C) Nuclear entry of phospho-p70S6K was stimulated in dKO myocardium, whereas cellular distribution of SRF did not change significantly (data represent counts of 200 nuclei on 4HPF sections). (D) Fractional synthesis rate (% per 48 h) of total cardiac muscle proteins was determined by injecting D5-F i.p. into mice of each genotype (n=6). Total cardiac muscle protein synthesis is elevated in dKO myocardium (P=0.05 in dKO versus WT, mice aged between 6 and 16 months). (E) Serum level of creatinine was increased in dKO mice (n=6 in each group, P=0.01 in dKO versus WT mice, mice aged between 6 and 16 months).
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f6: Elevated muscle protein synthesis after inactivation of MuRF1 and MuRF2. (A) The translation elongation factor EEF1G (interacting with MuRF1 and MuRF2; see Figure 3), its binding protein elF3a subunit INT6, and the translation-promoting p70S6 kinase are upregulated in dKO myocardium. (B) Immunohistochemistry with phospho-specific antibodies demonstrated the upregulation of the activated forms of p70S6K and its substrate S6 (phospho-p70S6K and phospho-S6, respectively) in dKO myocardium, suggesting activation of the AKT/mTOR pathway. Scale bar, 20 μm. (C) Nuclear entry of phospho-p70S6K was stimulated in dKO myocardium, whereas cellular distribution of SRF did not change significantly (data represent counts of 200 nuclei on 4HPF sections). (D) Fractional synthesis rate (% per 48 h) of total cardiac muscle proteins was determined by injecting D5-F i.p. into mice of each genotype (n=6). Total cardiac muscle protein synthesis is elevated in dKO myocardium (P=0.05 in dKO versus WT, mice aged between 6 and 16 months). (E) Serum level of creatinine was increased in dKO mice (n=6 in each group, P=0.01 in dKO versus WT mice, mice aged between 6 and 16 months).

Mentions: Possibly, the massive hypertrophic phenotype of dKO mice might be caused by reduced multi-ubiquitination and degradation of total muscle proteins (linked to inactivation of MuRF1/2 E3-ubiquitin-ligase activities). To test for reduced ubiquitination/degradation, we determined the levels of multi-ubiquitinated proteins on western blot panels displaying the different MuRF1/2 genotypes. Total levels of multi-ubiquitinated proteins did not change upon MuRF1/2 inactivation (Figure 5A). Among the family of ubiquitin-related modifiers, we only noted for SUMO4 an upregulation of SUMO4ylated species after MuRF1/2 inactivation (Figure 5A). Next, we speculated that, as an alternative mechanism, MuRF1 and MuRF2 might regulate the translational machinery directly (e.g., via interaction with INT6, EEF1G, GFM1; see Figure 3). When testing the myocardial expression of MuRF1/2-associated translation factors, we found that EEF1G and INT6 (subunits of EF-1 and elF3a, respectively; see Belle et al, 1995; Morris et al, 2007) were upregulated specifically after deletion of all four MuRF1/2 alleles (Figure 6A). Consistent with a general translational activation, we found upregulation of p70S6K (Figures 6A) and its activated phosphorylated form phospho-p70S6K, as well as its substrate phospho-S6 (markers for an activated Akt/mTor pathway; see Figure 6B). Phospho-p70S6K was recruited to the nucleus (Figure 6B), thus mimicking changes observed in exercise-induced translational activation (Koopman et al, 2006). For SRF, we noted no nuclear recruitment after MuRF1/2 deletions (Figure 6C).


Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2.

Witt CC, Witt SH, Lerche S, Labeit D, Back W, Labeit S - EMBO J. (2007)

Elevated muscle protein synthesis after inactivation of MuRF1 and MuRF2. (A) The translation elongation factor EEF1G (interacting with MuRF1 and MuRF2; see Figure 3), its binding protein elF3a subunit INT6, and the translation-promoting p70S6 kinase are upregulated in dKO myocardium. (B) Immunohistochemistry with phospho-specific antibodies demonstrated the upregulation of the activated forms of p70S6K and its substrate S6 (phospho-p70S6K and phospho-S6, respectively) in dKO myocardium, suggesting activation of the AKT/mTOR pathway. Scale bar, 20 μm. (C) Nuclear entry of phospho-p70S6K was stimulated in dKO myocardium, whereas cellular distribution of SRF did not change significantly (data represent counts of 200 nuclei on 4HPF sections). (D) Fractional synthesis rate (% per 48 h) of total cardiac muscle proteins was determined by injecting D5-F i.p. into mice of each genotype (n=6). Total cardiac muscle protein synthesis is elevated in dKO myocardium (P=0.05 in dKO versus WT, mice aged between 6 and 16 months). (E) Serum level of creatinine was increased in dKO mice (n=6 in each group, P=0.01 in dKO versus WT mice, mice aged between 6 and 16 months).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2168395&req=5

f6: Elevated muscle protein synthesis after inactivation of MuRF1 and MuRF2. (A) The translation elongation factor EEF1G (interacting with MuRF1 and MuRF2; see Figure 3), its binding protein elF3a subunit INT6, and the translation-promoting p70S6 kinase are upregulated in dKO myocardium. (B) Immunohistochemistry with phospho-specific antibodies demonstrated the upregulation of the activated forms of p70S6K and its substrate S6 (phospho-p70S6K and phospho-S6, respectively) in dKO myocardium, suggesting activation of the AKT/mTOR pathway. Scale bar, 20 μm. (C) Nuclear entry of phospho-p70S6K was stimulated in dKO myocardium, whereas cellular distribution of SRF did not change significantly (data represent counts of 200 nuclei on 4HPF sections). (D) Fractional synthesis rate (% per 48 h) of total cardiac muscle proteins was determined by injecting D5-F i.p. into mice of each genotype (n=6). Total cardiac muscle protein synthesis is elevated in dKO myocardium (P=0.05 in dKO versus WT, mice aged between 6 and 16 months). (E) Serum level of creatinine was increased in dKO mice (n=6 in each group, P=0.01 in dKO versus WT mice, mice aged between 6 and 16 months).
Mentions: Possibly, the massive hypertrophic phenotype of dKO mice might be caused by reduced multi-ubiquitination and degradation of total muscle proteins (linked to inactivation of MuRF1/2 E3-ubiquitin-ligase activities). To test for reduced ubiquitination/degradation, we determined the levels of multi-ubiquitinated proteins on western blot panels displaying the different MuRF1/2 genotypes. Total levels of multi-ubiquitinated proteins did not change upon MuRF1/2 inactivation (Figure 5A). Among the family of ubiquitin-related modifiers, we only noted for SUMO4 an upregulation of SUMO4ylated species after MuRF1/2 inactivation (Figure 5A). Next, we speculated that, as an alternative mechanism, MuRF1 and MuRF2 might regulate the translational machinery directly (e.g., via interaction with INT6, EEF1G, GFM1; see Figure 3). When testing the myocardial expression of MuRF1/2-associated translation factors, we found that EEF1G and INT6 (subunits of EF-1 and elF3a, respectively; see Belle et al, 1995; Morris et al, 2007) were upregulated specifically after deletion of all four MuRF1/2 alleles (Figure 6A). Consistent with a general translational activation, we found upregulation of p70S6K (Figures 6A) and its activated phosphorylated form phospho-p70S6K, as well as its substrate phospho-S6 (markers for an activated Akt/mTor pathway; see Figure 6B). Phospho-p70S6K was recruited to the nucleus (Figure 6B), thus mimicking changes observed in exercise-induced translational activation (Koopman et al, 2006). For SRF, we noted no nuclear recruitment after MuRF1/2 deletions (Figure 6C).

Bottom Line: The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues.Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis.Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany.

ABSTRACT
The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.

Show MeSH
Related in: MedlinePlus