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Effects of endotoxaemia on protein metabolism in rat fast-twitch skeletal muscle and myocardium.

Murton AJ, Alamdari N, Gardiner SM, Constantin-Teodosiu D, Layfield R, Bennett T, Greenhaff PL - PLoS ONE (2009)

Bottom Line: It is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically observed in fast-twitch skeletal muscle during endotoxaemia.The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P<0.05), but was no different from controls in the myocardium.In summary, these findings suggest that the myocardium does not undergo the same catabolic response as skeletal muscle during early endotoxaemia, partly due to the absence of transcriptional and signalling events in the myocardium typically associated with increased muscle proteolysis and the suppression of protein synthesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK. andrew.murton@nottingham.ac.uk

ABSTRACT

Background: It is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically observed in fast-twitch skeletal muscle during endotoxaemia.

Methodology/principal findings: To investigate this further, and to determine if there is any divergence in the response of skeletal muscle and myocardium in the mechanisms that are thought to be largely responsible for eliciting changes in protein content, Sprague Dawley rats were implanted with vascular catheters and administered lipopolysaccharide (LPS; 150 microg kg(-1) h(-1)) intravenously for 2 h, 6 h or 24 h (saline administered control animals were also included), after which the extensor digitorum longus (EDL) and myocardium were removed under terminal anaesthesia. The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P<0.05), but was no different from controls in the myocardium. At the same time point, a significant increase in MAFbx/atrogin-1 and MuRF1 mRNA (3.7+/-0.7- and 19.5+/-1.9-fold increase vs. controls, respectively; P<0.05), in addition to protein levels of alpha1-3, 5-7 subunits of the 20S proteasome, were observed in EDL but not myocardium. In contrast, elevations in phosphorylation of p70 S6K residues Thr(421)/Ser(424), and 4E-BP1 residues Thr(37)/Thr(46) (P<0.05), consistent with an elevation in translation initiation, were seen exclusively in the myocardium of LPS-treated animals.

Conclusions/significance: In summary, these findings suggest that the myocardium does not undergo the same catabolic response as skeletal muscle during early endotoxaemia, partly due to the absence of transcriptional and signalling events in the myocardium typically associated with increased muscle proteolysis and the suppression of protein synthesis.

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mRNA levels for proteins associated with proteolysis in muscle and myocardium following LPS infusion.Bars denote fold change in mRNA levels of A) MAFbx/atrogin-1, B) MuRF1, C) 20S proteasome subunit α1, D) TNFα and E) IL-6, compared to corresponding control values in response to either a 2 h, 6 h or 24 h i.v. LPS infusion. A value>1 indicates greater than control mRNA levels and <1 is lower then control mRNA levels. Protein levels of 20S proteasome subunits α1–3 and 5–7 were measured to confirm mRNA results for subunit alpha-1 (panel F). Values represent mean±SEM. n = 6–8 per group. By two-way ANOVA, significant differences were observed in the EDL for the main effects of treatment in all measures (P≤0.02), and for time and treatment-time interaction in all measures bar TNFα and IL-6 mRNA levels (P≤0.02). In the myocardium, significant differences for the main effect of treatment were observed in all measures bar MAFbx/atrogin-1 mRNA levels and proteasome subunit protein levels (P≤0.03). Significant differences in main effects of time and treatment-time interaction were only observed for 20S proteasome subunit α1 and IL-6 mRNA levels in the myocardium (P≤0.002). * indicates different from corresponding control (P<0.05); † different from 2 h LPS-treated muscle (P<0.05); ‡ different from 6 h LPS-treated muscle (P<0.05).
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pone-0006945-g003: mRNA levels for proteins associated with proteolysis in muscle and myocardium following LPS infusion.Bars denote fold change in mRNA levels of A) MAFbx/atrogin-1, B) MuRF1, C) 20S proteasome subunit α1, D) TNFα and E) IL-6, compared to corresponding control values in response to either a 2 h, 6 h or 24 h i.v. LPS infusion. A value>1 indicates greater than control mRNA levels and <1 is lower then control mRNA levels. Protein levels of 20S proteasome subunits α1–3 and 5–7 were measured to confirm mRNA results for subunit alpha-1 (panel F). Values represent mean±SEM. n = 6–8 per group. By two-way ANOVA, significant differences were observed in the EDL for the main effects of treatment in all measures (P≤0.02), and for time and treatment-time interaction in all measures bar TNFα and IL-6 mRNA levels (P≤0.02). In the myocardium, significant differences for the main effect of treatment were observed in all measures bar MAFbx/atrogin-1 mRNA levels and proteasome subunit protein levels (P≤0.03). Significant differences in main effects of time and treatment-time interaction were only observed for 20S proteasome subunit α1 and IL-6 mRNA levels in the myocardium (P≤0.002). * indicates different from corresponding control (P<0.05); † different from 2 h LPS-treated muscle (P<0.05); ‡ different from 6 h LPS-treated muscle (P<0.05).

Mentions: In the EDL there were markedly increased levels of MAFbx/atrogin-1 and MuRF1 mRNA within 6 h after the onset of LPS infusion (Fig. 3A & 3B respectively). Furthermore, interesting differences in the time course of change in the levels of these two ligases were observed. Thus, elevated levels of MAFbx/atrogin-1 mRNA at 6 h plateaued, whereas in contrast, MuRF1 mRNA levels were substantially elevated from the 6 h to the 24 h time-point. In addition, LPS administration resulted in the increased levels of the α1 proteasome subunit mRNA in the EDL at all examined time-points relative to control animals, but the change was particularly pronounced 24 h after the start of the LPS-infusion (Fig. 3C). Since the mRNA expression of individual subunits of the 20S proteasome can vary in response to a catabolic stimulus [30], the protein levels of subunits α1–3 and 5–7 were also determined. In agreement with findings for the α1 subunit, maximal protein levels for subunits α1–3 and 5–7 were observed in the EDL following 24 h LPS administration (Fig. 3F). While only animals subjected to 24 h LPS treatment underwent implantation of arterial catheters into the distal abdominal aorta, the reproducibility of the catabolic response to LPS in skeletal muscle to previous findings where arterial catheters were not used [3], suggest that the results observed were not due to prior surgical intervention.


Effects of endotoxaemia on protein metabolism in rat fast-twitch skeletal muscle and myocardium.

Murton AJ, Alamdari N, Gardiner SM, Constantin-Teodosiu D, Layfield R, Bennett T, Greenhaff PL - PLoS ONE (2009)

mRNA levels for proteins associated with proteolysis in muscle and myocardium following LPS infusion.Bars denote fold change in mRNA levels of A) MAFbx/atrogin-1, B) MuRF1, C) 20S proteasome subunit α1, D) TNFα and E) IL-6, compared to corresponding control values in response to either a 2 h, 6 h or 24 h i.v. LPS infusion. A value>1 indicates greater than control mRNA levels and <1 is lower then control mRNA levels. Protein levels of 20S proteasome subunits α1–3 and 5–7 were measured to confirm mRNA results for subunit alpha-1 (panel F). Values represent mean±SEM. n = 6–8 per group. By two-way ANOVA, significant differences were observed in the EDL for the main effects of treatment in all measures (P≤0.02), and for time and treatment-time interaction in all measures bar TNFα and IL-6 mRNA levels (P≤0.02). In the myocardium, significant differences for the main effect of treatment were observed in all measures bar MAFbx/atrogin-1 mRNA levels and proteasome subunit protein levels (P≤0.03). Significant differences in main effects of time and treatment-time interaction were only observed for 20S proteasome subunit α1 and IL-6 mRNA levels in the myocardium (P≤0.002). * indicates different from corresponding control (P<0.05); † different from 2 h LPS-treated muscle (P<0.05); ‡ different from 6 h LPS-treated muscle (P<0.05).
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Related In: Results  -  Collection

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pone-0006945-g003: mRNA levels for proteins associated with proteolysis in muscle and myocardium following LPS infusion.Bars denote fold change in mRNA levels of A) MAFbx/atrogin-1, B) MuRF1, C) 20S proteasome subunit α1, D) TNFα and E) IL-6, compared to corresponding control values in response to either a 2 h, 6 h or 24 h i.v. LPS infusion. A value>1 indicates greater than control mRNA levels and <1 is lower then control mRNA levels. Protein levels of 20S proteasome subunits α1–3 and 5–7 were measured to confirm mRNA results for subunit alpha-1 (panel F). Values represent mean±SEM. n = 6–8 per group. By two-way ANOVA, significant differences were observed in the EDL for the main effects of treatment in all measures (P≤0.02), and for time and treatment-time interaction in all measures bar TNFα and IL-6 mRNA levels (P≤0.02). In the myocardium, significant differences for the main effect of treatment were observed in all measures bar MAFbx/atrogin-1 mRNA levels and proteasome subunit protein levels (P≤0.03). Significant differences in main effects of time and treatment-time interaction were only observed for 20S proteasome subunit α1 and IL-6 mRNA levels in the myocardium (P≤0.002). * indicates different from corresponding control (P<0.05); † different from 2 h LPS-treated muscle (P<0.05); ‡ different from 6 h LPS-treated muscle (P<0.05).
Mentions: In the EDL there were markedly increased levels of MAFbx/atrogin-1 and MuRF1 mRNA within 6 h after the onset of LPS infusion (Fig. 3A & 3B respectively). Furthermore, interesting differences in the time course of change in the levels of these two ligases were observed. Thus, elevated levels of MAFbx/atrogin-1 mRNA at 6 h plateaued, whereas in contrast, MuRF1 mRNA levels were substantially elevated from the 6 h to the 24 h time-point. In addition, LPS administration resulted in the increased levels of the α1 proteasome subunit mRNA in the EDL at all examined time-points relative to control animals, but the change was particularly pronounced 24 h after the start of the LPS-infusion (Fig. 3C). Since the mRNA expression of individual subunits of the 20S proteasome can vary in response to a catabolic stimulus [30], the protein levels of subunits α1–3 and 5–7 were also determined. In agreement with findings for the α1 subunit, maximal protein levels for subunits α1–3 and 5–7 were observed in the EDL following 24 h LPS administration (Fig. 3F). While only animals subjected to 24 h LPS treatment underwent implantation of arterial catheters into the distal abdominal aorta, the reproducibility of the catabolic response to LPS in skeletal muscle to previous findings where arterial catheters were not used [3], suggest that the results observed were not due to prior surgical intervention.

Bottom Line: It is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically observed in fast-twitch skeletal muscle during endotoxaemia.The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P<0.05), but was no different from controls in the myocardium.In summary, these findings suggest that the myocardium does not undergo the same catabolic response as skeletal muscle during early endotoxaemia, partly due to the absence of transcriptional and signalling events in the myocardium typically associated with increased muscle proteolysis and the suppression of protein synthesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK. andrew.murton@nottingham.ac.uk

ABSTRACT

Background: It is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically observed in fast-twitch skeletal muscle during endotoxaemia.

Methodology/principal findings: To investigate this further, and to determine if there is any divergence in the response of skeletal muscle and myocardium in the mechanisms that are thought to be largely responsible for eliciting changes in protein content, Sprague Dawley rats were implanted with vascular catheters and administered lipopolysaccharide (LPS; 150 microg kg(-1) h(-1)) intravenously for 2 h, 6 h or 24 h (saline administered control animals were also included), after which the extensor digitorum longus (EDL) and myocardium were removed under terminal anaesthesia. The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P<0.05), but was no different from controls in the myocardium. At the same time point, a significant increase in MAFbx/atrogin-1 and MuRF1 mRNA (3.7+/-0.7- and 19.5+/-1.9-fold increase vs. controls, respectively; P<0.05), in addition to protein levels of alpha1-3, 5-7 subunits of the 20S proteasome, were observed in EDL but not myocardium. In contrast, elevations in phosphorylation of p70 S6K residues Thr(421)/Ser(424), and 4E-BP1 residues Thr(37)/Thr(46) (P<0.05), consistent with an elevation in translation initiation, were seen exclusively in the myocardium of LPS-treated animals.

Conclusions/significance: In summary, these findings suggest that the myocardium does not undergo the same catabolic response as skeletal muscle during early endotoxaemia, partly due to the absence of transcriptional and signalling events in the myocardium typically associated with increased muscle proteolysis and the suppression of protein synthesis.

Show MeSH
Related in: MedlinePlus