Korean J Physiol Pharmacol.  2009 Dec;13(6):491-496. 10.4196/kjpp.2009.13.6.491.

p38 MAPK Participates in Muscle-Specific RING Finger 1-Mediated Atrophy in Cast-Immobilized Rat Gastrocnemius Muscle

• Kim J
• Won KJ
• Lee HM
• Hwang BY
• Bae YM
• Choi WS
• Song H
• Lim KW
• Lee CK
• Kim B

• KMID: 2285386
• DOI: http://doi.org/10.4196/kjpp.2009.13.6.491

Abstract

Skeletal muscle atrophy is a common phenomenon during the prolonged muscle disuse caused by cast immobilization, extended aging states, bed rest, space flight, or other factors. However, the cellular mechanisms of the atrophic process are poorly understood. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK) in the expression of muscle-specific RING finger 1 (MuRF1) during atrophy of the rat gastrocnemius muscle. Histological analysis revealed that cast immobilization induced the atrophy of the gastrocnemius muscle, with diminution of muscle weight and cross-sectional area after 14 days. Cast immobilization significantly elevated the expression of MuRF1 and the phosphorylation of p38 MAPK. The starvation of L6 rat skeletal myoblasts under serum-free conditions induced the phosphorylation of p38 MAPK and the characteristics typical of cast-immobilized gastrocnemius muscle. The expression of MuRF1 was also elevated in serum-starved L6 myoblasts, but was significantly attenuated by SB203580, an inhibitor of p38 MAPK. Changes in the sizes of L6 myoblasts in response to starvation were also reversed by their transfection with MuRF1 small interfering RNA or treatment with SB203580. From these results, we suggest that the expression of MuRF1 in cast-immobilized atrophy is regulated by p38 MAPK in rat gastrocnemius muscles.

Keyword

Cast immobilization; MuRF1; p38 MAPK; Skeletal muscle atrophy; Starvation

MeSH Terms

Aging
Animals
Atrophy
Bed Rest
Fingers
Imidazoles
Immobilization
Muscle, Skeletal
Muscles
Myoblasts
Myoblasts, Skeletal
p38 Mitogen-Activated Protein Kinases
Phosphorylation
Protein Kinases
Pyridines
Rats
RNA, Small Interfering
Space Flight
Starvation
Transfection
Imidazoles
Protein Kinases
Pyridines
RNA, Small Interfering
p38 Mitogen-Activated Protein Kinases

Figure

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  Fig. 1. Morphological characterization of gastrocnemius muscle from cast-immobilized rat hindlimb. Gastrocnemius muscle weights (A, n=8), ratios of muscle weight to body weight (B, n=8), and cross-sectional areas (C, n=7) were measured 14 days after cast immobilization. The muscle fibers were visualized with H&E staining, as described in the Materials and methods. (D) Immunohistological analysis of rats at day 14 after cast immobilization. MuRF1 expression (red) was visualized with a phase-contrast microscope (×400). Representative result of six independent experiments. ∗Significantly different from the noncasted control (p<0.05).

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  Fig. 2. Expression of MuRF1 and p38 MAPK in gastrocnemius muscles from cast-immobilized rats. Immunoblotting analysis of MuRF1 (A) and p38 MAPK (B) in the gastrocnemius muscle. Protein expression and phosphorylation were examined using anti-nonphospho- and anti-phosphospecific antibodies, respectively. The statistical results were obtained from the upper panels. The levels of MuRF1 expression and p38 MAPK phosphorylation in the noncasted muscle group were considered to be 100% (n=4). ∗Significantly different from the non-casted controls (p<0.05). Non, non-casted.

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  Fig. 3. Changes in the characteristics of rat L6 skeletal myoblasts starved in serum-free medium. (A) Changes in the cell size. The cells were cultured in serum-free medium for the indicated times and visualized with H&E staining. The cell sizes were analyzed under a phase-contrast microscope (×100, n=8). (B) The expression of MuRF1. The cells were starved in serum-free medium for 24 h and the lysates were subjected to immuno-blotting analysis with anti-MuRF1 and anti-β-actin antibodies. The statistical results were obtained from the upper panel. (C) p38 MAPK phosphorylation in cells starved in serumfree medium. The phosphorylation and expression of p38 AMPK were examined using anti-phospho and anti-nonphospho antibodies, respectively. The basal level of phosphorylation in nonstarved control cells was considered to be 100% (n=5).

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  Fig. 4. Changes in the size of L6 myoblasts knocked down with MuRF1 siRNA. (A) MuRF1 knockdown using MuRF1 siRNA. The cells were transfected with MuRF1 siRNA. Extracts from the cells were immunoblotted with anti-MuRF1 and anti-β-actin antibodies (n=4). (B) Changes in cell size caused by transfection with MuRF1 siRNA. After transfection of the cells with MuRF1 siRNA, the cell sizes were measured under a phase-contrast microscope (×100). ∗Significant differences between siRNA-treated cells and nonsilenced controls (n=8; p<0.05). Control, nonstarved control cells; Starv, starvation; + Non-si, nonsilencing; + siRNA, MuRF1 siRNA.

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  Fig. 5. Effects of p38 MAPK on MuRF1 expression and cell size in serum-starved L6 myoblasts. (A) Effect of p38 MAPK inhibitor on MuRF1 expression. Cells were starved for 12 h in serum-free medium with 0.1% DMSO or 10 μM SB203580. Cell lysates were immunoblotted with anti-MuRF1 and anti-β-actin antibodies. The statistical results were obtained from the upper panel. MuRF1 expression in nonstarved cells was considered to be 100% (n=4). (B) Effect of p38 MAPK inhibitor on cell size. The cells were starved for 12 h in serum-free medium with 0.1% DMSO or 10 μM SB203580 and the cell sizes were measured under a phase-contrast microscope (×100; n=8). ∗Significant differences between the starved and nonstarved control groups (p<0.05).

Reference

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