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Mice deficient in ribosomal protein S6 phosphorylation suffer from muscle weakness that reflects a growth defect and energy deficit.

Ruvinsky I, Katz M, Dreazen A, Gielchinsky Y, Saada A, Freedman N, Mishani E, Zimmerman G, Kasir J, Meyuhas O - PLoS ONE (2009)

Bottom Line: Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a) a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b) a reduced content of ATP and phosphocreatine, two readily available energy sources.However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG) in the muscle.Interestingly, a similar role has been assigned for ribosomal protein S6 kinase 1, even though it regulates myoblast growth in an rpS6 phosphorylation-independent fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, IMRIC, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT

Background: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6(P-/-)), are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic beta-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests.

Methodology/principal findings: A large variety of experimental methodologies, including morphometric measurements of histological preparations, high throughput proteomic analysis, positron emission tomography (PET) and numerous biochemical assays, were used in an attempt to establish the mechanism underlying the relative weakness of rpS6(P-/-) muscles. Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a) a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b) a reduced content of ATP and phosphocreatine, two readily available energy sources. The abundance of three mitochondrial proteins has been shown to diminish in the knockin mouse. However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG) in the muscle.

Conclusions/significance: This study establishes rpS6 phosphorylation as a determinant of muscle strength through its role in regulation of myofiber growth and energy content. Interestingly, a similar role has been assigned for ribosomal protein S6 kinase 1, even though it regulates myoblast growth in an rpS6 phosphorylation-independent fashion.

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Related in: MedlinePlus

Soleus relative mass and CSA of its fibers are diminished in rpS6P−/− mice.(A) The relative soleus weight from male mice was monitored at different ages and presented as the percentage of whole body weight. Each point represents mean±SEM, (bars are smaller than the symbol's size). * P<0.02, ** P<0.0001 versus rpS6P+/+ mice. (n = 4 to 52 soleus samples per each time point). (B) Number of myonuclei inside sarcolemma per fiber section. (C) Number of fibers comprising soleus muscles. (D) Cross-sectional area (CSA) of fibers. Muscles were excised from three 2-mo-old male mice for each genotype and immediately frozen. 5 µm-thick transverse cross-sections were collected along the entire length of the muscle at 100 µm intervals with a cryostat and stained with amylase/diastase in (B) in haematoxylin/eosin in (C) and reticulin in (D). Digital images obtained from the sections with the largest CSA were used for analysis. Numbers of fibers and nuclei per fiber were assessed by manual counting, and CSA was calculated from digital images. Values are presented as a mean±SEM. In (B) and (C) n = 3 and 4 age-matched male mice for each genotype, respectively. In (D) n = 1000 fibers from 3 age-matched male mice for each genotype. The SEM bars in (C) and (D) are invisible, because they are smaller than the width of the upper line in each column.
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pone-0005618-g002: Soleus relative mass and CSA of its fibers are diminished in rpS6P−/− mice.(A) The relative soleus weight from male mice was monitored at different ages and presented as the percentage of whole body weight. Each point represents mean±SEM, (bars are smaller than the symbol's size). * P<0.02, ** P<0.0001 versus rpS6P+/+ mice. (n = 4 to 52 soleus samples per each time point). (B) Number of myonuclei inside sarcolemma per fiber section. (C) Number of fibers comprising soleus muscles. (D) Cross-sectional area (CSA) of fibers. Muscles were excised from three 2-mo-old male mice for each genotype and immediately frozen. 5 µm-thick transverse cross-sections were collected along the entire length of the muscle at 100 µm intervals with a cryostat and stained with amylase/diastase in (B) in haematoxylin/eosin in (C) and reticulin in (D). Digital images obtained from the sections with the largest CSA were used for analysis. Numbers of fibers and nuclei per fiber were assessed by manual counting, and CSA was calculated from digital images. Values are presented as a mean±SEM. In (B) and (C) n = 3 and 4 age-matched male mice for each genotype, respectively. In (D) n = 1000 fibers from 3 age-matched male mice for each genotype. The SEM bars in (C) and (D) are invisible, because they are smaller than the width of the upper line in each column.

Mentions: We have previously noticed that compromised glucose homeostasis in rpS6P−/− mice is associated with smaller β-cell size and reduced content of pancreatic insulin [14]. Hence, we set out to examine the possibility that the apparent diminished muscle strength in the knockin mice might reflect a decrease in total muscle mass. Indeed, monitoring the total soleus weight in 2 to 3.5 month-old mice has demonstrated that the relative soleus weight is reduced by about 25% in rpS6P−/− mice in comparison to that of rpS6P+/+ mice (Fig. 2A). Likewise, the weight of gastrocnemious muscles in 2-mo-old rpS6P−/− male mice (n = 10) is 76% of that of their wild-type counterparts (74.5±3.6 [n = 10] and 98.3±4.2 [n = 9] mg, respectively). The optimal force of a muscle is largely determined by its physiological cross-sectional area (CSA) [16]. This latter variable reflects, among others, the CSA and/or number of individual fibers. Morphometrically, soleus fibers from rpS6P−/− and rpS6P+/+ mice contain a similar number of myonuclei, testifying for the fusion of a similar number of myoblasts during fiber differentiation (Fig. 2B). Moreover, soleus muscles from both genotypes consist of a similar number of fibers (Fig. 2C). However, soleus muscle fibers of rpS6P−/− show a 25% decrease in their CSA compared to that of rpS6P+/+ mice (Fig. 2D), quite similarly to findings in S6K-deficient mice [7]. It appears, therefore, that the decrease in the myofibers CSA can fully account for the diminished relative weight of the soleus in rpS6P−/− mice, and this in turn might explain, at least partly the reduced muscle strength.


Mice deficient in ribosomal protein S6 phosphorylation suffer from muscle weakness that reflects a growth defect and energy deficit.

Ruvinsky I, Katz M, Dreazen A, Gielchinsky Y, Saada A, Freedman N, Mishani E, Zimmerman G, Kasir J, Meyuhas O - PLoS ONE (2009)

Soleus relative mass and CSA of its fibers are diminished in rpS6P−/− mice.(A) The relative soleus weight from male mice was monitored at different ages and presented as the percentage of whole body weight. Each point represents mean±SEM, (bars are smaller than the symbol's size). * P<0.02, ** P<0.0001 versus rpS6P+/+ mice. (n = 4 to 52 soleus samples per each time point). (B) Number of myonuclei inside sarcolemma per fiber section. (C) Number of fibers comprising soleus muscles. (D) Cross-sectional area (CSA) of fibers. Muscles were excised from three 2-mo-old male mice for each genotype and immediately frozen. 5 µm-thick transverse cross-sections were collected along the entire length of the muscle at 100 µm intervals with a cryostat and stained with amylase/diastase in (B) in haematoxylin/eosin in (C) and reticulin in (D). Digital images obtained from the sections with the largest CSA were used for analysis. Numbers of fibers and nuclei per fiber were assessed by manual counting, and CSA was calculated from digital images. Values are presented as a mean±SEM. In (B) and (C) n = 3 and 4 age-matched male mice for each genotype, respectively. In (D) n = 1000 fibers from 3 age-matched male mice for each genotype. The SEM bars in (C) and (D) are invisible, because they are smaller than the width of the upper line in each column.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682700&req=5

pone-0005618-g002: Soleus relative mass and CSA of its fibers are diminished in rpS6P−/− mice.(A) The relative soleus weight from male mice was monitored at different ages and presented as the percentage of whole body weight. Each point represents mean±SEM, (bars are smaller than the symbol's size). * P<0.02, ** P<0.0001 versus rpS6P+/+ mice. (n = 4 to 52 soleus samples per each time point). (B) Number of myonuclei inside sarcolemma per fiber section. (C) Number of fibers comprising soleus muscles. (D) Cross-sectional area (CSA) of fibers. Muscles were excised from three 2-mo-old male mice for each genotype and immediately frozen. 5 µm-thick transverse cross-sections were collected along the entire length of the muscle at 100 µm intervals with a cryostat and stained with amylase/diastase in (B) in haematoxylin/eosin in (C) and reticulin in (D). Digital images obtained from the sections with the largest CSA were used for analysis. Numbers of fibers and nuclei per fiber were assessed by manual counting, and CSA was calculated from digital images. Values are presented as a mean±SEM. In (B) and (C) n = 3 and 4 age-matched male mice for each genotype, respectively. In (D) n = 1000 fibers from 3 age-matched male mice for each genotype. The SEM bars in (C) and (D) are invisible, because they are smaller than the width of the upper line in each column.
Mentions: We have previously noticed that compromised glucose homeostasis in rpS6P−/− mice is associated with smaller β-cell size and reduced content of pancreatic insulin [14]. Hence, we set out to examine the possibility that the apparent diminished muscle strength in the knockin mice might reflect a decrease in total muscle mass. Indeed, monitoring the total soleus weight in 2 to 3.5 month-old mice has demonstrated that the relative soleus weight is reduced by about 25% in rpS6P−/− mice in comparison to that of rpS6P+/+ mice (Fig. 2A). Likewise, the weight of gastrocnemious muscles in 2-mo-old rpS6P−/− male mice (n = 10) is 76% of that of their wild-type counterparts (74.5±3.6 [n = 10] and 98.3±4.2 [n = 9] mg, respectively). The optimal force of a muscle is largely determined by its physiological cross-sectional area (CSA) [16]. This latter variable reflects, among others, the CSA and/or number of individual fibers. Morphometrically, soleus fibers from rpS6P−/− and rpS6P+/+ mice contain a similar number of myonuclei, testifying for the fusion of a similar number of myoblasts during fiber differentiation (Fig. 2B). Moreover, soleus muscles from both genotypes consist of a similar number of fibers (Fig. 2C). However, soleus muscle fibers of rpS6P−/− show a 25% decrease in their CSA compared to that of rpS6P+/+ mice (Fig. 2D), quite similarly to findings in S6K-deficient mice [7]. It appears, therefore, that the decrease in the myofibers CSA can fully account for the diminished relative weight of the soleus in rpS6P−/− mice, and this in turn might explain, at least partly the reduced muscle strength.

Bottom Line: Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a) a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b) a reduced content of ATP and phosphocreatine, two readily available energy sources.However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG) in the muscle.Interestingly, a similar role has been assigned for ribosomal protein S6 kinase 1, even though it regulates myoblast growth in an rpS6 phosphorylation-independent fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, IMRIC, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT

Background: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6(P-/-)), are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic beta-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests.

Methodology/principal findings: A large variety of experimental methodologies, including morphometric measurements of histological preparations, high throughput proteomic analysis, positron emission tomography (PET) and numerous biochemical assays, were used in an attempt to establish the mechanism underlying the relative weakness of rpS6(P-/-) muscles. Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a) a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b) a reduced content of ATP and phosphocreatine, two readily available energy sources. The abundance of three mitochondrial proteins has been shown to diminish in the knockin mouse. However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG) in the muscle.

Conclusions/significance: This study establishes rpS6 phosphorylation as a determinant of muscle strength through its role in regulation of myofiber growth and energy content. Interestingly, a similar role has been assigned for ribosomal protein S6 kinase 1, even though it regulates myoblast growth in an rpS6 phosphorylation-independent fashion.

Show MeSH
Related in: MedlinePlus