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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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Muscle strength is impaired in rpS6P−/− mice.(A to C) Age-matched (4 and 7 month) male mice (n = 13 for WT and 12 for rpS6P−/− mice) were subjected to screening, according to the SHIRPA behavioral protocol (see details in “Experimental Procedures”). (A) Grip strength. Mice were allowed to grip a grid and a gentle horizontal backwards pull through their tail was applied. Higher scores indicate greater grip strength. An unbiased observer, blinded to the genotype, performed the experiment in a blind fashion; (B) Wire maneuver. Results represent time in seconds required for a mouse that is hung from a wire with its forearms to elevate its hind limbs and grip the wire. (C) Rota-rod performance. Mice were placed on a moving cylinder, which was gradually accelerated from an initial speed of 4 rpm to a maximum of 40 rpm. Latency to fall from the rota-rod is presented in seconds. Motor performance was measured in three 10 min sessions (time 0, 1 h and 24 h). In each trial, the time in seconds until falling off was recorded. *P<0.0005 for each trial versus rpS6P+/+ mice. (D) Endurance test. 5 rpS6p+/+ and 4 S6P−/− age-matched (7–9 weeks) male mice were allowed to run on the treadmill set with a slope of 12.5 degree and a speed of 20 m/min. The results represent the total running time with two attempts to pause. Results of all experiments are presented as average±SEM, and the absence of SEM bars for some measurements simply reflects a value close to zero that is graphically invisible.
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pone-0005618-g001: Muscle strength is impaired in rpS6P−/− mice.(A to C) Age-matched (4 and 7 month) male mice (n = 13 for WT and 12 for rpS6P−/− mice) were subjected to screening, according to the SHIRPA behavioral protocol (see details in “Experimental Procedures”). (A) Grip strength. Mice were allowed to grip a grid and a gentle horizontal backwards pull through their tail was applied. Higher scores indicate greater grip strength. An unbiased observer, blinded to the genotype, performed the experiment in a blind fashion; (B) Wire maneuver. Results represent time in seconds required for a mouse that is hung from a wire with its forearms to elevate its hind limbs and grip the wire. (C) Rota-rod performance. Mice were placed on a moving cylinder, which was gradually accelerated from an initial speed of 4 rpm to a maximum of 40 rpm. Latency to fall from the rota-rod is presented in seconds. Motor performance was measured in three 10 min sessions (time 0, 1 h and 24 h). In each trial, the time in seconds until falling off was recorded. *P<0.0005 for each trial versus rpS6P+/+ mice. (D) Endurance test. 5 rpS6p+/+ and 4 S6P−/− age-matched (7–9 weeks) male mice were allowed to run on the treadmill set with a slope of 12.5 degree and a speed of 20 m/min. The results represent the total running time with two attempts to pause. Results of all experiments are presented as average±SEM, and the absence of SEM bars for some measurements simply reflects a value close to zero that is graphically invisible.

Mentions: During routine handling of adult mice (older than 6 weeks) our attention was drawn to the diminished force displayed by rpS6P−/− mice, relative to that of rpS6P+/+ mice, when attempted to escape. To more objectively quantify this difference, we implemented SHIRPA protocol designed for comprehensive phenotype assessment [15]. Semi-quantitative grip-strength assessment implied a diminished muscular tone of rpS6P−/− mice (Fig. 1A). This inferiority was corroborated by a wire-maneuver test that showed that rpS6P−/− mice lagged behind rpS6P+/+ mice when attempted to elevate their hind limbs to a horizontal wire (Fig. 1B). Next we observed that the ability of rpS6P−/− mice to withstand a prolonged effort was severely impaired, as exemplified by significantly shorter time spent on the Rota-Rod (Fig. 1C). However, since poor performance in this test might reflect a defect in sensorimotor coordination, rather than muscle weakness, we examined the mice on a rodent treadmill. Fig. 1D demonstrates that the endurance of the knockin mice is profoundly diminished, relative to their wild-type counterparts. Taken together, these results attest to the role of rpS6 phosphorylation in normal muscle function.


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)

Muscle strength is impaired in rpS6P−/− mice.(A to C) Age-matched (4 and 7 month) male mice (n = 13 for WT and 12 for rpS6P−/− mice) were subjected to screening, according to the SHIRPA behavioral protocol (see details in “Experimental Procedures”). (A) Grip strength. Mice were allowed to grip a grid and a gentle horizontal backwards pull through their tail was applied. Higher scores indicate greater grip strength. An unbiased observer, blinded to the genotype, performed the experiment in a blind fashion; (B) Wire maneuver. Results represent time in seconds required for a mouse that is hung from a wire with its forearms to elevate its hind limbs and grip the wire. (C) Rota-rod performance. Mice were placed on a moving cylinder, which was gradually accelerated from an initial speed of 4 rpm to a maximum of 40 rpm. Latency to fall from the rota-rod is presented in seconds. Motor performance was measured in three 10 min sessions (time 0, 1 h and 24 h). In each trial, the time in seconds until falling off was recorded. *P<0.0005 for each trial versus rpS6P+/+ mice. (D) Endurance test. 5 rpS6p+/+ and 4 S6P−/− age-matched (7–9 weeks) male mice were allowed to run on the treadmill set with a slope of 12.5 degree and a speed of 20 m/min. The results represent the total running time with two attempts to pause. Results of all experiments are presented as average±SEM, and the absence of SEM bars for some measurements simply reflects a value close to zero that is graphically invisible.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682700&req=5

pone-0005618-g001: Muscle strength is impaired in rpS6P−/− mice.(A to C) Age-matched (4 and 7 month) male mice (n = 13 for WT and 12 for rpS6P−/− mice) were subjected to screening, according to the SHIRPA behavioral protocol (see details in “Experimental Procedures”). (A) Grip strength. Mice were allowed to grip a grid and a gentle horizontal backwards pull through their tail was applied. Higher scores indicate greater grip strength. An unbiased observer, blinded to the genotype, performed the experiment in a blind fashion; (B) Wire maneuver. Results represent time in seconds required for a mouse that is hung from a wire with its forearms to elevate its hind limbs and grip the wire. (C) Rota-rod performance. Mice were placed on a moving cylinder, which was gradually accelerated from an initial speed of 4 rpm to a maximum of 40 rpm. Latency to fall from the rota-rod is presented in seconds. Motor performance was measured in three 10 min sessions (time 0, 1 h and 24 h). In each trial, the time in seconds until falling off was recorded. *P<0.0005 for each trial versus rpS6P+/+ mice. (D) Endurance test. 5 rpS6p+/+ and 4 S6P−/− age-matched (7–9 weeks) male mice were allowed to run on the treadmill set with a slope of 12.5 degree and a speed of 20 m/min. The results represent the total running time with two attempts to pause. Results of all experiments are presented as average±SEM, and the absence of SEM bars for some measurements simply reflects a value close to zero that is graphically invisible.
Mentions: During routine handling of adult mice (older than 6 weeks) our attention was drawn to the diminished force displayed by rpS6P−/− mice, relative to that of rpS6P+/+ mice, when attempted to escape. To more objectively quantify this difference, we implemented SHIRPA protocol designed for comprehensive phenotype assessment [15]. Semi-quantitative grip-strength assessment implied a diminished muscular tone of rpS6P−/− mice (Fig. 1A). This inferiority was corroborated by a wire-maneuver test that showed that rpS6P−/− mice lagged behind rpS6P+/+ mice when attempted to elevate their hind limbs to a horizontal wire (Fig. 1B). Next we observed that the ability of rpS6P−/− mice to withstand a prolonged effort was severely impaired, as exemplified by significantly shorter time spent on the Rota-Rod (Fig. 1C). However, since poor performance in this test might reflect a defect in sensorimotor coordination, rather than muscle weakness, we examined the mice on a rodent treadmill. Fig. 1D demonstrates that the endurance of the knockin mice is profoundly diminished, relative to their wild-type counterparts. Taken together, these results attest to the role of rpS6 phosphorylation in normal muscle function.

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