Downloaded - Cell Physiology

Transcription

Am J Physiol Cell Physiol 300: C456–C465, 2011.
First published December 22, 2010; doi:10.1152/ajpcell.00124.2010.
Muscle-specific overexpression of NCOATGK, splice variant of O-GlcNAcase,
induces skeletal muscle atrophy
Ping Huang,1 Shiuh-Rong Ho,2 Kai Wang,3 Bryan C. Roessler,4 Fengxue Zhang,5 Yong Hu,2
Damon B. Bowe,1 Jeffrey E. Kudlow,1,2,3,5 and Andrew J. Paterson3,4,5
1
Department of Pharmacology and Toxicology, 2Department of Cell Biology, 3Department of Physiology and Biophysics,
Graduate Biomedical Sciences Program, and 5Department of Medicine, Division of Endocrinology, Diabetes and
Metabolism, University of Alabama at Birmingham, Birmingham, Alabama
4
Submitted 7 April 2010; accepted in final form 17 December 2010
O-GlcNAcylation; Goto-Kakizaki; transgenic; cell death; proteasome;
O-linked ␤-N-acetylglucosamine
is a frequent complication of chronic
diseases, including cancer, sepsis, acquired immunodeficiency
syndrome, and diabetes (38, 43). The loss of myofibrillar
proteins (myosin, actin) and consequent cell death are primary
triggers for lean body mass decrease. Although increased
activity of the proteolytic system plays an important role in the
development of muscle atrophy (17, 36), the activation of cell
death systems performs an irreversible executer of muscle
atrophy. The activation of caspases and mitochondrial proteins
involved in the apoptotic pathway has been shown to increase
in rat skeletal muscle undergoing cancer cachexia (2), has
induced diabetes (28), and has been seen in other experimental
conditions causing muscle loss (35, 40). Furthermore, the
abnormal activation and accumulation of proapoptotic factors,
p53 and BAX, are associated with hyperglycemia-induced
apoptosis in myocytes (16). Contrarily, the avoidance of the
accumulation and activation of these proapoptotic factors may
ward against myocyte death and muscle loss.
The apoptotic destruction of islet in diabetic subjects has
been linked to the protein posttranslational modification of
O-linked ␤-N-acetylglucosamine (O-GlcNAc) (31). This pro-
SKELETAL MUSCLE ATROPHY
Address for reprint requests and other correspondence: A. J. Paterson, Dept.
of Medicine, Univ. of Alabama at Birmingham, Birmingham, AL 35294
(e-mail: [email protected]).
C456
tein modification (O-GlcNAcylation) involves the addition of
O-GlcNAc to Ser and Thr residues and is fueled via the
hexosamine biosynthetic pathway (HBP). It has been reported
that hyperglycemia and free fatty acids can increase flux
through the HBP and induce ␤-cell apoptosis (13, 39). Additionally, streptozotocin, an inhibitor of O-GlcNAcase and diabetorgenic agent, causes increased O-GlcNAc modification
and induces apoptosis in cardiomyocytes (5) and Schwann
cells (12). The Bastide group has reported that a number of
contractile muscle proteins are O-GlcNAcylated (22) and that
variations in O-GlcNAc can regulate muscle protein homeostasis, playing a role in the development of muscular atrophy
(9, 10, 24). Our laboratory has shown that O-GlcNAc modification of the proteasome can inhibit function, causing accumulation of short half-life proteins, including some proapoptotic factors (7, 19, 30, 41, 42, 56). Yang et al. (51) have also
shown that O-GlcNAcylation of p53 prevents its degradation
via the ubiquitin-proteasome system (UPS).
The O-GlcNAc modification is dynamic, and its addition to
proteins is catalyzed by O-GlcNAc transferase (OGT) and is
removed by O-GlcNAcase (NCOAT) (21, 27, 33). Both proteins are expressed ubiquitously but with varying enzyme
activity. Skeletal muscle, brain, and pancreas have higher
levels of O-GlcNAcase activity compared with other tissues
(14), and OGT has the same tissue distribution; in particular,
OGT is highly expressed in the pancreas (1, 20). The NCOAT
gene has been linked to type II diabetes in Mexican Pima
Indians (15, 29). Furthermore, our laboratory has cloned a
splice-variant of the NCOAT gene (NCOATGK) from the brain
of the spontaneously diabetic Goto-Kakizaki (GK) rat, lacking
exon 8 and disrupting O-GlcNAcase catalytic activity (45).
The overexpression of NCOATGK retarded mammary ductal
branching (3, 48) and caused premature cataracts in transgenic
mice (47), functionally behaving as a dominant-negative
NCOAT. However, the effect of NCOATGK on other glucosesensitive tissues, such as skeletal muscle, has yet to be determined.
In this current study, we report on the effects of overexpressing NCOATGK in the skeletal muscle of transgenic mice
utilizing the muscle creatine kinase (MCK) promoter (37).
Induced expression resulted in the development of severe
muscle atrophy due to myocyte apoptosis, occasionally causing
the death of the animal. We demonstrated that proteasome
function decreased in the affected cells concomitant with
increased cellular O-GlcNAc levels and that some proapoptotic
factors, such as p53 and caspase 3, accumulated. Our study
provides evidence to link the nutrient status of a muscle cell
0363-6143/11 Copyright © 2011 the American Physiological Society
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on October 26, 2016
Huang P, Ho SR, Wang K, Roessler BC, Zhang F, Hu Y, Bowe
DB, Kudlow JE, Paterson AJ. Muscle-specific overexpression of
NCOATGK, splice variant of O-GlcNAcase, induces skeletal muscle
atrophy. Am J Physiol Cell Physiol 300: C456 –C465, 2011. First
published December 22, 2010; doi:10.1152/ajpcell.00124.2010.—The
protein O-linked ␤-N-acetylglucosamine (O-GlcNAc) modification
plays an important role in skeletal muscle development and physiological function. In this study, bitransgenic mice were generated that
overexpressed NCOATGK, an O-GlcNAcase-inactive spliced variant
of the O-GlcNAcase gene, specifically in skeletal muscle using the
muscle creatine kinase promoter. Expression of the chimeric enhanced
green fluorescent protein-NCOATGK transgene caused an increase of
cellular O-GlcNAc levels, along with the accumulation and activation
of proapoptotic factors in muscles of bitransgenic mice. The consequence of overexpressing the transgene for a 2-wk period was muscle
atrophy and, in some cases, resulted in the death of male mice. Muscle
atrophy is a common complication of many diseases, some of which
correlate markedly with high cellular O-GlcNAc levels, such as
diabetes. Our study provides direct evidence linking muscle atrophy
and the disruption of O-GlcNAcase activity.
O-GLcNAc INDUCES MUSCLE ATROPHY
with the disruption of homeostasis and atrophy, through the
O-GlcNAc modification.
MATERIALS AND METHODS
The tetracycline-responsive element (TRE)-enhanced green fluorescent protein (EGFP)-NCOATGK transgene was modified from the TRENCOATGK transgene used previously (48) by inserting the EGFP sequence from plasmid pEGFP-C1 (Invitrogen) between the NCOAT
untranslated region and NCOATGK. The 5.6-kb ClaI-XhoI fragment
containing the entire transgene cassette was purified and microinjected as described above. Founder animals were screened by PCR
using oligonucleotides designed to anneal to NCOAT (5=-CCTTGCCAACTTCCCTTCTCTG-3=) and the SV40 polyadenylation
sequence terminating the transgene (5=-GTCCTTGGGGTCTTCTACCTTTCTC-3=).
MCK-rtTA mice were cross-bred with TRE-EGFP-NCOATGK
mice to generate bitransgenic animals, which were genotyped by PCR
as above, using tail-snip DNA as previously described (31). Doxycycline (Dox) was administered to mice via a grain-based rodent diet
containing Dox (6 g/kg) (catalog no. F4096; Bio-Serv, Frenchtown,
NJ) 4 wk after birth. To terminate transgene expression, the Dox diet
was replaced with the normal rodent diet. All mice were handled in an
accredited university animal resources facility with the approval of the
Institutional Animal Care and Use Committee in accordance with
institutional animal care policies.
Fig. 1. Schematic of transgenes and characterization of muscle creatine kinase (MCK)-reverse tetracycline-responsive transcriptional activator (rtTA) transgenic
mice. A and B: schematics of the transgene constructs for the rtTA driven by the MCK promoter (A) and the tetracycline-responsive element (TRE)-LacZ
transgene (B) (18, 25). C: ␤-Galactosidase activity assays showing ␤-Gal expression levels of three MCK-rtTA founder transgenic mice and controls on a
doxycycline (Dox) diet. RLU, relative light units. D: ␤-Gal expression in various tissues taken from line 1 transgenic founder, with or without Dox induction.
E–G: frozen sections of skeletal muscle from wild-type (WT) mice (control) and bitransgenic (MCK/LacZ) mice with Dox were stained using X-gal (blue), the
substrate of ␤-galactosidase.
AJP-Cell Physiol • VOL
300 • MARCH 2011 •
www.ajpcell.org
Mice. To generate the skeletal muscle-specific transgene, the 1.5-kb
cDNA fragment containing the MCK promoter was synthesized by
PCR using oligonucleotides: 5=-GATGAATTCCTGGCTGGCTGGCTCCTG-3= and 5=-GACTGAATTCATCGATGGCACCTCCCAGGCAGAAG-3=, with mouse liver genomic DNA as template, and cloned
into BlueScript KS(⫺) at the EcoRI site. The MCK gene was excised
from BlueScript KS(⫺) with ClaI-EcoRI and ligated into the K14tetracycline-responsive transcriptional activator (rtTA) transgene
(50), replacing the K14 sequence using standard cloning techniques. The 4.1-kb KpnI- KpnI fragment containing the entire
transgene cassette was isolated, and the purified DNA was microinjected into one-cell B6xSJL mouse zygotes at a concentration of
2 ng/ml, carried out at the University of Alabama at Birmingham
Transgenic Mouse Facility. Founder animals were screened by PCR
using oligonucleotides designed to anneal to MCK (5=-CAGTGAGCAAGTCAGCCCTTG-3=) and rtTA (5=-CTTCTTTAGCGACTTGATGCTC-3=).
C457
C458
anti-O-GlcNAc (RL2) (1:250 dilution) and anti-p53 (1:100 dilution;
Santa Cruz).
Plastic sections were prepared from muscle tissue fixed in 4%
paraformaldehyde-PBS for 4 h at 4°C and embedded in Technovit
8100 (Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer’s protocol, then cut into 5-␮m-thick sections and observed
under a fluorescent microscope.
Western blot analysis. Muscle samples from bitransgenic mice that
had been treated with or without Dox administration, or from other
control mice, were homogenized in ice-cold homogenization buffer,
as described previously (32). Protein content was determined using
the Bio-Rad assay. Aliquots containing equal amounts of protein were
prepared and separated on 10% SDS-PAGE. The mouse monoclonal
antibodies against RL2, p53 (Santa Cruz), Bax (Santa Cruz), active
caspase 3 (Cell Signaling), GFP (Sigma), and ␤-actin (Sigma), and
polyclonal antibodies against caspase 9 and p21WAF1 (NeoMarkers)
were used in the analysis.
Proteasome activity assays (fluorogenic peptide substrate assays).
Proteasome activity assays were conducted following the method
described previously (56). Skeletal muscle harvested from the hind
legs of Dox-treated transgenic mice was homogenized in proteasome lysis solution (56), and aliquots (20 ␮l) of these muscle
extracts containing 30 ␮g proteins were incubated at 37°C for 90
min with 2 ␮l of the fluorogenic substrate succinyl-Leu-Leu-ValTyr-4-methylcoumary-7-amide (suc-LLVY-AMC) (100 ␮M). The
reaction was stopped by addition of 900 ␮l SDS (1%). The activity
was determined as the increase in fluorescence of the products
Fig. 2. Schematic of the TRE-enhanced green fluorescent protein (EGFP)-NCOATGK transgene and characterization of transgene expression. A: transgene
construct consisting of the TRE upstream of the EGFP in frame with the NCOATGK gene. UTR, untranslated region. B and C: tissue sections of skeletal muscle
from control or Dox-treated bitransgenic (MCK/EGFP-NCOATGK) mice were prepared in Technovit 8100, and EGFP expression was observed under a
fluorescent microscope. D: Northern blot analysis of total cellular RNA (15 ␮g) from skeletal muscle of bitransgenic mice fed Dox for 12 h, 24 h (1d), and 48
h (2d), hybridized with a 32P-labeled NCOAT fragment, identifying the 3.5-kb transcript representing the chimera GFP-NCOATGK mRNA. E: Western blot
analysis of 40 ␮g protein from homogenized muscle extracts of wild-type mice, bitransgenic mice with Dox (⫹), or Dox-treated mice followed by withdrawal
from Dox (⫾). The 130-kDa EGFP-NCOATGK protein was detected in muscle of Dox-treated bitransgenic mice, identified using antibody to GFP. F: Western
blot analysis of 40 ␮g tissue lysates from mouse tissues as indicated, probing with antibodies to GFP and actin. C, control; T, transgene.
300 • MARCH 2011 •
www.ajpcell.org
␤-Galactosidase activity assays and ␤-Gal staining. Confirmation
of rtTA transgene expression in muscle was accomplished by breeding the MCK-rtTA founder lines with a tetracycline-responsive LacZ
transgenic mouse line (tetOLacZ) obtained from Dr. J. Segre (18, 25).
Bitransgenic mice were administered Dox-water, drinking water containing 2 mg/ml Dox-HCl (RPI, Mt. Prospect, IL) sweetened with 1
ml Sweet’N Low per 100 ml water, for 3 days. The mice were then
killed and the muscle tissues were collected and either homogenized
for ␤-galactosidase (␤-Gal) activity assays or embedded in optimal
cutting temperature compound (OCT, Tissue-Tek, Torrance, CA) and
frozen. Five-micrometer sections were cut and affixed to glass slides.
␤-Galactosidase was determined using the Galacto-Star system
(Tropix, Bedford, MA), following the protocol provided by the
manufacturer.
Northern blot analysis. Total RNA was prepared from the muscle
using the guanidine isothiocyanate extraction method and separated on denaturing formaldehyde gels, as described previously
(34). Northern blots were hybridized with 32P-labeled NCOAT
cDNA probes (34).
Immunohistochemistry. Paraffin sections were prepared from muscle tissue dissected from adult mice, fixed in PBS-buffered formalin,
and embedded in paraffin. Sections were cut at 5-␮m thickness and
stained with hematoxylin and eosin for histopathological evaluation.
Immunohistochemistry was conducted following the protocol outlined
by Vector Laboratories (Burlingame, CA). Muscle paraffin sections
were deparaffinized and subjected to antigen examination. A M.O.M
kit (Vector) was employed when using mouse monoclonal antibodies,
RESULTS
Characterization of inducible muscle-specific EGFPNCOATGK bitransgenic mice. From our previous studies, overexpression of the O-GlcNAcase splice variant NCOATGK
caused developmental defects in mammary and lens tissues of
transgenic mice (47, 48), along with significant increases
in cellular O-GlcNAc levels. The role of O-GlcNAcylation in
muscle development (10) and the importance of this tissue in
the regulation of glucose metabolism and insulin resistance (4,
11) are well established. The following studies were designed
to determine the role of O-GlcNAcase, the enzyme that removes the O-GlcNAc modification, in skeletal muscle. The
tetracycline-inducible system was used in which the MCK
promoter drove expression of the reverse Tet-activator protein,
rtTA, in skeletal muscle (Fig. 1A). Three MCK-rtTA transgenic founder mice were identified by PCR, as described in
MATERIALS AND METHODS, and each was bred with the TRE-LacZ
transgenic line (Fig. 1B) to determine expression efficiency of
the rtTA protein. As determined by the ␤-galactosidase assay,
founder line 1 was clearly the greatest expresser (Fig. 1C) and
was the transgenic line used in the subsequent experiments. An
examination of other tissues in the Dox-treated bitransgenic
mice showed undetectable LacZ expression in uterus and
kidney, with barely detectable activity in the heart and skeletal
muscle from the untreated animals (Fig. 1D). Immunohistochemistry ␤-Gal staining of paraffin-embedded muscle sections confirmed LacZ expression in the Dox-treated bitransgenic animals (Fig. 1, E–G). These studies demonstrated the
tight transcriptional control of the tetracycline system and the
target specificity of the MCK promoter, that virtually all
transgene expression occurs solely in skeletal muscle and only
when induced by Dox.
Fig. 3. Phenotype of MCK-EGFP-NCOATGK mice. A: male littermates on the Dox diet and a comparison of body size of wild-type control and bitransgenic mice
at 6 wk of age. B: representative pictures of hind leg of male control and bitransgenic mice. C: weights of the hind legs of male mice, 2 wk following Dox
treatment. D: percent body weight change of 25-wk-old male mice consisting of gene negative controls and bitransgenic littermates with or without Dox. Tg,
transgenic; nTg, nontransgenic. E: weights of tissues taken from mice in D at euthanasia. Statistics were derived from 12 male mice for study in C and ⬃30 male
mice for studies in D and E, using a nonparametric Student’s t-test assuming unequal variance. Standard error bars are shown with the P values where appropriate.
300 • MARCH 2011 •
www.ajpcell.org
from hydrolysis of the peptide. The fluorescence was measured
using a Barnstead/Turner fluorometer quipped with 360-nm excitation and 450-nm emission filters. As a control, an aliquot of each
lysate was also preincubated with the proteasome inhibitor MG132
(10 ␮M) for 30 min before addition of the proteasome peptide
substrate.
Statistical analysis. Experiments involving transgenic mice were
done in replicate, using three mice for each condition involving
Western blot and histochemical analyses, and as many as 30 mice
were used in the body and tissue weight measurements. Error bars on
graphs correspond to mean values ⫾ SE. Assays for proteasome
activity and densitometry measurements of O-GlcNAc levels were
analyzed using paired t-tests, while statistical significance on the
weight data was determined by a parametric, unpaired t-test, assuming
equal variance and normal distribution as determined with GraphPad
Prism software.
C459
C460
ing the temporal expression of the transgene under the control
of Dox induction.
Overexpression of EGFP-NCOATGK causes skeletal muscle
atrophy in mouse. We next examined the effect of overexpressed EGFP-NCOATGK on the mouse phenotype using ⬃30
mice, consisting of a mixture of bitransgenic males with or
without Dox, and wild-type male mice. All mice appeared
normal in terms of body mass and behavior before supplementation of their diet with Dox. Between 1 and 2 wk of Dox
treatment, bitransgenic mice experienced progressive weight
loss compared with their littermate controls, with significant
changes in their posture and gait (Fig. 3A). The loss of body
weight generally occurred after 10 days and became highly
significant after 14 days (Fig. 3D). This weight loss can be
attributed to the dramatic loss of skeletal muscle mass, shown
here in the hind legs (Fig. 3B) and also in the front legs of these
mice (not shown). Interestingly, our previous studies have
shown that the male mice were affected more than female
mice, resulting in ⬃80% male mortality between 2 and 4 wk of
Dox treatment (data not shown). The sex-sensitive effect of
EGFP-NCOATGK expression in muscle is not currently
known, and further study is needed to investigate this phenomenon. Although the exact cause of death is unclear, examina-
Fig. 4. Histology of bitransgenic muscle. A–E: representative cross sections of muscle tissues from 25-wk-old (A and B) or 6-wk-old (C–E) mice treated with
Dox for 2 wk. Following 2 wk of treatment, Dox was replaced with the regular diet for half the mice, which were allowed to recover for 2 more weeks (E).
Sections were stained with hematoxylin and eosin. Magnification, ⫻7.5 or ⫻100. F: measurement of myofiber area from 6 groups of mice treated as above,
counting 400 fibers from each group, principally from gastrocnemius. Areas were calculated using ImageJ software to obtain mean values ⫾ SE. A Student’s
t-test was used to establish significant differences (*P ⬍ 0.01).
300 • MARCH 2011 •
www.ajpcell.org
The TRE-EGFP-NCOATGK transgenic mouse line was generated by a similar method (48), with the addition of an EGFP
cDNA sequence as depicted in Fig. 2A. This transgenic line
was bred with the MCK-rtTA transgenic mouse to generate
bitransgenic litters identified by PCR genotyping, as described
in MATERIALS AND METHODS. The EGFP-NCOATGK transgene
was activated by administering a Dox diet for 2 wk, starting 4
wk after birth. Skeletal muscle tissues were dissected from a
mixture of wild-type, single transgenic, and bitransgenic mice
with or without Dox and embedded in Technovit 8100, and
examined under a fluorescent microscope. Expression of
EGFP-NCOATGK fusion protein was clearly apparent in the
tissues (Fig. 2, B and C). The activation of transgene transcription was confirmed by Northern blotting, and was both time
dependent (Fig. 2D), and dose dependent (data not shown)
upon Dox treatment (drinking water).
Western blot analysis, using an anti-GFP antibody, determined the expression of the EGFP-NCOATGK protein in skeletal muscle (Fig. 2F). Only the Dox-treated bitransgenic animals expressed the protein, confirming the tight control of the
tetracycline-inducible system functioning at the level of protein
expression. Withdrawal of the Dox diet for 2 wk showed a
significant reduction in EGFP-NCOATGK levels, demonstrat-
more than twofold compared with those of control mice (Fig.
4F). To reverse the atrophy of skeletal muscle in bitransgenic
mice, we replaced the Dox diet with normal rodent food and
analyzed the muscle tissue after 2 wk. The pause in expression
of NCOATGK almost restored the myofibers to their pre-Dox
state (Fig. 4E). A reduction in accumulation of nuclei in the
center of myofibers indicated the successful rescue from atrophy. These data so far demonstrate that overexpression of
EGFP-NCOATGK protein in skeletal muscle alone can cause a
dramatic decrease in muscle mass, particularly in male mice,
and that this tissue loss appears to be part of the apoptotic
process.
EGFP-NCOATGK expression causes O-GlcNAc-dependent
inhibition of proteasome activity. It has been reported that
O-GlcNAc modification of the Rpt2 subunit of the proteasome
complex inhibits ATPase activity and proteasome function in
cell culture (56) and in transgenic mice (3, 47). We therefore
examined proteasome function in skeletal muscle as a function
of protein O-GlcNAc levels. Western blot analysis of muscle
tissues extracted from Dox-treated bitransgenic mice showed a
general increase in O-GlcNAcylated proteins as determined by
immunoblotting with anti-O-GlcNAc antibody (RL2) (Fig. 5, A
and B). The increase in O-GlcNAc is likely a result of
NCOATGK, which is catalytically inactive, blocking the endogenous O-GlcNAcase from removing the O-GlcNAc modi-
Fig. 5. Inhibition of proteasome with excessO-linked ␤-N-acetylglucosamine (O-GlcNAc) modification. A: Western blot analysis of protein extracts (40 ␮g) from
muscle of wild-type and bitransgenic mice on Dox, using anti-O-GlcNAc antibody, RL2. The extracts from bitransgenic mice 2 wk following Dox withdrawal
are also shown (⫾). B: densitometry for the O-GlcNAc blot shown in A, showing SE error bars for 3 separate experiments. C and D: muscle paraffin sections
of wild-type (C) and bitransgenic mice on Dox (D) were subjected to immunostaining using anti-O-GlcNAc antibody. E: the proteasome LLVY peptidase activity
assays performed on muscle extracts, from multiple mice, giving mean values ⫾ SE. A Student’s t-test was used to establish significant differences (*P ⬍ 0.01).
300 • MARCH 2011 •
www.ajpcell.org
tion of the principle tissues appeared normal and, excluding the
gastrocnemius calf muscle, the transgenic tissue weights did
not differ significantly from the control animals (Fig. 3E). This
was not surprising since the transgene is not measurably
expressed in these tissues (Fig. 2F). Withdrawal of Dox before
the affected animals reached the critical end point could prevent the death of male bi-transgenic mice, advocating a regulatory role for O-GlcNAcylation in the development of muscle
atrophy.
Histology of skeletal muscle of EGFP-NCOATGK-expressing
mice. To further study the effects of NCOATGK expression in
muscle, hematoxylin and eosin-stained paraffin sections of the
midcalf region, including the medial and lateral gastrocnemius,
soleus, and extensor digitorum longus muscles, were examined. The total cross-sectional areas are depicted for a control
and transgenic mouse (Fig. 4, A and B), showing the large
difference in muscle mass with comparable bone size. Myofiber degeneration and cell loss were accompanied by the disassembly of myofiber structure (Fig. 4D). A necrotic focus
invaded by phagocytic cells can be seen in the center of the
image. Also apparent was a greater presence of dead myofibers
with reduced structure and an increased number of dying
myofibers with accumulations of nuclei attempting to reverse
the cell death process. The myofiber cross-sectional area of
bitransgenic mice after 2 wk of Dox induction was reduced by
C461
C462
sue dissected from control and Dox-treated bitransgenic mice
were stained for p53 (Fig. 6). Significant p53 accumulation was
apparent in the tissues from EGFP-NCOATGK-expressing
mice compared with controls (Fig. 6, B and E), and this
observation was compatible with the observed decrease in
proteasome activity (Fig. 5E). The predominant localization of
p53 to the nucleus was characteristic of the onset of apoptosis
(Fig. 6F). The withdrawal of Dox from the bitransgenic mice
halts expression of the dominant-negative O-GlcNAcase, causing a decrease in cell O-GlcNAc and reduction in p53 levels
(Fig. 6H). Western blot analysis confirmed the accumulation of
p53 protein in skeletal muscle (Fig. 7A), and consistent with
the earlier results, withdrawal of Dox caused a reduction in p53
protein. These data so far support our hypothesis that EGFPNCOATGK overexpression causes p53-dependent cell death,
and one possible mechanism may be through the hyper-OGlcNAc-mediated proteasome blockade.
The proapoptotic factors BAX and p21 are downstream
genes of the p53 gene. As seen with p53, both BAX and p21
were undetectable in control animals and, like p53, accumulated in the Dox-treated bitransgenics (Fig. 7, B and C).
Withdrawal of Dox diminished the levels of these proteins. The
accumulation of p53 and BAX proteins stimulates the release
of cytochrome c from mitochondria, promoting the formation
of the “apoptosome,” which cleaves and activates procaspases.
The procaspase form of caspase 9 (p46) was cleaved to the
activated form (p34) in Dox-treated bitransgenic mice (Fig.
7D). Consequently, the downstream caspase 3 was also processed to its active form (p19) (Fig. 7E). Neither caspase 9 nor
caspase 3 was detectable in muscle from the control animals. In
summary, the overexpression of NCOATGK in skeletal muscle
tissues resulted in the accumulation of proapoptotic factors that
normally are cleared by the UPS, and increased caspase apop-
Fig. 6. Immunostaining of proapoptotic factor p53 in muscle of bitransgenic mice. Paraffin sections from the muscles of wild-type
mice (A–C) and bitransgenic mice with Dox
(D–F) and then Dox withdrawal (⫾) (G–I)
were subjected to anti-p53 immunostaining
(green). DAPI (blue) staining for nuclei.
300 • MARCH 2011 •
www.ajpcell.org
fication from cellular proteins (48). Withdrawal of Dox from
the bitransgenic mice returned levels of O-GlcNAc to almost
that of control mice (Fig. 5B). Immunostaining muscle tissue
sections with RL2 showed moderate O-GlcNAc staining in the
EGFP-NCOATGK-expressing tissues (Fig. 5, C and D), confirming the results seen in the Western blot.
The proteasome peptidase activity was determined in
skeletal muscle protein extracts from control and Doxtreated bitransgenic mice in the chymotrypsin-like LLVY
peptide activity assay, as described in MATERIALS AND METHODS
(Fig. 5E). Increased O-GlcNAc modification in the EGFPNCOATGK-expressing mice corresponded to a 30% decrease
in proteasome activity, demonstrating that NCOATGK-mediated impairment of proteasome activity occurred either directly
through Rpt2 O-GlcNAcylation or indirectly through some
intermediate interacting protein. Since reduced proteasome
activity is known to increase the levels of proapoptotic factors
such as p53 and BAX, an O-GlcNAc-mediated blockade of the
UPS provides a link between O-GlcNAcylation and cell deathdependent muscle atrophy.
Accumulation of proapoptotic factors and activation of
caspases in skeletal muscle of bitransgenics. The abnormal
activation and accumulation of proapoptotic factors p53 and
BAX have been associated with the inhibition and cell death of
myocytes grown in high glucose concentrations (16). Moreover, the UPS is the major mechanism responsible for the
degradation and removal of these proapoptotic factors (7, 30).
To investigate whether impaired proteasome function, through
NCOATGK-mediated hyper-O-GlcNAc modification, leads to
the accumulation of proapoptotic factors and the activation of
the caspase-apoptotic pathway, we determined the expression
of these proteins in skeletal muscle from EGFP-NCOATGKexpressing bitransgenic mice. Paraffin sections of muscle tis-
C463
totic markers. The removal of NCOATGK expression restores
O-GlcNAc and proapoptotic proteins to normal levels, rescuing cells from death.
DISCUSSION
The O-GlcNAcase gene has been linked to type II diabetes
(15, 29). We have cloned a spliced variant of this gene from the
diabetic GK rat, abbreviated NCOATGK, which has all the
properties of NCOAT (OGT-binding site and histone acetyltransferase domain) minus the O-GlcNAcase activity (3, 45,
48). We also reported that the overexpression of NCOATGK
caused disrupted lens fiber cell differentiation and cataracts in
mice (47). In the current study, we explored the effect of
NCOATGK on mouse skeletal muscle to demonstrate that
disruption of O-GlcNAcase activity induced muscle atrophy in
transgenic mice.
Two weeks of overexpression of the EGFP-NCOATGK chimera protein in bitransgenic mice produced dramatic symptoms, including moderate body weight loss, which was mostly
attributed to severe muscle mass reduction, and impaired
mobility and gait, with 70 – 80% morbidity in male mice. We
are not sure of the cause of lethality for the bitransgenic male
mice since the gross pathology and mass of the organs appeared normal. One explanation could be the lack of food
intake caused by an inability to chew due to muscle loss,
although the addition of moistened food was only partially
helpful. Additional studies are needed to help explain the
sexual dimorphism regarding O-GlcNAcylation-induced atrophy. Interestingly, a few male bitransgenic mice survived the
challenge of overexpression of EGFP-NCOATGK, regaining
their health with time. The withdrawal of Dox from the
bitransgenic mouse diet slows the expression of EGFPNCOATGK in muscle, reverses the symptoms of muscle loss,
and is able to prevent the lethality experienced in the male
bitransgenic mice, evidence that overexpression of NCOATGK
was the major cause of this muscle atrophy. Reducing
NCOATGK expression and restoring the function of wild-type
O-GlcNAcase likely saves the animal from muscle loss before
a critical end point is reached.
The pathology of the affected bitransgenic skeletal muscle
may not resemble true cachexia of cancer patients, and the
accelerated symptoms seen in the mice may have other contributing factors associated with downregulating the O-GlcNAcase enzyme. The abnormal presence of higher-than-normal
O-GlcNAcylated proteins may itself be toxic, although this is
unlikely since increases in glucose and glucosamine above
physiological levels lead to increased O-GlcNAc and yet in
some systems appear to be protective, as reported by
Chatham’s group (6, 57) and others (49, 54). Also, from our
previous studies (3, 47), and in this work, the levels of
O-GlcNAcylated protein are not extraordinarily elevated in the
general cellular proteome. However, there are clearly multiple
factors driving myofibers into apoptosis, and one approach in
deciphering their action will be a proteomic study of OGlcNAc-modified proteins.
From the studies of Fiordaliso et al. (16), the abnormal
activation and accumulation of proapoptotic factors p53 and
BAX were associated with hyperglycemia-induced myocyte
death. As we observed in the tetracycline-inducible, MCKrtTA/TRE-EGFP-NCOATGK bitransgenic mice, overexpression of EGFP-NCOATGK in skeletal muscle also resulted in
the accumulation of p53, BAX, and p21, with the activation of
300 • MARCH 2011 •
www.ajpcell.org
Fig. 7. Accumulation of proapoptotic factors and activation of caspases in muscle of bitransgenic mice. Western
blots of protein extracts (40 ␮g) of wild-type (control)
and bitransgenic mice with Dox treatment (⫹) or 2 wk
following Dox withdrawal (⫾). Blots were probed with
antibodies to p53 (A), BAX (B), p21 (C), caspase 9 (D),
activated caspase 3 (E), and ␤-actin (F).
C464
ACKNOWLEDGMENTS
We thank Dr. Trenton R. Schoeb for assistance with the pathological
analysis of the transgenic mouse tissues.
GRANTS
This work was supported by funding from National Institutes of Health
Grants CA095021 and DK043652.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
REFERENCES
1. Akimoto Y, Kreppel LK, Hirano H, Hart GW. Localization of the
O-linked N-acetylglucosamine transferase in rat pancreas. Diabetes 48:
2407–2413, 1999.
2. Belizário JE, Lorite MJ, Tisdale MJ. Cleavage of caspases-1, -3, -6, -8
and -9 substrates by proteases in skeletal muscles from mice undergoing
cancer cachexia. Br J Cancer 84: 1135–1140, 2001.
3. Bowe DB, Sadlonova A, Toleman CA, Novak Z, Hu Y, Huang P,
Mukherjee S, Whitsett T, Frost AR, Paterson AJ, Kudlow JE. OGlcNAc integrates the proteasome and transcriptome to regulate nuclear
hormone receptors. Mol Cell Biol 26: 8539 –8550, 2006.
4. Buse MG, Robinson KA, Marshall BA, Hresko RC, Mueckler MM.
Enhanced O-GlcNAc protein modification is associated with insulin resistance in GLUT1-overexpressing muscles. Am J Physiol Endocrinol
Metab 283: E241–E250, 2002.
5. Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. Hyperglycemiainduced apoptosis in mouse myocardium: mitochondrial cytochrome Cmediated caspase-3 activation pathway. Diabetes 51: 1938 –1948, 2002.
6. Champattanachai V, Marchase RB, Chatham JC. Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2. Am J
Physiol Cell Physiol 294: C1509 –C1520, 2008.
7. Chen F, Chang D, Goh M, Klibanov SA, Ljungman M. Role of p53 in
cell cycle regulation and apoptosis following exposure to proteasome
inhibitors. Cell Growth Differ 11: 239 –246, 2000.
8. Cieniewski-Bernard C, Bastide B, Lefebvre T, Lemoine J, Mounier Y,
Michalski JC. Identification of O-linked N-acetylglucosamine proteins in
rat skeletal muscle using two-dimensional gel electrophoresis and mass
spectrometry. Mol Cell Proteomics 3: 577–585, 2004.
9. Cieniewski-Bernard C, Montel V, Stevens L, Bastide B. O-GlcNAcylation, an original modulator of contractile activity in striated muscle. J
Muscle Res Cell Motil 30: 281–287, 2009.
10. Cieniewski-Bernard C, Mounier Y, Michalski JC, Bastide B. OGlcNAc level variations are associated with the development of skeletal
muscle atrophy. J Appl Physiol 100: 1499 –1505, 2006.
11. Daniels MC, Ciaraldi TP, Nikoulina S, Henry RR, McClain DA.
Glutamine:fructose-6-phosphate amidotransferase activity in cultured human skeletal muscle cells: relationship to glucose disposal rate in control
and non-insulin-dependent diabetes mellitus subjects and regulation by
glucose and insulin. J Clin Invest 97: 1235–1241, 1996.
12. Delaney CL, Russell JW, Cheng HL, Feldman EL. Insulin-like growth
factor-I and over-expression of Bcl-xL prevent glucose-mediated apoptosis in Schwann cells. J Neuropathol Exp Neurol 60: 147–160, 2001.
13. Donath MY, Gross DJ, Cerasi E, Kaiser N. Hyperglycemia-induced
beta-cell apoptosis in pancreatic islets of Psammomys obesus during
development of diabetes. Diabetes 48: 738 –744, 1999.
14. Dong DL, Hart GW. Purification and characterization of an O-GlcNAc
selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol. J Biol
Chem 269: 19321–19330, 1994.
15. Farook VS, Bogardus C, Prochazka M. Analysis of MGEA5 on
10q24.1-q243 encoding the beta-O-linked N-acetylglucosaminidase as a
candidate gene for type 2 diabetes mellitus in Pima Indians. Mol Genet
Metab 77: 189 –193, 2002.
16. Fiordaliso F, Leri A, Cesselli D, Limana F, Safai B, Nadal-Ginard B,
Anversa P, Kajstura J. Hyperglycemia activates p53 and p53-regulated
genes leading to myocyte cell death. Diabetes 50: 2363–2375, 2001.
17. Franch HA, Price SR. Molecular signaling pathways regulating muscle
proteolysis during atrophy. Curr Opin Clin Nutr Metab Care 8: 271–275,
2005.
18. Furth PA, St Onge L, Böger H, Gruss P, Gossen M, Kistner A, Bujard
H, Hennighausen L. Temporal control of gene expression in transgenic
mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 91:
9302–9306, 1994.
19. Han I, Kudlow JE. Reduced O glycosylation of Sp1 is associated with
increased proteasome susceptibility. Mol Cell Biol 17: 2550 –2558, 1997.
20. Hanover JA, Lai Z, Lee G, Lubas WA, Sato SM. Elevated O-linked
N-acetylglucosamine metabolism in pancreatic beta-cells. Arch Biochem
Biophys 362: 38 –45, 1999.
21. Hart GW, Housley MP, Slawson C. Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446: 1017–1022,
2007.
22. Hédou J, Bastide B, Page A, Michalski JC, Morelle W. Mapping of
O-linked beta-N-acetylglucosamine modification sites in key contractile
proteins of rat skeletal muscle. Proteomics 9: 2139 –2148, 2009.
24. Hedou J, Cieniewski-Bernard C, Leroy Y, Michalski JC, Mounier Y,
Bastide B. O-linked N-acetylglucosaminylation is involved in the Ca2⫹
activation properties of rat skeletal muscle. J Biol Chem 282: 10360 –
10369, 2007.
25. Jaubert J, Patel S, Cheng J, Segre JA. Tetracycline-regulated transactivators driven by the involucrin promoter to achieve epidermal conditional gene expression. J Invest Dermatol 123: 313–318, 2004.
300 • MARCH 2011 •
www.ajpcell.org
caspase 9 and caspase 3, suggesting that apoptosis may contribute to muscle atrophy through O-GlcNAcylation. Tumor
suppressor p53 and its downstream genes, p21 and BAX, are
normally degraded by the proteasome complex. Zhang et al.
and others showed that increasing cellular O-GlcNAcylation
reduced proteasome proteolytic function (47, 55, 56). Indeed,
overexpression of NCOATGK in skeletal muscle of the bitransgenic mice also caused an elevation of cellular protein-OGlcNAc modification as well as impairment of proteasome
proteolytic activity. These results support our hypothesis that
O-GlcNAcylation-mediated proteasome blockade, caused by
the inhibition of O-GlcNAcase, induces muscle atrophy in
mouse skeletal muscle. Although proteasome inhibition by
O-GlcNAc may serve as a major cause of the accumulation of
proapoptotic factors and the activation of the caspase cascade,
it is also possible that NCOATGK and O-GlcNAc have a
pleiotropic effect on muscle cells, such as regulating transcription (52, 53), affecting protein-protein interaction between p53
and its negative regulator mdm2 (26), the direct regulation of
p53 itself (51), and contractile protein function (8 –10, 22, 24,
46). While advocating O-GlcNAc modification of the proteasome as a principle regulator of cell apoptosis and maintenance, an alternative cell death mechanism that has some
mechanistic overlap with the proteasome, autophagy (44),
might also be involved in NCOATGK-induced muscle atrophy
in the MCK-EGFP-NCOATGK transgenic mice. A role for
autophagy in O-GlcNAc-mediated muscle atrophy should be a
consideration in future studies.
Hyper-O-glycosylation is a characteristic seen in a number
of diseases, including muscle atrophy, cancer, and uncontrolled
diabetes. Muscle atrophy and the associated reduction in
strength contributes significantly to the discomfort of patients
encumbered by these diseases. In this current study, we extend
our previous findings of expressing the dominant-negative
form of O-GlcNAcase, NCOATGK, in mammary and lens. The
overexpression of NCOATGK in a glucose (O-GlcNAc)-sensitive tissue, such as skeletal muscle, resulted in significant
muscle atrophy, with at least some of the characteristic markers
of apoptosis. This work supports the linkage of muscle atrophy
with O-GlcNAc-mediated apoptosis and suggests that increased cellular O-GlcNAc exacerbates this condition by
blocking proteasome function.
42. Su K, Yang X, Roos MD, Paterson AJ, Kudlow JE. Human Sug1/p45
is involved in the proteasome-dependent degradation of Sp1. Biochem J
348: 281–289, 2000.
43. Sun Z, Liu L, Liu N, Liu Y. Muscular response and adaptation to diabetes
mellitus. Front Biosci 13: 4765–4794, 2008.
44. Tasdemir E, Maiuri M, Galluzzi L, Vitale I, Djavaheri-Mergny M,
D’Amelio M, Criollo A, Morselli E, Zhu C, Harper F, Nannmark U,
Samara C, Pinton P, Vicencio J, Carnuccio R, Moll U, Madeo
F, Paterlini-Brechot P, Rizzuto R, Szabadkai G, Pierron G, Blomgren
K, Tavernarakis N, Codogno P, Cecconi F, Kroemer G. Regulation of
autophagy by cytoplasmic p53. Nat Cell Biol 10: 676 –687, 2008.
45. Toleman C, Paterson AJ, Whisenhunt TR, Kudlow JE. Characterization of the histone acetyltransferase (HAT) domain of a bifunctional
protein with activatable O-GlcNAcase and HAT activities. J Biol Chem
279: 53665–53673, 2004.
46. Walgren JL, Vincent TS, Schey KL, Buse MG. High glucose and
insulin promote O-GlcNAc modification of proteins, including ␣-tubulin.
Am J Physiol Endocrinol Metab 284: E424 –E434, 2003.
47. Wang K, Ho SR, Mao W, Huang P, Zhang F, Schwiebert EM, Kudlow
JE, Paterson AJ. Increased O-GlcNAc causes disrupted lens fiber cell
differentiation and cataracts. Biochem Biophys Res Commun 387: 70 –76,
2009.
48. Whisenhunt TR, Yang X, Bowe DB, Paterson AJ, Van Tine BA,
Kudlow JE. Disrupting the enzyme complex regulating O-GlcNAcylation
blocks signaling and development. Glycobiology 16: 551–563, 2006.
49. Wu T, Zhou H, Jin Z, Bi S, Yang X, Yi D, Liu W. Cardioprotection of
salidroside from ischemia/reperfusion injury by increasing N-acetylglucosamine linkage to cellular proteins. Eur J Pharmacol 613: 93–99, 2009.
50. Xie W, Paterson AJ, Chin E, Nabell LM, Kudlow JE. Targeted
expression of a dominant negative epidermal growth factor receptor in the
mammary gland of transgenic mice inhibits pubertal mammary duct
development. Mol Endocrinol 11: 1766 –1781, 1997.
51. Yang WH, Kim JE, Nam HW, Ju JW, Kim HS, Kim YS, Cho JW.
Modification of p53 with O-linked N-acetylglucosamine regulates p53
activity and stability. Nat Cell Biol 8: 1074 –1083, 2006.
52. Yang X, Su K, Roos MD, Chang Q, Paterson AJ, Kudlow JE.
O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its
transcriptional capability. Proc Natl Acad Sci USA 98: 6611–6616, 2001.
53. Yang X, Zhang F, Kudlow JE. Recruitment of O-GlcNAc transferase to
promoters by corepressor mSin3A: coupling protein O-GlcNAcylation to
transcriptional repression. Cell 110: 69 –80, 2002.
54. Zachara NE, Hart GW. O-GlcNAc a sensor of cellular state: the role of
nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress. Biochim Biophys Acta 1673: 13–28, 2004.
55. Zachara NE, Hart GW. O-GlcNAc modification: a nutritional sensor that
modulates proteasome function. Trends Cell Biol 14: 218 –221, 2004.
56. Zhang F, Su K, Yang X, Bowe DB, Paterson AJ, Kudlow JE. OGlcNAc modification is an endogenous inhibitor of the proteasome. Cell
115: 715–725, 2003.
57. Zou L, Yang S, Champattanachai V, Hu S, Chaudry IH, Marchase
RB, Chatham JC. Glucosamine improves cardiac function following
trauma-hemorrhage by increased protein O-GlcNAcylation and attenuation of NF-␬B signaling. Am J Physiol Heart Circ Physiol 296: H515–
H523, 2009.
300 • MARCH 2011 •
www.ajpcell.org
26. Kubbutat MH, Jones SN, Vousden KH. Regulation of p53 stability by
Mdm2. Nature 387: 299 –303, 1997.
27. Kudlow JE. Post-translational modification by O-GlcNAc: another way to
change protein function. J Cell Biochem 98: 1062–1075, 2006.
28. Lee SW, Dai G, Hu Z, Wang X, Du J, Mitch WE. Regulation of muscle
protein degradation: coordinated control of apoptotic and ubiquitin-proteasome systems by phosphatidylinositol 3 kinase. J Am Soc Nephrol 15:
1537–1545, 2004.
29. Lehman DM, Fu DJ, Freeman AB, Hunt KJ, Leach RJ, Johnson-Pais
T, Hamlington J, Dyer TD, Arya R, Abboud H, Goring HH, Duggirala
R, Blangero J, Konrad RJ, Stern MP. A single nucleotide polymorphism in MGEA5 encoding O-GlcNAc-selective N-acetyl-beta-D glucosaminidase is associated with type 2 diabetes in Mexican Americans.
Diabetes 54: 1214 –1221, 2005.
30. Li B, Dou QP. Bax degradation by the ubiquitin/proteasome-dependent
pathway: involvement in tumor survival and progression. Proc Natl Acad
Sci USA 97: 3850 –3855, 2000.
31. Liu K, Paterson AJ, Chin E, Kudlow JE. Glucose stimulates protein
modification by O-linked GlcNAc in pancreatic beta cells: linkage of
O-linked GlcNAc to beta cell death. Proc Natl Acad Sci USA 97:
2820 –2825, 2000.
32. Liu K, Paterson AJ, Zhang F, McAndrew J, Fukuchi K, Wyss JM,
Peng L, Hu Y, Kudlow JE. Accumulation of protein O-GlcNAc modification inhibits proteasomes in the brain and coincides with neuronal
apoptosis in brain areas with high O-GlcNAc metabolism. J Neurochem
89: 1044 –1055, 2004.
33. Love DC, Hanover JA. The hexosamine signaling pathway: deciphering
the “O-GlcNAc code”. Sci STKE 2005: re13, 2005.
34. McAndrew J, Paterson AJ, Asa SL, McCarthy KJ, Kudlow JE.
Targeting of transforming growth factor-alpha expression to pituitary
lactotrophs in transgenic mice results in selective lactotroph proliferation
and adenomas. Endocrinology 136: 4479 –4488, 1995.
35. McClung JM, Kavazis AN, DeRuisseau KC, Falk DJ, Deering MA,
Lee Y, Sugiura T, Powers SK. Caspase-3 regulation of diaphragm
myonuclear domain during mechanical ventilation-induced atrophy. Am J
Respir Crit Care Med 175: 150 –159, 2007.
36. Pepato MT, Migliorini RH, Goldberg AL, Kettelhut IC. Role of
different proteolytic pathways in degradation of muscle protein from
streptozotocin-diabetic rats. Am J Physiol Endocrinol Metab 271: E340 –
E347, 1996.
37. Reisz-Porszasz S, Bhasin S, Artaza JN, Shen R, Sinha-Hikim I, Hogue
A, Fielder TJ, Gonzalez-Cadavid NF. Lower skeletal muscle mass in
male transgenic mice with muscle-specific overexpression of myostatin.
Am J Physiol Endocrinol Metab 285: E876 –E888, 2003.
38. Sartorelli V, Fulco M. Molecular and cellular determinants of skeletal
muscle atrophy and hypertrophy. Sci STKE 2004: re11, 2004.
39. Shimabukuro M, Zhou YT, Levi M, Unger RH. Fatty acid-induced beta
cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci
USA 95: 2498 –2502, 1998.
40. Siu PM, Alway SE. Mitochondria-associated apoptotic signalling in
denervated rat skeletal muscle. J Physiol 565: 309 –323, 2005.
41. Su K, Roos MD, Yang X, Han I, Paterson AJ, Kudlow JE. An
N-terminal region of Sp1 targets its proteasome-dependent degradation in
vitro. J Biol Chem 274: 15194 –15202, 1999.
C465

Downloaded - Cell Physiology

Transcription

Similar documents

Saturday, April 17th 5-9pm California Car Cover`s

Calf muscle tears - Performance Medicine

flyer for arasys

anatomically engineered running tights

Cuddly Critters Classroom Schedule

Clinically effective solution for muscle rehabilitation.

CollectorsShow July5

RS-4i Plus - RS Medical

Is it possible to gain 30 lbs of muscle in just 60 days?

So how do you strengthen your determination muscle?