| HOME | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|---|
| ||||||||||||||||||||||||||||||||
a Department of Anatomy, College of Medicine
b Institute on Aging, University of South Florida, Tampa
Stephen E. Alway, Division of Exercise Physiology, Department of Human Performance and Applied Exercise Science, School of Medicine, West Virginia University, P.O. Box 9227, Morgantown, WV 26506-9227 E-mail: salway{at}wvu.edu.
Decision Editor: Jay Roberts, PhD
 |
Abstract |
|---|
 Top Abstract MethodsResultsDiscussionReferences |
|---|
GLYCERALDEHYDE-3-PHOSPHATE dehydrogenase (GAPDH) is an enzyme in the glycolytic pathway that catalyzes the oxidation and phosphorylation of glyceraldehyde-3-phosphate by NAD+ and Pi. In skeletal muscle, the flux through the glycolytic pathway is dependent upon the fiber type composition of that muscle. Therefore, when assessing changes in glycolytic enzymes in a muscle it is important to consider the fiber-type composition of that muscle. Some previous studies have provided evidence indicating that rat skeletal muscle glycolytic capacity declines with age (1)(2)(3), whereas other data indicate that no change occurs (3)(4)(5). Of those studies only the one by Duan and coworkers (3) analyzed different muscles composed of various fiber types. They found, for example, that lactate dehydrogenase activity was lower in medial gastrocnemius muscle of aged compared to younger rats, whereas there was no age-related change in soleus, plantaris, and superficial or deep gastrocnemius muscles. To shed light on these discrepancies, additional studies are needed to determine age-related metabolic changes that occur in muscles and how those changes are affected by fiber-type composition. This information is important for understanding conditions such as muscle fatigue and atrophy that may occur with age. The purpose of the present study was to determine if a particular enzyme in the glycolytic pathway, GAPDH, changes in various rat hindlimb muscles with age.
Traditionally, GAPDH mRNA has been used as an internal control in experiments that measure mRNA in skeletal muscle (e.g., 6,7). GAPDH has been chosen because it is constitutively expressed in muscle at fairly high levels and because it is assumed that its expression is relatively constant under various conditions. Therefore, a second purpose of this study was to determine whether or not GAPDH mRNA is a proper internal control for mRNA studies of aging muscle. Data are shown which indicate that cyclophilin mRNA may be an alternative internal control for studies of aging muscle.
|
Methods |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
50% mortality (8). Animals were housed in pathogen-free conditions, two per cage at 20–22°C with a 12-hour light/dark cycle, and received rat chow and water ad libitum. All animals appeared healthy, e.g., they were able to ambulate normally and food and water consumption was standard. Animals were then anesthetized with pentobarbital sodium (55 mg/kg intraperitoneally) and gastrocnemius, plantaris, soleus, and extensor digitorum longus (EDL) muscles were excised from the right hindlimbs. Animals were euthanized with an overdose of pentobarbital sodium. Plantaris, soleus, and EDL muscles were quickly cut into three pieces and immediately frozen in liquid nitrogen. The deep, red portion of the gastrocnemius muscle was separated from the whole muscle, cut into three pieces and quickly frozen. All tissues were stored at -80°C.
mRNA Analyses
GAPDH RNA probe was made from a reverse-transcribed–polymerase chain reaction (RT-PCR) product. Total RNA was extracted from rat gastrocnemius muscle and 5 µg was reverse-transcribed in a total volume of 20 µL (Superscript II RNase H- Reverse Transcriptase, Life Technologies, Gaithersburg, MD). A 110-bp segment of GAPDH was amplified using 1 µL of cDNA, 50 ng of each primer (5'-AAATGGGGTGATGCTGGTG and 3'-GAGATGATGACC CTTTTGG), 250 µM dNTPs, 1x polymerase chain reaction buffer (Sigma Chemical Co, St. Louis, MO), and 2 units Taq DNA polymerase (Sigma Chemical Co, St. Louis, MO) using an annealing temperature of 54.2°C. The resulting GAPDH RT-PCR product was cloned using pCR-Script Amp SK(+) Cloning Kit (Stratagene, La Jolla, CA). The insert was verified by DNA sequencing (Molecular Biology Core Facility at the H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida) and was >99% similar to the previously reported rat GAPDH sequence (9). RNA probes were made by in vitro transcription as described previously (10) using BrightStar BIOTINscript Kit (Ambion, Austin, TX). The resulting GAPDH probe had 10% of the biotinylated nucleotides.
RNA probes were also generated to detect cyclophilin mRNA and 28S-rRNA (Rat Internal Standard Kit, Ambion). Transcriptions were done under the control of T7 polymerase and the resulting cyclophilin and 28S probes had 100% and 2% of the biotinylated nucleotides, respectively. All RNA probes were gel-purified, extracted, solubilized in 20 µL of RNase-free H2O and stored in 5 µL aliquots at -80°C. Aliquots were diluted 100-fold prior to use.
For Northern blots, total RNA was extracted from muscles (100 mg piece of each muscle) using 1 mL of TriReagent (Molecular Research Center, Cincinnati, OH) per muscle and a mechanical homogenizer. Extracted RNA was solubilized in 20 µL of RNase-free H2O, quantitated in duplicate by absorbance at 260 nm, and then stored at -80°C. Total RNA was fractionated on 1% formaldehyde-agarose gels and transferred to nylon using a rapid downward transfer system (Turboblotter, Schleicher & Schuell, Keene, NH). Following transfer, membranes were stained with methylene blue (Molecular Research Center, Cincinnati, OH) for 10 seconds, rinsed several times in distilled H2O and then dried on filter paper. RNA was immobilized by ultraviolet crosslinking and membranes were cut such that the upper part contained the 28S-rRNA bands and the lower part contained the 18S-rRNA bands. Membranes were prehybridized for 3 hours at 60°C in 5x Denhardt's solution, 5x sodium chloride/sodium citrate buffer (SSC), 1% sodium dodecyl sulfate (SDS), 50% formamide, and 0.1 mg/mL tRNA. Hybridization was done overnight at 60°C in fresh prehybridization buffer containing 10% dextran sulfate and RNA probe (10 µL GAPDH or cyclophilin RNA probe/mL buffer for lower membranes and 10 µL 28S RNA probe/mL buffer for upper membranes). Following hybridization, membranes were washed at room temperature twice in 2x SSC and 0.1% SDS, twice in 2x SSC and 0.25% SDS, and then once at 42°C in 2x SSC and 0.25% SDS. Signals were detected on film by chemiluminescence (BrightStar BioDetect Nonisotopic Detection, Ambion) and then quantitated (Bio Image, Genomic Solutions, Ann Arbor, MI). GAPDH bands were located just beneath the 18S-rRNA band at 1.4-kb and cyclophilin bands were located at 0.7 kb.
Western Blotting
Muscle GAPDH protein levels were determined by Western blotting. A 20–30-mg piece of muscle was homogenized in 19 volumes of 10 mM sodium phosphate buffer (pH 7.6) on ice. Insoluble material was pelleted by centrifugation at 5000 g for 10 minutes at 4°C and the supernatant was freeze-thawed. Protein concentration of the supernatant was determined spectrophotometrically using BCA Protein Assay and bovine serum albumin standards (Pierce, Rockford, IL). Twenty-five micrograms of protein from each sample was separated on 12% acrylamide sodium dodecyl sulfate–polyacrylamide gels. Proteins were blotted to nitrocellulose and probed with anti–glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (Chemicon International, Temecula, CA) diluted 1:1000 in I-Block (Tropix, Bedford, MA) for 30 minutes at 25°C. Single bands corresponding to a molecular mass of 37 kDa were detected by chemiluminescence (CDP-Star, Tropix). These bands were quantitated using Bio Image (Genomic Solutions).
Enzyme Activity
GAPDH activity in muscles was determined at 25°C by measuring NADH oxidation (11). The assay mixture contained 0.2 mM ß-NADH, 6 mM glycerate-3-phosphate, 0.9 mM EDTA, and 1.7 mM MgSO4 in 82.5 mM triethanolamine buffer (pH 7.6). Muscle sample (8 µL of supernatant used in Western blots) was added to the assay mixture, and the reaction was started by the addition of 1.1 mM ATP and 14.8 U/mL phosphoglyceric phosphokinase. The total assay volume was 0.5 mL. Absorbance at 340 nm was read every 5 seconds for 1 minute. Each sample was assayed in triplicate.
Statistics
Two-way analysis of variance tests with Student-Newman-Keuls post hoc tests were used to determine differences in mRNA, protein, and enzyme activity levels. Student's t tests were used to determine if differences in 28S and cyclophilin RNA levels existed between muscles from adult and senescent rats. An 
level of .05 was used for all tests.
|
Results |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
|
|
.69; Fig. 1). 28S signals were therefore used to confirm that samples were loaded onto gels and transferred to membranes equally (each 28S signal did not vary from the mean of the other 28S signals on that membrane by more than 8%). Cyclophilin mRNA levels in plantaris and soleus muscles did not differ between adult and senescent rats (all ps .42; Fig. 3).
|
50% of that in adult muscles, whereas those in red gastrocnemius and soleus muscles did not differ between adult and senescent rats (main effect of age, p < .001; Fig. 4 and Fig. 5). GAPDH protein levels were different between muscles (main effect of muscle type, p < .001) and age-related differences in GAPDH protein levels were dependent upon the muscle type (interaction between age and muscle type, p = .032).
|
|
|
|
Discussion |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
It is likely that many of the observed differences in GAPDH between different muscles and between muscles from animals of different ages are related to the fiber-type compositions and age-related changes in fiber-type composition, respectively. In general, there is a transition to slower, more oxidative fibers with aging (13). Soleus muscle of adult rats is composed of 90% slow-twitch oxidative (SO) fibers and 10% fast-twitch oxidative glycolytic (FOG) fibers (14). With age there is a slight increase to an even greater percentage of SO fibers in this muscle (13). GAPDH protein level and enzyme activity in soleus muscle was the lowest of any of the muscles studied and did not differ between adult and senescent rats. This indicates that glycolytic capacity does not change with age in oxidative muscles. In contrast, red gastrocnemius, plantaris, and EDL muscles of adult rats have mixed fiber-type compositions (1–35% SO fibers, 20–56% FOG fibers, and 9–79% FG fibers) (14). With age the fiber-type compositions of these muscles shift to more SO and FOG fibers and less FG fibers (13). In general, we found that GAPDH protein level and enzyme activity in those muscles were lower in senescent rats, suggesting that glycolytic capacity is diminished with age in muscles containing a substantial number of glycolytic fibers.
Age-related changes in enzyme molecules have been well documented and have been attributed to a wide variety of post-translational modifications (15). One specific mechanism of the age-related decrease in GAPDH enzyme activity has been shown to lie in the four NAD+ binding sites of the enzyme (16). Conformational changes occur in the NAD+ binding domains of GAPDH enzyme from muscle of older animals, which result in higher dissociation constants and ultimately reduced enzymatic activity (16). From those findings it was concluded that age-related decreases in GAPDH activity occur solely through post-translational processes. In contrast, our data show that when GAPDH activity is normalized to GAPDH protein levels in the muscles, enzyme activity per se is not affected by age. Therefore, our findings suggest that the decreased enzyme activity is the result of transcriptional modification, and possibly pretranslational and translational modifications as well, especially in muscles containing glycolytic fibers.
In the present study we provide evidence that GAPDH mRNA, a commonly used internal control in mRNA analyses of muscle, is reduced with age in several rat muscles. Internal controls in methods such as Northern blotting, ribonuclease protection assays, and RT-PCR are necessary in order to account for sample to sample variations that occur, for example, during RNA isolation, loading, transferring and blotting, or amplification. An internal control is valid only if it is not affected by the experimental treatment, e.g., GAPDH mRNA levels in muscles must not change as a function of age. Only when this condition is met can it be assumed that variations observed in the internal controls are the result of experimental errors and not due to the experimental treatment. Our data indicate that GAPDH mRNA is not a valid internal control for many experiments designed to analyze mRNA in aging rat muscle.
GAPDH mRNA levels may change with age differently in other experimental animals. Musaro and coworkers (6) did not report a difference in GAPDH mRNA levels in aging mouse muscles. However, they apparently pooled several mouse hindlimb muscles for the mRNA experiments, but statistical analyses to verify that GAPDH mRNA levels did not differ across age groups were not reported. Muscle GAPDH mRNA levels may also change as the result of experimental manipulations. It has been shown that denervation (17), chronic stimulation (18), and overload-induced hypertrophy (19) cause GAPDH mRNA levels in skeletal muscles to decline, whereas hindlimb suspension results in increases (20). However, the occurrence of these changes may depend upon the muscle being studied. For example, Mozdziak and colleagues (7) reported no change in GAPDH mRNA levels in overloaded rat soleus muscle whereas Tsika and colleagues (19) reported a 200% decrease in GAPDH mRNA levels in overloaded rat plantaris muscle.
ß-Actin, -tubulin, and the rRNAs 18S and 28S are other "housekeeping" genes whose transcripts are frequently used as internal controls in mRNA analyses of human, mouse, and rat tissues (Ambion and Clontech of Palo Alto, CA, have probes/primers for these genes). Using Northern blot analyses we have found that levels of 18S and 28S rRNAs do not differ in muscles of adult and senescent rats. However, because of the abundance of these rRNAs they are not optimal internal controls for many experiments. An alternative internal control for mRNA analyses in studies of aging muscle that we have found to be satisfactory is cyclophilin. Cyclophilin is a constitutively expressed gene that encodes a cytosolic protein essential for the correct folding of several proteins. We have analyzed cyclophilin mRNA by Northern blotting, as well as by ribonuclease protection assay and RT-PCR (unpublished results), and found that levels are constant in soleus and plantaris muscles of rats from 4 to 37 months of age. In summary, internal controls for mRNA analyses in skeletal muscles should be selected based upon the experimental manipulations used; cyclophilin may be a good alternative in lieu of the traditional GAPDH.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received March 4, 1999
Accepted August 17, 1999
|
References |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  D. Constantin, D. Constantin-Teodosiu, R. Layfield, K. Tsintzas, A. J. Bennett, and P. L. Greenhaff PPAR{delta} agonism induces a change in fuel metabolism and activation of an atrophy programme, but does not impair mitochondrial function in rat skeletal muscle J. Physiol., August 15, 2007; 583(1): 381 - 390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. H. Mortensen, L. Frandsen, P. Schjerling, E. Nishimura, and N. Grunnet PGC-1{alpha} and PGC-1beta have both similar and distinct effects on myofiber switching toward an oxidative phenotype Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E807 - E816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Li, L. I. Sinoway, and Y.-C. Ng Aging augments interstitial K+ concentrations in active muscle of rats J Appl Physiol, April 1, 2006; 100(4): 1158 - 1163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Y. Kostrominova, D. E. Dow, R. G. Dennis, R. A. Miller, and J. A. Faulkner Comparison of gene expression of 2-mo denervated, 2-mo stimulated-denervated, and control rat skeletal muscles Physiol Genomics, July 14, 2005; 22(2): 227 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Siu, D. A. Donley, R. W. Bryner, and S. E. Alway Myogenin and oxidative enzyme gene expression levels are elevated in rat soleus muscles after endurance training J Appl Physiol, July 1, 2004; 97(1): 277 - 285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Alway, H. Degens, G. Krishnamurthy, and C. A. Smith Potential role for Id myogenic repressors in apoptosis and attenuation of hypertrophy in muscles of aged rats Am J Physiol Cell Physiol, July 1, 2002; 283(1): C66 - C76. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Alway, H. Degens, D. A. Lowe, and G. Krishnamurthy Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2002; 282(2): R411 - R422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. G. Cintado, F. M. Pinto, P. Devillier, A. Merida, and M. L. Candenas Increase in Neurokinin B Expression and in Tachykinin NK3 Receptor-Mediated Response and Expression in the Rat Uterus with Age J. Pharmacol. Exp. Ther., December 1, 2001; 299(3): 934 - 938. [Abstract] [Full Text]
|
|
|
|
|
|
M. Inobe, I. Inobe, G. R. Adams, K. M. Baldwin, and S.'I. Takeda Effects of microgravity on myogenic factor expressions during postnatal development of rat skeletal muscle J Appl Physiol, May 1, 2002; 92(5): 1936 - 1942. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||
| HOME | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|---|
