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J Appl Physiol 99: 2149-2158, 2005. First published July 28, 2005; doi:10.1152/japplphysiol.00513.2005
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Resting and load-induced levels of myogenic gene transcripts differ between older adults with demonstrable sarcopenia and young men and women

Jeong-su Kim,1,2 David J. Kosek,1,2 John K. Petrella,1,2 James M. Cross,3 and Marcas M. Bamman1,2

Departments of 1Physiology and Biophysics and 3Surgery, The University of Alabama at Birmingham, and 2Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center, Birmingham, Alabama

Submitted 3 May 2005 ; accepted in final form 25 July 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Regenerative capacity appears to be impaired in sarcopenic muscle. As local growth factors and myogenic regulatory factors (MRFs) modulate repair/regeneration responses after overload, we hypothesized that resistance loading (RL)-induced expression of MRFs and muscle IGF-I-related genes would be blunted in older (O) males (M) and females (F) with demonstrable sarcopenia vs. young (Y) adults. Y (20–35 yr, 10 YF, 10 YM) and O (60–75 yr, 9 OF, 9 OM) underwent vastus lateralis biopsy before and 24 h after knee extensor RL. Sarcopenia was assessed by cross-sectional area of type I, IIa, and IIx myofibers. Transcript levels were assessed by relative RT-PCR and analyzed by age x gender x load repeated-measures ANOVA. O were sarcopenic based on type II atrophy with smaller type IIa (P < 0.05) and IIx (P < 0.001) myofibers. Within-gender cross-sectional area differences were more marked in F (OF < YF: IIa 21%, IIx 42%). Load effects (P < 0.05) were seen for four of seven mRNAs as IGF-IEa (34%), myogenin (53%), and MyoD (20%) increased, and myf-6 declined 10%. Increased IGF-IEa was driven by O (48%) and/or M (43%). An age x gender x load interaction was found for MyoD (P < 0.05). An age x load interaction for type 1 IGF receptor (P < 0.05) was driven by a small increase in O (16%, P < 0.05). A gender x load interaction (P < 0.05) was noted for IGF binding protein-4. Age effects (P < 0.05) resulted from higher MyoD (54%), myf-5 (21%), and IGF binding protein-4 (17%) in O and were primarily localized to F at baseline (OF > YF; MyoD 94%, myf-5 47%, P < 0.05). We conclude that RL acutely increases mRNA expression of IGF-IEa and myogenin, which may promote growth/regeneration in both Y and O. Higher resting levels of MRFs in OF vs. YF suggest elevated basal regenerative activity in sarcopenic muscle of OF.

sarcopenia; myogenic regulatory factors; insulin-like growth factor-I; resistance exercise

THE AGE-RELATED GRADUAL LOSS of skeletal muscle mass (sarcopenia) leads to declines in strength and power, which contribute to falls and impaired mobility in the elderly (reviewed in Refs. 27, 36). Aged muscle is more susceptible to contraction-induced injury (34, 50) and regenerates or recovers more slowly (14, 44). Loss of myofiber integrity and general weakness, along with impaired regenerative capacity, are thought to be primary causes of age-related myofiber atrophy in association with altered function of muscle progenitor (satellite) cells (22). Intermittent mechanical loading via resistance exercise training increases muscle size and strength; however, the efficacy of this type of training in older adults may be influenced by age-related changes in the adaptive capabilities of muscle.

Activation of nominally quiescent satellite cells is integral to the processes of repair or regeneration and growth in adult skeletal muscle (2, 31). These processes are modulated by a variety of local factors, including the family of transcription factors known as myogenic regulatory factors (MRFs) (MyoD, myf-5, myogenin, and myf-6) (35, 42). MRFs induce myoblast differentiation and regulate transcription of numerous muscle-specific genes [e.g., nicotinic acetylcholine receptor, {beta}-tropomyosin, myosin heavy chain (MHC), desmin, troponin C, creatine kinase] (10, 15, 16). Paracrine/autocrine growth factors modulate proliferative responses of satellite cells to local stimuli; among them, insulin-like growth factor (IGF)-IEa and/or its muscle-specific isoform, mechano-growth factor (MGF = IGF-IEc), has received significant attention based on the findings that IGF-I stimulates not only proliferation, but also differentiation and fusion, as satellite cells donate nuclei to myofibers undergoing growth and/or repair (1, 23). Acute resistance loading (RL) has been shown to induce mRNA expression of MRFs (41) and IGF-I isoforms (5, 26) in humans. We have recently demonstrated that acute RL markedly upregulates MGF mRNA expression and simultaneously downregulates levels of inhibitory factors, such as myostatin, which impairs satellite cell proliferation and differentiation, and p27kip, which inhibits early (G1-S) cell cycle progression (33). However, these responses appeared to be most favorable in young men (33). Several investigators have shown that the muscles of young men acutely respond to mechanical load with upregulation of factors thought to be important in the hypertrophic process (11, 26, 33, 41, 47, 48). On the other hand, it has been shown in both rats (44) and humans (26) that myogenic responses to a single bout of RL are reduced in aged muscle. The efficacy of long-term resistance training is not universal, as the hypertrophy adaptation appears to be influenced by age and/or gender (6, 18, 25, 27, 28, 45). We speculate that age differences in the acute responses (of local growth factors and/or myogenic transcription factors) to RL may partially account for a limited hypertrophy adaptation in sarcopenic muscle.

Therefore, the aims of the present study were to test the hypotheses that 1) acute RL using a regimen known to induce myofiber hypertrophy when performed 2–3 days/wk for several weeks (3 sets x 8–12 repetitions to volitional fatigue of squat, leg press, and knee extension) would upregulate mRNA expression of muscle IGF-IEa and MRFs (MyoD, myogenin, myf-5, myf-6); and 2) load-mediated responses would be blunted in older adults with demonstrable sarcopenia vs. younger men and women. We studied additional transcripts involved in local IGF-I regulation, including its primary receptor (type I or IGF-R1) and tissue binding protein (IGFBP-4) to further assess the influence of RL on the autocrine/paracrine IGF-I system. MGF mRNA results in these subjects were published previously (33). Expression levels of gene transcripts were determined by relative RT-PCR using 18S ribosomal RNA as an internal standard, and myofiber size measurements were assessed by immunofluorescence microscopy using antibodies against MHC isoforms.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects.   We published a detailed description of the subjects in a recent report (33). Briefly, 38 healthy, nonresistance-trained adults were recruited into two age groups, with age ranges of 60–75 yr for the older group (9 males, OM; 9 females, OF) and 20–35 yr for the younger group (10 males, YM; 10 females, YF). All subjects underwent a detailed health history appraisal, and all older subjects passed a comprehensive physical exam and a diagnostic graded treadmill stress test conducted by a geriatrician and a cardiologist, respectively. Subjects with obesity (body mass index > 30 kg/m2), any musculoskeletal disorder, or any leg resistance training experience within the past 5 yr were excluded. The study was approved by the Institutional Review Boards of both the University of Alabama at Birmingham and the Birmingham Veterans Affairs Medical Center. Written, informed consent was obtained before participation in the research.

Resistance loading stimulus.   To study acute myogenic responses to loading, we have adopted a RL stimulus typical of hypertrophy programs when performed two to three times per week for several weeks (7, 33). The "stimulus" consisted of three sets x 8–12 repetitions to volitional fatigue for each of three movements that load the knee extensors bilaterally (squat, leg press, knee extension). The effect of the stimulus was assessed in vastus lateralis biopsies obtained 24 h after the loading bout. Before this bout, subjects underwent a preexercise vastus lateralis muscle biopsy and were exposed to the exercises on four occasions (on alternate days) consisting of 1) an introduction and familiarization; 2) a second familiarization, including practice one-repetition maximum (1-RM) strength tests; 3) 1-RM assessment followed by one set x 8–12 repetitions of each exercise performed at 70% of 1 RM; and 4) two sets x 8–12 repetitions to volitional fatigue. All sets were separated by 90-s rest intervals. This progressive protocol was designed to prepare subjects for the full loading stimulus performed during the fifth exposure to the resistance exercises.

Muscular power and fatigability.   Our tests of power and fatigue are unique in that the primary dependent variable of interest is concentric velocity (external load is held constant). Our laboratory detailed these methods recently (40). Both power and fatigability were determined at a load equivalent to 40% of maximum isometric voluntary contraction (MVC) force. Because strength and maximum velocity both decrease with aging, age-related declines in muscle power are of greater magnitude than declines in strength alone (9, 13, 43). Using a constant external load that is consistent across individuals as the same percentage of MVC force enabled us to study age differences in the velocity-specific component of power.

Bilateral MVC was measured first while seated on the constant-load dynamometer by using a calibrated load cell (attached in lieu of the weight stack cable) at a knee angle of 0.942 rad (54 ± 1°) of flexion, as measured by electrogoniometry (model SG150, Biometrics, Gwent, UK). After practice attempts, MVC was accepted as the peak force obtained in two trials. Contractions were 5 s in duration and were separated by 60-s rest intervals. The weight stack load providing a resistance force equal to 40% of MVC force was determined by using a load cell-derived force regression equation for the dynamometer’s weight stack (R2 = 0.993). To assess concentric power, subjects completed three full-range-of-motion knee extension contractions by performing the concentric phase as rapidly as possible, while the eccentric phase was controlled. Knee electrogoniometry was used to measure the time interval from 0.873 rad (50°) to 0.349 rad (20°) of knee flexion during the concentric phase of each repetition (knee angle sampling rate = 500 Hz). Work (J), angular velocity (rad/s), and power (W) were determined as previously described. (40). The repetition yielding peak power was used for analysis. Power results were adjusted for thigh lean mass (TLM) to yield specific power. Fatigability was tested in a separate trial (after 2–3 min of rest) using the same external load (40% of MVC force). Ten repetitions were performed in succession. As in the power test, each concentric phase was performed as rapidly as possible followed by a controlled eccentric phase. Velocity, work, and power were computed for each repetition as described above. We define fatigue as a decline in maximum concentric velocity from the fastest repetition (typically repetition 2 or 3) to the tenth repetition.

Body composition.   Dual-energy X-ray absorptiometry (DEXA) with a Lunar Prodigy (model no. 8743, GE Lunar, Madison, WI) was used to determine TLM, total body lean mass, and percent body fat. Analyses were conducted according to manufacturer’s instructions using enCORE 2002 software (version 6.10.029). All but one YM completed a DEXA scan (n = 37 for DEXA analyses).

Muscle biopsy and tissue preparation procedures.   All muscle biopsies were performed in the Pittman General Clinical Research Center at the University of Alabama at Birmingham. Muscle samples were collected from vastus lateralis muscle by percutaneous needle biopsy using a 5-mm Bergstrom biopsy needle under suction, as previously described (21). The baseline biopsy was taken from the left leg, and the postexercise biopsy was taken from the right leg 24 h after the bilateral loading bout to avoid any residual effects. At the bedside, visible connective and adipose tissues were removed with the aid of a dissecting microscope. A portion of the sample to be used for RNA isolation was immediately weighed and snap-frozen in liquid nitrogen. A separate portion for immunohistochemistry was mounted cross sectionally on cork in optimum cutting temperature mounting medium mixed with tracaganth gum and frozen in liquid nitrogen-cooled isopentane. All samples were stored at –80°C until analyses.

Total RNA isolation.   The procedure of RNA isolation has been described in detail previously (33). Briefly, frozen muscle samples (average 35 mg) were homogenized, and total RNA was extracted by using the TRI Reagent (Molecular Research Center, Cincinnati, OH), followed by precipitation with isopropanol, two ethanol washes, drying, and suspension in nuclease-free water at a ratio of 0.8 µl/mg muscle. Fluorometric analysis (TD-700, Turner Designs, Sunnyvale, CA) was performed to determine RNA concentration using the RiboGreen RNA Quantitation Kit (Molecular Probes, Eugene, OR), as described previously (33). RNA samples were stored at –80°C.

RT-PCR.   As described (33), 1 µg of RNA was reverse transcribed in a total volume of 20 µl using SuperScript II Reverse Transcriptase (Invitrogen, GIBCO-BRL) with a mix of oligo(dT) (100 ng/reaction) and random primers (200 ng/reaction). After 50-min incubation at 45°C, the RT reaction mixtures were heated at 90°C for 5 min to discontinue the reaction and then stored at –80°C for subsequent PCR analyses.

A relative RT-PCR method using 18S ribosomal RNA as an internal standard (Invitrogen, Life Technology, GIBCO-BRL, Carlsbad, CA) was used to determine relative expression levels of mRNAs for MyoD, myf-5, myogenin, myf-6, IGF-IEa, IGF-R1, and IGFBP-4. Table 1 indicates the primer sequences for the specific target mRNAs. Primers were designed using the Primer Select computer program (DNAStar, Madison, WI) and custom-made by Invitrogen (GIBCO). Interaction tests between target mRNA primers and the alternate 18S primers were conducted, and primer sets were selected for use after confirming no interaction.


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  		Table 1. Sequences of the specific sets of primers used

 
For each PCR reaction, 18S (with a 324-bp product) was coamplified with each target cDNA (mRNA) to express each as a ratio of target mRNA/18S. The 18S primers were mixed with competimers to ensure that 18S and each target mRNA coamplified in the linear range. The ratio of this primer-to-competimer mixture was optimized in preliminary experiments and ranged from 1:15 to 1:50, depending on the abundance of the target mRNA. A representative linearity test to determine PCR cycle number is shown in Fig. 1. In this myf-5 linearity test, 34 was chosen as the optimum number of cycles as 18S and myf-5 levels deviated from linearity past 39 and 37 cycles, respectively (not shown).



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  		Fig. 1. Validation of PCR cycle number. A representative linearity test was done for the coamplification of 18S and Myf-5 mRNA products as a function of PCR cycle number. Regression analyses show that amplification of each product was highly linear across this range of 31–37 cycles. The order of PCR products on the image corresponds with the order of PCR cycle numbers in the scatterplots below.

 
The conditions of both RT and PCR reactions were standardized by using the same premixed reagents for all 76 samples to be compared. Based on the number of PCR reactions, a PCR premix was prepared, as described previously (33). For each PCR, 1 µl of RT product (cDNA) was added into 24 µl of premix and topped with 50 µl of mineral oil (Sigma-Aldrich, St. Louis, MO). PCR was carried out in a DNA Engine (PTC-200) Peltier Thermal Cycler (MJ Research, Waltham, MA) with an initial denaturing step of 3 min at 96°C, followed by specific cycles (29–34 cycles, depending on the results of linearity tests for each target mRNA and 18S) of 1 min at 96°C, 45 s at specific annealing temperatures (57–62°C, depending on primers), 45 s at 75°C, and a final step of 3 min at 72°C. Immediately following PCR, 25 µl of PCR product (22 µl of the reaction mixture diluted with 3 µl of loading buffer) were separated by electrophoresis (100-V constant) in a 2% agarose gel for 1.5 or 2 h [depending on the band separation between 18S (324 bp) and the specific target gene due to the size of each mRNA product]. Gels were run with molecular weight markers (100-bp Hyper Ladder IV, Genesee Scientific, San Diego, CA) to confirm the expected size of each mRNA. To eliminate age group, gender, or RL bias, each 20-well gel contained pre-post paired samples for subjects within each group (i.e., YM, YF, OM, OF) with the different subject groups loaded in random order on each gel. Ethidium bromide (0.1 µg/ml) was premixed in the 2% agarose gel, and images were captured under UV in a BioRad ChemiDoc imaging system (Hercules, CA). Band densitometry was performed by using BioRad Quantity One software. Parameters for image development were described in detail previously (33).

Immunofluorescence microscopy.   Muscle mounts were cut in 6-µm serial cross sections at –22°C using a Leica CM1900 cryostat microtome and placed on three-well black mask plus slides (Erie Scientific). Slides were kept in a humidified chamber throughout the staining protocol. All applied solutions were removed by aspiration before administration of the succeeding solution. Sections were fixed for 45 min at room temperature in 3% neutral-buffered formalin. Following fixation, sections were washed for 2 x 5 min with 1x PBS (all subsequent PBS wash steps were 3 x 5 min). Sections were blocked with 5% goat serum in PBS for 20 min at room temperature followed by a wash step. Primary and secondary antibodies were diluted in 1% goat serum in PBS. Anti-MHC type I (anti-MHC I) primary antibody (Ab) (mouse MAb NCL-MHCs, NovoCastra Laboratories, 1:100) was applied for 30 min at 37°C. After a wash step, sections were incubated with ALEXA 594-conjugated goat anti-mouse secondary Ab (Pierce Biotechnologies, 1:200) for 30 min at 37°C. Sections were washed and again blocked (5% goat serum in PBS) for 20 min at room temperature. To locate sarcolemmae for myofiber sizing, a wash step was followed by incubation with anti-laminin mouse MAb (VP-L551, NovoCastra Laboratories, 1:80) for 30 min at 37°C, a wash step, and incubation with ALEXA 488-conjugated goat anti-mouse secondary Ab (Pierce Biotechnologies, 1:200) for 30 min at 37°C. Slides were then washed and subjected to a third and final block (5% goat serum in PBS) for 20 min at room temperature. After a wash step, sections were incubated with the final primary Ab (anti-MHC IIa mouse MAb, University of Iowa Hybridoma Bank, 1:80) for 30 min at 37°C, washed, and incubated with ALEXA 488-conjugated goat anti-mouse secondary Ab (Pierce Biotechnologies, 1:200) for 30 min at 37°C. Nuclei were revealed by a Hoecsht 33258 DNA counterstain (Molecular Probes, 1:10,000 in PBS) for 2 min at room temperature. Slides underwent a final aspiration and were mounted with 1% paraphenylene diamine, and 90% glycerol in PBS. Slides and coverslips were bound together by use of nail polish and stored protected from light at –20°C.

High-resolution (48-bit TIFF) images were captured at x10 and x20 using an Olympus MagnaFire SP camera (S99810) and software online with an Olympus BX51 fluorescent microscope. Image analysis was performed by using Image-Pro Plus 5.0 software. All analyses were conducted by a single analyst blinded to age and gender of each sample. Myofiber-type distribution was determined from 935 ± 47 myofibers per sample. We confirmed MHC isoform specificities of these MAbs by Western blot (not shown). Myofibers positive for MHC I and negative for MHC IIa were classified as type I, fibers positive for MHC IIa and negative for MHC I were classified as type IIa, and fibers negative for both MHC I and MHC IIa were classified as type IIx. With this technique, hybrid myofibers (e.g., coexpression of I/IIa or IIa/IIx) are revealed by both color and intensity. Myofibers coexpressing more than one MHC isoform were excluded from analyses. For cross-sectional area (CSA) measurements, each myofiber was manually traced along its laminin-stained border. CSA (in µm2) was calibrated by using a stage micrometer, and only those fibers determined to be cross sectional based on a roundness factor <1.639 were included in the analysis (roundness = perimeter2/4{pi} area; perfect circle = 1.0, pentagon 1.163, square 1.266, equilateral triangle 1.639). The number of randomly selected myofibers by type included in CSA analyses were 60 ± 2 type I, 60 ± 2 type IIa, and 50 ± 2 type IIx. Five subjects did not have sufficient type IIx distribution to obtain IIx CSA.

Statistical analysis.   Data are reported as means ± SE. Between-group differences in preexercise descriptive variables were tested by using age x gender ANOVA. All variables measured before and after loading were analyzed by using age x gender x load repeated-measures ANOVA. For each ANOVA model with a significant main or interaction effect, Tukey honestly significant difference tests were performed post hoc to localize the effect(s). Zero-order correlations were tested between descriptive variables [myofiber size by type, myofiber-type distribution, lean body mass, TLM] and resting levels, as well as load-mediated changes, for each transcript studied. Statistical significance was accepted at P < 0.05 for all tests.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The four age/gender groups are defined as YF (young females), YM (young males), OF (older females), and OM (older males) on Figs. 1–4 and throughout the remaining text. A detailed description of these subjects first appeared in our previous report on myostatin expression and cell cycle regulation (33), and descriptive characteristics such as age, height, weight, and body composition are shown again in this report for clarity (Table 2). The new findings presented in Table 2 include myofiber size and distribution data. These results confirm that the older adults were in fact sarcopenic, based on the hallmark attribute of type II myofiber atrophy. Type I myofiber size and distribution did not differ by age or gender. Main age and gender effects were found for type IIa myofiber size, and post hoc tests revealed that these fibers were 18% smaller in older muscle (P < 0.05), 27% smaller in women (P < 0.001), and a marked 39% smaller in OF compared with YM (P < 0.001). Age differences in type IIx myofiber size were even greater in magnitude. Type IIx myofibers were 35% smaller in older adults (P < 0.001) and 37% smaller in women (P < 0.001). Post hoc tests showed that OF possessed significantly smaller type IIx myofibers than each of the other three groups, whereas type IIx myofiber size in YM was greater than in each of the remaining three groups (P < 0.05 all). Type IIx myofibers were 60% smaller in OF compared with YM. Within-gender differences were significant, as type IIx myofibers were 42% smaller in OF vs. YF, and 28% smaller in OM vs. YM (P < 0.05). A main age effect and age x gender interaction were found for type IIa myofiber distribution, which was higher in OM than in YM (P < 0.05).



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  		Fig. 4. Relative RT-PCR results for IGF-IEa (A), IGF-R1 (B), and IGF binding protein (BP)-4 (C) mRNA expression using 18S ribosomal RNA as an internal standard. In each panel, the image above the histogram displays an example of pre- and postloading mRNA expression levels for an individual subject within each group (YM, YF, OM, OF) run on the same gel. The order of samples on the image corresponds with the order of bars in the histogram below. A: levels of IGF-IEa mRNA were elevated (34%) 24 h after acute resistance loading (main loading effect, P < 0.005). B: for IGF-R1 mRNA levels, an age x load interaction revealed an increase exclusively in older adults (16%, P < 0.05). C: a main age effect (P < 0.01) for IGFBP-4 mRNA expression was driven by 30% higher levels in OF vs. YF. A gender x load interaction (P < 0.05) suggested slightly reduced levels of IGFBP-4 in women after loading. Values are means ± SE.

 

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  		Table 2. Summary of descriptive characteristics and age differences identified

 
Knee extensor specific strength and specific power were both lower in older vs. young adults (P < 0.05), and older adults fatigued more rapidly than the young (P < 0.05) (Table 2). Absolute power was significantly related to type IIa (r = 0.69, P < 0.001) and type IIx (r = 0.82, P < 0.001) CSAs and to the area distribution of type IIx myofibers (r = 0.62, P < 0.001). Power production, even after adjusting for muscle mass (i.e., specific power), was significantly related to the CSAs of type IIa (r = 0.48, P < 0.01) and type IIx (r = 0.53, P < 0.01) myofibers.

The results of specific transcript expression analyses are shown in Figs. 2–4. Main load effects (P < 0.05) were found for four of seven transcripts as mRNA levels increased after loading for myogenin (53%, Fig. 2A), MyoD (20%, Fig. 3A), and IGF-IEa (34%, Fig. 4A) and decreased for myf-6 (–10%, Fig. 2B). Age effects (P < 0.05) resulted from higher mRNA expression of MyoD (54%, Fig. 3A), myf-5 (21%, Fig. 3B), and IGFBP-4 (17%, Fig. 4C) in older vs. young adults.



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  		Fig. 2. Relative RT-PCR results for myogenin (A) and Myf-6 (B) mRNA expression using 18S ribosomal RNA as an internal standard. In each panel, the image above the histogram displays an example of pre- and postloading mRNA expression levels for an individual subject within each group (young males, YM; young females, YF; older males, OM; and older females, OF) run on the same gel. The order of samples on the image corresponds with the order of bars in the histogram below. A: an increase in myogenin mRNA levels (53%) was found 24 h after acute resistance loading (main loading effect, P < 0.001). Pre-post loading changes within groups were noted in YM (P < 0.001) and YF (P < 0.01) only. B: a small but significant reduction in myf-6 mRNA expression (–10%) was found (main loading effect, P < 0.05). Values are means ± SE.

 


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  		Fig. 3. Relative RT-PCR results for MyoD (A) and Myf-5 (B) mRNA expression using 18S ribosomal RNA as an internal standard. In each panel, the image above the histogram displays an example of pre- and postloading mRNA expression levels for an individual subject within each group (YM, YF, OM, OF) run on the same gel. The order of samples on the image corresponds with the order of bars in the histogram below. A: levels of MyoD mRNA expression increased (20%) 24 h after acute resistance loading (main loading effect, P < 0.05). Older adults expressed 54% more MyoD mRNA than young (main age effect, P < 0.05), driven by low levels in YF. An age x gender x load interaction for MyoD mRNA (P < 0.05) was caused by increases in OM and YF. B: levels of myf-5 mRNA were not altered by resistance loading. Older adults expressed 21% more myf-5 mRNA than young (main age effect, P < 0.05) as a result of age differences in women. Values are means ± SE.

 
Myogenin and myf-6 mRNA results are shown in Fig. 2, A and B, respectively. While acute RL resulted in a small but significant 10% reduction in myf-6 mRNA expression (Fig. 2B) (P < 0.05), myogenin mRNA levels (Fig. 2A) were elevated by 53% after RL (P < 0.00001). For myogenin, a trend toward age x load interaction (P = 0.069) suggested higher load-mediated increases in young (63%) vs. old (40%). Results for MyoD and myf-5 mRNAs are shown in Fig. 3, A and B, respectively. Resting MyoD levels (Fig. 3A) were considerably low in YF vs. the other three groups. This was a surprising but fairly consistent finding among YF. This resulted in significantly higher MyoD levels in the older group (54%, P < 0.05). A main loading effect indicated that levels of MyoD mRNA expression increased by 20% (P < 0.05) 24 h after acute RL. However, this was exclusive to YF and OM, leading to an age x gender x load interaction (P < 0.05). Levels of myf-5 mRNA were not altered by RL, but an age effect indicated 21% higher myf-5 in older vs. younger muscles (Fig. 3B). This age difference was primarily driven by women, as YF tended to express lower levels of myf-5 than the other three groups, and the age difference between the two groups of women was significant (47% greater expression in OF vs. YF, P < 0.05). An apparent age x gender interaction (P = 0.051) for myf-5 was also driven by the YF.

Myogenin and MyoD may be preferentially expressed in type I and type II myofibers, respectively, with MyoD expression being most closely associated with the IIx phenotype (20, 37). We found no relationship between type I myofiber distribution and myogenin expression but did note a modest relationship between type I myofiber size and resting myogenin mRNA levels; however, type I CSA accounted for only 14% of the variance in resting myogenin mRNA (r = 0.376 or r2 = 0.14, P < 0.05). Conversely, the distribution of type IIx myofibers accounted for a small but significant proportion of the variance (13%) in resting MyoD mRNA levels (r = 0.363or r2 = 0.13, P < 0.05). MyoD expression was not related to any other measure of type II muscle.

The results of load-induced changes in components of the local tissue IGF-I system are shown in Fig. 4. After acute RL, levels of IGF-IEa mRNA (Fig. 4A) expression were increased 34% (P < 0.005). Based on a post hoc analysis, RL-mediated increases in IGF-IEa were mainly led by older adults (+48%, P < 0.05) and/or men (+43%, P < 0.05). Interestingly, the magnitude of RL-mediated change in IGF-IEa expression was inversely related to type IIx myofiber size (r = –0.40, P < 0.05). An age x load interaction for IGF-R1 mRNA expression (Fig. 4B) revealed a small, but significant increase exclusively in older adults (16%, P < 0.05). Basal expression of IGF-R1 was inversely related to the sizes of type IIa (r = –0.41, P < 0.05) and type IIx (r = –0.45, P < 0.05) myofibers. Although no RL-induced changes (P > 0.05) were detected in IGFBP-4 mRNA levels (Fig. 4C), OM and OF expressed 17% more IGFBP-4 than young subjects (P < 0.01), and OF expressed 30% more than YF (P < 0.01). A gender x load interaction (P < 0.05) was found in the levels of IGFBP-4 mRNA expression, as IGFBP-4 tended to decrease only in women after RL.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In the present investigation, our first aim was to test the hypothesis that acute RL would upregulate mRNA expression of growth and/or myogenic factors, and our second aim was to determine whether these RL-mediated responses would be blunted in older adults with demonstrable sarcopenia (type II myofiber atrophy). Overall, we found that acute RL induced marked upregulation of myogenin and IGF-IEa levels, whereas a significant but less impressive increase was noted for MyoD. Based on average percent changes, the myogenin response appeared to be most prominent in YM and lowest in OF (YM 80%, YF 50%, OM 52%, OF 31%), leading to a strong trend toward age x load interaction (P = 0.069). The IGF-IEa response was blunted in YF and highest in OF (YM 45%, YF 7%, OM 41%, OF 56%). On the other hand, load-mediated increases in MyoD expression were most notable in YF (110%) and OM (34%), leading to the significant age x gender x load interaction.

Effects of acute RL on MRFs.   MRFs (MyoD, myf-5, myogenin, and myf-6) analyzed in the present study are well-known markers of myoblast/satellite cell differentiation in intact adult muscle and modulate the transcription of muscle-specific genes (10, 15, 16, 32, 35, 38, 42). Psilander and colleagues (41) demonstrated that mRNA levels of myogenin, MyoD, and myf-6 were significantly upregulated after a single bout of heavy RL in YM. Quantitative PCR analyses in rats have also revealed marked elevations after a bout of eccentric exercise (39). RL-mediated elevations of myogenin and MyoD suggest induction of myogenesis involving satellite cell activation (32, 35, 42). We found robust increases in myogenin mRNA after mechanical load but only a minor induction of MyoD. Differences between the current findings and those of Psilander et al. (41) may have resulted from differences in the timing of postloading muscle biopsy. Our analyses were limited to the 24-h time point, whereas Psilander et al. tracked changes serially (in YM only), beginning immediately after the loading bout, and found that dynamic changes in expression occurred very early for some transcripts (e.g., MyoD).

Sabourin and Rudnicki (42) have previously proposed that MRFs are divided into two groups, suggesting that MyoD and myf-5 are primary MRFs required for the commitment of proliferating somitic cells to the myogenic lineage (i.e., during the determination step), and myogenin and myf-6 are secondary MRFs required for terminal differentiation of myoblasts into myocytes and multinucleated myotubes during differentiation. This model suggests little to no redundancy in function among these two classes of MRFs. Kassar-Duchossoy et al. (32), however, recently provided evidence of redundancy in function by showing that myf-6 can act at the determination step, as skeletal muscle does develop in Myf-5/MyoD double-null mice, if myf-6 expression is not inhibited. Whether or not redundancy occurs, it is clear from a number of studies that MRFs are required for myogenesis during development, and it is likely that MRF upregulation following loading in adult muscle facilitates repair or regeneration and growth of mature myofibers. For example, after a protocol of eccentric treadmill running, mouse soleus was found to exhibit clusters of mononucleated myf-5/MyoD positive cells, suggesting regeneration via satellite cell activation (4). Our data indicate that myogenin mRNA levels are enhanced markedly by RL and that this effect is somewhat blunted in older sarcopenic muscle. Changes in MyoD 24-h post-RL were less impressive, but we do not have the tissue samples to determine whether MyoD or other factors responded at earlier time points postloading.

Effects of acute RL on muscle IGF-I and associated transcripts.   Satellite cell activity is also mediated by IGF-I, which is thought to exist in at least two primary isoforms in skeletal muscle: systemic or liver form (IGF-IEa) and a splice variant found in muscle and termed MGF (MGF = IGF-IEc) (23). IGF-I binding to IGF-R1 activates the insulin receptor substrate-1-phosphatidylinositol 3-kinase-p70S6 kinase pathway, leading to translation initiation/protein synthesis and enhanced cell differentiation, and activates the Ras-MAPK-ERK pathway, leading to increased cell proliferation (1). IGFBP-4 has a high affinity for IGF-I, and, therefore, a high concentration of IGFBP-4 in muscle would be expected to inhibit ligand binding to IGF-R1 (19). Load-mediated increases in muscle IGF-I and/or IGF-R1 with a concomitant reduction in IGFBP-4 would theoretically enhance IGF-R1 activation. We have previously shown that IGF-I mRNA is increased, and IGFBP-4 mRNA is reduced 48 h after a single bout of heavy eccentric RL in young subjects (5). In the present study, the elevations in IGF-IEa mRNA (all but YF) and IGF-R1 mRNA (older adults only) suggest upregulation of local IGF-I action following acute RL. Interestingly, our previous observation of enhanced MGF expression at this 24-h time point was most robust in YM (33), which supports other work (26), while the IGF-IEa response reported herein was quite strong in OM and OF. Increased IGF-IEa mRNA after RL may aid in overcoming any inhibitory effects of IGFBP-4, particularly in these older adults who expressed IGFBP-4 levels that were significantly higher than in young subjects. Haddad and colleagues (24) recently demonstrated upregulation of both IGF-I and IGFBP-4 mRNA expression in response to 5 wk of unilateral knee extensor resistance training, while IGF-R1 was not altered.

Age differences in resting and regenerating muscle.   For three transcripts (MyoD, myf-5, IGFBP-4), resting levels were highest among OF and lowest in YF, leading to significant overall age group differences. Similarly, our laboratory has also noted greater resting myogenin protein concentration in older vs. young adults (7). Our findings of higher resting myogenic gene expression levels in older muscles are supported by recent human (26) and rodent (22) studies. Our laboratory has previously suggested that such age differences may be indicative of a failing compensatory effort during gradually progressive degeneration and denervation processes in aging muscles undergoing sarcopenia (7). Our myofiber size results support this concept, as OF appeared to be more sarcopenic than OM based on within-gender differences in the magnitudes of age-related type IIa and type IIx atrophy. Heightened levels of expression and/or activity at rest within pathways thought to be important for myogenesis may also lead to a blunted regeneration/growth potential on stimulation by mechanical load (22, 26, 29, 30, 46). For example, the load-mediated increase in myogenin mRNA was more than 2.5-fold higher in YM vs. OF, and we reported similar findings for MGF expression recently (33). Reduced regenerative capacity has been reported in skeletal muscle of old rats following a single-loading bout, manifested as blunted MyoD protein and gene expression responses, lower amino acid uptake, and blunted mitotic activity in aged muscle after acute loading (44). These combined results suggest that age-related sarcopenia is partially due to impaired regenerative capacity during muscle utilization (22, 44).

Exogenous IGF-I and/or a stimulus sufficient to increase local, endogenous tissue IGF-I production may be therapeutic in older muscle to serve as a catalyst for satellite cell activation. Viral-mediated IGF-I expression has been shown to promote ~15% increases in muscle mass and strength in young adult mice and a 27% increase in strength in old mice (8). We found a rather marked load-mediated increase in IGF-IEa, a slight increase in IGF-R1 mRNA among older individuals, and a drop in IGFBP-4 expression in OF, suggesting that RL may be an effective means of enhancing endogenous IGF-I availability. Whether this leads to enhanced IGF-I action (e.g., increased protein synthesis and satellite cell activation) remains in question.

Time course of molecular responses following different loading stimuli.   Previous reports on transcriptional responses of growth and/or myogenic factors to acute loading are quite variable. Responses of these genes appear to vary by the different exercise protocols and/or biopsy time points tested. The timing of postloading muscle biopsy is critical in these types of studies. The 24-h window studied in the present investigation revealed several time effects at the mRNA level, but it is clearly too early to detect changes in most proteins (7). Yang et al. (49) recently demonstrated that peaks of myogenic gene induction were varied and generally occurred 4–8 h after a single bout of RL and, by 24-h expression levels, returned to preexercise levels. We detected significant elevations in four transcripts at 24 h, which may have resulted from a higher loading stimulus (9 sets at 80% 1 RM) compared with Yang’s protocol (3 sets at 70% 1 RM). Psilander et al. (41) conducted an exercise protocol involving eight sets of 8–12 repetitions, and biopsy samples were collected serially (pre- and 0, 1, 2, 6, 24, and 48 h postexercise). They observed peak mRNA responses of MRFs at 0–6 h postexercise. Others have also noted increases in MRF (MyoD, myogenin) mRNAs within 6 h postexercise (47). Psilander et al. (41), however, also reported a marked downregulation of IGF-IEa mRNA following a bout of RL in young men and suggested that this may be a phenomenon during the initial part of recovery from resistance exercise. Both we (33) and Hameed et al. (26) have observed elevated MGF mRNA expression after loading. However, in contrast to our present data, Hameed et al. reported no significant load-induced effect on MyoD and IGF-IEa mRNA in biopsies collected 2.5 h after 10 sets of unilateral knee extension at 80% 1 RM (26). Furthermore, Bickel et al. (12) demonstrated in nine men and women that a single bout of intensive isometric contractions induced by neuromuscular electrical stimulation upregulated levels of MyoD, myogenin, cyclin D1, and IGFBP-4 mRNAs but decreased IGF-I expression and did not alter MGF and IGF-R1 mRNA levels. The peak induction of MRF mRNAs was observed 12–24 h after stimulation. It is clear that not only volume and/or intensity of exercise, but also the specific mode of exercise plays an important role in determining loading responses, which further depend on the optimal biopsy time point(s) to capture these events. These protocol variations all contribute to differences among these studies, making it difficult to compare findings. The selected biopsy time point(s) is a potential limitation of this and other studies of age differences in acute gene expression responses. For example, it is entirely possible that changes in the levels of some transcripts followed a different (e.g., prolonged) time course in older adults, which we were not able to capture with a single biopsy 24 h postloading.

Obviously, the aim of these types of studies is to characterize load-mediated changes in molecular markers of myogenic processes, with the underlying assumption that these molecular events may shed light on mechanisms of regeneration and eventual myofiber growth with repetitive loading. Direct measures of satellite cell activation in these human acute loading studies are rare. However, one such study was recently published (17). Crameri et al. (17) assessed satellite cell activation by immunohistochemical staining of mononuclear cells positive for neural cell adhesion molecule and fetal antigen 1 and found increased numbers of these cells 4 and 8 days after a single, high-intensity bout of knee extensor exercise. These findings were interpreted as increased satellite cell proliferation. However, expression levels of myogenin and neonatal MHC were unchanged, suggesting the single-exercise bout did not lead to terminal differentiation. Although it is generally accepted that IGF-I promotes satellite cell activation and muscle hypertrophy, its requirement is in question based on recent data in rodents. Adams et al. (3) found, using an electrical stimulation model of overload in rats, that, while IGF-I mRNA levels were increased by isometric and concentric but not eccentric exercise training, all three training modes resulted in similar increases in muscle mass.

In conclusion, the findings of the present study clearly demonstrate that our acute RL model upregulated transcriptional levels of growth and myogenic factors in muscles of both young and older humans. Elevated levels of myogenic transcripts (MyoD and myf-5) in aged muscle, especially in OF vs. YF, support the concept of an ongoing degeneration/regeneration process in muscles undergoing sarcopenia. Elevated levels of IGFBP-4 in aged human muscle, especially in OF, may limit actions of IGF-IEa in non-overloaded muscle. However, a greater load-mediated induction of IGF-IEa and IGF-R1 mRNA expression in aged muscle (vs. young adults) may facilitate activity of the muscle IGF-I system to promote regeneration/growth. Future studies should determine, by direct assessment, if indeed satellite cells are activated by acute loading in aging human muscle.


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 ABSTRACT
 METHODS
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 DISCUSSION
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Funding for this work was provided by National Institute on Aging Grant R01 AG-17896 (M. M. Bamman) and General Clinical Research Center Grant M01 RR-00032.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We are indebted to the research subjects for invaluable contributions to this work. We thank S. C. Tuggle and S. Hall for administering the RL bouts and strength tests, and V. Hill for efforts in subject recruitment.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. M. Bamman, UAB Dept. of Physiology and Biophysics, Muscle Research Laboratory, GRECC/11G, Veterans Affairs Medical Center, 1530 3rdAve. South, Birmingham, AL 35294–0001 (e-mail: mbamman{at}uab.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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