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Human Performance Research Center, Brigham Young University, Provo, Utah
Submitted 20 December 2005 ; accepted in final form 26 January 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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myosin heavy chain; strength training
Desmin is a 52-kDa cytoskeletal protein that connects myofibrils at their Z lines and connects myofibrils to the sarcolemma through costameres. It also integrates the myofibrils with nuclei, mitochondria, and microtubules (24). As the predominant protein of the intermediate-filament system, desmin is largely responsible for the lateral transmission of force from contracting sarcomeres to the muscle exterior (8, 13, 30). In desmin-null mice (5, 34) and eccentrically injured wild-type mice (6, 15, 25, 26, 31) desmin is absent or decreased, and the effective transmission of force is compromised. Additionally, desmin-null mice demonstrate impaired voluntary treadmill running and endurance capacity (19).
The response of desmin to a progressive resistance training regimen is unknown; however, two studies indicate that an increase in desmin protein content occurs after a chronic stimulus. For instance, after 8 wk of sprint cycle training, desmin protein content was increased by 60% (40); and in rabbits exposed to several weeks of chronic low-frequency stimulation, desmin levels increased after 1 and 2 wk and reached a plateau by 3 wk of stimulation (4). Furthermore, eccentric exercise plays an important role in the strength and hypertrophy adaptations to resistance training (12, 18, 21), and desmin cytoskeletal alterations have been shown to occur after a single bout of severe eccentric exercise. In animals, a rapid loss of desmin immunostaining beginning as early as 5 min to as late as 7 days postexercise (6, 16, 25, 26, 31) was followed by an increase in desmin protein content occurring from 3 to 7 days postexercise (6). In humans, focal irregularities in the desmin staining pattern were followed by increased staining intensities for desmin and actin 2–3 and 7–8 days after downstairs running (42–44). In addition, desmin protein content was increased 14 days after downhill running in humans (14). Eccentric overload may provide an additional potent stimulus for desmin adaptation.
Given the data that desmin is elevated 7 and 14 days after a single bout of eccentric exercise (6, 14), we were interested in how long such an increase might persist. Furthermore, the increase in desmin seen after a single bout of eccentric exercise raises the possibility that the initial bout of exercise may be responsible for an increase in desmin seen after training. However, Z-disc streaming has also been reported in resistance-trained individuals (17, 33, 35), suggesting that cytoskeletal remodeling may continue to occur throughout resistance training. We, therefore, asked the following questions with regard to desmin and resistance training. Does desmin protein content increase after resistance training? Is this increase due to the initial single bout of exercise, or is it a cumulative effect of subsequent training bouts?
Dystrophin (427 kDa) is also important for lateral force transmission by providing a critical connection at the sarcolemma between the extracellular matrix, the sarcolemma, and the cytoskeleton (11, 27, 30). Perturbations to the dystrophin protein are responsible for a family of muscular dystrophies in humans in which normal muscle function is lost, the most severe of which is Duchenne muscular dystrophy in which dystrophin is completely absent.
An understanding of the response of dystrophin to exercise is incomplete. Limited research in dystrophic muscle has shown that moderate resistance training in humans and voluntary wheel running in mdx mice is beneficial to muscle function (3, 20, 39). However, the muscular response of dystrophin to resistance training in nondiseased muscle is unknown. Like desmin, disruption and loss of dystrophin protein have been seen immediately after eccentric overload in which there was a concomitant decrease in muscular force (7, 22, 23, 27). Furthermore, changes in dystrophin protein content have been seen when resistance training was used as a countermeasure to muscle atrophy incident to long-term bed rest. In this model, muscle atrophy resulted in an increase in dystrophin, which was eliminated by resistance training in the soleus muscle but not in the vastus lateralis muscle (9). These results do not provide a clear understanding of a potential dystrophin response to resistance training in a muscle hypertrophy model, but they do suggest that dystrophin may be modulated.
The relationship of desmin and dystrophin adaptation to other known adaptations of resistance training is unknown. Therefore, we have characterized the adaptation of desmin, dystrophin, MHC composition, muscle strength, and single-fiber muscle CSA to both a single bout of exercise and 8 wk of progressive resistance training. We hypothesized that desmin would increase as a result of both the single bout of exercise and resistance training but that the increases incident to training would be of a greater magnitude. We further hypothesized that dystrophin would show a transient decrease after the single bout of exercise but would increase as a result of resistance training.
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METHODS |
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Subjects. Subjects had not participated in a strength training program or any type of regular exercise training for at least 2 yr before the study. They were asked to refrain from additional physical activity throughout the study. Each subject's age, height, weight, and percent body fat were recorded. Percent body fat was measured with whole body plethysmography (Bod Pod, Life Measurements, Concord, CA). The university institutional review board for human subjects approved the study, and subjects gave their informed consent before participation.
Resistance exercises. Our resistance protocol consisted of three lower body exercises, the knee extension, the leg press, and the hamstring curl. All exercises were two-legged exercises. Because the vastus lateralis was our only muscle of interest, the hamstring curl was included to balance the vastus lateralis exercises as a benefit to the subjects.
Single-bout exercise. The single-bout exercise session began with a 10-min warm-up on a stationary bicycle at a self-selected pace (Monark, Stockholm, Sweden). Subjects were then tested for their 1 RM for each of the following exercises in the following order: leg press, knee extension, and hamstring curl. The 1 RM was determined in the following manner. Each subject performed 1) five repetitions of an estimated 50% 1 RM load, 2) three repetitions of an estimated 70% 1-RM load, 3) one repetition of an estimated 85% 1 RM, and 4) one repetition of an estimated 100% 1 RM. This resistance was increased (5-kg leg extension, 7.5-kg leg press) until a 1 RM was established. Three minutes of rest were given between sets, and 5 min of rest were given between 1-RM testing of different exercises. Twenty minutes after determination of the 1 RM, each subject engaged in 3 sets of 10 repetitions at 80% of 1 RM for each exercise, with 3 min of rest between sets and 5 min of rest between exercises. The 1-RM testing and the experimental exercise session were combined in this manner to avoid the experimental session becoming a second bout of exercise on a subsequent day and to avoid a repeated-bout effect that might influence the desmin protein response. Furthermore, no familiarization sessions were performed prior to this first experimental session. Each subject's 1 RM was also determined at week 8, after the biopsy.
Strength training program. For the training group, the first training session was identical to the protocol for the single-bout group. Subsequent to the initial training bout, subjects engaged in a progressive resistance strength training program three times per week for 8 wk. Each exercise session began with a 10-min warm-up at a self-selected pace on a stationary bicycle. The training program consisted of 1 warm-up set of 10 repetitions at 60% 1 RM and 3 sets of 10 repetitions at 80% 1 RM for the leg press, knee extension, and hamstring curl exercises. If a subject was unable to complete 10 repetitions on the final set, he remained at that resistance for the next exercise session. When a subject could complete all 10 repetitions on the final set without assistance, the resistance was increased (5 kg for the knee extension and hamstring curl, 7.5 kg for the leg press) for the next exercise session. Three minutes of rest were given between sets, and 5 min of rest were given between exercises. Each subject's 1 RM was also determined at weeks 1, 2, 4, and 8 after the biopsy and immediately before the exercise session.
Muscle biopsies. Muscle biopsies (100–120 mg) were obtained with a Bergstrom needle (suction applied). The muscle specimens were trimmed of any connective tissue and immediately frozen in liquid nitrogen until analysis. The week 0 muscle biopsy was taken from the vastus lateralis muscle of the right leg. Each succeeding biopsy was taken from the opposite leg of the preceding biopsy and 2 cm proximal to a previous biopsy. A total of five biopsies per subject were taken over 8 wk.
Protein extraction.
A 
20-mg sample of muscle was homogenized with a Polytron homogenizer (1 mg tissue/18 µl of sample buffer) in sample buffer (6 mg/ml EDTA, 0.06 M Tris, 1% SDS, 15% glycerol, and 5% 
-mercaptoethanol). Samples were centrifuged at 13,000 g for 2 min and then heated to 95°C for 2 min. Total protein content was determined spectrophotometrically (Bio-Rad RC DC protein assay) according to the manufacturer's directions. This assay is based on the Lowry method but modified to be reducing agent and detergent compatible.
Desmin and actin immunoblotting. Desmin and actin were measured using immunoblotting procedures as previously reported (40). Briefly, aliquots of the homogenate sample (7 µg of total protein) were loaded onto a 12% polyacrylamide minigel. Gels were run at 200 V for 45 min. Proteins were transferred to nitrocellulose paper for 50 min at 350 mA. Proteins on the blots were blocked in 5% milk PBST (phosphate-buffered saline plus 0.1% Tween 20) solution for 1 h. Blots were incubated overnight at 4°C (16 h) with both anti-desmin (D33, 1:1,000; Dako, Carpenteria, CA) and anti-actin (AC-40, 1:500,000, Sigma-Aldrich, St. Louis, MO) antibodies. Blots were incubated with the secondary antibody, anti-mouse IgG labeled with horseradish peroxidase (1:1,500; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Protein bands were detected with chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) (40), and quantified with densitometry (Un-Scan-It gel software, version 4.3, Silk Scientific, Orem, UT). A representative sample from each time point was included on each blot. Blots were run in triplicate; thus each sample appeared on three separate blots, and an average of these three values was taken. Desmin and actin data are presented as a percentage change from the week 0 value.
Desmin and actin bands were verified to be within the linear range for the antibody dilutions used. We constructed standard curves for both desmin (P < 0.0001, r2 = 0.93) and actin (P < 0.001, r2 = 0.94). The actual mass of desmin loaded ranged from 0.015 to 0.13 µg of protein, so that 173 of 180 samples fell within the standard curve range (0.02–0.12 µg). Removal of these outliers did not change statistical significance so that data are shown without these samples. The actual mass of actin protein loaded ranged from 0.41 to 1.79 µg, and all samples were within the standard curve range (0.4–4.8 µg).
Dystrophin immunoblotting. Aliquots of homogenate sample (14 µg of total protein) were loaded onto a 4% polyacrylamide minigel. All immunoblotting procedures were done as described above. The primary antibody was anti-dystrophin (MANDRA1, 1:2,000; Abcam, Cambridge, MA), and the secondary antibody was rabbit anti-mouse IgG labeled with peroxidase (1:1,500; Abcam). Band densities were detected with chemiluminescence and quantified with densitometry as described above. Data are presented as a percent change from the week 0 value.
MHC analysis. To determine MHC isoform distribution, aliquots of 2.5 µl (1:60 dilution) were loaded onto a 28-lane polyacrylamide gel (3% stacking gel, 5% separating gel). Gels were run overnight (17 h) at 140 V and then silver stained (29). MHC isoform bands (I, IIa, IIx) were identified based on known migration patterns and quantified with densitometry (Un-Scan-It gel software). Each band was expressed as a percentage of the total band density of all three bands.
CSA. Muscle fiber CSA was determined by cutting transverse sections (10 µm) on a cryostat-microtome (Cryo-Cut II, American Optical, Buffalo, NY) at –20°C then staining them using the NADH tetrazolium reductase method (28). Sections were viewed under a light microscope and captured on a computer with imaging software (NIH Public Domain v. 1.62 Software). Muscle fiber types I and II were determined based on staining intensity. Muscle fiber CSAs of type I and II fibers were determined using computerized planimetry. At least 60 muscle fibers representing types I and II for each sample were measured and mean CSA was calculated for each fiber type.
Statistics. The statistical design was a two-group repeated measures at unequal time intervals. The independent variable was exercise group (single bout or training). The dependent variables were desmin protein content, actin protein content, MHC isoforms I, IIa, and IIx, total protein concentration, CSA, and strength measurements (1 RM) for leg press, knee extension, and hamstring curl exercises. A two-group repeated-measures ANOVA was used to analyze each dependent variable. A Tukey-Kramer post hoc test was used for individual pairwise comparisons. A Pearson product-moment correlation matrix was used to test relationships between the desmin-to-actin ratio and percentage of MHC. For all analyses, significance was set at P < 0.05. Data are reported as means ± SE, unless otherwise indicated.
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RESULTS |
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Total protein. Total protein did not change after a single bout of exercise, or after 8 wk of strength training (Table 1) The values presented for total protein are consistent with previous reports from the literature (10, 37).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the present subjects, a significant increase in desmin is first seen at week 4 of the resistance training program, although a nonsignificant trend to increase is evident as early as week 1. Previous studies from an eccentric overload model have shown increases in desmin as early as 3–14 days. Our data are consistent with other reports that indicate that the desmin cytoskeletal structure has the ability to adapt rather quickly (6, 14, 16, 25, 26, 31, 42–44). This early adaptation could ensure that a functionally adequate cytoskeletal structure was in place before or in conjunction with increases in the force-generating capacity of the muscle (40). Interestingly, the increase in desmin appears to plateau after week 4, suggesting that desmin cytoskeleton adaptations could be mostly completed in the early stages of a resistance training program. A similar plateau in desmin was seen in rabbits exposed to chronic low-frequency stimulation for 3 wk, in which desmin was increased at weeks 1 and 2 but did not further increase at week 3 (4). Notwithstanding these data, it is possible that subsequent adaptation in desmin could also occur in conjunction with more significant hypertrophy and strength gains beyond 8 wk, although the present study cannot address this possibility.
MHC adaptations have generally been shown to occur sometime after 4 wk of strength training (2, 32, 38). Our data are consistent with these studies and indicate that desmin cytoskeletal modifications occur before contractile protein adaptations. The increase in desmin at week 4 temporally preceded a subsequent shift in MHC composition at week 8, in which there was increase in MHC IIa and a decrease in MHC IIx isoform percents. Thus an increase in the desmin cytoskeleton may provide a reinforced structure for lateral force transmission before modifications within the proteins that generate force in the muscle (41–44). In addition, we found a significant negative correlation between desmin and the percentage of MHC IIx in untrained muscle, which relationship was still evident when data from trained muscle were included. These correlations suggest that muscle containing a smaller percentage of MHC IIx may have a greater desmin protein content and that this relationship may be true regardless of training status. Information on fiber-type differences in desmin content is limited. Two animal studies have suggested greater desmin concentrations in the soleus muscle than either the extensor digitorum longus or the tibialis anterior muscles (4, 10). In contrast, in humans, one study has reported greater staining intensities for desmin in type II vs. type I muscle fibers (43).
The desmin response seen after severe eccentric contractions may be a result of repair processes associated with severe muscle damage. Maximal eccentric contractions in rodents resulted in a 200% increase in desmin content at 7 days postexercise (6), and in humans desmin was increased by 150% 14 days after a 30-min downhill run (14). Our single bout was a heavy-resistance protocol consisting of both concentric and eccentric contractions at 80% 1 RM for a concentric contraction. We chose this protocol to be representative of the resistance exercise that an untrained individual might be exposed to in an exercise training situation. In the present subjects, desmin was unchanged after a single bout of resistance exercise. This lack of acute change may be related to both the nature and intensity of the exercise. Although it was considered a heavy resistance protocol, our single bout did not represent a severe and maximal eccentric overload and was not specifically designed to elicit muscle damage as used in the two studies cited above.
Differences in the desmin response to eccentric overload may also be related to species. Rodent studies have consistently shown a rapid and significant loss in desmin immunostaining after eccentric overload (6, 16, 25, 26, 31); however, desmin loss after eccentric overload has not been replicated in human studies. Instead, Yu et al. (41–44) report a more subtle remodeling of the desmin cytoskeleton after eccentric exercise in humans, characterized by focal irregularities in the desmin staining pattern, increased staining intensities for desmin and actin, and nascent sarcomeres at areas of desmin irregularities 2–3 and 7–8 days after downstairs running. These authors propose that the severe muscle damage often seen in animal studies may not be present in humans but that active cytoskeletal remodeling does occur. A more subtle cytoskeletal remodeling that occurred repeatedly with chronic overload could explain the increase in desmin we report in our resistance training group, and it could account for the lack of detectable change to desmin with the single-bout group.
One limitation of the present study is our inability to isolate the specific contributions of concentric and eccentric contractions to the increased desmin during resistance training. The 82% increase in desmin after resistance training of both concentric and eccentric contractions reported presently is quite similar in magnitude to the 60% increase reported previously from our laboratory after 8 wk of concentric-only sprint cycling (40). These two studies raise the possibility that desmin could adapt similarly to repeated muscle contraction regardless of contraction type; however, future research is indeed warranted.
A loss in dystrophin protein content has been shown to occur after eccentric overload (22, 27). As with desmin, we did not detect modifications in dystrophin content after the single bout of exercise. Our inability to detect a loss of dystrophin may be related to the intensity of the single-bout exercise as discussed above for desmin. Or, given the observation that dystrophin loss and disruption have only been demonstrated in animals, the phenomenon may be species related. Timing of our measurements may also be an issue. Lovering and De Deyne (27) reported transient changes in dystrophin demonstrated by a reduction in dystrophin content immediately postcontraction and at day 3 after eccentric overload which subsequently recovered by day 7. It is therefore possible in our study that any acute modulation of the dystrophin protein was resolved before our first biopsy at week 1.
In contrast to muscle contraction, prolonged muscular inactivity resulting in muscle atrophy has been shown to cause an increase in dystrophin concentrations in nondiseased human skeletal muscle. After 84 days of strict bed rest, dystrophin content was increased and desmin content was unchanged in both the soleus and vastus lateralis muscles (9). However, in a diseased state, a decrease in dystrophin protein and alteration of the dystrophin glycoprotein complex contribute to muscle atrophy incident to cancer cachexia (1). In our muscle hypertrophy model, desmin was increased and dystrophin was unchanged. It has been hypothesized that desmin increases as the force-generating capacity of the muscle increases to enable the efficient transmission of force (40). However, muscle disuse in which there was a decrease in muscle CSA and force did not decrease desmin protein content (9). These data indicate that desmin and dystrophin may respond differently to the same stimulus. Whereas dystrophin structure appears to be adequate to withstand the forces placed on it during 8 wk of resistance training, desmin structure is apparently reinforced to withstand these forces. Future research is needed to elucidate how this increased desmin protein is incorporated into the existing desmin structure, and what other proteins are involved in this cytoskeletal remodeling.
In summary, desmin protein content was increased in response to resistance training, whereas dystrophin content was unchanged. Furthermore, neither dystrophin nor desmin was changed during 8 wk after a single bout of exercise. Our data provide a previously uncharacterized desmin response to resistance training in which the desmin cytoskeleton is reinforced as the muscle increases in strength during the first 4 wk of resistance training. We conclude that this early increase in desmin may be necessary to support initial increases in strength as well as future muscle remodeling related to MHC isoform alterations and muscle fiber hypertrophy. Additionally, whereas dystrophin alterations are not needed to support the early functional and structural responses to resistance exercise, the adaptations in the desmin protein occur in response to repeated bouts of exercise.
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FOOTNOTES |
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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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This article has been cited by other articles:
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  D. J. Kosek and M. M. Bamman Modulation of the dystrophin-associated protein complex in response to resistance training in young and older men J Appl Physiol, May 1, 2008; 104(5): 1476 - 1484. [Abstract] [Full Text] [PDF]
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O. R. Seynnes, M. de Boer, and M. V. Narici Early skeletal muscle hypertrophy and architectural changes in response to high-intensity resistance training J Appl Physiol, January 1, 2007; 102(1): 368 - 373. [Abstract] [Full Text] [PDF]
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