Human skeletal muscle expresses mainly three myosin heavy-chain (MHC) isoforms: MHC I, MHC IIa, and MHC IIx, with each isoform giving rise to special contractile properties for the individual fibers, resulting in the capacity for varying properties for the muscle as a whole (16). Each fiber may express only one isoform (pure fibers) or a combination of two or three isoforms, with the latter commonly referred to as hybrid fibers (20,22). Hybrid fibers have contractile properties in between those that would be found for pure fibers of the same MHC isoforms (16,25,28).
It is accepted that endurance athletes have a predominance of type I and IIa fibers identified by ATPase stains (12). However, both the type and volume of training may play significant roles in determining how many hybrid fibers exist. Few studies have thoroughly investigated the effect of specific exercise types and volumes on muscle hybridicity in humans. One study has shown that the gastrocnemius muscle of distance runners (3000-10,000 m) had 6% I/IIa and no IIa/IIx hybrid fibers (13). In another study, the vastus lateralis muscle of cross-country skiers had a high incidence of I/IIa hybrid fibers (± 36%) and only 4% IIa/IIx hybrid fibers (15). Presently, it is unclear why these two studies, both of endurance athletes, differ so much in the reported incidence of I/IIa hybrid fibers. Even if different muscle groups were studied, both these muscles are active with endurance exercise, and one could speculate that the volume of training was higher in the cross-country skiers promoting conversion of IIa fibers to I/IIa hybrid fibers. However, female track and field athletes participating in events not longer than 400 m are also reported to have a high proportion of hybrid fibers (34%), of which 12% were I/IIa, 16% IIa/IIx, and 6% I/IIa/IIx hybrid fibers in their gastrocnemius (18). Interestingly, these athletes had a hybrid fiber composition similar to that found in the untrained females of that study. There are two ways to interpret the findings in the latter study. It is possible that the volume of a particular type of training was not sufficient for fibers to convert to the most appropriate phenotype, or the overall volume of training may have been low. An alternative interpretation is that the fibers were responding to different stimuli (a variety of training intensities) and that this aspect of training promotes the existence of hybrid fibers.
The role of hybrid fibers in skeletal muscle is still unclear. In a recent review, Stephenson (23) suggests that a hybrid fiber might be a "fine-tuned" fiber to optimize levels of both power output and fatigue resistance within a broad range, resulting in a continuum of contractile properties in muscles with a high proportion of hybrids. Caiozzo et al. (10) suggest that the intermediate mechanical properties of motor units that contain hybrid fibers could provide a smoother transition in muscle function when switching activation between motor units. However, other research supports the idea that hybrid fibers are transitional and influenced primarily by training volume (21,29). Indeed, a recent study of recreationally active nonrunners who undertook 13 wk of running training for a marathon event suggests that it is possible to increase the relative quantity of MHC I and decrease the relative quantity of I/IIa hybrid fibers, supporting the possibility of full conversion from fast to slow fiber type in response to increased physical activity in human subjects (25).
The purpose of this study, therefore, was to investigate the occurrence of hybrid fibers in human subjects who varied in training volume and type. In the study design, both endurance runners and recreationally active subjects, with a wide variety of preferred racing distances or recreational sports participation, respectively, were included to facilitate an investigation of the relationship between training volume and fiber hybridicity. Given the variation in preferred racing distance between subjects within the group of runners, it was possible to test the hypothesis that hybrid fiber occurrence is also related to running speed, particularly that runners with shorter preferred racing distances would have a higher proportion of type IIa/IIx fibers. Training volume could be assessed in both runners and nonrunners, and intensity of training and racing was assessed indirectly in runners by calculating the average preferred racing distance for each runner, taking into account track, road, and cross-country races. Furthermore, it was hypothesized that the runners with a higher training volume would have a greater proportion of type I MHC within their I/IIa hybrid fibers than would those with lower training volumes, and that this might provide indirect evidence for fiber-type conversion from type IIa to type I in human subjects.
Thirteen middle-distance runners (age, 22 ± 3 yr; body mass, 63 ± 10 kg; height, 176 ± 9 cm) and nine recreationally active subjects performing no systematic running (nonrunners: age, 25 ± 2 yr; body mass, 70 ± 12 kg; height, 178 ± 8 cm) were recruited, and each of them signed an informed consent form. The study was approved by the University of Stellenbosch ethics committee for research on human subjects (subcommittee C). Inclusion criteria for runners were as follows: ability to complete a 10-km road race in less than 35 min (mean 10-km personal best in previous 3 months: 33.3 ± 1.4 min); training ≥ 35 km·wk−1; no other sport participation; and participation in competitive events longer than 800 m as preferred track distance and not longer than 21.1 km as preferred road-racing distance. Six runners were Caucasian, and seven were Xhosa-speaking black South Africans; the sedentary to recreationally active group comprised seven Caucasian and two Xhosa-speaking black South Africans. Given the genetic diversity within African populations, black subjects were restricted to those with parents and grandparents speaking Xhosa as their first language (27).
Each individual retrospectively completed a detailed questionnaire on the amount and type of training or exercise performed in a typical period of 4 wk. Runners were requested to report a preferred racing distance for each subdiscipline: track, cross-country, and road. If two or more preferred racing distances were reported, the average was calculated and reported as average preferred racing distance (PRDA) (refer to Table 1). Exercise quantity was calculated per week, and the 4-wk average was expressed either as kilometers per week (runners) or hours of exercise per week (nonrunners).
Subjects completed an incremental test on a treadmill (RunRace, Technogym, Italy) until exhaustion. The initial speed was 14 or 7 km·h−1 for runners and nonrunners, respectively. Speed was increased every 30 s by 0.5 km·h−1. Breath-by-breath samples were analyzed for volume, oxygen, and carbon dioxide contents (Jaeger OxyCon Pro, Germany), and heart rate was monitored throughout the test (Polar, Finland). The test was repeated if respiratory exchange ratio was not elevated to at least 1.10 and maximal heart rate was not within 5 bpm of the age-predicted maximum (220 bpm − age).
A medical doctor experienced in the technique performed the muscle biopsies. Local anesthetic (Xylotox, Adcock Ingram) was administered to the vastus lateralis, and a small cut was made using a scalpel blade. A sterile trephine needle (Stille, Sweden) was inserted into the midportion of the muscle, and, with the addition of suction, tissue was removed with a quick cutting action of the needle (7). Muscle specimens were rapidly frozen in liquid nitrogen and stored at −87°C until single-fiber analysis.
Analysis of single muscle fibers.
Muscle samples were freeze-dried overnight, and individual muscle fibers were dissected in a humidity-controlled room. A total of 2639 fibers (mean, 120 ± 41 per subject) were dissected. Each fiber was transferred to a capillary tube containing 30 μL of a denaturing buffer (10% glycerol, 5% β-mercaptoethanol, 2.3% sodium dodecyl sulfate (SDS), and 62.5 mM Tris-HCl, pH 6.80). Fibers were allowed to denature overnight at room temperature (8). Gel electrophoresis was carried out according to Talmadge and Roy (24), with β-mercaptoethanol added to the upper running buffer to a concentration of 0.03 M before electrophoresis (9). Electrophoresis was carried out at 70 V for 24 h at 4°C and was subsequently silver stained (PlusOne silver stain kit, Amersham, Sweden). Identification of bands was done according to the extent of mobility (Fig. 1), as previously proven by Pereira Sant'Ana et al. (19) using isoform-specific antibodies. For the group of runners, each hybrid fiber (total of N = 122) was scanned and analyzed for densitometric profile (UN-SCAN-IT gel, Silk Scientific Corporation) to determine the relative amount of each isoform present. These data were used firstly to categorize each hybrid fiber into one of two subtypes, each containing more (> 50%) of the opposite predominant isoform (Table 2), and then to categorize each hybrid fiber into one of five subtypes (20th percentiles).
All values are reported as means ± standard deviation (SD). Where applicable, runners were subdivided into groups, each with a PRDA > 8 km and PRDA < 8 km. Furthermore, runners were also subdivided into two groups, those having a training volume > 70 km·wk−1 and those < 65 km·wk−1. An ANOVA with a Bonferroni correction was applied for statistical comparison between the two runner groups and nonrunners. The P < 0.05 confidence level was used to indicate statistical significance. Correlations were performed using the Pearson's correlation coefficient, in each group separately. Where appropriate, one-phase exponential curve-fitting was applied, using nonlinear regression analysis that minimized the sum of squares of the actual distance of points from the curve (not weighted; not forced through 0). Goodness of fit is reported as R 2 values. Iterations proceeded until the change in the sum of squares between two consecutive iterations was less than 0.01%. To present the distribution of MHC isoform proportions within hybrid fibers, all the hybrid fibers of runners were pooled into one of two groups related to training volume. Thereafter, the distribution was plotted in bins of 1-20, 21-40, 41-60, 61-80, and 81-99%, according to the relative proportion of MHC I for the I/IIa hybrid fibers and relative to the proportion of MHC IIa for the IIa/IIx hybrid fibers. The data were normalized for the total number of fibers dissected for each group.
Training data and treadmill test results.
Table 1 indicates in detail the exercise patterns of individual runners and nonrunners. On average, runners trained a distance of 83.2 ± 31.7 km·wk−1, and nonrunners exercised, on average, 4.1 ± 4.9 h·wk−1. Runners had a PRDA of 10.1 ± 5.4 km. Furthermore, runners had a significantly higher peak treadmill velocity (21.4 ± 1.1 vs 13.6 ± 0.8 km·h−1, P< 0.01) and peak rate of oxygen consumption during the test (68.2 ± 3.6 vs 42.8 ± 3.2 mL·min−1·kg−1, P < 0.01) compared with nonrunners.
Single fibers: comparison between groups.
Clear distinctions were evident in the mobility of the MHC isoforms of each single fiber (Fig. 1). Fibers expressing pure MHC I, MHC IIa, and MHC IIx, as well as hybrid fibers expressing both MHC I and MHC IIa (I/IIa) and MHC IIa and MHC IIx (IIa/IIx), were identified in both groups and expressed as a percentage of total number of fibers. Runners had more fibers expressing pure MHC I than nonrunners (48.9 ± 15.6 vs 33.2 ± 14.9%, respectively, P < 0.05), and no difference in fibers expressing pure MHC IIa (runners: 39.9 ± 15.1 vs nonrunners: 38.7 ± 12.6%). In contrast, runners had a lower proportion of fibers expressing pure MHC IIx than did nonrunners (0.8 ± 2 vs 8.2 ± 10.0%, respectively, P < 0.05). Similarly, the proportion of IIa/IIx hybrid fibers was higher in nonrunners than in runners (nonrunners: 13.6 ± 7.3 vs runners: 6.6 ± 7.1%, P < 0.05). Although the proportion of I/IIa hybrid fibers did not differ between these two groups (nonrunners: 4.9 ± 6.4 vs runners: 3.8 ± 2.0%), the total proportion of fibers that contained more than one MHC isoform (total hybrids) was still significantly higher in nonrunners compared with runners (19.0 ± 9.0 vs 10.4 ± 5.9% respectively, P < 0.05).
However, when the runners were divided into two more homogenous subgroups according to their preferred racing distances, it was those with a PRDA > 8 km who had more MHC I fibers than nonrunners, whereas those with a PRDA < 8 km did not differ from either group (Fig. 2). All three groups had similar proportions of MHC IIa, but nonrunners tended (P = 0.07) to have higher proportions of MHC IIx than did the runners with the longer PRDA. Nonrunners and runners with PRDA < 8 km had similar proportions of IIa/IIx hybrid fibers, but both groups had significantly more than did the runners with longer PRDA (P < 0.05 for both). Proportions of I/IIa hybrid fibers did not differ between the groups.
Single fibers: relationships with exercise patterns.
In runners, PRDA and training volume were related (Fig. 3), but not all data points fitted within the 95% confidence limits, indicating that these two variables are not simply interchangeable. The percentage of pure fibers did not correlate significantly with the volume of exercise in either runners (type I: r = 0.54; type IIa: r = −0.22; type IIx: r = −0.34) or nonrunners (type I: r = 0.31; type IIa: r = 0.41; type IIx: r = −0.37), although there was a tendency for the percentage of pure type I fibers to correlate positively with training volume in runners (P = 0.06). PRDA, which influences both the volume of training and speed of running, also did not correlate with proportions of pure fibers (type I: r = 0.50; type IIa: r = −0.18; type IIx: r= −0.32). However, the proportions of hybrid fibers were significantly related to exercise pattern, with I/IIa and IIa/IIx hybrid fibers of runners correlating linearly with training volume (r = 0.60, P < 0.05, Fig. 4A; r = −0.80, P < 0.01, Fig. 4B, respectively) and PRDA (r = 0.77, P < 0.01, Fig. 4C; r = −0.83, P < 0.01, data not presented graphically). Exponential curve fitting improved the relationship between IIa/IIx hybrid fibers and PRDA in runners (R 2 = 0.88, Fig. 4D, as opposed to R 2 = 0.69 for thelinear relationship). However, in the case of the relationship between hybrid IIa/IIx fibers and training volume, exponential curve fitting did not improve the fit (R 2 = 0.66) compared with the linear relationship (R 2 = 0.64).
In nonrunners, the IIa/IIx hybrid fibers correlated with exercise volume (r = −0.72, P < 0.05), and, in this case, the exponential curve fitting yielded a better fit (R 2 = 0.82), whereas the I/IIa hybrids showed no linear or exponential relationship.
Proportions of MHC within hybrid fibers.
The proportion of IIa/IIx hybrid fibers (percentage of total fibers dissected) expressing predominantly MHC IIa (greater than 50%) was higher in nonrunners compared with runners (P < 0.05), with no difference between the two groups in the percentage of these hybrids with predominantly MHC IIx (Table 2). Similarly, runners and nonrunners did not differ for predominance of either MHC IIa or MHC I in the I/IIa hybrid fibers when the hybrid fibers were categorized simply into two subgroups. However, when the hybrid fibers were identified according to five categories of relative proportions of the slower of the two MHC isoforms within given hybrid fibers (1-20, 21-40, 41-60, 61-80, or 81-99%), trends emerged.
The histograms in Figure 5 illustrate the frequency distributions for the slower of the two MHC isoforms expressed in the two hybrid fiber populations (i.e., MHC I or MHC IIa proportions for I/IIa or IIa/IIx hybrid fibers, respectively), with the runners subdivided according to exercise pattern. The distribution of MHC IIa proportion in the IIa/IIx hybrid fibers is relatively normal in the runners training < 65 km·wk−1 (the same runners who preferred racing distances < 8 km) (Fig. 5A). However, in the runners with a longer training and racing distance, no fibers were found that expressed more than 60% MHC IIa. In contrast, when considering the influence of training pattern on the distribution of MHC I within the I/IIa hybrid fibers, a skewed distribution pattern was observed for both groups of runners (Fig. 5B): the frequency distribution was skewed in favor of a greater proportion of MHC I in the hybrids of those runners with training volume > 70 km·wk−1, but it was skewed in the opposite direction for those training < 65 km·wk−1.
This study addressed a variety of issues: i) whether training volume would account for the proportion of hybrid fibers in a group of athletes all participating in competitive distance running but with varying habitual training and in a nonhomogenous group of subjects either sedentary or recreationally active in a variety of sports; ii) whether a variable that influences both the volume of training and the speed of running would correlate better or worse with the proportions of hybrid fibers in runners; iii) whether the relationships would be more significant for the proportion of IIa/IIx than the I/IIa hybrid fibers; iv) whether the selection of subject cohorts with a range of training volumes could shed light on how much training might promote the formation of I/IIa hybrid fibers, which are less well understood than the IIa/IIx hybrid fibers; and v) whether quantification of the relative amounts of the different MHC isoforms within each hybrid fiber could illuminate whether hybrid fibers tend to be in transition or are clustered midway between two isoforms.
Significant differences between runners and nonrunners were found for the mean proportions of IIx fibers and IIa/IIx hybrid fibers, confirming existing knowledge. The literature provides ample evidence that regular exercise training reduces the proportion of type IIx fibers (4,11,21,29). Several studies have indicated that the proportion of IIa/IIx hybrid fibers is decreased in trained athletes and decreases in previously sedentary individuals with a training intervention (4,13,15,21,29). A main finding of the current study was that in both populations, exercise volume (kilometers per week and hours per week) was significantly related to IIa/IIx hybrid fiber proportions, despite the fact that one group trained systematically in a single sport for competition, whereas the other group had individuals who did not exercise or who exercised only for recreational reasons. In both populations, a higher volume of exercise resulted in fewer IIa/IIx hybrid fibers. The current study design allows for some refinement of the existing conclusion that increased exercise volume decreases the proportion of IIa/IIx hybrid fibers. Our data indicate specifically that training > 70 km·wk−1 consistently resulted in < 7% IIa/IIx hybrid fibers in distance runners. An application of this finding could be that training volume should not exceed 70 km·wk−1 if the velocity and power provided by these fibers are required for superior performance in shorter, middle-distance running events.
Previous data suggest that high proportions of IIa/IIx hybrid fibers (>15%) are mainly attributable to inactivity, resulting from paralysis, weightlessness, and detraining (2,5,6,17), whereas Harber et al. (13) found none of these hybrid fibers in gastrocnemius of endurance-trained track athletes racing between 3000 and 10,000 m. However, Andersen et al. (4) report that well-trained sprinters (100 m) had 12.9 ± 5.0% IIa/IIx hybrid fibers in their vastus lateralis, and our data suggest that even with training distances between 35 and 65 km·wk−1, these hybrids are in evidence in endurance runners (14.4 ± 4.6%, Fig. 2). We also show, for the first time, that taking into account the preferred speed of racing (i.e., PRDA) improves the prediction of how many IIa/IIx hybrid fibers will exist in vastus lateralis. This finding may support the concept of fine tuning skeletal muscle functional capacity through multiple different fiber types expressing differing proportions of MHC isoforms to accommodate both endurance and high power demands. When the runners were subdivided into two groups, the IIa/IIx hybrid fibers seemed to cluster predominantly midway between the two pure types, not tending toward a full conversion to type IIa in those runners with higher running speeds and relatively shorter training distances (Fig. 5A). In this subgroup, these fast hybrid fibers might not be simply transitional.
In nonrunners, there were fewer subjects participating in large volumes of exercise, but the data indicate that possibly 10 h or more of exercise per week would be required to clearly reduce IIa/IIx hybrid fiber proportions in athletes participating in sports with less continuous effort than distance running. This finding may be influenced by either the intensity or the duration of the rest periods of discontinuous-type exercise, as previously suggested by Campos et al. (11). The current study did not attempt to quantify, directly or indirectly, the exercise intensity or rest periods of nonrunners. Other authors have shown an increase in type IIa/IIx hybrid fibers as a result of high-power output training using either sprint cycling or resistance training as modalities (1,11). It is possible that the higher percentage of IIa/IIx hybrid fibers in nonrunners was also, in part, a function of the required power output in the chosen sports or the daily physical activities.
An important finding in the group of runners was that it was not only the type IIa/IIx hybrid fibers that correlated with training volume and PRDA, but also the I/IIa hybrid fibers (Fig. 4). Previous findings in human subjects after bed rest provide compelling evidence that a lack of physical exercise results in the expression of high proportions of I/IIa hybrid fibers (3,26). In contrast, Andersen et al. (4) report that well-trained sprinters (100 m) had only 0.2 ± 0.2% I/IIa hybrid fibers in biopsies taken from the vastus lateralis muscle after a competitive season. Holden (14) has concluded that little or no evidence currently exists to confirm that type IIa or IIx fibers convert to type I as a result of training. In the current study, the relationship between the proportion of pure slow-twitch muscle fibers and training distance was weak (P < 0.06), but the I/IIa hybrid fiber data (along with the previously published data on highly trained endurance skiers who had approximately 36% I/IIa hybrid fiber composition in the vastus lateralis muscle (15)) indicate that higher volumes of endurance training increase the existence of I/IIa hybrid fibers. In endurance runners who are training consistently at speeds suitable for races around 10 km (and in cross-country skiers), it is likely that both type I and type IIa fibers are recruited. With little or no recovery during a training session, there may be an increased duration of exposure of the intramuscular milieu to the signals promoting the expression of MHC type I, even in type IIa fibers, thus promoting the existence of more type I/IIa hybrid fibers than might be found in marathon runners training at lower speeds or sprinters who include long rest periods. In contrast, the inconsistent and intermittent activity patterns of the nonrunners in our study may explain the wide variety of type I/IIa hybrid expression, which could not be related statistically to training volume.
Caiozzo et al. (10) have suggested that the variety and continuum of hybrid fibers observed in rodents in response to various physiological conditions that affect MHC expression may lead to a spectrum of functional capacities, including small and finely tuned steps. Our data on the MHC distribution within the type IIa/IIx hybrid fibers in the runners training < 65 km·wk−1 suggest that a similar observation and conclusion are possible in human subjects. However, the proportions of MHC I within the type I/IIa hybrid fibers exhibit a skewed distribution, with more of these hybrid fibers containing higher MHC I proportions in those runners doing greater volumes of training (Fig. 5B). Therefore, the conversion of individual fibers in response to running training may not result in a well-distributed functional continuum for these hybrids. Also, Trappe et al.'s (25) data indicate that the response of pure type I fibers to marathon training is to improve maximal velocity of contraction without any change in MHC expression. Taken together with our data, this may be interpreted to mean that the significance of a greater expression of type I MHC may not be related solely to mechanics but, rather, to other concomitant changes such as smaller diffusion distances, altered metabolic profiles, improved economy, and resistance to fatigue.
In the current study, the proportions of pure fiber types did not correlate with either training volume or intensity. Therefore, although our data show that the proportions of hybrid fibers containing the IIa and IIx MHC isoforms, or a combination of fast and slow MHC isoforms, are both strongly influenced by recent training patterns, we suggest that the proportions of pure fiber types are also influenced by additional factors, most likely a longer training history.
This study is the first to show a linear decrease in IIa/IIx hybrid fibers as exercise volume increases in runners, but an exponential decrease with increasing preferred racing distance. This finding led specifically to the conclusion that in endurance runners, the proportion of IIa/IIx hybrid fibers is influenced by training volume and is further modulated by running speeds typically associated with the preferred racing distance. Furthermore, training distances below 65 km·wk−1 did not appreciably decrease the proportion of IIa/IIx hybrid fibers, despite the endurance nature of the training, whereas training distances above 70 km·wk−1 were clearly associated with a low proportion of the fast-twitch hybrid fibers. This is also the first study to show a positive linear relationship between I/IIa hybrid fibers and increasing training volume in runners and greater proportions of MHC I within the I/IIa hybrid fibers in those runners training more than 70 km·wk−1.
This study was supported by the Harry Crossley grant (K. H. Myburgh) and the South African National Research Foundation and the Swedish International Development Agency's bilateral program (K. H. Myburgh and B. Essen-Gustavsson). We would like to thank the Swedish Institute for a scholarship awarded to Tertius Kohn during a study period in Sweden. A special thank you goes to Bengt Saltin and Jesper Andersen for their financial and technical support during the study period of Dr. Kohn at the Copenhagen Muscle Research Centre, Denmark. The authors want to sincerely thank the subjects who participated in this study.
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Keywords:©2007The American College of Sports Medicine
SINGLE FIBERS; ELECTROPHORESIS; MYOSIN HEAVY CHAIN; TRAINING VOLUME