476 Physiology & Biochemistry
Authors
P. T. Reidy, J. M. Hinkley, T. A. Trappe, S. W. Trappe, M. P. Harber
Affiliation
Human Performance Laboratory, Ball State University, Muncie, United States
Key words ▶ endurance exercise ● ▶ actin ● ▶ myosin ● ▶ myosin light chain ● ▶ water content ● ▶ myofiber ●
Abstract
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Evidence suggests that myofibers from endurance trained skeletal muscle display unique contractile parameters. However, the underlying mechanisms remain unclear. To further elucidate the influence of endurance training on myofiber contractile function, we examined factors that may impact myofilament interactions (i. e., water content, concentration of specific protein fractions, actin and myosin content) or directly modulate myosin heavy chain (MHC) function (i. e., myosin light chain (MLC) composition) in muscle biopsy samples from highly-trained competitive (RUN) and recreational (REC) runners. Muscle water content was lower (P < 0.05) in RUN (73 ± 1 %) compared to REC (75 ± 1 %) and total muscle and myofibrillar protein concentration was higher (P < 0.05) in RUN, which may indicate differ-
Introduction accepted after revision June 27, 2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1351334 Published online: November 14, 2013 Int J Sports Med 2014; 35: 476–481 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0172-4622 Correspondence Dr. Matthew Harber Human Performance Laboratory Ball State University Muncie, IN47306 United States Tel.: + 1/765/285 9840 Fax: + 1/765/285 9840 [email protected]
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The influence of endurance exercise training on skeletal muscle metabolic properties has been well studied [33], and there is increasing evidence that endurance exercise influences skeletal muscle contractile function at the whole muscle and the single-fiber levels [10, 16–18, 34, 43, 47]. Previous cross-sectional and longitudinal studies in human and rodent models have revealed a general trend towards an increased shortening velocity (V0) [16, 18, 34, 43, 47] and a decrease in normalized force production (P0/CSA) [16–18] at the myofiber level with endurance exercise training. Interestingly, these changes in contractile function are independent of myosin heavy chain (MHC) isoform, which is the primary determinant of myofiber contractile function [5]. The underlying mechanisms for this increased V0 and a decreased P0/CSA are unclear, but are likely due to factors that could directly influence contractile myofilament interactions (e. g., muscle
ences in myofilament spacing. Content of the primary contractile proteins, myosin (0.99 ± 0.08 and 1.01 ± 0.07 AU) and actin (1.33 ± 0.09 and 1.27 ± 0.09 AU) in addition to the myosin to actin ratio (0.75 ± 0.04 and 0.80 ± 0.06 AU) was not different between REC and RUN, respectively, when expressed relative to the amount of myofibrillar protein. At the single-fiber level, slow-twitch MHC I myofibers from RUN contained less (P < 0.05) MLC 1 and greater (P < 0.05) amounts of MLC 3 than REC, while MLC composition was similar in fast-twitch MHC IIa myofibers between REC and RUN. These data suggest that the distinctive myofiber contractile profile in highly-trained runners may be partially explained by differences in the content of the primary contractile proteins and provides unique insight into the modulation of contractile function with extreme loading patterns.
water content, actin and myosin content) or modulate myosin heavy chain contractile function (e. g., myosin light chain [MLC] composition). Increasing muscle water could partially explain the increase in V0 and decrease in P0/CSA through altering the myofilament spacing [28]. Additionally, changes in contractile protein concentrations (i. e., myosin) have been associated with changes in muscle fiber force and power [18, 46], specifically through alterations in normalized force (i. e., P0/CSA) [5]. MLC composition has also received considerable attention for regulating maximal V0 of single muscle fibers from rabbits [12], endurance trained rats [34] and humans [18, 47]. However, this relationship is tenuous in human skeletal muscle [24, 41, 44]. Surprisingly, given the multitude of data showing alterations in contractile function in endurance trained muscle, few investigations have examined the composition of the muscle proteins that primarily influence myofiber contractile function. Therefore, to provide further insight into the
Reidy PT et al. Endurance Training and Muscle Protein Composition … Int J Sports Med 2014; 35: 476–481
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Protein Composition of Endurance Trained Human Skeletal Muscle
Physiology & Biochemistry
influence of endurance training on myofiber contractile function, we examined factors related to myofiber contractile function such as the concentration of the main protein fractions (i. e., total, myofibrillar, sarcoplasmic), content of the primary contractile proteins (i. e., actin and myosin), regulators of MHC function (e. g., MLC composition) and muscle water content that may influence myofilament spacing in recreational and highlytrained competitive runners that have distinct differences in myofiber contractile parameters [16]. We hypothesized that highly-trained competitive runners will have a greater water content and less myofibrillar protein than recreational runners as an explanation for the differences in myofiber function between these cohorts.
477
glass homogenizer. Samples were centrifuged at 21 000 g for 30 min at 4 °C. The supernatant was taken as the sarcoplasmic protein fraction, and the remaining pellet was re-suspended in 40 volumes of the same buffer and taken as the myofibrillar fraction. Aliquots of the homogenate (mixed protein), sarcoplasmic and myofibrillar protein fractions were then measured for protein concentration, in triplicate, using the bicinchoninic acid assay (Thermo Scientific, Rockford, IL) with bovine serum albumin used as the protein standard [42]. The amount of protein in each of the 3 fractions was normalized to the wet weight and dry weight of the muscle. Aliquots of the myofibrillar fraction were used for analysis of myosin content and actin content.
Materials & Methods
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Subjects 15 individuals volunteered for participation and were divided into 2 groups based on their distance running background and aptitude. 7 subjects (n = 7; age, 20 ± 1 yrs; height, 177 ± 2 cm; weight, 64 ± 3 kg; VO2max, 4.6 ± 0.2 l · min − 1, 71 ± 1 ml∙kg − 1∙min − 1) were classified as highly trained competitive runners (RUN). These individuals were collegiate varsity cross-country athletes and were examined before the fall competitive season and ~12 weeks after the conclusion of the spring season. Subjects averaged 95 ± 2 km/wk of total running with an average training pace of 15.5 ± 0.8 km/h and an 8-km run time of 25:51 ± 0:07 min. The other 8 individuals (n = 8; age, 22 ± 1 yrs; height, 176 ± 2 cm; weight, 72 ± 3 kg; VO2max, 3.4 ± 0.3 l · min − 1, 46 ± 2 ml∙kg − 1∙min − 1) were classified as recreationally active (REC) with limited running experience. These subjects ran no more than 4 days/wk with a weekly running volume < 25 km/wk. All volunteers were informed of all the risks and procedures associated with the investigation and provided written informed consent as approved by the Institutional Review Board at Ball State University in accordance with the ethical standards of the journal [20].
Subjects, muscle biopsy and sample preparation Biopsies were obtained 8–12 h after the previous exercise session. These athletes were counseled to consume adequate dietary carbohydrates for optimal glycogen re-synthesis between training bouts as a normal part of their high frequency training program. After resting supine for 30 min, to account for fluid shifts [3], percutaneous needle biopsies were obtained from the lateral head of the gastrocnemius muscle from all subjects [4]. The muscle specimen was immediately divided into longitudinal sections, frozen in liquid nitrogen, or processed for single muscle fiber physiology [16].
Myosin and actin content were determined by quantitative gel electrophoresis [25, 42]. Aliquots of the myofibrillar fraction from each of the REC and RUN subjects were diluted with sodium dodecyl sulfate (SDS) buffer (2 % SDS, 125 mM Tris HCl (pH 6.8), 12.5 % glycerol, 5 % 2-mercaptoethanol, 0.005 % bromophenol blue) with an equal volume of 2x SDS Tris-HCl buffer. The difference was made up with 1x SDS Tris-HCl buffer to dilute to a concentration of 0.8 μg/μl. A 20 μl aliquot was added to 180 μl of 1x SDS Tris-HCl buffer for a final dilution of 0.08 μg/μl. Samples where then heated at 60 °C for 5 min to further denature the proteins. To determine MLC composition of individual slow-twitch MHC I and fast-twitch MHC IIa muscle fibers, ~100 myofibers were isolated from each muscle biopsy sample [48]. MHC composition was determined for each individual fiber in order to determine fiber-type specific MLC profile (see below). Following MHC identification, ~10 MHC I and 10 MHC IIa myofibers per subject were analyzed for MLC composition.
Protein separation To determine myosin (whole chain myosin) and actin content as well as MHC and MLC profile, samples (myofibrillar fractions or single fibers) were run through SDS-PAGE consisting of a 4 % stacking gel with a 10 % (myosin), 6–12 % (actin), 5 % (MHC), or 12 % (MLC) separating gel at 4 °C. The actin 6–12 % gradient separating gel was polymerized overnight at 4 °C. MLC gels were run at 300 Volts until 15 min after the dye front was no longer visible (~5 h) [48]. For myosin and actin, electrophoresis was performed at a constant current of 20 mA per gel in the stacking gel (for 1 h), and 25 mA per gel in the separating gel (for 2 h) with a Tris-glycine electrode buffer at 4 °C for a total of 3 h (Hoeffer SE 600, Amersham Pharmacia Biotech, Piscataway, NJ, USA). Following gel electrophoresis, gels were silver-stained or with the actin gels, Coomassie Blue R-250 stained. Each actin and MLC gel was loaded in duplicate and myosin gel in triplicate with a one-point standard loaded on all the gels for normalization purposes.
Myosin, actin, and MLC content Muscle water content, tissue homogenization, and protein fractionation The wet weight of a muscle sample (~10 mg) was determined on a precision microbalance at − 30 °C and was subsequently freezedried for 72 h. The dry weight of each muscle sample was then determined at − 30 °C. Muscle water content was calculated from the difference in dry and wet weight for each muscle sample and expressed as percentage of initial wet weight. Each muscle sample was then homogenized in 40 volumes of cold homogenization buffer (250 mM sucrose, 100 mM potassium chloride, 20 mM imidazole and 5 mM EDTA; pH 6.8) in a ground
After staining, a digital image of each myosin, MHC, MLC and actin gel was captured using an imaging system (Alpha Innotech Corp. ChemiImager 4400, San Leandro, CA) to quantify the relative density of each band. Images were transferred to a personal computer and were analyzed using Image J (Version Beta 4.02 for Windows) by determining absorbance of a whole band and comparing this to a known standard. All measurements were made in blinded fashion by the same investigator. An average of the duplicate or triplicate densities was taken to represent each standard and sample. We compared differences in the content of myosin and actin between REC and RUN. Additionally, a myosin
Reidy PT et al. Endurance Training and Muscle Protein Composition … Int J Sports Med 2014; 35: 476–481
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Sample preparation
478 Physiology & Biochemistry
Table 1 Concentration of skeletal muscle protein fractions, normalized for muscle wet weight, for recreational (REC) and competitive runners (RUN). Skeletal muscle protein fractions of recreational (REC) and
1.2
Total
Sarcoplasmic
Myofibrillar
147 ± 4 165 ± 4*
51 ± 3 54 ± 3
82 ± 3 106 ± 6*
Myosin (AU)
competitive runners (RUN) REC RUN
Fig. 1 Relative myosin content a actin content b and the myosin to actin ratio c in skeletal muscle of recreational (REC) and competitive runners (RUN). Values are means ± SE.
a 1.6
0.8
0.4
Values are Means ± SE. μg of protein per mg muscle wet weight. *p < 0.05 compared with REC
0.0
to actin ratio was calculated from the normalized myosin and actin values for each sample [40]. The MLC isoforms in the single muscle fibers were identified according to final relative migration position and compared with molecular weight standard (SigmaMarker M-4038) following SDS-PAGE and silver staining. MLC images were transferred to a personal computer and analyzed with FluorChem SP.
Myosin:Actin Ratio (AU)
c
▼
Skeletal muscle water content and protein fraction composition
Skeletal muscle myosin light chain composition MHC I myofibers from RUN contained less MLC 1 and greater ▶ Fig. 2a). Inversely, the amounts of MLC 3 (P < 0.05) than REC (● MLC composition of the MHC IIa myofibers contained more MLC ▶ Fig. 2b). The ratio of MLC 1 and less MLC 3 (P < 0.05) than REC (● 3 to MLC 2 was greater in RUN than REC (P < 0.05) in the MHC I ▶ Fig. 3). However, no strong correlative assomyofibers only (● ciation was found between any MLC ratio (MLC 3/2, MLC 3/1) and V0 (FL/sec) of MHC I or MHC IIa myofibers in RUN or REC ▶ Fig. 4a, b). However, a strong correlative association was (●
REC
RUN
1.2 0.8
0.4 0.0
Water content was lower (P < 0.05) in RUN (73 ± 1 %) compared to REC (75 ± 1 %). Relative to muscle wet weight, the total and myofibrillar protein fractions were greater (P < 0.05) in RUN ▶ Table 1). compared to REC (●
Myosin heavy chain composition of single fibers analyzed for MLC profile were 65 % vs. 50 % MHC I, 32 % vs. 38 % MHC IIa and 3 % vs. 12 % total hybrid MHC myofibers from RUN and REC, respectively.
RUN
1.6
found in the MLC ratio (MLC 3/2) and V0 (FL/sec) of MHC I ▶ Fig. 4a). myofibers when both groups were combined (●
Discussion
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The purpose of this investigation was to examine protein composition in endurance trained skeletal muscle to identify factors associated with the unique myofiber contractile profile of highly trained run athletes [16]. The primary findings from this research were that: 1. Muscle water content was greater in REC compared to RUN; 2. Protein concentrations of total mixed and myofibrillar protein fractions were greater in RUN compared to REC; 3. The content of myosin and actin, within the myofibrillar protein fraction, were similar in RUN and REC. However, factoring in the higher myofibrillar protein concentration in RUN, it appears that total myosin and actin content are higher in RUN; 4. MLC composition may only partly explain the higher contractile velocity of MHC I myofibers in RUN and appears to be unrelated to differences in MHC IIa myofibers. These data suggest that endurance trained human skeletal muscle contains a higher content of the primary contractile proteins, providing insight into the differences in myofiber contractile function with endurance training. We originally hypothesized that muscle water content would be higher in RUN based on the unique contractile profile previously observed and because oxidative muscle tissues have greater
Reidy PT et al. Endurance Training and Muscle Protein Composition … Int J Sports Med 2014; 35: 476–481
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Actin (AU)
0.0
Results
Single fiber myosin heavy chain composition
REC
0.8 0.4
Significance was set at P ≤ 0.05 for each analysis. The RUN versus REC comparison was conducted with an unpaired t-test. Correlation of the functional data and 3/2 MLC ratio in MHC I and MHC IIa myofibers was performed using GraphPad Prism version 5.04 for Windows, GraphPad Software (La Jolla California USA). Data are presented as mean ± SE.
Myosin content was not different between REC and RUN, being ▶ Fig. 1a). Similarly, 0.99 ± 0.08 and 1.01 ± 0.07 AU, respectively (● actin content was not different between REC and RUN, being ▶ Fig. 1b). The rela1.33 ± 0.09 and 1.27 ± 0.09 AU, respectively (● tive content of myosin to actin, determined as the myosin to actin ratio, was similar between REC and RUN as well, being ▶ Fig. 1c). 0.75 ± 0.04 and 0.80 ± 0.06, respectively (●
RUN
1.2
Statistics
Skeletal muscle myosin and actin content
REC
b 1.6
Physiology & Biochemistry
40
*
30
*
20 10 0
MLC 1
MLC 2 REC MHC I
2.5 2.0 1.5 1.0 0.5 0.0
MLC 3 RUN MHC I
0.0
0.2
0.4 3/2 Ratio
b 50
*
*
30
b
20 10 0
ALL RUN REC
MLC 1
MLC 2 REC MHC IIa
MLC 3 RUN MHC IIa
Fig. 2 Myosin light chain composition in MHC I a and MHC IIa b single muscle fibers from recreational (REC) and competitive runners (RUN). Values are means ± SE. *p < 0.05 compared with REC.
Shortening Velocity (Vo)
Percent
40
MLC 3/2 ratio
1.2 0.8
r = 0.70, p= 0.02 r = –0.25, p= 0.69 r = 0.01, p= 0.99
4.0
2.0
0.0 1
2 3/2 Ratio ALL RUN REC
1.6
0.8
6.0
0
2.0
0.6
3
4
r = 0.08, p= 0.80 r = –0.02, p= 0.98 r = 0.66, p= 0.15
Fig. 4 Correlation of shortening velocity (Vo) and MLC3/MLC2 ratio measured from single MHC l a and MHC IIa b myofibers in recreational (REC) and competitive runners (RUN). p < 0.05.
*
0.4 0.0
MHC I
MHC IIa REC
RUN
Fig. 3 Myosin light chain 3/2 ratio in MHC I and MHC IIa single muscle fibers from recreational (REC) and competitive runners (RUN). Values are means ± SE. *p < 0.05 compared with REC.
water contents than muscle tissues displaying primarily glycolytic properties [30, 37]. A higher muscle water content would potentially lead to a greater V0 and decreased P0/CSA through alteration in myofilament spacing [28]. However, the current data does not support this contention. Two potential factors influencing muscle water content in endurance runners are muscle glycogen levels and hydration status. Since the runners were instructed to consume adequate carbohydrate and biopsies were obtained 8–12 h after the previous exercise session, we do not suspect that transient glycogen depletion influenced muscle water content. Furthermore, there is not a consistent relationship with glycogen and muscle water storage in humans [5], and glycogen re-synthesis can occur
tduring dehydration [4]. As such, it is not likely that levels of glycogen use in RUN would sufficiently influence the water content between our groups. A plausible factor explaining the differences in muscle water content in highly trained runners could be hydration status. Heavy aerobic training can result in voluntary dehydration several hours post-exercise [15]. Costill et al. demonstrated decreased intramuscular water content with increasing dehydration as an effort to maintain circulating plasma volume [8]. The lower water content found in RUN could be a result of transient dehydration occurring during the muscle biopsy sampling. RUN trained more often than REC and also at greater absolute training intensities, suggesting potential for more frequent and variable fluctuations in plasma volume and muscle water content. We measured global muscle water content, which does not account for differences in the extracellular and intracellular muscle water compartments. Further examination of the water compartments within muscle may be necessary to provide further insight into the relationship between muscle water content and myofiber contractile function with endurance training. The unique contractile characteristics of isolated myofibers from endurance trained muscle (elevated V0 and reduced P0/CSA) are
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Percent
50
Shortening Velocity (Vo)
a
a 60
479
thought to be consistent with a lower concentration of myofibrillar protein. Fast-twitch muscle fibers have a higher specific tension (P0/CSA) relative to slow-twitch fibers and this coincides with a higher amount of myofibrillar protein [5, 7]. Furthermore, P0/CSA is reduced and V0 is increased following 12 weeks of aerobic exercise training, and this was linked with reductions in myofibrillar protein concentration [18]. Our examination of the myofibrillar fraction in the current study did not support this pattern. Moreover, the competitive runners had a greater myofibrillar protein concentration, suggesting that other factors are contributing to the unique contractile profile. The disconnect between myofibrillar protein content and contractile function is supported by evidence from our laboratory that the relative proportion of protein fractions in skeletal muscle is typically unaltered relative to the changing size of muscle after aging and extreme perturbations such as unloading [45]. To gain more specific insight into the relationship between protein composition and contractile function, we examined the content of the main force-producing proteins, actin and myosin. Interestingly, we found comparable myosin content between the different phenotypes of runners which is in agreement with previous work in rats exhibiting an unaltered actomyosin complex [1] or myofibrillar protein content [35] with endurance training and is consistent with our previous literature that reveals no differences or changes in myosin content in mixed-muscle protein across multiple activity/inactivity paradigms characterized by changes in myofiber contractile function [45]. Similar to these previous reports, we demonstrate the same amount of myosin and actin per amount of myofibrillar protein. However, when coupled with more myofibrillar protein per unit muscle, this suggests more myosin and actin per unit muscle. This is a unique finding that warrants further inquiry. The lack of a difference in actin content between groups or comparisons is consistent with previous research examining exercise training in rats [1], unloading in humans [21, 25], fiber type comparisons [7] and in aging human skeletal muscle [42]. Actin concentration decreases in skeletal muscle after damaging eccentric exercise in rats [23] but not in humans [39]. Although, alterations in the actin architecture have been found in unloading [31], actin concentration does not appear to be influenced by endurance exercise training. Although actin turnover is increased in endurance trained muscle [36], it may be more resistant to breakdown in the normally loaded state than in the unloaded state. Reasons for this are unclear, but may be related to a specific degradation pathway. Heat shock protein expression has been shown to be elevated in response to endurance exercise [26] and has demonstrated a protective effect regarding the myofibrillar proteins [49], potentially indicating that quantity of myofibrillar proteins are preserved and actually higher in our cohort of runners. Myosin light chains have been associated with myofiber contractile function since they have been shown to alter V0 [12]. Additionally, animal data [34] and some evidence in humans [47] suggests a higher MLC3f-to-MCL2s (3:2 ratio) with endurance exercise training correlates with increased V0. The single fiber analysis of the MLC composition demonstrated that RUN contained less MLC1 and more MLC3 in MHC I myofibers than REC. In the MHC I myofibers of RUN, this contributed a greater MLC 3:2 ratio which would be typically associated with increased V0. However, we were unable to demonstrate a correlative association in this analysis. The higher proportion of fast MLC isoforms in isolated MHC I myofibers may offer limited explanation regarding the higher V0 in well-trained endurance runners. Most
of the support for MLC induced changes in myofiber function during endurance training has been conducted in rats [34] with little consensus in human tissue for endurance exercise [18, 41, 47] or other analysis [24, 44]. This study and another cross-section comparison in runners [47] provide insufficient evidence, in human muscle tissue, toward an endurance exercise training-related connection between MLC composition and myofiber function. Furthermore, due to the cross-sectional nature of these studies, it is unclear whether the differences in MLC composition at the single fiber level observed in the trained runners can be attributed to a genetic element, to several years of training at a high level or a combination of these factors. Given this incongruence in human skeletal muscle it is most likely that a variable other that MLC composition can better explain the blatant differences in myofiber function between runners. Further in-depth analysis of the in-vitro phosphorylation of MLCs [14], post-translational modifications [5, 22] and calcium kinetics [6, 9, 11] could possibly provide additional explanation for regulation of myofiber function. Of particular interest is that the improved muscle function with aerobic exercise training has been connected to markers of calcium kinetics and muscle energy homeostasis in animal literature [2, 6, 9, 38] and human skeletal muscle [2, 13, 27, 29, 50]. In response to exercise training, skeletal muscle fibers alter their contractile properties, independent of MHC isoform, to meet the functional demands of the exercise stimulus [16–19, 43]. Cellular adaptations to resistance training are primarily attributed to myofiber size (i. e., quantitative), while endurance training remodels the contractile properties (i. e., normalized force, contraction velocity) more independently of size. However, the underlying mechanism for the unique contractile function in endurance trained myofibers is largely unexplained. The current data suggest that the content and concentration of the primary contractile proteins does not completely explain this contractile phenotype, suggesting that alterations in the inherent functional properties of the contractile proteins may occur. Lessons from comparative physiology suggest that contractile force and speed can be manipulated through altering the number of crossbridges generating force during the contraction cycle, leading to extreme changes in function that cannot be explained by the type or amount of proteins present [32]. Our findings further highlight the remarkable plasticity of skeletal muscle in response to chronically performed exercise and validate the need for further inquiry into the underlying mechanisms.
Acknowledgements
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This research was funded by NIH grant AG32127.
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handling in human chronic heart failure. Med Sci Sports Exerc 2010; 42: 847–855 Polak JF, Jolesz FA, Adams DF. NMR of skeletal muscle. Differences in relaxation parameters related to extracellular/intracellular fluid spaces. Invest Radiol 1988; 23: 107–112 Riley DA, Bain JL, Romatowski JG, Fitts RH. Skeletal muscle fiber atrophy: altered thin filament density changes slow fiber force and shortening velocity. Am J Physiol 2005; 288: C360–C365 Rome LC. Design and function of superfast muscles: new insights into the physiology of skeletal muscle. Annu Rev Physiol 2006; 68: 193–221 Saltin B, Gollnick PD. Skeletal muscle adaptability: significance for metabolism and performance. In: Peachey LD et al (ed.). Handbook of physiology, section 10: skeletal muscle. Bethesda, Md: American Physiological Society, 1983; p 555–631 United States; 1983 Schluter JM, Fitts RH. Shortening velocity and ATPase activity of rat skeletal muscle fibers: effects of endurance exercise training. Am J Physiol 1994; 266: C1699–C1673 Seene T, Alev K, Kaasik P, Pehme A. Changes in fast-twitch muscle oxidative capacity and myosin isoforms modulation during endurance training. J Sports Med Phys Fitness 2007; 47: 124–132 Seene T, Kaasik P, Alev K, Pehme A, Riso EM. Composition and turnover of contractile proteins in volume-overtrained skeletal muscle. Int J Sports Med 2004; 25: 438–445 Sjogaard G, Saltin B. Extracellular and Intracellular Water Spaces in Muscles of Man at Rest and with Dynamic Exercise. Am J Physiol 1982; 243: R271–R280 Thomas MM, Vigna C, Betik AC, Tupling AR, Hepple RT. Initiating treadmill training in late middle age offers modest adaptations in Ca2 + handling but enhances oxidative damage in senescent rat skeletal muscle. Am J Physiol 2010; 298: R1269–R1278 Thompson HS, Maynard EB, Morales ER, Scordilis SP. Exercise-induced HSP27, HSP70 and MAPK responses in human skeletal muscle. Acta Physiol Scand 2003; 178: 61–72 Thompson LV, Durand D, Fugere NA, Ferrington DA. Myosin and actin expression and oxidation in aging muscle. J Appl Physiol 2006; 101: 1581–1587 Trappe S, Creer A, Minchev K, Slivka D, Louis E, Luden N, Trappe T. Human soleus single muscle fiber function with exercise or nutrition countermeasures during 60 days of bed rest. Am J Physiol 2008; 294: R939–R947 Trappe S, Gallagher P, Harber M, Carrithers J, Fluckey J, Trappe T. Single muscle fibre contractile properties in young and old men and women. J Physiol 2003; 552: 47–58 Trappe S, Harber M, Creer A, Gallagher P, Slivka D, Minchev K, Whitsett D. Single muscle fiber adaptations with marathon training. J Appl Physiol 2006; 101: 721–727 Trappe S, Williamson D, Godard M, Porter D, Rowden G, Costill D. Effect of resistance training on single muscle fiber contractile function in older men. J Appl Physiol 2000; 89: 143–152 Trappe T. Influence of aging and long-term unloading on the structure and function of human skeletal muscle. Appl Physiol, Nutr Metabol 2009; 34: 459–464 Widrick JJ, Knuth ST, Norenberg KM, Romatowski JG, Bain JL, Riley DA, Karhanek M, Trappe SW, Trappe TA, Costill DL, Fitts RH. Effect of a 17 day spaceflight on contractile properties of human soleus muscle fibres. J Physiol 1999; 516 (Pt 3): 915–930 Widrick JJ, Trappe SW, Blaser CA, Costill DL, Fitts RH. Isometric force and maximal shortening velocity of single muscle fibers from elite master runners. Am J Physiol 1996; 271: C666–C675 Williamson DL, Godard MP, Porter DA, Costill DL, Trappe SW. Progressive resistance training reduces myosin heavy chain coexpression in single muscle fibers from older men. J Appl Physiol 2000; 88: 627–633 Willoughby DS, Priest JW, Nelson M. Expression of the stress proteins, ubiquitin, heat shock protein 72, and myofibrillar protein content after 12 weeks of leg cycling in persons with spinal cord injury. Arch Phys Med Rehabil 2002; 83: 649–654 Zoladz JA, Grassi B, Majerczak J, Szkutnik Z, Korostynski M, Karasinski J, Kilarski W, Korzeniewski B. Training-induced acceleration of O(2) uptake on-kinetics precedes muscle mitochondrial biogenesis in humans. Exp Physiol 2013; 98: 883–898
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Physiology & Biochemistry
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Authors
P. T. Reidy, J. M. Hinkley, T. A. Trappe, S. W. Trappe, M. P. Harber
Affiliation
Human Performance Laboratory, Ball State University, Muncie, United States
Key words ▶ endurance exercise ● ▶ actin ● ▶ myosin ● ▶ myosin light chain ● ▶ water content ● ▶ myofiber ●
Abstract
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Evidence suggests that myofibers from endurance trained skeletal muscle display unique contractile parameters. However, the underlying mechanisms remain unclear. To further elucidate the influence of endurance training on myofiber contractile function, we examined factors that may impact myofilament interactions (i. e., water content, concentration of specific protein fractions, actin and myosin content) or directly modulate myosin heavy chain (MHC) function (i. e., myosin light chain (MLC) composition) in muscle biopsy samples from highly-trained competitive (RUN) and recreational (REC) runners. Muscle water content was lower (P < 0.05) in RUN (73 ± 1 %) compared to REC (75 ± 1 %) and total muscle and myofibrillar protein concentration was higher (P < 0.05) in RUN, which may indicate differ-
Introduction accepted after revision June 27, 2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1351334 Published online: November 14, 2013 Int J Sports Med 2014; 35: 476–481 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0172-4622 Correspondence Dr. Matthew Harber Human Performance Laboratory Ball State University Muncie, IN47306 United States Tel.: + 1/765/285 9840 Fax: + 1/765/285 9840 [email protected]
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The influence of endurance exercise training on skeletal muscle metabolic properties has been well studied [33], and there is increasing evidence that endurance exercise influences skeletal muscle contractile function at the whole muscle and the single-fiber levels [10, 16–18, 34, 43, 47]. Previous cross-sectional and longitudinal studies in human and rodent models have revealed a general trend towards an increased shortening velocity (V0) [16, 18, 34, 43, 47] and a decrease in normalized force production (P0/CSA) [16–18] at the myofiber level with endurance exercise training. Interestingly, these changes in contractile function are independent of myosin heavy chain (MHC) isoform, which is the primary determinant of myofiber contractile function [5]. The underlying mechanisms for this increased V0 and a decreased P0/CSA are unclear, but are likely due to factors that could directly influence contractile myofilament interactions (e. g., muscle
ences in myofilament spacing. Content of the primary contractile proteins, myosin (0.99 ± 0.08 and 1.01 ± 0.07 AU) and actin (1.33 ± 0.09 and 1.27 ± 0.09 AU) in addition to the myosin to actin ratio (0.75 ± 0.04 and 0.80 ± 0.06 AU) was not different between REC and RUN, respectively, when expressed relative to the amount of myofibrillar protein. At the single-fiber level, slow-twitch MHC I myofibers from RUN contained less (P < 0.05) MLC 1 and greater (P < 0.05) amounts of MLC 3 than REC, while MLC composition was similar in fast-twitch MHC IIa myofibers between REC and RUN. These data suggest that the distinctive myofiber contractile profile in highly-trained runners may be partially explained by differences in the content of the primary contractile proteins and provides unique insight into the modulation of contractile function with extreme loading patterns.
water content, actin and myosin content) or modulate myosin heavy chain contractile function (e. g., myosin light chain [MLC] composition). Increasing muscle water could partially explain the increase in V0 and decrease in P0/CSA through altering the myofilament spacing [28]. Additionally, changes in contractile protein concentrations (i. e., myosin) have been associated with changes in muscle fiber force and power [18, 46], specifically through alterations in normalized force (i. e., P0/CSA) [5]. MLC composition has also received considerable attention for regulating maximal V0 of single muscle fibers from rabbits [12], endurance trained rats [34] and humans [18, 47]. However, this relationship is tenuous in human skeletal muscle [24, 41, 44]. Surprisingly, given the multitude of data showing alterations in contractile function in endurance trained muscle, few investigations have examined the composition of the muscle proteins that primarily influence myofiber contractile function. Therefore, to provide further insight into the
Reidy PT et al. Endurance Training and Muscle Protein Composition … Int J Sports Med 2014; 35: 476–481
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Protein Composition of Endurance Trained Human Skeletal Muscle
Physiology & Biochemistry
influence of endurance training on myofiber contractile function, we examined factors related to myofiber contractile function such as the concentration of the main protein fractions (i. e., total, myofibrillar, sarcoplasmic), content of the primary contractile proteins (i. e., actin and myosin), regulators of MHC function (e. g., MLC composition) and muscle water content that may influence myofilament spacing in recreational and highlytrained competitive runners that have distinct differences in myofiber contractile parameters [16]. We hypothesized that highly-trained competitive runners will have a greater water content and less myofibrillar protein than recreational runners as an explanation for the differences in myofiber function between these cohorts.
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glass homogenizer. Samples were centrifuged at 21 000 g for 30 min at 4 °C. The supernatant was taken as the sarcoplasmic protein fraction, and the remaining pellet was re-suspended in 40 volumes of the same buffer and taken as the myofibrillar fraction. Aliquots of the homogenate (mixed protein), sarcoplasmic and myofibrillar protein fractions were then measured for protein concentration, in triplicate, using the bicinchoninic acid assay (Thermo Scientific, Rockford, IL) with bovine serum albumin used as the protein standard [42]. The amount of protein in each of the 3 fractions was normalized to the wet weight and dry weight of the muscle. Aliquots of the myofibrillar fraction were used for analysis of myosin content and actin content.
Materials & Methods
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Subjects 15 individuals volunteered for participation and were divided into 2 groups based on their distance running background and aptitude. 7 subjects (n = 7; age, 20 ± 1 yrs; height, 177 ± 2 cm; weight, 64 ± 3 kg; VO2max, 4.6 ± 0.2 l · min − 1, 71 ± 1 ml∙kg − 1∙min − 1) were classified as highly trained competitive runners (RUN). These individuals were collegiate varsity cross-country athletes and were examined before the fall competitive season and ~12 weeks after the conclusion of the spring season. Subjects averaged 95 ± 2 km/wk of total running with an average training pace of 15.5 ± 0.8 km/h and an 8-km run time of 25:51 ± 0:07 min. The other 8 individuals (n = 8; age, 22 ± 1 yrs; height, 176 ± 2 cm; weight, 72 ± 3 kg; VO2max, 3.4 ± 0.3 l · min − 1, 46 ± 2 ml∙kg − 1∙min − 1) were classified as recreationally active (REC) with limited running experience. These subjects ran no more than 4 days/wk with a weekly running volume < 25 km/wk. All volunteers were informed of all the risks and procedures associated with the investigation and provided written informed consent as approved by the Institutional Review Board at Ball State University in accordance with the ethical standards of the journal [20].
Subjects, muscle biopsy and sample preparation Biopsies were obtained 8–12 h after the previous exercise session. These athletes were counseled to consume adequate dietary carbohydrates for optimal glycogen re-synthesis between training bouts as a normal part of their high frequency training program. After resting supine for 30 min, to account for fluid shifts [3], percutaneous needle biopsies were obtained from the lateral head of the gastrocnemius muscle from all subjects [4]. The muscle specimen was immediately divided into longitudinal sections, frozen in liquid nitrogen, or processed for single muscle fiber physiology [16].
Myosin and actin content were determined by quantitative gel electrophoresis [25, 42]. Aliquots of the myofibrillar fraction from each of the REC and RUN subjects were diluted with sodium dodecyl sulfate (SDS) buffer (2 % SDS, 125 mM Tris HCl (pH 6.8), 12.5 % glycerol, 5 % 2-mercaptoethanol, 0.005 % bromophenol blue) with an equal volume of 2x SDS Tris-HCl buffer. The difference was made up with 1x SDS Tris-HCl buffer to dilute to a concentration of 0.8 μg/μl. A 20 μl aliquot was added to 180 μl of 1x SDS Tris-HCl buffer for a final dilution of 0.08 μg/μl. Samples where then heated at 60 °C for 5 min to further denature the proteins. To determine MLC composition of individual slow-twitch MHC I and fast-twitch MHC IIa muscle fibers, ~100 myofibers were isolated from each muscle biopsy sample [48]. MHC composition was determined for each individual fiber in order to determine fiber-type specific MLC profile (see below). Following MHC identification, ~10 MHC I and 10 MHC IIa myofibers per subject were analyzed for MLC composition.
Protein separation To determine myosin (whole chain myosin) and actin content as well as MHC and MLC profile, samples (myofibrillar fractions or single fibers) were run through SDS-PAGE consisting of a 4 % stacking gel with a 10 % (myosin), 6–12 % (actin), 5 % (MHC), or 12 % (MLC) separating gel at 4 °C. The actin 6–12 % gradient separating gel was polymerized overnight at 4 °C. MLC gels were run at 300 Volts until 15 min after the dye front was no longer visible (~5 h) [48]. For myosin and actin, electrophoresis was performed at a constant current of 20 mA per gel in the stacking gel (for 1 h), and 25 mA per gel in the separating gel (for 2 h) with a Tris-glycine electrode buffer at 4 °C for a total of 3 h (Hoeffer SE 600, Amersham Pharmacia Biotech, Piscataway, NJ, USA). Following gel electrophoresis, gels were silver-stained or with the actin gels, Coomassie Blue R-250 stained. Each actin and MLC gel was loaded in duplicate and myosin gel in triplicate with a one-point standard loaded on all the gels for normalization purposes.
Myosin, actin, and MLC content Muscle water content, tissue homogenization, and protein fractionation The wet weight of a muscle sample (~10 mg) was determined on a precision microbalance at − 30 °C and was subsequently freezedried for 72 h. The dry weight of each muscle sample was then determined at − 30 °C. Muscle water content was calculated from the difference in dry and wet weight for each muscle sample and expressed as percentage of initial wet weight. Each muscle sample was then homogenized in 40 volumes of cold homogenization buffer (250 mM sucrose, 100 mM potassium chloride, 20 mM imidazole and 5 mM EDTA; pH 6.8) in a ground
After staining, a digital image of each myosin, MHC, MLC and actin gel was captured using an imaging system (Alpha Innotech Corp. ChemiImager 4400, San Leandro, CA) to quantify the relative density of each band. Images were transferred to a personal computer and were analyzed using Image J (Version Beta 4.02 for Windows) by determining absorbance of a whole band and comparing this to a known standard. All measurements were made in blinded fashion by the same investigator. An average of the duplicate or triplicate densities was taken to represent each standard and sample. We compared differences in the content of myosin and actin between REC and RUN. Additionally, a myosin
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Sample preparation
478 Physiology & Biochemistry
Table 1 Concentration of skeletal muscle protein fractions, normalized for muscle wet weight, for recreational (REC) and competitive runners (RUN). Skeletal muscle protein fractions of recreational (REC) and
1.2
Total
Sarcoplasmic
Myofibrillar
147 ± 4 165 ± 4*
51 ± 3 54 ± 3
82 ± 3 106 ± 6*
Myosin (AU)
competitive runners (RUN) REC RUN
Fig. 1 Relative myosin content a actin content b and the myosin to actin ratio c in skeletal muscle of recreational (REC) and competitive runners (RUN). Values are means ± SE.
a 1.6
0.8
0.4
Values are Means ± SE. μg of protein per mg muscle wet weight. *p < 0.05 compared with REC
0.0
to actin ratio was calculated from the normalized myosin and actin values for each sample [40]. The MLC isoforms in the single muscle fibers were identified according to final relative migration position and compared with molecular weight standard (SigmaMarker M-4038) following SDS-PAGE and silver staining. MLC images were transferred to a personal computer and analyzed with FluorChem SP.
Myosin:Actin Ratio (AU)
c
▼
Skeletal muscle water content and protein fraction composition
Skeletal muscle myosin light chain composition MHC I myofibers from RUN contained less MLC 1 and greater ▶ Fig. 2a). Inversely, the amounts of MLC 3 (P < 0.05) than REC (● MLC composition of the MHC IIa myofibers contained more MLC ▶ Fig. 2b). The ratio of MLC 1 and less MLC 3 (P < 0.05) than REC (● 3 to MLC 2 was greater in RUN than REC (P < 0.05) in the MHC I ▶ Fig. 3). However, no strong correlative assomyofibers only (● ciation was found between any MLC ratio (MLC 3/2, MLC 3/1) and V0 (FL/sec) of MHC I or MHC IIa myofibers in RUN or REC ▶ Fig. 4a, b). However, a strong correlative association was (●
REC
RUN
1.2 0.8
0.4 0.0
Water content was lower (P < 0.05) in RUN (73 ± 1 %) compared to REC (75 ± 1 %). Relative to muscle wet weight, the total and myofibrillar protein fractions were greater (P < 0.05) in RUN ▶ Table 1). compared to REC (●
Myosin heavy chain composition of single fibers analyzed for MLC profile were 65 % vs. 50 % MHC I, 32 % vs. 38 % MHC IIa and 3 % vs. 12 % total hybrid MHC myofibers from RUN and REC, respectively.
RUN
1.6
found in the MLC ratio (MLC 3/2) and V0 (FL/sec) of MHC I ▶ Fig. 4a). myofibers when both groups were combined (●
Discussion
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The purpose of this investigation was to examine protein composition in endurance trained skeletal muscle to identify factors associated with the unique myofiber contractile profile of highly trained run athletes [16]. The primary findings from this research were that: 1. Muscle water content was greater in REC compared to RUN; 2. Protein concentrations of total mixed and myofibrillar protein fractions were greater in RUN compared to REC; 3. The content of myosin and actin, within the myofibrillar protein fraction, were similar in RUN and REC. However, factoring in the higher myofibrillar protein concentration in RUN, it appears that total myosin and actin content are higher in RUN; 4. MLC composition may only partly explain the higher contractile velocity of MHC I myofibers in RUN and appears to be unrelated to differences in MHC IIa myofibers. These data suggest that endurance trained human skeletal muscle contains a higher content of the primary contractile proteins, providing insight into the differences in myofiber contractile function with endurance training. We originally hypothesized that muscle water content would be higher in RUN based on the unique contractile profile previously observed and because oxidative muscle tissues have greater
Reidy PT et al. Endurance Training and Muscle Protein Composition … Int J Sports Med 2014; 35: 476–481
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Actin (AU)
0.0
Results
Single fiber myosin heavy chain composition
REC
0.8 0.4
Significance was set at P ≤ 0.05 for each analysis. The RUN versus REC comparison was conducted with an unpaired t-test. Correlation of the functional data and 3/2 MLC ratio in MHC I and MHC IIa myofibers was performed using GraphPad Prism version 5.04 for Windows, GraphPad Software (La Jolla California USA). Data are presented as mean ± SE.
Myosin content was not different between REC and RUN, being ▶ Fig. 1a). Similarly, 0.99 ± 0.08 and 1.01 ± 0.07 AU, respectively (● actin content was not different between REC and RUN, being ▶ Fig. 1b). The rela1.33 ± 0.09 and 1.27 ± 0.09 AU, respectively (● tive content of myosin to actin, determined as the myosin to actin ratio, was similar between REC and RUN as well, being ▶ Fig. 1c). 0.75 ± 0.04 and 0.80 ± 0.06, respectively (●
RUN
1.2
Statistics
Skeletal muscle myosin and actin content
REC
b 1.6
Physiology & Biochemistry
40
*
30
*
20 10 0
MLC 1
MLC 2 REC MHC I
2.5 2.0 1.5 1.0 0.5 0.0
MLC 3 RUN MHC I
0.0
0.2
0.4 3/2 Ratio
b 50
*
*
30
b
20 10 0
ALL RUN REC
MLC 1
MLC 2 REC MHC IIa
MLC 3 RUN MHC IIa
Fig. 2 Myosin light chain composition in MHC I a and MHC IIa b single muscle fibers from recreational (REC) and competitive runners (RUN). Values are means ± SE. *p < 0.05 compared with REC.
Shortening Velocity (Vo)
Percent
40
MLC 3/2 ratio
1.2 0.8
r = 0.70, p= 0.02 r = –0.25, p= 0.69 r = 0.01, p= 0.99
4.0
2.0
0.0 1
2 3/2 Ratio ALL RUN REC
1.6
0.8
6.0
0
2.0
0.6
3
4
r = 0.08, p= 0.80 r = –0.02, p= 0.98 r = 0.66, p= 0.15
Fig. 4 Correlation of shortening velocity (Vo) and MLC3/MLC2 ratio measured from single MHC l a and MHC IIa b myofibers in recreational (REC) and competitive runners (RUN). p < 0.05.
*
0.4 0.0
MHC I
MHC IIa REC
RUN
Fig. 3 Myosin light chain 3/2 ratio in MHC I and MHC IIa single muscle fibers from recreational (REC) and competitive runners (RUN). Values are means ± SE. *p < 0.05 compared with REC.
water contents than muscle tissues displaying primarily glycolytic properties [30, 37]. A higher muscle water content would potentially lead to a greater V0 and decreased P0/CSA through alteration in myofilament spacing [28]. However, the current data does not support this contention. Two potential factors influencing muscle water content in endurance runners are muscle glycogen levels and hydration status. Since the runners were instructed to consume adequate carbohydrate and biopsies were obtained 8–12 h after the previous exercise session, we do not suspect that transient glycogen depletion influenced muscle water content. Furthermore, there is not a consistent relationship with glycogen and muscle water storage in humans [5], and glycogen re-synthesis can occur
tduring dehydration [4]. As such, it is not likely that levels of glycogen use in RUN would sufficiently influence the water content between our groups. A plausible factor explaining the differences in muscle water content in highly trained runners could be hydration status. Heavy aerobic training can result in voluntary dehydration several hours post-exercise [15]. Costill et al. demonstrated decreased intramuscular water content with increasing dehydration as an effort to maintain circulating plasma volume [8]. The lower water content found in RUN could be a result of transient dehydration occurring during the muscle biopsy sampling. RUN trained more often than REC and also at greater absolute training intensities, suggesting potential for more frequent and variable fluctuations in plasma volume and muscle water content. We measured global muscle water content, which does not account for differences in the extracellular and intracellular muscle water compartments. Further examination of the water compartments within muscle may be necessary to provide further insight into the relationship between muscle water content and myofiber contractile function with endurance training. The unique contractile characteristics of isolated myofibers from endurance trained muscle (elevated V0 and reduced P0/CSA) are
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Percent
50
Shortening Velocity (Vo)
a
a 60
479
thought to be consistent with a lower concentration of myofibrillar protein. Fast-twitch muscle fibers have a higher specific tension (P0/CSA) relative to slow-twitch fibers and this coincides with a higher amount of myofibrillar protein [5, 7]. Furthermore, P0/CSA is reduced and V0 is increased following 12 weeks of aerobic exercise training, and this was linked with reductions in myofibrillar protein concentration [18]. Our examination of the myofibrillar fraction in the current study did not support this pattern. Moreover, the competitive runners had a greater myofibrillar protein concentration, suggesting that other factors are contributing to the unique contractile profile. The disconnect between myofibrillar protein content and contractile function is supported by evidence from our laboratory that the relative proportion of protein fractions in skeletal muscle is typically unaltered relative to the changing size of muscle after aging and extreme perturbations such as unloading [45]. To gain more specific insight into the relationship between protein composition and contractile function, we examined the content of the main force-producing proteins, actin and myosin. Interestingly, we found comparable myosin content between the different phenotypes of runners which is in agreement with previous work in rats exhibiting an unaltered actomyosin complex [1] or myofibrillar protein content [35] with endurance training and is consistent with our previous literature that reveals no differences or changes in myosin content in mixed-muscle protein across multiple activity/inactivity paradigms characterized by changes in myofiber contractile function [45]. Similar to these previous reports, we demonstrate the same amount of myosin and actin per amount of myofibrillar protein. However, when coupled with more myofibrillar protein per unit muscle, this suggests more myosin and actin per unit muscle. This is a unique finding that warrants further inquiry. The lack of a difference in actin content between groups or comparisons is consistent with previous research examining exercise training in rats [1], unloading in humans [21, 25], fiber type comparisons [7] and in aging human skeletal muscle [42]. Actin concentration decreases in skeletal muscle after damaging eccentric exercise in rats [23] but not in humans [39]. Although, alterations in the actin architecture have been found in unloading [31], actin concentration does not appear to be influenced by endurance exercise training. Although actin turnover is increased in endurance trained muscle [36], it may be more resistant to breakdown in the normally loaded state than in the unloaded state. Reasons for this are unclear, but may be related to a specific degradation pathway. Heat shock protein expression has been shown to be elevated in response to endurance exercise [26] and has demonstrated a protective effect regarding the myofibrillar proteins [49], potentially indicating that quantity of myofibrillar proteins are preserved and actually higher in our cohort of runners. Myosin light chains have been associated with myofiber contractile function since they have been shown to alter V0 [12]. Additionally, animal data [34] and some evidence in humans [47] suggests a higher MLC3f-to-MCL2s (3:2 ratio) with endurance exercise training correlates with increased V0. The single fiber analysis of the MLC composition demonstrated that RUN contained less MLC1 and more MLC3 in MHC I myofibers than REC. In the MHC I myofibers of RUN, this contributed a greater MLC 3:2 ratio which would be typically associated with increased V0. However, we were unable to demonstrate a correlative association in this analysis. The higher proportion of fast MLC isoforms in isolated MHC I myofibers may offer limited explanation regarding the higher V0 in well-trained endurance runners. Most
of the support for MLC induced changes in myofiber function during endurance training has been conducted in rats [34] with little consensus in human tissue for endurance exercise [18, 41, 47] or other analysis [24, 44]. This study and another cross-section comparison in runners [47] provide insufficient evidence, in human muscle tissue, toward an endurance exercise training-related connection between MLC composition and myofiber function. Furthermore, due to the cross-sectional nature of these studies, it is unclear whether the differences in MLC composition at the single fiber level observed in the trained runners can be attributed to a genetic element, to several years of training at a high level or a combination of these factors. Given this incongruence in human skeletal muscle it is most likely that a variable other that MLC composition can better explain the blatant differences in myofiber function between runners. Further in-depth analysis of the in-vitro phosphorylation of MLCs [14], post-translational modifications [5, 22] and calcium kinetics [6, 9, 11] could possibly provide additional explanation for regulation of myofiber function. Of particular interest is that the improved muscle function with aerobic exercise training has been connected to markers of calcium kinetics and muscle energy homeostasis in animal literature [2, 6, 9, 38] and human skeletal muscle [2, 13, 27, 29, 50]. In response to exercise training, skeletal muscle fibers alter their contractile properties, independent of MHC isoform, to meet the functional demands of the exercise stimulus [16–19, 43]. Cellular adaptations to resistance training are primarily attributed to myofiber size (i. e., quantitative), while endurance training remodels the contractile properties (i. e., normalized force, contraction velocity) more independently of size. However, the underlying mechanism for the unique contractile function in endurance trained myofibers is largely unexplained. The current data suggest that the content and concentration of the primary contractile proteins does not completely explain this contractile phenotype, suggesting that alterations in the inherent functional properties of the contractile proteins may occur. Lessons from comparative physiology suggest that contractile force and speed can be manipulated through altering the number of crossbridges generating force during the contraction cycle, leading to extreme changes in function that cannot be explained by the type or amount of proteins present [32]. Our findings further highlight the remarkable plasticity of skeletal muscle in response to chronically performed exercise and validate the need for further inquiry into the underlying mechanisms.
Acknowledgements
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This research was funded by NIH grant AG32127.
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