Vastus lateralis myofiber CSA
Vastus lateralis myofiber CSA means (Fig. 1, Table 1) were increased after RET as revealed by difference by time (P < 0.05) with no treatment or interaction effects (P > 0.10). Mean fiber area of all fiber types was increased ~800–900 μm2 after RET (P < 0.05). However, there was no effect of treatment (P = 0.967). Mean MHC I CSA was increased ~500 μm2 after WP and PB treatment and ~750 μm2 after supplementation of MDP after RET (P < 0.05). There was also no effect of treatment (P = 0.721). Individual treatment changes revealed significant increases after treatment with PB and MDP (P < 0.05) and a trend for an increase (P = 0.083) after treatment with WP. Mean MHC IIa CSA was increased ~900–1000 μm2 after RET (P < 0.05) with no ANCOVA effect of treatment (P = 0.921). Mean fiber area (MFA) of all hybrid fiber types increased ~1000–1100 μm2 after RET (P < 0.05) with no effect of treatment (P = 0.906). PRO (PB + WP) treatment displayed significant increases in all fiber types (P < 0.05). No significant effect of PRO vs MDP treatment was observed in any fiber type (P > 0.423).
Analysis of CSA relative frequency distribution demonstrated that all treatments displayed myofiber growth (rightward shift) (see Figure, Supplemental Digital Content 2, Relative frequency of vastus lateralis MHC I, II, and all myofibers pooled by select CSA bins, http://links.lww.com/MSS/A867). However, few papers report that MHC II fiber types are responsive to protein supplementation (12,19) (see Table, Supplemental Digital Content 3, Summary of all protein supplement studies with a placebo group directly assessing muscle mass during RET in young adults, http://links.lww.com/MSS/A868). Thus, we explored the frequency distributions of MHC II myofibers to determine whether slight patterns for differences between treatments not observed with CSA means were evident. CSA bins were expanded to reflect changes in large myofibers (CSA bins in myofibers larger than 6000, 7000, 7500, and 8000 μm2) and also smaller fibers (CSA bins in myofibers sized 1000 to 5000 μm2) after RET (see Figure, Supplemental Digital Content 4, Change in the relative frequency of larger vastus lateralis MHC II myofibers by select cross-sectional area bins, http://links.lww.com/MSS/A869). Similar to CSA means, this analysis revealed time differences (P < 0.001) with no treatment or interaction effects (P > 0.10), except the smaller bin (myofibers sized 1000 to 5000 μm2), which had a treatment effect (P = 0.031). This treatment difference was shown as a greater absolute frequency of smaller myofibers at pretraining (P = 0.020) and posttraining (P = 0.095) for MDP vs PRO treatment. All treatments demonstrated clear decreases (P < 0.001) in the frequency of smaller MHC IIa myofibers (P < 0.007). Treatment with (PRO: pooled PB and WP) resulted in clear increases (P < 0.001) in the frequency of larger MHC IIa myofibers, whereas increases after treatment with MDP were less evident (P = 0.064 to P = 0.535). When examining these larger myofiber CSA bins, only tendencies approaching statistical significance (P = 0.098–0.194) were observed for an effect of protein (PRO) treatments vs MDP treatment.
Effect size and sample size estimations for significant effects of PRO supplementation during RET on body composition, strength, and muscle mass are shown in Supplemental Digital Content (see Table, Supplemental Digital Content 5, Effect size and sample size estimations for significant effects of PRO supplementation, http://links.lww.com/MSS/A870).
Vastus lateralis SC content
Vastus lateralis myofiber Pax7+ SC content displayed a main effect of time (P < 0.05), which was evident by a ~50% increase in abundance after RET (Fig. 2, Table 2). There were no interactions or effects of treatment with any of the SC outcomes. Because the responses in the PB and WP treatments were identical, the treatments were pooled as PRO and tested against changes in MDP. Mean fiber SC content (SC/fiber), proportion (% SC/myonuclei), and domain (SC/mm2) increased after RET (P < 0.05) with no effect of treatment (P > 0.588). This increase was driven primarily by changes in MHC II myofibers. MHC II SC content (SC/fiber), proportion (% SC/myonuclei), and domain (SC/mm2) increased after RET (P < 0.05), and there was no effect of treatment (P > 0.575). MHC I SC content (SC/fiber) was not globally altered across time (P < 0.05), but there was a trend for an effect of PRO treatment versus MDP treatment (P = 0.073). MHC I SC proportion (% SC/myonuclei) and domain (SC/mm2) were unchanged after RET (P > 0.10). SC domain (SC/mm2) displayed a trend for an effect of PRO treatment vs MDP treatment (P = 0.072). MHC I SC proportion (% SC/myonuclei) displayed a weak trend for an effect of PRO treatment vs MDP treatment (P = 0.099).
Vastus lateralis myonuclei
Vastus lateralis myofiber myonuclear content and domain (Fig. 2, Table 3) were altered by RET. Pre- to postchanges in all treatments pooled together demonstrated an increase (P < 0.05) with RET. Thus, a main effect of time was seen for MHC I, MHC IIa, and mean myonuclei content (P < 0.05). There were no interactions or effects of treatment with any of the myonuclei outcomes (P > 0.10). Because the responses in the PB and WP treatments were identical, the treatments were pooled as PRO and tested against changes in MDP Mean myonuclei content (MyoN/fiber) increased after RET (P < 0.05) with no effect of PRO vs MDP treatment (P = 0.602). The changes in MHC II fibers exerted the greatest influence on the mean myofiber MyoN response. The effect of time was observed as a trend for an overall increase from pre- to posttraining (P = 0.096) in MHC II fibers, which was seen as an increased change score (P < 0.05) with no PRO vs MDP treatment effect (P = 0.378). MHC I myonuclei content did not show changes from pre- to posttraining, except with PRO (P < 0.05) and a PRO treatment effect vs MDP was not evident (P = 0.143).
Myonuclear domain (Fig. 2, Table 3) (MHC II myofibers and all myofibers pooled) demonstrated a slight increase after RET (P < 0.05), whereas MHC I myofibers were not significant (P > 0.10). There were no interactions or treatment effects (P > 0.10). Overall increases from pre- to posttraining were evident in MHC II myofibers (P < 0.05) and demonstrated as a trend (P = 0.086) when all myofiber types were pooled. Both PRO and MDP treatments increased myonuclear domain (P < 0.05) in MHC II myofibers. When pooling all fiber types, a significant increase was indicated in treatment with PRO (P < 0.05) and a trend after treatment with MDP (P = 0.084). This increase was likely due to greater statistical power by grouping a greater number of myofibers with PRO. No changes were observed in MHC I fiber myonuclear domain (P > 0.10). There was no significant effect in the change of PRO over MDP in all fibers pooled (P = 0.746), MHC I (P = 0.880), or MHC II (P = 0.379) myofibers.
Associations and statistical results between measures of muscle hypertrophy (lean mass, myofiber CSA, and muscle thickness) and SC, myonuclei, and myonuclear domain are shown in Supplemental Digital Content (see Tables, Supplemental Digital Content 6, Correlation analysis between myofiber cross-sectional area and myonuclear number, http://links.lww.com/MSS/A871; and Supplemental Digital Content 7, secondary correlation analysis, http://links.lww.com/MSS/A872). Myonuclei number per fiber was highly correlated with fiber size at each time point and in all fiber types and with the increase in myofiber size. Myofiber number per fiber change was well correlated to CSA change and also to the change in SC per fiber. Those who had smaller myonuclear domains at pretraining experienced expansion of their myonuclear domain at posttraining. Also, those participants with the largest CSA changes also experienced the greatest expansion of their myonuclear domain. Absolute values of lean mass but not change values correlated with myofiber size. There was a moderate association for MFA change to positively correlate with all SC per fiber change, which was prominent in MHC I, but not MHC II fibers.
This is the first study reporting the role of protein supplementation and protein supplementation type on fiber-type–specific adaptations of myofiber growth, SC, and myonuclei during traditional progressive resistance training using shortening and lengthening contractions. We demonstrated a similar increase in myofiber CSA, SC content, and myonuclear addition for all three treatment groups. This study was a follow-up analysis to our initial clinical trial (38), where we demonstrate minimal trends for group differences in whole-body and arm-specific lean mass (PB > MDP), yet no differences in the increases in leg lean mass or vastus lateralis muscle thickness. Collectively, these data suggest that the additional lean mass in the PB group was accrued in other locations (arms or trunk) and/or that leg hypertrophy had peaked for all treatments after 3 months of RET with our protocol. This indicates that protein supplementation during RET did not enhance muscle-specific adaptations in the lower limb.
Unfortunately, most of the studies claiming an effect of protein to enhance muscle adaptations to RET rely on whole-body lean mass. Indeed, in examination of the literature, we have previously highlighted that ~62 to ~140 participants (depending on the clinical trial, effect size = 0.24–0.67) would be needed to find a statistical effect of protein supplementation on whole-body lean mass or fat-free mass (39). Similar to these studies, we report an effect for protein supplementation to increases whole-body lean mass as compared with placebo (effect size = 0.565), yet we found similar changes in more direct measures of muscle hypertrophy (ultrasound muscle thickness (38), leg lean mass, and myofiber-type–specific CSA) after RET. As we previously highlighted (38,39), the use and interpretation of whole-body lean mass DXA data to act as the sole measure of muscle hypertrophy is suspect and would likely have limited functional relevance on force production.
Our WP treatment demonstrated similar adaptations when compared with MDP. Contrary to popular dogma, it is not unusual to observe no effect of protein supplementation, in particular WP, over placebo on lean mass or myofiber CSA (39). A meta-analysis determined that protein supplementation during RET in young adults will produce greater increases in vastus lateralis CSA, ~250 μm2, yet that analysis only included data from four studies and is in conflict with results from another meta-analysis (41). We are aware of only three studies demonstrating greater changes in vastus lateralis myofiber CSA (2,12,19) and two studies with magnetic resonance imaging (13,21) comparing protein versus carbohydrate placebo supplementation during RET. In one of the vastus lateralis myofiber CSA studies, the placebo group started with higher CSA and did not experience hypertrophy after RET (2), whereas the other two studies demonstrated this effect only in MHC II fibers (12,19). In comparison, five other studies demonstrated equivalent increases in vastus lateralis myofiber CSA in protein-supplemented treatments (WP, n = 13; milk, n = 11; EAA, n = 11) and carbohydrate placebo treatments (6,9,23,31,32). In addition, studies using magnetic resonance imaging of the biceps (10) or latissimus dorsi (33) and ultrasound (3,4,22,23,48) of the thigh muscles have clearly shown the same pattern: no effect of protein supplementation (whey) to enhance vastus lateralis muscle hypertrophy. Given these findings, it is no surprise that only one study on protein supplementation showed an enhancement of strength, although myofiber CSA was not different with protein supplementation (9). The remainder of the studies demonstrate identical increases in strength with protein supplementation compared with carbohydrate placebo (2,6,10,12,13,19,21–23,31–33), similar to our observations, in part. Indeed, in examination of the literature, we have previously highlighted that ~40 to 500 participants (depending on the clinical trial) would be needed to find a statistical effect of protein supplementation on myofiber CSA (39). We show in this trial that (see Figure, Supplemental Digital Content 4, Change in the relative frequency of larger vastus lateralis MHC II myofibers by select cross-sectional area bins, http://links.lww.com/MSS/A869) 115 to >1000 participants (effect size: mean fiber area = −0.174, MHC I = −0.375, MHC II = 0.052) would be required. Also, 550 participants would be needed to find a statistical effect (effect size = −0.174) of protein supplementation on vastus lateralis muscle thickness. These data further illustrate the minimal effect of protein supplementation to enhance thigh, in particular, vastus lateralis muscle strength and hypertrophy during RET.
Analysis of mean CSA, the predominant method utilized in these types of clinical trials, can obscure subtle changes in myofiber hypertrophy. Recently, Farup et al. (12) completed an elegant study comparing the effect of WP supplementation on isolated lengthening or shortening contractions of skeletal muscle. They demonstrated that myofiber CSA was enhanced in MHC II fibers with WP supplementation during shortening, but not lengthening contractions. They conducted a follow-up analysis by demonstrating a tendency (P < 0.10) for protein supplementation to result in a shift toward a greater frequency of larger myofibers (>8000 μm2) and a lower frequency of smaller fibers (>1000 < 5000 μm2) posttraining, compared with posttraining whey-supplemented eccentric training. Although we did not observe a difference in the CSA means between protein-supplemented and carbohydrate placebo treatments, we similarly demonstrated that protein supplementation displayed a pattern for a slightly greater improvement (nonsignificant) in the frequency of MHC II bins with larger fibers versus the MDP. This suggests that protein supplementation may play a very limited role in expanding MHC II size during RET. However, we stress that this effect is minimal, and given the low statistical confidence seen in these examples, we believe this effect is limited to a subpopulation of myofibers/individuals that is likely an example of responder/nonresponder clustering. The functional relevance of this finding is unknown. However, a minimal effect of protein supplementation to increase whole-body lean mass (not limb/appendicular lean mass) after RET does exist, which we have speculated (39) may likely include nonforce producing lean mass (e.g., trunk muscle). This would result in increased body weight with similar changes in muscle mass. Thus, to maintain the mass-to-strength ratio, this pattern with MHC II myofibers may provide support for increased muscle force to serve as a compensatory mechanism and offset the increased weight. In partial support of this concept, we demonstrated improved isokinetic torque in the protein-supplemented treatments only, suggesting a possible role for the changes in these MHC II fibers with protein supplementation.
Although, SC is not necessary to support hypertrophy through myonuclear addition (14), they are involved in the magnitude of muscle growth (5,14,37). Given that protein supplementation was also thought to influence the magnitude of muscle growth, we sought to examine the role between SC and protein supplementation. Very little research has examined the acute effects of protein/amino acids on the enhancement of SC content after RE. We were aware of one study that used a severe 4-d protein restriction protocol to compare normal (~90 g) versus very low (~11 g) of protein per day, to find no effect on skeletal muscle SC content during the 3-d recovery period after RE (43). It is hard to find relevancy in that study design to our findings other than an overall lack of effect of protein to enhance myogenic adaptations. Olsen et al. (32) first demonstrated that chronic RET with protein supplementation may provide a slight enhancement of the SC pool compared with RET alone. On the basis of basic science and preclinical findings, we anticipated that protein supplementation would enhance SC activity and content through mTORC1 (17,40) and particularly on MHC II fibers (1,11). Instead, we demonstrated similar increases in SC content between treatments, which were driven primarily through increases in MHC II fibers. However, we did demonstrate a significant increase in SC number per fiber for MHC I fibers with protein supplementation but not with an MDP. This resulted in a trend for an effect of protein (P = 0.073) over MDP, which was also seen when expressed as SC per square millimeter and proportion of SC/MyoN. Interestingly, MHC I, but not MHC II, SC number per myofiber change was correlated with CSA change. Farup et al. demonstrated similar findings, to ours, after 3 months of RET with protein supplementation in MHC I, but not MHC II fibers, suggesting that protein supplementation may provide greater expansion of the SC pool in this fiber type to regulate myofiber growth. Taken together, these findings are somewhat contradictory, although they may be explained as differences between preclinical and clinical research. MHC II fibers are thought to be most responsive to heavy strength training (47), yet the training program we utilized was whole-body, high-intensity training, which likely recruited all fiber types. We also discovered that those who had lower initial SC content in MHC I fibers experienced the greatest change in MHC I SC per fiber (r = −0.529, P < 0.001) and MHC I myonuclei per fiber (r = −0.383, P = 0.006). However, this effect was absent in MHC II fibers. These data suggest that myonuclear addition was a primary fate of SC in MHC I fibers. Our data are in agreement with Bellany et al. (5) but in contrast with a previous report (37) in the literature, suggesting that a higher pretraining SC content is a characteristic of high-responders to RET. We are unsure as to why this difference exists in the literature, but we suspect that the differences could be explained by the use of different markers of SC (NCAM for Petrella et al. and Pax7 in this manuscript and with Bellany et al.)
Myonuclear accretion occurred with RET, as has been previously demonstrated (36), but was not different by treatment as has been demonstrated elsewhere (33,44). A significant increase was seen with PB and WP but not MDP treatment. Others have suggested that CSA changes greater than ~15% are needed before changes in myonuclear number occur (25,36). Here we demonstrated 15%–20%, ~20%, and 20%–30% increases in CSA of MHC I, II, and hybrid fibers, respectively, suggesting that our larger sample size included enough participants with substantial changes in CSA to detect changes in myonuclear number with RET. Myonuclear number was highly correlated with fiber size at each time point and in all fiber types (r = 0.724–0.826, P < 0.001), illustrating remarkable control of the myonuclear domain, as others have shown (20,24,26,29,30).
Even with such tight coupling of myonuclear number to myofiber size, we observed a slight but significant expansion of the myonuclear domain, ~150 μm2 per myonucleus, after 3 months of RET. In fact, a significant, inverse relationship (r = −0.634, P < 0.001) was demonstrated, indicating that those with smaller initial myonuclear domains experienced the greatest change in myonuclear domain over the course of the training. This effect was most evident in MHC II fibers, highlighting their remarkable plasticity to this contractile stimulus. Maintenance of this expanded domain was likely assisted by increased total RNA content (translational capacity), and through increases in myonuclear size, as demonstrated by Cabric et al. (7) in human skeletal muscle after 3 wk of electrical stimulation. This would suggest enhanced transcriptional capacity per myonucleus.
Certainly, many studies, including those from our laboratory, have clearly demonstrated a robust effect of protein/amino acids to stimulate the early metabolic response of muscle growth (i.e., muscle protein synthesis) (39). The question persists as to why these effects are not as readily discovered in physiological outcomes after chronic exposure to such a stimulus (35). Our hypothesis is that physiological adaptation may best explain the insensitivity to protein supplementation typically seen in chronic exercise studies. Farup et al. (11) demonstrated that WP supplementation after eccentric exercise accelerated the SC pool expansion compared with consumption of carbohydrate placebo. However, by 168 h postexercise (11) and after 12 wk of training (12), the SC pool was identical between treatments. For novice exercisers, peak SC activity occurs after 2 wk of RET (18). Also, some evidence suggests that the majority of the SC pool expansion occurs early, 1–4 wk into RET, during dietary supplementation (32). These data suggest that protein supplementation may provide an enhancement early during exercise training, but additional protein is unlikely to confer added benefit to further promote muscle growth as adaptation occurs. Interestingly, this time frame is also when most myofiber damage and remodeling is likely to occur. Although attractive, this hypothesis has not yet been clearly proven (34,35). Protein metabolism also becomes more efficient after resistance training (80, 81), which provides further support that in the presence of a well-balanced diet, muscle hypertrophy, and strength are not further augmented by protein supplementation (35).
A limitation to this study is that several samples from the WP group were not suitable for immunohistochemical analysis and as a result the sample size of that group was smaller than the size of the other treatments. It is possible that we were slightly underpowered in our ability to determine certain exercise effects (myonuclear domain or number); however, statistical analysis clearly demonstrated an absence of treatment differences in most outcomes, suggesting that sample size was not an issue in delineating treatment effects. It was not feasible for us to sample at earlier time points throughout the training, although this may have provided greater insight into the effect of protein supplementation. This would have allowed a preferential examination of SC content, myonuclear domain, and myonuclear addition during RET. Also, although many of the inferences were made using correlational analyses, a major strength of this study is that a cohort of this size makes correlational analysis possible and generates additional research questions.
The majority of similar studies were collected after biopsies at 24–48 h after the last exercise session; however, we took our samples at 72 h postexercise. It could be hypothesized that this 72-h time point was examining the acute effects of the last exercise session. We found only one paper that examined the acute response (in the trained state) demonstrating an increase in SC content at 72 h after exercise and a return to basal–pretraining values at 96 h (4 d) posttraining. This recent study may suggest that our postbiopsy effects could be due to the acute exercise response. However, there are several studies in the literature with conflicting results showing that increases in SC content are detected 4 d post-RET (28,42,46) and after 10 d of detraining (25). These conflicting data suggest that the timing of sampling for SC studies should be an important consideration in designing these studies.
Daily supplementation of protein during RET did not enhance muscle adaptations in the vastus lateralis as demonstrated by the nearly identical increases in muscle strength, hypertrophy (whole muscle and myofiber-type specific), MHC II SC content, and overall myonuclear addition. When results from the soy–dairy PB and WP treatments were pooled, very modest effects of protein supplementation existed to enhance MHC I SC content, isokinetic torque, and a slight expansion of a greater proportion of larger MHC II fibers over placebo after RET. We conclude that protein supplementation during RET has a modest effect on promoting a larger gain in whole-body lean mass as compared with exercise training without protein supplementation. However, protein supplementation does not enhance RE-induced increases in myofiber hypertrophy, SC content or myonuclear addition in young healthy men. We propose that as long as protein intake is adequate during muscle overload the adaptations in muscle growth and function will not be influenced by protein supplementation. Future work should focus on the effectiveness of protein supplementation or increasing daily protein intake in clinical populations undergoing significant muscle atrophy.
The (PAX7 and BA.D5) developed by A. Kawakami and S. Schiaffino were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the National Institutes of Health (NIH) and maintained at the Department of Biology, University of Iowa, Iowa City, IA 52242. The authors thank the Clinical Research staff of the Institute for Translational Science Clinical Research Center at UTMB for assisting in screening and consenting patients and participants and for assisting in data collection. They also thank DPT Samantha Dillon, DPT Matthew Nguyen, SPT Benjamin Brightwell, Camille Brightwell, and SPT Jennifer Thedinga for their assistance in supervising the exercise training of research participants and Michael Borack, Jared Dickinson, Melissa Markofski, and Syed Husaini for assistance during the clinical portion of the study. They also thank Dr. Marinel Ammenheuser for editing the manuscript.
This project was supported by a grant from DuPont Protein Solutions with assistance from NIH R01 AR49877, T32-HD07539, and NIDRR H133P110012, in part by an NIH Clinical and Translational Science Award UL1TR000071 from the National Center for Advancing Translational Sciences and from NIH/NIA grant P30 AG024832.
This was a subset of the trial registered at clinicaltrials.gov as NCT01749189.
P. T. R., M. S. B., R. R. D., S. I., M. B. C., R. M., K. J., E. V., and B. B. R. have no conflicts of interest.
B. B. R., E. V., P. T. R., R. R. D., M. B. C., and R. M. designed the research; P. T. R., C. S. F., B. B. R., S. I., M. S. B., and R. R. D. conducted research; B. B. R., E. V., P. T. R., S. H. H., R. R. D., C. S. F., K. J., M. B. C., and R. M. reviewed the manuscript; P. T. R., R. R. D., M. S. B., K. J., and B. B. R. analyzed data; and P. T. R. and B. B. R. wrote the manuscript and had primary responsibility for final content. M. B. C. and R. M. were not involved with conducted research or laboratory analysis. All authors read and approved the final manuscript.
The authors declare that this study was funded by Dupont Nutrition and Health. Representatives from Dupont Nutrition and Health were not involved with data collection and laboratory analysis.
The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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MUSCLE; HYPERTROPHY; MYOFIBER; PROTEIN TYPE; WHEY; SOY; RESISTANCE; EXERCISE; TRAINING
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