Skeletal muscle myosin heavy chain protein fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy

Abstract

We sought to examine how resistance exercise (RE), cycling exercise, and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation in humans. In the first study (1boutRE), younger adult men (n=8; 5±2 years of RE experience) performed a lower body RE bout with vastus lateralis (VL) biopsies obtained immediately before, 3-, and 6-hours post-exercise. In the second study (10weekRT), VL biopsies were obtained in untrained younger adults (n=36, 18 men and 18 women) before and 24 hours (24h) after their first/naïve RE bout. These participants also engaged in 10 weeks (24 sessions) of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined from a third acute cycling study (n=7) and a fourth study involving two weeks of leg immobilization (n=20, 15 men and 5 women) to determine how MyHC fragmentation was affected. In the 1boutRE study, the fragmentation of all MyHC isoforms (MyHC_Total) increased 3 hours post-RE (~ +200%, p=0.018) and returned to pre-exercise levels by 6 hours post-RE. Immunoprecipitation of MyHC_Total revealed ubiquitination levels remained unaffected at the 3- and 6-hour post-RE time points. Interestingly, a greater increase in magnitude for MyHC type IIa versus I isoform fragmentation occurred 3-hours post-RE (8.6±6.3-fold versus 2.1±0.7-fold, p=0.018). In all 10weekRT participants, the first/naïve and last RE bouts increased MyHC_Total fragmentation 24h post-RE (+65% and +36%, respectively; p<0.001); however, the last RE bout response was attenuated compared to the first bout (p=0.045). The first/naïve bout response was significantly elevated in females only (p<0.001), albeit females also demonstrated a last bout attenuation response (p=0.002). Although an acute cycling bout did not alter MyHC_Total fragmentation, ~8% VL atrophy with two weeks of leg immobilization led to robust MyHC_Total fragmentation (+108%, p<0.001), and no sex-based differences were observed. In summary, RE and disuse atrophy increase MyHC protein fragmentation. A dampened response with 10 weeks of resistance training, and more refined responses in well-trained men, suggest this is an adaptive process. Given the null polyubiquitination IP findings, more research is needed to determine how MyHC fragments are processed. Moreover, further research is needed to determine how aging and disease-associated muscle atrophy affect these outcomes, and whether MyHC fragmentation is a viable surrogate for muscle protein turnover rates.

Keywords: immunoblotting; myosin heavy chain; proteolysis; resistance exercise; skeletal muscle.

Figure 1.. Summary of human studies

Schematic (drawn using Biorender.com) illustrates the study logistics and participant number for each study whereby MyHC analyses occurred. More details related to each study can be found in-text.

Figure 2.. Evidence of post-exercise myofibril protein fragmentation in 1boutRE study participants

As discussed in-text, preliminary 1boutRE experiments were performed on two well-trained participants’ myofibril isolates aiming to examine the presence of titin using 4-15% SDS-PAGE gels and Coomassie staining. In both participants, visual myofibril protein fragments were observed in the myosin heavy chain (MyHC) kilodalton region 3 hours following the resistance exercise bout. Conversely, the rapid disappearance of these fragments was evident by the 6-hour post-exercise time point.

Figure 3.. Post-exercise MyHC_Total fragmentation in 1boutRE participants

Data from well-trained 1boutRE men (n=8) show that significant total myosin heavy chain (MyHC_Total) fragmentation is evident in the myofibril fraction 3 hours following a resistance exercise bout (panel a); however, the rapid (and significant) disappearance of these fragments was evident by the 6-hour post-exercise time point. Also notable is the high presence of MyHC_Total fragments in the cytoplasmic fraction in several participants (panel b); however, this did not reach statistical significance. Representative immunoblots are shown for 2 of 8 participants, and data are presented as mean and standard deviation values with repeated measures (RM) ANOVA p-values.

Figure 4.. Type I versus IIa MyHC isoform fragmentation in 1boutRE participants

Data from well-trained 1boutRE men (n=8) show that significant myosin heavy chain (MyHC) fragmentation of the type I and IIa isoforms is evident in the myofibril fraction 3 hours following the resistance exercise bout (panel a); however, as with MyHC_Total fragments, the rapid (and significant) disappearance of I and IIa fragments was evident by the 6-hour post-exercise time point. Also notable were the different patterns of fragmentation between isoforms, with lighter molecular weight type I isoform fragments appearing post-RE versus heavier type IIa fragments. Representative immunoblots are shown for 2 of 8 participants, and data are presented as mean and standard deviation values with two-way (isoform*time) ANOVA time and interaction p-values. Panel b shows lane profiles of type I and IIa isoform fragmentation from two different participants where “*” indicates fragments detected by analysis software.

Figure 5.. Total myofibril protein and MyHC_Total poly-ubiquitination in 1boutRE participants

Data from well-trained 1boutRE men (n=8) show that total myofibril protein polyubiquitination levels remain unaltered post-exercise (panel a). Additionally, the polyubiquitination signal on immunoprecipitated MyHC fragments (spanning ~15-50 kD) remained unaltered 3- and 6-hours post-exercise when data were normalized to the IP: MyHC signal (panel b). Representative immunoblots are shown for 2 of 8 participants, and data are presented as mean and standard deviation values with one-way repeated measures (RM) ANOVA p-values.

Figure 6.. 24-hour post-exercise MyHC_Total fragmentation in the untrained and trained states in 10weekRT participants

Data from all 10weekRT participants (n=36) show that significant total myosin heavy chain (MyHC_Total) fragmentation is evident in the whole tissue lysate 24 hours following the first/naïve resistance exercise bout (panel a). While this same 24-hour post-exercise response occurs following 10 weeks of training (24 leg extensor sessions), it is significantly attenuated. Sex analysis in 10weekRT participants (18 men and 18 women) show that the 24-hour first bout RE responses in panel a are largely driven by females (panel b). Representative immunoblots are shown for 3 participants, and data are presented as mean and standard deviation values with two-way (training state*time) ANOVA main effect and interaction p-values.

Figure 7.. MyHC_Total fragmentation is absent following a cycling exercise bout

Data from cycling study participants (n=7) show that total myosin heavy chain (MyHC_Total) fragmentation is not significantly altered 2- and 8-hours following a 60-minute cycling exercise bout (panel a). Representative immunoblots are shown for 2 participants.

Figure 8.. MyHC_Total fragmentation increases following two weeks of disuse atrophy

Data from two-week disuse participants (n=20) show that VL muscle atrophy occurs with lower-limb immobilization (determined by ultrasound, panel a), and that this coincides with significant total myosin heavy chain (MyHC_Total) fragmentation (panel b). Representative immunoblots are shown for 3 participants, and data are presented as mean and standard deviation values with dependent t-test p-values.