Our laboratory has performed various experiments examining the proteomic alterations that occur with mechanical overload (MOV)-induced skeletal muscle hypertrophy. In the current study we first sought to determine how 10 weeks of resistance training in 15 college-aged females affected protein concentrations in different tissue fractions. Training, which promoted significantly lower body muscle- and fiber-level hypertrophy, notably increased sarcolemmal/membrane protein content (+10.1%, p<0.05). Sarcolemmal/membrane protein isolates were queried using mass spectrometry-based proteomics, ∼10% (38/387) of proteins associated with the sarcolemma were up-regulated (>1.5-fold, p<0.05), and one of these targets (the intermediate filament vimentin; VIM) warranted further mechanistic investigation. VIM expression was first examined in the plantaris muscles of 4-month-old C57BL/6J mice following 10- and 20-days of MOV via synergist ablation. Relative to Sham (control) mice, VIM mRNA and protein content was significantly higher in MOV mice and immunohistochemistry indicated that VIM was predominantly present in the extracellular matrix (ECM). The 10- and 20-day MOV experiments were replicated in Pax7-DTA (tamoxifen-induced, satellite cell depleted) mice, which reduced the presence of VIM in the ECM. Finally, a third set of 10- and 20-day MOV experiments were performed in C57BL/6 mice intramuscularly injected with either AAV9-scrambled (control) or AAV9-VIM shRNA. While VIM shRNA mice presented with lower VIM in the ECM (∼50%), plantaris masses in response to MOV were similar between the injection groups. However, VIM shRNA mice presented with appreciably more MyHC emb-positive fibers with centrally located nuclei, indicating a regenerative phenotype. Using an integrative approach, we propose that skeletal muscle VIM is a mechanosensitive target predominantly localized to the ECM, and satellite cells are involved in its expression. Moreover, a disruption in VIM expression during MOV leads to dysfunctional skeletal muscle hypertrophy.
Skeletal muscle is a highly dynamic tissue that can undergo marked changes such as an increase in size (i.e., hypertrophy). The most potent stimulus for skeletal muscle hypertrophy is mechanical overload, either through progressive resistance training in humans or synergist ablation in rodents. Mechano-sensitive protein signaling pathways are responsible for relaying the mechanical perturbations and tension placed on skeletal muscle into downstream signals for adaptations to occur. A major pathway for mechanotransduction involves transmembrane proteins (integrins) and various accessory proteins that interact with the extracellular matrix (ECM), cytoskeletal network, and sarcolemma to propagate signals that enhance muscle protein synthesis.
Cytoskeletal and intermediate filaments not only provide structural support to the cell, but are also involved in processes like signal transmission, mechanotransduction, and gene regulation. Intermediate filaments in muscle cells have also been linked to development, regeneration, and growth. Desmin is a commonly studied muscle-specific gene that has been implicated in several processes that govern skeletal muscle homeostasis and is responsive to mechanical loading. In contrast, the intermediate filament vimentin (VIM) is predominately expressed in immature developing myofibers and decreases in mature myofibers. Interestingly, increased VIM expression is linked to active and proliferating satellite cells and appears to be critical for myofiber regeneration. VIM also plays a key role in several signaling pathways including mTORC1. Most notably, while not in skeletal muscle, a recent study reported that VIM-knockout cells had a reduction in mTORC1 signaling, blunted protein synthesis, and affected cell size. However, the association of VIM with skeletal muscle hypertrophy has not been investigated, and its role in hypertrophy remains to be established.
Taken together, skeletal muscle hypertrophy in response to mechanical loading is dependent on several tightly regulated pathways and it appears that various ECM, cytoskeletal and intermediate filament-related proteins play important roles in this process. Furthermore, VIM has been shown to regulate cell size and protein synthesis response in other cell types. Herein, we performed shotgun proteomics on the sarcolemmal/ECM fraction of human skeletal muscle and show that: i) 10 weeks of resistance training increases VIM protein expression, and ii) a significant positive correlation exists between training-induced VIM protein levels and changes in mean muscle fiber cross-sectional area. Through a series of experiments in mice subjected to mechanical overload, we show that VIM expression in the ECM is upregulated during synergist ablation-induced skeletal muscle hypertrophy, and this response is dampened in mice lacking satellite cells. Finally, viral-mediated knockdown experiments in mice subjected to synergist ablation show that a blunting of VIM expression does not affect gross measures of hypertrophy in skeletal muscle but may delay/impair the regeneration and remodeling processes. Collectively, these data support that the upregulation of VIM occurs with mechanical overload-induced skeletal muscle hypertrophy, this process is partially dependent upon satellite cells, and a sufficient upregulation of VIM is needed to support proper skeletal muscle hypertrophy.
Human muscle was from a study that was approved by the Auburn University Institutional Review Board (IRB) (Protocol # 19-249 MDR 1907), conformed to the standards set by the latest revisions of the Declaration of Helsinki, and was registered as a clinical trial (NCT04707963). The intent of the trial was to determine whether daily peanut protein supplementation (SUP), or no supplementation (control group [CON]), affected resistance training-related adaptations. In the present study we utilized the remaining muscle samples, which included nine women from the SUP group and six women from the CON group. Our sub-sample showed no differences between SUP and CON mean fiber cross-sectional area (fCSA), P = 0.264), vastus lateralis cross-sectional area (by ultrasonography) (P = 0.421), thigh cross-sectional area (by peripheral computed tomography) (P = 0.855), as previously demonstrated. Therefore, we combined data from the SUP and CON groups to perform the analyses presented herein. The samples were from 15 females (age: 21 ± 2 years, body mass index: 24.6 ± 3.7 kg/m 2) who had not participated in resistance training programs, who were free of obvious cardiovascular or metabolic disease or other conditions contraindicating participation in exercise programs or donating muscle biopsies, and who were not pregnant or trying to become pregnant.
Baseline assessments included muscle imaging assessments and vastus lateralis biopsies obtained after an overnight fast. Immediately afterwards, participants engaged in familiarization sessions with the RT protocol and test procedures. Approximately 3-5 days following familiarization sessions, the participants initiated an RT program with 2 sessions per week for 10 weeks. Seventy-two hours after the last RT session all assessments (including the procurement of a muscle biopsy) were repeated. For more details related to the study design and resistance training program, readers are referred to Sexton et al.
Pre- and post-intervention vastus lateralis muscle biopsies were performed from the right leg. Initially the participants laid down on an athletic table where the upper thigh was shaved and cleaned with 70% isopropanol. A subcutaneous injection of 1% lidocaine (0.8 mL) was administered. After 5 minutes, the area was cleaned with chlorhexidine solution. Thereafter, a 7 mm-width pilot incision was made through the dermis with a sterile No. 11 surgical blade (AD Surgical; Sunnyvale, CA, USA). A sterile 5-gauge biopsy needle was then inserted into the pilot incision, through the muscle fascia, and ∼2 cm into the muscle (for a total depth of ∼4-7 cm) where a 40-80 mg sample was collected while applying suction. Following biopsies, tissue was rapidly teased of blood and connective tissue. A portion of the tissue (∼10–20mg) was preserved in optimal cutting temperature (OCT) media for histology (Tissue-Tek®, Sakura Finetek Inc.; Torrance, CA, USA), slowly frozen in liquid nitrogen-cooled isopentane, and subsequently stored at −80°C. Another portion of the tissue (∼30–50mg) was placed in pre-labeled foils, flash frozen in liquid nitrogen, and subsequently stored at −80°C for other molecular analyses described below. Tissue triage procedures were performed over a 3-minute period.
The resistance training program was described in detail previously. Briefly, resistance training bouts consisted of participants performing a 45° leg press, leg extension, hex-bar deadlifts, flat barbell bench press, and wide-grip cable pulldown exercises. The same exercise order was followed for all training sessions, and two day per week training sessions consisted of performing 4 sets of 10 repetitions for each exercise one day per week, and 5 sets of 6 repetitions for each exercise one day per week. Two-minute rest intervals between sets and exercises were allotted throughout the training intervention. The training load was progressively increased on a session-by-session basis using the rating of perceived exertion (RPE) scale of 1-10 (“really easy” to “really hard”) so that each set was performed close to concentric muscle failure.
Isolation of sarcoplasmic and sarcolemmal protein were initially isolated from muscle tissue using a high-fidelity membrane protein extraction kit according to the manufacturer’s recommendations (Mem-PRE Plus Membrane Protein Extraction Kit; Thermo Fisher Scientific; Waltham, MA, USA). This process yielded solubilized sarcoplasmic and sarcolemmal protein isolates.
The protein concentrations of both fractions were determined in duplicate on the same day as protein isolations to minimize freeze-thaw effects using a commercially available assay (bicinchoninic acid [BCA] Protein Assay Kit; Thermo Fisher Scientific). Thereafter, the fractions were stored at -80°C until subsequent analyses. The duplicate coefficient of variation (CV) values for the sarcoplasmic and sarcolemmal proteins readings were 2.3% and 2.4%, respectively.
Sarcolemmal protein isolates were shipped to a commercial vendor for proteomics using a nanoLC-MS/MS platform (Creative Proteomics; Shirley, NY, USA). Briefly, the samples were treated with 50 mM ammonium bicarbonate and the resultant solutions were transferred to Microcon devices YM-10 (Millipore; Burlington, MA, USA). The samples were then centrifuged at 12,000 g at 4°C for 10 minutes. Protein concentrates were again treated with 50 mM ammonium bicarbonate and centrifuged again. Concentrates were then reduced using 10 mM DTT at 56°C for 1 hour and alkylated with 20 mM indole-3-acetic acid at room temperature in dark for 1 hour. Following reduction reactions, and one wash with 50 mM ammonium bicarbonate and centrifugation (12,000g at 4°C for 10 minutes), concentrates were incubated with 100 μL of 50 mM ammonium bicarbonate and free trypsin (ratio of 1:50) at 37°C overnight. The samples were then centrifuged at 12,000 g at 4°C for 10 minutes, and 100 μL of 50 mM ammonium bicarbonate was added to Microcon devices and centrifuged (for a total of two washes). Peptides were then isolated, lyophilized to near dryness, and resuspend in 20 μL of 0.1% formic acid for LC-MS/MS analysis.
Nanoflow ultra-high-performance liquid chromatography (UHPLC) was performed using an Ultimate 3000 nano UHPLC system (Thermo Fisher Scientific). Associated hardware included a trapping column (PepMap C18, 100
