A novel deep proteomic approach in human skeletal muscle unveils distinct molecular signatures affected by aging and resistance training.

The skeletal muscle proteome alterations to aging and resistance training have been reported in prior studies. However, conventional proteomics in skeletal muscle typically yields wide protein abundance ranges that mask the detection of lowly expressed proteins. Thus, we adopted a novel deep proteomics approach whereby myofibril (MyoF) and non-MyoF fractions were separately subjected to protein corona nanoparticle complex formation prior to digestion and Liquid Chromatography Mass Spectrometry (LC-MS). Specifically, we investigated MyoF and non-MyoF proteomic profiles of the vastus lateralis muscle of younger (Y, 22±2 years old; n=5) and middle-aged participants (MA, 56±8 years old; n=6). Additionally, MA muscle was analyzed following eight weeks of resistance training (RT, 2d/week). Across all participants, the number of non-MyoF proteins detected averaged to be 5,645±266 (range: 4,888-5,987) and the number of MyoF proteins detected averaged to be 2,611±326 (range: 1,944-3,101). Differences in the non-MyoF (8.4%) and MyoF (2.5%) proteomes were evident between age cohorts, and most differentially expressed non-MyoF proteins (447/543) were more enriched in MA versus Y. Biological processes in the non-MyoF fraction were predicted to be operative in MA versus Y including increased cellular stress, mRNA splicing, translation elongation, and ubiquitin-mediated proteolysis. RT in MA participants only altered ~0.3% of MyoF and ~1.0% of non-MyoF proteomes. In summary, aging and RT predominantly affect non-contractile proteins in skeletal muscle. Additionally, marginal proteome adaptations with RT suggest more rigorous training may stimulate more robust effects or that RT, regardless of age, subtly alters basal state skeletal muscle protein abundances.

The effects of resistance training on denervated myofibers, senescent cells, and associated protein markers in middle-aged adults.

Denervated myofibers and senescent cells are hallmarks of skeletal muscle aging. However, sparse research has examined how resistance training affects these outcomes. We investigated the effects of unilateral leg extensor resistance training (2 days/week for 8 weeks) on denervated myofibers, senescent cells, and associated protein markers in apparently healthy middle-aged participants (MA, 55 ± 8 years old, 17 females, 9 males). We obtained dual-leg vastus lateralis (VL) muscle cross-sectional area (mCSA), VL biopsies, and strength assessments before and after training. Fiber cross-sectional area (fCSA), satellite cells (Pax7+), denervated myofibers (NCAM+), senescent cells (p16+ or p21+), proteins associated with denervation and senescence, and senescence-associated secretory phenotype (SASP) proteins were analyzed from biopsy specimens. Leg extensor peak torque increased after training (p < .001), while VL mCSA trended upward (interaction p = .082). No significant changes were observed for Type I/II fCSAs, NCAM+ myofibers, or senescent (p16+ or p21+) cells, albeit satellite cells increased after training (p = .037). While >90% satellite cells were not p16+ or p21+, most p16+ and p21+ cells were Pax7+ (>90% on average). Training altered 13 out of 46 proteins related to muscle-nerve communication (all upregulated, p < .05) and 10 out of 19 proteins related to cellular senescence (9 upregulated, p < .05). Only 1 out of 17 SASP protein increased with training (IGFBP-3, p = .031). In conclusion, resistance training upregulates proteins associated with muscle-nerve communication in MA participants but does not alter NCAM+ myofibers. Moreover, while training increased senescence-related proteins, this coincided with an increase in satellite cells but not alterations in senescent cell content or SASP proteins. These latter findings suggest shorter term resistance training is an unlikely inducer of cellular senescence in apparently healthy middle-aged participants. However, similar study designs are needed in older and diseased populations before definitive conclusions can be drawn.

Resistance training in humans and mechanical overload in rodents do not elevate muscle protein lactylation.

Although several reports have hypothesized that exercise may increase skeletal muscle protein lactylation, empirical evidence in humans is lacking. Thus, we adopted a multi-faceted approach to examine if acute and subchronic resistance training (RT) altered skeletal muscle protein lactylation levels. In mice, we also sought to examine if surgical ablation-induced plantaris hypertrophy coincided with increases in muscle protein lactylation. To examine acute responses, participants' blood lactate concentrations were assessed before, during, and after eight sets of an exhaustive lower body RT bout (n = 10 trained college-aged men). Vastus lateralis biopsies were also taken before, 3-h post, and 6-h post-exercise to assess muscle protein lactylation. To identify training responses, another cohort of trained college-aged men (n = 14) partook in 6 weeks of lower-body RT (3x/week) and biopsies were obtained before and following the intervention. Five-month-old C57BL/6 mice were subjected to 10 days of plantaris overload (OV, n = 8) or served as age-matched sham surgery controls (Sham, n = 8). Although acute resistance training significantly increased blood lactate responses ∼7.2-fold (p < 0.001), cytoplasmic and nuclear protein lactylation levels were not significantly altered at the post-exercise time points, and no putative lactylation-dependent mRNA was altered following exercise. Six weeks of RT did not alter cytoplasmic protein lactylation (p = 0.800) despite significantly increasing VL muscle size (+3.5%, p = 0.037), and again, no putative lactylation-dependent mRNA was significantly affected by training. Plantaris muscles were larger in OV versus Sham mice (+43.7%, p < 0.001). However, cytoplasmic protein lactylation was similar between groups (p = 0.369), and nuclear protein lactylation was significantly lower in OV versus Sham mice (p < 0.001). The current null findings, along with other recent null findings in the literature, challenge the thesis that lactate has an appreciable role in promoting skeletal muscle hypertrophy.

Hip thrust and back squat training elicit similar gluteus muscle hypertrophy and transfer similarly to the deadlift.

We examined how set-volume equated resistance training using either the back squat (SQ) or hip thrust (HT) affected hypertrophy and various strength outcomes. Untrained college-aged participants were randomized into HT (n = 18) or SQ (n = 16) groups. Surface electromyograms (sEMG) from the right gluteus maximus and medius muscles were obtained during the first training session. Participants completed 9 weeks of supervised training (15-17 sessions), before and after which gluteus and leg muscle cross-sectional area (mCSA) was assessed via magnetic resonance imaging. Strength was also assessed prior to and after the training intervention via three-repetition maximum (3RM) testing and an isometric wall push test. Gluteus mCSA increases were similar across both groups. Specifically, estimates [(-) favors HT (+) favors SQ] modestly favored the HT versus SQ for lower [effect ±SE, -1.6 ± 2.1 cm2; CI95% (-6.1, 2.0)], mid [-0.5 ± 1.7 cm2; CI95% (-4.0, 2.6)], and upper [-0.5 ± 2.6 cm2; CI95% (-5.8, 4.1)] gluteal mCSAs but with appreciable variance. Gluteus medius + minimus [-1.8 ± 1.5 cm2; CI95% (-4.6, 1.4)] and hamstrings [0.1 ± 0.6 cm2; CI95% (-0.9, 1.4)] mCSA demonstrated little to no growth with small differences between groups. mCSA changes were greater in SQ for the quadriceps [3.6 ± 1.5 cm2; CI95% (0.7, 6.4)] and adductors [2.5 ± 0.7 cm2; CI95% (1.2, 3.9)]. Squat 3RM increases favored SQ [14 ± 2 kg; CI95% (9, 18),] and hip thrust 3RM favored HT [-26 ± 5 kg; CI95% (-34, -16)]. 3RM deadlift [0 ± 2 kg; CI95% (-4, 3)] and wall push strength [-7 ± 12N; CI95% (-32, 17)] similarly improved. All measured gluteal sites showed greater mean sEMG amplitudes during the first bout hip thrust versus squat set, but this did not consistently predict gluteal hypertrophy outcomes. Squat and hip thrust training elicited similar gluteal hypertrophy, greater thigh hypertrophy in SQ, strength increases that favored exercise allocation, and similar deadlift and wall push strength increases.

Proteolytic markers associated with a gain and loss of leg muscle mass with resistance training followed by high-intensity interval training.

We recently reported that vastus lateralis (VL) cross-sectional area (CSA) increases after 7 weeks of resistance training (RT, 2 days/week), with declines occurring following 7 weeks of subsequent treadmill high-intensity interval training (HIIT) (3 days/week). Herein, we examined the effects of this training paradigm on skeletal muscle proteolytic markers. VL biopsies were obtained from 11 untrained college-aged males at baseline (PRE), after 7 weeks of RT (MID), and after 7 weeks of HIIT (POST). Tissues were analysed for proteolysis markers, and in vitro experiments were performed to provide additional insights. Atrogene mRNAs (TRIM63, FBXO32, FOXO3A) were upregulated at POST versus both PRE and MID (P < 0.05). 20S proteasome core protein abundance increased at POST versus PRE (P = 0.031) and MID (P = 0.049). 20S proteasome activity, and protein levels for calpain-2 and Beclin-1 increased at MID and POST versus PRE (P < 0.05). Ubiquitinated proteins showed model significance (P = 0.019) with non-significant increases at MID and POST (P > 0.05). in vitro experiments recapitulated the training phenotype when stimulated with a hypertrophic stimulus (insulin-like growth factor 1; IGF1) followed by a subsequent AMP-activated protein kinase activator (5-aminoimidazole-4-carboxamide ribonucleotide; AICAR), as demonstrated by larger myotube diameter in IGF1-treated cells versus IGF1 followed by AICAR treatments (I+A; P = 0.017). Muscle protein synthesis (MPS) levels were also greater in IGF1-treated versus I+A myotubes (P < 0.001). In summary, the loss in RT-induced VL CSA with HIIT coincided with increases in several proteolytic markers, and sustained proteolysis may have driven this response. Moreover, while not measured in humans, we interpret our in vitro data to suggest that (unlike RT) HIIT does not stimulate MPS. NEW FINDINGS: What is the central question of this study? Determining if HIIT-induced reductions in muscle hypertrophy following a period of resistance training coincided with increases in proteolytic markers. What is the main finding and its importance? Several proteolytic markers were elevated during the HIIT training period implying that increases in muscle proteolysis may have played a role in HIIT-induced reductions in muscle hypertrophy.

Hip thrust and back squat training elicit similar gluteus muscle hypertrophy and transfer similarly to the deadlift.

Purpose: We examined how set-volume equated resistance training using either the back squat (SQ) or hip thrust (HT) affected hypertrophy and various strength outcomes. Methods: Untrained college-aged participants were randomized into HT or SQ groups. Surface electromyograms (sEMG) from the right gluteus maximus and medius muscles were obtained during the first training session. Participants completed nine weeks of supervised training (15-17 sessions), before and after which we assessed muscle cross-sectional area (mCSA) via magnetic resonance imaging and strength via three-repetition maximum (3RM) testing and an isometric wall push test. Results: Glutei mCSA growth was similar across both groups. Estimates [(-) favors HT; (+) favors SQ] modestly favored the HT compared to SQ for lower [effect ± SE, -1.6 ± 2.1 cm2], mid [-0.5± 1.7 cm2], and upper [-0.5 ± 2.6 cm2], but with appreciable variance. Gluteus medius+minimus [-1.8 ± 1.5 cm2] and hamstrings [0.1 ± 0.6 cm2] mCSA demonstrated little to no growth with small differences between groups. Thigh mCSA changes were greater in SQ for the quadriceps [3.6 ± 1.5 cm2] and adductors [2.5 ± 0.7 cm2]. Squat 3RM increases favored SQ [14 ± 2.5 kg] and hip thrust 3RM favored HT [-26 ± 5 kg]. 3RM deadlift [0 ± 2 kg] and wall push strength [-7 ± 13 N] similarly improved. All measured gluteal sites showed greater mean sEMG amplitudes during the first bout hip thrust versus squat set, but this did not consistently predict gluteal hypertrophy outcomes. Conclusion: Nine weeks of squat versus hip thrust training elicited similar gluteal hypertrophy, greater thigh hypertrophy in SQ, strength increases that favored exercise allocation, and similar strength transfers to the deadlift and wall push.

A novel deep proteomic approach in human skeletal muscle unveils distinct molecular signatures affected by aging and resistance training.

We examined the myofibrillar (MyoF) and non-myofibrillar (non-MyoF) proteomic profiles of the vastus lateralis (VL) muscle of younger (Y, 22±2 years old; n=5) and middle-aged participants (MA, 56±8 years old; n=6), and MA following eight weeks of knee extensor resistance training (RT, 2d/week). Shotgun/bottom-up proteomics in skeletal muscle typically yields wide protein abundance ranges that mask lowly expressed proteins. Thus, we adopted a novel approach whereby the MyoF and non-MyoF fractions were separately subjected to protein corona nanoparticle complex formation prior to digestion and Liquid Chromatography Mass Spectrometry (LC-MS) analysis. A total of 10,866 proteins (4,421 MyoF and 6,445 non-MyoF) were identified. Across all participants, the number of non-MyoF proteins detected averaged to be 5,645±266 (range: 4,888-5,987) and the number of MyoF proteins detected averaged to be 2,611±326 (range: 1,944-3,101). Differences in the non-MyoF (8.4%) and MyoF (2.5%) proteome were evident between age cohorts. Further, most of these age-related non-MyoF proteins (447/543) were more enriched in MA versus Y. Several biological processes in the non-MyoF fraction were predicted to be operative in MA versus Y including (but not limited to) increased cellular stress, mRNA splicing, translation elongation, and ubiquitin-mediated proteolysis. Non-MyoF proteins associated with splicing and proteostasis were further interrogated, and in agreement with bioinformatics, alternative protein variants, spliceosome-associated proteins (snRNPs), and proteolysis-related targets were more abundant in MA versus Y. RT in MA non-significantly increased VL muscle cross-sectional area (+6.5%, p=0.066) and significantly increased knee extensor strength (+8.7%, p=0.048). However, RT modestly altered the MyoF (~0.3%, 11 upregulated and two downregulated proteins) and non-MyoF proteomes (~1.0%, 56 upregulated and eight downregulated proteins, p<0.01). Further, RT did not affect predicted biological processes in either fraction. Although participant numbers were limited, these preliminary results using a novel deep proteomic approach in skeletal muscle suggest that aging and RT predominantly affects protein abundances in the non-contractile protein pool. However, the marginal proteome adaptations occurring with RT suggest either: a) this may be an aging-associated phenomenon, b) more rigorous RT may stimulate more robust effects, or c) RT, regardless of age, subtly affects skeletal muscle protein abundances in the basal state.

The effects of resistance training to near failure on strength, hypertrophy, and motor unit adaptations in previously trained adults.

Limited research exists examining how resistance training to failure affects applied outcomes and single motor unit characteristics in previously trained individuals. Herein, resistance-trained adults (24 ± 3 years old, self-reported resistance training experience was 6 ± 4 years, 11 men and 8 women) were randomly assigned to either a low-repetitions-in-reserve (RIR; i.e., training near failure, n = 10) or high-RIR (i.e., not training near failure, n = 9) group. All participants implemented progressive overload during 5 weeks where low-RIR performed squat, bench press, and deadlift twice weekly and were instructed to end each training set with 0-1 RIR. high-RIR performed identical training except for being instructed to maintain 4-6 RIR after each set. During week 6, participants performed a reduced volume-load. The following were assessed prior to and following the intervention: (i) vastus lateralis (VL) muscle cross-sectional area (mCSA) at multiple sites; (ii) squat, bench press, and deadlift one-repetition maximums (1RMs); and (iii) maximal isometric knee extensor torque and VL motor unit firing rates during an 80% maximal voluntary contraction. Although RIR was lower in the low- versus high-RIR group during the intervention (p < 0.001), total training volume did not significantly differ between groups (p = 0.222). There were main effects of time for squat, bench press, and deadlift 1RMs (all p-values < 0.05), but no significant condition × time interactions existed for these or proximal/middle/distal VL mCSA data. There were significant interactions for the slope and y-intercept of the motor unit mean firing rate versus recruitment threshold relationship. Post hoc analyses indicated low-RIR group slope values decreased and y-intercept values increased after training suggesting low-RIR training increased lower-threshold motor unit firing rates. This study provides insight into how resistance training in proximity to failure affects strength, hypertrophy, and single motor unit characteristics, and may inform those who aim to program for resistance-trained individuals.

Different Resistance Exercise Loading Paradigms Similarly Affect Skeletal Muscle Gene Expression Patterns of Myostatin-Related Targets and mTORC1 Signaling Markers.

Although transcriptome profiling has been used in several resistance training studies, the associated analytical approaches seldom provide in-depth information on individual genes linked to skeletal muscle hypertrophy. Therefore, a secondary analysis was performed herein on a muscle transcriptomic dataset we previously published involving trained college-aged men (n = 11) performing two resistance exercise bouts in a randomized and crossover fashion. The lower-load bout (30 Fail) consisted of 8 sets of lower body exercises to volitional fatigue using 30% one-repetition maximum (1 RM) loads, whereas the higher-load bout (80 Fail) consisted of the same exercises using 80% 1 RM loads. Vastus lateralis muscle biopsies were collected prior to (PRE), 3 h, and 6 h after each exercise bout, and 58 genes associated with skeletal muscle hypertrophy were manually interrogated from our prior microarray data. Select targets were further interrogated for associated protein expression and phosphorylation induced-signaling events. Although none of the 58 gene targets demonstrated significant bout x time interactions, ~57% (32 genes) showed a significant main effect of time from PRE to 3 h (15↑ and 17↓, p < 0.01), and ~26% (17 genes) showed a significant main effect of time from PRE to 6 h (8↑ and 9↓, p < 0.01). Notably, genes associated with the myostatin (9 genes) and mammalian target of rapamycin complex 1 (mTORC1) (9 genes) signaling pathways were most represented. Compared to mTORC1 signaling mRNAs, more MSTN signaling-related mRNAs (7 of 9) were altered post-exercise, regardless of the bout, and RHEB was the only mTORC1-associated mRNA that was upregulated following exercise. Phosphorylated (phospho-) p70S6K (Thr389) (p = 0.001; PRE to 3 h) and follistatin protein levels (p = 0.021; PRE to 6 h) increased post-exercise, regardless of the bout, whereas phospho-AKT (Thr389), phospho-mTOR (Ser2448), and myostatin protein levels remained unaltered. These data continue to suggest that performing resistance exercise to volitional fatigue, regardless of load selection, elicits similar transient mRNA and signaling responses in skeletal muscle. Moreover, these data provide further evidence that the transcriptional regulation of myostatin signaling is an involved mechanism in response to resistance exercise.

Biomechanical, Anthropometric and Psychological Determinants of Barbell Bench Press Strength.

The purpose of this study was to improve our understanding of the relative contributions of biomechanical, anthropometric, and psychological factors in explaining maximal bench press (BP) strength in a heterogeneous, resistance-trained sample. Eighteen college-aged participants reported to the laboratory for three visits. The first visit consisted of psychometric testing. The second visit assessed participants' anthropometrics, additional psychometric outcomes, and bench press one repetition maximum (1RM). Participants performed isometric dynamometry testing for horizontal shoulder adduction and elbow extension at a predicted sticking point joint position. Multiple linear regression was used to examine the relationships between the biomechanical, anthropometric, and psychological variables and BP 1RM. Our primary multiple linear regression accounted for 43% of the variance in BP strength (F(3,14) = 5.34, p = 0.01; R2 = 0.53; adjusted R2 = 0.43). The sum of peak isometric net joint moments from the shoulder and elbow had the greatest standardized effect (0.59), followed by lean body mass (0.27) and self-efficacy (0.17). The variance in BP 1RM can be similarly captured (R2 = 0.48) by a single principal component containing anthropometric, biomechanics, and psychological variables. Pearson correlations with BP strength were generally greater among anthropometric and biomechanical variables as compared to psychological variables. These data suggest that BP strength among a heterogeneous, resistance-trained population is explained by multiple factors and is more strongly associated with physical than psychological variables.

Muscle Fiber Type Transitions with Exercise Training: Shifting Perspectives.

Human muscle fibers are generally classified by myosin heavy chain (MHC) isoforms characterized by slow to fast contractile speeds. Type I, or slow-twitch fibers, are seen in high abundance in elite endurance athletes, such as long-distance runners and cyclists. Alternatively, fast-twitch IIa and IIx fibers are abundant in elite power athletes, such as weightlifters and sprinters. While cross-sectional comparisons have shown marked differences between athletes, longitudinal data have not clearly converged on patterns in fiber type shifts over time, particularly between slow and fast fibers. However, not all fiber type identification techniques are created equal and, thus, may limit interpretation. Hybrid fibers, which express more than one MHC type (I/IIa, IIa/IIx, I/IIa/IIx), may make up a significant proportion of fibers. The measurement of the distribution of fibers would necessitate the ability to identify hybrid fibers, which is best done through single fiber analysis. Current evidence using the most appropriate techniques suggests a clear ability of fibers to shift between hybrid and pure fibers as well as between slow and fast fiber types. The context and extent to which this occurs, along with the limitations of current evidence, are discussed herein.

Loading Recommendations for Muscle Strength, Hypertrophy, and Local Endurance: A Re-Examination of the Repetition Continuum.

Loading recommendations for resistance training are typically prescribed along what has come to be known as the "repetition continuum", which proposes that the number of repetitions performed at a given magnitude of load will result in specific adaptations. Specifically, the theory postulates that heavy load training optimizes increases maximal strength, moderate load training optimizes increases muscle hypertrophy, and low-load training optimizes increases local muscular endurance. However, despite the widespread acceptance of this theory, current research fails to support some of its underlying presumptions. Based on the emerging evidence, we propose a new paradigm whereby muscular adaptations can be obtained, and in some cases optimized, across a wide spectrum of loading zones. The nuances and implications of this paradigm are discussed herein.

Isolated Leucine and Branched-Chain Amino Acid Supplementation for Enhancing Muscular Strength and Hypertrophy: A Narrative Review.

Branched-chain amino acids (BCAA) are one of the most popular sports supplements, marketed under the premise that they enhance muscular adaptations. Despite their prevalent consumption among athletes and the general public, the efficacy of BCAA has been an ongoing source of controversy in the sports nutrition field. Early support for BCAA supplementation was derived from extrapolation of mechanistic data on their role in muscle protein metabolism. Of the three BCAA, leucine has received the most attention because of its ability to stimulate the initial acute anabolic response. However, a substantial body of both acute and longitudinal research has now accumulated on the topic, affording the ability to scrutinize the effects of BCAA and leucine from a practical standpoint. This article aims to critically review the current literature and draw evidence-based conclusions about the putative benefits of BCAA or leucine supplementation on muscle strength and hypertrophy as well as illuminate gaps in the literature that warrant future study.