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.

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.

A novel imaging method (FIM-ID) reveals that myofibrillogenesis plays a major role in the mechanically induced growth of skeletal muscle.

An increase in mechanical loading, such as that which occurs during resistance exercise, induces radial growth of muscle fibers (i.e. an increase in cross-sectional area). Muscle fibers are largely composed of myofibrils, but whether radial growth is mediated by an increase in the size of the myofibrils (i.e. myofibril hypertrophy) and/or the number of myofibrils (i.e. myofibrillogenesis) is not known. Electron microscopy (EM) can provide images with the level of resolution that is needed to address this question, but the acquisition and subsequent analysis of EM images is a time- and cost-intensive process. To overcome this, we developed a novel method for visualizing myofibrils with a standard fluorescence microscope (fluorescence imaging of myofibrils with image deconvolution [FIM-ID]). Images from FIM-ID have a high degree of resolution and contrast, and these properties enabled us to develop pipelines for automated measurements of myofibril size and number. After extensively validating the automated measurements, we used both mouse and human models of increased mechanical loading to discover that the radial growth of muscle fibers is largely mediated by myofibrillogenesis. Collectively, the outcomes of this study offer insight into a fundamentally important topic in the field of muscle growth and provide future investigators with a time- and cost-effective means to study it.

Approximately 45% of human body mass is made of skeletal muscle. These muscles contract and relax to provide the mechanical forces needed for breathing, moving, keeping warm and performing many other essential processes. Both sedentary and active adults lose approximately 30-40% of this muscle mass by the age of 80, increasing their risk of disease, disability and death. As a result, there is much interest in developing therapies that can restore, maintain and increase muscle mass in older individuals. Muscles are made of multiple fibers that are in turn largely composed of smaller units known as myofibrils. Previous studies have shown that performing resistance training or other exercise that increases the mechanical loads placed on muscles stimulates muscle growth. This growth is largely due to increased girth of the existing muscle fibers. However, it remained unclear whether this was due to myofibrils growing in size, increasing in number, or a combination of both. To address this question, Jorgenson et al. developed a fluorescence imaging method called FIM-ID to count the number and measure the size of myofibrils within cross-sections of skeletal muscle. Using FIM-ID to study samples of mouse and human muscle fibers then revealed that increasing mechanical loads on muscles increased the number of myofibrils and this was largely responsible for muscle fiber growth. FIM-ID mostly relies on common laboratory instruments and free open-source software is used to count and measure the myofibrils. Jorgenson et al. hope that this will allow as many other researchers as possible to use FIM-ID to study myofibrils in the future. A better understanding of how the body controls the number of myofibrils may lead to the development of therapies that can mimic the effects of exercise on muscles to maintain or even increase muscle mass in human patients.

Making the case for resistance training in improving vascular function and skeletal muscle capillarization.

Through decades of empirical data, it has become evident that resistance training (RT) can improve strength/power and skeletal muscle hypertrophy. Yet, until recently, vascular outcomes have historically been underemphasized in RT studies, which is underscored by several exercise-related reviews supporting the benefits of endurance training on vascular measures. Several lines of evidence suggest large artery diameter and blood flow velocity increase after a single bout of resistance exercise, and these events are mediated by vasoactive substances released from endothelial cells and myofibers (e.g., nitric oxide). Weeks to months of RT can also improve basal limb blood flow and arterial diameter while lowering blood pressure. Although several older investigations suggested RT reduces skeletal muscle capillary density, this is likely due to most of these studies being cross-sectional in nature. Critically, newer evidence from longitudinal studies contradicts these findings, and a growing body of mechanistic rodent and human data suggest skeletal muscle capillarity is related to mechanical overload-induced skeletal muscle hypertrophy. In this review, we will discuss methods used by our laboratories and others to assess large artery size/function and skeletal muscle capillary characteristics. Next, we will discuss data by our groups and others examining large artery and capillary responses to a single bout of resistance exercise and chronic RT paradigms. Finally, we will discuss RT-induced mechanisms associated with acute and chronic vascular outcomes.

The effects of a sugar-free amino acid-containing electrolyte beverage on 5-kilometer performance, blood electrolytes, and post-exercise cramping versus a conventional carbohydrate-electrolyte sports beverage and water.

OBJECTIVE: The purpose of this study was to examine the acute effects of a multi-ingredient, low calorie dietary supplement (MIDS, XTEND® Healthy Hydration) on 5-kilometer (5-km) time trial performance and blood electrolyte concentrations compared to a carbohydrate-electrolyte beverage (CE, GATORADE® Thirst Quencher) and distilled water (W). METHODS: During visit 1 (V1), participants (10 men and 10 women, 20-35 years old, BMI ≤ 29 kg/m2, recreationally active) reported to the laboratory whereby the following tests were performed: i) height and weight measurements, ii) body composition analysis, iii) treadmill testing to measure maximal aerobic capacity, and iv) 5-km time trial familiarization. The second visit (V2) was one week after V1 in the morning (0600 - 0900) and participants arrived 12-14 h fasted (no food or drink). The first battery of assessments (V2-T1) included nude body mass, urine specific gravity (USG), a profile of mood states (POMS) questionnaire, and the completion of a visual analogue scale (VAS) questionnaire to quantify cramping. Then heart rate (HR), blood pressure (BP), total body hydration (via bioelectrical impedance spectroscopy [BIS]) were examined. Finally, a measurement of blood markers via finger stick was performed. Participants consumed a randomized beverage (16 fl. oz. of MIDS, 16 fl. oz. of W, or 16 fl. oz. of CE) within 3 min followed by a 45-min rest. Following the rest period, a second battery (V2-T2) was performed whereby participants' USG was assessed and they completed the POMS and VAS questionnaires, and HR, BP, and blood markers were measured. The participants then performed a 5-km treadmill time trial. Immediately following the 5-km time trial, participants completed a third testing battery (V2-T3) that began with blood markers, HR and BP assessments, followed by nude body weight assessment, and the POMS and VAS questionnaires. After 60 min, a fourth battery (V2-T4) was performed that included HR, BP, and blood markers. After sitting quietly for another 60 min a fifth battery assessment was performed (V2-T5) that included participants' USG, POMS and VAS questionnaires, HR, BP, blood markers, and total body hydration. Visits 3 (V3) and 4 (V4) followed the same protocol except a different randomized drink (16 oz. of CE, MIDS, or W) was consumed; all of which were separated by approximately one week. RESULTS: No differences occurred between conditions for 5-km time trial completion, indirect calorimetry outcomes during 5-km time trials, USG, or nude mass measurements (p > 0.05 for all relevant statistical tests). However, blood potassium and the sodium/potassium ratio displayed significant interactions (p < 0.05), and post hoc testing indicated these values were better maintained in the MIDS versus other conditions. Post-exercise cramp prevalence was greater in the CE (p < 0.05) and trended higher with W (p = 0.083) compared to the MIDS condition. Post-exercise cramp severity was also elevated with the W and CE beverages (p < 0.05) but not the MIDS (p = 0.211). CONCLUSIONS: The MIDS did not affect 5-km time trial performance but exhibited favorable effects on blood electrolyte and post-exercise self-reporting cramp outcomes compared to the CE and W drinks.

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.

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.

A Novel Imaging Method (FIM-ID) Reveals that Myofibrillogenesis Plays a Major Role in the Mechanically Induced Growth of Skeletal Muscle.

An increase in mechanical loading, such as that which occurs during resistance exercise, induces radial growth of muscle fibers (i.e., an increase in cross-sectional area). Muscle fibers are largely composed of myofibrils, but whether radial growth is mediated by an increase in the size of the myofibrils (i.e., myofibril hypertrophy) and/or the number of myofibrils (i.e., myofibrillogenesis) is not known. Electron microscopy (EM) can provide images with the level of resolution that is needed to address this question, but the acquisition and subsequent analysis of EM images is a time- and cost-intensive process. To overcome this, we developed a novel method for visualizing myofibrils with a standard fluorescence microscope (FIM-ID). Images from FIM-ID have a high degree of resolution and contrast, and these properties enabled us to develop pipelines for automated measurements of myofibril size and number. After extensively validating the automated measurements, we used both mouse and human models of increased mechanical loading to discover that the radial growth of muscle fibers is largely mediated by myofibrillogenesis. Collectively, the outcomes of this study offer insight into a fundamentally important topic in the field of muscle growth and provide future investigators with a time- and cost-effective means to study it.

Cross-sectional analysis of racial differences in hydration and neighborhood deprivation in young adults.

BACKGROUND: Inadequate hydration is associated with cardiovascular and kidney disease morbidity and all-cause mortality. Compared with White individuals, Black individuals exhibit a higher prevalence of inadequate hydration, which may contribute to racial health disparities. However, the underlying reasons for these differences in hydration remain unclear. OBJECTIVE: This cross-sectional study aimed to investigate whether neighborhood deprivation contributes to racial differences in hydration status. METHODS: We assessed 24 Black and 30 White college students, measuring 24-hour urine osmolality, urine flow rate, urine specific gravity, and plasma copeptin concentration. Participants recorded their food and fluid intake for 3 d to assess total water intake from food and beverages. Neighborhood socioeconomic deprivation was measured using a tract-level Area Deprivation Index. RESULTS: Black participants exhibited higher urine osmolality (640 [314] compared with 440 [283] mOsm/kg H2O, respectively, P = 0.006) and lower urine flow rate (1.06 [0.65] compared with 1.71 [0.89] ml/min, respectively, P = 0.009) compared with White participants, indicating greater hypohydration among Black participants. Black participants reported lower total water intake from food and beverages than White participants (2.3 ± 0.7 compared with 3.5 ± 1.1 L/day, respectively, P < 0.01). Black participants exhibited higher copeptin than White participants (6.3 [3.1] compared with 4.5 [2.3] pmol/L, P = 0.046), and urine osmolality mediated 67% of the difference (P = 0.027). Black participants reported greater cumulative exposure to neighborhood deprivation during childhood (ages 0-18 y). Furthermore, neighborhood deprivation during childhood was associated with urine specific gravity (P = 0.031) and total water intake from food and beverages (P = 0.042) but did not mediate the racial differences in these measures. CONCLUSION: Our data suggest that compared with White young adults, Black young adults are hypohydrated and exhibit higher plasma copeptin concentration, and that greater neighborhood deprivation is associated with chronic underhydration irrespective of race. This trial was registered at clinicaltrials.gov as NCT04576338.

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.

Resistance training diminishes mitochondrial adaptations to subsequent endurance training in healthy untrained men.

We investigated the effects of performing a period of resistance training (RT) on the performance and molecular adaptations to a subsequent period of endurance training (ET). Twenty-five young adults were divided into an RT+ET group (n = 13), which underwent 7 weeks of RT followed by 7 weeks of ET, and an ET-only group (n = 12), which performed 7 weeks of ET. Body composition, endurance performance and muscle biopsies were collected before RT (T1, baseline for RT+ET), before ET (T2, after RT for RT+ET and baseline for ET) and after ET (T3). Immunohistochemistry was performed to determine fibre cross-sectional area (fCSA), myonuclear content, myonuclear domain size, satellite cell number and mitochondrial content. Western blots were used to quantify markers of mitochondrial remodelling. Citrate synthase activity and markers of ribosome content were also investigated. RT improved body composition and strength, increased vastus lateralis thickness, mixed and type II fCSA, myonuclear number, markers of ribosome content, and satellite cell content (P < 0.050). In response to ET, both groups similarly decreased body fat percentage (P < 0.0001) and improved endurance performance (e.g. V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ , and speed at which the onset of blood lactate accumulation occurred, P < 0.0001). Levels of mitochondrial complexes I-IV in the ET-only group increased 32-66%, while those in the RT+ET group increased 1-11% (time, P < 0.050). Additionally, mixed fibre relative mitochondrial content increased 15% in the ET-only group but decreased 13% in the RT+ET group (interaction, P = 0.043). In conclusion, RT performed prior to ET had no additional benefits to ET adaptations. Moreover, prior RT seemed to impair mitochondrial adaptations to ET. KEY POINTS: Resistance training is largely underappreciated as a method to improve endurance performance, despite reports showing it may improve mitochondrial function. Although several concurrent training studies are available, in this study we investigated the effects of performing a period of resistance training on the performance and molecular adaptations to subsequent endurance training. Prior resistance training did not improve endurance performance and impaired most mitochondrial adaptations to subsequent endurance training, but this effect may have been a result of detraining from resistance training.

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.

Resistance Training Diminishes Mitochondrial Adaptations to Subsequent Endurance Training.

We investigated the effects of performing a period of resistance training (RT) on the performance and molecular adaptations to a subsequent period of endurance training (ET). Twenty-five young adults were divided into RT+ET (n=13), which underwent seven weeks of RT followed by seven weeks of ET, and ET-only (n=12), which performed seven weeks of ET. Body composition, endurance performance, and muscle biopsies were collected before RT (T1, baseline for RT+ET), before ET (T2, post RT for RT+ET and baseline for ET), and after ET (T3). Immunohistochemistry was performed to determine fiber cross-sectional area (fCSA), myonuclear content, myonuclear domain size, satellite cell number, and mitochondrial content. Western blots were used to quantify markers of mitochondrial remodeling. Citrate synthase activity and markers of ribosome content were also investigated. Resistance training improved body composition and strength, increased vastus lateralis thickness, mixed and type II fCSA, myonuclear number, markers of ribosome content, and satellite cell content (p<0.050). In response to ET, both groups similarly decreased body fat percentage and improved endurance performance (e.g., VO 2 max, and speed at which the onset of blood lactate accumulation occurred during the VO 2 max test). Levels of mitochondrial complexes I-IV in the ET-only group increased 32-66%, while the RT+ET group increased 1-11%. Additionally, mixed fiber relative mitochondrial content increased 15% in the ET-only group but decreased 13% in the RT+ET group. In conclusion, RT performed prior to ET had no additional benefits to ET adaptations. Moreover, prior RT seemed to impair mitochondrial adaptations to ET. KEY POINTS SUMMARY: Resistance training is largely underappreciated as a method to improve endurance performance, despite reports showing it may improve mitochondrial function.Although several concurrent training studies are available, in this study we investigated the effects of performing a period resistance training on the performance and molecular adaptations to subsequent endurance training.Prior resistance training did not improve endurance performance and impaired most mitochondrial adaptations to subsequent endurance training, but that seemed to be a result of detraining from resistance training.

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.

Skeletal Muscle DNA Methylation and mRNA Responses to a Bout of Higher versus Lower Load Resistance Exercise in Previously Trained Men.

We sought to determine the skeletal muscle genome-wide DNA methylation and mRNA responses to one bout of lower load (LL) versus higher load (HL) resistance exercise. Trained college-aged males (n = 11, 23 ± 4 years old, 4 ± 3 years self-reported training) performed LL or HL bouts to failure separated by one week. The HL bout (i.e., 80 Fail) consisted of four sets of back squats and four sets of leg extensions to failure using 80% of participants estimated one-repetition maximum (i.e., est. 1-RM). The LL bout (i.e., 30 Fail) implemented the same paradigm with 30% of est. 1-RM. Vastus lateralis muscle biopsies were collected before, 3 h, and 6 h after each bout. Muscle DNA and RNA were batch-isolated and analyzed using the 850k Illumina MethylationEPIC array and Clariom S mRNA microarray, respectively. Performed repetitions were significantly greater during the 30 Fail versus 80 Fail (p < 0.001), although total training volume (sets × reps × load) was not significantly different between bouts (p = 0.571). Regardless of bout, more CpG site methylation changes were observed at 3 h versus 6 h post exercise (239,951 versus 12,419, respectively; p < 0.01), and nuclear global ten-eleven translocation (TET) activity, but not global DNA methyltransferase activity, increased 3 h and 6 h following exercise regardless of bout. The percentage of genes significantly altered at the mRNA level that demonstrated opposite DNA methylation patterns was greater 3 h versus 6 h following exercise (~75% versus ~15%, respectively). Moreover, high percentages of genes that were up- or downregulated 6 h following exercise also demonstrated significantly inversed DNA methylation patterns across one or more CpG sites 3 h following exercise (65% and 82%, respectively). While 30 Fail decreased DNA methylation across various promoter regions versus 80 Fail, transcriptome-wide mRNA and bioinformatics indicated that gene expression signatures were largely similar between bouts. Bioinformatics overlay of DNA methylation and mRNA expression data indicated that genes related to "Focal adhesion," "MAPK signaling," and "PI3K-Akt signaling" were significantly affected at the 3 h and 6 h time points, and again this was regardless of bout. In conclusion, extensive molecular profiling suggests that post-exercise alterations in the skeletal muscle DNA methylome and mRNA transcriptome elicited by LL and HL training bouts to failure are largely similar, and this could be related to equal volumes performed between bouts.

Changes in vastus lateralis fibre cross-sectional area, pennation angle and fascicle length do not predict changes in muscle cross-sectional area.

NEW FINDINGS: What is the central question of this study? Do changes in myofibre cross-sectional area, pennation angle and fascicle length predict vastus lateralis whole-muscle cross-sectional area changes following resistance training? What is the main finding and its importance? Changes in vastus lateralis mean myofibre cross-sectional area, fascicle length and pennation angle following a period of resistance training did not collectively predict changes in whole-muscle cross-sectional area. Despite the limited sample size in this study, these data reiterate that it remains difficult to generalize the morphological adaptations that predominantly drive tissue-level vastus lateralis muscle hypertrophy. ABSTRACT: Myofibre hypertrophy during resistance training (RT) poorly associates with tissue-level surrogates of hypertrophy. However, it is underappreciated that, in pennate muscle, changes in myofibre cross-sectional area (fCSA), fascicle length (Lf ) and pennation angle (PA) likely coordinate changes in whole-muscle cross-sectional area (mCSA). Therefore, we determined if changes in fCSA, PA and Lf predicted vastus lateralis (VL) mCSA changes following RT. Thirteen untrained college-aged males (23 ± 4 years old, 25.4 ± 5.2 kg/m2 ) completed 7 weeks of full-body RT (twice weekly). Right leg VL ultrasound images and biopsies were obtained prior to (PRE) and 72 h following (POST) the last training bout. Regression was used to assess if training-induced changes in mean fCSA, PA and Lf predicted VL mCSA changes. Correlations were also performed between PRE-to-POST changes in obtained variables. Mean fCSA (+18%), PA (+8%) and mCSA (+22%) increased following RT (P < 0.05), but not Lf (0.1%, P = 0.772). Changes in fCSA, Lf and PA did not collectively predict changes in mCSA (R2 = 0.282, adjusted R2 = 0.013, F3,8  = 1.050, P = 0.422). Moderate negative correlations existed for percentage changes in PA and Lf (r = -0.548, P = 0.052) and changes in fCSA and Lf (r = -0.649, P = 0.022), and all other associations were weak (|r| < 0.500). Although increases in mean fCSA, PA and VL mCSA were observed, inter-individual responses for each variable and limitations for each technique make it difficult to generalize the morphological adaptations that predominantly drive tissue-level VL muscle hypertrophy. However, the small subject pool is a significant limitation, and more research in this area is needed.