Effects of aging and lifelong aerobic exercise on expression of innate immune components in skeletal muscle of women.

This study examined the effects of aging and lifelong aerobic exercise on innate immune system components in the skeletal muscle of healthy women in the basal state and after an unaccustomed resistance exercise (RE) challenge. We also made exploratory between-sex comparisons with our previous report on men. Three groups of women were studied: young exercisers (YE, n = 10, 25 ± 1 yr, V̇o2max: 44 ± 2 mL/kg/min), lifelong aerobic exercisers with a 48 ± 2 yr training history (LLE, n = 7, 72 ± 2 yr, V̇o2max: 26 ± 2 mL/kg/min), and old healthy nonexercisers (OH, n = 10, 75 ± 1 yr, V̇o2max: 18 ± 1 mL/kg/min). Ten Toll-like receptors (TLRs)1-10, TLR adaptors (Myd88, TRIF), and NF-κB pathway components (IκBα, IKKß) were assessed at the mRNA level in vastus lateralis biopsies before and 4 h after RE [3×10 repetitions, 70% 1-repetition maximum (1RM)]. Basal TLR1-10 expression was minimally influenced by age or LLE in women (TLR9 only; OH > YE, +43%, P < 0.05; OH > LLE, +30%, P < 0.10) and was on average 24% higher in women versus men. Similarly, basal adaptor expression was not influenced (P > 0.05) by age or LLE in women but was on average 26% higher (myeloid differentiation primary response 88, Myd88) and 23% lower [Toll interleukin (IL)-1 receptor-containing adaptor-inducing interferon-γ, TRIF] in women versus men. RE-induced changes in women, independent of the group, in TLR3, TLR4, TLR6 (∼2.1-fold, P < 0.05), Myd88 (∼1.2-fold, P < 0.10), and IκBα (∼0.3-fold, P < 0.05). Although there were some similar RE responses in men (TLR4: 2.1-fold, Myd88: 1.2-fold, IκBα: 0.4-fold), several components responded only in men to RE (TLR1, TLR8, TRIF, and IKKß). Our findings support the sexual dimorphism of immunity, with women having greater basal skeletal muscle TLR expression and differential response to unaccustomed exercise than men.NEW & NOTEWORTHY We recently reported that aging increases basal expression of many Toll-like receptors (TLRs) in men and lifelong aerobic exercise does not prevent this effect. In addition, a resistance exercise (RE) challenge increased the expression of many TLRs. Here we show that basal TLR expression is minimally influenced by aging in women and findings support the sexual dimorphism of immunity, with women having greater basal skeletal muscle TLR expression and a differential response to unaccustomed exercise than men.

Fast and slow muscle fiber transcriptome dynamics with lifelong endurance exercise.

We investigated fast and slow muscle fiber transcriptome exercise dynamics among three groups of men: lifelong exercisers (LLE, n = 8, 74 ± 1 yr), old healthy nonexercisers (OH, n = 9, 75 ± 1 yr), and young exercisers (YE, n = 8, 25 ± 1 yr). On average, LLE had exercised ∼4 day·wk-1 for ∼8 h·wk-1 over 53 ± 2 years. Muscle biopsies were obtained pre- and 4 h postresistance exercise (3 × 10 knee extensions at 70% 1-RM). Fast and slow fiber size and function were assessed preexercise with fast and slow RNA-seq profiles examined pre- and postexercise. LLE fast fiber size was similar to OH, which was ∼30% smaller than YE (P < 0.05) with contractile function variables among groups, resulting in lower power in LLE (P < 0.05). LLE slow fibers were ∼30% larger and more powerful compared with YE and OH (P < 0.05). At the transcriptome level, fast fibers were more responsive to resistance exercise compared with slow fibers among all three cohorts (P < 0.05). Exercise induced a comprehensive biological response in fast fibers (P < 0.05) including transcription, signaling, skeletal muscle cell differentiation, and metabolism with vast differences among the groups. Fast fibers from YE exhibited a growth and metabolic signature, with LLE being primarily metabolic, and OH showing a strong stress-related response. In slow fibers, only LLE exhibited a biological response to exercise (P < 0.05), which was related to ketone and lipid metabolism. The divergent exercise transcriptome signatures provide novel insight into the molecular regulation in fast and slow fibers with age and exercise and suggest that the ∼5% weekly exercise time commitment of the lifelong exercisers provided a powerful investment for fast and slow muscle fiber metabolic health at the molecular level.NEW & NOTEWORTHY This study provides the first insights into fast and slow muscle fiber transcriptome dynamics with lifelong endurance exercise. The fast fibers were more responsive to exercise with divergent transcriptome signatures among young exercisers (growth and metabolic), lifelong exercisers (metabolic), and old healthy nonexercisers (stress). Only lifelong exercisers had a biological response in slow fibers (metabolic). These data provide novel insights into fast and slow muscle fiber health at the molecular level with age and exercise.

Exercise microdosing for skeletal muscle health applications to spaceflight.

Findings from a recent 70-day bedrest investigation suggested intermittent exercise testing in the control group may have served as a partial countermeasure for skeletal muscle size, function, and fiber-type shifts. The purpose of the current study was to investigate the metabolic and skeletal muscle molecular responses to the testing protocols. Eight males (29 ± 2 yr) completed muscle power (6 × 4 s; peak muscle power: 1,369 ± 86 W) and V̇o2max (13 ± 1 min; 3.2 ± 0.2 L/min) tests on specially designed supine cycle ergometers during two separate trials. Blood catecholamines and lactate were measured pre-, immediately post-, and 4-h postexercise. Muscle homogenate and muscle fiber-type-specific [myosin heavy chain (MHC) I and MHC IIa] mRNA levels of exercise markers (myostatin, IκBα, myogenin, MuRF-1, ABRA, RRAD, Fn14, PDK4) and MHC I, IIa, and IIx were measured from vastus lateralis muscle biopsies obtained pre- and 4-h postexercise. The muscle power test altered (P ≤ 0.05) norepinephrine (+124%), epinephrine (+145%), lactate (+300%), and muscle homogenate mRNA (IκBα, myogenin, MuRF-1, RRAD, Fn14). The V̇o2max test altered (P ≤ 0.05) norepinephrine (+1,394%), epinephrine (+1,412%), lactate (+736%), and muscle homogenate mRNA (myostatin, IκBα, myogenin, MuRF-1, ABRA, RRAD, Fn14, PDK4). In general, both tests influenced MHC IIa muscle fibers more than MHC I with respect to the number of genes that responded and the magnitude of response. Both tests also influenced MHC mRNA expression in a muscle fiber-type-specific manner. These findings provide unique insights into the adaptive response of skeletal muscle to small doses of exercise and could help shape exercise dosing for astronauts and Earth-based individuals.NEW & NOTEWORTHY Declines in skeletal muscle health are a concern for astronauts on long-duration spaceflights. The current findings add to the growing body of exercise countermeasures data, suggesting that small doses of specific exercise can be beneficial for certain aspects of skeletal muscle health. This information can be used in conjunction with other components of existing exercise programs for astronauts and might translate to other areas focused on skeletal muscle health (e.g., sports medicine, rehabilitation, aging).

NASA SPRINT exercise program efficacy for vastus lateralis and soleus skeletal muscle health during 70 days of simulated microgravity.

The efficacy of the NASA SPRINT exercise countermeasures program for quadriceps (vastus lateralis) and triceps surae (soleus) skeletal muscle health was investigated during 70 days of simulated microgravity. Individuals completed 6° head-down-tilt bedrest (BR, n = 9), bedrest with resistance and aerobic exercise (BRE, n = 9), or bedrest with resistance and aerobic exercise and low-dose testosterone (BRE + T, n = 8). All groups were periodically tested for muscle (n = 9 times) and aerobic (n = 4 times) power during bedrest. In BR, surprisingly, the typical bedrest-induced decrements in vastus lateralis myofiber size and power were either blunted (myosin heavy chain, MHC I) or eliminated (MHC IIa), along with no change (P > 0.05) in %MHC distribution and blunted quadriceps atrophy. In BRE, MHC I (vastus lateralis and soleus) and IIa (vastus lateralis) contractile performance was maintained (P > 0.05) or increased (P < 0.05). Vastus lateralis hybrid fiber percentage was reduced (P < 0.05) and energy metabolism enzymes and capillarization were generally maintained (P > 0.05), while not all of these positive responses were observed in the soleus. Exercise offsets 100% of quadriceps and approximately two-thirds of soleus whole muscle mass loss. Testosterone (BRE + T) did not provide any benefit over exercise alone for either muscle and for some myocellular parameters appeared detrimental. In summary, the periodic testing likely provided a partial exercise countermeasure for the quadriceps in the bedrest group, which is a novel finding given the extremely low exercise dose. The SPRINT exercise program appears to be viable for the quadriceps; however, refinement is needed to completely protect triceps surae myocellular and whole muscle health for astronauts on long-duration spaceflights.NEW & NOTEWORTHY This study provides unique exercise countermeasures development information for astronauts on long-duration spaceflights. The NASA SPRINT program was protective for quadriceps myocellular and whole muscle health, whereas the triceps surae (soleus) was only partially protected as has been shown with other programs. The bedrest control group data may provide beneficial information for overall exercise dose and targeting fast-twitch muscle fibers. Other unique approaches for the triceps surae are needed to supplement existing exercise programs.

Prostaglandin and myokine involvement in the cyclooxygenase-inhibiting drug enhancement of skeletal muscle adaptations to resistance exercise in older adults.

Twelve weeks of resistance training (3 days/wk) combined with daily consumption of the cyclooxygenase-inhibiting drugs acetaminophen (4.0 g/day; n = 11, 64 ± 1 yr) or ibuprofen (1.2 g/day; n = 13, 64 ± 1 yr) unexpectedly promoted muscle mass and strength gains 25-50% above placebo (n = 12, 67 ± 2 yr). To investigate the mechanism of this adaptation, muscle biopsies obtained before and ∼72 h after the last training bout were analyzed for mRNA levels of prostaglandin (PG)/cyclooxygenase pathway enzymes and receptors [arachidonic acid synthesis: cytosolic phospholipase A(2) (cPLA(2)) and secreted phospholipase A(2) (sPLA(2)); PGF(2α) synthesis: PGF(2α) synthase and PGE(2) to PGF(2α) reductase; PGE(2) synthesis: PGE(2) synthase-1, -2, and -3; PGF(2α) receptor and PGE(2) receptor-4], cytokines and myokines involved in skeletal muscle adaptation (TNF-α, IL-1ß, IL-6, IL-8, IL-10), and regulators of muscle growth [myogenin, myogenic regulatory factor-4 (MRF4), myostatin] and atrophy [Forkhead box O3A (FOXO3A), atrogin-1, muscle RING finger protein 1 (MuRF-1), inhibitory κB kinase ß (IKKß)]. Training increased (P < 0.05) cPLA(2), PGF(2α) synthase, PGE(2) to PGF(2α) reductase, PGE(2) receptor-4, TNF-α, IL-1ß, IL-8, and IKKß. However, the PGF(2α) receptor was upregulated (P < 0.05) only in the drug groups, and the placebo group upregulation (P < 0.05) of IL-6, IL-10, and MuRF-1 was eliminated in both drug groups. These results highlight prostaglandin and myokine involvement in the adaptive response to exercise in older individuals and suggest two mechanisms underlying the enhanced muscle mass gains in the drug groups: 1) The drug-induced PGF(2α) receptor upregulation helped offset the drug suppression of PGF(2α)-stimulated protein synthesis after each exercise bout and enhanced skeletal muscle sensitivity to this stimulation. 2) The drug-induced suppression of intramuscular PGE(2) production increased net muscle protein balance after each exercise bout through a reduction in PGE(2)-induced IL-6 and MuRF-1, both promoters of muscle loss.

Local anesthetic effects on gene transcription in human skeletal muscle biopsies.

INTRODUCTION: We examined if epinephrine in the local anesthetic to help control incision-related bleeding interferes with molecular measurements obtained with the Duchenne-Bergström percutaneous needle biopsy technique for sampling human skeletal muscle. METHODS: Three groups received 2.5-3.0 ml of 1% lidocaine in 2 injections: (1) 0.5-1.0 ml superficially, which varied among the groups according to (i) -Epi; intra- and subcutaneous without epinephrine, (ii) +Epi -Fascia; intra- and subcutaneous with epinephrine, avoiding the fascia, and (iii) +Epi +Fascia; intra- and subcutaneous with epinephrine, directing a small amount (∽0.2 ml) into the fascia area; and (2) ∽2.0 ml without epinephrine into the fascia area for all subjects. A muscle biopsy was obtained 5-10 min later for IL-6 and MuRF-1 mRNA levels. RESULTS: IL-6 mRNA levels were low in -Epi and +Epi -Fascia, but ∽300-fold higher in +Epi +Fascia. MuRF-1 mRNA levels were similar among the groups. CONCLUSIONS: Lidocaine with epinephrine can confound intramuscular measurements from needle biopsies, but this can be avoided with a careful injection approach.

Amino acid infusion alters the expression of growth-related genes in multiple skeletal muscles.

BACKGROUND: Exercise and nutritional interventions have been examined independently as countermeasures to offset the loss of skeletal muscle mass with unloading, yet a protocol to completely preserve the soleus has not been identified. Little is known regarding the combined effect of exercise and nutrition on factors regulating skeletal muscle growth. The purpose of this investigation was to evaluate the influence of amino acid (AA) infusion on myogenic (MRF-4, MyoD, and Myogenin), proteolytic (MuRF-1, Atrogin-1, FOXO3A, Calpain-1, Calpain-2, Caspase-3, Cathepsin L1), and cytokine (IL-6, IL-8, and IL-15) mRNA transcripts in two skeletal muscles that respond distinctly to microgravity unloading. METHODS: Muscle biopsies were obtained from the vastus lateralis (VL) and soleus of eight male subjects prior to and after 4 h of AA infusion for analysis of mRNA expression. All subjects performed a standardized exercise bout (45-min treadmill run) 24 h prior to the AA infusion. RESULTS: In the VL, proteolytic factors MuRF-1 and FOXO3A were reduced (44 +/- 9 and 28 +/- 6%, respectively) in response toAA infusion. In the soleus, mRNA transcripts of myogenic factor MRF-4 (91 +/- 36%) and cytokines IL-6, IL-8, and IL-15 were elevated while the proteolytic marker FOXO3A mRNA was reduced by 19 +/- 9%. DISCUSSION: These data suggest that the expression of genes related to skeletal muscle remodeling is altered during acute AA infusion 24 h post-exercise. It appears that increased amino acid availability in concert with exercise may create an intramuscular environment favorable for the prevention of muscle atrophy associated with unloading, which may be particularly beneficial for the soleus.

Human skeletal muscle fiber type specific protein content.

The aim of this project was to develop a method to assess fiber type specific protein content across the continuum of human skeletal muscle fibers. Individual vastus lateralis muscle fibers (n = 264) were clipped into two portions: one for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fiber typing and one for Western blot protein identification. Following fiber type determination, fiber segments were combined into fiber type specific pools (∼20 fibers/pool) and measured for total protein quantity, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), citrate synthase (CS), and total p38 content. GAPDH content was 64, 54, 160, and 138% more abundant in myosin heavy chain (MHC) I/IIa, MHC IIa, MHC IIa/IIx, and MHC IIx fibers, respectively, when compared with MHC I. Inversely, CS content was 528, 472, 242, and 47% more abundant in MHC I, MHC I/IIa, MHC IIa, and MHC IIa/IIx fibers, respectively, when compared with MHC IIx. Total p38 content was 87% greater in MHC IIa versus MHC I fibers. These data and this approach establish a reliable method for human skeletal muscle fiber type specific protein analysis. Initial results show that particular proteins exist in a hierarchal fashion throughout the continuum of human skeletal muscle fiber types, further highlighting the necessity of fiber type specific analysis.

Influence of acetaminophen and ibuprofen on skeletal muscle adaptations to resistance exercise in older adults.

Evidence suggests that consumption of over-the-counter cyclooxygenase (COX) inhibitors may interfere with the positive effects that resistance exercise training has on reversing sarcopenia in older adults. This study examined the influence of acetaminophen or ibuprofen consumption on muscle mass and strength during 12 wk of knee extensor progressive resistance exercise training in older adults. Thirty-six individuals were randomly assigned to one of three groups and consumed the COX-inhibiting drugs in double-blind placebo-controlled fashion: placebo (67 ± 2 yr; n = 12), acetaminophen (64 ± 1 yr; n = 11; 4 g/day), and ibuprofen (64 ± 1 yr; n = 13; 1.2 g/day). Compliance with the resistance training program (100%) and drug consumption (via digital video observation, 94%), and resistance training intensity were similar (P > 0.05) for all three groups. Drug consumption unexpectedly increased muscle volume (acetaminophen: 109 ± 14 cm(3), 12.5%; ibuprofen: 84 ± 10 cm(3), 10.9%) and muscle strength (acetaminophen: 19 ± 2 kg; ibuprofen: 19 ± 2 kg) to a greater extent (P < 0.05) than placebo (muscle volume: 69 ± 12 cm(3), 8.6%; muscle strength: 15 ± 2 kg), when controlling for initial muscle size and strength. Follow-up analysis of muscle biopsies taken from the vastus lateralis before and after training showed muscle protein content, muscle water content, and myosin heavy chain distribution were not influenced (P > 0.05) by drug consumption. Similarly, muscle content of the two known enzymes potentially targeted by the drugs, COX-1 and -2, was not influenced (P > 0.05) by drug consumption, although resistance training did result in a drug-independent increase in COX-1 (32 ± 8%; P < 0.05). Drug consumption did not influence the size of the nonresistance-trained hamstring muscles (P > 0.05). Over-the-counter doses of acetaminophen or ibuprofen, when consumed in combination with resistance training, do not inhibit and appear to enhance muscle hypertrophy and strength gains in older adults. The present findings coupled with previous short-term exercise studies provide convincing evidence that the COX pathway(s) are involved in the regulation of muscle protein turnover and muscle mass in humans.

Effect of a cyclooxygenase-2 inhibitor on postexercise muscle protein synthesis in humans.

Nonselective blockade of the cyclooxygenase (COX) enzymes in skeletal muscle eliminates the normal increase in muscle protein synthesis following resistance exercise. The current study tested the hypothesis that this COX-mediated increase in postexercise muscle protein synthesis is regulated specifically by the COX-2 isoform. Sixteen males (23 +/- 1 yr) were randomly assigned to one of two groups that received three doses of either a selective COX-2 inhibitor (celecoxib; 200 mg/dose, 600 mg total) or a placebo in double-blind fashion during the 24 h following a single bout of knee extensor resistance exercise. At rest and 24 h postexercise, skeletal muscle protein fractional synthesis rate (FSR) was measured using a primed constant infusion of [(2)H(5)]phenylalanine coupled with muscle biopsies of the vastus lateralis, and measurements were made of mRNA and protein expression of COX-1 and COX-2. Mixed muscle protein FSR in response to exercise (P < 0.05) was not suppressed by the COX-2 inhibitor (0.056 +/- 0.004 to 0.108 +/- 0.014%/h) compared with placebo (0.074 +/- 0.004 to 0.091 +/- 0.005%/h), nor was there any difference (P > 0.05) between the placebo and COX-2 inhibitor postexercise when controlling for resting FSR. The COX-2 inhibitor did not influence COX-1 mRNA, COX-1 protein, or COX-2 protein levels, whereas it did increase (P < 0.05) COX-2 mRNA (3.0 +/- 0.9-fold) compared with placebo (1.3 +/- 0.3-fold). It appears that the elimination of the postexercise muscle protein synthesis response by nonselective COX inhibitors is not solely due to COX-2 isoform blockade. Furthermore, the current data suggest that the COX-1 enzyme is likely the main isoform responsible for the COX-mediated increase in muscle protein synthesis following resistance exercise in humans.

Muscle protein synthesis and gene expression during recovery from aerobic exercise in the fasted and fed states.

The purpose of this investigation was to assess mixed-muscle fractional synthesis rate (FSR) and the expression of genes involved in skeletal muscle remodeling after aerobic exercise in the fasted and fed states. Eight recreationally active males (25 ± 1 yr; Vo(2 max): 52 ± 2 ml·kg(-1)·min(-1)) performed 60-min of cycle ergometry at 72 ± 1% Vo(2 max) on two occasions in a counter-balanced design. Subjects ingested a noncaloric placebo (EX-FAST) or a beverage containing (per kg body wt): 5 kcal, 0.83 g carbohydrate, 0.37 g protein, and 0.03 g fat (EX-FED) immediately and 1 h after exercise. FSR was assessed at rest and following exercise with the use of a l-[ring (2)H(5)]-phenylalanine infusion combined with muscle biopsies at 2 and 6 h postexercise. mRNA expression was assessed at 2 and 6 h postexercise via real-time RT-PCR. FSR was higher (P < 0.05) after exercise in both EX-FAST (0.112 ± 0.010%·h(-1)) and EX-FED (0.129 ± 0.014%·h(-1)) compared with rest (0.071 ± 0.005%·h(-1)). Feeding attenuated the mRNA expression (P < 0.05) of proteolytic factors MuRF-1 (6 h) and calpain-2 (2 and 6 h) postexercise but did not alter FOXO3A, calpain-1, caspase3, or myostatin mRNA expression compared with EX-FAST. Myogenic regulatory factor (MRF4) mRNA was also attenuated (P < 0.05) at 2 and 6 h postexercise in EX-FED compared with EX-FAST. These data demonstrate that a nonexhaustive bout of aerobic exercise stimulates skeletal muscle FSR in the fasted state and that feeding does not measurably enhance FSR between 2 and 6 h after aerobic exercise. Additionally, postexercise nutrient intake attenuates the expression of factors involved in the ubiquitin-proteosome and Ca(2+)-dependent protein degradation pathways. These data provide insight into the role of feeding on muscle protein metabolism during recovery from aerobic exercise.

Effects of aging and lifelong aerobic exercise on expression of innate immune components in human skeletal muscle.

The purpose of this investigation was to evaluate the effects of aging and lifelong exercise on skeletal muscle components of the innate immune system. Additionally, the effects of an acute resistance exercise (RE) challenge were explored. Three groups of men were studied: young exercisers (YE: n = 10, 25 ± 1 yr; V̇o2max: 53 ± 3 mL/kg/min; quadriceps size: 78 ± 3 cm2), lifelong aerobic exercisers with a 53 ± 1 yr training history (LLE; n = 21, 74 ± 1 yr; V̇o2max: 34 ± 1 mL/kg/min; quadriceps size: 67 ± 2 cm2), and old healthy nonexercisers (OH: n = 10, 75 ± 1 yr; V̇o2max: 22 ± 1 mL/kg/min, quadriceps size: 56 ± 3 cm2). Vastus lateralis muscle biopsies were obtained in the basal state and 4 h after RE (3 × 10 reps, 70% of 1 repetition maximum) to assess Toll-like receptors (TLR)1-10, TLR adaptors (Myd88 and TRIF), and NF-κB pathway components (IκΒα and IKKß) mRNA expression. Basal TLR3, TLR6, and TLR7 tended to be higher (P ≤ 0.10) with aging (LLE and OH combined). In general, RE increased expression of TLR1 and TLR8 (P ≤ 0.10) and TLR3 and TLR4 (P < 0.05), although TLR3 did not respond in OH. Both TLR adaptors also responded to the exercise bout; these were primarily (Myd88, main effect P ≤ 0.10) or exclusively (TRIF, P < 0.05) driven by the OH group. In summary, aging appears to increase basal expression of some innate immune components in human skeletal muscle, and lifelong aerobic exercise does not affect this age-related increase. An exercise challenge stimulates the expression of several TLRs, while the TLR adaptor response appears to be dysregulated with aging and maintained with lifelong exercise. Partially preserved muscle mass, coupled with a notable immunity profile, suggests lifelong exercisers are likely better prepared for a stress that challenges the immune system.NEW & NOTEWORTHY Findings from this investigation provide novel insight into the effect of aging and lifelong aerobic exercise on structural components of the innate immune system in skeletal muscle of humans. Data presented here suggest aging increases basal expression of select Toll-like receptors (TLRs), and lifelong exercise does not impact this age-related increase. Additionally, acute exercise stimulates gene expression of several TLRs, while the adaptor response is likely dysregulated with aging and maintained with lifelong exercise.

Effects of aging and lifelong aerobic exercise on basal and exercise-induced inflammation in women.

Low muscle mass and frailty are especially prevalent in older women and may be accelerated by age-related inflammation. Habitual physical activity throughout the life span (lifelong exercise) may prevent muscle inflammation and associated pathologies, but this is unexplored in women. This investigation assessed basal and acute exercise-induced inflammation in three cohorts of women: young exercisers (YE, n = 10, 25 ± 1 yr, [Formula: see text]: 44 ± 2 mL/kg/min, quadriceps size: 59 ± 2 cm2), old healthy nonexercisers (OH, n = 10, 75 ± 1 yr, [Formula: see text]: 18 ± 1 mL/kg/min, quadriceps size: 40 ± 1 cm2), and lifelong aerobic exercisers with a 48 ± 2 yr aerobic training history (LLE, n = 7, 72 ± 2 yr, [Formula: see text]: 26 ± 2 mL/kg/min, quadriceps size: 42 ± 2 cm2). Resting serum IL-6, TNF-α, C-reactive protein (CRP), and IGF-1 were measured. Vastus lateralis muscle biopsies were obtained at rest (basal) and 4 h after an acute exercise challenge (3 × 10 reps, 70% 1-repetition maximum) to assess gene expression of cytokines (IL-6, TNF-α, IL-1ß, IL-10, IL-4, IL-1Ra, TGF-ß), chemokines (IL-8, MCP-1), cyclooxygenase enzymes (COX-1, COX-2), prostaglandin E2 synthases (mPGES-1, cPGES) and receptors (EP3-4), and macrophage markers (CD16b, CD163), as well as basal macrophage abundance (CD68+ cells). The older cohorts (LLE + OH combined) demonstrated higher muscle IL-6 and COX-1 (P ≤ 0.05) than YE, whereas LLE expressed lower muscle IL-1ß (P ≤ 0.05 vs. OH). Acute exercise increased muscle IL-6 expression in YE only, whereas the older cohorts combined had the higher postexercise expression of IL-8 and TNF-α (P ≤ 0.05 vs. YE). Only LLE had increased postexercise expression of muscle IL-1ß and MCP-1 (P ≤ 0.05 vs. preexercise). Thus, aging in women led to mild basal and exercise-induced inflammation that was unaffected by lifelong aerobic exercise, which may have implications for long-term function and adaptability.NEW & NOTEWORTHY We previously reported a positive effect of lifelong exercise on skeletal muscle inflammation in aging men. This parallel investigation in women revealed that lifelong exercise did not protect against age-related increases in circulating or muscle inflammation and that preparedness to handle loading stress was not preserved by lifelong exercise. Further investigation is necessary to understand why lifelong aerobic exercise may not confer the same anti-inflammatory benefits in women as it does in men.

Effects of aging and lifelong aerobic exercise on basal and exercise-induced inflammation.

Age-associated chronic basal inflammation compromises muscle mass and adaptability, but exercise training may exert an anti-inflammatory effect. This investigation assessed basal and exercise-induced inflammation in three cohorts of men: young exercisers [YE; n = 10 men; 25 ± 1 yr; maximal oxygen consumption (V̇o2max), 53 ± 3 mL·kg-1·min-1; quadriceps area, 78 ± 3 cm2; means ± SE], old healthy nonexercisers (OH; n = 10; 75 ± 1 yr; V̇o2max, 22 ± 1 mL·kg-1·min-1; quadriceps area, 56 ± 3 cm2), and lifelong exercisers with an aerobic training history of 53 ± 1 yr (LLE; n = 21; 74 ± 1 yr; V̇o2max, 34 ± 1 mL·kg-1·min-1; quadriceps area, 67 ± 2 cm2). Resting serum IL-6, TNF-α, C-reactive protein, and IGF-1 levels were measured. Vastus lateralis muscle biopsies were obtained at rest (basal) and 4 h after an acute exercise challenge (3 × 10 repetitions, 70% 1-repetition maximum) to assess gene expression of cytokines [IL-6, TNF-α, IL-1ß, IL-10, IL-4, interleukin-1 receptor antagonist (IL-1Ra), and transforming growth factor-ß (TGF-ß)], chemokines [IL-8 and monocyte chemoattractant protein-1 (MCP-1)], cyclooxygenase enzymes [cyclooxygenase-1 and -2 (COX-1 and COX-2, respectively), prostaglandin E2 synthases [microsomal prostaglandin E synthase 1 (mPGES-1) and cytosolic prostaglandin E2 synthase (cPGES)] and receptors [prostaglandin E2 receptor EP3 and EP4 subtypes (EP3 and EP4, respectively), and macrophage markers [cluster of differentiation 16b (CD16b) and CD163], as well as basal macrophage abundance (CD68+ cells). Aging led to higher (P ≤ 0.05) circulating IL-6 and skeletal muscle COX-1, mPGES-1, and CD163 expression. However, LLE had significantly lower serum IL-6 levels (P ≤ 0.05 vs. OH) and a predominantly anti-inflammatory muscle profile [higher IL-10 (P ≤ 0.05 vs. YE), TNF-α, TGF-ß, and EP4 levels (P ≤ 0.05 vs. OH)]. In OH only, acute exercise increased expression of proinflammatory factors TNF-α, TGF-ß, and IL-8 (P ≤ 0.05). LLE had postexercise gene expression similar to YE, except lower IL-10 (P ≤ 0.10), mPGES-1, and EP3 expression (P ≤ 0.05). Thus, although aging led to a proinflammatory profile within blood and muscle, lifelong exercise partially prevented this and generally preserved the acute inflammatory response to exercise seen in young exercising men. Lifelong exercise may positively impact muscle health throughout aging by promoting anti-inflammation in skeletal muscle.NEW & NOTEWORTHY This study assessed a unique population of lifelong aerobic exercising men and demonstrated that their activity status exerts an anti-inflammatory effect in skeletal muscle and circulation. Furthermore, we provide evidence that the inflammatory response to acute exercise is dysregulated by aging but preserved with lifelong exercise, which might improve skeletal muscle resilience to unaccustomed loading and adaptability into late life.

Effect of consecutive repeated sprint and resistance exercise bouts on acute adaptive responses in human skeletal muscle.

We examined acute molecular responses in skeletal muscle to repeated sprint and resistance exercise bouts. Six men [age, 24.7 +/- 6.3 yr; body mass, 81.6 +/- 7.3 kg; peak oxygen uptake, 47 +/- 9.9 mlxkg(-1)xmin(-1); one repetition maximum (1-RM) leg extension 92.2 +/- 12.5 kg; means +/- SD] were randomly assigned to trials consisting of either resistance exercise (8 x 5 leg extension, 80% 1-RM) followed by repeated sprints (10 x 6 s, 0.75 Nxm torquexkg(-1)) or vice-versa. Muscle biopsies from vastus lateralis were obtained at rest, 15 min after each exercise bout, and following 3-h recovery to determine early signaling and mRNA responses. There was divergent exercise order-dependent phosphorylation of p70 S6K (S6K). Specifically, initial resistance exercise increased S6K phosphorylation ( approximately 75% P < 0.05), but there was no effect when resistance exercise was undertaken after sprints. Exercise decreased IGF-I mRNA following 3-h recovery ( approximately 50%, P = 0.06) independent of order, while muscle RING finger mRNA was elevated with a moderate exercise order effect (P < 0.01). When resistance exercise was followed by repeated sprints PGC-1alpha mRNA was increased (REX1-SPR2; P = 0.02) with a modest distinction between exercise orders. Repeated sprints may promote acute interference on resistance exercise responses by attenuating translation initiation signaling and exacerbating ubiquitin ligase expression. Indeed, repeated sprints appear to generate the overriding acute exercise-induced response when undertaking concurrent repeated sprint and resistance exercise. Accordingly, we suggest that sprint-activities are isolated from resistance training and that adequate recovery time is considered within periodized training plans that incorporate these divergent exercise modes.

Effect of acute resistance exercise and sex on human patellar tendon structural and regulatory mRNA expression.

Tendon is mainly composed of collagen and an aqueous matrix of proteoglycans that are regulated by enzymes called matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Although it is known that resistance exercise (RE) and sex influence tendon metabolism and mechanical properties, it is uncertain what structural and regulatory components contribute to these responses. We measured the mRNA expression of tendon's main fibrillar collagens (type I and type III) and the main proteoglycans (decorin, biglycan, fibromodulin, and versican) and the regulatory enzymes MMP-2, MMP-9, MMP-3, and TIMP-1 at rest and after RE. Patellar tendon biopsy samples were taken from six individuals (3 men and 3 women) before and 4 h after a bout of RE and from a another six individuals (3 men and 3 women) before and 24 h after RE. Resting mRNA expression was used for sex comparisons (6 men and 6 women). Collagen type I, collagen type III, and MMP-2 were downregulated (P < 0.05) 4 h after RE but were unchanged (P > 0.05) 24 h after RE. All other genes remained unchanged (P > 0.05) after RE. Women had higher resting mRNA expression (P < 0.05) of collagen type III and a trend (P = 0.08) toward lower resting expression of MMP-3 than men. All other genes were not influenced (P > 0.05) by sex. Acute RE appears to stimulate a change in collagen type I, collagen type III, and MMP-2 gene regulation in the human patellar tendon. Sex influences the structural and regulatory mRNA expression of tendon.

Proteolytic gene expression differs at rest and after resistance exercise between young and old women.

BACKGROUND: Skeletal muscle atrophy in rodents is associated with increased gene expression of proteolytic markers muscle-RING-finger protein 1 (MuRF-1) and atrogin-1. In humans with age-related muscle atrophy, known as sarcopenia, little is known about these key proteolytic biomarkers. Therefore, the purpose of this investigation was 2-fold: (i) measure messenger RNA (mRNA) expression of proteolytic genes MuRF-1, atrogin-1, forkhead box (FOXO)3A, and tumor necrosis factor-alpha (TNF-alpha) in young and old women at rest, and (ii) measure these proteolytic genes in response to an acute resistance exercise (RE) bout, a known hypertrophic stimulus. METHODS: A group of old women (OW: n =6, 85+/-1 years, thigh muscle =89+/-4 cm(2)) and young women (YW: n=8, 23+/-2 years, thigh muscle = 122+/-6 cm(2)) performed three sets of 10 knee extensions at 70% of one-repetition maximum. Muscle biopsies were taken from the vastus lateralis before and 4 hours after RE. Using real-time reverse transcription-polymerase chain reaction (RT-PCR), mRNA was amplified and normalized to GAPDH. RESULTS: At rest, OW expressed higher mRNA levels of MuRF-1 (p=.04) and FOXO3A (p=.001) compared to YW. In response to RE, there was an age effect (p=.01) in the induction of atrogin-1 (OW: 2.5-fold). Both YW and OW had an induction (p=.001) in MuRF-1 (YW: 3.6-fold; OW: 2.6-fold) with RE. CONCLUSIONS: These data show that the regulation of ubiquitin proteasome-related genes involved with muscle atrophy are altered in very old women (>80 years). This finding is manifested both at rest and in response to RE, which may contribute to the large degree of muscle loss with age.

Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle.

The aim of this study was to examine the time course induction of select proteolytic [muscle ring finger-1 (MuRF-1), atrogin-1, forkhead box 3A (FOXO3A), calpain-1, calpain-2], myostatin, and cytokine (IL -6, -8, -15, and TNF-alpha) mRNA after an acute bout of resistance (RE) or run (RUN) exercise. Six experienced RE (25 +/- 4 yr, 74 +/- 14 kg, 1.71 +/- 0.11 m) and RUN (25 +/- 4 yr, 72 +/- 5 kg, 1.81 +/- 0.07 m) subjects had muscle biopsies from the vastus lateralis (RE) or gastrocnemius (RUN) before, immediately after, and 1, 2, 4, 8, 12, and 24 h postexercise. RE increased (P < 0.05) mRNA expression of MuRF-1 early (3.5-fold, 1-4 h), followed by a decrease in atrogin-1 (3.3-fold) and FOXO3A (1.7-fold) 8-12 h postexercise. Myostatin mRNA decreased (6.3-fold; P < 0.05) from 1 to 24 h postexercise, whereas IL-6, IL-8, and TNF-alpha mRNA were elevated 2-12 h. RUN increased (P < 0.05) MuRF-1 (3.6-fold), atrogin-1 (1.6-fold), and FOXO3A (1.9-fold) 1-4 h postexercise. Myostatin was suppressed (3.6-fold; P < 0.05) 8-12 h post-RUN. The cytokines exhibited a biphasic response, with immediate elevation (P < 0.05) of IL-6, IL-8, and TNF-alpha, followed by a second elevation (P < 0.05) 2-24 h postexercise. In general, the timing of the gene induction indicated early elevation of proteolytic genes, followed by prolonged elevation of cytokines and suppression of myostatin. These data provide basic information for the timing of human muscle biopsy samples for gene expression studies involving exercise. Furthermore, this information suggests a greater induction of proteolytic genes following RUN compared with RE.

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers.

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research.NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men.

Aspirin as a COX inhibitor and anti-inflammatory drug in human skeletal muscle.

Although aspirin is one of the most common anti-inflammatory drugs in the world, the effect of aspirin on human skeletal muscle inflammation is almost completely unknown. This study examined the potential effects and related time course of an orally consumed aspirin dose on the inflammatory prostaglandin E2 (PGE2)/cyclooxygenase (COX) pathway in human skeletal muscle. Skeletal muscle biopsies were taken from the vastus lateralis of 10 healthy adults (5 male and 5 female, 25 ± 2 yr old) before (Pre) and 2, 4, and 24 h after (Post) a standard dose (975mg) of aspirin and partitioned for analysis of 1) in vivo PGE2 levels in resting skeletal muscle and 2) ex vivo skeletal muscle PGE2 production when stimulated with the COX substrate arachidonic acid (5 µM). PGE2 levels in vivo and PGE2 production ex vivo were generally unchanged at each time point after aspirin consumption. However, most individuals clearly showed suppression of PGE2, but at varying time points after aspirin consumption. When the maximum suppression after aspirin consumption was examined for each individual, independent of time, PGE2 levels in vivo (184 ± 17 and 104 ± 23pg/g wet wt at Pre and Post, respectively) and PGE2 production ex vivo (2.74 ± 0.17 and 2.09 ± 0.11pg·mg wet wt-1·min-1 at Pre and Post, respectively) were reduced ( P < 0.05) by 44% and 24%, respectively. These results provide evidence that orally consumed aspirin can inhibit the COX pathway and reduce the inflammatory mediator PGE2 in human skeletal muscle. Findings from this study highlight the need to expand our knowledge regarding the potential role for aspirin regulation of the deleterious influence of inflammation on skeletal muscle health in aging and exercising individuals. NEW & NOTEWORTHY This study demonstrated that orally consumed aspirin can target the prostaglandin/cyclooxygenase pathway in human skeletal muscle. This pathway has been shown to regulate skeletal muscle metabolism and inflammation in aging and exercising individuals. Given the prevalence of aspirin consumption, these findings may have implications for skeletal muscle health in a large segment of the population.