Lauren G. MacNeil

Gene expression profiling in human skeletal muscle during recovery from eccentric exercise

We used cDNA microarrays to screen for differentially expressed genes during recovery from exercise-induced muscle damage in humans. Male subjects (n = 4) performed 300 maximal eccentric contractions, and skeletal muscle biopsy samples were analyzed at 3 h and 48 h after exercise. In total, 113 genes increased 3 h postexercise, and 34 decreased. At 48 h postexercise, 59 genes increased and 29 decreased. On the basis of these data, we chose 19 gene changes and conducted secondary analyses using real-time RT-PCR from muscle biopsy samples taken from 11 additional subjects who performed an identical bout of exercise. Real-time RT-PCR analyses confirmed that exercise-induced muscle damage led to a rapid (3 h) increase in sterol response element binding protein 2 (SREBP-2), followed by a delayed (48 h) increase in the SREBP-2 gene targets Acyl CoA:cholesterol acyltransferase (ACAT)-2 and insulin-induced gene 1 (insig-1). The expression of the IL-1 receptor, a known regulator of SREBP-2, was also elevated after exercise. Taken together, these expression changes suggest a transcriptional program for increasing cholesterol and lipid synthesis and/or modification. Additionally, damaging exercise induced the expression of protein kinase H11, capping protein Z alpha (capZalpha), and modulatory calcineurin-interacting protein 1 (MCIP1), as well as cardiac ankryin repeat protein 1 (CARP1), DNAJB2, c-myc, and junD, each of which are likely involved in skeletal muscle growth, remodeling, and stress management. In summary, using DNA microarrays and RT-PCR, we have identified novel genes that respond to skeletal muscle damage, which, given the known biological functions, are likely involved in recovery from and/or adaptation to damaging exercise.

Eccentric exercise activates novel transcriptional regulation of hypertrophic signaling pathways not affected by hormone changes.

Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17β-estradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 single-leg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P<0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RT-PCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P<0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P<0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2.

Effect of creatine supplementation during cast-induced immobilization on the preservation of muscle mass, strength, and endurance.

Johnston, APW, Burke, DG, MacNeil, LG, and Candow, DG. Effect of creatine supplementation during cast-induced immobilization on the preservation of muscle mass, strength, and endurance. J Strength Cond Res 23(1): 116-120, 2009-Muscle and strength loss will occur during periods of physical inactivity and immobilization. Creatine supplementation may have a favorable effect on muscle mass and strength independently of exercise. The purpose of this study was to determine the effects of creatine supplementation on upper-limb muscle mass and muscle performance after immobilization. Before the study, creatine-naïve men (n = 7; 18-25 years) were assessed for lean tissue mass (dual-energy X-ray absorptiometry), strength (1-repetition maximum [1RM] isometric single arm elbow flexion/extension), and muscle endurance (maximum number of single-arm isokinetic elbow flexion/extension repetitions at 60% 1RM). After baseline measures, subjects had their dominant or nondominant (random assignment) upper limb immobilized (long arm plaster cast) at 90° elbow flexion. Using a single-blind crossover design, subjects received placebo (maltodextrin; 4 × 5 g·d−1) during days 1-7 and creatine (4 × 5 g·d−1) during days 15-21. The cast was removed during days 8-14 and 22-29. The dependent measures of lean tissue mass, strength, and endurance were assessed at baseline, postcast, and after the study. During immobilization, compared with isocaloric placebo, creatine supplementation better maintained lean tissue mass (Cr +0.9% vs. PLA −3.7%, p < 0.05), elbow flexor strength (Cr −4.1% vs. PLA −21.5%, p < 0.05), and endurance (Cr −9.6% vs. PLA −43%, p < 0.05), and elbow extensor strength (Cr −3.8% vs. PLA −18%, p < 0.05) and endurance (Cr −6.5% vs. PLA −35%, p < 0.05). These results indicate that short-term creatine supplementation attenuates the loss in muscle mass and strength during upper-arm immobilization in young men.

Long-term Aerobic Exercise Is Associated With Greater Muscle Strength Throughout the Life Span

Aging is associated with a progressive decline in muscle strength, muscle mass, and aerobic capacity, which reduces mobility and impairs quality of life in elderly adults. Exercise is commonly employed to improve muscle function in individuals of all ages; however, chronic aerobic exercise is believed to largely impact cardiovascular function and oxidative metabolism, with minimal effects on muscle mass and strength. To study the effects of long-term aerobic exercise on muscle strength, we recruited 74 sedentary (SED) or highly aerobically active (ACT) men and women from within three distinct age groups (young: 20-39 years, middle: 40-64 years, and older: 65-86 years) and tested their aerobic capacity, isometric grip and knee extensor strength, and dynamic 1 repetition maximum knee extension. As expected, ACT subjects had greater maximal oxygen uptake and peak aerobic power output compared with SED subjects (p < .05). Grip strength relative to body weight declined with age (p < .05) and was greater in ACT compared with SED subjects in both hands (p < .05). Similarly, relative maximal isometric knee extension torque declined with age (p < .05) and was higher in ACT versus SED individuals in both legs (p < .05). Absolute and relative 1 repetition maximum knee extension declined with age (p < .05) and were greater in ACT versus SED groups (p < .05). Knee extensor strength was associated with a greater amount of leg lean mass in the ACT subjects (p < .05). In summary, long-term aerobic exercise appears to attenuate age-related reductions in muscle strength in addition to its cardiorespiratory and metabolic benefits.

17β-estradiol attenuates exercise-induced neutrophil infiltration in men.

17β-estradiol (E2) attenuates exercise-induced muscle damage and inflammation in some models. Eighteen men completed 150 eccentric contractions after random assignment to placebo (Control group) or E2 supplementation (Experimental group). Muscle biopsies and blood samples were collected at baseline, following 8-day supplementation and 3 h and 48 h after exercise. Blood samples were analyzed for sex hormone concentration, creatine kinase (CK) activity and total antioxidant capacity. The mRNA content of genes involved in lipid and cholesterol homeostasis [forkhead box O1 (FOXO1), caveolin 1, and sterol regulatory element binding protein-2 (SREBP2)] and antioxidant defense (SOD1 and -2) were measured by RT-PCR. Immunohistochemistry was used to quantify muscle neutrophil (myeloperoxidase) and macrophage (CD68) content. Serum E2 concentration increased 2.5-fold with supplementation (P < 0.001), attenuating neutrophil infiltration at 3 h (P < 0.05) and 48 h (P < 0.001), and the induction of SOD1 at 48 h (P = 0.02). Macrophage density at 48 h (P < 0.05) and SOD2 mRNA at 3 h (P = 0.01) increased but were not affected by E2. Serum CK activity was higher at 48 h for both groups (P < 0.05). FOXO1, caveolin 1 and SREBP2 expression were 2.8-fold (P < 0.05), 1.4-fold (P < 0.05), and 1.5-fold (P < 0.001) and higher at 3 h after exercise with no effect of E2. This suggests that E2 attenuates neutrophil infiltration; however, the mechanism does not appear to be lesser oxidative stress or membrane damage and may indicate lesser neutrophil/endothelial interaction.

Exercise‐stimulated interleukin‐15 is controlled by AMPK and regulates skin metabolism and aging

Aging is commonly associated with a structural deterioration of skin that compromises its barrier function, healing, and susceptibility to disease. Several lines of evidence show that these changes are driven largely by impaired tissue mitochondrial metabolism. While exercise is associated with numerous health benefits, there is no evidence that it affects skin tissue or that endocrine muscle‐to‐skin signaling occurs. We demonstrate that endurance exercise attenuates age‐associated changes to skin in humans and mice and identify exercise‐induced IL‐15 as a novel regulator of mitochondrial function in aging skin. We show that exercise controls IL‐15 expression in part through skeletal muscle AMP‐activated protein kinase (AMPK), a central regulator of metabolism, and that the elimination of muscle AMPK causes a deterioration of skin structure. Finally, we establish that daily IL‐15 therapy mimics some of the anti‐aging effects of exercise on muscle and skin in mice. Thus, we elucidate a mechanism by which exercise confers health benefits to skin and suggest that low‐dose IL‐15 therapy may prove to be a beneficial strategy to attenuate skin aging.

Supplementation with α-Lipoic Acid, CoQ10, and Vitamin E Augments Running Performance and Mitochondrial Function in Female Mice

Antioxidant supplements are widely consumed by the general public; however, their effects of on exercise performance are controversial. The aim of this study was to examine the effects of an antioxidant cocktail (α-lipoic acid, vitamin E and coenzyme Q10) on exercise performance, muscle function and training adaptations in mice. C57Bl/J6 mice were placed on antioxidant supplement or placebo-control diets (n = 36/group) and divided into trained (8 wks treadmill running) (n = 12/group) and untrained groups (n = 24/group). Antioxidant supplementation had no effect on the running performance of trained mice nor did it affect training adaptations; however, untrained female mice that received antioxidants performed significantly better than placebo-control mice (p ≤ 0.05). Furthermore, antioxidant-supplemented females (untrained) showed elevated respiratory capacity in freshly excised muscle fibers (quadriceps femoris) (p ≤ 0.05), reduced oxidative damage to muscle proteins (p ≤ 0.05), and increased expression of mitochondrial proteins (p ≤ 0.05) compared to placebo-controls. These changes were attributed to increased expression of proliferator-activated receptor gamma coactivator 1α (PGC-1α) (p ≤ 0.05) via activation of AMP-activated protein kinase (AMPK) (p ≤ 0.05) by antioxidant supplementation. Overall, these results indicate that this antioxidant supplement exerts gender specific effects; augmenting performance and mitochondrial function in untrained females, but does not attenuate training adaptations.

Elevated Mitochondrial Oxidative Stress Impairs Metabolic Adaptations to Exercise in Skeletal Muscle

Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in Sod2 +/- mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training Sod2 +/- mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised Sod2 +/- mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.

Oxidative stress and Nrf2 signaling in McArdle disease

McArdle disease (MD) is a metabolic myopathy due to myophosphorylase deficiency, which leads to a severe limitation in the rate of adenosine triphosphate (ATP) resynthesis. Compensatory flux through the myoadenylate deaminase > > xanthine oxidase pathway should result in higher oxidative stress in skeletal muscle; however, oxidative stress and nuclear factor erythroid 2-related factor 2 (Nrf2) mediated antioxidant response cascade in MD patients have not yet been examined. We show that MD patients have elevated muscle protein carbonyls and 4-hydroxynonenal (4-HNE) in comparison with healthy, age and activity matched controls (P < 0.05). Nuclear abundance of Nrf2 and Nrf2-antioxidant response element (ARE) binding was also higher in MD patients compared with controls (P < 0.05). The expressions of Nrf2 target genes were also higher in MD patients vs. controls. These observations suggest that MD patients experience elevated levels of oxidative stress, and that the Nrf2-mediated antioxidant response cascade is up-regulated in skeletal muscle to compensate.

Daily chocolate milk consumption does not enhance the effect of resistance training in young and old men: a randomized controlled trial

Older and younger men completed 12 weeks of resistance training and ingested either 500 mL of chocolate milk or placebo daily. Training increased strength in both age groups (p < 0.05), with no supplementation effect. Type I muscle fibre area increased with training (p = 0.008) with no effect of age or supplementation. Type II fibre area increased (p = 0.014) in young men only with no supplementation effect. Chocolate milk did not enhance skeletal muscle hypertrophy following training.