Gianni Parise

Analysis of global mRNA expression in human skeletal muscle during recovery from endurance exercise

To search for novel transcriptional pathways that are activated in skeletal muscle after endurance exercise, we used cDNA microarrays to measure global mRNA expression after an exhaustive bout of high‐intensity cycling (∼75 min). Healthy, young, sedentary males performed the cycling bout, and skeletal muscle biopsies were taken from the vastus lateralis before, and at 3 and 48 h after exercise. We examined mRNA expression in individual muscle samples from four subjects using cDNA microarrays, used repeated‐measures significance analysis of microarray (SAM) to determine statistically significant expression changes, and confirmed selected results using real‐time RT‐PCR. In total, the expression of 118 genes significantly increased 3 h postcycling and 8 decreased. At 48 h, the expression of 29 genes significantly increased and 5 decreased. Many of these are potentially important novel genes involved in exercise recovery and adaptation, including several involved in 1) metabolism and mitochondrial biogenesis (FOXO1, PPARδ, PPARγ, nuclear receptor binding protein 2, IL‐6 receptor, ribosomal protein L2, aminolevulinate δ‐synthase 2); 2) the oxidant stress response (metalothioneins 1B, 1F, 1G, 1H, 1L, 2A, 3, interferon regulatory factor 1); and 3) electrolyte transport across membranes [Na+‐K+‐ATPase (β3), SERCA3, chloride channel 4]. Others include genes involved in cell stress, proteolysis, apoptosis, growth, differentiation, and transcriptional activation, as well as all three nuclear receptor subfamily 4A family members (Nur77, Nurr1, and Nor1). This study is the first to characterize global mRNA expression during recovery from endurance exercise, and the results provide potential insight into 1) the transcriptional contributions to homeostatic recovery in human skeletal muscle after endurance exercise, and 2) the transcriptional contributions from a single bout of endurance exercise to the adaptive processes that occur after a period of endurance exercise training.

Low-load high volume resistance exercise stimulates muscle protein synthesis more than high-load low volume resistance exercise in young men.

Background We aimed to determine the effect of resistance exercise intensity (% 1 repetition maximum—1RM) and volume on muscle protein synthesis, anabolic signaling, and myogenic gene expression. Methodology/Principal Findings Fifteen men (21±1 years; BMI = 24.1±0.8 kg/m2) performed 4 sets of unilateral leg extension exercise at different exercise loads and/or volumes: 90% of repetition maximum (1RM) until volitional failure (90FAIL), 30% 1RM work-matched to 90%FAIL (30WM), or 30% 1RM performed until volitional failure (30FAIL). Infusion of [ring-13C6] phenylalanine with biopsies was used to measure rates of mixed (MIX), myofibrillar (MYO), and sarcoplasmic (SARC) protein synthesis at rest, and 4 h and 24 h after exercise. Exercise at 30WM induced a significant increase above rest in MIX (121%) and MYO (87%) protein synthesis at 4 h post-exercise and but at 24 h in the MIX only. The increase in the rate of protein synthesis in MIX and MYO at 4 h post-exercise with 90FAIL and 30FAIL was greater than 30WM, with no difference between these conditions; however, MYO remained elevated (199%) above rest at 24 h only in 30FAIL. There was a significant increase in AktSer473 at 24h in all conditions (P = 0.023) and mTORSer2448 phosphorylation at 4 h post-exercise (P = 0.025). Phosporylation of Erk1/2Tyr202/204, p70S6KThr389, and 4E-BP1Thr37/46 increased significantly (P<0.05) only in the 30FAIL condition at 4 h post-exercise, whereas, 4E-BP1Thr37/46 phosphorylation was greater 24 h after exercise than at rest in both 90FAIL (237%) and 30FAIL (312%) conditions. Pax7 mRNA expression increased at 24 h post-exercise (P = 0.02) regardless of condition. The mRNA expression of MyoD and myogenin were consistently elevated in the 30FAIL condition. Conclusions/Significance These results suggest that low-load high volume resistance exercise is more effective in inducing acute muscle anabolism than high-load low volume or work matched resistance exercise modes.

Resistance exercise‐induced increases in putative anabolic hormones do not enhance muscle protein synthesis or intracellular signalling in young men

We aimed to determine whether exercise‐induced elevations in systemic concentration of testosterone, growth hormone (GH) and insulin‐like growth factor‐1 (IGF‐1) enhanced post‐exercise myofibrillar protein synthesis (MPS) and phosphorylation of signalling proteins important in regulating mRNA translation. Eight young men (20 ± 1.1 years, BMI = 26 ± 3.5 kg m−2) completed two exercise protocols designed to maintain basal hormone concentrations (low hormone, LH) or elicit increases in endogenous hormones (high hormone, HH). In the LH protocol, participants performed a bout of unilateral resistance exercise with the elbow flexors. The HH protocol consisted of the same elbow flexor exercise with the contralateral arm followed immediately by high‐volume leg resistance exercise. Participants consumed 25 g of protein after arm exercise to maximize MPS. Muscle biopsies and blood samples were taken as appropriate. There were no changes in serum testosterone, GH or IGF‐1 after the LH protocol, whereas there were marked elevations after HH (testosterone, P < 0.001; GH, P < 0.001; IGF‐1, P < 0.05). Exercise stimulated a rise in MPS in the biceps brachii (rest = 0.040 ± 0.007, LH = 0.071 ± 0.008, HH = 0.064 ± 0.014% h−1; P < 0.05) with no effect of elevated hormones (P= 0.72). Phosphorylation of the 70 kDa S6 protein kinase (p70S6K) also increased post‐exercise (P < 0.05) with no differences between conditions. We conclude that the transient increases in endogenous purportedly anabolic hormones do not enhance fed‐state anabolic signalling or MPS following resistance exercise. Local mechanisms are likely to be of predominant importance for the post‐exercise increase in MPS.

Beneficial effects of creatine, CoQ10, and lipoic acid in mitochondrial disorders

Mitochondrial disorders share common cellular consequences: (1) decreased ATP production; (2) increased reliance on alternative anaerobic energy sources; and (3) increased production of reactive oxygen species. The purpose of the present study was to determine the effect of a combination therapy (creatine monohydrate, coenzyme Q10, and lipoic acid to target the above‐mentioned cellular consequences) on several outcome variables using a randomized, double‐blind, placebo‐controlled, crossover study design in patients with mitochondrial cytopathies. Three patients had mitochondrial encephalopathy, lactic acidosis, and stroke‐like episodes (MELAS), four had mitochondrial DNA deletions (three patients with chronic progressive external ophthalmoplegia and one with Kearns–Sayre syndrome), and nine had a variety of other mitochondrial diseases not falling into the two former groups. The combination therapy resulted in lower resting plasma lactate and urinary 8‐isoprostanes, as well as attenuation of the decline in peak ankle dorsiflexion strength in all patient groups, whereas higher fat‐free mass was observed only in the MELAS group. Together, these results suggest that combination therapies targeting multiple final common pathways of mitochondrial dysfunction favorably influence surrogate markers of cellular energy dysfunction. Future studies with larger sample sizes in relatively homogeneous groups will be required to determine whether such combination therapies influence function and quality of life. Muscle Nerve, 2006

Resistance exercise training decreases oxidative damage to DNA and increases cytochrome oxidase activity in older adults.

Regular resistance exercise increases muscle strength and induces muscle fibre hypertrophy in older adults. Although the underlying causes of aging remain unclear, like acute exercise, aging is associated with oxidative stress. In ageing, however, oxidative stress is closely associated with mitochondrial dysfunction as proposed by the mitochondrial theory of aging. The effect of regular resistance exercise upon mitochondrial function and oxidative stress in older adults is unknown. Twenty-eight older men and women (approximately 68.5+/-5.1 yr) performed whole-body resistance exercise training for 14 weeks. Muscle biopsies were taken before and 72 h following the last exercise bout from the vastus lateralis. Urine samples were also taken at the time of tissue collection. Resistance exercise training was associated with a decrease in 8-OHdG (Pre: 10783+/-5856, Post: 8897+/-4030 ng g(-1) creatinine; p<0.05). Protein content for CuZnSOD, MnSOD, and catalase, and enzyme activities for citrate synthase, mitochondrial ETC complex I+III, and complex II+III were not significantly different from baseline. However, complex IV activity was significantly higher after training as compared to before training (Pre: 2.2+/-0.5, Post: 2.9+/-0.9 micromol min(-1) g(-1)ww; p<0.05), as was the ratio of complex IV to complex I (Pre: 11.1+/-9.3, Post: 14.5+/-10.3; p<0.05). There were no apparent changes in normal mtDNA content or visible mtDNA deletion products as a function of training. These data suggest that regular resistance exercise decreases oxidative stress, but does not affect mtDNA. Moreover, increases in complex IV of the electron transport chain may have an indirect antioxidant effect in older adults and may improve function in daily activities.

Co-expression of IGF-1 family members with myogenic regulatory factors following acute damaging muscle-lengthening contractions in humans

Muscle regeneration following injury is dependent on the ability of muscle satellite cells to activate, proliferate and fuse with damaged fibres. This process is controlled by the myogenic regulatory factors (MRF). Little is known about the temporal relation of the MRF with the expression of known myogenic growth factors (i.e. IGF‐1) in humans following muscle damage. Eight subjects (20.6 ± 2.1 years; 81.4 ± 9.8 kg) performed 300 lengthening contractions (180 deg s−1) of their knee extensors in one leg on a dynamometer. Blood and muscle samples were collected before and at 4 (T4), 24 (T24), 72 (T72) and 120 h (T120) post‐exercise. Mechano growth factor (MGF), IGF‐1Ea and IGF‐1Eb mRNA were quantified. Serum IGF‐1 did not change over the post‐exercise time course. IGF‐1Ea and IGF‐1Eb mRNA increased ∼4‐ to 6‐fold by T72 (P < 0.01) and MGF mRNA expression peaked at T24 (P= 0.005). MyoD mRNA expression increased ∼2‐fold at T4 (P < 0.05). Myf5 expression peaked at T24 (P < 0.05), while MRF4 and myogenin mRNA expression peaked at T72 (P < 0.05). Myf5 expression strongly correlated with the increase in MGF mRNA (r2= 0.83; P= 0.03), while MRF4 was correlated with both IGF‐1Ea and ‐Eb (r2= 0.90; r2= 0.81, respectively; P < 0.05). Immunofluorescence analysis showed IGF‐1 protein expression localized to satellite cells at T24, and to satellite cells and the myofibre at T72 and T120; IGF‐1 was not detected at T0 or T4. These results suggest that the temporal response of MGF is probably related to the activation/proliferation phase of the myogenic programme as marked by an increase in both Myf5 and MyoD, while IGF‐1Ea and ‐Eb may be temporally related to differentiation as marked by an increase in MRF4 and myogenin expression following acute muscle damage.

Effect of creatine and weight training on muscle creatine and performance in vegetarians.

PURPOSE To compare the change in muscle creatine, fiber morphology, body composition, hydration status, and exercise performance between vegetarians and nonvegetarians with 8 wk of creatine supplementation and resistance training. METHODS Eighteen VG and 24 NV subjects (19-55 yr) were randomly assigned (double blind) to four groups: VG + creatine (VGCr, N=10), VG + placebo (VGPl, N=8), NV + creatine (NVCr, N=12), and NV + placebo (NVPl, N=12). Before and at the end of the study, muscle biopsies were taken from the vastus lateralis m, body composition was assessed by DXA, and strength was assessed using 1-RM bench press and leg press. Subjects participated in the same 8-wk resistance-training program. Creatine dosage was based on lean tissue mass (0.25 g.kg(-1) LTM.d(-1) x 7 d; 0.0625 g.kg(-1) LTM.d(-1) x 49 d). RESULTS Biopsy samples indicated that total creatine (TCr=free Cr + PCr) was significantly lower in VG compared with NV at baseline (VG=117 mmol.kg(-1); NV=130 mmol.kg(-1); P<0.05). For Cr subjects, there was a greater increase in PCr, TCr, bench-press strength, isokinetic work, Type II fiber area, and whole-body lean tissue compared with subjects on placebo (P<0.05). Vegetarians who took Cr had a greater increase in TCr, PCr, lean tissue, and total work performance than nonvegetarians who took Cr (P<0.05). The change in muscle TCr was significantly correlated with initial muscle TCr, and the change in lean tissue mass and exercise performance. These findings confirm an ergogenic effect of Cr during resistance training and suggest that subjects with initially low levels of intramuscular Cr (vegetarians) are more responsive to supplementation.

Direct measurement of high-energy phosphate compounds in patients with neuromuscular disease.

Several neuromuscular disorders are associated with reductions in intramuscular adenosine triphosphate (ATP) and/or phosphocreatine (PCr). These alterations have been primarily characterized using 31P–magnetic resonance spectroscopy (31P‐MRS). We prospectively measured total creatine, PCr, and ATP in muscle biopsies from 81 patients: normal controls (n = 33), mitochondrial cytopathy (n = 8), neuropathic (n = 3), dystrophy/congenital myopathies (n = 7), inflammatory myopathy (n = 12), and miscellaneous myopathies (n = 18) using direct biochemical analysis. Intramuscular concentrations of PCr and ATP were lower for the dystrophy/congenital myopathy, inflammatory myopathy, and mitochondrial disease patients with ragged red fiber (RRF) as compared with normal controls (P < 0.05). Total creatine was lower for the dystrophy/congenital myopathy group as compared with the normal control group (P < 0.05). These values compare favorably to results from other studies using 31P‐MRS and provide external validation for the values obtained using that method. Given the reductions in high‐energy phosphate compounds in these patients, there is the potential for therapeutic intervention with creatine monohydrate supplementation.

Association of interleukin-6 signalling with the muscle stem cell response following muscle-lengthening contractions in humans.

Background The regulation of muscle stem cells in humans in response to muscle injury remains largely undefined. Recently, interleukin-6 (IL-6) has been implicated in muscle stem cell (satellite cell)-mediated muscle hypertrophy in animals; however, the role of IL-6 in the satellite cell (SC) response following muscle-lengthening contractions in humans has not been studied. Methodology/Principal Findings Eight subjects (age 22±1 y; 79±8 kg) performed 300 maximal unilateral lengthening contractions (3.14 rad.s−1) of the knee extensors. Blood and muscle samples were collected before and at 4, 24, 72, and 120 hours post intervention. IL-6, IL-6 receptor (IL-6Rα), cyclin D1, suppressor of cytokine signling-3 (SOCS3) mRNA were measured using quantitative RT-PCR and serum IL-6 protein was measured using an ELISA kit. JAK2 and STAT3 phosphorylated and total protein was measured using western blotting techniques. Immunohistochemical analysis of muscle cross-sections was performed for the quantification of SCs (Pax7+ cells) as well as the expression of phosphorylated STAT3, IL-6, IL-6Rα, and PCNA across all time-points. The SC response, as defined by an amplification of Pax7+ cells, was rapid, increasing by 24 h and peaking 72 h following the intervention. Muscle IL-6 mRNA increased following the intervention, which correlated strongly (R2 = 0.89, p<0.002) with an increase in serum IL-6 concentration. SC IL-6Rα protein was expressed on the fiber, but was also localized to the SC, and IL-6+ SC increased rapidly following muscle-lengthening contractions and returned to basal levels by 72 h post-intervention, demonstrating an acute temporal expression of IL-6 with SC. Phosphorylated STAT3 was evident in SCs 4 h after lengthening contraction, and the downstream genes, cyclin D1 and SOCS3 were significantly elevated 24 hours after the intervention. Conclusions/Significance The increased expression of STAT3 responsive genes and expression of IL-6 within SCs demonstrate that IL-6/STAT3 signaling occurred in SCs, correlating with an increase in SC proliferation, evidenced by increased Pax7+/PCNA+ cell number in the early stages of the time-course. Collectively, these data illustrate that IL-6 is an important signaling molecule associated with the SC response to acute muscle-lengthening contractions in humans.

Resistance training, sarcopenia, and the mitochondrial theory of aging

Skeletal muscle aging is associated with a significant loss of muscle mass, strength, function, and quality of life. In addition, the healthcare cost of aging and age-related disease is growing, and will continue to grow as a larger proportion of our population reaches retirement age and beyond. The mitochondrial theory of aging has been identified as a leading explanation of the aging process and describes a path leading to cellular senescence that includes electron transport chain deficiency, reactive oxygen species production, and the accumulation of mitochondrial DNA deletions and mutations. It is also quite clear that regular resistance exercise is a potent and effective countermeasure for skeletal muscle aging. In this review, we discuss age-related sarcopenia, the mitochondrial theory of aging, and how resistance exercise may directly affect key components of the mitochondrial theory. It is clear from the data discussed that regular resistance training can effectively disturb processes that contribute to the progression of aging as it pertains to the mitochondrial theory.