Overview
The methods and ethics of the present study have been approved by the Institutional Review Board of the University of Thessaly (ref. number 1387) and procedures are in accordance with the 1975 Declaration of Helsinki, as revised in 2000.
Participants’ characteristics and eligibility
We will initially recruit 40-60 young males. In organized meetings, participants will be informed by the investigators about the purpose of the study, the experimental procedures and all the possible risks and benefits associated. Volunteers will participate in the study if they: 1) are young males aged 18-30 years; 2) are non-smokers; 3) abstain from any vigorous physical activity during the study and ≥4 weeks prior to the study; 4) have no recent history of musculoskeletal injury, lower limb trauma and metabolic diseases; 5) abstained from consumption of alcohol, caffeine, any type of nutritional supplements, NSAIDs and medication before (≥6 months) and throughout the experimental period; and 6) have no allergies or intolerance to N-acetylcysteine (NAC).
Participants recruitment and screening
Participants will be recruited via media advertisements and posters. All volunteers will complete a health history questionnaire and a written consent form will be acquired from each participant by the investigators. A preliminary power analysis (based on previous studies that used NAC administration to investigate its effects on EIMD), using the G*Power 3.0.10 program, showed that a minimum number of 10 participants per group is needed to achieve a statistical power greater than 0.90 at an α error of 0.05 and effect size >0.55 among repeated measurements [21]. However, the total number of participants depends also on potential dropouts according to the following formula: n’ = n/(1-d) [22]. Thus, the final number of participants to be recruited with a dropout rate of 10% would be n’ =10 / (1-0.1) = 11.1. Therefore, ≥12 participants per group (LR vs. HR) will be selected from the initial sample to participate in the clinical trial.
Study design
A randomized, two-trial (placebo vs. N-acetylcysteine), cross-over, double-blind, repeated measures design will be implemented. Participants who will fulfill the inclusion criteria will have their body mass and height, resting metabolic rate (RMR), body composition, muscle strength, soreness and maximal oxygen consumption (VO2max) measured. Since protein and antioxidant consumption as well as systematic physical activity and/or exercise may affect SC responses and/or redox status, daily protein and antioxidant intake of volunteers will be monitored for a week [23-25]. A 2-week adaptation period (only before the first trial) will be applied to adjust (through a balanced diet using RMR values and daily energy expenditure measurements) protein and antioxidant intake at the levels required by current RDAs (a protein intake of 0.8-1 g protein/kg/day; 900 mg/day for vitamin A; 90 mg/day for vitamin C; 600 IU/d for vitamin D; 15 mg/day for vitamin E; 11 mg/d for Zinc; 400 mg/d for Magnesium and 55 mg/d for selenium) [26,27]. This dietary protocol will be designed and implemented by a registered dietician. During the same time-frame, participants’ daily physical activity levels will be monitored and familiarization with experimental procedures will be provided.
Evidence exists that skeletal muscle growth and myogenic potential is directly related to its basal SCs content [23,28]. As such, volunteers will provide a resting (they will abstain from any exercise or intense physical activity for at least five days prior to sampling) muscle biopsy sample (baseline sampling) from their vastus lateralis muscle (of their dominant limb) and based on its SCs content will be stratified to either high (HR) or low (LR) respondents. Participants will be stratified as high (HR) or low (LR) respondents using a K-means cluster analysis. Blood sampling and performance measurements will also be performed at baseline.
Following stratification to HR and LR, volunteers will then perform two trials in a randomized order: a) placebo ingestion and b) NAC ingestion. Placebo or NAC will be consumed before (a 7-day loading phase) and after (immediately post-exercise and for eight consecutive days thereafter) an intense eccentric exercise protocol. During both trials, participants will follow the balanced daily dietary protocol of the adaptation period. However, daily dietary intake will be recorded and analyzed during each trial in an attempt to minimize deviations from the prescribed diet. A 4-week washout period will be implemented between trials (dietary intake during this period will also be adjusted according to that applied during the adaptive period). During the entire experimental period, participants will be asked to abstain from any strenuous physical activity or exercise. Muscle biopsies and blood samples will be collected after overnight fasting before the exercise protocol (pre-exercise sampling) as well as at 2- and 8-days post-exercise. Muscle strength and delayed onset of muscle soreness (DOMS) will be evaluated at the same time points. All measurements and collection of biological samples will be performed at the same time of day, in both trials, to prevent circadian rhythm variations. Figure 1 indicates the SPIRIT flow diagram of the SpEED trial, Figure 2 shows the CONSORT diagram of the study and Figure 3 illustrates the experimental flowchart.
Exercise protocol
Participants will perform a protocol consisting of 300 eccentric unilateral maximal contractions (20 sets, 15 repetitions/set, 30-second rest between sets) of the quadriceps muscles on an isokinetic dynamometer (Cybex 770, USA) at a speed of 30 °/s. A different limb will be used in each trial. Before the protocol a standard warm up will precede involving 8-min cycling on a cycle ergometer (Monark 834, 154 ERGOMED C, Sweden) at a speed of 70 rpm/min and at 50 W, followed by 5-min stretching exercises. Knee extensors will be isolated using straps in the shoulders, hips and thigh. This protocol has been described in the literature to effectively induce a significant level of skeletal muscle damage and myofibrillar disruption as documented with electron microscopy and immunohistochemistry [21,29].
Supplementation protocol
Participants will consume either NAC or placebo in a random order. A dosage of 40 mg NAC/kg/day will be administered orally in 3 daily doses (equally distributed). This dosage successfully increases total cysteine and cystine levels in plasma and is below the upper safe limit of 5 g/day [30,31]. N-acetylcysteine in a powder form will be diluted in a 250-mL drink containing 248 ml water and 2 ml of natural, non-caloric, flavoring-sweetener containing sucralose (Flavdrops, My Protein, UK). The placebo supplement will contain 248 ml water and 2 ml of the flavoring-sweetener substance and it will be prepared to be identical to NAC in terms of taste and smell apart from the NAC content. Participants and investigators will be blinded to the supplementation condition. In both trials, each participant will be asked 15 times (once a day) if they realize whether the drink they consumed was the placebo or the experimental one. Responses will be recorded, and correct or incorrect answers will be measured. The presence of any adverse side effects will be monitored in both trials via questionnaires by an unblinded (for ethical reasons) investigator [21].
Anthropometric measurements
Standing body mass and height will be measured on a beam balance equipped with stadiometer (Beam Balance-Stadiometer, SECA, Vogel & Halke, Hamburg, Germany) while participants wearing light clothing as described previously [32]. Body mass index will be calculated as mass per height squared. Dual energy X-ray absorptiometry scanner (GE Healthcare, Lunar DPX-NT) will be utilized for body composition assessment. On each testing day the equipment will be calibrated using a LS phantom in accordance to standard procedures. Participants will be asked to remain still and they will be scanned in the supine position using the total body analysis under scanning conditions automatically selected by the software (standard, thick, thin). Total and regional fat mass (g), lean mass (kg) and body fat (% and kg) values will be obtained. GE enCORE software will be utilized for all DXA scans and analyses.
Resting metabolic rate
For RMR assessment, resting VO2/CO2 values will be measured in the morning (07:00–09:00) after overnight fasting utilizing an open-circuit type indirect calorimeter with a ventilated hood system (Vmax Encore 29, BEBJO296, Yorba Linda, CA, USA) and the 24-h RMR will be calculated as previously described [33].
Physical activity assessment
Habitual physical activity (PA) will be monitored over a 7-day period using the ActiGraph, GT3X+ accelerometers (ActiGraph, Pensacola, FL, USA). Participants will be taught, by an experienced researcher, how to wear the adjustable belt on the waist with the accelerometer monitor on the right side of the hip and they will be asked to wear it throughout the day for seven consecutive days, apart from bathing, swimming and sleep. To be included in the analysis, participants will have to complete four full days of wearing time (i.e., ≥four days with ≥10 wear hours/day). From the data obtained, nonwear time will be calculated and daily activity levels and sedentary time will be expressed as steps per day and time spent at sedentary, light, moderate, vigorous and moderate-to-vigorous PA [34,35]. ActiLife 6 software will be used to initialize accelerometers and download data using 60-s epoch length.
Dietary intake analysis
Participants will be instructed by a registered dietitian on how to estimate food/fluid servings and sizes and how to complete a 7-day diet recall during both trials and the washout period to ensure that they will follow the same dietary regimen. Specifically, participants will be provided with colored images showing different food portions and detailed instructions that they will use to weight their food. When possible, the name of the brand and/or manufacturer will be recorded. Diet recalls will be analyzed using the Science Fit Diet 200 A (Science Technologies, Athens, Greece) dietary software for data regarding total energy (kcal), carbohydrate, fat, protein (g/kg/day & g/day), vitamin A (mg/day), vitamin C (mg/day), vitamin D (IU/day), vitamin E (mg/day), selenium (mg/day), zinc (mg/day), and magnesium (mg/d).
Maximal oxygen consumption
Maximal oxygen consumption (VO2max) will be measured using open-circuit spirometry with an automated pulmonary gas exchange system (Vmax Encore 29, BEBJO296, Yorba Linda, CA, USA) via the breath-by-breath analysis during a graded exercise test on a treadmill (Stex 8025 T, Korea) until volitional fatigue, according to procedures previously described [36].
Muscle strength and soreness
Knee extensors’ (KE) maximal eccentric and concentric peak torque of the exercised limb will be measured on an isokinetic dynamometer (Cybex 770, USA) at 60 °/s as described elsewhere [37]. Delayed onset of muscle soreness (DOMS) of KE of the exercised limb will be evaluated by palpation of the belly and distal region after participants have performed three full squat repetitions. Then, participants will rate their soreness level on a scale from 1 to 10 (1 = no pain, 10 = extremely sore). DOMS assessment will be carried out by the same investigator [38].
Blood sampling and biochemical assays
Following an overnight fasting blood samples will be drawn from the antecubital vein by venipuncture with a 20-gauge disposable needle equipped with a Vacutainer tube holder (Becton Dickinson) with the participants in a supine position. For serum separation, blood samples will be allowed to clot at room temperature and then will be centrifuged (1370 g, 10 min, 4 C). The supernatant will be aliquoted into eppendorf tubes for subsequent analysis of CRP, TNF-α, IL-6, IL-8, IL-10 (inflammation), creatine kinase (CK) activity (muscle damage), total antioxidant capacity (TAC) (oxidative stress) and cortisol (hormonal response). Another blood portion will be collected in EDTA-containing tubes and will be centrifuged at 1370 g, 10 min, 4 C to collect the plasma. Plasma samples will be used for the measurement of protein carbonyls (PC) (protein oxidation) and malondialdehyde (MDA) (lipid peroxidation). Packed erythrocytes (RBCs) will be obtained after lysis of the plasma samples for the measurement of reduced and oxidized glutathione (GSH; GSSG), catalase (CAT) and hemoglobin (Hb) (RBCs’ redox status). All samples will be aliquoted in multiple eppendorf tubes and stored at – 80 °C until analysis. A small portion of whole blood (2 ml) will be collected in tubes containing EDTA for a complete blood count analysis on an automated hematology analyzer (Mythic 18, Orphee SA, Geneva, Switzerland). All assays will be performed in duplicate.
Muscle biopsy sampling
Percutaneous needle muscle biopsies will be obtained after an (~10 h) overnight fast (baseline) using the Bergstrom technique with the application of manual suction from the mid-portion of the vastus lateralis muscle under local anesthetic (xylocaine 1%) [39]. Subjects will be asked to refrain from any physical activity at least 96 hours prior to muscle biopsy sampling. Subsequent muscle biopsies (Pre, 2- and 8-days post-exercise) will be spaced 5 cm apart to diminish a repeated biopsy effect. Upon excision, adipose tissue and blood will be carefully removed and muscle samples suited for histology will be aligned and immediately be mounted in optimal cutting temperature (OCT) compound, immersed in nitrogen-cooled isopentane and stored at –80°C. Embedded samples will be sectioned (7 μm) at –20°C using a cryostat and stored at –80°C. Muscle samples suited for western blotting, mRNA, muscle thiols and oxidative stress analyses will directly be frozen in liquid nitrogen, and stored at –80°C.
Histological analyses
Sections will be stained with hematoxylin and eosin (H&E) in order to quantify damaged myofibers [40]. Fibers indicating loss of the physiological outline, sarcolemma damage, mononuclear cells infiltration and central nuclei will be expressed as a percentage of the total number of fibers.
Immunofluorescence
Muscle cross sections (7μm) will be allowed to air dry at room temperature for 30 minutes. For fiber-type-specific SCs analyses samples will be stained with appropriate primary and secondary antibodies against specific antigens such as, Pax7, MyoD, myosin heavy chain type II, and laminin as described previously [41-43]. For fiber-type-specific M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage quantification, muscle cross sections will be stained with appropriate primary and secondary antibodies against CD11b+, CD206+ and laminin as described previously [44]. Nuclei will be visualized with 4’,6-diamidino-2-phenylindole (DAPI) contained in the mounting media prior to coverslipping. The specificity of staining will be verified using negative controls. Slides will be viewed using the Olympus BX2 microscope equipped with a high-resolution fluorescent camera. Images will be captured and analyzed using the manufacture’s software. All images will be obtained with the 20X objective. Fiber cross sectional area (CSA), fiber type distribution (% Type I and II fibers) myonuclei content (DAPI+ cells), fiber-type-specific satellite cell content and activation status (i.e. Pax7+/MyoD− and Pax7+/MyoD+) and M1 (CD11b+ cells) and M2 (CD206+ cells) macrophage content will be determined. The satellite cells content and activation will be determined via the colocalization of Pax7+ and DAPI and/or the colocalization of Pax7, MyoD and DAPI within the laminin border. Macrophage content will be determined via the colocalization of CD11b+ and DAPI for M1 macrophages and CD206+ and DAPI for M2 macrophages.
Quantitative RT-PCR
Total RNA will be isolated from 10-20 mg of frozen muscle tissue using the NucleoSpin RNA Plus kit (MACHERY-NAGEL), according to the manufacturer’s instructions, at a final volume of 80-120 μL. RNA concentration (ng/mL) and purity (260/280) will be measured spectophotometrically (Hitachi UV/VIS; Hitachi Instruments Inc.). Then samples will be reverse transcribed using a PrimeScript 1st strand cDNA synthesis kit (Takara) in 20 μl reaction volumes, according to the protocol of the manufacturer. Quantitative RT-PCR reactions will run in triplicates containing RT Sybr Green qPCR Master Mix. Primers for Myf5, myogenin, MRF4, myostatin and GAPDH will be purchased and mRNA expression levels will be calculated using the 2 - ΔΔCt method. Fold changes from baseline will be calculated using the ΔΔCt method and normalization will be performed using the housekeeping gene GAPDH [45].
Western blotting
Changes in protein expression levels of glutathione peroxidase 3 (GPX-3), superoxide dismutase 2 (SOD-2) and thioredoxin (Trx-1) (related to SCs homeostasis), IGF-1, Notch1 and Wnt3 (related to SCs mobilization) will be analyzed by immunoblotting. Muscle samples will be homogenized in lysis buffer and then centrifuged (13,000 rpm, 4°C, 10 min) and the supernatant will be collected. Total protein concentration will be determined using the Bradford method (Bradford Protein Assay; Bio-Rad). 20 mg of protein will be loaded in gradient precast gels (Mini-PROTEAN TGX Gels; Bio-Rad) and will be subjected to SDS-PAGE electrophoresis at room temperature. Afterwards, proteins will be transferred to transblot stacks using the Trans-blot Turbo transfer system (Bio-Rad), blocked for 1 hour and incubated with primary antibodies overnight at 4 °C. Membranes will be washed in TBS-T solution and will be incubated with appropriate secondary antibodies for 1 hour at room temperature. Following another washing step (in TBS-T), membranes will be visualized by chemiluminescence and quantified using densitometry. Normalization will be performed with the housekeeping protein GAPDH.
Muscle thiols and OXS markers
Muscle samples will be homogenized in PBS containing protein inhibitors as described previously [46]. After homogenization, the samples will be centrifuged (12000 g, 4°C, 30 min) and the supernatant will be collected. Muscle reduced and oxidized glutathione (GSH, GSSG), protein carbonyls (PC), and malondialdehyde (MDA) will be measured as indices of muscle’s redox status. All measurements will be performed spectophotometrically (Hitachi UV/VIS; Hitachi Instruments Inc.) as described elsewhere [46]. All assays will be performed in duplicate.
Statistical analyses
K-means cluster analysis will be utilized to efficiently define two separate groups of subjects (LR and HR group) from the total sample, based on the SCs content of their vastus lateralis muscle of their dominant leg [23]. This type of analysis is a form of partitional clustering and is a multivariate method used to identify homogeneous groups (i.e., clusters) of cases based on a common trait [28,47]. Data normality will be examined using the Kolmogorov-Smirnov and the Shapiro-Wilk test. If our data sets follow normal distribution, parametric tests will be applied. Baseline comparisons on the LR and HR group (anthropometrics, body composition, strength, VO2max, dietary profile, physical activity) will be performed using One-Way Anova. Anova repeated measures will be used to determine a time-effect in each trial accompanied by Bonferroni correction for pairwise comparisons. Two-Way Anova will be used to determine a trial-effect with Bonferroni correction for pairwise comparisons. If the data normality is violated, non-parametric tests will be applied. Baseline comparisons will be performed using Kruskal–Wallis test. Friedman test will be used to determine time-effect in each trial accompanied by Wilcoxon signed-rank test for pairwise comparisons. Kruskal–Wallis test will be used to determine trial-effect accompanied by Mann-Whitney for pairwise comparisons. Pearson’s correlation analysis will also be used to examine possible relations among variables. Correlation coefficients of r < 0.2, 0.2 < r < 0.7 and r > 0.7 will be defined as small, moderate, and high, respectively. The level of statistical significance will be set at p < 0.05. Effect sizes (ES) and confidence intervals (CI) will be calculated on results of all dependent variables using the Hedge’s g method, corrected for bias. ES will be interpreted as none, small, medium-sized, and large for values 0.00–0.19, 0.20–0.49, 0.50–0.79, and ≥0.8, respectively. Statistical analyses will be performed using the SPSS 20.0 software (IBM SPSS Statistics).