Redox Regulation of Satellite Cells and Skeletal Muscle Healing

Study Overview
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Brief Summary
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Skeletal muscle stem cells (Satellite cells) are indispensable for muscle growth and remodeling following myofibril damage. Skeletal muscle trauma is present in numerous catabolic conditions, characterized by elevated proteolysis and muscle wasting such as, cancer cachexia and muscular dystrophy, which result in physical capacity impairment and a deteriorated quality of life. Recent studies performed in animals and cell cultures indicate that the increased levels of inflammation and oxidative stress and the reduction of antioxidant defense may blunt the satellite cells response and myogenic programming during muscle healing. However, evidence regarding the effects of redox status on satellite cells and muscle myogenic potential in humans is lacking. Exercise-induced muscle damage bears striking similarities with the aforementioned conditions, which makes it a valuable tool to investigate the redox-dependent regulation of satellite cells during muscle healing. Thus, the objectives of the present study are to examine the effects of redox status perturbation (via N-acetylcysteine administration) on intracellular pathways responsible for satellite cells responses at rest and following aseptic muscle trauma induced by damaging exercise.

Detailed Description
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A total number of 40-60 males, young individuals aged 18-30 years, will be initially enrolled in the study. Then, participants will be allocated to either a 1) Low Respondents (LR) or a 2) High Respondents (HR) group based on the basal satellite cells content of their vastus lateralis muscle of their dominant leg. In a double-blind, crossover, repeated measures design, participants will consume either Placebo (PLA) or N-acetylcysteine (NAC) before (7-day loading phase), on exercise day and for 8 consecutive days following a single bout of intense exercise (300 eccentric contractions at 30 deg/sec in an isokinetic dynamometer). In both conditions, blood samples and muscle biopsies will be collected at baseline, before the exercise protocol and at 2- and 8-days post-exercise. Muscle performance and soreness will also be assessed at the same time points. Before the first trial, participants' dietary intake (via diet recalls) and physical activity (via accelerometry) will be analyzed and nutritional plans will be provided to participants in order to normalize their antioxidant and protein intake. A 4-week washout period will be implemented between trials. Blood samples will be analyzed for inflammation and oxidative stress markers. Muscle samples will be analyzed for satellite cells responses and myogenic potential, protein levels of intracellular signaling proteins, muscle thiols and antioxidant enzymes activity.

Official Title
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The Effect of Redox Potential on the Regulation of Satellite Cells and Skeletal Muscle Healing Following Exercise-Induced Muscle Damage

Conditions
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Satellite Cells, Redox Status, Exercise-induced Muscle Trauma, Aseptic Inflammation

Intervention / Treatment
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* Dietary Supplement: N-Acetylcysteine
* Dietary Supplement: Placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: No recent history of musculoskeletal injury Non-smokers. Abstain from any vigorous physical activity during the study Abstain from consumption of caffeine, alcohol, performance-enhancing or antioxidant supplements, NSAIDs and medications before (at least 6 months) and during the study. Exclusion Criteria: A known NAC intolerance or allergy A recent febrile illness A recent history of muscle lesion and/or lower limb trauma Presence of metabolic diseases Use of anti-inflammatory medication. Use of medication interacting with muscle metabolism.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 30 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Crossover Assignment
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: N-Acetylcysteine<br>N-Acetylcysteine supplementation: Orally, 40 mg/kg per day in 3 doses (250 ml each) for 7 consecutive days and immediately post-exercise. The remaining 8 days, 40mg/kg per day in 3 doses (250 ml each). | Dietary Supplement: N-Acetylcysteine<br>* N-Acetylcysteine in a powder form diluted in a 250 ml drink containing 248 ml water and 2 ml of natural, non-caloric, flavoring-sweetener containing sucralose.<br>|
| Active Comparator: Placebo<br>Placebo administration: Orally 750 ml per day in 3 doses (250 ml each) for 7 consecutive days and immediately post-exercise. The remaining 8 days, 750 ml per day in 3 doses (250 ml each). | Dietary Supplement: Placebo<br>* Placebo consisted of 248 ml water and 2 ml of natural, non-caloric, flavoring-sweetener containing sucralose.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in muscle satellite cells number (i.e. Pax7+ cells) and activation status (i.e. Pax7+/MyoD+ cells) | Satellite cells number and activation status, will be assessed in muscle via immunohistochemistry. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Change in muscle myogenic mRNA expression | mRNA expression levels of Myogenic factor 5 (Myf5), myogenin and Myogenic factor 6 (Myf6/MRF4) and myostatin will be assessed in muscle using Real-Time Polymerase Chain Reaction (RT-PCR). | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Change in muscle inflammatory state | Pro-inflammatory (M1+) and anti-inflammatory (M2+) macrophages will be measured in muscle using immunohistochemistry. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Change in intracellular antioxidant enzymes in muscle | Protein levels of Glutathione peroxidase 3 (GPx3), Superoxide dismutase 1 (SOD1) and Thioredoxin (Trx1) will be measured using western blotting. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Change in muscle thiol content | Concentration levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) will be measured spectophotometrically. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Change in intracellular signaling proteins in muscle | Protein levels of IGF-1, Notch1 and Wnt3 will be measured using western blotting. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Change in skeletal muscle damage levels | Skeletal muscle damage will be quantified via histochemistry using H&E staining. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Resting metabolic rate (RMR) | RMR will be assessed after an overnight fast with participants in a supine position following a 15-min stabilization period by taking 30 consecutive 1-min VO2/CO2 measurements using a portable open-circuit indirect calorimeter with a ventilated hood system following a standard calibration protocol. | At baseline | 
| Body composition | Body composition will be measured using a dual-energy x-ray absorptiometry scanner (DXA). | At baseline | 
| Maximal oxygen consumption (VO2max) | VO2max will be assessed during continuous incremental running to volitional fatigue on a treadmill with a pulmonary gas exchange system (Oxycon Mobile; Sensor-Medics Corporation). | At baseline | 
| Isokinetic strength | Maximal knee extensor eccentric peak torque at 60 degrees will be assessed on an isokinetic dynamometer. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Change in delayed onset of muscle soreness (DOMS) | Muscle soreness will assessed during palpation of the muscle belly and the distal region of relaxed vastus medialis, vastus lateralis and rectus femoris following three repetitions of of a full squat. Subjects will rate their DOMS on a visual analogue scale (0-10). | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Physical activity | Level of habitual physical activity will be assessed using accelerometry (ActiGraph GT3X-BT accelerometer). | At baseline. | 
| Dietary intake | Daily dietary intake will be assessed using 7-day diet recalls. | At baseline. | 
| Interleukin-1β (IL-1β) in blood | Concentration of IL-1β will be measured in plasma. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Interleukin-6 (IL-6) in blood | Concentration of IL-6 will be measured in plasma. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Interleukin-8 (IL-8) in blood | Concentration of IL-6 will be measured in plasma. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Interleukin-10 (IL-10) in blood | Concentration of IL-10 will be measured in plasma. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Cortisol in blood | Concentration of cortisol will be measured in serum. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Protein carbonyls (PC) | Concentration of protein carbonyls will be measured in muscle. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Malondialdehyde (MDA) | Concentration of malondialdehyde will be measured in muscle. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Total antioxidant capacity (TAC) | Concentration of TAC will be measured in serum. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Catalase | Concentration of catalase will be measured in red blood cell lysate. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| Creatine Kinase (CK) | Concentration of CK will be measured in serum. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 
| White blood cell count in blood | White blood cell count will be measured in blood. White blood cell count will be measured in blood. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Pax7, MyoD, Antioxidants, Myogenic potential, Skeletal muscle healing