Redox Regulation of Satellite Cells and Skeletal Muscle Healing

Not Applicable

Completed

• Conditions: Aseptic Inflammation; Redox Status; Exercise-induced Muscle Trauma; Satellite Cells
• Interventions: Dietary Supplement: Placebo; Dietary Supplement: N-Acetylcysteine

• Registration Number: NCT03711838
• Lead Sponsor: University of Thessaly

Brief Summary

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

A total number of 40-60 young males aged 18-30 will be initially enrolled in the study. Then, participants will be allocated to either a 1) Low glutathione (GSH-low) or a 2) High glutathione (GSH-high) group based on the basal glutathione levels of their vastus lateralis muscle. 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 each trial, participants' dietary intake will be analyzed via diet recalls. Physical activity will be analyzed only at baseline via accelerometry. 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 cell responses and myogenic potential, protein levels of intracellular signaling proteins, muscle thiols and antioxidant enzyme activity.

Study Timeline

• Study First Submitted: 2018-10-11
• Study First Submitted QC: 2018-10-16
• Study First Posted: 2018-10-19
• Study Start: 2019-07-22
• Primary Completion: 2022-05-01
• Study Completion: 2022-08-01
• Status Verified: 2024-11
• Last Update Submitted: 2024-11-16
• Last Update Posted: 2024-11-20

Recruitment & Eligibility

• Status: COMPLETED
• Sex: Male
• Target Recruitment: 45

Inclusion Criteria

1. No recent history of musculoskeletal injury
2. Non-smokers.
3. Abstain from any vigorous physical activity during the study
4. 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

1. A known NAC intolerance or allergy
2. A recent febrile illness
3. A recent history of muscle lesion and/or lower limb trauma
4. Presence of metabolic diseases
5. Use of anti-inflammatory medication.
6. Use of medication interacting with muscle metabolism.

Study & Design

• Study Type: INTERVENTIONAL
• Study Design: CROSSOVER

Arm && Interventions

Group	Intervention	Description
Placebo	Placebo	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).
N-Acetylcysteine	N-Acetylcysteine	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).

Primary Outcome Measures

Name	Time	Method
Change in intracellular antioxidant enzymes in muscle	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Protein levels of Glutathione peroxidase 3 (GPx3), Superoxide dismutase 1 (SOD1) and Thioredoxin (Trx1) will be measured using western blotting.
Change in muscle satellite cells number (i.e. Pax7+ cells) and activation status (i.e. Pax7+/MyoD+ cells)	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Satellite cells number and activation status, will be assessed in muscle via immunohistochemistry.
Change in muscle inflammatory state	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Pro-inflammatory (M1+) and anti-inflammatory (M2+) macrophages will be measured in muscle using immunohistochemistry.
Change in muscle thiol content	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) will be measured spectophotometrically.
Change in intracellular signaling proteins in muscle	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Protein levels of IGF-1, Notch1 and Wnt3 will be measured using western blotting.
Change in skeletal muscle damage levels	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Skeletal muscle damage will be quantified via histochemistry using H\&E staining.
Change in muscle myogenic mRNA expression	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	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).

Secondary Outcome Measures

Name	Time	Method
Body composition	At baseline	Body composition will be measured using a dual-energy x-ray absorptiometry scanner (DXA).
White blood cell count in blood	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	White blood cell count will be measured in blood. White blood cell count will be measured in blood.
Interleukin-8 (IL-8) in blood	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of IL-6 will be measured in plasma.
Resting metabolic rate (RMR)	At baseline	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.
Change in delayed onset of muscle soreness (DOMS)	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	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).
Physical activity	At baseline.	Level of habitual physical activity will be assessed using accelerometry (ActiGraph GT3X-BT accelerometer).
Dietary intake	At baseline.	Daily dietary intake will be assessed using 7-day diet recalls.
Maximal oxygen consumption (VO2max)	At baseline	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).
Cortisol in blood	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of cortisol will be measured in serum.
Protein carbonyls (PC)	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of protein carbonyls will be measured in muscle.
Malondialdehyde (MDA)	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of malondialdehyde will be measured in muscle.
Isokinetic strength	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Maximal knee extensor eccentric peak torque at 60 degrees will be assessed on an isokinetic dynamometer.
Interleukin-1β (IL-1β) in blood	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of IL-1β will be measured in plasma.
Interleukin-6 (IL-6) in blood	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of IL-6 will be measured in plasma.
Total antioxidant capacity (TAC)	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of TAC will be measured in serum.
Interleukin-10 (IL-10) in blood	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of IL-10 will be measured in plasma.
Catalase	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of catalase will be measured in red blood cell lysate.
Creatine Kinase (CK)	At baseline, before the exercise protocol and at days 2 and 8 following exercise.	Concentration of CK will be measured in serum.

Trial Locations

Locations (1)

Laboratory of Exercise Biochemistry, Exercise Physiology,and Sports Nutrition, School of Physical Education and Sport Science, University of Thessaly; 🇬🇷 Trikala, Thessaly, Greece