Redox Regulation of Satellite Cells and Skeletal Muscle Healing

Sponsor

University of Thessaly (Other)

Overall Status

Active, not recruiting

CT.gov ID

NCT03711838

Collaborator

(none)

Enrollment

Location

Arms

41.3

Anticipated Duration (Months)

1.1

Patients Per Site Per Month

Study Details

Study Description

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.

Condition or Disease	Intervention/Treatment	Phase
Satellite Cells Redox Status Exercise-induced Muscle Trauma Aseptic Inflammation	Dietary Supplement: N-Acetylcysteine Dietary Supplement: Placebo	N/A

Detailed Description

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.

Study Design

Study Type:

Interventional

Actual Enrollment :

45 participants

Allocation:

Randomized

Intervention Model:

Crossover Assignment

Masking:

Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)

Primary Purpose:

Basic Science

Official Title:

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

Actual Study Start Date :

Jul 22, 2019

Anticipated Primary Completion Date :

Jun 30, 2022

Anticipated Study Completion Date :

Dec 31, 2022

Arms and Interventions

Arm	Intervention/Treatment
Experimental: 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).	Dietary Supplement: N-Acetylcysteine 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.
Active Comparator: 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).	Dietary Supplement: Placebo Placebo consisted of 248 ml water and 2 ml of natural, non-caloric, flavoring-sweetener containing sucralose.

Arm

Intervention/Treatment

Experimental: 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).

Dietary Supplement: N-Acetylcysteine

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.

Active Comparator: 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).

Dietary Supplement: Placebo

Placebo consisted of 248 ml water and 2 ml of natural, non-caloric, flavoring-sweetener containing sucralose.

Outcome Measures

Primary Outcome Measures

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 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).
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 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 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.

Secondary Outcome Measures

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.
Body composition [At baseline]
Body composition will be measured using a dual-energy x-ray absorptiometry scanner (DXA).
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.

Eligibility Criteria

Criteria

Ages Eligible for Study:

18 Years to 30 Years

Sexes Eligible for Study:

Male

Accepts Healthy Volunteers:

Yes

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.

Contacts and Locations

Locations

	Site	City	State	Country	Postal Code
1	Laboratory of Exercise Biochemistry, Exercise Physiology,and Sports Nutrition, School of Physical Education and Sport Science, University of Thessaly	Trikala	Thessaly	Greece	42100