Impact of Muscle Temperature on Muscle Growth and Breakdown: Cooling During Resistance Training

Study Overview
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Brief Summary
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The purpose of this study is to examine the effect of human skeletal muscle temperature during resistance exercise on myogenic and proteolytic signaling. Subjects will perform bilateral resistance exercise bouts at an intensity that should stimulate a muscle growth response in the Vastus Lateralis. During the resistance exercise, the subjects will receive a cold (10°C) intervention on the experimental limb, while receiving a neutral temperature intervention (22°C) on the control limb.

Detailed Description
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A total of 2 visits to the laboratory will be required following the signing of informed consent. All visits will take place at the University of Nebraska Exercise Physiology Laboratory. Initial Visit ( 1.5-2 hours): Height and weight will be measured using a medical scale. Body fat will be assessed with hydrostatic weighing using an electronic load cell based system (Exertech, Dresbach, MN) correcting for residual lung volume, or Bioelectrical Impedance Analysis body composition analyzer (InBodyUSA, Cerritos, CA) if needed. Subjects will then perform a 12 Repetition (12RM) Max Test for bilateral seated leg press and seated leg extension using the following protocol on isotonic exercise machines at a weight designed to create failure between 8 and 12 repetitions. Following a short warm-up on a cycle ergometer, the subjects proceed with a bilateral leg press warm-up consisting of one set of ten repetitions at a self-determined light load, then one set of 5 repetition at a self-determined moderate load, and one set of 1 repetition at a predicted 12 rep maximum as predicted in relation to their Fat Free Mass. During the warm-up subjects will be coached on form, and observed for their ability to complete the repetition max test. Weight for the test will be altered to suit the subject's ability to complete approximately 12 repetitions. Once the desired weight is determined, subjects will be asked to complete as many repetitions as possible until the subject can no longer complete a repetition using a full range of motion. An identical testing protocol is then performed for the leg extension. This data will be used to estimate a 12RM workload, that would be appropriate for hypertrophic growth using the Watham equation . Experimental Visit ( 6.5-8 hours): Participants will be instructed to arrive to the lab having fasted overnight, and they will be asked to avoid strenuous activity, alcohol consumption, tobacco use, and drug use for the 24 hour period leading up to experimental trial. Subjects will complete the following experimental protocol briefly described: consume a small meal, a pre-exercise muscle biopsy, precool for 30 minutes, complete the exercise intervention, immediately post-exercise biopsy, 4 hour recovery with cooling, and post-recovery biopsy. At three time points (pre-exercise, immediately post-exercise, and post-4-hour recovery), skin temperature, muscle temperature, blood flow, and muscle tissue biopsies will be taken. Details are as followed: Meal When subjects arrive a consistent meal will be provided to standardize dietary intake. Food choices will be given to participants to create a meal between 700-800 calories, containing approximately 25-35 g of protein, 75-85 g of carbohydrates, and 35-45 g of fat. These foods will be made-up of commercially available pre-packaged foods. They will have approximately 30 minutes to consume their meal. Cooling protocol Subjects will have Game Ready Med 4 elite thigh wraps around each thigh. This system circulates cooled liquid through a wrap specifically fitted for different body segments. One thigh will be cooled while the other thigh will be wrapped with the cuff, but not have any liquid circulating through it. These wraps do provide slight compression and thus it is important to apply the wraps to both legs. Each leg is assigned to either cold experimental group or control, will be randomized and counter-balanced between subjects. The liquid temperature circulating through the thermal wraps will be set to 10°C. The thermal wraps will be worn during a 30 minute cool down period prior to the exercise bout, during the exercise intervention, and during a 4 hour recovery period. Exercise Intervention The subjects will complete an intervention designed to stimulate the muscle growth signaling pathway (hypertrophy) in the Vastus Lateralis (outer thigh), via bilateral seated leg press and bilateral seated leg extension on isotonic exercise machines. Subjects will perform four sets of bilateral leg press exercises at the weight determined to be their 12 RM from the repetition max test. This should allow completion to failure of 8-12 repetitions. So, if more than 12 reps are able to be completed, weight will be added to the resistance for the subsequent sets. Conversely, if less than 8 reps lead to failure, the weight should be reduced for the subsequent sets. Constant dialogue with subjects will give investigators the ability to monitor set weights to maximize a hypertrophic response. Subjects will be allowed 2 minute of rest between each set. Upon completion of 4 sets, the subjects will be given 5 minutes of rest prior to repeating this protocol for bilateral leg extensions. Biopsies A total of 6 muscle biopsies (1 per leg) will be obtained from the vastus lateralis (outer thigh) muscle. One from each leg for each of the 3 collection time-points, pre-exercise, immediately post-exercise, and post-4-hour recovery. Briefly, the area above the vastus lateralis muscle belly will be shaved and cleaned with alcohol. 1% xylocaine is then injected under the skin with a 25 ga hypodermic needle. The area is then further disinfected with betadine, and a sterile fenestrated drape is placed over the biopsy site. Using sterile techniques, a small incision is made through the skin ( 5mm) and the Bergstrom biopsy needle is inserted through the incision into the belly of the muscle. The Bergstrom needle then takes a small clip of muscle ( 50mg) and is removed from the muscle and stored in a chemical stabilizer. Slight pressure is held over the incision for approximately 2 minutes and is then closed using steri-strips and a band-aid with antibiotic ointment. Intramuscular Temperature Using the incision from the muscle biopsy, a hypodermic ( 26ga) thermocouple (Physitemp Instruments LLC, Clifton, NJ) that is smaller than the needle used to inject the xylocaine will be inserted into the belly of the muscle to measure intramuscular temperature. The thermocouple readings stabilize in approximately 3 seconds and the thermocouple is then removed. Recordings for intramuscular temperature will be collected during the biopsy process following the intervention. Skin Temperature Skin Temperature will be measured on the surface of each thigh using a skin thermistor (Physitemp Instruments LLC, Clifton, NJ), infrared digital thermometer, and/or using an infrared thermal camera. Measurements will be taken at the same time as the intramuscular. The thermistor is simply placed on the skin for approximately 3 seconds and is then recorded. The infrared thermal camera can measure and record surface temperatures for a field of multiple surfaces.

Official Title
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Impact of Muscle Temperature on Muscle Growth and Breakdown: Cooling During Resistance Training

Conditions
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Exercise Training

Intervention / Treatment
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* Device: Muscle Cooling
* Other: Resistance Exercise


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Any male or female who is 19-45 of age and is considered low risk for physical activity based on the American College of Sports Medicine (ACSM) guidelines recreationally active during the 6 weeks prior to participation by meeting the ACSM minimum activity guidelines Exclusion Criteria: Any person outside of the age range of 19-45 not considered 'low risk' on the ACSM Risk Stratification Form not meeting the exercise requirements for the preceding 6 weeks will be excluded. self-report of a history of joint or muscular injury that would prevent them from safely completing the required activities self-report of taking prescription drugs or supplements that may interfere with this research self-report allergy to Xylocaine pregnant breast feeding self-report sensitivity to heat or cold

Ages Eligible for Study
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Minimum Age: 19 Years
Maximum Age: 45 Years

Sexes Eligible for Study
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All

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: Other
Allocation: Randomized
Intervention Model: Crossover Assignment
Interventional Model Description: Each Subject acts as their own control in a de facto repeated measures design, where there is a Single bilateral exercise bout, with experimental and control assignment to individual limbs.
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Muscle Cooling<br>The intervention will be applying a cooling wrap during the bilateral resistance exercise. | Device: Muscle Cooling<br>* Cooling wrap will be set to 10°C<br>Other: Resistance Exercise<br>* Resistance Machines (Leg Press and Knee extension). 12 rep Max<br>|
| Placebo Comparator: Control<br>There will be no temperature alteration for this leg during bilateral resistance exercise. | Other: Resistance Exercise<br>* Resistance Machines (Leg Press and Knee extension). 12 rep Max<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Western Blot for mammalian target of rapamycin signaling | phosphorylation state of mTOR | immediately post-exercise | 
| Western Blot for mammalian target of rapamycin signaling | phosphorylation state of mTOR | 4-hours post-exercise | 
| Western Blot for Ribosomal protein S6 kinase beta-1 signaling | phosphorylation state of p70s6K1 | 4-hours post-exercise | 
| Western Blot for Ribosomal protein S6 kinase beta-1 signaling | phosphorylation state of p70s6K1 | immediately post-exercise | 
| Western Blot for Eukaryotic translation initiation factor 4E-binding protein 1 signaling | phosphorylation state of 4E-BP1 | immediately post-exercise | 
| Western Blot for Eukaryotic translation initiation factor 4E-binding protein 1 signaling | phosphorylation state of 4E-BP1 | 4-hours post Exercise | 
| RT-qPCR for F-Box Protein 32 (FBXO32) | mRNA response of FBXO32 | 4-hours post Exercise | 
| RT-qPCR for Forkhead Box O3 (FOXO3a) | mRNA response FOXO3a | 4-hours post Exercise | 
| RT-qPCR for Tripartite Motif Containing 63 (TRIM63) | mRNA response of TRIM63 | 4-hours post Exercise | 
| RT-qPCR for Growth/Differentiation Factor 8 (MSTN) | mRNA response of MSTN | 4-hours post Exercise | 
| RT-qPCR for Myogenic Factor 5 (MYF5) | mRNA response of MYF5 | 4-hours post Exercise | 
| RT-qPCR for Myogenic Factor 6 (MYF6) | mRNA response of MYF6 | 4-hours post Exercise | 
| RT-qPCR for Myocyte Enhancer Factor 2A (MEF2a) | mRNA response of MEF2a | 4-hours post Exercise | 
| RT-qPCR for Myogenic Differentiation 1 (MYOD1) | mRNA response of MYOD1 | 4-hours post Exercise | 
| RT-qPCR for Myogenin (MYOG) | mRNA response of MYOG | 4-hours post Exercise | 
| RT-qPCR for Ribosomal Protein S3 (RPS3) | mRNA response of RPS3 | 4-hours post Exercise | 
| RT-qPCR for Ribosomal Protein L3 Like (RPL3L) | mRNA response of RPL3L | 4-hours post Exercise | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Muscle Temp | Intramuscular Temperature | immediately post Exercise | 
| Muscle Temp | Intramuscular Temperature | 4-hours post Exercise | 
| Skin Temp | Surface Temperature of the skin | immediately post Exercise | 
| Skin Temp | Surface Temperature of the skin | 4-hours post Exercise | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Resistance Exercise, Muscle Cooling