Resistance Training, Detraining, and Retraining Study 2024 (TraDeRe2024)
NCT Number: NCT06573086
Acronym: TraDeRe
Status: RECRUITING
Study Type: INTERVENTIONAL
Phases: NA
Conditions: Hypertrophy|Exercise Training|Skeletal Muscle Atrophy|Healthy
Interventions: OTHER: Resistance Training
Sponsor: University of Jyvaskyla
Collaborators: Academy of Finland
Sex: ALL
Age: ADULT
Enrollment: 90
Study Design: Allocation: NON_RANDOMIZED|Intervention Model: PARALLEL|Masking: SINGLE (PARTICIPANT)|Primary Purpose: BASIC_SCIENCE
Start Date: 2024-08-19
Completion Date: 2025-06-15
Locations: University of Jyväskylä, Jyväskylä, Central Finland, 40014, Finland

Summary:
The goals of our research project are to identify factors explaining inter-individual variation in responses to resistance training (RT) and the baseline determinants underlying an individual's sensitivity to respond to RT. Thus, investigators aim to gain a deeper understanding of RT adaptation processes. The main questions investigators aim to answer are:

* Can the physiological responses of one RT intervention be extrapolated to a subsequent RT intervention?
* If so, what are the mechanisms underlying differing skeletal muscle growth responses in low, and high responders of skeletal muscle hypertrophy?
* If so, do the low responders of skeletal muscle growth respond more favourably when the amount of RT is increased?

To examine these main research questions, high (n=30), low (n=30), and moderate (n=30) responders of skeletal muscle growth in response to RT (intervention I, NCT05874986) are reallocated into a subsequent RT intervention (intervention II) after a detraining period.

In this intervention II, reallocated participants will be:

* Resistance training with supervision for 12 weeks
* Consuming deuterium oxide for the assessment of muscle protein synthesis
* Consuming D3-3-methylhistidine for the assessment of muscle protein breakdown
* Consuming D3-creatine for the examination of whole-body skeletal muscle mass
* Providing a spot urine sample six (6) times, and urine collection for 24 hours performed twice, after each drink
* Providing a saliva sample two (2) times per week, before and after drinking D2O, for a duration of 6 weeks, and additionally 24 and 48 hours after the first 3 boluses.
* Providing a muscle biopsy four (4) times during the study
* Providing a blood sample fourteen (14) times during the study
* Assessed for body composition and body volume four times (4) during the study
* Participating in muscle size, and strength measurements ten (10) times during the study, of which four (4) include neuromuscular measurements
* Asked to answer questionnaires related to e.g. stress, physical activity, sleep, and diet
* Participating in an acute resistance exercise (RE) and associated recovery assessments for the next five consecutive days following RE.

Primary Outcome Measures: Change in m. vastus lateralis (VL) cross-sectional area (CSA) with ultrasound (US), VL CSA (cm\^2) is determined at mid-thigh using a B-mode axial plane US (Venue Fit R4, GE Medical Systems, USA) with a L4-20t-RS linear-array probe (48,43 mm width) in extended-ﬁeld-of-view mode (6/12 MHz, 39 frames per second).

Additionally, VL CSA (cm\^2) will also be determined at baseline with different US, at mid-thigh using a B-mode axial plane US (model SSD-α10, Aloka, Tokyo, Japan) with a 13 MHz linear-array probe (60 mm width) in extended-ﬁeld-of-view mode (23 Hz sampling frequency). Aloka-US was used in the intervention I, but GE-US in intervention II due to the progressive malfunctioning of Aloka. Therefore, both machines are used in baseline measurements to calculate the plausible inter-US variability.

The CSA will be measured using the polygon function in ImageJ software. Per each measurement, three images will be analysed from which the average from the two closest ones will be calculated., Baseline (test and retest), week 6, week 12
Secondary Outcome Measures: Muscle protein synthesis (MPS) with the Combined Oral Stable Isotope Assessment of Muscle (COSIAM), A baseline saliva sample is collected to determine the deuterium labelling of plasma proteins. Then, participants ingest 3ml/kg deuterium oxide (D2O, 70-Atom%) divided into three boluses and 1 ml/kg top-up dose twice a week throughout the MPS examination period. When muscle proteins are built, the accumulation of deuterium-labelled alanine amino acid in the VL can be used to measure local fractional sarcoplasmic, myofibrillar, mitochondrial, and collagen MPS rates via gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-Pyrolysis-IRMS). The outcome will be expressed as %/day. 30 mg of VL muscle biopsy is used for MPS analysis. To estimate alanine precursor enrichment (APE), saliva samples are collected before and after each D2O drink consumption and 24 and 48 hours after the 3 x bolus to monitor body water enrichment via thermal conversion elemental analyser (TC/EA)-IRMS. Mass spectrometry analyses will be performed at the University of Nottingham, United Kingdom., From baseline to week 6 (duration of 6-weeks)|Muscle protein breakdown (MPB) with the Combined Oral Stable Isotope Assessment of Muscle (COSIAM), Before each measurement day, participants ingest 10 mg of stable isotopically labelled D-3-methylhistidine (D-3-MH) dissolved in 50 millilitres of water. When muscle proteins containing D-3-MH are degraded, D-3-MH is released into circulation. As D-3-MH cannot be reused for protein synthesis, D-3-MH can be used as a biomarker for muscle protein breakdown (MPB). The enrichment of labelled D-3-MH is then measured from the blood samples to assess MPB. The outcome will be expressed as the tracer dilution (k) rate of D3-3-MH and measured with repeated plasma sampling. 20-24 hours after the ingestion of D3-3MH, five plasma samples will be collected within three hours, each separated by 45 minutes. Plasma samples will be analysed at the University of Nottingham, United Kingdom., Week 0, Week 6|Change in whole-body skeletal muscle mass (SMM) with the Combined Oral Stable Isotope Assessment of Muscle (COSIAM), First, a baseline urine sample is collected to measure D3-creatinine enrichment. Then, after the bladder is empty, participants consume 30 mg of labelled D3-creatine dissolved in 50 millilitres of water. The amount of D-3 creatine that spills over in urine samples after 24 hours of ingestion will then be used to determine the amount of D-3 creatine retained in the body by 24-hour urine collection. Then, as the amount of creatine-to-creatinine conversion remains similar in the total muscle pool, the comparison of both non-labelled and labelled creatinine can be used to estimate the whole-body muscle creatine pool from the urine samples gathered 48 to 72 hours post-ingestion. This enables the calculation of whole-body skeletal muscle mass with a simple equation and the following outcome will be expressed as kilograms (kg). Urine samples will be analysed at the University of Nottingham, United Kingdom., Week 0, Week 6|Change in muscle thickness (MT) of the elbow flexors after 6 and 12-week resistance training, MT of m. biceps brachii and m. brachialis is measured using a B-mode axial plane ultrasound (Venue Fit R4, GE Medical Systems, USA) with a L4-20t-RS linear-array probe (48,43 mm width)., Baseline (test and retest), week 6, week 12|Change in lower and upper limb maximal strength after 6 and 12-week resistance training, Maximal voluntary concentric muscle strength of leg extensors is determined in a horizontal leg press device (David 210) via a one-repetition maximum (1RM) test. Moreover, the maximal voluntary concentric muscle strength of elbow flexors is also measured using barbell bicep curl performed in Scott bench. Maximum strength tests are performed according to the National Strength and Conditioning Association (NCSA) guidelines.

Both concentric muscle strengths are expressed as kilograms (kg)., Baseline (test and retest), week 6, week 12,|Change in corticoreticular excitability after resistance training with transcranial magnetic stimulation (TMS), Electrical stimulation of the brachial plexus is performed during rest to elicit the maximum M-wave (M-max) of the biceps brachii. M-max is obtained by increasing the stimulator output intensity until the electromyography (EMG) response plateau. M-max is recorded using surface EMG. Next, single TMS pulses are delivered during tempo-controlled dynamic barbell bicep curls performed with a 30% load of one repetition maximum (1 RM). The TMS pulses are administered when the elbow joint flexion angle reaches 110 degrees. The stimulation intensity will be 100% of the maximum output. The protocol consists of two phases: the first involves four sets of five repetitions, followed by one set of 40 repetitions. Using TMS, investigators analyze the motor-evoked potential (MEP) amplitude, contralateral (cMEPs) and ipsilateral (iMEPs), the iMEP/cMEP amplitude ratio (ICAR), latency, and the silent period, all extracted from the EMG signal., Baseline (test and retest), week 6, week 12|Change in ribosome biogenesis, A portion of the VL muscle biopsies will be used for ribosomal analyses. Ribosome biogenesis and changes in messenger RNA and protein expression will be determined from the muscle biopsy samples by measuring ribosomal RNA and its regulators using real-time quantitative polymerase chain reaction (PCR) and proteomics., Baseline, week 6, week 12|Change in muscle fiber capillarization, A portion of the VL muscle biopsies will be used for immunohistochemical analysis. CD56 is used as an antibody for capillaries with a specific secondary fluorophore-labelled antibody. The structural quantification will be performed automatically using an artificial intelligence (AI)SDS PA-based software in Fiji., Baseline, week 6, week 12|Change in muscle fiber cross-sectional area (CSA) during the intervention period, A portion of the VL muscle biopsies will be used for immunohistochemical analysis. MANDYS8 antibody is used for staining muscle fiber borders with a specific secondary fluorophore-labelled antibody. Muscle fiber CSA will be quantified using an AI-based software in Fiji., Baseline, week 6, week 12|Change in myonuclear content of the muscle fibers, A portion of the VL muscle biopsies will be used for immunohistochemical analysis. PCM1 is used as an antibody for myonuclei with a specific secondary fluorophore-labelled antibody. Myonuclear content will be quantified using AI-based software in Fiji., Baseline, week 12|Change in satellite cell content during the intervention of the muscle fibers, A portion of the VL muscle biopsies will be used for immunohistochemical analysis. Pax7 is used as an antibody for satellite cells with a specific secondary fluorophore-labelled antibody. Satellite cell content will be quantified using AI-based software in Fiji., Baseline, week 12|Change in macrophage content of the muscle fibers, A portion of the VL muscle biopsies will be used for immunohistochemical analysis of macrophage content with the methods and primary (CD11b, CD206) and fluorophore-labelled secondary antibodies commonly utilized in our laboratory. Macrophage content will be quantified using AI-based software in Fiji., Baseline, week 6, week 12|Changes in anaerobic enzymes, Will be measured by Western blotting and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)., Baseline, week 6, week 12|Changes in sarcoplasmic protein contents, Will be measured by Western blotting and SDS-PAGE., Baseline, week 6, week 12|Changes in multiomics during the intervention, Blood and muscle samples are used for the OMIC analyses. DNA extraction will be performed from blood and muscle samples in our Faculty.

For genomic analysis, DNA samples will be sent to the Institute for Molecular Medicine Finland to produce single nucleotide polymorphisms using the advanced genotyping array (Illumina Infinium Global Screening Array-24v2.0 BeadChip). Then, quantitative genome-wide polygenic risk score (PRS) will be quantified using known genomic loci. Epigenetic analysis for DNA methylation will be assessed by Illumina's Infinium Methylation EPIC BeadChip (Illumina, San Diego, CA, USA). Transcriptomic profiling will be performed by RNA sequencing. Proteome profiling will be performed using large format difference in-gel electrophoresis (DiGE). Lastly, a high-throughput serum Nuclear Magnetic Resonance (NMR) metabolomics platform will be used for the absolute quantification of serum lipids and metabolite profiles., Baseline, week 12|Satellite cell isolation, From muscle biopsy samples, satellite cells will be isolated, and fibroblast-free primary satellite cell cultures will be established using the methods recently applied in our laboratory. Cultures of differentiated myotubes can be later utilized in mechanistic studies to examine specific molecular networks revealed by OMIC analyses., Week 6|Change in whole body fat mass after 6 and 12-week resistance training, Fat body mass (kg) is measured at morning after overnight fasting by bioimpedance device (InBody 770, Biospace Co. Seoul, Korea)., Baseline (test and retest), week 6, week 12|Change in whole body fat-free mass after 6 and 12-week resistance training, Fat-free body mass (kg) is measured at morning after overnight fasting by bioimpedance device (InBody 770, Biospace Co. Seoul, Korea), Baseline (test and retest), 6-weeks, 12-weeks|Change in whole-body volume (cm^3) after 6 and 12-weeks of resistance training, The 3D optical body (3DO) scan is used to measure whole-body volume, the volume of the body segments, and waist, hip, and thigh circumference. For 3DO, Fit3D ProScanner (Redwood City, CA, USA) is used., Baseline (test and retest), week 6, week 12|Change in blood count determined from the venous blood sample obtained in the morning after overnight fasting before, mid, and after 12-week resistance training, Full blood count is measured by hematology analyzer (Sysmex KX-21N, Sysmex Corp., Japan), Baseline, week 6, week 12|Change in C-reactive protein (CRP) determined from the venous blood sample obtained in the morning after overnight fasting before, mid, and after 12-week resistance training, Serum CRP is measured by high-sensitivity ELISA kit (Quantikine HS, R\&D Systems, Minneapolis, USA)., Baseline, week 6, week 12|Self-reported measure of physical activity, Investigated by the Global Physical Activity Questionnaire (GPAQ), a standardized 16-question questionnaire that assesses categories of low, moderate, and vigorous physical activity (in metabolic equivalent \[MET\] minutes per week) in three different domains: activity at work, travel to and from places, and leisure activities. Also, sedentary behavior (minutes per week) is assessed., Baseline, week 12|Sleep self-assessment, Investigated by The Pittsburgh Sleep Quality Index (PSQI) questionnaire which consists of questions of a four-point Likert scale (0-3), with higher scores representing greater sleep difficulties., Baseline, week 12|Self-report of eating disorder behaviors and attitudes, Investigated by the Eating Disorder Examination Questionnaire (EDE-Q), which assesses the extent, frequency, and severity of eating disorder-related behaviors on a seven-point Likert scale or occurrence over a 28-day period. Higher scores represent higher levels of eating disorders., Baseline, week 12|Self-estimated energy availability, Investigated by the Low Energy Availability Questionnaire (LEAF-Q for females, LEAM-Q for men) which identifies persons at risk for low energy availability by utilizing subsets of gastrointestinal symptoms, injury frequency, and menstrual dysfunction (in women). A score ≥8 indicates that an individual is at risk for low energy availability., Week 12|Self-measure of perceived stress, Investigated by Perceived Stress Scale (14 items); from 0 (never) to 4 (very often). The scores are obtained by reverse scoring the positively stated items (7 items) and then summing the points across all 14 items., Baseline, week 6, week 12|Recovery of muscle swelling after acute resistance exercise (RE), Vastus Lateralis (VL) muscle thickness (MT) is determined mid-thigh using a B-mode axial plane US (Venue Fit R4, GE Medical Systems, USA). VL MT is measured as the distance between superficial and deep aponeuroses., Week 13: after 24, 48, 72, 96, 120 hours of RE|Recovery of muscle strength after acute resistance exercise (RE), Maximal voluntary concentric muscle strength of leg extensors (kg) is determined in horizontal leg press device (David 210) via a one-repetition maximum (1RM) test. Maximum strength test is performed according to the NCSA guidelines., Week 13: after 24, 48, 72, 96, 120 hours of RE|Subjective muscle soreness after acute resistance exercise (RE), Subjective muscle soreness is determined by a questionnaire and rated on a Likert scale of 0 (= no pain) to 5 (= maximum pain) for the overall muscle soreness of the quadriceps muscles during palpation of the muscle belly and walking up and down stairs, squatting, and sitting down., Week 13: after 24, 48, 72, 96, 120 hours of RE