Trial Review

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Trial registered on ANZCTR


Registration number
ACTRN12621001062819
Ethics application status
Approved
Date submitted
21/06/2021
Date registered
12/08/2021
Date last updated
23/03/2023
Date data sharing statement initially provided
12/08/2021
Date results information initially provided
23/03/2023
Type of registration
Retrospectively registered

Titles & IDs
Public title
Molecular networks underlying exercise-induced mitochondrial biogenesis in humans
Scientific title
Transcriptomic and proteomic changes associated with high intensity interval training-induced mitochondrial biogenesis within healthy adult human skeletal muscle
Secondary ID [1] 304548 0
None
Universal Trial Number (UTN)
U1111-1266-8459
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Metabolic disorders 322427 0
Condition category
Condition code
Metabolic and Endocrine 320080 320080 0 0
Normal metabolism and endocrine development and function
Musculoskeletal 320522 320522 0 0
Normal musculoskeletal and cartilage development and function

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
High-intensity interval training (HIIT) exercise.
Participants will train 4 times per week throughout the 8-week period. Each exercise session will consist of four 4-minute intervals at an intensity of 80% of a participants peak power output on a stationary bike. Each interval will be seperated by 2-minutes of rest for a total session time of 22 minutes. Each training session will be performed at the Exercise Physiology laboratory at Footscray Park Campus (Victoria University) one-on-one with a researcher to ensure compliance. This intensity has been selected as it has previously been shown to elicit a large response within muscle to generate mitochondrial biogenesis.

Muscle biopsies and blood samples will be collected from participants by an experienced medical doctor during the first HIIT session (at timepoints before, in the middle of, and at timepoints 0, 3, 6, 9, 12, 24, and 48 hours after exercise completion) at the beginning of training and after the completion of the 8-weeks of training.
Intervention code [1] 320899 0
Lifestyle
Intervention code [2] 321239 0
Treatment: Other
Comparator / control treatment
Samples collected prior to training will be used as a control (before the start of the first HIIT session). An additional control will be a number of participants who will have the same number and timing of muscle biopsies but will not complete the HIIT session.
Control group
Active

Outcomes
Primary outcome [1] 327954 0
Mitochondrial dynamics measured as protein and phosphoprotein content changes in different subcellular compartments (via mass spectrometry-based proteomics) and gene expression (via RNA sequencing). Although this study will be exploratory, confirmation of biomolecular changes will occur through targeted analyses (quantitative PCR & immunoblotting). All measurements are from skeletal muscle obtained from the vastus lateralis.
Timepoint [1] 327954 0
Before, in the middle of, and 0, 3, 6, 9, 12, 24, and 48 hours after the first exercise session in week 1.
Secondary outcome [1] 397077 0
Mitochondrial content measured as mitochondrial volume are from a composite of electron microscopy (EM) images, citrate synthase activity through an enzymatic assay, and mtDNA copy number through quantitative PCR. All three measurements of mitochondrial content are from skeletal muscle obtained from the vastus lateralis.
Timepoint [1] 397077 0
Before the first exercise session in week 1 and 72h after the last exercise session in week 8.
Secondary outcome [2] 397078 0
Mitochondrial respiratory function in permeabilised fibres obtained from the vastus lateralis assessed with an oxygen respirometer that tracks changes in oxygen levels when substrates are added to the permeabilised fibres. These substrates include malate, pyruvate, ADP of varying concentrations, succinate, cytochrome c, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and antimycin A.
Timepoint [2] 397078 0
Before the first exercise session in week 1 and 72h after the last exercise session in week 8.
Secondary outcome [3] 398686 0
Changes in the physiological fitness of participants. This will be assessed as a composite of changes in VO2max, power at lactate threshold, and peak power output values taken from an exercise test prior to the beginning the study and after 8-weeks of training on a cycling ergometer.
Timepoint [3] 398686 0
Before the start of the 8-weeks of training and following the completion of the 8-weeks of training.

Eligibility
Key inclusion criteria
- Aged 18-40 years old, Men & Women
- Be generally ‘healthy’: Non-smoker, free from pre-existing medical conditions (e.g., heart rhythm disturbance, elevated blood pressure, diabetes), cardiovascular abnormalities, respiratory conditions and musculoskeletal injuries.
- Not taking prescription medication (e.g., beta-blockers, anti-arrhythmic drugs, statins or insulin sensitising drugs).
- Undertaken less than the current weekly physical activity guidelines for adults (150 minutes of moderate intensity physical activity, or 75 minutes of vigorous intensity physical activity) for at least 3 months prior to the study.
- Have a body mass index (BMI) between 18.5-34.9 kg/m2
Minimum age
18 Years
Maximum age
40 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
- Current muscle or ligament injury of the lower body.
- Current or previous cardiovascular or respiratory condition or abnormality.
- Current metabolic disease (e.g. diabetes)

Study design
Purpose of the study
Treatment
Allocation to intervention
Non-randomised trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)

Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Masking / blinding
Open (masking not used)
Who is / are masked / blinded?



Intervention assignment
Single group
Other design features
Phase
Not Applicable
Type of endpoint/s
Efficacy
Statistical methods / analysis
Physiological and performance data will be analysed using a two-way ANOVA with repeated measures for time to investigate the influence of sex and time. Statistical significance will be defined as a P-value less than 0.05. A Bonferonni correction will be used to correct P-values for the multiple comparisons, helping to lower the rate of false positives.
The RNA-Seq reads will be quality controlled and trimmed using Trim Galore v0.3.7 and Cutadapt v1.4.2. Pre-processed reads will be mapped to the latest annotated human genome using Rsubread and gene coverages will be computed with 'featureCounts' and the 'Gencode' annotation. Gene lists will be filtered to achieve a read coverage for each gene larger than 0.1 reads per kilo base million in at least five samples. Differential expression will be computed using edgeR with the 'glmQLFit/glmQLFTest' modelling framework and the following models 'y~Timepoint + Subject' will be used for each of the groups. Genes with a false discovery rate (FDR) < 5% will be considered differentially expressed. Quantitative Realtime PCR (qPCR), will be used to verify gene expression results of a panel of known and novel exercise regulated genes identified by the RNA-Seq analysis.
The raw mass spectrometry data will initially be processed in the MaxQuant software. Intensity data will be normalised to remove variation arising from labelling, batch, and handling differences using novel algorithms developed at the University of Melbourne. Log2 transformed normalised intensities will be assigned to experimental groups based on the sex and time point of the participants using the Perseus software. Proteins with large standard deviation and/or excessive missing values (>30%) for a time point will be removed. Differentially regulated proteins will be identified by a modified ANOVA incorporating permutation-based FDR statistics, which tests for changes in the means between time points as well as reproducibility within each time point. Significantly regulated proteins and their respective phosphosites (<5% FDR, >1.5 fold change) will be analysed for enriched terms (e.g. using the GO, & KEGG databases) using the FunRich software, and the DAVID online tool as previously performed by his group. Clusters of enriched terms and their interconnection will be visualised by Cytoscape and the Enrichment Map plugin.

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
Actual
14/06/2021
Date of last participant enrolment
Anticipated
16/05/2022
Actual
22/08/2022
Date of last data collection
Anticipated
25/07/2022
Actual
2/12/2022
Sample size
Target
40
Accrual to date
Final
40
Recruitment in Australia
Recruitment state(s)
VIC
Recruitment postcode(s) [1] 34417 0
3011 - Footscray
Recruitment postcode(s) [2] 34418 0
3000 - Melbourne
Recruitment postcode(s) [3] 37511 0
3065 - Fitzroy

Funding & Sponsors
Funding source category [1] 308917 0
Government body
Name [1] 308917 0
Australian Research Council
Country [1] 308917 0
Australia
Funding source category [2] 308920 0
University
Name [2] 308920 0
Victoria University
Country [2] 308920 0
Australia
Primary sponsor type
University
Name
Victoria University
Address
Institute of Sport, Exercise & Active Living
College of Sport and Exercise Science
70/104 Ballarat Rd, Footscray 3011
Melbourne VIC, Australia
Country
Australia
Secondary sponsor category [1] 309839 0
University
Name [1] 309839 0
The Australian Catholic University
Address [1] 309839 0
Mary MacKillop Institute for Health Research
5/215 Spring St, Melbourne 3000
Melbourne VIC, Australia
Country [1] 309839 0
Australia

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 308809 0
VU Human Research Ethics Committee (VUHREC)
Ethics committee address [1] 308809 0
Institute of Sport, Exercise & Active Living
College of Sport and Exercise Science
70/104 Ballarat Rd, Footscray 3011
Melbourne VIC, Australia
Ethics committee country [1] 308809 0
Australia
Date submitted for ethics approval [1] 308809 0
25/11/2020
Approval date [1] 308809 0
28/05/2021
Ethics approval number [1] 308809 0
HRE20-212

Summary
Brief summary
Mitochondria are compartments within our cells that facilitate various important processes, including that of energy generation. Mitochondria rapidly adapt to changing needs within the cell through a process called mitochondrial biogenesis, by which, new components are added to the network of mitochondria. Mitochondrial biogenesis is stimulated in skeletal muscle by exercise, however, the signalling that allows this to occur is poorly understood.
This project aims to uncover the different signalling pathways activated by exercise in skeletal muscle using novel techniques combined with advanced statistical analysis. Analysis will be performed on quadriceps muscle biopsies collected from forty untrained men and women (equal numbers of each). Using samples both prior, in the middle, and at multiple time points after exercise, we will capture all changes in gene expression and protein localisation. This data will be combined with measurements of participants' fitness before and after training to generate a timeline and predictive model of mitochondrial biognesis signalling.
Expanding the knowledge of mitochondrial biology will have many ramifications for both athletic and biomedical disciplines. Recently, links between the function of mitochondria and life expectancy have been discovered, with numerous diseases characterised by mitochondrial dysfunction, such as diabetes, cardiomyopathy and various neurological disorders on the rise globally. We hope by understanding how exercise triggers mitochondrial biogenesis, exercise can be used to a greater effect as therapy for these diseases. By applying this methodology to different exercise intensities and durations, better exercise prescription for athletes can also be determined. Additionally, the identification of novel mitochondrial biogenesis regulators may enable new targeted drugs that aid treatment of mitochondrial dysfunction or mimic exercise in those who cannot do exercise.
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 111966 0
Prof David Bishop
Address 111966 0
Victoria University
Ballarat Rd
Footscray Park Campus
Footscray, Melbourne
3011, VIC
Australia
Country 111966 0
Australia
Phone 111966 0
+61 399199471
Fax 111966 0
Email 111966 0
[email protected]
Contact person for public queries
Name 111967 0
Prof David Bishop
Address 111967 0
Victoria University
Ballarat Rd
Footscray Park Campus
Footscray, Melbourne
3011, VIC
Australia
Country 111967 0
Australia
Phone 111967 0
+61 399199471
Fax 111967 0
Email 111967 0
[email protected]
Contact person for scientific queries
Name 111968 0
Mr Dale Taylor
Address 111968 0
Victoria University
Ballarat Rd
Footscray Park Campus
Footscray, Melbourne
3011, VIC
Australia
Country 111968 0
Australia
Phone 111968 0
+61 426297186
Fax 111968 0
Email 111968 0
[email protected]

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
Yes
What data in particular will be shared?
Main outcome variables; physiological measurements (BMI, VO2max, power at lactate threshold, enzymatic assays)
When will data be available (start and end dates)?
As soon as possible after publication for at least 5 years
Available to whom?
Case by case basis by principal investigator discretion
Available for what types of analyses?
For meta-analyses
How or where can data be obtained?
By emailing PI
[email protected]


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
No additional documents have been identified.