Precision High-Intensity Training Through Epigenetics (PHITE)

Overview:

The US military relies on physical training to increase and maintain force readiness. In this
study, we hypothesize that epigenetic modification is a primary mechanism by which specific
physical exercise training prescriptions transduce warfighter genotype into warfighting
phenotype. Our aim with this program is 3- fold. We will (1) identify training methodologies
that modify epigenetic responses; (2) characterize epigenetic regulation of physiological
processes, pathways and mechanisms associated with moderate and high-intensity physical
training, and (3) produce real-time biomarkers of cardiorespiratory and neuromuscular
performance that predict physical training outcomes. We will satisfy these goals through the
following six research projects:

I. Phenotyping the Exercise Dose-Response

1. Determine the effect of exercise Intensity on neuromuscular and cardiorespiratory
performance

2. Assess the impact of baseline, pre-training phenotype on acute responsiveness and
adaptability to different intensity exercise regimens

3. Determine the effect of exercise intensity on mechanisms regulating muscle protein
metabolism, myofiber hypertrophy, and muscle oxidative capacity

II. Real-time Genetic and Epigenetic Biomarkers of Performance

1. Identify muscle miRNA that are differentially expressed in response to varying exercise
intensities

2. Identify circulating muscle-derived miRNA that are differentially expressed in response
to varying exercise intensities

3. Genotype individual athletic potential for biomarker optimization

IV. Epigenomic Signatures of Human Performance

1. Identify and correlate exercise induced methylation changes in blood and muscle

2. Functionally link changes in gene expression signatures and physiologic outcomes

3. Define nuclear receptor induced epigenetic signatures in metabolically active tissues

V. Exercise Induced Histone Modifications and Chromatin Remodeling

1. Profile histone modification and histone subunit usage signatures

2. Assess chromatin as a signal integration platform for long-term stability

VI. Integrative Epigenomics

1. Categorize baseline performance and group subjects

2. Characterize epigenetic changes in response to varied exercise intensities

3. Develop forecasting models from miRNA biomarkers of performance

4. Develop epigenetic regulatory network models using Deep Belief Networks

UAB will enroll healthy, untrained men and women who resemble the US soldier. They
participate in an exercise dose response training trial that lasts approximately 12
weeks.

There are 2 randomly assigned exercise prescriptions: MOD - 3 days per week on treadmill
or stationery cycle ergometer, combined with 2 days per week of resistance training; HI
- 3 days per week high intensity interval training similar to military high intensity
tactical training, combined with high intensity resistance training 2 days per week.

Physical performance is evaluated pre-training, mid-training and post-training.
Additionally, body composition and cognitive performance are evaluated pre-training and
post-training. Muscle biopsies and blood draws are performed at pre-training and
post-training acute exercise response bouts, and samples are sent to additional study
sites for specific evaluations.

Recruitment:

Participants are recruited through IRB approved advertisements.

Informed Consent:

Once an individual is identified; meets the inclusion/exclusion criteria (pre-screen);
and expresses interest in the study, a member of the research team provides the
individual with a consent form to review. A member of the team follows up with the
individual to further explain the study and provide an opportunity for the prospective
participant to ask any questions. If the person decides to participate, he/she is
scheduled for the initial visit. He/she is encouraged to ask any further questions prior
to giving written informed consent.

Participant Procedures:

MOD exercise regimen - 3 days per week of endurance exercise at 70% heart rate reserve
for 30 mins on a stationary cycle or treadmill, combined with resistance exercise on 2
of the 3 days. The resistance training prescription involves 3 sets x 10-14 repetitions
for each movement, with 1 minute rest between set. The movements include leg press or
squat, knee extension, chest press, overhead press, seated row, wide grip pull down, ab
crunch, heel raise, triceps push down, bicep curls. Resistance loads are progressed when
14 repetitions are achieved for 2 of 3 sets. Heart rate is monitored throughout each
session.

HI Exercise regimen - 3 days per week of maximum intensity interval exercise, combined
with resistance exercise on 2 of the 3 days. Instead of moderate intensity endurance
exercise, participants in this group perform 10 high-intensity intervals (30 sec of
work, 30 sec of rest) including box jumps, burpees, split squat jumps, kettle bell
swings, cycling sprints, battle ropes, wall ball, dips, etc. The resistance training
prescription involves 3 sets x 8-10 repetitions for the same movements as MOD, but with
supersets of two movements. Resistance loads are progressed when 10 repetitions are
achieved for 2 of 3 sets. Rest periods are 30-45 seconds between supersets. Heart rate
is monitored throughout each session.

DXA Scan -The dual energy x-ray absorptiometry (DXA) procedure for determination of body
composition (muscle, fat, bone) involves minor x-ray exposure. DXA will be performed at
weeks 0, and 12. The effective dose exposure is comparable to 4 days of background
radiation in Birmingham.

Urine Pregnancy Test - All females who have had at least one menstrual cycle have a
urine pregnancy test at initial assessment and prior to each DEXA scan. All females who
have had at least one menstrual cycle are asked questions pertaining to their menstrual
cycle. Because hormone levels may have significant impact on biospecimen analysis, the
acute exercise bout with muscle and blood collections after the intervention period will
be timed to occur in the same phase of the menstrual cycle as the pre-training acute
response exercise bout.

Muscle Sampling - A total of 7 muscle samples are collected by percutaneous needle
muscle biopsy from the vastus lateralis using established procedures:

3 collected before and after the first acute response exercise bout in the pre-training
state (pre-exercise, 3 h post-exercise, 24 h post-exercise); 3 collected before and
after the acute response exercise bout in the post-training state (pre-exercise, 3 h
post-exercise, 24 h post-exercise);

1 collected at a follow up visit 4 weeks after the conclusion of the intervention
period.

Blood Sampling - A total of 9 blood samples are collected by venipuncture using
established procedures:

4 collected before and after the first acute response exercise bout in the pre-training
state (pre-exercise, 15 min post-exercise, 3 h post-exercise, 24 h post-exercise); 3
collected before and after the acute response exercise bout in the post-training state
(pre-exercise, 15 min post-exercise, 3 h post-exercise, 24 h post-exercise);

1 collected at a follow up visit 4 weeks after the conclusion of the intervention
period.

Exercise Performance Evaluations - 1-repetition maximum (1RM) strength on upper and
lower body movements, peak aerobic capacity (VO2peak) on a cycle ergometer, Wingate
anaerobic power test, peak muscle power of the knee extensors using a
computer-controlled dynamometer, and vertical jump test are conducted before and after
the intervention period. Some performance tests are also conducted mid-intervention
(approximately week 6).

Neurocognitive and Neuropsychological Profile - Participants complete the Multiple
Aptitude Battery-2nd Edition (MAB-II), and the MicroCog Neuropsychological Test
(MicroCog) to measure cognitive and psychological phenotype. Each subject completes the
MAB-II one time (at baseline) and the MicroCog two times (pre- and post-intervention
period). The data will be used to assess the impact of pre-training phenotype on acute
responsiveness and adaptability to the two different exercise prescriptions.

Dietary Intake - Dietary intake will be monitored using a validated 24 hour recall at
weeks 0 and 12.

Quality Assurance:

Exercise Sessions: Subjects will be carefully instructed in proper methods and are
monitored continuously. All exercise sessions are performed under full supervision by
experienced, certified staff [Certified Strength and Conditioning Specialist (National
Strength and Conditioning Association) and/or Certified Exercise Specialist (American
College of Sports Medicine)].

Biospecimens Collection: All muscle biopsies and blood draws are performed by trained,
professional research staff who are experienced and locally certified.

DXA scans: All DXA scans are performed and analyzed by trained technicians at UAB.

Genetic Testing: Genetic testing of samples will be linked to a unique participant ID
that will not identify a participant in any way. Linkages to personal health information
are kept secure at the clinical site (UAB).

The research will be conducted by well trained and experienced personnel who will be
identified to the IRB prior to working on this study.

PI will be ultimately responsible for data and safety monitoring: (i) Keeping all study
documents updated and available for inspection by the sponsor, UAB IRB, and other
authorized reviewers; (ii) Ensuring that appropriate mechanisms to protect the safety of
study participants are being followed; (iii) Ensuring adherence to protocol
requirements; and (iv) Ensuring that data are accurate, complete, and secure. All
documents pertaining to the conduct of the study will be kept on file by the PIs.

Even though this trial does not meet the requirements for a Data and Safety Monitoring
Board, we will follow a proactive, Data and Safety Monitoring Plan as described below.

1. Weekly reviews at investigators/staff team meetings of recruitment progress notes,
updates/reports from the exercise facility, laboratories, and data management
staff, and newly available data.

2. PIs' quarterly review of safety and interim data analysis.

3. Annual review performed by the UAB IRB.

4. Semi-annual patient safety review by the study team.

All non-serious AEs will be documented, and a summary delivered to the UAB IRB at
pre-determined intervals (at least annually). Any SAEs will be reported to the UAB IRB
within 3 days of their discovery. Aggregate reports of AEs/SAEs will be delivered to the
sponsor as mandated. If an AE (serious or non-serious) occurs, the PI will be
immediately notified. Depending on the event, follow up may require additional tests or
medical procedures as indicated, and/or referral to a specialist. The study physicians
will make such decisions. Depending on the type of AE/SAE, the PIs may opt to
discontinue the subject from the study. The PIs will be ultimately responsible for
evaluating each AE for relationship to the protocol, seriousness and expectedness, any
impact on the risk-to-benefit ratio, and whether modifications are needed to the
informed consent document and/or protocol. The PIs will also have the responsibility of
determining whether the protocol must be suspended until satisfactory modifications are
made; however, ultimate authority in such cases will rest with the UAB IRB.

Inclusion Criteria:

-Healthy 18-27 year olds who are not in the US armed forces

Exclusion Criteria:

- History of regular endurance or resistance training in past 12 months

- BMI>30

- Inability to tolerate intense exercise

- Diabetes

- Uncontrolled hypertension

- Unstable or exercise induced angina pectoris or myocardial ischemia

- Vascular disease

- Neurologic disease

- Musculoskeletal disorder

- Mental health disorder

- Any chronic or infectious disease that would preclude full participation

- Lidocaine allergy

- Pregnancy