Searching for non-invasive Magnetic Resonance-based markers for CrAT activity and carnitine availability

Previous studies have shown that mitochondrial function is essential at the
transitions from rest to steady state exercise, exhibiting a short activation
delay to supply ATP for muscle contraction. A delayed mitochondrial activation
rate at the onset of exercise is associated with muscle metabolic instability,
poor exercise tolerance and early muscle fatigue. As a likely underlying
mechanism, it is proposed the existence of an Acetyl group deficiency, referred
as metabolic inertia. In this context, acetylcarnitine is pointed out to as a
key factor, as acetylcarnitine breakdown (regulated by the action of CrAT
protein) may deliver Acetyl-CoA groups to the tricarboxylic acid cycle (TCA
cycle) under conditions of high demand; for example at the onset of exercise
(Acetylcarnitine on-kinetic). Indeed, increments of acetylcarnitine content in
the resting state before exercise modulates the mitochondrial activation speed
and CrAT activity may determine the duration of metabolic inertia. To date, the
only possibility to get information on CrAT activity is by taking a muscle
biopsy and perform enzyme activity assays ex vivo. By dynamic MRS scanning, it
might be possible to deduce information that reflects CrAT activity, thereby
making the biopsy sampling unnecessary.
It is expected that CrAT protein activity modulates mitochondrial activation
during exercise. As phosphocreatine (PCr) kinetic at the beginning of exercise
might reflect mitochondrial activation, it is expected that CrAT protein
activity is correlated with PCr on-kinetic at the beginning of exercise.
Furthermore, another interesting metabolic parameter that can influence
mitochondrial activation is muscle tissue oxygenation and intrinsic
mitochondrial function. Thus, those parameters will be assessed in the current
proposal. We hypothesize that muscle tissue oxygenation correlates with
mitochondrial activation. Finally, it will be tested whether CrAT protein
activity and carnitine availability in skeletal muscle determines maximal
acetylcarnitine formation upon exercise.

Primary objective:

To investigate the correlation between skeletal muscle phosphocreatine (PCr)
on-kinetic at the beginning of exercise and CrAT protein abundance/activity in
healthy young males characterized by a wide range of maximal aerobic capacity.

Secondary objective:

To investigate the correlation of CrAT protein abundance/activity and and
muscle tissue oxygenation in healthy young males in a wide range of maximal
aerobic capacity

To investigate whether phosphocreatine (PCr) on-kinetic at the beginning of
exercise and CrAT protein abundance/activity is associated with ex vivo
skeletal muscle mitochondrial function in healthy young males in a wide range
of maximal aerobic capacity.

To explore whether maximal acetylcarnitine formation upon exercise is
correlated with CrAT protein activity/content and carnitine availability from
muscle biopsies

In this cross sectional study 13 subjects will visit the University 3 different
days, for screening and for the exercise test days.

Screening; to assess eligibility.
-Informed consent
-Medical history questionnaire
-MRI contra-indications form
-Fasted blood samples to determine metabolic health
-Maximal aerobic capacity
- Maximal one-legged extension
- Electrocardiogram at resting

MRS+Exercise test day 1:
- Body composition by Bod pod technique
- Muscle tissue oxygenation before and after one-legged exercise

Exercise test day 2:
-Acetylcarnitine levels at resting and post cycling exercise
- Standardized cycling test 30 minutes total; 10 minutes at 30% Wmax, 10
minutes at 50% Wmax and 10 minutes at 70% Wmax
- PCr kinetic during and post exercise.
- Maximal acetylcarnitine formation upon exercise
- One muscle biopsy before exercise
-Indirect calorimetry throughout cycling test
-4 Blood samples during exercise and 4 blood samples during recovery post
exercise (8 blood sample sin total; ~10 ml each sample)

The results of this research will provide a novel approach to measure CrAT
protein activity and carnitine availability by using a non-invasive Magnetic
Resonance-based methodology in humans. Indeed, by measuring the kinetic of
phosphocreatine (PCr) during exercise may give us information about
mitochondrial activation, process on which CrAT protein activity might play a
pivotal role to determine the exercise tolerance and early muscle fatigue
during exercise in humans.

The risk involved in this research are very small. Adverse effects will hardly
occur.
The exercise test days comprise non-invasive and invasive measurements. The
used techniques are safe, but the muscle biopsies can cause some discomfort and
may result in a local bruise or hematoma. Likewise, blood sampling can cause a
local hematoma. The risk of infection and or prolonged bleeding is very low due
to state of the art technique and sterility measures. Magnetic Resonance
Imaging can result in unexpected medical findings.