Presentation text

Good afternoon everyone, today, we are going to explore the controversies surrounding the involvement of high
intensity exercise on mitochondrial biogenesis. Some exercises seem to stimulate mitochondrial biogenesis. But
what about high intensity exercises?

I will first make a little introduction about the mitochondria, then, I’ll move on defining mitochondrial biogenesis,
HIIT and study the mitochondrial protein synthesis. Anna will clarify some methodological issues then Héloïse and
Marie will talk about mitochondrial content and respiratory function. Finally, Madina will present other
considerations of mitochondrial biogenesis and conclude.

First, let's start with a reminder about mitochondria.

Mitochondria are organelles that produce cellular energy through oxidative phosphorylation. They have 2
membranes, and their sizes vary from 0.1 to 5 micrometres in skeletal muscle.

Sub-optimal characteristics of mitochondria are often associated with a pathological condition as they are involved in
many essential cellular functions related to metabolism.

A correlation could be made between endurance performance and mitochondria. Their content and respiratory
function are related to maximal oxygen consumption, time trial performance and lactate threshold.

There is no widely accepted definition for mitochondrial biogenesis, however they define it as "the making of new
components of the mitochondrial reticulum".

It has been suggested that mitochondrial biogenesis can be assessed by measuring the rate of mitochondrial protein
synthesis (mitoPS). However, it is necessary to establish whether exercise-induced changes in mitoPS can provide
quantitative or qualitative information about subsequent training-induced changes in the content of the
mitochondrial reticulum.

In our paper, the authors decided to consider high-intensity exercise to be intervals performed at more than 75% of
maximal power (Wmax) achieved during a graded exercise test from 8 to 12 minutes. They therefore considered
continuous moderate-intensity exercise to be continuous exercise performed at an intensity below 75% of Wmax.

The aim of this study was to assess changes in mitoPS in response to high-intensity endurance exercise. A single
session (45 min, 75% V O2 max) unilateral cycling study showed an increase in mitoPS 4h after training in trained and
untrained individuals. Differences in the methods used influence the changes inferred. For example, changes in
mitoPS are sometimes inferred from changes in protein synthesis in the sarcoplasmic fraction (sarcoPS). During
fractionation, most skeletal muscle mitochondria are retained in the sarcoplasmic fraction and changes in sarcoPS
should indirectly reflect changes in mitoPS. However, there are other cellular components such as quantification of
protein synthesis rates in this fraction.

Measurement of the rate of synthesis of mixed muscle proteins and their sub-fractions are usually quantified by
infusion of stable isotope labelled amino acid tracers. This technique is limited to short durations, controlled
environments and feeding strategies.

Now, I am leaving the floor to Anna who will talk about methodological issues.

It’s important to remember that there are several methodological issues to consider.
The first one is the time course of molecular changes. The diagram below portrays the evolution of a mitochondrial
biogenesis component over time.
The variations are not set and constant; rather, they differ over time. According to study, the timing of the muscle
sampling has a considerable influence on the findings, so it is important to gather muscle samples collected at
multiple different time to draw accurate conclusions on the effects of exercise on mitochondrial biogenesis.
The second issue that is important to consider is the fibre-specific effect.
Type 1 fibre are fatigue resistant, and will be mobilized especially during low intensity exercise when type 2 fibres
provide bigger and more powerful forces, but fatigue quickly.
it is believed that the results of high-intensity exercise would be exclusive to each type of fibre.
We can see that AMPK activation is fiber type specific in human skeletal muscle same goes for glycogen.

Then there is the impact of gender. Due to a lack of female subjects, most studies have been performed on males;
however, the findings are expected to be the same. For example, the citrate synthase activity increases equally for
gender

However, we know that there is gender-based differences into the mitochondrial biogenesis.
For example, differences in mRNA content of metabolic related genes, women have higher mRNA abundance for
genes involved in fat metabolism and a higher% area of type I fibres.
Some results suggested that the response of PGC-1α mRNA expression to exercise may depend on the ratio of
progesterone to oestrogen. So, it varies according to the menstrual cycle phase for women.
we still need more research to fully know whether there is gender-based effect in case of high intensity exercises.

And lastly, the Relative exercise intensity and the “first bout” effect. It is important to consider the reference point
used to calculate relative exercise intensity.
A study demonstrates that the markers of exercise stress were greater in untrained subject compared with trained
subject when the exercise was performed at the same relative percentage of peak oxygen consumption. In contrast,
the metabolic and cardiac stress in trained and untrained subjects were similar during exercise at 95% lactate
threshold (LT).
In order to compare exercise-induced changes in individuals with different aerobic capacities, it may be more
appropriate to select a work rate relative to the LT.
A second critical methodological problem is the effect of hiring people who are inexperienced with high-intensity
exercise.. Results showed that changes in PGC-1 mRNA in response to high-intensity exercise are often decreased
with each following exercise, even though the exercise intensity is retained.
Introducing participants with exercise that will be conducted prior to performing will benefit resolving some of the
contradictory results published in the literature.

Those are the four main methodological challenges that we face.

Now let’s hear Héloïse talk about mitochondrial content and respiratory function

We can suppose that the mitochondrial content and the respiratory function increase concomitantly but not always.

John Holloszy has concluded that the training can increase the mitochondrial content. Then, the transmission
electron microscopy has confirmed these results.

Recently a study showed that HIIT increases the expression of anti-apoptotic genes and modify the anti-apoptotic/
pro-apoptotic ratio. One of those apoptotic factors is the AIF, also known as apoptosis inducing factor that is
important for the life and death of the cell.

This factor is encoded by the gene apoptosis inducing factor mitochondria associated 1, also called AIFM1.

The protein is required for maturation of the mitochondrial respiratory complex I. Also, the protein displays NADH
oxidoreductase.

When it is time for the cell to die, the protein is translocated to the nucleus to induce the chromosome condensation
and fragmentation to permit apoptosis in a way caspase-independent and induce mitochondria to release
cytochrome c and caspase 9.

The gene is in the X chromosome, in the q arm: Xq26.1.

The gene size is 36.471 Kilobases with a transcribed region of 2.215 Kilobases. This transcribed region codes for 613
amino acids and contains 16 coding exons. Nowadays, 5 isoforms of this gene have been described: AIF, AIFexB,
AIFsh, AIFsh2 and AIFsh3.
Translation start (ATG, in green) and stop (TGA / TAA, in red) codons are indicated, and the predicted protein
product is shown at the right of the diagram. The blue lines indicate the splicing’s.

The protein contains a mitochondria localization signal when it is in a precursor form. When the precursor is
addressed to the mitochondria, the precursor is cleaved in a mature form with a transmembrane domain. The
protein is anchored to the inner membrane by this domain with the C-term domain exposed to the intermembrane
space and the N-term exposed to the matrix. Currently, the protein is useful to maintain the complex I of the
mitochondrial respiratory chain. After an apoptotic insult, the protein is cleaved by active calpains and cathepsins to
give a proapoptotic protein, the truncated AIF. This protein is a cytosolic protein because the cleavage removes the
TM domain. When the protein is in the cytosol, it can go to the nucleus thanks to the nucleus localization sequence
and induce the apoptosis by chromosomic fragmentation and condensation.

I am now pleased to hand over to Marie to talk about tertiary structure of the protein and pathologies associated to
mutations.

There are 3 major domains in the protein:

• The FAD-binding domain is composed by residues 128 to 262 and 401 to 480

• The NADH-binding domain is composed by residues 263 to 400

These 2 are in yellow because they are both oxidoreductase domains.

• The C-terminal domain is composed by residues 481 to 608

This domain is in blue, it is the domain where the proapoptotic activity of the protein resides.

In pink, it is the AIF cofactor Flavin Adenine Dinucleotide.

If the gene is muted by a transition where the guanine in 1013 position of the coding DNA is substituted by adenine
(c.1013G>A), it induces combined oxidative phosphorylation deficiency 6, also called COXPD6.

The symptoms of this pathology are:

• In the muscular system, we have muscle weakness and atrophy, mitochondrial DNA depletion about 20 to 35% in
skeletal muscle, decreased activity of mitochondrial respiratory complex enzymes and ragged-red fibbers

• In the respiratory system, we have respiratory insufficiency due to muscle weakness

• In the nervous system, we have psychomotor regression and retardation, hypotonia, decreased spontaneous
movements, tetraplegia, seizures, hyporeflexia or areflexia and sensory and motor axonal polyneuropathy

• In a systemic scale, we have increased lactate and pyruvate

The disease is very rare, the prevalence is under 1/1 000 000 in the world

The mutant protein has an inferior Km for NADH, so it is reduced 4 times easier by NADH than the normal protein.
The half-life is reduced but the protein is still able to dimerize. So, the redox capacity isn’t affected but there will be
more apoptotic cells.

There is a mutation that causes the Charcot-Marie Tooth disease X-linked recessive, also known as Cowchock
syndrome. The mutation is a transversion where the adenine in position 1478 is substituted by a thymine in the
coding DNA (c.1478A>T). In the amino acid sequence, it induces a missense mutation. Normally, it is a glutamic acid,
but the mutation changes this one by a valine.

The symptoms are very different than the first disease:

• At ears level, we have hearing loss and auditory neuropathy

• At eyes level, we have abnormal smooth pursuit, abnormal saccades, poor vision, and retinopathy

• At feet level, we have pes cavus and hammertoes that is a toe that is bent permanently downward

• In the muscle system, we have distal muscle weakness due to peripheral neuropathy, neurogenic atrophy seen on
muscle biopsy, increased numbers of abnormally shaped mitochondrial, and decreased complex IV activity

• In the nervous system, we have cerebellar ataxia and atrophy, limb and gait ataxia, cognitive impairment,
dysarthria, titubation, tremor, dysmetria, bradykinesia, hyporeflexia, Babinski reflex, and axonal motor &sensory
neuropathy

• In a systemic scale, we have increased creatine kinase and transaminases because the elevation of transaminases
is sometimes due to muscular necrosis and the augmentation of creatine kinase is sometimes due to muscular
lesions.

There is two other mutation, one that causes spondylo-epi-metaphyseal dysplasia and the other causes deafness,
neither of the two will be detailed.

At this point, I would like to turn to Madina to give other considerations and conclude.
Most of the research on mitochondrial adaptations to training has assigned groups to one type of training—either
MICT (moderate intensity continuous training), HIIT (high intensity interval training), or SIT (sprint interval training).

However, this type of training differs from that of endurance athletes following a combination of moderate intensity
and high-intensity exercise training. And that’s how some of the largest increases in aerobic fitness and
mitochondrial respiration have been reported.

There is also evidence that training twice every day or twice every second day may be more effective than a daily
training, to increase citrate synthase (CS) activity.

According to short duration training studies, high-intensity exercise training is more effective to increase
mitochondrial respiratory function than moderate-intensity training. Even when training volume is matched

There is a need to make longer-duration and time-course studies

• to understand how best to distribute different types of training across days, weeks, and months to optimize
mitochondrial adaptations

• determine the greater increases in mitochondrial respiration in response to high-intensity compared with
moderate-intensity exercise training when the training is continued beyond 10 weeks.

In healthy and untrained males, only two weeks of high intensity training increased mitochondrial respiration by
22%. If this rate of change were to continue, we could achieve values recorded by elite athletes in only 3-4 months.
But the rate of increase seems to slow as training duration lengthens. However, there is a need for Time-course
studies to confirm this hypothesis

The ability of skeletal muscle to respond differently to a stimulus if it has previously been encountered is called
skeletal muscle memory. It was suggested that the muscle memory could be related to epigenetics which leads to a
modification of gene expression as a result of structural modifications of DNA, without changing the underlying DNA
sequence.

And there is also the DNA HYPOMETHYLATION which, conversely to DNA METHYLATION, leads to enhanced gene
expression.

Despite much research and improved understanding there has been in the last 50 years about mitochondrial
adaptations to training, there are still many conflicting findings. A consensus hasn’t been reached about the role of
high intensity exercise in promoting mitochondrial biogenesis. However, in this review, we have highlighted that,
many fundamental questions need attention before we can inch closer to solving the more complex issues.

And we would like to conclude by emphazing these fundamental questions

1) How should we define mitochondrial biogenesis

2) When should we take muscle biopsies to best capture the molecular events associated with mitochondrial
biogenesis

3) Which mitochondrial characteristics are most important for human health and performance

There is also a need to discern « what’s a high intensity exercise » exactly and how to define it.

We are now coming to the end of the presentation, thank you all for your attention and we welcome your
questions.