Transcript Fibre types

Molecular Exercise Physiology
Fibre Phenotype Regulation
Presentation 4
Henning Wackerhage
Learning outcomes
At the end of this presentation you should be able to:
• Explain how fibre types can be visualised and describe the
features of different fibre types. Also be able to critically
comment on the fibre type concept.
• Explain how fibre types and proteins therein respond to
changes in contractile activity;
• Explain how the calcineurin and ERK1/2 pathway may
regulate proteins within muscle fibres.
Fibre Phenotype Regulation
Part 1
Fibre types
Fibre types
Muscle consist of thousands of muscle fibres. For example there are
about half a million muscle fibres in the vastus lateralis of a young
male. Muscle fibres are thin but can be very long; i.e. a fibre can
probably span the whole length of a muscle from tendon to tendon.
Muscle fibres are thus the largest cells of the human body.
Muscle fibres are heterogenous: At the end of the 19th century,
“translucent” and “opaque” fibres were distinguished by microscopy.
Fast muscle(s) (fibres) have a higher ATPase activity
Barany et al. (1965, 1967) then found that fast and slow muscles had
different ATPase activities which correlated with the speed of
contraction. Thus, muscles that can split ATP quickly can contract
quicker. ATPase activity is thus a good measure to distinguish
between muscle fibres. Barany et al.’s data are shown on the next
slide. The figure shows that there is a close correlation between
contraction speed (v) and myosin ATPase activity.
Fibre types
Later, Guth and Samaha (1969) and Brooke and Kaiser (1970)
developed histological stains for ATPase which allowed them to
distinguish between muscle fibres with different ATPase activity: I
(slow), IIa (intermediate) and IIb (fast).
ATPase histochemistry
How does the ATPase fibre stain work? ATPase is an enzyme that
splits ATP into ADP and Pi. Thus, muscle sections are incubated with
an ATP solution and the ATPase in the section will split ATP into ADP
and Pi. The more ATPase, the more ADP and Pi will be produced
during incubation. The invisible Pi is then visualised as a dark stain
with calcium chloride, cobalt chloride and ammonium sulphate.
The result of an ATPase stain is shown above. Slow (type I) fibres are
white and fast IIa and IIb fibres are stained black.
ATPase histochemistry
The ATPase activity also depends strongly on the pH of a so-called
preincubation solution as is shown in the table below. The table also
features IIx fibres which are another fibre type that was discovered
later. It was also found that the fast muscles fibres in human muscle
are IIx rather than IIb fibres.
NBT stain for a mitochondrial enzyme
Fibres also differ in their mitochondrial content. Mitochondria can
be stained with a so-called nitro blue tetrazolium (NBT) stain.
Enzymes in the mitochondria convert the substrate in a brown blue
dye. The more mitochondria, the darker the slider after the
incubation period. In this weeks practical, you will both perform an
ATPase and a NBT stain.
Myosin heavy chain
Myosin is the key motor that converts the chemical energy from
the ATPase reaction into mechanical energy (i.e. muscle
contraction) and heat. The ATPase function of myosin was
discovered by Engelhardt and Lyubimova in 1939. Later it was
discovered that myosin consisted of light and heavy chains and
that there were different myosin heavy chains.
The following slide shows an experiment that visualises the
different MHCs in fast and slow muscles.
SDS gel electrophoresis for myosin heavy chain
isoforms
Myosin heavy chains were separated by an electrical field in a gel
and then stained with a protein stain. The I (slow), IIa
(intermediated) and IIx and IIb (fast) myosin heavy chain
isoforms can be distinguished.
M. soleus
Diaphragm
M. gastrocnemius
Task
What is the standard technique to measure the concentration of a
protein?
Summary
Muscle fibres can be distinguished into type I, IIa, IIx and IIb
fibres based on the predominant MHC isoform, mitochondrial
content, colour and contraction speed among other. The table
below gives an overview.
Fibre type
I
IIa
Functional description
slow
intermediate fast
Predominant MHC isoform
I
IIa
IIb (IIx)1)
Mitochondrial content
high
high
low
Contraction speed
slow
intermediate high
Endurance
high
intermediate low
1)Human
IIb (IIx)1)
fast fibres are usually not reactive to mammalian MHC IIb
antibodies. Fast human fibres predominantly express MHC IIx.
MHC Myosin heavy chain.
Fibre types: an oversimplification
It is now generally recognised that fibre types occur not as three
(or four) distinct forms but that they present a continuum based
on combinations of MHC isoform content, oxidative and glycolytic
enzymes, myoglobin, and calcium-handling proteins.
Thus, the differentiation of three different fibre types is an
oversimplification.
Fibre Phenotype Regulation
Part 2
Fibre type and training
Fibre type variations
Soon the ATPase stain developed around 1970 and other stains
were used for human muscle samples in order to detect variations
in fibre types between individuals and to see whether there is a link
between fibre type percentages and performance and to see
whether training can affect the percentages of fibre types.
Landmark studies by Gollnick and Saltin
Landmark studies were performed by Gollnick and Saltin’s groups in
the early 1970s. In the first study, Gollnick et al. (1972) found that
endurance athletes had more slow twitch (type I) fibres than
untrained subjects in their vastus lateralis. In the second study,
Gollnick et al. (1973) reported that endurance training for 1 h at 4
days per week for 5 months had no significant effect on the
percentages of fast and slow fibres. However, they had only
investigated 6 subjects and thus large differences could not be
detected.
Later studies did investigate the variability of fibres in a muscle and
the effect of training on fibre types in more detail.
Fibre percentages differ between athletes
% type I fibres
This study shows the % of type I fibres in track athletes. The data
suggest a relationship between the length of the event and the
percentage of slow fibres.
70
60
50
40
30
20
10
0
Untrained
Sprint
Middle
Long
distance distance
Costill et al. (1976)
Slow fibres increase with time
The Costill group also investigated whether the percentage of fibres
changes over a 20-year period. The authors found a significant
increase in type I fibres in fit and untrained subjects but not highly
endurance-trained subjects were the type I fibre percentage was high
already. The increase maybe partially due to a loss of type II muscle
fibres (Lexell et al. 1988).
80
70
*
*
*
1973
1993
60
50
40
Highly
trained
Fit
Untrained Total
Trappe et al. (1995)
Fibre type variations
Type IIx-to-IIa shifts by endurance training
The Gollnick et al. (1973) paper suggested that training did not
stimulate changes from type I to type II fibres. However, later
studies suggested that there was a decrease in IIx and an increase
in IIa fibres (Jansson & Kaijser, 1977;Ingjer, 1979). In addition,
most training studies are not long enough to allow a conclusive
answer regarding whether type II fibres can be turned into type I
fibres by endurance training over years.
Promotion of type II fibres in most denervation models
No muscle activity usually results in an increase of type II fibres
and a decrease of type I fibres. In rat soleus and adductor longus
muscles, MHC I is gradually exchanged by MHC IIx mRNA and
protein starting at day 15-30 up to day 90 (Huey et al., 2001). A
longitudinal study in human beings with spinal cord injury suggest
that slow MHC isoform fibres drop between 1 and 20 months after
trauma and that by 70 months fibres almost exclusively express
type II MHC isoforms (Burnham et al., 1997).
Chronic electrical low-frequency stimulation
Chronic electrical stimulation can convert type II into type I
fibres
Stimulating a motor nerve continuously transforms the a type II to
type I fibre type change in the innervated muscle. The extent of
transformation depends on the stimulation dose.
Sutherland et al. (1998) stimulated rabbit tibialis anterior muscle
with 2.5, 5 or 10 Hz for 10 months. In the control muscle, there are
nearly no type I fibres (tibialis anterior is a fast muscle) and 10 Hz
stimulation > 95% of fibres are type I fibres. Stimulation with 2.5
or 5% stimulates a limited increase of type I fibres. Nuhr et al.
(2003) stimulated human muscle with 15 Hz via surface electrodes
(less effective than nerve stimulation). The found an approximately
20% decrease in the relative concentration of MHCIId/x (from 28%
to 22%) and an approximately 10% increase in the relative
concentration of MHCI (from 30% to 34%). Although they
measured MHC isoform content and did not count fibres, these data
suggest that a type II to type I expression change may be possible
in human muscle if the dose is high enough.
Metabolic enzymes respond as well
Chronic electrical stimulation also affects the expression of
oxidative and glycolytic enzymes in rabbit tibialis anterior muscle.
Mitochondrial, oxidative
enzymes
Glycolytic enzymes
Henriksson et al. (1986)
Summary
(1)
(1) Inactivation (paraplegia, denervation)
partially transforms type I fibres into fast type II.
(2) Chronic electrical stimulation turns fast
type IIb/x fibres into intermediate IIa or slow type
I fibres  complete fast-to-slow transformation can
be achieved.
(3) Endurance and strength training mainly
turn very fast type IIb/x into intermediate type IIa
fibres  incomplete fast-to-slow transformation.
(2)
(3)
Electrode (10 Hz stimulation)
Motor nerve
Fibre Phenotype Regulation
Part 3
Signal transduction
Calcineurin hypothesis
Before 1998, virtually nothing was known about the regulation of
the adaptation to endurance exercise. In 1998, Eva Chin
published a landmark paper where her research group linked
the exercise signal calcium (calcium makes muscles contract) to
the induction of “slow” genes via the calcineurin pathway. The
paper is:
Chin et al. A calcineurin-dependent transcriptional pathway
controls skeletal muscle fibre type. Genes & Dev. 2499-2509,
1998.
Eva Chin and co-workers demonstrated that a blockade of
calcineurin with cyclosporin A led to an increased in the
percentage of fast fibres. This suggested that the non-blocked,
exercise/calcium activated pathway would promote a fast-toslow fibre phenotype conversion. The cyclosporin A blockade
data are shown on the next slide.
Calcineurin hypothesis
Fast fibres (%)
40
Mean
30
Blockade of the calcineurin
pathway with cyclosporin A
increased the type II fibre
percentage
in
rat
soleus
muscle.
20
Mean
10
0
Control
Cyclosporin A
treated
Chin et al. (1998)
Calcineurin hypothesis
Chin et al. hypothesised that the calcineurin pathway would be a
“molecular mechanism by which different patterns of motor nerve
activity promote the specialised characteristics of slow and fast
myofibres”.
A schematical overview over the hypothesized function of the
calcineurin pathway is shown on the next slide.
Calcineurin hypothesis
Muscle
fibre
Sarcoplasmic reticulum
Slow type I
genes
[Ca2+]
Calmodulin
CnB
NFAT
NFAT
CnA
NFAT
P
Nucleus
Whenever we exercise, calcium is released from the sarcoplasmic
reticulum. Calcium binds to calmodulin which in turn activates calcineurin
(Cn). Calcineurin consists of a regulatory (CnB) and catalytic subunit
(CnA). Calcineurin is a protein phosphatase and activated calcineurin
dephosphorylates the nuclear factor of activated T-cells (NFAT).
Dephosphorylation of NFAT exposes a nuclear localisation signal (NLS)
which leads to the import of NFAT into the nucleus. NFAT then binds to its
transcription factor binding site and increases the expression of “slow”
genes that respond e.g. to endurance training.
Task
Carry out a literature search for other functions of the calcineurin
pathway.
ERK1/2 pathway
Various studies showed that the calcineurin story was more
difficult that previously imagined.
For example, Swoap et al. (2001) reported that the calcineurin
pathway also upregulated some fast genes, contradicting Chin et
al.’s hypothesis.
In 2000 Murgia reported that the activation of the ERK signal
transduction pathway by its upstream activator Ras could induce
slow fibres in regenerating muscle.
Murgia et al.: Ras is involved in nerve-activity-dependent
regulation of muscle genes. Nature Cell Biology. 2 142-147,
2000.
Some of their results are shown on the next slide.
ERK1/2 pathway
Figure.
Regenerating
soleus (a) without and (b)
activated RasV12-ERK1/2.
Light stains indicate the
expression of slow myosin
heavy chain I.
Figure: Murgia et al. (2000) activated with transgenic methods
RasV12, an upstream activator of the extracellular-signal regulated
kinase (ERK1/2) pathway in denervated, regenerating soleus muscle.
The muscle normally regenerates as a fast muscle that does not
express slow myosin heavy chain I, which is shown in figure a.
RasV12 activation, however, let to an increase in the expression of
myosin heavy chain I in many fibres which is shown on the right (light
grey and white fibres).
ERK1/2 pathway
Several researchers have shown that the ERK1/2 pathway is
activated by exercise in isolated and intact rat and human skeletal
muscle.
However, the signals that lead to an activation of ERK1/2 are
unknown. The following slide shows data from the study of Yu et al.
(2003), who reported an increased ERK1/2 phosphorylation in
response to exercise.
Exercise and ERK1/2 phosphorylation
Western blot with
phospho-specific
ERK1/2 antibody
Figure. The signal transduction protein ERK1/2 is more
phosphorylated in response to exercise in both untrained and
trained subjects. ERK1/2 is an example for the phosphorylation and
activation of a signal transduction protein by exercise.
Yu et al. (2003)
ERK1/2 pathway
What is the ERK1/2 pathway? The ERK1/2 pathway belongs to the
mitogen activated protein kinase (MAPK) signal transduction pathways
that sense signals such as mitogens and oxidative stress. MAPK
pathways are protein kinase cascades where one kinase
phosphorylates the next one downstream until the last kinase (such as
ERK1/2) usually enters the nucleus.
The major three MAPK pathways are ERK1/2, p38 and JNK.
Researchers at the University of Dundee, such as Sir Philip Cohen,
have contributed landmark papers to analyse these pathways. The
following slide shows a schematical overview over the ERK1/2 pathway.
ERK1/2 pathway
Exercise ?
signal or
stress
Muscle
fibre
Ras
Slow type I
genes
Raf
P
MEK1/2
P
P
P
ERK1/2
P
P
ERK1/2
?
Nucleus
The ERK1/2 pathway is a cascade of kinases that each phosphorylate
each other. Ras phosphorylates Raf which phosphorylates MEK1/2 which
phosphorylates ERK1/2. ERK1/2 phosphorylation leads to the import of
ERK1/2 into the nucleus. Inside the nucleus, ERK1/2 is believed to
activate transcription factors but the targets in muscle are unknown. This
leads to the expression of slow genes.
Summary
The three major human muscle fibre types are I (slow) IIa
(intermediate) and IIx (fast). Rodents have also a IIb isoform.
However, the fibre type complex is problematic because many
transition forms exist. Fibres are a mix of 1000nds of proteins.
Very high amounts of contractile activity (chronic electrical stimulation)
can achieve II-to-I fibre type transformations. Training studies suggest
that IIx fibres decrease and IIa fibres increase. Denervation studies
usually cause a I-to-II fibre type transformation.
Calcineurin and ERK1/2 are both activated in contracting muscle and
promote the expression of at least some “slow” muscle genes. It was
shown last week that the induction of the transcriptional co-factor PGC1 by AMPK can also promote the formation of slow muscle fibres.
To conclude, fibre type transformations are probably mediated by
several pathways including the calcineurin, ERK1/2 and AMPK-PGC-1
pathways. Remember, there are hundreds of slow proteins that need to
be up-regulated and equally hundreds of fast proteins that need to be
down-regulated. It ain’t simple!
The End