Spieren: myogenese, plasticiteit en regeneratie

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Spieren myogenese, plasticiteit en regeneratie

• Frank Wuytack
• Laboratorium voor Fysiologie
• Faculteit Geneeskunde
• K.U.LEUVEN

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Skeletal muscle cells ( muscle fibers) are
large postmitotic syncytia resulting from fusion
of myoblasts, which show a high degree of
plasticity and a robust capacity for regeneration
Muscle fibers are not fixed entities but
dynamic structures with an extraordinary adaptive
potential
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Different muscle phenotypes Muscles are composed
of different fibers I? I? IIA
IIX IID IIB
slow fast
fast oxidative oxidative/
glycolytic
glycolytic
hybrid fibers
tension cost
Phosphorylation potential ATP/ADPfree
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The most useful scheme to delineate fiber types
is based om myosin heavy chains (11 isoforms in
mammalian muscle) As body mass increases
expression of slow MHC increases MHC1? rat is
faster than MHC1? human MLC are also
different Fiber type transitions
innervation exercise training loading/unl
oading hormones aging
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Part 1 Myogenesis
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mesoderm
somatic mesoderm prechordial plate
lateral dorsal
HEAD MUSCLES mesoderm
mesoderm
HEART CELLS early somites
dermamyotome sclerotome
dermatome myotome SKELETAL
ELEMENTS
DERMIS SKELETAL MUSCLE
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Differentiation of skeletal muscle cells
Precursor cells in somites Balls of
epithelial-like cells paraxial mesoderm form in
the mouse at day 8 post coitum
to limb
Precursor mesodermal cells ? myoblasts ?
myocyte ? myotubes ? myofibers
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myogenesis
Muscle regulatory factors
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Differentiation regulated expression of a
distinct set of cell-type specific genes
Regulation of muscle differentiation can be
reduced to the question
How is muscle-specific gene expression regulated?
MUSCLE PROGRAM set of muscle-specific
proteins and protein isoforms
Contractile proteins MHC / MLC actin trop
onin / tropomyosin Metabolic enzymes creatine
phosphokinase LDH EC coupling
apparatus DHPR RYR SERCA
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Microarray chip
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Muscle regulatory factors/genes (MRFs)
Fibroblasts treated with 5-azacytidine
differentiate into myotubes ? WHY?
Answer blocks DNA methylation ? many genes
become demethylated ? become active ? WHICH OF
THESE GENES DRIVES MYO-
GENIC DETERMINA- TION?
Answer myoD (myogenic determination gene D)
myoD MASTER GENE
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myoD is a master gene which can drive myogenic
differentiation
What is the structure of the protein encoded by
MyoD? Four related genes are found in the
vertebrate genome ? why four?
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MRFs are basic- helix-loop-helix DNA-binding
proteins
basic helix-loop-helix
transactivation
transactivation
NH2
COOH
binds to CANNTG protein-protein
interactions (E box in DNA) forms (homo)
heterodimers of promoter with ubiquitously
expressed (once every 256nt) E2-A or E2-B
proteins other bHLH also in neuronal and
hematopoietic cells but MRFs strictly limited to
skeletal muscle
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Basic DNA-binding
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Muscle enhancer-binding factors (MEFs)
MADS MEF
NH2
COOH
transactivating
MRF interacting domain ? MEF cannot induce
myogenic conversion only enhances MRFs to do
so MEFs are also found in non-muscle cells DNA
binding / dimerization ? MEF forms
homodimers MEF necessary for
differentiation ? for determination
E box
MRF
E
MEF
MEF
muscle-specific gene
MEF box
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• ? Direct MRF effect some muscle specific genes
• (muscle creatine kinase, acetylcholine receptor,
  MyoD)
• Indirect MRF effect gene expression is
  controlled by other
• genes that have MRF binding sites in their
  promoter (MEF)

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Invertebrates (Caenorhabditis elegans,
Drosophila, sea urchins) have only one MRF
gene Vertebrates 4 genes Myf 5 MyoD
myogenin MRF-4
Establish myoblasts Chromatin rearrangement
Terminal differentiation
8 9 10 11 12 13 14
15 16 17 days pc
Myf-5 MyoD Myogenin MRF-4 MEF-2
In early somites
Repressed until birth
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Four cellular basic helix loop helix factors
regulate the muscle differentiation program in
vertebrates
Differentiation is the regulated expression of a
distinct set of cell-type specific genes
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Gene targeting (KO experiments)
Myf-5 normal muscle delay of myotome
formation, aberrant migration
of muscle cell precursors, MyoD ?compensates
MyoD normal muscle abnormal ribs
(lateral sclerotome does not develop)
Myf-5 ? compensates Combined
MyoD/Myf-5 KO no myoblasts,
no differentiated muscles Myogenin
differentiation defects in myoblasts
muscle-forming regions contain many
mononucleated cells ( unfused
myoblasts?) MRF-4 only mild phenotype,
myogenin overexpression KI experiments Knock
In experiments show that myogenin cannot
compensate for Myf-5 or Myo D
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Inhibitors of differentiation (Ids)
growth differentiation
(proliferation) General property of neurons,
adipocytes, blood cells, keratinocytes
HOW? Myogenic regulatory factors are extremely
sensitive to growth factors FGF (fibroblast
growth factor) activates C-kinase ?
phosphorylates DNA domain MRFs stimulates
production of Ids Ids are related to MRFs but
lack basic DNA-binding domain
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Control of myogenic bHLH gene expression by
extracellular proteins
Morphogens are molecules that specify different
cell fates in a concentration-dependent
manner Wnt, sonic hedgehog and bone
morphogenetic proteins can induce the expression
of bHLH factors
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Signaling from the notochord controls patterning
of the embryonic central nervous system
Gene sonic hedgehog produces inducer
protein Mutations in this gene in mice and humans
cause loss of midline structures cyclops (single
eye and nostril)
induction
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The Hedgehog family
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Part 2 Plasticity
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Tijdens postnatale groei ? seriële toevoeging van
nieuwe sarcomeren aan spieruiteinden o.i.v.
groeifactoren In volgroeide spieren ? toevoegen
wegnemen van sarcomeren Groei ? toename van
proteïnen ? toename van RNA (ribosomaal) ?
krachtontwikkeling ? Oorzaak van groei ?
lengteverandering ? ? electrische
activiteit ? ? neurotrofe invloeden ?
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Myostatin (GDF-8) adapts muscle size to body size
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Myostatin a negative regulator of muscle growth
inhibits myoblast proliferation by increasing the
p21 levels
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The cell cycle can be halted in G1 by Cdk
inhibitor proteins
How p53 arrests the cell cycle in G1
Control for DNA damage before replication
brakes at checkpoints tumor suppressors
recessive
myostatin
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Gebruik van spieren leidt tot Hypertrofie
(regeling grotendeels t.h.v. translatie,
gebeurt relatief snel) Verandering
spierfenotype MHC-I (trage vorm) ? MHC-II
(snelle vorm) ? (regeling t.h.v. transcriptie
gebeurt trager) training voor sprint gebeurt
best in korte intensieve sessies om opregeling
trage MHC te voorkomen
DNA mRNA matuur proteïne
proteolytische fragmenten
transcriptie translatie
proteolyse (calpaine
proteasomen)
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Muscle protein synthesis per day
0
5
10
Rabbit anterior tibialis muscle (fast)
control
unstretched
control
stretched
control
stretched stimulated
Modified from Goldspink G. (1999) J. Anat. 194
323 -334
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Spierfenotypes Spieren zijn
opgebouwd uit verschillende soorten vezels die
o.m. verschillende myosine isovormen exprimeren
? traag oxidatieve vezels type I
economisch vermoeidheidsresistent ?
snelle vezels (type II) ?IIA redelijk
snel tonische activiteit ?IIX IID snelle
contractie glycolytisch vlug vermoeid ?2B
zeer snel (kleinere dieren)
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human
Adult skeletal genes Speed of contraction
Chrom 17 emb.MyHC ?neo.MyHC ? MyHCIIA ? MyHCIIX
?( MyHCIIB)
Thyroid hormone
Chrom 14
? MyHC ?MyHC
Cardiac genes
Repetitive use and overload
Chrom 16 two smooth muscle MyHC
From Goldspink G. (1999) J. Anat. 194 323 -334
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Muscle fiber composition differs among individuals
Fast type IIb superfast
Quadriceps femoris
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Wat bepaalt het spierfenotype ?

• ? default vorm is snel (IIx)
• ? activiteit / ?belasting/ chronische lage freq.
  stim. ? traag
• EMG studies tonen aan dat bij staan of lopen de
  soleus
• 90 van de tijd actief is
• Vele andere spieren slechts 5 ? het is
  fysiologisch
• zinvol dat voortdurend actieve spieren
  economisch
• werken
• Inactiviteit/ ?belasting/ fasische hoge freq.
  Stim. ? snel
• (minder efficiënt)
• Thyroid hormoon ? snel

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IIa
Overload ? transformation to slow Nerve
stimulation ? transformation to slow
(cardiomyoplasty) Corticosteroids (triamcinolon)
? selective atrophy of fast Clenbuterol (??2
agonist) ? selective hypertrophy of fast
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young muscle more fast fibers
Elderly muscle motor nerves die Note type
grouping more slow fibers
smaller irregular fibers
selective decrease in fast ? motor neurons
sarcopenie
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Clenbuterol (?2 agonist) Induces hypertrophy of
fast contracting fibers and transformation of
slow to fast fibers IIa (mechanism undefined) Mor
e muscle (illegal veterinary medicinal use
only approved for bronchodilatation in horses and
calves in EC reports of residue-induced food
poisoning in France, Spain and Italy) but has
little therapeutic effect on functional
muscle properties and has deleterious effects on
cardiac muscle structure and performance
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Cardiomyoplasty (1985) (aortomyoplasty)
Skeletal muscle can be electrically conditioned
to become fatigue resistant adaptive fiber
transformation (IIx ? IIa fibers) Avoid
transformation to type I (loss of power and
speed)
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What determines muscle protein isoform expression?
Expression of some genes is not dependent on
innervation (SERCA) Expression of genes
dependent on innervation trophic factors
released by nerve endings local effect
(positioning of Ach receptors) excitation-transc
ription coupling (MHCI) (also in denervated
electrically-stimulated muscle depends on
stimulation pattern)
MHCI expression is mediated by myogenin
? calcineurin (via NF-AT blocked by
cyclosporin, FK506) Ras
MAPK (ERK)
(Murgia et al., Nature Cell Biol.(
2000) 2, 142-147)
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Growth hormone induces muscle growth
2.32 m
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De GH/IGF-1 as voornaamste regelaar van
weefselmassa
hypothalamus
GHRH
hypofyse
IGF-1 variant MGF (mechano growth factor)
GH
lever
autocrien
IGF-1
IGF-1
spier
andere weefsels
IGF-1
endocrien
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IGF-I (endocrien lever) IGF-I/MGF
(auto/paracrien spier) niet geglycosyleerd ?
labieler? specifiek IGF-I bindend proteïne
(BP5) voorkomen endocriene (insuline- achtige
) effecten intermittente training
werkt spier rekken induceert MGF
productie niet in mdx muizen (Duchenne
dystrofie model)
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Tijdens intensieve arbeid is het grootste
deel van het circulerend IGF-I afkomstig van de
spieren en het meeste IGF-I wordt gebruikt door
de spieren Met stijgende leeftijd wordt de
bijdrage van in spieren geproduceerd IGF-I
belangrijker BLIJF AKTIEF Spierdystrofie is een
gevolg van dalende locale productie van IGF-I
(MGF) Duchenne dystrofie autosomaal
recessieve dystrofie door merosine
mutatie
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(No Transcript)
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MGF cDNA injectie in spier induceert
spiergroei 25 ø ? in 2 weken tijd Activeert
satellietcellen
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IGF-1
proliferating
Committed cells
Satellite cells occur at day 17 pc Are
responsible for post natal growth Quiescent cells
do not express MRFs Are activated upon injury or
stress The total number remains constant
after cycles of degeneration/regeneration,
but the proliferative potential decreases
Sabourin LA, Rudnicki MA Clin. Genet. (2000) 57,
16-25
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Embryonic stem cells
Fertilized egg (zygote) totipotent forms ?
embryonal cells ?
extraembryonal cells Embryonic stem
cells (ES) pluripotent form only embryonal


cells Adult stem cells skeletal muscle
(somatic) haematopoietic stem cells
many unexpected others
liver
adult olfactory bulb, hippocampus
form neurons,
oligodendrocytes,
astrocytes ES cells can be
easily genetically manipulated Human embryonic
stem cells can be cultured since end 1998
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ES cells can form different tissues adult stem
cells show remarkably versatile stem cell
transitions
Stromal bone marrow cells ? muscle (or other
mesenchymal cells) ? glia when injected in
brain Hematopoietic stem cells can form liver
cells
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Anabolic steroids Adreno-Spermin is dynamogenic
(1924)
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Anabolic steroids
Synthetic compounds based on the natural male
sex hormone testosterone with maximized anabolic
effect minimized androgenic effect
Oral steroids (stanozolol Ben Johnson,
dianabol) doses up to 200mg/day short-lived
liver toxicity! Injectable
(intramuscular) steroids (nandrolone) 200 mg/
2 to 3 weeks
Cycling (6 to 12 week cycles) Stacking (combining
several steroids) Pyramiding (gradually
increasing, then tapering off)
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Ectopic expression of proteins (somatic gene
therapy)

• MHC type IIb expression ? making a faster muscle?
• Utrophin replacing dystrophin
• Rescue of cardiac ? actin deficient mice by
• smooth muscle ? actin (but hypodynamic
  hypertrophied hearts)
• SERCA1 expression in failing heart
• Induction by disease therapy
• adenovirusbased phospholamban antisense
  expression
• controlled by atrial natriuretic factor (ANF)

Induced by adenovirus infection
(transgenes overexpressing smooth muscle ? actin
in heart show reduced contractility)
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References Florini J.R., Ewton D.Z. Coolican
S. A. (1996) Growth hormone and the insulin-like
growth factor system in myogenesis. Endocrine
Reviews 17 481-517 Goldspink G . (1999) Changes
in muscle mass and phenotype and the expression
of autocrine and systemic growth factors by
muscle in response to stretch and overload. J.
Anat. 194 323-334 Pette D. Staron R.S. (2001)
Transitions of muscle fiber phenotypic profiles.
Histochem. Cell Biol. 115 359-372 Sonksen P.H.
(2001) Hormones and sport. Insulin, growth
hormone and sport. J. Endocrinol. 170 13-25