The human muscle proteome in
aging
Cecilia Gelfi
Sarcopenia: the mystery of muscle
loss
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It account for
a decrease of muscle mass
a decrease in velocity
a decrease in Po/CSA
a decrease in strength
Factors involved:
physical inactivity
motor unit remodeling
decrease of hormone levels
decrease in protein synthesis
Human muscle structure
Fiber classification:
type 1 slow, oxidative
type 2A fast, oxidative
type 2X fast, glycolitic
type 1-2A, mixed fiber
type 2A-2X, mixed fiber
Functional parameters under debate
‰Most contractile and energetic parameters show wide
variations among fibers of the same muscle
‰differences in velocity among different fiber types are actually related to myosin
heavy chain isoforms but the variability in velocity within the same type remain
unknown
maximum shortening velocity (Vo)
40
30
20
10
Vo (L/s)
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
Number of fibers
50
MHC Distribution
Pool EL
Pool YA
….………………...….MHC 2X
…………………….…MHC 2A
.……………….….….MHC 1
1%
13%
31%
48%
39%
68%
Pool EL
MHC 1
MHC 2A
MHC 2X
Pool YA
MHC Distribution
13%
YA-1
10%
YA-2
YA-3
18%
38%
21%
48%
66%
42%
44%
17%
YA-4
12%
YA-5
41%
10%
YA-6
45%
46%
44%
43%
42%
YA
EL-1
EL-2
0%
EL-3
0%
4%
32%
38%
36%
60%
62%
68%
EL-4
EL-5
3%
0%
34%
5%
EL-6
0%
37%
63%
63%
95%
EL
MHC 1
MHC 2A
MHC 2X
MUSCOLO
VASTO LAT. UMANO
Mappa standard 3-10 NL
con divisione in classi funzionali
delle proteine identificate
Aggiornamento del: 31-03-05
Legenda:
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Prot. contrattili-strutturali
Prot. metabolismo ossidativo
(TCA, Fosforilazione ox., β−ox.acidi grassi)
Prot. metabolismo anaerobico
(glicolisi, gluconeogenesi, high energy phosphate buff.)
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Prot. di trasporto
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Prot. stress ossidativo
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Prot. biosintesi-degradazione
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Prot. cell signalling
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Altre funzioni
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*** FAI CLICK SULLE CROCETTE PER COLLEGARTI A SWISSPROT ***
Two Dimensional Difference In Gel Electrophoresis
(2D-DIGE)
The samples are minimal labelled
2D-Dige increases
qualitative and quantitative
accuracy of 2-DE results
The sensitivity is
125 pg per spot
A pooled internal standard is
included in all gels
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Does the functional changes induced by different
conditions such as aging and bed rest reflect the
same variability at the muscle proteomic level?
Does the functional impairment induced by
dystrophies with different genetic origin reflect
the same changes in muscle proteome?
Which are, if any, the specific markers?
SDS and 2D-Dige for sarcopenia and
muscle impairment assessment
• Aging
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6 subjects moderately active , 70-75 yrs old
6 subjects not specifically trained, 18-25 yrs old
• Bed rest
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4 subjects ( 25-35 yrs old) before and after 55 days bed rest
• FSHD (unknown genetic origin)
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9 patients
9 normal subjects sex and aged matched
• Dysferlin deficit (defect in sarcolemmal repair)
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6 patients
6 normal subjects sex and aged matched
MHC and Fiber Typing
1%
AGING
IIX
elderly
31%
IIX
13%
yeung
IIA I
IIA
I
48%
39%
68%
t
CONTROL
IIX
14%
BEDREST
IIX
I
14%
DYSFERLINOPATHY
I
IIX
5%
51%
I
38%
51%
57%
35%
35%
IIA
IIA
IIA
FSHD
CONTROL
27 Kb
21-26 Kb
16-19 Kb
10 Kb
Physiological sarcopenia vs FSHD
Av. Ratio FSHD/Control
Av. Ratio Aging
V YA /V EL
5
4
3
2
1
1
-5
V EL /V YA
2
OXIDATIVE
-4
-3
-2
-1
0
1
2
3
4
3
1
1
1
1
1
1
GLYCOLITIC
1
1
CONTRACTILES
1
1
TRANSPORT
1
1
1
OTHER
SYNTHESIS/DEGRAD
OXIDATIVE STRESS
n
Loss of muscle mass induced by aging and bed rest
Av. Ratio Aging
V YA /V EL
5
4
3
2
1
1
V EL /V YA
2
Av Ratio bed rest/control
3
-2.5
OXIDATIVE
GLYCOLITIC
CONTRACTILES
TRANSPORT
OTHERS
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
Physiological sarcopenia vs muscle impairment
induced by dysferlin deficency
5
V YA /V EL
4
3
Av. Ratio Aging
1
2
1
2
V EL /V YA
Av. Ratio Disferlinopatie/Control
3
-3
-2
-1
OXIDATIVE
SYNTHESIS/DEGRAD
GLYCOLYTIC
CONTRACTILES
TRANSPORT
OTHER
OXYDATIVE S TRESS
0
1
2
3
Muscle Phospho-proteome
Myosin Light Chains
1sb
1f
2s2
2s1
2s
2f2
Bed Rest
2f1
2f
Aging
Dysferlinophaty
FSHD
10
2f2
5
2f1 2f2
0
-5
2s
2f 2f1 2f2
2s
2f
2s1
2s2
2s2
2f
-10
-15
-20
-25
-30
-35
Conclusions
• 2D-DIGE approach enables the simultaneous assessment
of contractile proteins ,their isoforms, a large number of
metabolic and signalling markers. The overall evaluation
allows to characterize muscle tissue and it represents an
essential tool in muscle research.
• The phosphorylation signal of regulatory MLC’s
combined with MHC evalution reveal a function
compatible with the modulation of the contraction velocity
and could be a marker of muscle function
Aknowledgements
Proteomic Unit
‰
Marilena Ripamonti
‰ Agnese Viganò
‰ Sara De Palma
‰ Daniele Capitanio
‰ Manuela Moriggi
Finantial supports:
Telethon , FIRB, CARIPLO, ASI
‰
Robin Wait
Imperial College London
‰ Paolo Cerretelli
University of Milano
‰ Joern Rittweger
Manchester University
‰ Roberto Bottinelli
University of Pavia
‰ Enzo Ricci
Catholic University, Rome
‰ Lucia Morandi
Carlo Besta Neurological
Institute, Milano