The human muscle proteome in aging

Cecilia Gelfi

Sarcopenia: the mystery of muscle loss

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

Fiber classification:type 1 slow, oxidative

type 2A fast, oxidative

type 2X fast, glycolitic

type 1-2A, mixed fiber

type 2A-2X, mixed fiber

Human muscle structure

maximum shortening velocity (Vo)

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500

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Vo (L/s)

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Most contractile and energetic parameters show widevariations 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

Functional parameters under debate

….………………...….MHC 2X…………………….…MHC 2A.……………….….….MHC 1

Pool EL Pool YA

Pool YAPool EL MHC 1MHC 2AMHC 2X

68%

31%

1%

48%

39%

13%

MHC Distribution

66%

21%

13%

48%

42%

10%

38%

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YA

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32%

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62%

38%

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60%

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63%

34%

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37%

0%

63%

5% 0%

95%

EL

EL-1 EL-2 EL-3

EL-4 EL-5 EL-6

YA-1 YA-2 YA-3

YA-4 YA-5 YA-6

MHC 1MHC 2AMHC 2X

MHC Distribution

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Aggiornamento del: 31-03-05

Mappa standard 3-10 NLcon divisione in classi funzionalidelle proteine identificate

MUSCOLO MUSCOLO VASTO LAT. UMANOVASTO LAT. UMANO

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Prot. metabolismo ossidativo(TCA, Fosforilazione ox., β−ox.acidi grassi)

Prot. contrattili-strutturali

Prot. metabolismo anaerobico(glicolisi, gluconeogenesi, high energy phosphate buff.)

Prot. di trasporto

Prot. stress ossidativo

Altre funzioni

Prot. biosintesi-degradazione

Legenda:

+ Prot. cell signalling

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Two Dimensional Difference In Gel Electrophoresis (2D-DIGE)

2D-Dige increasesqualitative and quantitative accuracy of 2-DE results

The sensitivity is 125 pg per spot

A pooled internal standard is included in all gels

The samples are minimal labelled

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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• 6 subjects moderately active , 70-75 yrs old• 6 subjects not specifically trained, 18-25 yrs old• Bed rest• 4 subjects ( 25-35 yrs old) before and after 55 days bed rest• FSHD (unknown genetic origin)• 9 patients • 9 normal subjects sex and aged matched• Dysferlin deficit (defect in sarcolemmal repair)• 6 patients • 6 normal subjects sex and aged matched

BEDREST

FSHD

DYSFERLINOPATHY

MHC and MHC and Fiber TypingFiber Typing

CONTROL

21-26 Kb27 Kb 10 Kb16-19 Kb

AGING

elderly yeung

68%

31%

1%

48%

39%

13%

IIIA

IIX IIX

IIA I

t

51%

35%

14%

57%

38%

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51%

35%

14%IIX IIXIIX II I

IIA IIA IIA

CONTROL

Av. Ratio FSHD/Control-5 -4 -3 -2 -1 0 1 2 3 4

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1

1111

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11

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SYNTHESIS/DEGRAD

Physiological sarcopenia vs FSHD

1 2 312345

Av. Ratio Aging VEL/VYAVYA/VEL

GLYCOLITICGLYCOLITIC

OXIDATIVEOXIDATIVE

CONTRACTILESCONTRACTILES

TRANSPORTTRANSPORT

OTHER

OXIDATIVE STRESSOXIDATIVE STRESS

1 2 312345

Av. Ratio Aging VEL/VYAVYA/VEL

GLYCOLITICGLYCOLITIC

OXIDATIVEOXIDATIVE

CONTRACTILESCONTRACTILES

TRANSPORTTRANSPORT

OTHER

OXIDATIVE STRESSOXIDATIVE STRESS

Av Ratio bed rest/control

-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2

Loss of muscle mass induced by aging and bed rest

1 2 312345

Av. Ratio Aging VEL/VYAVYA/VEL

OXIDATIVE

GLYCOLITICGLYCOLITIC

CONTRACTILESCONTRACTILES

TRANSPORTTRANSPORT

OTHERSOTHERS

1 2 312345

Av. Ratio Aging VEL/VYAVYA/VEL

OXIDATIVE

GLYCOLITICGLYCOLITIC

CONTRACTILESCONTRACTILES

TRANSPORTTRANSPORT

OTHERSOTHERS

Av. Ratio Disferlinopatie/Control

-3 -2 -1 0 1 2 3

SYNTHESIS/DEGRAD

Physiological sarcopenia vs muscle impairment induced by dysferlin deficency

1 2 312345Av. Ratio Aging V EL/VYAVYA/VEL

OXIDATIVEOXIDATIVE

GLYCOLYTICGLYCOLYTIC

CONTRACTILESCONTRACTILES

TRANSPORTTRANSPORT

OTHEROTHEROXYDATIVE STRESSOXYDATIVE STRESS

1 2 312345Av. Ratio Aging V EL/VYAVYA/VEL

OXIDATIVEOXIDATIVE

GLYCOLYTICGLYCOLYTIC

CONTRACTILESCONTRACTILES

TRANSPORTTRANSPORT

OTHEROTHEROXYDATIVE STRESSOXYDATIVE STRESS

Muscle Phospho-proteome

Myosin Light Chains

2f22f1 2f

2s2 2s1

2s

1f 1sb

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-25

-20

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Bed Rest Aging Dysferlinophaty FSHD

2f1 2f2 2s2f 2f1 2f2

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2s2

2f 2s1 2s2

2f

2f2

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 velocityand could be a marker of muscle function

AknowledgementsAknowledgements

Marilena RipamontiAgnese Viganò Sara De PalmaDaniele CapitanioManuela Moriggi

Robin WaitImperial College London

Paolo CerretelliUniversity of Milano

Joern RittwegerManchester University

Roberto BottinelliUniversity of Pavia

Enzo RicciCatholic University, Rome

Lucia MorandiCarlo Besta Neurological

Institute, Milano

Finantial supports:

Telethon , FIRB, CARIPLO, ASI

Proteomic Unit