skeletal muscle fatigue - aalborg universitet course aau2011... · skeletal muscle fatigue: role of...

1

Skeletal muscle fatigue:role of glycogen availability and subcellular localization within fibre types

Niels Ørtenblad, Ass. Prof.

Institute of Sports Science and Clinical BiomechanicsUniversity of Southern Denmark, Odense, Denmark.

Swedish Winter Sport Research Centre, Mid Sweden University.

Aalborg University,November 2011

contractile elements (myofibrils)

intracellular space

[Ca2+](1-2 mM)

sarcolemma

extracellular space

ATPNa+ K+

Na,K-ATPase 5-10%SR Ca-ATPase 30-40%

Contractile apparaust 50-60%

Skeletal muscle excitations-contractions coupling

Ca2+

3

6

[Ca2+](100 nM) ATP

ADP

[Ca2+](10 20 M)

SR Ca2+-ATPase


Ca2+

Ca2+

Ca2+

Ca2+

t-tu

bu

le

4

[Ca2+](1 µM)

5

Ca2+ buffers(calmodulin,parvalbumin)

DHPRVoltage sensitive

channels

2

t-tu

bu

le

Na+

1ADP

ATP

mitokondriaCa2+

(10-20 mM)

sarcoplasmic reticulum (SR)RyR

(Ca2+ release channels)

Ca2+

channels

Ca2+

2

ATP

Energy utilization must be meet by an equal energy production

ATP production ATP utilization

ADP + Pi

Liver glycogen Adipose tissue

Energy stores for ATP production

Fatty acidsGlucose O2 CO2

Lactate + H+

Oxidative Ph h l ti

Fat

Pyruvate Phosphorylation

PCr Creatine

ATP

ATP

ATP

Glycogen

3

The glycogen granule

Glycogen phosphorylasePhosphorylase kinase

Muscle fibre/cell

EM-pic 6300x

sarcolemmaInterGly

71%

nucleus

mitochondriaIntraGly

29%

I-band 25%

A-band 4%

TG

4

C

SSN

Subcellular localization of skeletal muscle glycogen

Intra Glycogen

IMF Glycogen

M

Arm muscle (m. triceps brachii)

77-84%

8 12%8 11% SS

Myo

Intra Glycogen

SR

T-systemIMF Glycogen

Z

Z: Z-line: M: M-band.Scale bare = 0.5 μm. Original x 40,000 magnification.

8-12%8-11%

Scale bar = 5 μm. Original x 5,000 magnification.

Nielsen, Holmberg, Schrøder, Saltin & Ørtenblad, J Physiol 2011

TEM image of sarcomer (x40,000 magnification)TEM image of sarcomer (x40,000 magnification)

IMF Glycogen

Intra Glycogen

SR

M

Z


Z: Z-line: M: M-band.Scale bare = 0.5 μm. Original x 40,000 magnification.

5

Cross sectional image of a skinned fibre

I-band

A-band

Glycogen and muscle function

6

Clear relationship between glycogen concentration in human skeletal muscle on endurance during exercise at 75% VO2max

126 min

189 min

Bergström et al. (1967) Acta Physiol Scand 71

59 min

Exhaustion following long-term exercise is associated with low muscle glycogen levels

H t l (1967) A t Ph i l SHermansen et al. (1967) Acta Physiol Scan

Hermansen et al. (1967) Acta Physiol. Scan.

The underlying mechanism for the role of glycogen on endurance has primarily been recognised as an impaired energy metabolism.

However, this idea has been challenged during the last decade.

7

Role of glycogen content on SR vesicle Ca2+ release rateand contractile properties i rat soleus muscle

2 5

3

3,5

4

se r

ate

min

-1)

*

SR vesicle Ca2+ release rates

3.2 ± 0.3

2.8 ± 0.22.6 ± 0.2

Resting muscle glycogen concentrations following 14 days of different diets

dw

-1 )

175

200 165 ± 20

0

,5

1

1,5

2

2,5

Ca

2+

rele

as

( µ

mol

.g

-1.

CHO FAT FAT, fastedMu

scle

gly

cog

en

( m

mo

l .kg

d

0

25

50

75

100

125

150

175

*

**

CHO FAT FAT, fasted

70% CHO 20% CHO 20% CHO

85 ± 4

50 ± 3

Values are means ± SE; n=7-8 in each group* Significantly lower than CHO (P<0.01)** Significantly lower than CHO and FAT (P<0.01)

10%fat20% prot

60%fat20% prot

60%fat20% protfasted 24h

Ørtenblad et al. unpublished


intracellular space

1

[Ca2+](1-2 mM)

sarcolemma

extracellular space

Ca2+

3

6

[Ca2+](100 nM) ATP

ADP

[Ca2+](10 20 M)

SR Ca2+-ATPase


Ca2+

Ca2+

Ca2+

Ca2+

t-tu

bu

le

4

[Ca2+](1 µM)

5



channels

2

t-tu

bu

le

Na+

1

Low Gly

÷

mitokondriaCa2+

(10-20 mM)



Ca2+

channels

Ca2+

8

Before race (Pre)Biopsy arm + leg

After race (Post)Biopsy arm + leg

4 hBiopsy arm + leg

22 hBiopsy arm + leg

~1 h raceclassic style

5 skiers optimal CHO5 skiers water (÷ CHO) All skiers optimal CHO

10 Norwegian elite skiers (VO2max 72 [ 67;79 ]), 700 training hour per year

Total muscle glycogen content in arm

M /

kg d

w)

500

600

1.5 h race Recovery

Not sign. dif. from Con

(84%) (88%)

Ske

leta

l mu

scle

gly

cog

en c

on

ten

t (m

M

200

300

400

31%29%

59%

0

100

Con 0hwaterCHO

4h

Water+CHOCHO

22h

Ørtenblad et al. J Physiol (2011)

9

SR vesicle Ca2+ release rate

ol)

Muscle glycogen content

w)

500

600

100

sic

le C

a2+

re

leas

e ra

te (

% c

on

tro

86%

84%

77%

scl

e g

lyc

og

en c

on

ten

t (m

M/k

g d

w

200

300

400

500

35%

59%

29%29

%90

Criticallevel

0Con 0h 4h 22h

waterCHO

Con 0h 4h 22h

Re

lati

ve S

R v

es

Con 0h 4h 22h

waterCHO

Con 0h 4h 22h

Sk

ele

tal m

us

0

100

80

120

ol)

Association between muscle glycogen contents andmuscle Ca2+ release rate

80

90

100

110

SR

ves

icle

Ca2

+ re

leas

e ra

te (

% c

on

tro

60

70

0 100 200 300 400 500 600 700

Skeletal muscle glycogen content (mmol . Kg-1 dw)

Rel

ativ

e S

Ørtenblad et al. J Physiol (2011)

10


intracellular space

1

[Ca2+](1-2 mM)

sarcolemma

extracellular space

Ca2+

3

6

[Ca2+](100 nM) ATP

ADP

[Ca2+](10 20 M)

SR Ca2+-ATPase


Ca2+

Ca2+

Ca2+

Ca2+

t-tu

bu

le

4

[Ca2+](1 µM)

5



channels

2

t-tu

bu

le

Na+

1

Low Gly

÷

mitokondriaCa2+

(10-20 mM)



Ca2+

channels

Ca2+

A

Mi

Representative TEM images of the pre and post 1 hr of exhaustive exercise

pre exercise (A, C)

ole

mm

al r

egio

n

B

post exercise (B, D)

C

Mi

Z

sub

sarc

o

D

Mi

M

egio

n

Mi

Z

M

All images originate from an arm type I fiber. Glycogen is visualized as black dots. Mi, mitochondria. Z, Z-line. M, M-band. The arrows indicate the sarcolemma. Scale bar = 0.5 μm.

MiZ

myo

fib

rill

ar

re

Nielsen et al. (2011) J Physiol 589:2871-2885

11

C

Glycogen content in three subcellular localizations of arm skeletal muscle (m. triceps brachii) before (Pre) and after (Post) approximately 1 hr of exhaustive exercise

Intra Glycogen

IMF Glycogen77-84%

8-12%8-11%SS glycogen

IMF Glycogen

Z

0,120

Sub‐sarcolemmal

0 040

0,045

Inter‐myofibrillar

0 0090,010 #

Intra‐myofibrillar

#: Type I higher than Type II

0,000

0,020

0,040

0,060

0,080

0,100

1 2 3 4 5

SS g

lyco

gen

(µm

3µ

m-2

)

Pre Post

*

*

Decreased to

28%

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

1 2 3 4 5

IMF

glyc

ogen

(µm

3µ

m-3

)

Pre Post

*

*

Decreased to

29%

0,0000,0010,0020,0030,0040,0050,0060,0070,0080,009

1 2 3 4 5

Intr

a gl

ycog

en(µ

m3

µm

-3)

Pre Post

* *

Decreased to

14%


Type 1

Type 2

Intra Glycogen

IMF Glycogen

SR

M

Z


e ra

te (

% o

f )100

110

120

130

Intra Glycogen(μm3 μm-3 intramyofibrillar space)

SR

Ca2

+re

leas

eP

re)

50

60

70

80

90

00

0 0,004 0,008 0,012

Ørtenblad, Nielsen, Saltin & Holmberg, J Physiol (2010)

12

Hjerne

Low glycogen level affects the l bilit t l C 2+ f

Muskel

Rygmarvmuscle ability to release Ca2+ from

The sarcoplasmatic reticulum→ muscle fatigue

Glycogen and muscle fatigue

What is the mechanism ?

13

The mechanically skinned fibre technique

The role of glycogen on muscle function has been further investigated in the skinned fibre model where ATP and PCr are kept high and constant.

Intact part

Cuff

‘Skinned’ part

Rolled back membrane

50 μm

14

2 mm

TEM image of one fiber (x3,000 magnification)

TEM image of one fiber (x3,000 magnification)

40-50 musclefibres

50 µm

Force transducer

muscle fiber

15

Schematic representation of mammalian fibre ultrastructure and of the mechanical skinning procedure

mitochondria

sealed t-tubule

Part ofintact fibre

Part ofskinned fibre

SR

sarcolemma

t-tu

bu

leCuff

50 μm

In the mammalian muscle cell the paired long mitochondria are transversely located at the I-band level (I) wrapped around the contractile apparatus and in contact with the SR but clearly separated from the t-tubules.

Z

AI I

Z

Ørtenblad and Stephenson, J Physiol 2003

5.0 m 5.0

t-tubules

Sealed t-tubules containing fluo-3

Launikonis & Stephenson, 2002

16

AP in Voltage

SR Ca2+ Force

Depolarisation by ion substitution

AP in T-system

Voltagesensor

activation

SR Ca2+

releaseForce

production

Electrical stimulation

2 s

0.1 mN

VERSATILITY OF THE MECHANICALLY SKINNED MUSCLE FIBRE PREPARATION

T-systemsensor

activationrelease production

sarcolemmaintracellular space

contractile elements (myofibrils)1

K+, Na+, Cl-

Na+

AP in T-system

Voltagesensor

activation

SR Ca2+

releaseForce

production

Direct activation of Ca2+-release channels

+

DHPRs(VoltageSensors)

Ca2+

3

6

[Ca2+]ATP

ADP

[Ca2+](10-20 mM)

sarcoplasmic reticulum (SR)

SR Ca2+-ATPase

Ca2+ releasechannels

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

t-tu

bu

le

4

5

2

t-tu

bu

le

Ca2+

Na+

Na+

Na+

Na+

Na+

CaffeineLow Mg2+

÷


intracellular space

The mechanically skinned fibre preparation

T-system reseals

ATP

Global ATP and PCr kept high and constant

Ca2+

3

6

[Ca2+](100 nM) ATP

ADP

[Ca2+](10 20 M)

SR Ca2+-ATPase


Ca2+

Ca2+

Ca2+

Ca2+

t-tu

bu

le

4

[Ca2+](1 µM)

5


channels

2

t-tu

bu

le

Na+

1

PCr

mitokondriaCa2+

(10-20 mM)



Ca2+

channels

Ca2+

Is muscle function now affected ?Is the regulation of SR Ca2+ by glycogen simply energy dependent ?

17

0,8%

My

F

F

F

Inter- and intramyofibrillar glycogen in fibres from feed and fasted rats

135791357

0,0%

0,4%

Inter

yofíb

rillar glyc

og

envo

lum

e percen

t

Intra

F F F

F

F

F

F

Subpopulations of glycogen in the investigated 19 fibre segments. The glycogen content is expressed as a volume percent of the myofibrillar space.Inter: Intermyofibrillar glycogen; Intra: Intramyofibrillar glycogen; F: fibre from a 24hr fasted rat.

113151719 Fibre #

Average myofibrillar glycogen volume percent was 0.46 ± 0.07% (fastet 0.33 ± 0.1% and feed 0.58 ± 0.0.1%).)

Nielsen, Rix, Schrøder & Ørtenblad, J Physiol 2009

Fibre with initial high IntraGly (0.20%)

Role of Intramyofibrillar glycogen on muscle fibre endurance

IMF Glycogen

M

Endurance in ”Mechanically skinned” fibre where ATP and PCr can be kept highand constant, while stimulated just like in the intact fibre

Fibre with initial low IntraGly (0.02%)

Intra Glycogen

IMF Glycogen

M

Z

The run down protocol (0.8s every 3s) was stopped at the time when the tetanic force was reduced to 50% of the maximal tetanic force (tF50%).

Nielsen, Rix, Schrøder & Ørtenblad, J Physiol (2009)

18

C

Glycogen content in three subcellular localizations of arm skeletal muscle (m. triceps brachii) before (Pre) and after (Post) approximately 1 hr of exhaustive exercise

Intra Glycogen

IMF Glycogen77-84%

8-12%8-11%SS glycogen

IMF Glycogen

Z

0,120

Sub‐sarcolemmal

0 040

0,045

Inter‐myofibrillar

0 0090,010 #

Intra‐myofibrillar

0,000

0,020

0,040

0,060

0,080

0,100

1 2 3 4 5

SS g

lyco

gen

(µm

3µ

m-2

)

Pre Post

*

*

Decreased to

28%

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

1 2 3 4 5

IMF

glyc

ogen

(µm

3µ

m-3

)

Pre Post

*

*

Decreased to

29%

0,0000,0010,0020,0030,0040,0050,0060,0070,0080,009

1 2 3 4 5

Intr

a gl

ycog

en(µ

m3

µm

-3)

Pre Post

* *

Decreased to

14%


Type 1

Type 2

0.8

0.9

1.0

m f

orc

e)

Role of total glycogen on contractile properties

0.2

0.3

0.4

0.5

0.6

0.7

Fo

rce

(rel

ativ

e to

max

imu

m

TotalGly 0.60% TotalGly 0.049%

First tetanic contraction in the run down protocol (2 ms pulses at 10 Hz, 75 V cm-1 for 800 ms) in mechanically skinned fibres with initial high and low glycogen.TotalGly was 0.00601 and 0.00049 µm3 glycogen pr. myofibrillar space, high and low glycogen respectively.

0.0

0.1

1 sec

19

Triad

Inter-myofibrillar glycogen between the myofibrils is important for the relaxation rate

TEM image illustrating the definition of subpopulations of glycogen content in the myofibrillar space (scale bar = 2 µm, original x20,000 magnification

Intra-myofibrillar glycogen in the myofibrils is important for endurance and SR Ca2+ release

ATP

Low glycogen levels → SR Ca2+ release↓

Glycogen affects energy turn-over by modulating SR Ca2+ -release and thereby restrains the rate of ATP-utilisation

ATP

ATP production ATP utilization

ADP + Pi

+

20

How does glycogen control the delicate balance between energy utilization and energy production?

Glycogen can control the energy utilization by inhibiting the contraction

Glycogen thereby serves as both energy store and control site of energy utilization

Conclusions

It is indicated that glycogen has a structural role inIt is indicated that glycogen has a structural role in maintaining normal skeletal muscle function. Thus, glycogen might be more than just an energy reserve.

Furthermore, the results suggest different roles of the subpopulations of glycogen in the working muscle, with special emphasis on Intra-myofibrilar glycogen.

21

Thanks to collaboratorsJoachim Nielsen, post doc, University of Southern DenmarkHC Holmberg, Swedish Winter Sport Research Centre, Mid Sweden UniversitetetProf Henrik Daa Scrøder Odense University HospitalProf. Henrik Daa Scrøder, Odense University HospitalProf. Bengt Saltin, Copenhagen Muscle Research Centre, KU

Intracellular lipid contents and localization in T2Dlocalization in T2D

Effects of training

22

Effect of 10 weeks of aerobic training and type 2 diabetesCollaboration with Martin Mogensen, University of Copenhagen

Ergometer cycling 3‐4 times per week

T2D patients (♂, 53y, 108kg, n = 12) versus

Control subjects (♂, 53y, 110kg, n = 12)

Biopsies was obtained from m. vastus lateralisbefore and after 10 weeks of training

23

Aspects of muscle subcellular compartmentalization in type 2 diabetes

Aspects of muscle subcellular compartmentalization in type 2 diabetes

r2 = 0.62P = 0.002

skeletal muscle fatigue - aalborg universitet course aau2011... · skeletal muscle fatigue: role of...

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