Transcript Folie 1 - OROBOROS INSTRUMENTS

MiPschool 2008
Schröcken, July 2008
http://www.mitophysiology.org/index.php?id=mip-textbook
Mitochondrial
Respiratory Physiology.
Mitochondrial respiratory control:
Electron transport system, oxidative
phosphorylation and leak –
ETS, OXPHOS and LEAK.
Erich Gnaiger
Medical University
[email protected] Innsbruck, Austria
Excess Capacity
and Biochemical Threshold
H+
c
I
II
S
Biochemical threshold:
Cellular function is
buffered against a
specific enzymatic
defect.
Pathway flux
NADH
Excess capacity:
Insurance against a
specific enzymatic
injury.
H+
H+
Q
bc1
aa3
F1
F
O2
H+
H+
Excess capacity
2
1
Threshold
0
0.0
0.5
1.0
Enzymatic defect
Excess Capacity
and Biochemical Threshold
H+
c
I
NADH
II
S
Q
bc1
aa3
F1
F
Different excess capacities
imply tissue-specific
(in)sensitivity to enzymatic
defects in:
•
•
•
•
H+
H+
O2
H+
genetic mitochondrial disorders
aging
ischemia-reperfusion injury
degenerative diseases
H+
Cytochrome c Oxidase
H+
Antimycin A
c
aa3
KCN Titration
TMPD
Ascorbate
KCN
O2
F1
ADP
ATP
H+
Rel. inhibition of COX
1.00
Isolated step in intact
isolated mitochondria:
0.75
TMPD+Ascorbate
0.50
Cyanide titration
0.25
0.00
0
10
20
KCN concentration [µM]
Electron Transport Chain
and Cytochrome c Oxidase
H+
c
I
Q
bc1
aa3
F1
DH
Excess capacity
O2
NADH
Glutamate+Malate
1.00
ADP ATP
H
H+
+
2.5
Flux/Complex I
Rel. inhibition of COX
H+
H+
0.75
0.50
0.25
TMPD+Asc
0.00
0
10
20
KCN concentration [µM]
Excess capacity
2.0
1.5
1.0
0.5
Glu+Mal
0.0
0.00
0.25
0.50
0.75
1.00
Rel. inhibition of COX
0.5 mM TMPD + 2 mM ascorbate: 2-fold relative COX capacity
Electron Transport
System
H+
I
NADH
H+
H+
II
S
Q
F
III
bc1
c
IV
aa3
O2
F1
ADP ATP
H+ H+
Which metabolic state
represents electron
transport capacity?
A. Definition of ETS capacity.
B. Measurement in mitochondria and
permeabilized cells.
C. Measurement in intact cells.
Conventional Protocol
Derived from Bioenergetics
H+
c
I
Electron Transport Chain
H+
H+
Q
bc1
aa3
F1
DH
O2
ADP ATP
H
H+
+
Bioenergetic paradigm (1):
Respiratory capacity in State 3,
feeding electrons specifically into
complex I
Conventional Protocol
Derived from Bioenergetics
Electron Transport Chain
H+
H+
c
II
Q
bc1
aa3
F1
O2
Bioenergetic paradigm (1):
Respiratory capacity in State 3,
feeding electrons
specifically into
complex I, or
complex II
ADP ATP
H
H+
+
Conventional Protocol
Derived from Bioenergetics
Electron Transport Chain
H+
H+
c
II
Q
bc1
aa3
F1
O2
ADP ATP
H
H+
+
Bioenergetic paradigm (1):
Respiratory capacity in State 3,
feeding electrons specifically into
complex I, or
complex II (rotenone+succinate)
Then we are surprised to find ...
Intact versus
Permeabilized Cells
H+
c
I
Uncoupled
Coupled
Respiration / GMADP
Intact
DH
Permeabilized
H+ FCCP
H+
NADH
GM
II
Q
Succinate
bc1
aa3
F1
O2
ADP ATP
H+
H+
3
In permeabilized cells,
State 3 respiration
(Glutamate+Malate) is
short of representing
respiratory capacity of
intact uncoupled cells.
2
1
Fibroblasts NIH3T3
0
ROUTINE uncoupl. GMADP
Cell
State 3
Contoversy
on Isolated Mitochondria
H+
H+
H+
c
I
NADH
II
S
Q
bc1
aa3
F1
F
O2
COX excess capacity is high in isolated
mitochondria, with corresponding phenotypic
threshold.
•
•
•
•
H+
H+
Letellier et al (1994) Biochem. J. 302: 171.
Gnaiger et al (1998) BBA 1365: 249
Rossignol et al (2003) Biochem. J. 370: 751.
Antunes et al (2004) PNAS 101: 16774.
But low COX excess in intact cells „raises the
critical issue of how accurately the data
obtained with isolated mitochondria reflect the
in vivo situation“.
• Villani, Attardi (1997) PNAS 94: 1166.
Controversy
Living cells vs isolated mitochondria
Bioenergetic paradigm (2) of
substrate/uncoupler combinations
which yield maximum flux in:
• Intact cells:
Villani and Attardi (1997) PNAS
• Permeabilized muscle fibers:
Kunz et al (2000) JBC
• Isolated mitochondria:
Rasmussen et al (2001) AJP
Gold standard to
assess maximum
aerobic capacity in
cultured cells:
→Uncoupled flux
• Villani, Attardi (1997) PNAS 94: 1166
But intact cells do not
have uncoupled
mitochondria !
Respiration [pmol·s-1·10-6]
Oxidative Phosphorylation
in Top Gear
360
FCCP
Oligomycin
Routine
270
180
Ama
90
0
Rot
0
20
40
60
Time [min]
80
Oxidative Phosphorylation
in Top Gear – Mitochondrial Physiology
Gold standard to
assess maximum
aerobic capacity in
humans:
→VO2 max
Electron Transport
Coupled to ATP
Synthesis
OXPHOS and
Respiratory Capacity
H+
H+
H+
c
I
NADH
II
S
Q
bc1
aa3
F1
F
O2
H+
Oxidative Phosphorylation:
Coupling
O2
ATP
O2
Δp
H+
ATP
+
H
+
H
Mitochondrial Pathways
Convergent Redox and ET System
Glycolysis
Gp
GpDH
OXPHOS
PDH
H+
MDH
IDH
NADH
aa3
bc1
4:1
F1
II
ODH
O2
Succinate
II
SDH
c
I
Q
GDH
H+
H+
β-Oxidation
Convergent
ADP
H+
H+
ETF
Convergent
Linear
Coupled
p. 24
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Question 1
H+
I
ETS
NADH
H+
H+
II
S
Q
F
III
bc1
c
IV
aa3
O2
F1
ADP ATP
H+ H+
How do we measure
mitochondrial
electron transport capacity?
A. Mitochondria
B. Intact cells
MitoPathways
Succinate + Rotenone
H+
H+
c
I
II
S
P2i
Q
bc1
aa3
F1
F
O2
H+
H+
Oxaloacetate2NADH
Malate2Fumarate2II
FADH2
Succinate2-
P2i
Succinate2www.oroboros.at/index.php?id=mipnet-publications
MitoPathways
Pyruvate+Malate+Succinate, PMS
H+
PyruvateH+
Malate2-
c
I
PyruvateNADH
NADH
H+
Acetyl-CoA
Oxaloacetate2Pi2-
Fumarate2-
II
S
Q
aa3
bc1
F1
F
O2
H+
H+
H+
Citrate3-
NADH
Malate2-
H+
H+
HCO3-
NADH
Malate2-
2-Oxoglutarate2NADH
FADH2
Succinate2-
CO2
Malate2-
Pi2-
Succinate2-
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MitoPathways
Pyruvate+Malate, PM
PyruvateH+
c
Oxaloacetate2P2i
Q
I
bc1
Fumarate2-
O2
P2i
H+
Malate2HCO3
NADH
2-Oxoglutarate2-
NADH
FADH2
H+
H+
Citrate3-
NADH
Malate2-
aa3
F1
PyruvateNADH
H+
NADH
Acetyl-CoA
Malate2-
H+
H+
H+
Succinate2-
CO2
Malate2-
Complex II is
not active in
respiration on
pyruvate +
malate.
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MitoPathways
Glutamate+Malate+Succinate, GMS
GlutamateH+
Malate2GlutamateH+
Oxaloacetate2Pi2-
Aspartate-
NADH
Malate2-
2-Oxoglutarate2NADH
Fumarate2NADH
FADH2
Succinate2-
NH4+
Malate2-
CO2
GlutamateH+
Pi2-
GlutamateH+
Succinate2www.oroboros.at/index.php?id=mipnet-publications
High-Resolution Respirometry
in Permeabilized Cells
CI Substrates
Cytochrome c test: Intact
mitochondrial outer membrane
+ADP +c
+uncoupler
160
200
120
150
GM+Dig
250
O2 Concentration
200
80
100
ADP
40
c
F
F
50
0
0
0:20
0:30
0:40
0:50
Time [h:min]
endogen.
CI
CI
CI
ROUTINE
LEAK
OXPHOS
ETS
1:00
1:10
O2 Flow per cells
Permeab. GMN
High-Resolution Respirometry
in Permeabilized Cells
PC
GMN
+D
+c
+u
160
200
Rot
200
+S +Rot +Ama
250
GM+Dig
120
80
F
S
D
S1
40
c
F
150
Ama
O2 Concentration
CeR
F
100
50
0
0
0:20
0:30
0:40
0:50
1:00
1:10
Time [h:min]
endogen.
CI
CI
ROUTINE
LEAK
OXPHOS
CI
CI+II
ETS
CII
O2 Flow per cells
ETS capacity with CI+II substrates
Reference State
H+
I
ETS
NADH
H+
H+
II
S
Q
III
bc1
F
c
IV
aa3
O2
F1
ADP ATP
H+ H+
Maximum electron transport
capacity is obtained with convergent
CI+II electron input.
H+
I
NADH
c
Q
FADH2
II
S
H+
H+
F
bc1
CI+II:
aa3
F1
O2
H+
H+
1
Q-Junction Ratio
H+
I
ETS
NADH
H+
H+
II
S
Q
III
bc1
F
c
IV
aa3
O2
F1
ADP ATP
H+ H+
With CI substrates, respiration is
limited to 0.70 of ETS capacity.
H+
H+
H+
I
NADH
c
Q
bc1
CI:
aa3
0.70
F1
O2
H+
H+
CI+II:
1
Q-Junction Ratio
H+
I
ETS
NADH
H+
H+
II
S
Q
III
bc1
F
c
IV
aa3
O2
F1
ADP ATP
H+ H+
With CII substrates, respiration is
limited to 0.36 of ETS capacity.
H+
H+
CI+II:
1
c
Q
II
S
F
FADH2
bc1
aa3
F1
O2
+
+
CII:
0.36
Q-Junction Ratio
H+
I
ETS
NADH
H+
H+
II
S
Q
III
bc1
F
c
IV
aa3
O2
F1
ADP ATP
H+ H+
Convergent CI+II electron
input exerts an additive
effect in human fibroblasts.
H+
CI:
I
c
Q
FADH2
II
S
H+
H+
NADH
0.70
F
bc1
CI+II:
aa3
1
F1
O2
H+
H+
CII:
0.36
Electron Transport:
from Chain to System
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?
• Electron Transport Chain
??ETS
System,
Pyruvate
Succinate
Malate
H+
CIII
c CIV
aa3
H+
Isocitrate
NADH
H+
CI
Q
bc1
Uncoupler
Glutamate
3-Hydroxyacyl CoA
Fatty acyl CoA
3-Glycerol phosphate
O2
H+
Convergent Electron Flux and the Q-junction
The most frequent
misnomer in
bioenergetics:
Electron Transport Chain
ETS
Question 1
H+
I
ETS
NADH
H+
H+
II
S
Q
F
III
bc1
c
IV
aa3
O2
F1
ADP ATP
H+ H+
How do we measure
mitochondrial
electron transport capacity?
A. Mitochondria
B. Intact cells
High-Resolution Respirometry
in Intact Cells
200
250
160
120
Cells
80
40
200
150
100
50
FCCP
0
0:00
0:30
ROUTINE
1:00
LEAK
1:30
Time [h:min]
2:00
2:30
0
3:00
Respiration
[pmol∙s-1∙10-6 cells]
Uncoupling
Oligomycin
O2 Concentration
Fibroblasts NIH3T3
ETS
Gnaiger E (2008) In: Mitochondrial Dysfunction in Drug-Induced
Toxicity. (Dykens JA, Will Y, eds) John Wiley.
Mitochondrial Pathways
and Q-Junction
Perm. cells [pmol∙s-1∙10-6 cells]
ETS
CI+II:
Glutamate+Malate+Succinate
uncoupled
250
ETS capacities were
identical in intact and
permeabilized cells,
with convergent
electron flow through
Complexes I and II
(CI+II e-input).
200
150
100
50
0
0
50
100
150
200
250
Intact cells [pmol∙s-1∙10-6 cells]
Identical ETS Capacity in
Permeabilized and Intact Cells
A: Permeabilized Cells
B: Intact Cells
Control
Coupling
ETS
ROUTINE
LEAK
L/R
E
L
R
Oligomycin
L/E
ETS
R/E
uncoupler
E
Culture
medium
CI+II combined
1
0.09
0.32
0.09
0.29
0.29
186
1
199
pmol∙s-1∙10-6 cells
High-Resolution Respirometry
in Intact Cells
200
160
120
Cells
80
250
200
150
100
40
50
FCCP
0
0:00
0:30
ROUTINE
1:00
LEAK
1:30
Time [h:min]
2:00
ETS
State 3?
OXPHOS Capacity ?
0
3:00
2:30
O2
H+
Respiration
[pmol∙s-1∙10-6 cells]
Uncoupling
Oligomycin
O2 Concentration
Fibroblasts NIH3T3
ATP
H+
ADP
Question 2
H+
I
OXPHOS
NADH
H+
H+
II
S
Q
F
III
bc1
c
IV
aa3
O2
F1
ADP ATP
H+ H+
How do we measure
OXPHOS capacity?
A. Mitochondria
B. In intact cells ?
High-Resolution Respirometry
in Permeabilized Cells
OXPHOS capacity is less than ETS
ADP-stimulated
250
160
Omy
Digitonin
ADP
150
100
c
ADP
Succinate
40
F
FCCP
0
0:15
0:30
0:45
1:00
1:15
1:30
50
Rot
80
Glutamate
+Malate
120
200
0
1:45
Time [h:min]
CI
CI+II
CI+II
CII
O2 Flow per cells
ETS
OXPHOS
200
O2 Concentration
uncoupled
OXPHOS
Flux Control Diagrams for
Permeabilized and Intact Cells
A: Permeabilized Cells
B: Intact Cells
Control
Coupling
OXPHOS
LEAK
L/P
L
ADP
P
P/
E
uncoupler
L/E
ETS
L/R
E
0.10
0.20
0.10
0.50
L
R
Oligomycin
L/E
ETS
R/E
uncoupler
E
Culture
medium
Glutamate+Malate+Succinate
CI+II combined
ROUTINE
LEAK
1
0.09
0.32
0.09
R/P
0.58
0.29
1
R/E
0.29
Reserve capacity is
overestimated 2-fold
OXPHOS
H+
I
NADH
H+
H+
II
S
Q
F
III
bc1
c
IV
aa3
O2
F1
ADP ATP
H+ H+
The phosphorylation system
exerts strong control over
OXPHOS
O
ATP
2
in human
+
fibroblasts.
H
+
H
P
0.50
E
Question 3
H+
I
LEAK
NADH
H+
H+
II
S
Q
F
III
bc1
c
IV
aa3
O2
F1
ADP ATP
H+ H+
How do we express
respiratory coupling ratios?
A. Mitochondria
B. Intact cells
High-Resolution Respirometry
in Permeabilized Cells
L/E ratio but not L/P ratio reflects
the relative LEAK.
ADP-stimulated
250
160
Omy
Digitonin
ADP
100
c
ADP
40
Succinate
0
0:15
0:30
150
0:45
1:00
F
FCCP
1:15
1:30
50
Rot
80
Glutamate
+Malate
120
200
0
1:45
Time [h:min]
CI
CI
CI+II
CI+II
CII
O2 Flow per cells
ETS
OXPHOS
200
O2 Concentration
uncoupled
ETS Capacity versus
OXPHOS Capacity
Coupling
ADP
LEAK
OXPHOS
L/P
Control
L
ADP
P
ETS
P/E
uncoupler
L/E
E
Substrate
Glutamate
+Malate
L
0.25
P
0.55
E
Limitation by the
phosphorylation system
4.0
Respiratory Control Ratio (State 3/State 4)
is the inverse L/P ratio
Permeabilized Cells, NIH3T3 Fibroblasts
ETS Capacity versus
OXPHOS Capacity
Coupling
ADP
LEAK
OXPHOS
L/P
Control
L
ADP
P
ETS
P/E
uncoupler
L/E
E
Substrate
Glutamate
+Malate
L
0.25
0.14
P
0.55
E
L/E ratio
expresses uncoupling
7.1
Respiratory Control Ratio
should be the inverse L/E ratio
Permeabilized Cells, NIH3T3 Fibroblasts
Mitochondrial Pathways
and Q-Junction
Perm. cells [pmol∙s-1∙10-6 cells]
LEAK
250
ETS capacities and
LEAK respiration were
identical in intact and
permeabilized cells,
with convergent
electron flow through
Complexes I and II
(CI+II e-input)
CI+II: GMSE
CI+II: GMSL
CI: GML
200
150
100
50
CrE
0
0
50
100
150
200
250
Intact cells [pmol∙s-1∙10-6 cells]
LEAK
Mitochondrial Respiratory
Control: The Q-Junction
1. Convergent e-input at the
Q-junction corresponds to the
operation of the citric acid cycle.
2. The additive Q-junction effect
and phosphorylation limitation of
OXPHOS reveal an unexpected
diversity of mitochondrial
function.
Q-junction ratios: 0.97 to 0.5
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Mitochondrial Respiratory
Control: The Q-Junction
3. Interpretation of apparent excess
capacities of ET complexes and of
flux control coefficients is largely
dependent on the metabolic
reference state. Higher capacities
with CI+II substrates explain
apparent discrepancies between
mitochondria and intact cells.
p. 33
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Mitochondrial Respiratory
Control: The Q-Junction
4. Interpretation of excess
capacities of various components
of the respiratory chain and of
flux control coefficients is largely
dependent on the metabolic
reference state. Appreciation of
the concept of the Q-junction will
provide new insights into the
functional design of the
respiratory chain.
p. 33
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Mitochondrial Respiratory
Control: The Q-Junction
5. The relation between membrane
potential and flux is reversed
when an increase in flux is
effected by a change in substrate
supply.
MultiSensor O2k: TPMP+
Mitochondrial Pathways and Respiratory
p. 33
Control. OROBOROS MiPNet Publ. 2007
www.oroboros.at/index.php?id=mipnet-publications
OROBOROS INSTRUMENTS
high-resolution respirometry
Oxygraph-2k
www.oroboros.at
Faculty
Disclosure
Statement
O2
H+
Ca2+
TPP+
NO
High-Resolution Respirometry
State-of-the-art polarography
Mitochondrial Respiratory
Control: The Q-Junction
6. ROS production and reversed
electron flow from Complex II to
Complex I: Multiple substrate
supply plays a key role (Capel et
al 2005; Garait et al 2005). The
dependence of ROS production on
membrane potential and
metabolic state will have to be
investigated further based on the
concept of the Q-junction.
p. 33
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Coupling Control
LEAK, OXPHOS, ETS
rol
ng
A: Permeabilized Cells
ADP
LEAK
L/P
ate
M
S
OXPHOS
L
fast
0.09
0.10
0.25
0.14
0.20
0.10
0.55
(0.32)
LEAK
0.50
0.34
Routine
L/R
E
L
0.70
Culture
medium
R
Oligomycin
L/E
ETS
R/E
E
uncoupler
0.70
0.50
186 = 1
0.68
(0.35)
(0.12)
0.38
0.77
(1.2)
slow
uncoupler
L/E
0.98
combined
ETS
P/E
P
ADP
B: Intact Cells
0.32
0.29
P: Oxidative
Phosphorylation
0.29
0.09
0.09
199 = 1
0.36
0.93
0.36
L: LEAK
E: Electron Transport System
Odra Noel