Mitohealth Workshop, Bergen, Nov 8-9 2008
Respirometry,
monitoring mitochondrial function
Karl Johan Tronstad
[email protected]
Mitochondrial energy metabolism
CPT I
CPT II CT
β-oxidation
Ketone
bodies
NADH
FADH
Respiration
TCA
cycle
----+++ ΔpH og ΔΨ
Mitochondrial oxidative capacity
Single complex activities
Ubiquinol-cyt c
reductase
NADH:CoQ
reductase
2H+
ATP
synthase
Cyt c
oxidase
Succinate:CoQ
reductase
4H+
2H+
3H+
cyt
UQ
I
II
FADH2
NADH
NAD+
H+
c
III
Fo
IV
V
ETFP
FAD
½O2
FAD
Acyl-CoA
dehdrogenase
2H+
H2O
2H+
F1
ADP + Pi
3H+
Depletion of mitochondrial
membrane potential
Intermembrane Space
rotenone
Antimycin A
2H+
CN-
4H+
2H+
3H+
cyt
UQ
I
II
FADH2
NADH
NAD+
H+
Pyruvate
Matrix
III
Fo
IV
V
ETFP
FAD
½O2
FAD
Acyl-CoA
dehdrogenase
Succinate
c
2H+
H2O
2H+
F1
ADP + Pi
Ascorbate/TMPD
3H+
Respirometry
-measuring oxygen consumption
Samples?
• Isolated mitochondria
• Cells
• Biopsies
e.g. Permeabilized muscle fibers
=> intact/viable
Parameters?
•
•
•
•
Routine respiration
Respiratory capacity
Respiratory complex activities
Respiratory control
- uncoupling
- energy transduction
Measuring oxygen consumption
Oligo
Rout
FCCP
reox
Succ
Rot
Anti A
250
200
[O2] in chamber
(nmol/ml)
150
100
Aicar
50
0
Ctr (+G)
20
30
40
200
180
160
140
O2 flow per cells 120
(pmol/(s*Mill)
100
80
60
40
20
0
20
30
40
time (min)
50
60
70
50
60
70
Uncoupling
Heat
H+
Uncoupling effects of fatty acids
Control (DMSO)
TTA
10
50
100 µM
24
Succinate
25
20
15
10
rotenone
20
18
16
5
14
0
12
-5
10
0
500
1000
1500
time (s)
2000
2500
Palmitic acid
22
AntiA
Oxygen flux
pmol oxygen /(s*mill cells)
Oxygen flux
30
TTA
DMSO
0
20
40
60
80
FA concentration (µM)
Involvement of a cyclosporin A
sensitive pore?
Palmitic acid
TTA
35
Cyclosporin A
Fatty acid (50µM)
pmol oxygen/(s*mill cells)
30
25
20
15
succinate
10
Cells
+ digitonin
+ rotenon
5
0
-5
0
400
800
1200
time (s)
1600
2000
2400
Test trace
• Cultured cells (HL60/Jurkat)
•
•
•
•
Routine respiration
Complex I activity
Respiratory capacity
Flux ratios