Auxiliary HRR-Tools
Mitochondrial Physiology Network 17.15: 1-6 (2012-2013)
2013 OROBOROS
Version 3: 2013-01-15
Tissue Homogenates
for Diagnosis of
Mitochondrial
Respiratory Function:
Mouse Heart, Brain
and Liver
Andrea Eigentler,1 Mona Fontana-Ayoub,2 Erich
Gnaiger1,2
1
Medical University of Innsbruck
Department of Visceral, Transplant and Thoracic Surgery
D. Swarovski Research Laboratory, 6020 Innsbruck, Austria
2
OROBOROS INSTRUMENTS Corp
high-resolution respirometry
Schöpfstr 18, A-6020 Innsbruck, Austria
Email: erich.gnaiger@oroboros.at
www.oroboros.at
http://wiki.oroboros.at/index.php/K-Regio_MitoCom_Tyrol
1. Introduction
A high-quality preparation of tissue homogenate may represent an
optimum compromise for a variety of respirometric and fluorometric
studies. These considerations provided the rationale for initiating a study
with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized
approach to prepare homogenates of various tissues (e.g. heart, liver,
brain) with high reproducibility of mitochondrial yield and mitochondrial
function as evaluated with HRR.
2. Sample preparation
2.1.
Organ harvest
Heart, liver and brain are excised from the sacrificed mouse and added
into Falcon tubes containing sufficient ice-cold BIOPS (mouse heart) or
respiration medium (mouse liver and brain) to cover the entire tissue
sample. Keep on ice and minimize transportation and storage time as far
as possible.
instruments@oroboros.at
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MiPNet17.15
2.2.
PBI-Shredder: an auxiliary tool for HRR
2
Tissue preparation and homogenization (shredding)
Prepare tissue samples of about 4 mg wet weight (Ww) for mouse heart
muscle and 8 mg Ww for mouse brain and liver for two O2k-Chambers
(half the Ww if one Shredder Tube should be used for one O2k-Chamber).
Preparation of tissue homogenate and determination of the wet
weight (Ww) has to be done according to MiPNet17.02.
3. Experimental setup with the Oxygraph-2k
Respiration of all tissue homogenates was measured in respiration
medium at 37 °C and in a normoxygen range. For detailed describtion see
MiPNet17.02.
4. Experimental protocol
Substrate-uncoupler-inhibitor-titration (SUIT) protocols were used to
evaluate the mitochondrial function of mouse heart, mouse brain and
mouse liver homogenate.
All substrates, inhibitors and the uncoupler used in the protocols with
mouse heart, brain and liver homogenate can be found in MiPNet09.12.
5. Results
In the present methodological evaluation of tissue homogenates prepared
with the PBI-Shredder SG3, results are expressed per mg of tissue applied
for preparation per O2k-chamber (2 ml). Representative results of two
chambers are presented for each tissue homogenate.
6. Mouse heart homogenate
High-resolution respirometry
300
450
240
360
180
270
120
180
60
90
0
0:00
0:10
PMG
0:21
D2
D3
D4
0:32
Range
[h:min]:
1:05
c
S
0:43
D5 F0.25 F0.5 Rot
0:54
Ama
0
1:05
O2 Flux per mass (C) [pmol/(s*mg)]
6.1.
Mna
Fig. 1: SUIT protocol with homogenate of mouse myocardium. Overlay of O2ktraces of two chambers. Oxygen concentration [µM] (black and blue line) and
oxygen flux per mass [pmols-1mg-1] (red and green line). MiR05Cr, 37 °C.
OROBOROS INSTRUMENTS
OROBOROS Oxygraph-2k
MiPNet17.15
PBI-Shredder: an auxiliary tool for HRR
3
400
1.2
300
200
chamber C
chamber D
100
Flux control ratio
O2 flux per mass [pmol/(s*mg)]
-1
6.2. O2 Mouse
flux per
tissue
mass [pmols-1mgMouse
] and
FCRhomogenate
heart
homogenate
heart
1.0
0.8
0.6
chamber C
0.4
chamber D
0.2
0.0
0
Fig. 2: O2 flux per mass [pmols1mg-1] and flux control ratio normalized to
PMGSE and corrected for ROX, of chamber C and chamber D, in mouse heart
homogenate.
Coupling control ratios and substrate control ratios
FCR with ROX correction
CI
ETS
OXPHOS
0.59
LEAK
0.07
PMG
CI+II
CII
1.00
0.52
1.00
E
1.00
Flux control ratio
Coupling control
6.3.
P
L
PMGS
S
0.80
L
0.60
P
0.40
E
0.20
E
P
0.00
Substrate control
PMG
L
PMGS
S(Rot)_E
Fig. 3: Coupling control and substrate control ratios for mouse
homogenate, calculated as mean of chamber C and chamber D.
heart
7. Mouse brain (cortex) homogenate
High-resolution respirometry
250
70
200
56
150
42
100
28
50
14
0
0:00
0:11
PM
0:23
D1
D2
G
D3 D3
cc
0:35
Range
[h:min]: 1:10
S
D4 F0.5 F1
Rot
0:46
Ama
0:58
0
1:10
O2 Flux per mass (A) [pmol/(s*mg)]
7.1.
Mna
Fig. 4: Overlay of O2k-trace of chamber A and chamber B. Oxygen concentration
[µM] (black and blue line) and oxygen flux per mass [pmols-1mg-1] (red and
green line) of mouse brain (cortex) homogenate in MiR05.
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MiPNet17.15
4
O2 flux
per
tissue mass [pmols-1Mouse
mg-1]brain
andhomogenate
FCR
Mouse
brain
homogenate
7.2.
1.2
60
40
chamber A
20
chamber B
Flux control ratio
O2 flux per mass [pmol/(s*mg)]
PBI-Shredder: an auxiliary tool for HRR
1.0
0.8
0.6
chamber A
0.4
chamber B
0.2
0
0.0
Fig. 5: O2 flux per mass [pmols-1mg-1] and flux control ratio normalized to
PMGSE and corrected for ROX, of chamber A and chamber B, in mouse brain
(cortex) homogenate.
7.3.
Coupling control ratios and substrate control ratios
FCR with ROX correction
CI
ETS
OXPHOS
0.28
LEAK
0.16
PM
0.38
CI+II
CII
1.00
0.60
1.00
Flux control ratio
Coupling control
CI
E
0.99
P
L
PMG
PMGS
0.80
0.60
L
0.40
P
E
0.20
E
P
0.00
S
PM
PMG
Substrate control
L
PMGS
S(Rot)
Fig. 6: Coupling control and substrate control ratios for mouse brain (cortex)
homogenate, calculated as mean of chamber A and chamber B.
8. Mouse liver homogenate
High-resolution respirometry
250
160
200
128
150
96
100
64
50
32
0
0:00
0:13
Amr
HRPGM
0:27
D1
D2
D3
P
D4 S
0:41
Range
[h:min]: 1:23
reoxy
D5 F0.5 F1 reoxy
0:55
F1.5 Rot
Mna
0
1:23
1:09
Ama
H2O2
O2 Flux per mass (A) [pmol/(s*mg)]
8.1.
H2O2
Fig. 7: Overlay of O2k-trace of chamber A and chamber B. Oxygen concentration
[µM] (black and blue line) and oxygen flux per mass [pmols-1mg-1] (red and
green line) of mouse liver homogenate in MiR05.
OROBOROS INSTRUMENTS
OROBOROS Oxygraph-2k
MiPNet17.15
5
O flux per tissue mass [pmols-1mg-1] and FCR
2
Mouse
liver homogenate
Mouse liver homogenate
140
1.2
120
1.0
100
80
60
chamber A
40
chamber B
Flux control ratio
O2 flux per mass [pmol/(s*mg)]
8.2.
PBI-Shredder: an auxiliary tool for HRR
0.8
0.6
chamber A
0.4
20
0.2
0
0.0
chamber B
Fig. 8: O2 flux per mass [pmols-1mg-1] and flux control ratio normalized to
PMGSE and corrected for ROX, of chamber A and chamber B, in mouse liver
homogenate.
8.3.
Coupling control ratios and substrate control ratios
CI
CI
ETS
OXPHOS
034
LEAK
0.07
GM
0.47
CI+II
CII
1.00
0.67
0.86
1.00
E
P
L
GMP
GMPS
S
Flux control ratio
Coupling control
FCR with ROX correction
0.80
0.60
L
0.40
P
E
0.20
E
P
0.00
Substrate control
GM
GMP
L
GMPS
S(Rot)
Fig. 9: Coupling control and substrate control ratios for mouse liver homogenate,
calculated as mean of chamber A and chamber B.
9. Conclusions
The application of an additional tool to remove the serrated Shredder Ram
as well as the Shredder Cap after homogenization increased the
mitochondrial yield by washing out the homogenate completely from both
sides of the Lysis Disk.
The cytochrome c effect could be diminished in mouse heart
homogenate and no cytochrome c effect occurred in mouse liver and
mouse brain homogenate.
10. Acknowledgements
Contribution to K-Regio project
MitoCom Tyrol, funded in part by
the Tyrolian Government and the
European Regional Development
Fund (ERDF).
www.oroboros.at/?MitoCom-Tyrol
OROBOROS INSTRUMENTS
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MiPNet17.15
PBI-Shredder: an auxiliary tool for HRR
6
11. References
Mitochondr Physiol Network – MiPNet Manuals and Protocols
MiPNet09.12: Oxygraph-2k titrations. Mitochondria, permeabilized cells
and biopsies. Mitochondr Physiol Network 09.12.
MiPNet17.02: PBI-Shredder HRR-Set: Preparation of tissue homogenates
for diagnosis of mitochondrial respiratory function.
12. Author contributions and publication versions
Eigentler A performed experiments and wrote the
manuscripts.
Fontana-Ayoub
M
performed
experiments. Gnaiger E designed experiments and
edited the manuscript.
Results for fish (trout) homogenate of liver and heart
are published in MiPNet17.03: Doerrier C, Draxl A,
Wiethuechter A, Eigentler A, Gnaiger E (2012)
Mitochondrial respiration in permeabilized fibres
versus homogenate from fish myocardium and liver.
An application study with the PBI-Shredder.
Mitochondr Physiol Network 17.03.
Preliminary results for mouse heart homogenate
versus permeabilized fibres can be found in the
Appendix of MiPNet17.03.
OROBOROS INSTRUMENTS
OROBOROS Oxygraph-2k