Supplemental Materials and Methods
Myocardial Mitochondrial Dysfunction and Uncoupling
in Mice Lacking Adiponectin Receptor 1
Christoph Koentges, MS1 *, Alexandra König, MD1 *, Katharina Pfeil, MS1, Maximilian E
Hölscher, MD1, Tilman Schnick, MD2, Adam R Wende, PhD3, Andrea Schrepper, PhD5,
Maria C Cimolai, MS1, Sophia Kersting, MD1, Michael M Hoffmann, PhD1,6, Judith Asal,
BS1, Moritz Osterholt, MD5, Katja E Odening, MD1, Torsten Doenst, MD5, Lutz Hein, MD2,
E Dale Abel, MD, PhD4, Christoph Bode, MD1, and Heiko Bugger, MD1
1
Heart Center Freiburg University, Division of Cardiology and Angiology I, Freiburg, Germany
2
Institute of Experimental and Clinical Pharmacology, University of Freiburg, Freiburg, Germany
3
Department of Molecular and Cellular Pathology, University of Alabama at Birmingham,
Birmingham, AL, USA
4
Fraternal Order of Eagles Diabetes Research Center and Division of Endocrinology and Metabolism,
Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
5
Department of Cardiothoracic Surgery, Jena University Hospital, Jena, Germany
6
Institute for Clinical Chemistry and Laboratory Medicine, Freiburg University Hospital, Freiburg,
Germany
* Authors contributed equally
Corresponding author: Heiko Bugger, MD
E-mail: heiko.bugger@universitaets-herzzentrum.de
1
Isolated working heart perfusion: Isolated working heart perfusions were performed using 8
week-old male mutant mice and their respective C57BL/6J wildtype (WT) littermate controls.
Animals were weighed and anesthetized using 0.3 mg/g body weight thiopenthal i.p.. Before
excising the heart, mice were heparinized with an intracaval injection of 200 I.U. heparin.
Hearts were excised together with lungs and placed into Krebs-Henseleit Buffer (KHB) at
room temperature containing (in mmol/L) 128 NaCl, 5 KCl, 1 KH2PO4, 1.3 MgSO4, 15
NaHCO3, 2.5 CaCl2 and 5 Glucose. After removing the lungs, hearts were placed into ice cold
KHB for further preparation. Other non-cardiac tissue was removed and the aorta was
canulated and bound to a 20-gauge plastic canula. Perfusion was carried out using water
jacketed glass jars warmed to 38.5 °C resulting in a final myocardial perfusion temperature of
approx. 37 °C. Hearts were initially perfused in a retrograde Langendorff mode (50 mmHg
perfusion pressure) with KHB solution gassed with 95% O2 and 5% CO2 resulting in pH 7.4.
Subsequently, the left atrium was canulated within 60 s using a 18-gauge metal cannula and
sealed using 4/0 surgical suture. After switching to the working mode, hearts were perfused
for 60 min with 15 mmHg preload and 50 mmHg afterload using KHB with 0.4 mmol/L
palmitate bound to 3 % BSA. Aortic pressure changes were measured using a pressure
catheter placed inside the aortic cannula (Millar Micro-Tip, Millar Instruments, Houston,
TX). Aortic developed pressure was calculated as the difference of systolic and systemic
pressure. Heart rate was determined from the pressure traces by measuring the time interval
between peak systolic values. In conjunction with pressure measurements, aortic and coronary
flow measurements were obtained by collecting the flow from the afterload line and the
effluent dripping off the heart, respectively. To accomplish these flow measurements without
disrupting metabolic measurements, the perfusion apparatus was made air-tight. Graduated
syringes were connected and sealed within the perfusion apparatus and flow was determined
(at 20-min intervals) by measuring the time required to collect a 2 ml sample for coronary
flow and 5 ml sample for aortic flow. Cardiac output (ml/min) was calculated as the sum of
aortic and coronary flow. Cardiac power (mW/g) was calculated as the product of cardiac
output and afterload per dry heart weight. Cardiac work (ml*mmHg/min) was calculated as
product of cardiac output and aortic developed pressure per minute. Hydraulic work (J/min*g)
was calculated as the product of cardiac output and aortic developed pressure per wet heart
weight (WHW).
Myocardial oxygen consumption, cardiac efficiency and substrate oxidation rates:
Myocardial oxygen consumption (MVO2) was measured every 20 min during working heart
perfusions as the difference of percent oxygen concentration in pre- (arterial (aO2)) and
2
postcardial (venous (vO2)) buffer samples. Oxygen concentration in the samples was
measured using a fiber-optic oxygen sensor (Ocean Optics, Orlando, FL). The following
formulae were used to determine MVO2 (μl/min/gww) and cardiac efficiency (%): MVO2 =
(aO2-vO2) * (coronary flow/WHW) * (718/760) * (1000 * Bunsen coefficient); where Bunsen
coefficient for plasma is 0.0212, and where 718 and 760 mmHg are atmospheric pressures in
Freiburg and at sea level, respectively. Cardiac efficiency = hydraulic work/MVO2 * 100.
MVO2 was converted to µmol/min by multiplying by the conversion factor 0.0393 and then to
Joules (J/min) using the conversion of 1 µmol O2 = 0.4478 J as described by Suga et al. [1].
Palmitate oxidation was measured in the same perfusion by determining the amount of 3H2O
released from [9,10-3H] palmitate (specific activity, 500 GBq/mol). 3H2O was separated from
[9,10-3H] palmitate by mixing 500 µl perfusate sample with 1.88 ml Chloroform/Methanol
(1:2 v/v) (15 min incubation) followed by the addition of 625 µl chloroform (15 min
incubation). Then 2 mol/L HCl/KCl solution was added, mixed and incubated for at least 30
min until a polar and a non-polar phase appeared. 1.8 ml of the polar phase was transferred
into another tube and mixed with 1 ml of chloroform, 1ml of Methanol and 900 µl of
HCl/KCl solution, and mixed and incubated for 15 min after each step. After the last addition
and at least 30 min incubation, two 500 µl aliquots were taken from the upper layer and
counted for 3H. Palmitate oxidation rates were calculated from 3H2O production, taking into
account the dilution factor incurred from the process of separating 3H2O from [9,10-3H]
palmitate.
Glucose oxidation and glycolysis were measured in separately perfused hearts. Glucose
oxidation was determined by trapping 14CO2 released from [U-14C] glucose (specific activity,
300 MBq/mol) in 15 ml of hyamine hydroxide (Fischer Scientific, Schwerte, Germany). After
the addition of 4 ml UltimaGold scintillation cocktail, samples were counted for
14
C. Since
14
CO2 is also dissolved in the form of bicarbonate anion, a 3.5 ml sample was drawn every 20
min and injected under 1 ml of mineral oil to avoid CO2 gassing out. From this sample 750 µl
were taken and injected into 750 µl 9N H2SO4 in a sealed 10 ml BD vacutainer tube capped
with a hyamine hydroxide filter lined scintillation vial. Tubes were vortexed every 5 min for 1
h. In the end, the scintillation vial was disconnected from the vacutainer tube and after the
addition of 5 ml UltimaGold scintilation cocktail, counted for
14
C. Glycolytic flux was
determined by measuring the amount of 3H2O released from the metabolism of [5-3H] glucose
(specific activity 300 MBq/mol). To separate 3H2O from [5-3H] glucose and [U-14C] glucose,
perfusate samples were passed through an anion exchange resin (200-400 mesh Dowex AG-1X4) column pretreated with 1 mol/L NaOH and then converted to the borate form using 0.5
3
mol/L boric acid. Columns were washed 5 times, 100 µl buffer sample was loaded, and the
sample was eluted with 800 µl of H2O. After addition of 3 ml UltimaGold scintillation
cocktail samples were counted for 3H and 14C. The 3H2O measurements were corrected for the
column efficiency in retaining glucose by measuring the amount of [U-14C] glucose which
passed the column. This signal was equaled to the amount of [5-3H] glucose, which also
passes the column and was accounted to contribute to the 3H signal.
H2O2 generation: H2O2 production was measured using the Amplex Red Hydrogen
Peroxide/Peroxidase Assay Kit according to the manufacturer instructions (Molecular Probes,
Darmstadt, Germany). 20 µg of freshly isolated mitochondria contained in respiration buffer
(125 mmol/l KCl, 4 mmol/l KH2PO4, 14 mmol/l NaCl, 20 mmol/l HEPES, 1 mmol/l MgCl2,
0.02 mmol/l EGTA, 0.2% BSA; pH 7.2) were stimulated with glutamate (5 mmol/l)/malate (2
mmol/l), and fluorescence developed by its reaction with the reactant containing 40 µmol/l
Amplex Red and 8 U/ml horseradish peroxidase was measured for 30 min at 37°C (λexc: 530560 nm; λem: 590 nm).
4
Table S1. Sequences of forward and reverse primers used for RT-PCR.
Gene
Primer
α1-Tubulin
Forward
AAGGAGGATGCTGCCAATAA
Reverse
AGGTGAGCCAGAGCCAGT
Forward
GTCCACCATCACAGGAA
Reverse
GCTTGCCCTTCTCCTCCA
Forward
TACACACAGAGACGGGC
Reverse
CCCAAGAAGAACAAGCC
Forward
CAAACAAATAATGCTAATCCACACACC
Reverse
GCTGTAAGCCGGACTGCTAATG
Forward
AGCGACCAGATGAAGCAGTG
Reverse
TCCGCTCTCTGTCAAAGTGTG
Forward
CCATCCCAGGCCGACTAA
Reverse
CAGAGCATTGGCCATAGAATAACC
Forward
GGAAGTGCATCTGCTTGTCTC
Reverse
TAGGGACACCACCTCCAGAA
Forward
TGCCTTTACATCGTCTCCAA
Reverse
AGACCCCGTAGCCATCATC
Forward
CCAGCTGACCAAAGAAGCA
Reverse
GCAGCCTATCCAGTCATCGT
Forward
CAAGCATAAGACTGGACCAAA
Reverse
TTGTTGGCATCTGTGTAAGAGAATC
Forward
GGAGGACGGCAGAAGTACAA
Reverse
CAGGTTCAACAACCAGCAGA
Forward
GTCCACCGTGTATGCCTTCT
Reverse
TCACCATTCACTTCGCACTT
Forward
ATGCCCGATATGCTGAGTGT
Reverse
CGGCAGGTCCTTCTCTATCA
Forward
GCCAACAGACTGAGGAAGGAA
Reverse
ACACTGGCAAGGCTGGATT
AdipoR1
AdipoR2
ATPase6
Cat
Cox II
Cox Vb
Cpt1b
Cpt2
Cyt c
Errα
Gpx1
Gpx4
Hadhβ
Primer Sequence (5´-3`)
5
Lcad
Mcad
mt -Nd2
Ndufv1
Uqcrc1
Nrf1
Ppargc1α
Ppargc1β
Pparα
Sdh1
Sod2
Tfam
Txn1
Forward
ATGGCAAAATACTGGGCATC
Reverse
TCTTGCGATCAGCTCTTTCA
Forward
ACTGACGCCGTTCAGATTTT
Reverse
GCTTAGTTACACGAGGGTGATG
Forward
CGCCCCATTCCACTTCTG
Reverse
TTAAGTCCTCCTCATGCC
Forward
TGTGAGACCGTGCTAATGGA
Reverse
CATCTCCCTTCACAAATCGG
Forward
TGCCAGAGTTTCCAGACCTT
Reverse
CCAAATGAGACACCAAAGCA
Forward
CTTCAGAACTGCCAACCACA
Reverse
GCTTCTGCCAGTGATGCTAC
Forward
GTAAATCTGCGGGATGATGG
Reverse
AGCAGGGTCAAAATCGTCTG
Forward
TGAGGTGTTCGGTGAGATTG
Reverse
CCATAGCTCAGGTGGAAGGA
Forward
GAGAATCCACGAAGCCTACC
Reverse
AATCGGACCTCTGCCTCTTT
Forward
CCTTTCTGAGGCAGGGTTTA
Reverse
CAGTCGGAGCCTTTCACAGT
Forward
ACAACTCAGGTCGCTCTTCA
Reverse
GAACCTTGGACTCCCACAGA
Forward
CAAAAAGACCTCGTTCAGCA
Reverse
CTTCAGCCATCTGCTCTTCC
Forward
GCCAAAATGGTGAAGCTGAT
Reverse
TGATCATTTTGCAAGGTCCA
Abbreviations: ATPase6, ATP synthase F0 subunit 6; Cat, catalase; Cox II, cytochrome c
oxidase subunit 2; Cox Vb, cytochrome c oxidase subunit Vb; Cpt1b, carntine
palmitoyltransferase 1b ; Cpt2, carnitine palmitoyltransferase 2 ; Cyt c, cytochrome c ; Errα,
estrogen related receptor alpha ; Gpx1, glutathione peroxidase 1; Gpx4, glutathione
peroxidase 4; Hadhβ, hydroxyacyl-CoA dehydrogenase, β subunit ; Lcad, long chain acyl6
CoA dehydrogenase ; Mcad, medium chain acyl-CoA dehydrogenase ; mt-Nd2, NADH
dehydrogenase 2, mitochondrial; Ndufv1, NADH dehydrogenase [ubiquinone] flavoprotein 1;
Ndufa9, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 ; Uqcrc1,
ubiquinol cytochrome c reductase core protein 1; Nrf1, nuclear respiratory factor 1; Ppargc1α,
peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α); Ppargc1β;
peroxisome proliferator-activated receptor gamma coactivator 1 beta (PGC-1β); Pparα,
peroxisome proliferator-activated receptor α ; Sdh1, succinate dehydrogenase flavoprotein
subunit 1; Sod2, superoxide dismutase 2; Tfam, mitochondrial transcription factor A; Txn1,
thioredoxin 1.
7
Table S2: Heart weights normalized to tibia length or body weight in WT and AdipoR1(-/-)
mice at 10 weeks of age; n=10.
Heart weight [mg]
Tibia length [mm]
HW-TL ratio [mg/mm]
Body weight [g]
HW-BW ratio [mg/g]
121.9
17.0
7.3
23.3
5.2
WT
±
±
±
±
±
1.4
0.1
0.1
0.2
0.1
AdipoR1(-/-)
111.8 ± 2.6*
16.2 ± 0.1*
6.9 ± 0.2
23.1 ± 0.7
4.9 ± 0.1*
* p<0.05 versus WT
8
Table S4: Cardiolipin species detected in whole heart homogenates of wildtype and
AdipoR1(-/-) mice at 10 weeks of age, presented in ng/mg; n=3.
Species
C70:7
C70:6
C72:9
C72:8 (CL)
C72:7
C72:6
C72:5
C74:10
C74:9
C74:8
C74:7
C76:11
C76:10
Formula
18:2-18:2-18:2-16:1
18:2-18:2-18:2-16:0
18:2-18:2-18:1-16:1
18:2-18:2-18:2-18:3
20:4-18:2-18:2-16:1
20:4-20:4-16:1-16:0
18:2-18:2-18:2-18:2
18:2-18:2-18:2-18:1
18:2-18:2-18:1-18:1
18:2-18:2-18:1-18:0
18:2-18:1-18:1-18:1
20:4-18:2-18:2-18:2
22:6-18:2-18:2-16:0
22:6-18:2-18:1-16:1
20:4-20:3-18:2-16:1
20:2-18:2-18:2-18:2
20:1-18:2-18:2-18:2
20:2-18:2-18:2-18:1
22:5-18:2-18:2-18:2
22:6-18:2-18:2-18:1
22:6-18:2-18:1-18:1
m/z
1422.9
1424.9
1424.9
1446.9
1446.9
1446.9
1448.9
1450.9
1452.9
1455.0
1455.0
1472.9
1472.9
1472.9
1472.9
1477.0
1479.0
1479.0
1499.0
1498.9
1500.9
WT
23.0 ± 1.0
14.5 ± 0.5
AdipoR1(-/-)
24.8 ± 0.5
16.0 ± 0.7
17.1 ± 0.6
16.8 ± 1.7
549.2
218.6
64.7
4.0
±
±
±
±
10.6
5.6
1.1
0.4
673.9
229.6
68.4
3.9
±
±
±
±
45.8
8.5
4.1
0.3
38.5 ± 0.6
42.0 ± 3.6
65.1 ± 27.4
54.1 ± 0.5
25.3 ± 1.9
76.4 ± 31.4
65.1 ± 4.3
27.3 ± 3.1
149.9 ± 5.8
67.8 ± 4.1
152.2 ± 4.1
48.7 ± 1.8*
* p<0.05 versus WT
CL, tetralinoleoyl cardiolipin; m/z, mass-to-charge ratio.
9
Table S5: Heart weights normalized to tibia length or body weight in WT and AdipoR2(-/-)
mice at 10 weeks of age; n=10.
Heart weight [mg]
Tibia length [mm]
HW-TL ratio [mg/mm]
Body weight [g]
HW-BW ratio [mg/g]
118.4
16.8
7.0
25.8
4.6
WT
±
±
±
±
±
4.7
0.2
0.2
0.6
0.2
AdipoR2(-/-)
118.1 ± 6.9
16.5 ± 0.2
7.2 ± 0.3
24.3 ± 1.0
4.7 ± 0.3
10
Table S3: Echocardiographic parameters measured in WT and AdipoR1(-/-) mice 4 weeks following TAC or sham surgery; n=4-8.
Sham
heart rate (min⁻ ¹)
LVIDd (mm)
LVIDs (mm)
LVPWd (mm)
LVPWs (mm)
EF (%)
FS (%)
WT
529 ± 12
3.81 ± 0.06
2.83 ± 0.07
0.70 ± 0.03
0.92 ± 0.05
59 ± 2
25.7 ± 1.1
TAC
AdipoR1(-/-)
445 ± 10
4.36 ± 0.14
3.30 ± 0.12
0.66 ± 0.04
0.95 ± 0.02
57 ± 2
24.4 ± 1.1
WT
524 ±
4.58 ±
3.69 ±
0.72 ±
0.98 ±
47 ±
19.3 ±
20
0.04*
0.02*
0.03
0.04
1*
0.6*
AdipoR1(-/-)
467 ± 17$
4.57 ± 0.12§$
3.84 ± 0.13#§$
0.71 ± 0.01
0.95 ± 0.03
41 ± 2#§$&
16.8 ± 0.8#§$&
2-way ANOVA: § effect of TAC, $ effect of genotype.
T-test: * p<0.05 versus WT Sham, # p<0.05 versus AdipoR1 Sham, & p<0.05 versus WT TAC
11
Supplemental Figure legends:
Fig. S1: Glucose tolerance test (a) and serum levels of free fatty acids (b) and triglycerides (c)
in AdipoR1(-/-) mice and WT mice at 10 weeks of age; n=6. * p<0.05 versus WT
Fig. S2: Immunoblots of Ucp2 (a), Ucp3 (b) and complex V (c) of homogenates (Ucp2,
Ucp3) or isolated mitochondria (complex V) before immunoprecipitation (input), of
immunoprecipitates using specific antibodies (bound), of bead-free supernatant (unbound),
and of immunoprecipitates using unspecific IgG (IgG) of hearts of AdipoR1(-/-) mice and WT
mice at 10 weeks of age.
Fig. S3: Myocardial protein expression of complex V in isolated mitochondria of AdipoR1 (-/-)
mice and WT mice at 10 weeks of age; n=4. * p<0.05 versus WT
Fig. S4: H2O2 generation (a) and gene expression of mitochondrial antioxidant proteins (b) in
AdipoR1(-/-) mice and WT mice at 10 weeks of age; n=4 for H2O2 generation, n= 6-8 for gene
expression.
Fig. S5: Glucose tolerance test (a) and serum levels of free fatty acids (b) and triglycerides (c)
in AdipoR2(-/-) mice and WT mice at 10 weeks of age; n=6.
Fig. S6: Gene expression of AdipoR2 in AdipoR1(-/-) and WT hearts (a), and of AdipoR1 in
AdipoR2(-/-) and WT hearts (b); n=8. * p<0.05 versus WT
References
1.
Suga H (1990) Ventricular energetics. Physiol Rev 70:247-277
12