SUPPLEMENTAL DATA
Methods
Animal model
Study 1: Juvenile Wistar Han rats (weight 186.8±1.1g) were i.p. injected with 10 8 AH130 hepatoma cells and the tumor allowed to grow for 5 (n=6), 7 (n=10), 9 (n=9), 11 (n=8) or
13 (n=12) days. The tumor cells grow exponential until day 8/9 when they reach a plateau as
described elsewhere1. In the early phases of tumor growth, the protein mass of the body is
preserved, while in overt cachexia protein, mainly from muscle, is lost 2. High levels of proinflammatory cytokines and prostaglandins have been reported in this model,2, 3 as wells as
increased myostatin expression.4 Sham animals were sacrificed on day 7, 11 and 13 (all
n=5). The sham animals were combined for functional analyses (e.g. cardiac function, food
intake, activity), but for all body weight and body composition analyses their data is displayed
according to the sacrifice day. Before tumor inoculation, baseline body weight, body
composition, spontaneous locomotor activity, food and water intake, as well as cardiac
function (by echocardiography) were assessed. These analyses were repeated on the
respective days of sacrifice, but cardiac function analysis was extended to ECG and invasive
hemodynamics (Supplemental Figure 1). At the end of the study, plasma was collected,
organs were removed and weighed.
Study 2: The effects of cardiovascular medication on outcome were tested over a
period of maximal 16 days. Animals were treated with the 0.5 (n=13), 2 (n=14), 5 (n=23) or
50 mg/kg/d (n= 20) of the -1 specific b-blocker bisoprolol, (n=9), 0.4 (n=10), 1 (n=14) or 10
mg/kg/d (n=15) of the ACE-inhibitor imidapril, 2 (n=6), 5 (n=16) or 50 mg/kg/day (n=16) of
the aldosterone antagonist spironolactone or placebo (n=73). Again, body weight and
composition, activity and food intake as well as cardiac function (echocardiography) were
assessed at baseline. While locomotor activity, food intake and cardiac function were reassessed on day 10/11 after tumor inoculation, body weight and composition were analyzed
1
on day 16 or the day the individual animal was sacrificed prematurely due to reaching ethical
endpoints (hypotherima, apathy, persisting staggering, bleeding, persisting diarrhea, labored
breathing, cyanosis, complete lack of food intake, dehydration). All animal studies were in
compliance with the Guide for the Care and Use of Laboratory Animals as published by the
U.S. National Institutes of Health and were approved by our local Institutional Review
Boards.
Body composition
Body composition (fat and lean body mass) was analyzed with an NMR spectroscopy
device EchoMRI-700TM (Echo Medical Systems, Houston, TX) as described before.5
Locomotor activity
Animals were house individually and spontaneous movement was recorded by an
infrared monitoring system (Supermex, Muromachi, Tokyo, Japan) over a 24h period as
described before.6 Also food and water (study 1) intakes were monitored.
Echocardiography
Echocardiography was performed as described before,
7
using the high resolution
Vevo 770 (Visual Sonics, Toronto, Canada).
Electrocardiogram
A three-lead ECG was obtained from all animals at the end of study 1. ECG tracings
were recorded with Chart 5 software (ADIstruments, Spechbach, Germany) and analyzed
using the eMOUSE software (Mouse Specifics, Quincy, MA, USA).
Invasive Hemodynamics
Hemodynamic assessment was performed at the end of study 1, using a Millar
pressure catheter and Chart 5 software for analysis. After anesthesia with isoflurane (1.5%),
the catheter was inserted into the right carotic artery in the normally breathing animal. After
2
blood pressure (BP) recoding in the aorta the catheter was advanced into the LV and the
pressure time indices (dP/dtmin and dP/dtmax) were recorded.
Biomarkers and clinical parameters
Plasma was collected at the end of the study and brain natriuretic peptide (BNP)
tissue inhibitors of metalloproteinase-1 (TIMP-1), monocyte chemoattractant protein-1 (MCP1), adiponectin, troponin-I, troponin-T and fibrinogen were measured in plasma using a
Milliplex assay (Millipore) and Luminex-200 system. The plasma levels of cholesterol (total,
high density lipoprotein (HDL), low-density lipoprotein (LDL)), trigylerides, albumin, were
measured by a commercial laboratory (Labor28, Berlin, Germany). Plasma aldosterone
levels were determined by radioimmunoassay (Figure 6 A) or ELISA (Figure 6C). Plasma
levels of cortisol, noradrenaline and renin were measured by commercial enzyme linked
immunosorbent assay (ELISA) kits (Asbach Medical Products) according to manufacturer’s
protocols. Results were calculated using 4 parameter logistics.
Proteasome activity
The septum of the heart was homogenized in ice-cold lysis-buffer (10mM
tris(hydroxymethyl)aminomethane (Tris) pH 7,5, 1mM ethylenediaminetetraacetic acid
(EDTA), 2mM adenosinetrisphosphat (ATP), 20% Glycerin, 4mM dithiothreitol (DTT)),
briefly sonicated, centrifuged at 13.000 g for 15 min. 40 µg protein were incubated with the
fluorogenic substrates (benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-methylcoumarin [Z-LLEAMC] for trypsin-like activity, succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin [LLVYAMC] for chymotrypsin-like activity or benzoyl- Val-Gly-Arg-7-amidocoumarin [Bz-VGRAMC], Biomol, Hamburg, Germany) for peptidyl-glutamyl-protein-hydrolysing activity. The
change fluorescence intensity was measured with a fluorometer (Twinkle LB 970, Berthold,
3
Bad Wildbad, Germany) at 360 nm excitation and 460 nm emission in 5 min intervals for 1 h.
The activity, expressed as nmol/mg/min, was calculated by using free amidomethylcoumarin
(AMC) as working standard.
Caspase activity
The septum of the heart was homogenized in ice-cold lysis-buffer (100mM (4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 10% Sucrose, 0.1%
tergitol-type NP-40 (NP-40), 10mM DTT and complete mini protease inhibitor cocktail,
Boehringer, Germany). The homogenate was frozen on dry ice/ethanol and heated to 37°C
for three cycles. After centrifugation (20.000 g for 30 min) 100 µg protein was used for the
caspase-3 or caspase-6 activity measurement. The protein was pre-incubated in assay
buffer
(100mM
HEPES
pH
7.5,
10%
cholamidopropyl)dimethylammonio]-1-propanesulfonate
Sucrose,
(CHAPS),
2%
0.1%
3-[(3-
dimethlysufoxide
(DMSO), 10mM DTT with or without 50µM caspase-3-Inhibitor Ac-DEVD-CHO) at 37°C for
30 min. The fluorogenic substrate (50µM) Ac-DEVD-AMC was added for caspase-3. Assay
conditions were identical to the proteasome assay.
Western Blotting
Protein lysates were prepared according to standard protocols. We used primary
antibodies against FOXO3a (2497), pFOXO3a (9466), MuRF-1 (4305), Akt (9272), pAkt
(Ser473; 4051), pAkt (Thr308; 9275), p-p70S6K (Thr389; 9205), pSmad-2 (3101), 4E-BP1
(53H11; 9644), p4E-BP1 (Thr 37/47; 9459), p4E-BP1 (Ser65; 9451), GSK-3a (9338), GSK3b (27C10; 9315) pGSK-3and pGSK-3b (Ser9; 37F11; 9320), AMPK (2532), pAMPK
(2386), pPI3K p85 (Tyr458) / p55 (Tyr 199; 4228) all from Cell Signaling, myostatin (AF788,
R&D Systems), LC-3 (NEB100-2220, Novus Biologicals), p70S6K (sc-230; Santa Cruz) and
GAPDH (G9545; Sigma), cardiac MHC (Abcam), tropomyosin, troponin T (Sigma) as well as
appropriate secondary antibodies. Protein levels of IGF-1- and insulin-receptor were
determined by Luminex-assay (LHO 0491 and LHR 9111, respectively, Millipore).
4
Hydroxyproline assay
LV tissue was dried at 100°C for 24 hours and the dry weight determined. The tissue
was digested with 4N NaOH, neutralized and incubated with chloramineT (SIGMA) reagent
at 20°C for 20min. P-dimethylaminobenzaldehyde (SIGMA) was added and the mix
incubated at 65°C for 15 min. Absorbance was measured at 550 nm and the hydroxyproline
amount calculated using hydroxyproline as standard and data was normalized to dry tissue
weight.
Human samples, study 3
Aldosterone and BNP were measured in serum samples of patients with non-smallcell lung or colorectal cancer with or without cachexia as well as healthy controls (Labor28).
Twenty-two controls were compared to 32 patients with cancer (25 non-small cell lung
cancer (NSCLC), 7 colon cancer) or 20 patients with cancer cachexia (13 NSCLC, 7 colon
cancer). Mean age was in 63±2, 59±2 and 60±2 years for control, cancer and cancer
cachexia, respectively. Cadaver heart weight was assessed from patients who died of
NSCLC (cancer: n=4, cancer cachexia: n=3) gastrointestinal (cancer: n=4, cancer cachexia:
n=7) or pancreatic (cancer: n=4, cancer cachexia: n=4) cancer and compared to controls
(n=11), who died of non-cancer, non-cardiovascular related illness. Mean age was 56±5,
59±2 and 60±2 for control, cancer and cancer cachexia, respectively. For detailed patients
characteristics see Supplemental Table 1 and 2, respectively.
Histology
Rat and human heart samples were sectioned at 3 µm. Fibrosis was visualized by
AZAN and Sirius Red stains, according to standard protocols.
Neonatal cardiomyocytes
5
Isolation and cultivation of neonatal rat cardiomyocytes (NRCM) were performed as
described previously.8 NRCM were set serum free in Dulbecco's Modified Eagle's Medium
(DMEM) / medium 199 (M199) (4:1) for 24 h and subsequently stimulated with
spironolactone (1 M, SIGMA), bisoprolol (10 M, SIGMA), SB 415286 (10 M), SIGMA.
Aldosterone (1 M, SIGMA) or isoproterenol (10 M, SIGMA) were applied 1 h later. Five
days later NRCM were photographed and harvested for further analyses.
mRNA analyses
Isolation of total RNA from LV tissue and NRCM was performed by TRIZOL according
to the manufactorer’s protocol. cDNA synthesis and qRT-PCR were performed according to
standard procedures.9 qRT-PCR measurement was performed using the Stratagene
MX3005p QPCR System with a SYBR green dye method (Brilliant SYBR Green MastermixKit; Stratagene). Results were expressed as percent of control (control at 100%). Specific
primers used for real-time PCR were:
-MHC:5’-GGAAGAGCGAGCGGCGCATCAAGG-3’ 5’- CTGCTGGACAGGTTATTCCTCA-3’
-MHC: 5’-CAAGTTCCGCAAGGTGC-3’ 5’-AAATTGCTTTATTGTGTTTCT-3’
GAPDH: 5'-ACCACCATGGAGAAGGCTGG -3’ 5'-CTCAGTGTAGCCCAGGATGC -3’
Statistics
Data were analyzed using GraphPad PRISM 5.0 (GraphPad Software, Inc, La Jolla,
CA, USA). Results are shown as mean ± SEM. Data was tested for normal distribution using
the Kolmogorov Smirnov test. Data with normal distribution were analyzed with ANOVA
followed by Students t-test, while data without normal distribution were analyzed by KruskalWallace and Mann Whitney U-test. Correlation analysis was performed using Pearson’s test.
Survival was analyzed by Log-rank (Mantel-Cox) Test. A two-sided p-value <0.05 was
considered significant.
6
References
1.
Endo S, Shinoki T. Influence of reticuloendothelial function on the growth of
transplantable rat ascites hepatoma (AH-130). Tohoku J Exp Med 1968;94(1):81-101.
2.
Tessitore L, Costelli P, Baccino FM. Humoral mediation for cachexia in tumour-
bearing rats. Br J Cancer 1993;67(1):15-23.
3.
Costelli P, Garcia-Martinez C, Llovera M, Carbo N, Lopez-Soriano FJ, Agell N,
Tessitore L, Baccino FM, Argiles JM. Muscle protein waste in tumor-bearing rats is effectively
antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitindependent proteolytic pathway. J Clin Invest 1995;95(5):2367-72.
4.
Costelli P, Muscaritoli M, Bonetto A, Penna F, Reffo P, Bossola M, Bonelli G,
Doglietto GB, Baccino FM, Rossi Fanelli F. Muscle myostatin signalling is enhanced in
experimental cancer cachexia. Eur J Clin Invest 2008;38(7):531-8.
5.
Palus S, Akashi Y, von Haehling S, Anker SD, Springer J. The influence of age and
sex on disease development in a novel animal model of cardiac cachexia. Int J Cardiol
2009;133(3):388-93.
6.
Schmidt K, von Haehling S, Doehner W, Palus S, Anker SD, Springer J. IGF-1
treatment reduces weight loss and improves outcome in a rat model of cancer cachexia. J
Cachex Sarcopenia Muscle 2011;2(2):105-109.
7.
Akashi YJ, Palus S, Datta R, Halem H, Taylor JE, Thoene-Reineke C, Dong J, Thum
T, Culler MD, Anker SD, Springer J. No effects of human ghrelin on cardiac function despite
profound effects on body composition in a rat model of heart failure. Int J Cardiol
2009;137(3):267-75.
8.
Hilfiker-Kleiner D, Kaminski K, Kaminska A, Fuchs M, Klein G, Podewski E, Grote K,
Kiian I, Wollert KC, Hilfiker A, Drexler H. Regulation of proangiogenic factor CCN1 in cardiac
muscle: impact of ischemia, pressure overload, and neurohumoral activation. Circulation
2004;109(18):2227-33.
7
9.
Haghikia A, Missol-Kolka E, Tsikas D, Venturini L, Brundiers S, Castoldi M,
Muckenthaler MU, Eder M, Stapel B, Thum T, Haghikia A, Petrasch-Parwez E, Drexler H,
Hilfiker-Kleiner D, Scherr M. Signal transducer and activator of transcription 3-mediated
regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: a
key role for ubiquitin-conjugating enzymes. Eur Heart J 2011;32(10):1287-97.
8
SUPPLEMENTAL DATA
Control
N
11
NSCLC / GI / pancreatic
Cancer
Cancer cachexia
12
14
4/4/4
3/7/4
male / female
8/3
7/5
6/8
Age
56±5
59±2
60±2
Weight
92.8±5.8 79.6±3.1
51.9±2.7
BMI
30.5±1.9
17.8±0.5
27.70.8
Supplemental table 1: Basic characterization of patients whose cadaver heart weight and
dimensions were recorded. NSCLC: non-small cell lung cancer, GI: gastrointestinal cancer,
BMI: body mass index.
9
Control
N
22
NSCLC / colon
Cancer
Cancer cachexia
32
20
25 / 7
13 / 7
male / female
9 / 13
18 / 14
11 / 9
Age
63±2
59±2
60±2
Weight
75.7±3.5 70.5±4.1
72.9±1.8
BMI
26.1±0.8 23.3±1.2
25.9±0.7
Supplemental table 2: Basic characterization of patients whose plasma aldosterone and
BNP levels were analyzed. NSCLC: non-small cell lung cancer.
10
Tumor-bearing
n
Cell no. x10
9
Sham
Day 5
Day 7
Day 9
Day 11
Day 13
Day 7
Day 11
Day 13
6
10
6
8
12
5
5
5
3.09±0.45
4.21±0.23
6.04±0.46
4.98±0.42
5.36±0.36
-
-
-
858±17**
907±17
726±44**
672±22**
1064±25
1128±35
1218±22
gastrocemius 903±17
[mg]
tibialis [mg]
326±14
337±7***
349±8
282±18**
257±7***
422±4
419±16
434±9
soleus [mg]
77±9
80±3*
80±2
61±2**
71±4**
98±7
101±6
107±4
EDL [mg]
77±9
78±2**
84±2
59±6**
63±3***
94±3
100±3
109±3
White
1.80±0.15
1.29±0.03
1.27±0.09
0.71±0.24*
0.38±0.06**
1.25±0.07
1.60±0.03
1.83±0.07
202±16
1775±9
131±12
78±12**
104±6**
209±12
248±31
348±13
Liver [g]
8.64±0.11
8.78±0.25
9.071±0.24 6.76±0.47*
6.33±0.25**
8.75±0.31
9.38±0.44
10.23±0.20
Activity
63804±5035 44679±4237** 38790±1850 28091±6540** 15528±3068** 62452±3836 69920±4738 60280±3192
adipose
tissue [g]
Brown
adipose
tissue [mg]
[counts/24h]
Food [g/24h] 19.4±0.7
10.3±1.3**
7.5±1.2
2.7±1.9**
5.2±1.5***
21.8±0.9
22.4±0.8
23.4±0.6
Water
22.9±1.7
30.1±3.0
19.2±2.7
20.7±1.9*
25.1±0.6
27.2±1.8
29.7±1.3
27.8±0.7
[mL/24h]
Supplemental table 3: Organ weights on the respective day of necropsy. Quality of life indicators spontaneous activity, food intake and water
intake 24 hours before necropsy. *: p<0.05, **: p<0.01, ***: p<0.001 vs sham on respective days. EDL: extensor digitalis longus.
11
Supplemental Figure 1:
Schematic overview of the study design, a) timeline study, b) intervention study.
12
Supplemental figure 2: Representative electrocardiogram (ECG)-tracings, a loss of
conduction velocity and slower repolarization were observed with cancer cachexia
progression. *: p<0.05, **: p<0.01, ***: p<0.001 vs sham.
13
Supplemental figure 3:
Cardiac function was analyzed on day 11 of the 16 day protocol. When correlated with the
survival day of each placebo-treated rat, a correlation was found between cardiac function
and maximum survival, making cardiac function a predictor of survival.
14
Supplemental figure 4:
The heart failure marker brain derived natriuretic peptide (BNP) was significantly increased
on day 7 and still somewhat elevated on day 9. On day 11 and 13 BNP-levels were
significantly lower compared to sham, which may reflect an exhaustion of BNP synthesis.
Fibrinogen was increased starting day 11, MCP-1 starting day 7 and TIMP-1 starting day 5,
suggesting inflammation and remodeling. Troponin-T and –I were significantly increased on
day 11, suggesting necrosis of cardiomyocytes. Plasma albumin was reduced by cancer
starting day 5. *: p<0.05, **: p<0.01, ***: p<0.001 vs sham.
15
Supplemental figure 5:
Survival proportions. Animals were treated with the 0.5 (n=13), 2 (n=14), 5 (n=23) or 50
mg/kg/d (n= 20) of the b-1 specific beta-blocker bisoprolol, (n=9), 0.4 (n=10), 1 (n=14) or 10
mg/kg/d (n=15) of the ACE-inhibitor imidapril, 2 (n=6), 5 (n=16) or 50 mg/kg/day (n=16) of
the aldosterone antagonist spironolactone or placebo (n=73). The 50mg/kg/d spironolactone
group was chosen for further analysis, because of its superior effects on cardiac function
compared to the 5 mg/kg/d dose.
16
Supplemental figure 6:
An increased activity of the ubiquitin-proteasome system in left ventricular tissue was seen
on day 11, while its activity was reduced on day 5 and 13 compared to sham. The activity of
caspase-3 was lower on day 5 and increased on day 11 and 13 compared to sham.*: p<0.05,
**: p<0.01, ***: p<0.001 vs sham.
17
Supplemental figure 7:
CC increased the activity of the ubiquitin-proteasome system (UPS) in skeletal muscle
(gastrocnemius (GC)) compared to sham. Bisoprolol and spironolactone reduced UPS
activity, while imidapril only inhibited chymostrypsin-like activity of the UPS. PGHP: peptidylglutamyl-protein-hydrolysing. *: p<0.05, **: p<0.01, ***: p<0.001 vs sham.
18
Supplemental figure 8:
a) and b) Signaling was investigated on relevant days; day 7 because the animals met the
clinical definition of cachexia and day 11 because of the greatest cardiac wasting activity and
profound alterations of cardiac function. All data are given as % of the mean sham value a)
A reduced expression of the insulin (IR) and IGF-1 receptor was observed, which should
blunt anabolic signaling. b) Indeed, while we observed an up-regulation of Akt, both
phosphorylated Akt forms were significantly lower in tumor-bearing animals. The expression
of p70S6K was reduced with tumor progression, while the phosphorylated form was higher
on day 7. 4E-BP1 showed an increase of expression, while its phospho-forms were lower in
tumor-bearing animals. SMAD-2 was progressively reduced. Total GSK- and GSK- were
increased on day 7 and 11, while the phospho-forms were reduced on day 11. Total
FOXO3a was unchanged, while the phosphorylation was increased in tumor-bearing
animals. The expression of MuRF-1 was unchanged. N=5-10 per group. *: p<0.05, **:
p<0.01, ***: p<0.001 vs sham.
19