Online Appendix for the following JACC article
TITLE: Dietary Nitrate Supplementation Protects Against Doxorubicin-Induced
Cardiomyopathy by Improving Mitochondrial Function
AUTHORS: Shu-Guang Zhu, MD, PHD, Rakesh C. Kukreja, PHD, Anindita Das, PHD,
Qun Chen, MD, PHD, Edward J. Lesnefsky, MD, FACC, FAHA, Lei Xi, MD
APPENDIX
Detailed Methods
Animals
Adult male CF-1 outbred mice (weighed 30 to 42 g) were purchased from Harlan Sprague
Dawley Inc. (Indianapolis, Indiana). The animal experimental protocol was approved by the
Institutional Animal Care and Use Committee of the Virginia Commonwealth University. All
animal experiments were conducted under the guidelines on humane use and care of laboratory
animals for biomedical research published by the National Institutes of Health (No. 85-23,
Revised 1996).
1
Experimental protocol
As illustrated in Figure 1A, the adult male CF-1 mice were administered a single dose of DOX
intraperitoneally (15 mg/kg, dissolved in saline), which was purchased from Sigma-Aldrich, St.
Louis, MO; DOX group; n=8) or volume-matched saline (0.2 mL; Control group; n=8). Another
group of mice (Nitrate+DOX group; n=8) received chronic nitrate supplementation with NaNO3
added into their drinking water at the concentration of 1 g/L (12 mM) for 7 days before the DOX
injection on Day 8. The nitrate treatment regimen was continued throughout the post-DOX
period. Finally, a group of mice were given nitrate supplementation alone for the entire 13-day
experimental period (Nitrate group; n=8). We adopted the oral dose of NaNO3 that was
previously shown to be cardioprotective against ischemia-reperfusion injury in vivo (24). Five
days later (i.e. on Day 13), left ventricular (LV) function was assessed with echocardiography
under light anesthesia and subsequently with a Millar catheter inserted into the LV cavity under
surgical anesthesia. This 5-day post-DOX time point was chosen because it is more than 5 times
greater than the half-life of DOX in plasma and cardiac tissue in mice (32), which allows to
study the cumulative detrimental effects of DOX on LV function, rather than its direct
cytotoxicity.
To exclude/identify bioconversion of nitrate by bacteria in the mouth and lower
gastrointestinal tract, a subset of mice were subcutaneously implanted with a micro-osmotic
pump (Alzet® model 1002, 100 µL volume; supplied by Durect Corp., Cupertino, CA), which
continuously delivered NaNO3 solution (1 g/L concentration) or saline (as sham controls) at the
rate of 0.25 µL/hour for 13 days – the same treatment duration used for oral nitrate intake
(Figure 1A). The nitrate as well as saline infused mice received DOX injection (15 mg/kg, i.p.)
on Day 8 (n=7 per group).
2
Measurement of DOX cytotoxicity in vitro
As illustrated in Figure 1B, the CF-1 mice with or without 7 days of oral nitrate supplementation
were sacrificed on Day 8 and cardiomyocytes were isolated as reported previously (33). The
freshly isolated mouse cardiomyoctytes were exposed to either normal culture conditions or 1
µM DOX added into the cell culture medium. Myocyte necrosis and apoptosis were quantified
18 hours later with trypan blue exclusion and TUNEL assays, respectively, as previously
described (33).
Echocardiographic assessment of ventricular contractile function
Echocardiography was performed using the Vevo770TM imaging system (VisualSonics Inc.,
Toronto, Canada) as described previously (34). In brief, under light anesthesia (pentobarbital, 30
mg/kg, i.p.), mice were placed in the supine position. A 30-MHz probe was utilized to obtain Mmode from parasternal short-axis view at the level of the papillary muscles according to the
American Society of Echocardiography recommendation. The LV end-systolic and end-diastolic
diameters (i.e. LVESD and LVEDD) were measured. The LV fractional shortening (FS) was
subsequently calculated as follows: FS = (LVEDD-LVESD)/LVEDD×100. The ejection fraction
was calculated with the Teichholz formula.
Measurement of LV hemodynamics
Following the echocardiographic assessment, a micro-tip pressure-volume catheter transducer
(Millar instruments Inc., Houston, Texas; Model SPR-1045) was inserted into the right carotid
artery and advanced into LV cavity. After stabilization for 15 to 20 min, the signals were
continuously recorded with a MPVS-300 system (Millar Instruments, Houston, Texas) coupled
3
with a Powerlab 8/30 converter (AD Instruments, Colorado Springs, Colorado), stored and
displayed on a computer. LV systolic and end-diastolic pressures, maximal slope of systolic
pressure increment (+dP/dtmax) and diastolic pressure decrement (-dP/dtmax), heart rate, and aortic
blood pressure were recorded on a beat-by-beat basis.
Measurement of plasma levels of nitrate and nitrite
The blood samples were collected from the mice that underwent four different treatment
conditions (n=6 per group) and centrifuged to obtain the supernatant plasma. Plasma samples
were subsequently centrifuged using Amicon Ultra-4 centrifugal filter devices at 7500 g in 4oC
to eliminate large molecules (molecular weight >30 kDa) from the plasma. The plasma nitrate
and nitrite were measured with a SIEVERS nitric oxide analyzer (model 280NOA). The reducing
agents used were either vanadium (III) chloride (VCl3) in 1 M HCl (for nitrate) or 1% sodium
iodide (NaI) in glacial acetic acid (for nitrite). Five mL of a reagent plus 100 µL of 1:30 diluted
anti-foaming agent were loaded into the purge vessel for analysis. These reducing agents
converted nitrite and nitrate respectively to gaseous NO at 90°C, which was quantified by the
analyzer.
Isolation of cardiac mitochondria
The mitochondria were treated with protease (trypsin) to maximize the mitochondrial protein
yield. The heart was quickly isolated and placed in cold buffer A (composition in mM: 100 KCl,
50 MOPS [3 (N morpholino) propanesulfonic acid], 1 EGTA, 5 MgSO4, 1 ATP). The heart
tissue was homogenized in buffer A using a Polytron tissue homogenizer at a setting of 3.5 for
2.5 seconds. The homogenates were incubated with trypsin (5 mg/g tissue) for 10 min at 4oC,
4
followed by the addition of same volume buffer B [buffer A + 0.2% bovine serum albumin] and
centrifugation at 500 g for 10 min. The supernatant was recovered and centrifuged at 3000 g to
pellet mitochondria. The mitochondrial pellets were then washed with buffer B and KME (100
mM KCl, 50 mM MOPS, 0.5 mM EGTA) and resuspended in KME for analysis.
Measurement of mitochondrial oxidative phosphorylation and enzyme activities
Oxidative phosphorylation in the isolated mitochondria was studied using glutamate+malate
(complex I) and succinate (complex II) as substrates to localize defects within the ETC (35).
Maximal enzyme activities of the respective ETC complexes were measured. The isolated
mitochondria were treated with cholate and resuspended into buffer containing 75 mannitol, 220
sucrose, 1 EDTA (in mM, pH 7.4). Enzyme activities of complex I (NADH:duroquinone
oxidoreductase) and NADH dehydrogenase (proximal segment of complex I) were determined
(36).
Assessment of lipid peroxidation in cardiac tissues
Lipid peroxidation in the cardiac tissues was determined by measuring malondialdehyde and 4hydroxyalkenals using a colorimetric assay kit (Bioxytech LPO-586, Oxis International, Foster
City, CA). The frozen tissue samples (n=6 per group) were ground in liquid nitrogen into fine
powder and homogenized in ice cold PBS containing 5 mM butylated hydroxytoluene in
acetonitrile. The homogenate was centrifuged at 3000 g at 4°C for 10 min. 200 µL of clear
supernatant containing protein extracts were used for the assay following the manufacturer’s
instruction.
5
Measurement of H2O2 in mitochondria
The rate of H2O2 generation in isolated mitochondria (n=5 per group) was determined using the
oxidation of the fluorogenic indicator amplex red (25 μM) in the presence of horseradish
peroxidase (HRP, 0.1 U/ml) (37). Fluorescence was recorded in a fluorimeter (LS-55, Perkin
Elmer, Boston, MA) with 530 nm excitation and 590 nm emission wavelengths. Standard curves
obtained by adding known amounts of H2O2 to assay medium in the presence of the reactants
were linear up to 2 µM. The increased fluorescence intensity was converted to the concentration
of H2O2 (pmol/L) and the rate of H2O2 production was calculated as (pmol/min/mg protein)
using the delta change of H2O2 concentration over time. H2O2 production was initiated in
mitochondria using glutamate (10 mM) + malate (5 mM) as complex I substrate and succinate
(10 mM) + rotenone (2.4 M) as complex II substrate.
Statistical analysis
Data are presented as group mean±standard error (SEM). Statistical analysis was performed
using one-way ANOVA with subsequent Student-Newman-Keuls post hoc test for pair-wise
comparison among the four treatment groups. Unpaired t test was used for comparing two
treatment groups in the study with subcutaneous infusion of nitrate. Probability value of P<0.05
was considered significant.
6
Supplemental Figure 1. Experimental protocol
(A): In vivo study protocol to determine left ventricular contractile dysfunction and cardiac
mitochondrial respiratory chain damage caused by DOX with or without chronic nitrate
supplementation. (B): Protocol for in vitro studies in adult cardiomyocytes to assess the cell
viability following exposure to DOX with or without chronic nitrate supplementation.
A
Day 8
Day 13
I.P. injection of
Doxorubicin (15 mg/kg)
or Saline
Echocardiography
Millar catheter
Heart tissue samples
Blood samples
Day 1
Adult male CF-1
outbred mice
With or without 1 g/L NaNO3 in the animals’ drinking water
B
Day 8
Day 9
Heart isolation and
enzymatic digestion for
isolating and culturing
ventricular myocytes
Necrosis
(Trypan blue)
Apoptosis
(TUNEL)
Day 1
With or without Doxorubicin
(1 M) in culture medium
for 18 hours
Adult male CF-1
outbred mice
With or without 1 g/L NaNO3
in the animals’ drinking water
7
Supplemental Figure 2.
Effect of chronic subcutaneous infusion of nitrate on DOX-
induced ventricular dysfunction and plasma levels of nitrate and nitrite
The left ventricular function was accessed with Millar micro-tip catheter: i.e. systolic pressure
(A), end-diastolic pressure (B), positive, and negative dP/dtmax (C, D). Levels of nitrate (E) and
nitrite (F) were measured with Sievers NO analyzer. Data are Mean±SEM (n=7 per group).
* indicates P<0.05 versus DOX group. An arrow dashed line is inserted in each of the graphs to
indicate the average level of each parameter in the control mice without DOX treatment as
shown in Fig. 4 and Fig. 5.
A
B
125
10.0
Control Level
7.5
75
50
(mmHg)
LVEDP
Control
Level
5.0
2.5
25
O
D
it r
N
N
it r
at
e+
at
e+
D
D
D
O
X
O
X
0.0
X
0
O
X
LVSP
(mmHg)
100
C
D
10000
8000
7000
Control Level
Control Level
6000
-dP/dt
6000
4000
(mmHg/s)
dP/dt
(mmHg/s)
8000
5000
4000
3000
2000
2000
1000
0
X
F
5
70
*
40
30
Control
Level
20
( M)
4
50
Nitrite in Plasma
60
3
Control
Level
2
1
O
X
O
X
itr
N
N
itr
at
e
at
e+
D
+D
D
X
O
O
X
0
10
D
( M)
O
D
N
itr
at
e+
it r
N
E
Nitrate in Plasma
X
O
D
at
e+
D
D
O
X
O
X
0
8