Supplementary Material
Detailed Methods
All materials used were purchased from Sigma Aldrich unless otherwise stated.
Cardiomyocyte isolation
Animals received humane care in accordance with the United Kingdom Home Office Guide on the
Operation of Animal (Scientific Procedures) Act of 1986. The investigation conforms to the guidelines
from Directive 2010/63/EU of the European Parliament on the protection of animals used for
scientific purposes or the NIH guidelines. Male Sprague–Dawley rats were obtained from Charles
River UK Limited (Margate, UK) and anaesthetized by i.p. injection of 160 mg/kg pentobarbitone.
Ventricular cardiomyocytes were prepared from isolated, perfused hearts by collagenase II digestion
and plated on laminin-coated coverslips as previously described 1 and used within 24 h. Cells were
treated with either vehicle (DMSO), Ned-19, Ned-K, or pre-treated 10 min with 10 µmol / L
ryanodine, or 100 nmol / L thapsigargin + 10 mmol / L caffeine.
mPTP assay
We monitored induction of the mPTP opening in live cardiomyocytes using a previously described
model of oxidative stress
1-3
. For each treatment, a total of 10–25 cardiomyocytes were analyzed.
Briefly, cardiomyocytes were loaded with the fluorescent dye tetramethyl rhodamine methyl ester
(TMRM; 3 μM) for 15 min and then washed. Laser stimulation of TMRM generates reactive oxygen
species (ROS) within the mitochondria
4
thereby simulating mitochondrial ROS production during
reperfusion. Induction of mPTP opening is visualized as mitochondrial depolarization and the de
quenching of TMRM fluorescence as it relocates to the cytoplasm
5, 6
. The time until depolarization
and subsequent rigor contracture was recorded. After loading mitochondria with TMRM for 15 min,
cells were washed and treated for 10 min with Ned-19, Ned-K, 0.2 μmol / L ciclosporin A (CsA) or
vehicle (dimethyl sulfoxide, DMSO). 10 μmol / L ryanodine, and 100 nmol / L thapsigargin with 10
mmol / L caffeine. Cells were then scanned and recorded using a Leica SP5 confocal microscope
using the following settings: HeNe 543 nm laser at 40% power; gain 377V; time interval 2.63s; line
average 2; zoom 2. In experiments in which [Ca2+] was also measured, cells were co-loaded with 5
μM Fluo4-AM and 3 μM TMRM as above, and Fluo4 and TMRM fluorescence was measured over
time (Supplementary Fig 6), using the Argon 488 nm laser at 2% power and gain 1074V,
simultaneously with the HeNe laser. Emitted light was collected in the range 500-540nm and 580670nm. Lack of fluorescence bleed-through was confirmed. For each cell, the maximal fluorescence
of Fluo4 (Fmax) immediately before mPTP opening was divided by the starting fluorescence (F0), and
recorded as “Ca2+ increase (Fmax/F0)”.
Mitochondrial swelling assay
Male Sprague-Dawley rats were anaesthetised with pentobarbitone (100 mg/Kg) and the heart was
rapidly removed and perfused with ice-cold saline. The ventricles were cut into small pieces (180200 mg) and homogenised using a glass Potter-Elvehjem homogeniser in “isolation buffer”
containing 100 mM KCl , 50 mM MOPS, 1 mM EGTA, 5 mM MgSO4 and 1 mM ATP at pH 7.4. 0.5%
trypsin was added to aid tissue breakdown during shaking for 10 min, after which time, 0.2% fattyacid free bovine serum albumin (BSA) was added to homogenates to quench the protelolytic activity
of the trypsin. Homogenates were then centrifuged at 1000 g at 4˚C for 10 min to pellet unbroken
tissue and nuclei, supernatants were then collected and spun at 5000 g at 4˚C for 10 min to pellet
the mitochondrial fraction. The mitochondrial pellets were resuspended in 100 µl of isolation buffer
supplemented with 0.2% fatty-acid free BSA, and combined into one tube to form one pooled
sample of mitochondria per heart which was given a final spin at 5000 g for 10 min at 4˚C. The final
single pellet was resuspended in mitochondrial swelling buffer containing 150 mM KCSN, 20 mM
MOPS, 10 mM Tris and 2 mM NTA supplemented with 0.5 µM rotenone, 0.5 µM antimycin A and 0.2
µM Ca2+ ionophore A23187. Mitochondrial yield was estimated using a BCA protein assay using BSA
as the standard. Mitochondrial swelling was assessed by measuring the absorbance of the
mitochondrial suspension (0.5 mg/ml) at 520 nm after the addition of 500 µM free Ca2+ in the
presence or absence of cyclosporine A (1 µM). The effects of adding 10 µM Ned19 and 10 µM NedK
were investigated with DMSO as vehicle control. Data are expressed as the percentage change in
the initial absorbance over 17 min.
Confocal imaging of reperfusion-induced Ca2+ oscillations
Cells were loaded for 30 min at 37 °C with 5 µM fluo-4-AM in the presence of 0.1% Pluronic, in
imaging buffer (156 mmol / L NaCl, 3 mmol / L KCl, 2 mmol / L MgSO4.7H20, 1.25 mmol / L K2HPO4, 2
mmol / L CaCl2, 10 mmol / L HEPES, 10 mmol / L D-Glucose). Cells were washed with imaging buffer
and then the coverslip was mounted in a gassed, 37 °C chamber on the stage of the confocal
microscope. To simulate ischemia and reperfusion, the buffer on the cells was replaced with
ischemic buffer containing 128 mmol / L NaCl, 14.8 mmol / L KCl, 1.2 mmol / L MgSO4, 1.2 mmol / L
K2HPO4, 1 mmol / L CaCl2, 2.2 mmol / L NaHCO3, 10 mmol / L Na-lactate, that had been made hypoxic
by gassing with 95% N2 / 5% CO2 for pH 6.4 at 37 °C (adapted from 7). Cells were imaged for 60 min,
then the buffer was changed to normoxic buffer containing 118 mmol / L NaCl, 2.6 mmol / L KCl, 1.2
mmol / L MgSO4, 1.2 mmol / L K2HPO4, 1 mmol / L CaCl2, 22 mmol / L NaHCO3, 10 mmol / L DGlucose, gassed with 95% O2 / 5% CO2 for pH 7.4 at 37 °C and imaged for a further 20 min. Average
fluorescence value were collected using a ROI over each cell in the field of view. A Ca2+ oscillation
was defined as having occurred when the coefficient of variation in a moving window of 3 frames
was greater than 0.3 (i.e.:
> 0.3). The value 0.3 was empirically determined as
sufficient to exclude background Ca2+ variation in control cells, whilst detecting the fluorescent
excursions that occur during occasional spontaneous contractions. The average number of Ca2+
oscillations in each cell was evaluated during the first 7 min of reoxygenation. The number of Ca2+
oscillations over time (Fig 2G) was determined by averaging the number of oscillations detected in
all cells at each time point, and graphed with smoothing window of 1 second for clarity (Fig 2H).
Confocal imaging of Ca2+ sparks and transients
Cells were incubated for 10 min in imaging buffer containing the indicated drugs. Rapid line scans
were performed (3 line scans per cell at different regions of the cell) using confocal settings: sizewidth: 82 µm, voxel-width: 160.5nm, scan length: 2.56s, number of lines: 1024, resolution: 512x512,
laser: HeNe 488 nm at 10% power
“Sparkmaster” plugin
10
8, 9
. The images were then analyzed using ImageJ using the
to detect Ca2+ sparks, using settings: background (FI. U.): 12, criteria: 3.8,
number of intervals: 1. The spark detection algorithm defines potential sparks as being regions of
fluorescence that are two times the standard deviation above the mean. Such regions are
acknowledged as sparks if there are pixel values greater than the standard deviation multiplied by a
threshold factor plus the background mean
10
. Ca2+ transients were stimulated by electrical field
stimulation with platinum electrodes (square pulses, 1 Hz, 1 ms, 5 V/cm). The change in Ca2+
transients amplitude after drug treatment for 5 min was calculated by the formula (FmaxFbaseline)t=10min/(Fmax-Fbaseline)t=0min .
Confocal imaging of SR Ca2+ content
Each coverslip was incubated with fluo4-AM + 0.1% pluronic at 37 °C for 30 min in imaging buffer.
Drugs were added and incubated for 10 min as above. A camera (RT power supply, Diagnostic
Instruments, Inc., SPOT) linked to a fluorescence microscope (Nikon Eclipse TE200), and with the
SPOT Advanced software, was used to take a series of sequential images every 40 ms commencing
before the addition of the calcium release stimulant. 10 mmol / L Caffeine was used to trigger Ca2+
release from the sarcoplasmic reticulum. Drugs added were DMSO, Ned-19 at 100 μmol / L, 10 μmol
/ L, 1 μmol / L and 0.1 μmol / L, Ned-20 at 100 μmol / L , 10 μmol / L and 1 μmol / L , 10 μmol / L
ryanodine, and 100 nmol / L thapsigargin. Cells were first exposed to caffeine, then rinsed with
buffer and allowed to recover, then exposed to drug for 10 min before a second exposure to
caffeine, and the difference in total Ca2+ release determined using ImageJ.
Cell survival after simulated ischemia and reperfusion
After sIR, the cells were stained with 1 mg/ml propidium iodide (PI) of each well were counted and
scored as either rod-shaped (live), or hypercontracted and PI +ve (dead).
In vivo myocardial infarction
These experiments used C57Bl/6J male mice aged 8-12 weeks, or TPC1 knockout mice (B6;129S5Tpcn1Gt(OST359423)Lex) that were generated by Lexicon Genetics and have been previously
described11, 12. TPC1 mice were were backcrossed onto C57Bl/6J mice for 3–4 generations before
experiments. RT-PCR analysis and Western blot analysis demonstrated complete absence of TPC1
transcript and protein11, 12. For the in vivo model of ischaemia and reperfusion injury, mice were
anesthetized by intraperitoneal injection (0.01 mL/g) of a solution containing ketamine 10 mg/mL,
xylazine 2 mg/mL, and atropine 0.06 mg/mL. The left anterior descending (LAD) coronary artery was
occluded for 30 minutes followed by 120 minutes of reperfusion, at the end of which myocardial
infarct size was determined by triphenyl-tetrazolium chloride (TTC) and Evans blue staining to
demarcate the infarct area (IA) and the area at risk (AAR), respectively, while blinded to treatment.
Planimetry was then performed to calculate the infarct area as a percentage of area at risk using
Image J software. The infarct area was expressed as a percentage of the AAR (%IA/AAR). Mice were
randomly assigned to receive by intravenous injection either vehicle control (0.1 mL of 0.1%
dimethyl sulfoxide in saline), or vehicle containing 0.36 μg/kg Ned-19 or 0.36 μg/kg Ned-K, 5 minutes
before reperfusion (n=5 mice per treatment group). The in vivo doses were calculated on the basis of
being equivalent to the 10 μM concentration effective in vitro; however, the pharmacokinetic
properties of the drug in vivo are unknown.
Assessment of NAADP levels. C57Bl/6 male mice (8-12 weeks old) were given a lethal injection of
pentobarbitone (100 mg/Kg), the hearts were rapidly excised and perfused using a Langendorff
apparatus with Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 25 mM NaHCO3,
1.2 mM MgSO4, 11 mM glucose and 1.8 mM CaCl2). After stabilisation, hearts were subjected to 30
minutes of global ischaemia, followed by either 0, 5 or 10 minutes of reperfusion. The ventricles
were then immediately frozen in liquid nitrogen and stored at -80˚C until further use. NAADP was
extracted from ventricular homogenates using perchloric acid with differential centrifugation and
NAADP levels were quantified using an enzymatic cycling assay, as previously described 13.
Chemical synthesis
transNed-19 was made as described previously 14.
Formation of Ned-K ester.
CO2 Me
N
NH
L-trp-OMe ester
CN
N
N
H
OMe
A Reacti-vial containing an acetic acid (2 mL) solution of the free base of L-tryptophan methyl ester
(0.180 g, 0.82 mol), and 3-chloromethyl anisaldehyde (0.13 g, 0.68 mol) was heated with stirring at
75oC (block temperature) for an hour before the contents were cooled to room temperature, and
azeotroped with toluene (3 x 20 mL) to remove the acetic acid. The residue was treated with cold
sodium hydroxide (10%; 2 x 10 mL), and extracted with dichloromethane, before the organic phase
was dried over sodium sulfate, and concentrated to a yellow gum.
Diisopropylethylamine (0.35 mL, 2.0 mol) was added to a dichloromethane (3 mL) solution of the
residue from the reaction above, followed by the addition of the 2-(piperazinyl)benzonitrile (0.125 g,
0.68 mol), and the contents stirred for 6 hours, after which the reaction mixture was diluted with
dichloromethane (5 mL) and water (2 mL) and passed through a phase extractor. The organic phase
was concentrated under reduced pressure to a gum, which was chromatographed (SiO2;
dichloromethane : methanol = 19 : 1) to afford the desired ester as a white foam (122 mg, 27%); 1H
NMR (300 MHz. CDCl3): = 2.65 (br, 4H), 3.02 (br, 5H), 3.24 (dd, J = 15.4 and 5.1 Hz, 1H), 3.61 (ABq,
2H), 3.71 (s, 3H), 3.80 (s, 3H), 3.89 (dd, J = 8.4 and 5.1 Hz, 1H), 4.95 (s, 1H), 5.31 (s, 0.65H, NH), 6.80
(m, 2H), 6.95 (m, 2H), 7.15 (m, 3H), 7.30 (d, J = 7 Hz, 1H), 7.47 (ddd, J = 8.4, 7.4 and 1.8 Hz, 1H), 7.53
(m, 2H); 13C NMR (75 MHz, CDCl3): = 24.9, 51.2, , 51.8, 52.2, 53.0, 54.5, 55.6, 55.7, 105.8, 108.4,
110.2, 111.0, 118.2, 118.5, 118.7, 119.3, 121.6, 121.8, 126.0, 126.9, 128.2, 131.3, 133.4, 133.7,
134.0, 134.3, 136.4, 155.7, 157.4, 174.1.
Optical rotation value: -0.283o (5.70 mg in 2mL MeOH, 27oC).
Formation of Ned-K.
CO2 H
CO2 Me
N
NH
N
N
H
N
NH
CN
CN
N
N
H
OMe
OMe
A stirred THF (1 mL) solution of the ester (0.045 g, 0.084 mol) was treated with an aqueous solution
of lithium hydroxide (2M, 0.090 mL, 0.18 mol), before the contents were left to stir for 6 hours at
room temperature, and the residue diluted with water (2 mL), chilled in ice-bath and made neutral
with hydrochloric acid (2M). The resulting orange suspension was treated with methanol (3 mL),
before preactivated Amberlyst-15 resin (1.0 g) was added and the suspension stirred for an hour and
filtered through a phase extraction cartridge. The resin was washed with methanol, followed by
methanolic-ammonia (7M), and the ammonical fractions were collected and concentrated under
reduced pressure to obtain a yellow glass which was dried in-vacuo at 70oC; (0.031 g, 72%); 1H NMR
(300 MHz. CD3OD): = 2.67 (br, 4H), 2.95 (br, 4H), 3.25 (br, 1H), 3.43 (dd, J = 16.2 and 5.5 Hz, 1H),
3.67 (s, 2H). 3.84 (s, 3H), 4.02 (dd, J = 8.4 and 5.5 Hz, 1H), 5.98 (s, 1H), 6.92 (d, J = 8.1 Hz, 1H), 7.01
(m, 4H), 7.26 (m, 3H), 7.50 (m, 3H);
C NMR (75 MHz, CDCl3): = 24.05, 52.08, 53.80, 54.70, 56.19,
13
56.24, 56.28, 107.02, 109.01, 112.12, 112.43, 119.31, 120.27, 120.53, 123.48, 123.60, 126.19,
127.41, 127.84, 129.27, 132.02, 133.63, 135.29, 135.34, 138.69, 156.80, 160.58, 173.9. MS (+ESI) m/z
522 (M + H)+. HRMS: calcd for C31H32FN5O3 522.2500 [M + H]+, found 522.2505.
Optical rotation value: -0.135o (3.0 mg in 2mL MeOH, 23oC).
Sea urchin assay of Ca2+ release inhibition
Sea urchin egg homogenates (50%, v/v) were prepared according to standard methods as described
previously 15 and stored at -80 °C until required. For the sea urchin Ca2+ release assay, homogenates
were diluted to 2.5% (v/v) with intracellular-like medium supplemented with an ATP regeneration
system (1 mmol / L MgATP, 10 mmol / L creatine phosphate, 10 units/ml creatine phosphokinase).
Ca2+ release was measured by cuvette-based fluorimetry using the Ca2+ indicator fluo-3 (3 µM,
Invitrogen). All agonist and drugs (from Sigma) were used from 100 X stock solutions.
Statistics
All values are expressed as means ± S.E.M. Data were analyzed by one-way ANOVA followed where
significant by post-hoc analysis using Dunnett test for comparisons solely against control values, or
Tukey test for multiple comparisons.
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