Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist Rescue from complete heart block: Pharmacological and genetic evidence for direct s1p3 regulation of mouse cardiac conduction

Article abstract of DOI:10.1124/mol.115.100222

The molecular pharmacology of the G protein-coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3 antagonist, SPM-354, with in vivo activity, has been used,

Full text of DOI:10.1124/mol.115.100222

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2015/10/22/mol.115.100222.DC1.html
1521-0111/89/1/176–186$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.115.100222
Mol Pharmacol 89:176–186, January 2016
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist
Rescue from Complete Heart Block: Pharmacological and
Genetic Evidence for Direct S1P3 Regulation of Mouse
Cardiac Conduction s
M. Germana Sanna,1 Kevin P. Vincent,1 Emanuela Repetto,1 Nhan Nguyen,
Steven J. Brown, Lusine Abgaryan, Sean W. Riley, Nora B. Leaf, Stuart M. Cahalan,
William B. Kiosses, Yasushi Kohno, Joan Heller Brown, Andrew D. McCulloch, Hugh Rosen,
and Pedro J. Gonzalez-Cabrera
Departments of Chemical Physiology (M.G.S., E.R., N.N., H.R., P.J.G.-C.), Immunology (N.B.L.), and Molecular and Cellular
Neuroscience (S.M.C.), Scripps Research Institute Molecular Screening Center (S.J.B., L.A., S.W.R.), Microscopy Core (W.B.K.),
Scripps Research Institute, La Jolla, California; Kyorin Pharmaceutical Company, LTD, Tokyo, Japan (Y.K.); and Departments of
Bioengineering (A.D.M., K.P.V.) and Pharmacology, University of California, San Diego, California (J.H.B.)
Received June 8, 2015; accepted October 20, 2015
ABSTRACT
The molecular pharmacology of the G protein–coupled recep- described here. Homologous knockin of S1P3-mCherry is fully
tors for sphingosine 1-phosphate (S1P) provides important functional pharmacologically and is strongly expressed by
insight into established and new therapeutic targets. A new, immunohistochemistry confocal microscopy in Hyperpolar-
potent bitopic S1P3 antagonist, SPM-354, with in vivo activ- ization Activated Cyclic Nucleotide Gated Potassium Chan-
ity, has been used, together with S1P3-knockin and S1P3- nel 4 (HCN4)-positive atrioventricular node and His-Purkinje
knockout mice to define the spatial and functional properties fibers, with relative less expression in the HCN4-positive
of S1P3 in regulating cardiac conduction. We show that S1P3 sinoatrial node. In Langendorff studies, at constant pressure,
is a key direct regulator of cardiac rhythm both in vivo and in SPM-354 restored sinus rhythm in S1P-induced complete
isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl] heart block and fully reversed S1P-mediated bradycardia.
propane-1,3-diol in vivo and S1P in isolated hearts induced a S1P3 distribution and function in the mouse ventricular
spectrum of cardiac effects, ranging from sinus bradycardia cardiac conduction system suggest a direct mechanism for
to complete heart block, as measured by a surface electro- heart block risk that should be further studied in humans. A
cardiogram in anesthetized mice and in volume-conducted richer understanding of receptor and ligand usage in the
Langendorff preparations. The agonist effects on complete pacemaker cells of the cardiac system is likely to be useful in
heart block are absent in S1P3-knockout mice and are re- understanding ventricular conduction in health, disease, and
versed in wild-type mice with SPM-354, as characterized and pharmacology.
prodrug fingolimod [2-amino-2-[2-(4-octylphenyl)ethyl]propane-
1,3-diol (FTY720)] is an effective oral therapy for the treatment
of relapsing, remitting multiple sclerosis by altering lympho-
Introduction
Five high-affinity G protein–coupled receptors for sphingo-
sine 1-phosphate (S1P) have been identified (Rosen et al.,
cytic and central functions (Cohen et al., 2010; Kappos et al.,
2013), and the crystal structure of S1P1 has been solved
2010). Five (S1P1–5) subtypes that differ in spatial distribu-
(Hanson et al., 2012). This receptor cluster is medically
tion, coupling, and function can, singly or in combination, play
important because the nonselective Sphingosine 1-Phosphate
complex roles in the embryonic formation of the arterial
media, blood pressure regulation, and cardiac function
[reviewed by Mendelson et al. (2014)]. For example, FTY720
in humans is associated with significant sinus bradycardia
and prolongation of the QTc interval (Schmouder et al., 2006;
Kappos et al., 2010; Espinosa and Berger, 2011; Yagi et al.,
2014). FTY720 treatment has additionally revealed an
receptor (S1P-R; S1P1, S1P3, S1P4, and S1P5) modulator
This work was supported by the National Institutes of Health [Grant 5U54
MH084512 to H.R. and Grants 1RO1HL105242 and 1R03EB014593 to A.D.M]
and Kyorin Pharmaceuticals [Grant SFP-1499 to H.R.].
1M.G.S., K.P.V., and E.R. contributed equally to this work.
dx.doi.org/10.1124/mol.115.100222.
s
This article has supplemental material available at molpharm.
aspetjournals.org.
ABBREVIATIONS: AV, atrioventricular; AVN, atrioventricular node; bpm, beats per minute; CCS, cardiac conduction system; CF, coronary flow;
CHB, complete heart block; ECG, electrocardiogram; FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol; HCN4, cyclic nucleotide-gated
K+ channel 4; IHC, immunohistochemistry; KI, knockin; KO, knockout; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline 1 0.5%
Triton X-100; PCR, polymerase chain reaction; RT, room temperature; SAN, sinoatrial node; S1P, sphingosine 1-phosphate; WT, wild type.
176

S1P3 Regulation of Murine Complete Heart Block
177
and iodotrimethylsilane (117 ml, 0.822 mmol) was slowly added at 0°C
under an argon atmosphere. After stirring for 30 minutes at 0°C, the
reaction mixture was poured into water (10 ml), and then the resultant
mixture was stirred for 1 hour at 0°C. The precipitate was filtered,
washed with water, and dried in vacuo at 50°C. The crude residue was
dissolved in MeOH and stirred for 30 minutes at RT (room temper-
ature), and then the mixture was filtered and insoluble materials were
washed with MeOH (5 ml). The filtrate was concentrated in vacuo. The
residue was suspended in MeCN and stirred for 30 minutes at RT, and
then the resultant precipitate was filtered, washed with MeCN, and
dried in vacuo at 50°C to give SPM-354 (50 mg, 0.0973 mmol) as a
colorless powder (m.p.: 156–159°C). [a]D25: 27.67 (c 0.50, MeOH).
1H NMR (dimethylsulfoxide-d6-dTFA, 400 MHz):d 0.90 (3H, t, J 5 7.3
Hz), 1.26–1.40 (2H, m), 1.54–1.66 (2H, m), 1.70–1.82 (2H, m), 2.61–
2.75 (2H, m), 3.86–3.97 (2H, m), 7.10 (1H, d, J 5 7.9 Hz), 7.19 (1H, dd,
J 5 7.9, 1.8 Hz), 7.27 (1H, d, J 5 1.8 Hz), 7.34 (1H, d, J 5 7.9 Hz), 7.40
(1H, d, J 5 1.8 Hz), and 7.56 (1H, d, J 5 8.6 Hz). high-resolution
electrospray ionisation mass spectrometry (1): 514.08255 (calculated
for C20H25ClF3NO5PS, 514.08317). Analysis calculated for
C20H24ClF3NO5PS. 0.1H2 O: C, 46.74%; H, 4.71%; and N, 2.73%.
Found: C, 46.58%; H, 4.73%; and N, 2.72%. SPM-354 is reference
example 47 in WO2011004604 (Yasushi et al., 2011).
SPM-354 Selectivity. Receptor activation through beta-arrestin
recruitment (TANGO; Life Technologies, Carlsbad, CA) was used to
determine SPM-354 selectivity. U2OS cells stably expressing individ-
ual S1P receptors (S1P1–5) were seeded on 384-well plates at 10,000
cells per well and incubated for 48 hours according to the manufac-
turer’s protocol. SPM-354 in 1% fatty acid free bovine serum albumin
was added at the indicated concentrations to incubate for 1 hour. The
S1P or selective S1P3 agonist CYM-5541 concentrations were added to
the wells and incubated for 4 hours at 37°C, followed by the addition of
the LiveBLAzer reagent. Following 2 hours, the plates were read on an
EnVision (Perkin Elmer, Waltham, MA) plate reader to measure S1P
or CYM-5541 activity, blue (410 nm excitation, 460 emission) to green
(410 excitation, 535 emission), ratios. For Schild analyses in S1P1 and
S1P3 cells, the S1P or CYM-5541 concentration response curves were
fit using sigmoidal nonlinear regression in GraphPad Prism software
(GraphPad Software, Inc., La Jolla, CA), and the resulting EC50 values
were compared with no-ligand controls to generate the dose ratios.
These were graphed in the Schild’s plot versus the log [M] of SPM-354,
yielding the pA2 values.
increased incidence of atrioventricular (AV) disturbances
during the first dose, including AV block and asystole,
particularly in subgroups of patients with preexisting cardiac
conditions that require other therapeutics, i.e., beta blockers
and calcium channel blockers, to maintain adequate cardio-
vascular dynamics (Vanoli et al., 2014). Atropine reversal of
the sinus bradycardia (Kovarik et al., 2008) and the demon-
stration of sinus bradycardia with S1P1/5-selective agonists in
humans (Legangneux et al., 2013) as well as rodents (Fryer
et al., 2012) suggest that sinoatrial node (SAN) effects and
those events resulting from alterations in AV-nodal conduc-
tion might be distinctly regulated.
There is little evidence to date concerning actual protein
expression for the distinct S1P-R subtypes in the mammalian
cardiac conduction system (CCS), a highly differentiated,
neural-crest derived system responsible for initiating (SAN)
and propagating [atrioventricular node (AVN) and His-
Purkinje system] synchronous cardiac contractions. Pharma-
cologically, S1P-R activation by S1P, FTY720, and selective
S1P1/5 agonists all induce cardiac depression, both at the
level of the SAN (Guo et al., 1999) as well as by activating
cardiomyocyte S1P1 G protein-coupled inwardly-rectifying
potassium channel currents (Bunemann et al., 1995; Fryer
et al., 2012; Legangneux et al., 2013). At the AVN level, in
rats, FTY720 was recently shown to elevate the PR-interval
duration or total AV conduction time, which defines first-
degree AV block (Egom et al., 2015). In the same study, the
authors also demonstrated the presence of S1P1–3 transcripts
in AVN, suggesting a direct link between FTY720’s PR-
interval augmentation and S1P1–3 engagement in situ.
The analysis of in vivo S1P3 cardiac function can be
confounded by its high expression on vascular smooth muscle
(Forrest et al., 2004), where, functionally, it can result in
vasoconstriction to S1P (Salomone et al., 2003, 2008) or, in the
case with FTY720, an acute overall increase in total periph-
eral resistance (Fryer et al., 2012). Consistent with the role
of S1P3 in vasoconstriction, Murakami et al. (2010) demon-
strated that the coronary flow (CF) rate reduction observed
following S1P perfusion in rat Langendorff heart preparations
was fully sensitive to S1P3 antagonist reversal.
Animals. Experiments were conducted in accordance with the
National Institutes of Health Guide for the Care and Use of Lab-
oratory Animals, following approved protocols by the Institutional
The detailed understanding of S1P3 signaling in the CCS Animal Care and Use Committee at the Scripps Research Institute
(La Jolla, CA) and the University of California, San Diego. Wild-type
(WT) C57BL/6J male mice were obtained from the Scripps Research
Institute in-house breeding colony. Experiments with KO and KI mice
included 8–12 week old male, C57BL/6J background animals bred for
at least 20 generations. Isolated heart experiments were conducted
with 8–12 week old male C57BL/6J mice and obtained from the
Jackson Laboratory (Bar Harbor, ME) and age-matched male KO
mice.
remains incomplete. We have approached the problem of
defining receptor usage in distinct and highly differentiated
cell populations that are difficult to study ex vivo by building a
tool set of both genetic and pharmacological tools that together
define S1P3 distribution and function. In this report, we
characterize a novel bitopic S1P3 antagonist, SPM-354, which
competes for binding in both the orthosteric and allosteric
sites, as defined by the natural ligand and the selective
allosteric S1P3 agonist CYM-5541, respectively (Parrill
et al., 2000; Schürer et al., 2008; Jo et al., 2012). Using SPM-
In Vivo Electrocardiogram Recordings. Electrocardiograms
(ECGs) were recorded in Avertin (240 milligram per kilogram (mpk) i.p.)
anesthetized mice using subcutaneous inserted electrodes. Recordings
354, the S1P1 selective antagonist W146 (Sanna et al., 2006), were performed on a PowerLab 8/35 (AD Instruments, Dunedin, New
Zealand) data acquisition unit at a 4-kHz sampling rate and under a
heat lamp. Reversal of single-dose FTY720-induced bradycardia by
SPM-354 was assessed in mice preadministered i.p. with 20 mpk
FTY720 for 5 hours, following anesthesia and a baseline ECG period of
4–6 minutes. After baseline determination, mice were challenged i.p.
with either 40 mpk SPM-354 or vehicle (20% b-cyclodextrin; Sigma
Aldrich, St. Louis, MO), with continuous recording until 12 minutes.
For determination of the RR- and PR-interval duration, we included
those values at the end of baseline equilibration with those at the
and fluorescence-tagged S1P3-mCherry knockin (KI) mice in
concert with the previously described S1P3-knockout (KO)
mouse (Ishii et al., 2001; Yang et al., 2002), we combined these
systems to allow the roles of S1P3 causal expression and its
functional consequences in the CCS to be more clearly defined.
Materials and Methods
Synthesis of SPM-354. SPM-354 was synthesized as follows: 122 end of FTY720 treatment taken from LabChart Pro software (AD
mg, 0.164 mmol (Supplemental Fig. 1) was dissolved in MeCN (2 ml), Instruments).

178
Sanna et al.
For FTY720 complete heart block (CHB) reversal experiments, WT
mice pretreated with 20 mpk FTY720 for 5 hours were anesthetized as
above, implemented for ECG recording, and challenged at the onset of
Center for Antibody Development and Production by i.p. immuniza-
tion of Wistar rats with 50 mg of mCherry fluorescent protein
(Biovision, Milpitas, CA) in 200 ml of phosphate-buffered saline
the appearance of consecutive P-waves without a sequential QRS (PBS)/Sigma Adjuvant System (Sigma Aldrich) and two antigen
complex, with either 80 mpk SPM-354 i.p. or 1 mpk atropine, and boosts in PBS/adjutant 3 weeks apart. Antigen-positive hybridomas
recorded further to 12 minutes.
For FTY720-propranolol CHB experiments, WT, KI, or KO mice limited dilution subcloning. Antibodies were further purified by
were pretreated with 20 mpk FTY720 for 5 hours and challenged with protein G chromatography. Clone 16D7 rat anti-mCherry antibody
were expanded, and a clone, 16D7, was isolated after three rounds of
1 mpk (S)-(2)-propranolol (Sigma Aldrich) during the last hour of is commercially available at Life Sciences (Carlsbad, CA) and Kerafast
FTY720 incubation. Two minutes prior to anesthesia, mice were then (Boston, MA).
treated i.p. with a single 120-mpk dose of SPM-354 or an equal volume
of vehicle, and ECG recordings were further collected for 12 minutes. whole hearts from homozygous S1P3-KI mice or S1P3-KO mice were
Data are shown as the mean 6 S.E.M. Statistical analyses between rinsed in cold PBS, minced, and homogenized in RIPA buffer with the
S1P3-mCherry Expression via Western blotting. Excised
conditions were done using unpaired, two-tailed t tests (95% confi- HALT protease inhibitor cocktail (Thermo Scientific Pierce Protein
dence interval) or two-way repeated-measure analyses of variance Research Products, Waltham, MA). Lysate processing, protein
followed by a Bonferroni test (GraphPad Prism; GraphPad Inc.).
Isolated Heart Studies. For volume-conducted ECG studies, WT
or S1P3-KO mouse hearts were excised from mice anesthetized with
determination, SDS-PAGE, and blotting were performed as re-
ported by Cahalan et al. (2011). S1P3-mCherry expression was
detected using 30 mg of protein per lane with the anti-mCherry
2.5% isoflurane in 100% oxygen. After aortic cannulation, the hearts primary (1:1000) described above, followed by rat HRP secondary
were perfused retrograde with a constant pressure (70 mm Hg)
Langendorff setup using 37°C heated, oxygenated Krebs-Henseleit
solution, as reported (Liao et al., 2012). The hearts were placed in a
custom chamber containing 37°C warmed perfusate with two elec-
trodes and a ground lead for the recording of volume-conducted ECG.
The voltage signal was filtered and amplified, and 60-Hz noise was
removed (Hum Bug Noise Eliminator; Quest Scientific, North Van-
couver, Canada) before the signal was digitized at 5000 Hz. Parallel
measurements of the CF rate were made using an in-line flow probe
(Transonic Systems, Inc., Ithaca, NY), and the average flow rate (0.1 Hz
low-pass filtered) was recorded. Volume-conducted ECG and coronary
flow rate data were analyzed with custom software in MATLAB
(MathWorks, Natick, MA). ECGs were further digitally filtered
(250 Hz low pass) for display.
Drug delivery from a 0.5 mg/ml stock of S1P (Avanti Polar Lipids,
Inc., Alabaster, AL) in 50 mM Na2CO3–20% b2cyclodextrin, 5 mg/ml
SPM-354 stock in 20% b-cyclodextrin, 1 mg/ml W146 (Avanti Polar
Lipids, Inc.) in 50 mM Na2CO3–20% b2cyclodextrin, or 1 mM atropine
(Sigma Aldrich) was done by dissolving stocks into 3–5 ml volumes of a
prewarmed, gassed Krebs-Henseleit solution to be delivered to the
(Jackson ImmunoResearch Inc., West Grove, PA) with ECL Plus
(GE Healthcare Bio-Sciences, Pittsburgh, PA).
Perfusion, Tissue Sectioning, and Immunohistochemistry
of Mouse Heart. Sample preparation for staining was performed as
follows: mice were euthanized by CO2 asphyxiation and perfused
through the heart with Z-fix (Anatech LTD, Battle Creek, MI). Hearts
were harvested and fixed in Z-fix for 1 hour and placed into a 30%
sucrose/PBS solution for 48 hours at 4°C. Samples were frozen in
blocks of optimal cutting temperature (Tissue Tek) with dry ice and
ethanol. 40 to 100 mm sections were cut using a Microtome (Microm
HM 550; ThermoFisher Scientific, Waltham, MA). Immunohistochem-
istry (IHC) sections were permeabilized in PBS 1 0.5% Triton X-100
(PBST) for 2 hours at RT, blocked in PBST for 2 hours at RT with 5%
normal goat serum, washed three times in PBST, and incubated for
12 hours at 4°C with primary antibody (1:100) diluted in PBST 1 1%
normal goat serum. Primary incubation was followed by washing
(three times) in PBST, incubated with secondary antibody in PBST at
1:1000 dilution for 2 hours at RT, washed three times in PBS, stained
for 1 hour with DAPI (1:500), and mounted on Vectashield (Vector
Laboratories, Burlingame, CA) for confocal microscopy. The following
primary antibodies were used for staining: anti-mCherry, anti-green
perfusion line.
Generation of S1P3
mCherry/mCherry
Mice. Both the 59 (4.5 kilo- fluorescent protein (GFP) (Abcam, Cambridge, UK), CD31 (Abcam),
base) and 39 (3.5 kilobase) arms of Exon2 encoding for the mouse S1pr3 cyclic nucleotide-gated K1 channel 4 [HCN4 (cyclic nucleotide-gated
gene were cloned by polymerase chain reaction (PCR) from BAC K+ channel 4)] (Santa Cruz Biotechnology, Dallas, TX), and Pan-
(RP24-69B23, C.H.O.R.I) into the Psp72 backbone vector (Promega, Neuronal (Millipore, Darmstadt, Germany). Secondaries were Alexa
Madison, WI) and sequenced. mCherry (Clontec, Mountain View, CA)
and the loxP neomycin resistance cassette (CAN; a generous gift from
Mario Capecchi) were subsequently added downstream from the
coding sequence of S1pr3. The linearized construct was injected into
clone Bruce4 embryonic stem cells derived from C57BL/6J mice.
Homologous recombination was confirmed by Southern blot using
two different 59 probes. Correctly targeted C57BL/6J embryonic stem
cells were injected into recipient C57BL/6J (B6 albino) blastocysts.
The resulting chimeric animals were crossed to C57BL/6J mice.
Heterozygotes were mated to generate homozygous mice. Genotyping
was performed from genomic DNA extracted from tails by PCR using
two reverse and a common forward primer. PCR was performed using
Phire polymerase (NEB) with two reverse primers (BW) and a
common forward primer: P mCherry N1 686 BW, 59 TCA CCA TGG
TGG CGA CCG GTG GCT TGC A 39; S1pr3 11322 BW, 59 AGG CTG
AAA TGC GTT GGT GAC TCC TTG GGT 39; and S1pr3 11103 forward
primer, 59 ATG CAG CCT GCC CTC GAC CCA AGC AGA AGT 39. All mice
used were between 8 and 12 weeks of age. S1P3mCherry/mCherry mice were
555, Alexa 633, Phalloidin 488, and 49,6-diamidino-2-phenylindole-
nuclear stain (Life Technologies).
Confocal Microscopy. Confocal images were obtained with a
Zeiss LSM 780 laser scanning confocal microscope and processed with
Zen 2012 software (both Carl Zeiss Inc., San Diego, CA). Z stacks of
images (obtained at 0.3-mm optical slice increments) were collected
sequentially using a 63Â objective and then were maximum projected
into single flattened stacks for figure panels. In some cases, Z stacks
were additionally imported into IMARIS software (Bitplane Inc.,
Concord, MA) for three-dimensional rendering.
Results
SPM-354 Ligand. SPM-354 (Fig. 1A), a 2-propyl substitu-
ent of the previously described S1P3 antagonist SPM-242 (Jo
et al., 2012), was found to be 6- to 10-fold more potent than SPM-
242 for antagonizing S1P-S1P3 b-arrestin recruitment (Supple-
mental Fig. 2A) and 30-fold more potent on S1P3 versus S1P1
(Supplemental Fig. 2B). In S1P3 cells and similar to the bitopic
antagonist SPM-242, SPM-354 was shown to be noncompetitive
against the selective allosteric S1P3 agonist CYM-5541 (esti-
then crossed onto the S1P1eGFP/eGFP mice strain (Cahalan et al., 2011) to
mCherry/mCherry
generate the double homozygote mouse line S1P3
/
eGFP/eGFP
S1P1
.
Generation of Anti–mCherry Monoclonal Antibody for
Western Blottings and Immunohistochemistry. Rat anti-
mouse mCherry was generated at the Scripps Research Institute mated equilibrium dissociation constant 5 29.01 6 0.45;

S1P3 Regulation of Murine Complete Heart Block
179
Fig. 1. S1P3-dependent bradycardia by FTY720 treatment is associated with enhanced RR- and PR-interval duration that is reversed by SPM-354. (A)
Chemical structure of SPM-354. (B) Typical chronotropic responses in WT mice following 5-hour treatment with vehicle, FTY720, or the S1P1 agonist
CYM-5442. The recordings show that a single i.p. SPM-354 dose, but not antagonist vehicle, fully reverses FTY720-mediated bradycardia without having
a significant effect on heart rate when administered alone. The top graph shows that CYM-5442 does not significantly alter heart rate in WT mice at
5 hours postadministration. Absence of bradycardia by FTY720 in S1P3 KO mice (lower graph). Bar graphs show the mean 6 S.E.M. heart rates in WT
and S1P3 KO mice throughout baseline (BL), FTY720 (F), FTY720 + SPM-354 (F+S), or SPM-354 alone (S) conditions. Electrocardiographic waveforms in
FTY720-treated mice display enhanced RR- and PR-interval duration in wild-type (C) but not S1P3 KO (D) mice and partial restoration upon SPM-354
administration.
slope 5 1.34 6 0.32; n 5 3; Supplemental Fig. 2C). SPM-354 after FTY720 treatment (P 5 0.0023; Fig. 1C), indicative of
had a margin of selectivity of 1840-fold versus S1P2, 8000-fold first-degree heart block. In contrast, and as expected (Forrest
versus S1P4, and 20,000-fold versus S1P5 for inhibiting S1P–b- et al., 2004; Sanna et al., 2004), there were no measurable
arrestin activation. SPM-354 was chosen for in vivo studies over heart rate or ECG changes in KO mice treated with 20 mpk
SPM-242 due to its improved stability in vivo (the half-life in a FTY720 (Fig. 1B, bottom graph, red tracing; Fig. 1D), despite
mouse is 6.7 hours, Y. Kohno, personal communication), and KO mice having a similar heart rate modulation maxima
allowed for a more effective in vivo probe of S1P3 function.
versus WT mice following acute sympathetic and parasympa-
Role for S1P3 in Regulating Cardiac Rate and Rhythm thetic drug challenges (Supplemental Fig. 3).
In Vivo. First-dose FTY720 administration in humans is
Additional experiments with the selective S1P1 agonist
known to reduce heart rate and, in some cases, induce acute CYM-5442, which has therapeutic efficacy in the experimental
episodes of AV block (Vargas and Perumal, 2013). To further autoimmune encephalomyelitis models of multiple sclerosis
understand the contribution of S1P3 in cardiac rhythm, we (Gonzalez-Cabrera et al., 2008, 2012) produced no bradycardia
employed a surface ECG analysis under Avertin anesthesia in when measured 5 hours following 10 mpk i.p. administration
conjunction with FTY720 and SPM-354. Avertin was chosen (n 5 5; Fig. 1B, green tracing).
because it is well tolerated in mice (Roth et al., 2002; Chu
FTY720-induced bradycardia in WT mice was fully reversed
et al., 2006) and allowed for the capture of high-quality ECG in a bimodal fashion by the i.p. bolus injection of 40 mpk SPM-
waveforms. The heart rate plots show that administration of 354 (P 5 0.001; Fig. 1B, red tracing, light arrows), but not by
FTY720 to WT mice 5 hours prior to anesthesia produced 40 mpk i.p. injection of the S1P1 antagonist W146 (n 5 5;
significant bradycardia in all animals (n 5 7; P 5 0.0006; Fig. Supplemental Fig. 4). Furthermore, reversal of bradycardia
1B, top graph, red tracing) compared with water vehicle by SPM-354 administration was associated with a partial,
treatment (n 5 6; blue tracing). FTY720-induced bradycardia but not significant, restoration of PR-interval elevation by
in WT mice was calculated from the RR interval on the ECG FTY720 at the protocol’s end (P 5 0.17; Fig. 1C). Parallel
and showed a significantly increased PR-interval duration studies indicated that antagonist vehicle i.p. administration
from 49 6 3 milliseconds at baseline to 70 6 3 milliseconds in FTY720 bradycardiac mice did not alter heart rate

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Sanna et al.
throughout the conditions (P 5 0.148; n 5 6; Fig. 1B, top CHB failed to revert the abnormal ECG to the normal sinus
graph, dark arrows), and, importantly, 40 mpk SPM-354 rhythm (not shown).
treatment alone did not affect the basal heart rate (P 5 0.090;
n 5 4; Fig. 1, blue line, light arrow), even when dosed up to System. We postulated that the appearance of CHB was a
120 mpk (data not shown). direct consequence of S1P- to S1P3-mediated inhibition of
S1P3 Expression in the Mouse Cardiac Conduction
To our surprise, because mice are not known to be a highly ventricular conduction; therefore, we examined S1P3 expres-
arrythmogenic species (Boukens et al., 2014) and because no sion in cardiac tissues of KI mice by IHC confocal microscopy.
descriptions of this FTY720 effect have been published, ∼25% Wild-type mouse S1P3 was homologously replaced by KI of the
of the mice receiving FTY720/Avertin developed signs of CHB, coding sequence of mCherry to the immediate 39 end of the
which were characterized by prolonged periods of at least S1pr3 gene (Supplemental Fig. 5). Homozygous KI mice were
three consecutive P-waves in the absence of a propagated QRS viable and fertile and shown to express full-length S1P3-
ventricular conduction complex (ECG tracings, time point i; mCherry in whole heart lysates by Western blot when probed
Fig. 2, B and C). This indicates that atrial depolarization with a monoclonal anti-mCherry antibody (Supplemental Fig.
remains intact but does not propagate through the AVN His- 6A) and become lymphopenic to acute FTY720 treatment
Purkinje fibers to depolarize the ventricle. All the mice that (Supplemental Fig. 6C).
underwent CHB (N 5 11) also displayed profound bradycardia
For IHC studies, we used the HCN4 antibody to the cyclic
[102 6 30 beats per minute (bpm)] prior to establishment of nucleotide gated K channel since it selectively labels all
CHB. Importantly, in mice with established CHB, i.p. admin- components of the CCS in the adult mouse heart (Yamamoto
istration of 80 mpk SPM-354 (n 5 4; Fig. 2C), but not 1 mpk of et al., 2006; Harzheim et al., 2008; Kuratomi et al., 2009;
atropine (n 5 5; Fig. 2B), after the onset of CHB led to acute Baruscotti et al., 2011). A confocal low power view of a KI heart
rescue of QRS ventricular conduction within the first minute stained with anti-mCherry antibody for S1P3 (red), HCN4
and coupling to the atrial P-wave and restoration of sinus (green), and nuclei (blue) is shown in Fig. 3A, with fields for
rhythm within 2–3 minutes (bottom ECG tracing). SPM-354 subsequent high power imaging boxed (Supplemental Fig. 7
reversal of CHB was accompanied by a positive chronotropic includes the high magnification panels of Fig. 3A). Represen-
response, which averaged 370 6 28 bpm at the end of the tative merged confocal images of SAN and AVN are shown
protocol. Mice administered with atropine, on the other hand, (Fig. 3B) together with associated differential interference
sustained deep bradycardia (85 6 55 bpm at the protocol’s images (Supplemental Fig. 8 includes the high magnification
end) and CHB throughout the entire protocol. Moreover, panels of Fig. 3B). The central artery of the SAN is readily
b-cyclodextrin (vehicle) administration in two mice undergoing seen (a, Fig. 3B), with arterial smooth muscle expressing
Fig. 2. FTY720 treatment increases the susceptibil-
ity for developing CHB. Typical heart rate recordings
and detailed ECGs of wild-type mice undergoing CHB
following 5 hours of 20 mpk FTY720 administration
and anesthesia. (A) Protocol design. Single-dose
120 mpk SPM-354 administration (C), but not atro-
pine (B), at the onset of CHB results in rapid reversal
of FTY720-induced CHB within minutes. Detailed
ECG analysis of mice undergoing FTY720-induced
CHB has characteristic consecutive P-waves with
absent QRS complexes. Representative ECG wave-
forms in FTY720-treated mice who received either
SPM-354 or atropine while undergoing CHB. Same
animal ECG recordings indicate the presence of CHB
in both mice at time (i), and the outcome on cardiac
rhythm post–drug treatment (ii).

S1P3 Regulation of Murine Complete Heart Block
181
Fig. 3. Immunohistology of S1P3-mCherry in the mouse cardiac conduction system. Laser scanning confocal fluorescence images and differential
interferences (DICs) of whole heart, coronary artery, septum, and Purkinje fibers of S1P3-mCherry KI mouse. (A) Mapped and multistitched composite of
a typical whole heart section (10Â objective, 40 panels) stained for S1P3 (anti-mCherry/Alexa555, red), the cyclic nucleotide-gated K+ channel 4 (HCN4)
(green), and nuclei (DAPI-nuclear, blue). Signals strongly positive for S1P3-mCherry and HCN4 are clearly evident in the Purkinje-conduction system, as
indicated in the white rectangle box of the image. (B) Examples of high-resolution multistack and maximum projected confocal images (63Â) of the SA and
AV nodes are shown as both merged fluorescence and DIC image projections. S1P3 (red) is significantly less expressed with HCN4 in the SA node and
strongly expressed in the AV node. Nuclei are shown in blue, and the sinoatrial nodal artery is indicated (a). The IMARIS-rendered images are also shown
on the right panels. (C) Cross section of the AV bundle labeled with the same antibodies as in (A and B). (D) S1P3 is localized in Purkinje fibers and in
smooth muscle of the S1P3-mCherry KI mouse heart. Panels show high-resolution (63Â) multistack and maximum projected laser scanning confocal
fluorescence images of cardiac smooth muscle cells (i) and Purkinje fibers (ii and iii), labeled with S1P3 (anti-mCherry/Alexa555, red), S1P1 (anti-GFP/
Alexa633, green), and nuclei (DAPI, blue). S1P3 is expressed in heart coronary artery smooth muscle and Purkinje fibers, whereas S1P1 is localized in the
endothelium (i). The last panel series following the merged image shows a single snap shot image of a three-dimensional surface-rendered reconstruction
of the fluorescent signals created with the IMARIS software. Longitudinal sections of heart ventricular septum (ii) shows S1P3 (red) staining of heart
Purkinje fibers as projections interdigitating between ventricular myocytes. A magnified image (iii) of a single Purkinje fiber further shows axonal-like
projections wrapped around myocytes.
S1P3-mCherry. Obvious HCN4 staining of SAN is seen with morphology on differential interference and is strongly double
a small number of interdigitating S1P3-mCherry positive positive for both S1P3-mCherry and HCN4 (see Supplemen-
processes. The atrioventricular node shows a characteristic tal Fig. 8 for relative quantification of HCN4 and mCherry

182
Sanna et al.
merged images in AVN and SAN). The bundle of His, shown in which was differentiated from endothelial cells, as shown in
the cross section in Fig. 3C, is also strongly double labeled for samples colabeled with the endothelial marker CD31 (Sup-
both HCN4 and S1P3-mCherry (a three-dimensional isosur- plemental Fig. 9, A and B).
facing IMARIS image is shown on the rightmost panel of Fig.
Incidence of FTY720-Induced CHB Can Be Increased
3C). Additional IHC experiments using left ventricle heart to Almost 100% by Propranolol Administration. To
sections from double-cross, S1P1-GFP Â S1P3-mCherry, ho- assay the FTY720-induced CHB phenotype with a more
mozygous KI mice, showed S1P3-mCherry expression (red) in robust protocol, we included the nonselective b-androgen
Purkinje fibers as projections interdigitating between ventric- receptor antagonist propranolol, which is known to reduce
ular myocytes and coronary artery smooth muscle (Fig. 3D, heart rate and cardiac output in mice. FTY720-induced CHB
panel ii). A magnified image of a single Purkinje fiber further was demonstrated by administering both FTY720 and pro-
shows axonal-like projections wrapped around myocytes pranolol, as shown in Fig. 4A. Under this protocol, we studied
(Fig. 3D, panel iii). In contrast, S1P1-GFP expression was, as the actions of vehicle or SPM-354 administration 2 minutes
expected (Cahalan et al., 2011), predominantly found on prior to anesthesia. The establishment of CHB in FTY720/
vascular endothelium (Fig. 3D, panel i). mCherry positive propranolol-treated WT mice with b-cyclodextrin vehicle
projections could be double labeled with anti-mCherry mAb preadministration prior to anesthesia increased to almost
and Pan-neuronal markers (Supplemental Fig. 9C), support- 100% (eight out of nine mice displayed CHB throughout the
ing the neuronal origin of S1P3 projections and their identi- protocol; Table 1) and was associated with consistently low
fication as Purkinje fibers. In addition, and as reported heart rates, usually below 100 bpm, as represented in the red
(Mazurais et al., 2002; Forrest et al., 2004), we found strong tracing (Fig. 4B). In parallel experiments, intraperitoneal
discrete S1P3-mCherry expression in vascular smooth muscle, injection of SPM-354 (120 mpk) 2 minutes prior to anesthesia
Fig. 4. The incidence of FTY720-induced CHB in-
creased by propranolol coadministration requires S1P3
and is reversed by single-dose SPM-354 administration.
(A) Protocol schema. (B) Representative heart rate
recordings with magnified sections of the ECG at times
(i) and (ii) of the recordings in mice administered with
FTY720/propranolol and acutely pretreated with either
vehicle (red tracing) or SPM-354 (blue tracing) 2 minutes
prior to anesthesia. Vehicle-pretreated, FTY720/
propranolol mice displayed deep bradycardia and an
irregular heart rate throughout and had distinctive
CHB episodes characteristic of consecutive P-waves
with skipped QRS complexes (C). In comparison, SPM-
354 pretreated, FTY720/propranolol mice displayed a
steady heart rate, with no signs of CHB, as depicted in
the magnified ECG taken at times (i) and (ii) of the
recording (D). Table 1 includes the number of WT, S1P3
KO, and S1P3 KI mice that underwent CHB under the
protocol diagrammed in (A), the mean 6 S.E.M. heart
rates at the protocol’s end, and the outcome of vehicle
and/or SPM-354 administration on CHB establishment.

S1P3 Regulation of Murine Complete Heart Block
183
TABLE 1
of S1P to isolated WT hearts reduced CF rate in a
concentration-dependent manner (Supplemental Fig. 10,
A and B; N 5 4; EC50 of 0.80 6 0.14 mM), which amounted to
a 4.4-fold reduction of 80% maximal response versus
baseline CF rate (Table 2; normal ECG values). According
to previous reports of S1P-S1P3 mediated coronary vaso-
constriction (Murakami et al., 2010), a subsequent bolus of
3 mM SPM-354 during maximal 10 mM S1P CF rate re-
duction led to rapid reversal toward the baseline CF rate
and, in some cases, to above-baseline values (Supplemental
Fig. 10A). Complete tracking of the RR-interval duration in
Supplemental Fig. 10A shows that at 3 mM S1P, there is a
sharp increase in the RR-interval duration, accompanied by
segments of high interbeat variability, which appeared
sustained at 10 mΜ S1P, and, like CF, were rapidly reversed
to baseline values following 3 mM SPM-354 perfusion
(Table 2; normal ECG values).
Incidence of CHB and heart rates from in vivo FTY720 and propranolol
administration in WT, KI, and KO mice acutely pretreated with either
vehicle or SPM-354
FTY720 + Propranolola
Vehicleb
SPM-354b
Treatment
Group
Number of Mice
without CHB
(Mice Tested)
Number Mice
without CHB
(Mice Tested)
Heart
Rate
Heart
Rate
bpm
bpm
Wild type
Knockin
Knockout
1 (9)
2 (7)
5 (5)
N.D.
N.D.
350 6 6.0
7 (8)
4 (4)
N.D.
219 6 12
148 6 7.0
N.D.
N.D., not determined.
a4 hours and 1 hour, respectively.
bAcute 2-minute pretreatment.
Further analysis into S1P perfusion and cardiac rhythm
variability was done using 3 mM S1P or an approximate EC80
of the S1P-mediated reduction in the CF rate. In the Langen-
dorff, S1P-induced arrhythmias are readily seen (Fig. 5A),
with the sequential transition from normal sinus rhythm at
baseline to gradual and steady bradycardia (not shown), then
second-degree heart blocks with 2:1 and 3:1 atrioventricular
conduction (S1P), and finally to CHB, as shown by a complete
absence of QRS ventricular complexes. Figure 5A (vehicle)
demonstrates that vehicle perfusion to a heart with complete
absence of QRS ventricular complexes does not induce reversal
to normal rhythm, unlike subsequent perfusion with 3 mM SPM-
354 (end of tracing in Fig. 5A). Moreover, acute CHB (QRS
complex suppression) following 3 mΜ S1P perfusion was
atropine resistant (Fig. 5B); in contrast, in all three examples
shown, perfusion with 3 mΜ SPM-354 reverted the isolated CHB
to sinus rhythm, which was accompanied by both compensatory
tachycardia and restoration of the CF rate to near baseline
values (end of tracing in Fig. 5B; Table 2; CHB values).
To formally exclude a role for S1P1 in the CHB phenotype,
additional experiments tested the effects of S1P1 antagonist
W146 perfusion on S1P-induced CHB in WT hearts (Fig. 5C).
The data show that in established 3 mΜ S1P-induced CHB,
W146 perfusion at 10 mM does not modify the abnormal
cardiac rhythm, whereas subsequent SPM-354 perfusion at
3 mM fully restored CHB to a normal, steady waveform ECG in
all four hearts studied.
to WT mice with FTY720-propranolol nearly completely
prevented the establishment of CHB (seven out of eight mice
studied; Table 1) by maintaining sinus rhythm and an
adequate heart rate, as evidenced from the ECG pattern of
SPM-354–pretreated mice relative to vehicle (Fig. 4, C and D).
Table 1 shows that when this protocol was applied to KO mice,
there were no signs of CHB in the ECG (N 5 5), although KO
mice were slightly bradycardic due to propranolol admin-
istration. No differences in heart rate were noted to the
administration of vehicles between WT and KO mice (WT
baseline: 458 6 12; WT end of the recording: 443 6 8; KO
baseline: 445 6 17; KO end of recording: 423 6 10). The
FTY720 induction of nonlethal bradycardia and its reversal
by SPM-354 (Supplemental Fig. 6B) as well as SPM-354
prevention of FTY720-propranolol induced CHB (Table 1)
were also demonstrated in the S1P3 KI mice, providing
formal evidence for the normal pharmacological function of
the tagged receptor KI.
SPM-354 Reverses S1P-Induced Normal ECG Func-
tion and S1P-Induced CHB in Isolated, Perfused
Hearts. To differentiate between central or peripheral ac-
tions of SPM-354 CHB reversal in vivo, direct effects on
cardiac rate and rhythm were studied in Langendorff prepa-
rations, with S1P as the ligand. For these studies, we tracked
both the RR-interval duration along with modulation of the
CF rate by the drugs, the latter serving as an internal control
of S1P-S1P3 function and antagonist reversal (Murakami et al.,
2010). We found that perfusion of increasing concentrations
To conclude that S1P-induced CHB was dependent on S1P3,
we performed Langendorff experiments in hearts from S1P3
KO mice (Supplemental Fig. 11). Unlike WT (Supplemen-
tal Fig. 11A), bolus perfusion of 3 mM S1P to KO hearts
(Supplemental Fig. 11B) produced marginal elevations in the
RR-interval duration throughout the protocol, and, despite
inducing modest, albeit significant 30% acute inhibition in the
CF rate (Supplemental Fig. 11C), S1P did not elicit measur-
able cardiac arrhythmias or CHB in KO hearts (n 5 7).
TABLE 2
SPM-354 reverses S1P-induced RR interval augmentation and S1P-
mediated CFR reduction in isolated perfused hearts of WT mice
undergoing either normal ECG patterns or those displaying CHB
Baseline values were taken immediately following 5 minutes of heart equilibration
from start of perfusion.
Normal ECG
CHB
Treatment
RR Interval
CFR
RR Interval
CFR
Discussion
milliseconds
ml/min
milliseconds
ml/min
This combination of genetic and pharmacological analyses
in WT, receptor KO, and KI mice provides multiple lines of
evidence for S1P receptor subtype selective differential regu-
lation of atrial and ventricular pacemaking and conduction
events. S1P1 has been shown to regulate sinus bradycardia, is
coupled to G protein-coupled inwardly-rectifying potassium
Baseline
+ S1P
177 6 35
1.31 6 0.31
168 6 7.6
1.33 6 0.40
351 6 164* 0.27 6 0.15* 620 6 177** 0.25 6 0.05*
+ SPM-354 156 6 42a 1.32 6 0.86a 205 6 31*** 1.65 6 0.54a
aNot significant versus baseline.
*P , 0.05 versus baseline.
**P , 0.01 versus baseline.
***P , 0.05 versus S1P.

184
Sanna et al.
Fig. 5. SPM-354 reverses S1P-induced CHB in ex vivo mouse heart studies. Volume-conducted ECGs were measured in isolated, unpaced, Langendorff-
perfused WT mouse hearts under constant pressure. The four rows for each panel contain representative 3-second ECG tracings shown at baseline,
3 minutes following a bolus of 3 mM S1P, 3 minutes following a bolus of vehicle/W146, and 3 minutes following a bolus of SPM-354. After S1P introduction,
baseline sinus rhythm (A–C; top row) degenerates into second-degree AV block with a variable RR interval and eventually CHB (A and B; second row).
b-cyclodextrin vehicle (A; third row), 1 mM atropine (B; third row), and 10 mM W146 (C; third row) fail to rescue S1P-induced CHB, whereas SPM-354
(A–C; bottom row) restores AV conduction and returns the heart to a normal sinus rhythm.
channel (Guo et al., 1999), and is responsible for the atropine in those mice experiencing severe bradycardia (,200 bpm)
sensitive vagal sinus bradycardia (Kovarik et al., 2008; Fryer due to FTY720 coadministration along with the beta-blocker
et al., 2012; Legangneux et al., 2013). S1P3 in mice has rich propranolol. Cardiac arrhythmias induced by FTY720-
expression at the mRNA level on cells of neural crest origin propranolol treatment in WT mice included episodes of
(Meng and Lee, 2009) and was recently shown to be transcrip- CHB, where AV propagation of atrial depolarization is
tionally present along S1pr1 and S1pr2 transcripts in the absent. Notably, 2-minute prior treatment with single-dose
AVN of rats. We corroborate this finding in the KI mouse and SPM-354 in FTY720-propranolol pretreated WT and/or KI
find expression of S1P3-mCherry on neural crest–derived mice largely prevented the establishment of CHB, while
AVN, His bundles, and cardiac Purkinje fibers in addition to maintaining a fairly constant heart rate, albeit a low (ap-
finding a relatively high expression, as expected, on vascular proximately 200 bpm) and normal rhythm, throughout the
smooth muscle in the coronary arteries (Forrest et al., 2004).
In mice, S1P3 agonism by FTY720-P as well as the chiral
protocol.
Lack of arrhythmias in S1P3-KO mice by FTY720-
analog 2-amino-4-(4-(heptyloxy)-phenyl)-2-methylbutyl dihy- propranolol, coupled with the high S1P3 IHC expression in
drogen phosphate produce bradycardia in WT, but not in mouse CCS and prevention of CHB by the S1P3 antagonist
S1P3-deletant, mice (Forrest et al., 2004; Sanna et al., 2004). SPM-354 in WT and KI mice, provide support that arrythmo-
Based on the KI IHC findings and using proof-of-concept genesis is strongly dependent on cardiac-intrinsic S1P3 ago-
reversal of FTY720 bradycardia, we characterized a potent nism. However, in vivo limitations of the study precluding
S1P3 antagonist tool, SPM-354, to further query physiologic absolute interpretation of the “origin” of the FTY720-induced
functions of cardiac S1P3 modulation.
conduction defects include: 1) possible modulation of other
We confirmed that FTY720 does not induce bradycardia in cardiac receptors (the mouse heart expresses S1P1, S1P2, and
KO mice, whereas in bradycardic WT and/or KI mice pre- S1P5); 2) the known FTY720-mediated S1P3 bronchoconstric-
administered with FTY720, single-dose SPM-354 induced a tion in mice, as shown by Trifilief and Fozard (2012), which
significant positive, chronotropic response, as shown pre- would be relevant and potentially aggravated by coadminis-
viously by Murakami et al. (2010) with a separate S1P3 tration of the nonselective beta-blocker propranolol; and 3) use
antagonist.
of an anesthetic.
Most importantly, we show the first evidence of FTY720’s
The potential limitations are solved by measuring the
arrythmogenic potential in anesthetized WT mice, particularly cardiac rate and rhythm directly in isolated hearts. We chose

S1P3 Regulation of Murine Complete Heart Block
185
S1P for the Langendorff hung heart studies rather than pro- S1P-induced CHB is replicated in Langendorff preparations
drug FTY720 to avoid fingolimod’s intrinsic requirement for and reversed by SPM-354 and not atropine or the S1P1
kinase phosphorylation by sphingosine kinase-2 to become an antagonist W146.
active agonist of S1P3 and remove any potential for off-target
S1P3 distribution in the mouse heart thus provides an
effects of FTY720, such as sphingosine-1-phosphate lyase insight into a potential mechanism-based cardiac risk that
(Bandhuvula et al., 2005) and Transient receptor potential now prompts verification of S1P3 expression in the human
melastatin 7 (Qin et al., 2013). These Langendorff studies CCS. These new insights into S1P3 usage in the pacemaker
provide the first evidence for direct S1P3 cardiac intrinsic cells of the mouse heart will enhance understanding of ven-
induction of lethal cardiac arrhythmias, including CHB, which tricular conduction physiology and pathology, particularly as
are highly sensitive to reversal by the S1P3 antagonist SPM- pharmacological tools allow these data to be extended into
354. To add validation to SPM-354 as an antagonist of S1P3 larger animal species and humans.
with 30-fold higher potency over S1P1, we measured CF rate
reduction by S1P3 agonism and reversal thereof by SPM-354
Authorship Contributions
Participated in research design: Rosen, Gonzalez-Cabrera, Sanna,
Repetto, Cahalan, J. Brown, McCulloch, Vincent.
since the S1P-S1P3 axis reduces the CF rate via coronary
vasoconstriction. The data indicate that S1P-induced arrhyth-
mias presented sporadically, whereas CF rate reduction
maxima and RR-interval elevation functions of S1P were
universal. Moreover, SPM-354, and not the S1P1 antagonist
W146, fully reversed all three functions of S1P perfusion,
whereas CHB was atropine insensitive and rapidly reversed
by SPM-354, as similarly observed in the reversal of CHB with
FTY720 in vivo.
Conducted experiments: Gonzalez-Cabrera, Vincent, Sanna,
Repetto, Kiosses, S. Brown, Abgaryan, Riley, Leaf, Nguyen.
Contributed new reagents or analytic tools: Kohno, McCulloch.
Performed data analysis: Gonzalez-Cabrera, Vincent, Sanna,
Kiosses, S. Brown, Abgaryan, Riley.
Wrote or contributed to the writing of the manuscript: Rosen,
Gonzalez-Cabrera, Sanna, Vincent, S. Brown.
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Address correspondence to: Hugh Rosen, Department of Chemical
Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La
Jolla, CA 92037. E-mail: hrosen@scripps.edu or Pedro J. Gonzalez-Cabrera,
Department of Chemical Physiology, The Scripps Research Institute, 10550 N.
Torrey Pines Rd., La Jolla, CA 92037. E-mail: gonzalp@scripps.edu
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J