A sphingosine kinase 1 mutation sensitizes the myocardium
to ischemia/reperfusion injury
Zhu-Qiu Jina, Jianqing Zhanga, Yong Huangc, Holly E. Hoovera,
Donald A. Vesseyb, Joel S. Karlinera,d,⁎
aCardiology Section, VA Medical Center and University of California, San Francisco, CA 94121, U.S.A.
bLiver Study Unit, Veterans Affairs Medical Center and Department of Medicine, University of California, San Francisco, CA 94121, U.S.A.
cDrug Studies Unit, Department of Biopharmaceutical Sciences, University of California, San Francisco, San Francisco, CA 94143, U.S.A.
dCardiovascular Research Institute, University of California, San Francisco, CA 94143, U.S.A.
Received 9 January 2007; received in revised form 27 May 2007; accepted 31 May 2007
Available online 8 June 2007
Time for primary review 23 days
Objective: Sphingosine kinase (SphK) is a key enzyme in the synthesis of sphingosine 1-phosphate (S1P), a bioactive sphingolipid. SphK is
involved in ischemic preconditioning (IPC). To date no studies in genetically altered animals have examined the role of SphK1 in myocardial
ischemia/reperfusion (IR) injury and IPC.
Methods and results: Wild-type and SphK1 null mouse hearts were subjected to IR (50 min global ischemia and 40 min reperfusion) in a
Langendorff apparatus. IPC consisted of 2 min of global ischemia and 2 min of reperfusion for two cycles. At baseline, there were no
differences in left ventricular developed pressure (LVDP), ±dP/dtmax, and LV end-diastolic pressure (EDP) between SphK1 mutant and
wild-type (WT) mouse hearts. In the mutants, total SphK enzyme activity was reduced by 44% and S1P levels were decreased by 41%.
SphK1 null hearts subjected to IR exhibited more cardiac damage compared with WT: LVDP and ±dP/dtmax decreased, LVEDP
increased, and infarct size increased (n=6, Pb0.05). Apoptosis was markedly enhanced in SphK1 mutant IR mouse hearts. IPC was
cardioprotective in WT hearts, but this protection appeared to be ineffective in SphK1 null hearts. There was no change in infarct size in the
IPC+IR group compared to the IR group in the null hearts (50.1±5.0% vs 45.0±3.8%, n=6, P=NS). IPC remained ineffective in the null
hearts even when the index ischemia time was shortened by 10 min.
Conclusions: Deletion of the SphK1 gene sensitizes the myocardium to IR injury and appears to impair the protective effect of IPC. These
data provide the first genetic evidence that the SphK1-S1P pathway is a critical mediator of IPC and cell survival.
© 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Ischemia; Myocardial infarction; Sphingosine kinase; Sphingosine 1-phosphate; Cardioprotection; Signal transduction
This article is referred to in the Editorial by Nishino,
Webb and Marber (pages 3–4) in this issue.
Myocardial injury during reperfusion after ischemia
results from a complex cascade of events that involves free
radical generation, calcium overload and cytokine activation
. Recent studies have indicated that sphingolipid meta-
bolites also contribute to myocardial ischemia/reperfusion
(IR) injury . Thus, activation of neutral sphingomyelinase
and generation of ceramide and sphingosine occur early
during IR injury . It has been suggested that ceramide,
which is increased in myocardial IR injury , and
sphingosine mediate the actions of cytokines such as
TNFα and interleukin-1 on the heart [5,6]. Sphingosine-1-
phosphate (S1P), a product of sphingosine kinase (SphK)
activation, is an important intracellular signaling molecule
that regulates diverse cellular events, including survival,
Cardiovascular Research 76 (2007) 41–50
⁎Corresponding author. Cardiology Section (111C5), 4150 Clement
Street, San Francisco, CA 94121, U.S.A. Tel.: +1 415 221 4810x3171; fax:
+1 415 750 6959.
E-mail address: email@example.com (J.S. Karliner).
0008-6363/$ - see front matter © 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
by guest on June 12, 2013
growth, motility, differentiation, cytoskeletal reorganization,
and calcium mobilization . S1P is anti-apoptotic and the
ratio of S1P to ceramide has been proposed as a major
determinant of cell survival . S1P is a ligand for G-protein
coupled S1P receptors . It is cardioprotective in cultured
cardiac myocytes [10,11] and in isolated hearts via a PKCε-
independent pathway [12,13].
Ischemic preconditioning(IPC) is a short period of IR that
rescues hearts from subsequent long term IR injury. Reported
triggers are adenosine, bradykinin, opioids, and α1-agonists
coupled to G-proteins, mainly Gi . Among subsequent
mediators are activated PKCε and KATPchannel opening
followed by activation of prosurvival pathways . Recent
studies have shown that sphingolipids are involved in IPC.
Thus, IR injury increased ceramide content, whereas IPC had
the opposite effect . In contrast, S1P content was
enhanced after IPC . We have recently reported that
SphK mediates an alternative or parallel pathway of
myocardial IPC . Increased S1P formation was observed
after IPC while dimethylsphingosine, a selective SphK
inhibitor, abolished IPC-induced cardioprotection .
SphK is the key enzyme responsible for the formation of
S1P  and exhibits two isoforms in mouse heart [18,19].
intracellular S1P content and promotes cell growth and
survival [20,21]. Increased cell viability induced by SphK1
and subsequent S1P synthesis results from activation of a
prosurvival pathway that includes PI-3K/Akt and increased
bcl-2 expression followed by reduced cytochrome C release
and caspase activation [11,12,20,22]. In contrast, SphK2 is
considered to be pro-apoptotic (23). To date, no studies of
non-receptor member of the sphingolipid pathway has been
genetically altered. Thus, the primary objective of the present
study was to utilize mouse hearts harboring an inactivated
SphK1 gene to definitively determine the role of SphK1 in
2. Materials and methods
This study was conducted in accordance with the Guide
for the Care and Use of Laboratory Animals (National
Academic Press, Washington DC, 1996). All procedures
were approved by the Animal Care Subcommittee of the San
Francisco VA Medical Center.
2.1. SphK1 null mice
SphK1 null (KO) mice in which exons 3–6 of the SphK1
gene had been deleted were obtained from Drs. Shaun
Coughlin and Rajita Pappu (Cardiovascular Research
Institute, University of California, San Francisco). These
mice along with their wild-type littermates were used for all
studies reported herein. Male homozygous null (SphK1−/−)
and wild-type (WT) mice were generated by breeding
heterozygous (SphK1 +/−) mice. Genotyping using PCR to
confirm the absence of exons 3–6 of SphK1 DNA was
routinely performed on tail biopsies of 3–4-week-old mice.
Fig. 1 shows a typical PCR analysis.
2.2. Langendorff isolated perfused heart preparation
28–32 g) were heparinized (500 U/kg, IP) and anesthetized
excised, washed in ice-cold arresting solution (NaCl
120 mmol/L, KCl 30 mmol/L), and cannulated via the aorta
on a 20 gauge stainless steel blunt needle. Hearts were
perfused at 70 mmHg on a modified Langendorff apparatus
using Krebs–Henseleit solution at 37 °C as previously
described in our laboratory [12,17]. Platinum electrodes
connected to a stimulus generator (Grass Instruments, West
Warwick, RI) were used to pace hearts at 360 bpm.
2.3. Ischemia–reperfusion (IR) and ischemic preconditioning
For IR experiments, the protocol consisted of a 20 min
equilibration period, followed by 50 min of global ischemia
and 40 min of reperfusion. In some studies, the index
ischemia time was reduced to 40 min. For IPC, hearts were
equilibrated for 16 min and then subjected to two short
as described above. Hemodynamics were recorded as
previously described [12,17].
2.4. Infarct size measurement
After IR±IPC, a subset of wild-type and SphK1 null
hearts was infused with 15 ml 1% triphenyltetrazolium
chloride (Sigma) in phosphate-buffed saline at a rate of
1.5 ml/min as previously described . Hearts were then
removed from the cannula, weighed, and fixed overnight in
10% formalin. Hearts were removed from formalin and
stored at −20 °C until sectioning for analysis of LV infarct
size as previously described in our laboratory [12,17]. The
infarct size of each section was expressed as a fraction of the
area at risk defined as the total area of the left ventricle in this
global ischemia model.
Fig. 1. PCR analysis showing amplification of SphK1 from wild-type (+/+),
heterozygous (+/−), and null (−/−) mice. A=primer for identification of the
wild-type gene; B=primer for the detection of the null genotype.
42Z.-Q. Jin et al. / Cardiovascular Research 76 (2007) 41–50
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2.5. Creatine kinase (CK) determination
CK measurements were performed as previously de-
scribed in our laboratory (12). CK reagent was from Stanbio
Laboratory, Boerne, TX.
2.6. DNA fragmentation (DNA ladder)
IR hearts were placed in liquid nitrogen and stored at
−80 °C. Hearts were minced on ice in digestion buffer (NaCl
100 mM, Tris (pH 8.0) 10 mM, EDTA 25 mM, SDS 0.5%)
and proteinase K (400 μg/ml) added. After overnight
incubation at 55 °C, 25 μl of 10 mg/ml DNA-free RNase
1/20 volume of 5mol/L NaCl was added. DNAwas extracted
in a 1: 1 volume ratio of phenol: chloroform: isoamyl alcohol
(25: 24: 1) 3 times, and precipitated in isopropranol. DNA
was centrifuged and washed with 80% ethanol. Twenty μg of
DNA was loaded onto a 1.8% agarose gel, and DNA ladder
formation was detected under UV light.
2.7. Terminal deoxynucleotidyl transferase-mediated dUTP-
biotin in situ nick-end labeling (TUNEL)
Detection Kit, Fluorescein (Roche, Mannheim, Germany)
according to the manufacturer's instructions. Randomly
selected cardiomyocytes from 4 sections/heart were
Body weight (g)
Heart weight (mg)
Heart/body wt (mg/g)
166.4±14.7 156.2±13.9 161.0±5.8
WT: wild-type; KO: knockout; IR: ischemia/reperfusion; IPC: ischemic
above parameters between groups as assessed by one-way ANOVA.
Hemodynamic changes after IR and IPC followed by IR are shown in Fig. 2.
Fig.2. A. Panel A,left ventricular developedpressure (LVDP),its maximumincrease(Panel B)anddecrease (PanelC) of velocity (⁎Pb0.05vs WT-IR.#Pb0.05
vs WT-IPC. IR: ischemia/reperfusion; IPC: ischemic preconditioning followed by ischemic preconditioning. n=5–7/group. Panel E. Representative cross-
sections of wild-type (WT) and sphingosine kinase1 (SphK1) knockout (KO) mouse hearts after perfusion with 2, 3, 5-triphenyltetrazolium chloride. Visual
inspection identifies larger infarcts in WT-IR, KO-IR, and KO-IPC hearts compared to WT-IPC. Abbreviations as in Panel A. Infarct size (Panel F) expressed as
percentage of risk area and creatine kinase (CK) release (Panel G) in wild-type (WT) and sphingosine kinase1 (SphK1) knockout (KO) mouse hearts subjected to
50 min of global myocardial ischemia and 40 min of reperfusion. Data are expressed as mean±s.e.m.⁎Pb0.05 vs WT-IR.#Pb0.05 vs WT-IPC. IR: ischemia/
reperfusion; IPC: ischemic preconditioning followed by IR. n=6–7/group.
43 Z.-Q. Jin et al. / Cardiovascular Research 76 (2007) 41–50
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evaluated with fluorescence microscopy to determine the
number and percentage of cells exhibiting apoptosis. Nuclei
were stained blue with Hoechst 33258 (Molecular Probes,
Eugene, OR). Actin was stained red with fluorescent
stained green. For each section 10 fields were randomly
chosen and counted. The proportion of apoptotic nuclei was
determined from a total of 40 fields/heart.
2.8. Sphingosine kinase activity
Mice were anesthetized by intraperitoneal injection of pen-
and washed in cold buffer A (0.13 M KCl, 20 mM Hepes, pH
7.4, 1 mM EGTA, 1 μg/L leupeptin, 0.25 μg/L each of
chymostatin and pepstatin A). Tissue was minced, homoge-
and nuclei. The supernatant was centrifuged for 50 min at
100,000 ×g, decanted and designated as the cytosolic fraction.
The assay uses a chloroform/methanol/aqueous triso-
dium EDTA extraction system to separate reactant ([3H]
sphingosine) from product ([3H]S1P) as previously de-
scribed in our laboratory . A standard assay contains
(300–400 cpm/pmol), 5 mM ATP, 10 mM Mg2+, 100 mM
Tris, pH 8.0 (30 °C), and enzyme protein in a volume of
0.5 ml. A 0.1 ml aliquot is removed into 0.3 ml of methanol
and 0.6 ml of chloroform. The mixture is separated into two
phases by the addition of 0.3 ml of trisodium EDTA (pH 9)
followed by vortexing and centrifugation. The upper
aqueous phase containing the S1P is removed for liquid
scintillation counting. SphK activity is assayed by conver-
sion of [3H]-sphingosine to [3H]-S1P and is reported as
2.9. Sphingosine-1-phosphate measurement
Hearts were removed from the Langendorff apparatus,
plunged into cold perfusion buffer and cut into small pieces.
After chilling, they were rapidly blotted dry and weighed.
Minced heart was then homogenized in 1 ml ice-cold
chloroform: methanol (1:2) and incubated at −20 °C
overnight. Samples were then centrifuged at 15,000 rpm
for 10min, the supernatant decanted and 0.95ml CHCl3and
centrifuged at 15,000 rpm. The supernatant was removed
and 0.1 μg S1P was added to each tube. Samples were
lyophilized and resuspended in 0.3 ml of 70% acetonitrile
for analysis by combined liquid chromatography/dual
tandem mass spectrometry. The system was a Micromass
Quatro Ultima equipped with an electrospray source,
Shimadzu LC-10 AD pumps and Waters Intelligent Sample
Processor 717 Plus. The LC column was BDS C18
(4.6×50 mm, 5 μM particle size, Keystone). Conditions
described elsewhere .
2.10. Western Blot analysis
SphK protein was measured using standard SDS-PAGE
Western blotting as previously described in our laboratory
(17). Primary antibodies (Cell Signaling Technology, Inc.
Beverly, MA and Santa Cruz Biotechnology, Inc, Santa Cruz,
CA) were used to measure Akt phosphorylation (Ser 473),
total Akt, cytochrome C, and SphK2. Immunoreactive bands
were detected by enhanced chemiluminescence (ECL)
(Amersham Bioscience, Piscataway, NJ) and quantitated by
densitometric analysis of digitized autoradiograms with NIH
Image 1.61 software.
44Z.-Q. Jin et al. / Cardiovascular Research 76 (2007) 41–50
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Data are mean±SEM. The significance of the differences in
mean values for hemodynamics, infarct size, and CK release
between groups was evaluated by one-way ANOVA, followed
by post-hoc testing (Newman–Keuls). Differences in SphK
3.1. Baseline characteristics
There were no baseline differences in heart weight, heart
weight/body weight ratio, and left ventricular end-diastolic
pressure (LVEDP) between WT and KO mice. As shown in
Table 1, the left ventricular developed pressure (LVDP),
Fig. 3. Panel A. Representative photomicrographs of in situ detection of DNA fragments in SphK1 knockout (KO) and wild-type (WT) mouse hearts. Panel B:
Percent TUNEL-positive nuclei.⁎Pb0.05 vs all other groups, n=3/per group. Panel C. Electrophoretic analysis of internuclosomal DNA extracted from two
SphK1knockouthearts(lanes 1and2) andtwoseparatewild-type(lanes 3and4) mousehearts exposedto 50min of ischemiaand120 min ofreperfusion.Lane0
is a DNA size marker. One additional set of hearts showed identical results.
45Z.-Q. Jin et al. / Cardiovascular Research 76 (2007) 41–50
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+dP/dtmax, and −dP/dtmax were slightly higher in KO
mouse hearts, but these differences did not reach statistical
dial survival during IR injury and ischemic preconditioning
Both WTand KO hearts were subjected to 50 min of global
ischemia and 40 min of reperfusion. As shown in Fig. 2A,
recovery of LVDP at the end of IR was significantly lower in
KO mouse hearts than in WT hearts. +dP/dtmax and −dP/
(Fig. 2D). Thus, IR caused more serious impairment of both
the dysfunction observed in hearts from WT mice.
As shown in Fig. 2A–D, ischemic preconditioning (IPC)
increased cardiac performance in WT hearts. At the end of IR,
WT-IPC hearts exhibited improved LVDP and +dP/dtmax
and −dP/dtmax compared with the WTcontrol group. In WT
but this difference was not significant (Fig. 2D).
A prominent hemodynamic finding was that the cardio-
protective effect of IPC is apparently ineffective in KO hearts.
The recovery of LVDP at the end of IR was not different
between KO-IR hearts and KO-IPC hearts (Fig. 2A). LVEDP
differences in coronary flow between WT and KO hearts
subjected to IR or IR+IPC (data not shown).
As shown in Fig. 2E and F, infarct size, expressed as
percentage of risk area, was significantly larger in KO
hearts than in WT hearts. CK release at the end of IR also
was significantly higher in KO hearts than in WT hearts
IPC reduced CK release in WT hearts compared with WT-
these cardioprotective effects of IPC were significant
(Fig. 2E–G). Instriking contrast, the cardioprotection induced
by IPC was not evident in SphK1 null hearts. There was no
difference in CK release at the end of IR between KO-IPC
in KO-IPC hearts compared with KO-IR hearts (Fig. 2F).
When the index ischemia time was reduced to 40 min,
infarct size in the KO hearts declined to the level seen in the
Fig. 4. Sphingosine-1-phosphate improved left ventricular developed
pressure (LVDP) (A) and reduced infarction size (B) in SphK1 mutant
mouse hearts subjected to ischemia/reperfusion injury. See Methods and
Results for details.⁎Pb0.05 vs Control. n=4/group. RA=risk area.
46 Z.-Q. Jin et al. / Cardiovascular Research 76 (2007) 41–50
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WT-IR group (28.5±2.7% of the risk area). At this reduced
level of injury, IPC was still ineffective in producing
cardioprotection in the KO hearts (infarct size=29.3±1.6%,
P=NS vs IR alone, n=4/group).
3.3. SphK1 deletion sensitizes cardiac myocytes to apoptosis
induced by IR injury
At baseline, myocardial tissue from sham WT and SphK1
null mouse hearts exhibited virtually no TUNEL-positive
staining (Fig. 3A and B). To determine if there are differences
we extended the reperfusion time to 120 min. This maneuver
much more prevalent in SphK1 null IR hearts compared with
WT hearts (Fig. 3A and B). Similarly, in both WT and KO
hearts no DNA laddering was present at baseline. Consistent
with the induction of TUNEL-positivity, prolonged reperfu-
sion induced formation of nucleosome ladders SphK1 null IR
hearts, compared with WT-IR hearts (Fig. 3C).
3.4. Exogenous S1P is cardioprotective in SphK1 null
A 2 min infusion of 10 nM S1P given as pretreatment
[12,17] protected SphK1 null mouse hearts against IR injury.
As shown in Fig. 4, S1P significantly improved LVDP
recovery and reduced infarct size, consistent with previous
results from our laboratory in WT hearts [2,17].
fractions in WT hearts, with the majority of enzyme activity
detected in the cytosol. We also measured SphK activity in
was higher in spleen vs heart tissue. In the SphK1 null hearts,
SphK activity in heart tissue was decreased in the cytosol by
Fig. 5. Sphingosine kinase (SphK) activity and sphingosine-1-phosphate
(S1P) content in wild-type (WT) and sphingosine kinase1 (SphK1)
knockout (KO) mice. (A). SphK activity in the normal control WT and
KO hearts. (B). SphK activity in the normal control WT and KO spleens.
(C). S1P content in WT and KO hearts. Data are expressed as mean±SEM;
Fig. 6. Above: Representative western blots of sphingosine kinase (SphK)
type 2 protein expression in wild-type (WT) and sphingosine kinase1
(SphK1) knockout (KO) mouse hearts. Below: bar graphs quantitating the
data from WTand KO hearts, n=4/group. Data are expressed as mean±s.e.m.
⁎Pb0.05 vs WT. P=positive control.
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44%. It was also decreased in the membrane fraction. SphK
After IR, SphK activity in the WT hearts was decreased from
256±8pmol/min/gtissueinthe normalcontrol heartsto144±
39 pmol/min/g tissue in the IR injured hearts (n=4, Pb0.05).
min/g tissue vs 126±51 pmol/min/g tissue, n=4, P=NS).
As shown in Fig. 5C, S1P content detected by mass spec-
trometry was significantly decreased in the KO mouse hearts.
3.6. Effect of SphK1 deletion on cardiac SphK2 protein
Since SphK activity was still detected in SphK1 null
mouse hearts, we wondered whether residual SphK activity
was the result of an increase in SphK2 protein. As shown in
Fig. 6, SphK2 expression was significantly higher in SphK1
mutant mouse hearts than in WT hearts. To test the
hypothesis that the increase in SphK2 in the KO hearts
was responsible for impaired hemodynamics and increased
infarct size after IR injury, we used dimethysphingosine
(DMS), a known SphK inhibitor. DMS (3 μM) was perfused
for 5 min before IR injury in both WTand KO mouse hearts.
We found that there was no difference between WT and KO
groups with regard to infarct size (39.0±4.5%% in the DMS-
WT group vs 38.3±5.4% in the DMS-KO group, n=3/
group) and LVDP recovery (8.1±2.0% in the DMS-WT
group vs 8.0±5.7% in the DMS-KO group). This result
suggests that raised SphK2 in the SphK1 KO mouse hearts is
not associated with alterations in infarct size or hemody-
namic recovery after IR injury.
3.7. Phosphorylation of Akt (Ser 473) and cytochrome C
release after IPC and IR injury in wild-type and SphK1 null
Phosphorylation of the prosurvival signaling molecule
Akt (p-Akt, Ser 473) was measured in WT and KO hearts
during IPC and IR. At baseline, p-Akt was increased in KO
hearts (Fig. 7A). After IPC, p-Akt was enhanced in WT
hearts. No alteration of p-Akt was observed in KO hearts
(Fig. 7B). After IR injury, cytochrome C release into the
cytosol was increased by over 4-fold in KO hearts as
determined by western blotting and subsequent densitometry
from 12.8±2.6 to 57.8±7.6 arbitrary units (n=4, Pb0.05).
In these experiments, we sought additional evidence for
the role of SphKincardioprotection by testing the hypothesis
that genetic elimination of SphK1 alters the cardiac response
in which deletion of exons 3–6 of the SphK1 gene leads to
reduction of total SphK activity and a concurrent decrease in
serum and tissue S1P levels. These mice exhibit no evident
phenotype, breed normally, have normal vascular develop-
ment, and live a normal lifespan. We found no significant
SphK1 null mouse hearts. However, after 50 min of global
ischemia and 40 min of reperfusion, left ventricular
developed pressure and ±dP/dtmax were significantly
decreased in KO hearts. Infarct size, CK release, and
cytochrome C content in the cytosol were significantly
increased in the KO hearts after IR injury. Strikingly, IPC-
induced cardiac protection was apparently ineffective in the
KO hearts. Extensive apoptosis was present in the KO hearts
following prolongation of reperfusion injury. When the
extent of IR injury in the KO hearts was reduced by a shorter
index ischemia time, IPC was still ineffective. These results
provide the first evidence in a genetically modified animal
that SphK1 is an important lipid kinase mediating cell
survival and that SphK1 appears to be required for IPC in the
heart. We also recognize that these data, although highly
suggestive, do not establish an absolute causal relation
between the SphK1 gene mutation and impaired IPC.
Recently, Allende et al. reported that mice deficient in
SphK1 were still rendered lymphopenic by S1P1receptor
agonist FTY720 . Like us, they found mutant mice to be
fertile, long-lived, and also reported no histologic abnor-
malities in major organs. Although they performed no
functional cardiac studies, Allende et al. reported a greater
Fig. 7. (A). Representative immunoblots showing phosphorylation of Akt
(p-Akt, Ser 473) in wild-type (WT) and SphK1-knockout (KO) mouse
hearts. (B). p-Akt and total Akt expression in whole tissue homogenates of
normal control (NC) WT and KO hearts and hearts subjected to ischemic
preconditioning (IPC). N=4/group.⁎Pb0.05 vs WT-NC.
48Z.-Q. Jin et al. / Cardiovascular Research 76 (2007) 41–50
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than 60% reduction in serum S1P levels, but surprisingly in
contrast to our observations, no significant decrease in tissue
levels, including brain, heart, kidney, liver, spleen and testis.
A possible explanation for this apparent discrepancy could
be related to tissue sample preparation and methods of lipid
extraction. However, substantial reductions in SphK1
enzyme activity were observed in all of these organs,
suggesting a compensatory increase in SphK2 activity (see
below). Of note, in their SphK1 null mice, Allende et al.
reported no changes in the mRNA levels for the genes
encoding enzymes known to regulate S1P levels, including
S1P lyase, S1P phosphatase, or ceramidase .
Recent reports have further elucidated the role and
mechanisms of SphK1 in maintaining cell viability. In MCF-
downregulated SphK1 protein and activity and reduced the
percentage of viable cells, an effect reversed by S1P treatment
be necessary for induction of cell death . Knockdown of
SphK1 by siRNA caused cell cycle arrest and induced
apoptosis via effector caspase activation, cytochrome C
release and Bax oligomerization in the mitochondrial
membrane . The considerable residual SphK activity that
both we and Allende et al.  found is likely attributable to
has been implicated mitochondria-mediated apoptotic path-
the endoplasmic reticulum converted it from anti-apoptotic to
generated by SphK isoforms is critical to cell function . It
should be emphasized that these studies were done in vitro in
cell lines, and may not apply in vivo.
Our recent work has revealed additional possibilities. In
acutely ischemic hearts, SphK activity declines markedly and
remains depressed during recovery, while in hearts that have
been preconditioned, recovery of enzyme activity is much
more robust . S1P levels are altered in parallel , and
there may be a threshold concentration below which
prosurvival pathways are either not activated or are
suppressed. In isolated adult cardiac myocytes we have
found that internally generated S1P must be exported to act in
an autocrine or paracrine manner, and that this process
is impaired during simulated ischemia . Thus many
factors, which require further study, may contribute to the
IR injury .
Acute Akt activation is cardioprotective both in vitro
and in vivo [33,34]. In contrast, chronic activation is
deleterious . Enhanced chronic Akt phosphorylation is
found in the hearts of patients with advanced heart failure
 and restoration of PI3K rescues the deleterious effects
of chronic Akt activation in the heart during IR injury .
In this study, we found that the increased phosphorylation
of Akt occurred in SphK1 null mouse hearts at baseline.
This was associated with depressed cardiac function,
increased infarct size, and enhanced cytochrome C release
in SphK1 KO mouse hearts after IR injury.
Despite an abundance of residual SphK activity and serum
and tissue levels of S1P that would seem to be adequate to
evident in the SphK1 null mice even when the index ischemia
time was shortened. Such a result might be predicted,
presumably because all of the residual SphK activity can be
attributed to SphK2, which generates S1P that does not
transactivate S1P receptors . Our observation (Fig. 4) that
exogenously supplied S1P improves hemodynamics and
reduces infarct size, even in the SphK1 null mouse, is
consistent with this hypothesis. Of note is that specific
a proportional decrease of S1P and a concomitant increase of
ceramide in cardiac myoblasts that leads to apoptosis . In
isolated adult mouse cardiac myocytes subjected to hypoxic
stress, we have previously shown that S1P can trigger a
prosurvival pathway involving the S1P1receptor, Gi, and
phosphorylation of Akt, Bad, and GSK-3β, resulting in
reduced mitochondrial cytochrome C release . Moreover,
that VPC 23019, the selective S1P1 receptor antagonist,
abolished the protective effect induced by S1P and by a SphK
activator, the monoganglioside GM-1 .
As noted in the Results and Fig. 5, we measured SphK
activity in WT and SphK1 knockout Langendorff hearts
immediately prior to and after IR. SphK activity was reduced
in the KO hearts and was unchanged by IR injury. In contrast
this intervention reduced SphK activity by an average of
44% in the WT hearts. In addition, we have reported
elsewhere that SphK activity declined 61% during ischemia
and did not recover upon reperfusion . Preconditioning
reduced the decrease in SphK activity during ischemia by
half and upon reperfusion activity returned to normal .
Measurements of S1P content followed a similar pattern
organ cardiac preparation that myocardial damage is enhanced
after IR injury and that the cardioprotective intervention of
preconditioning appears to be impaired by deletion of the
SphK1 gene. These observations are consistent with accumu-
lating evidence, derived heretofore exclusively by the in vitro
study of cell lines, that SphK1 and SphK2 orchestrate opposing
functions that regulate cell fate. In addition, our data using this
genetically modified model provide the strongest evidence to
date for the critical role of SphK, specifically SphK1 activation,
in ischemic preconditioning.
This work was supported by NIH grant 1P01 HL068738-
The authors thank Drs. Shaun Coughlin and Rajita Pappu
for supplying the SphK1 null mice and Mr. Michael Kelley
for technical assistance.
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