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Cardiovascular Research 2006 71(4):725-734; doi:10.1016/j.cardiores.2006.06.010
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Copyright © 2006, European Society of Cardiology

Low dose N, N-dimethylsphingosine is cardioprotective and activates cytosolic sphingosine kinase by a PKC{varepsilon} dependent mechanism

Zhu-Qiu Jin and Joel S. Karliner*

Cardiology Section, VA Medical Center and Department of Medicine, University of California, San Francisco, CA 94121, USA

* Corresponding author. Cardiology Section (111C), 4150 Clement Street, San Francisco, CA 94121, USA. Tel.: +1 415 221 4810x3171; fax: +1 415 750 6950. Email address: joel.karliner{at}med.va.gov

Received 30 December 2005; revised 2 June 2006; accepted 6 June 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: N, N-Dimethylsphingosine (DMS) is recognized as an inhibitor of sphingosine kinase (SphK), a key enzyme responsible for the formation of sphingosine-1-phosphate (S1P). We previously showed that S1P was cardioprotective and that SphK was critical for myocardial ischemic preconditioning. Although DMS is an endogenous sphingolipid, its effect on cardiac function and cardioprotection at low concentration has not been studied.

Methods In Langendorff-perfused wild-type and protein kinase C (PKC){varepsilon}-null mouse hearts, cardiac function, infarction size, and SphK activity were measured.

Results: Pretreatment with 0.3 µM and 1 µM DMS for 10 min protected against ischemia/reperfusion injury. Cardiac function (LVDP, ±dP/dtmax) was improved and infarction size was reduced. The cardiac protection induced by DMS was abolished in PKC{varepsilon}-null mouse hearts. Administration of 1 µM DMS ex vivo increased cytosolic SphK activity. This enhanced SphK activity was abolished in PKC{varepsilon}-null mouse hearts. DMS also increased PKC{varepsilon} translocation from the particulate to the cytosolic fraction with no effect on PKC{alpha} distribution. Co-immunoprecipitation showed that SphK1 interacted with PKC{varepsilon} phosphorylated on Ser729. DMS also increased cytosolic Akt phosphorylation (Ser 473) and Akt translocation from a Triton-insoluble fraction to the cytosol.

Conclusions: DMS has a biphasic effect on cardioprotection. Higher concentrations (10 µM) are inhibitory, whereas a low concentration (0.3 µM and 1 µM) of DMS protects murine hearts against ischemia/reperfusion injury. DMS activates SphK in the cytosol via a PKC{varepsilon} dependent mechanism. The PKC{varepsilon}–SphK–S1P–Akt pathway is involved in the cardiac protection induced by DMS.

KEYWORDS Preconditioning; Protein kinase C; Lipid signaling; Ischemia; Reperfusion


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Bioactive sphingolipids are known to play a physiological role in cell growth, apoptosis and differentiation [1,2]. Among these sphingolipids, sphingosine-1-phosphate (S1P) has been implicated as a signaling molecule for cell survival, cytoskeletal rearrangement, and prevention of apoptosis [3–6]. By binding to cognate cell surface receptors S1P initiates signal transduction. In addition, S1P may also act as an intracellular second messenger [7]. It is cardioprotective against ischemia/reperfusion injury via a PKC{varepsilon}-independent pathway [8,9]. Sphingosine kinase (SphK), the key enzyme responsible for the formation of S1P, exhibits two isoforms in mouse heart [10]. SphK1 appears to be a protective kinase, as its overexpression increases intracellular S1P content and promotes cell growth and survival. Activation of SphK2 increases cytochrome C release from mitochondria and is proapoptotic [11]. In recent studies we have reported that SphK mediates an alternative or parallel pathway of myocardial ischemic preconditioning [12]. Moreover, increased formation of S1P also has been reported after ischemic preconditioning [12,13].

Sphingosine is a major product of ceramide catabolism [14], from which N, N-dimethylsphingosine (DMS) is formed by N-methylation catalyzed by a transferase enzyme [15]. DMS is a competitive SphK inhibitor in vitro (IC50=5.0 µM, [14,15]) and has been widely used experimentally. In a previous study, we reported that DMS decreased SphK activity, reduced cardiac function, and abolished preconditioning in isolated murine hearts at a concentration of 10 µM [12]. In vitro experiments suggested that DMS modulates transmembrane signaling via dual mechanisms [18]: 1) inhibition of protein kinase C activity and 2) enhancement of tyrosine kinase (particular EGF receptor kinase) activity. Endogenous DMS enhanced autophosphorylation of the EGF receptor and showed EGF-like effects [14]. EGF receptors and tyrosine kinases are involved in pharmacological preconditioning and cardiac protection against myocardial ischemia/reperfusion injury [19,20]. For example, Src and Lck tyrosine kinases are downstream of PKC{varepsilon} in the signaling cascade of ischemic preconditioning [21]. However, to our knowledge, very little is known about the potential role of DMS in myocardial ischemia/reperfusion injury and the possible signaling pathway involved.

After completion of our previous study indicating that 10 µM DMS abolished ischemic preconditioning by inhibiting SphK activity [12], we sought to determine the response to a reduced inhibitory concentration of DMS. To our surprise, we found that 0.3 and 1 µM DMS appeared to be cardioprotective. We therefore conducted the present experiments: 1) to examine the effect of ex vivo administration of a low concentration of DMS on murine heart function and its action against ischemia/reperfusion injury; and 2) to investigate the alteration of SphK and Akt activity in these processes in relation to PKC{varepsilon} activation and translocation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academic Press, Washington, DC, 1996), and all procedures were approved by the Animal Care Subcommittee of the San Francisco Department of Veterans Affairs Medical Center, both of which conform to NIH Guidelines.

2.1. Langendorff isolated perfused heart preparation
Male C57BL/6J mice (4 months of age, weighing 28–30 g) were heparinized (500 U/kg, IP) and anesthetized with sodium pentobarbital (60 mg/kg, IP). Hearts were rapidly 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 mm Hg on a modified Langendorff apparatus using Krebs–Henseleit solution containing (mmol/L) NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 24, glucose 5.5, Na pyruvate 5.0, and EDTA 0.5 bubbled with 95% O2/5% CO2 at 37 °C. Platinum electrodes connected to a stimulus generator (Grass Instruments, West Warwick, RI) were used to pace hearts at 360 beats per min.

2.2. Ischemia–Reperfusion (IR) and pharmacological preconditioning protocols
For IR experiments, the protocol consisted of 20 min of global ischemia and 30 min of reperfusion. We have previously found that this protocol produces a substantial infarction in control hearts [8,12]. In some experiments hearts were perfused with 1 µM N, N-dimethylsphingosine (DMS, Biomol, Plymouth Meeting, PA) for 10 min followed by washout for 7 min before initiating IR. In experiments examining the concentration–response to DMS (0.3 µM, 1 µM, and 3 µM) and the effects of an EGF receptor inhibitor (AG1478), longer periods of ischemia (50 min) and reperfusion (40 min) were used. DMSO (0.25%) was used as vehicle control. Hemodynamics [left ventricular (LV) developed pressure, LV±dP/dtmax, and coronary flow] were recorded as previously described [12].

2.3. Infarct size determination
After 20 min of global ischemia and 30 min of reperfusion, a subset of hearts in each group was infused with 15 ml of 1% triphenyltetrazolium chloride (TTC, Sigma) in phosphate-buffed saline at a rate of 1.5 ml/min as previously described [12]. Hearts were then removed from the cannula, weighed, and fixed overnight in 10% formalin. Hearts were removed from formalin and stored frozen at – 20 °C until sectioning for analysis of left ventricular infarct size. Hearts were sliced into 2 mm transverse section from apex to base and digitally photographed on each side (Camedia E-10, Olympus Camera). Computerized area analysis was performed with NIH Image software. The infarct size of each section was expressed as a fraction of the area at risk defined as the total area of the ventricle in this global ischemia model.

2.4. Cell fractionation
Isolated mouse hearts were mounted on a modified Langendorff apparatus as previously described in our laboratory [12]. After treatments, all the hearts were placed in liquid nitrogen and stored at – 80 °C. Hearts were thawed and homogenized in assay buffer: 200 µl 1 M Tris–HCl, pH 7.4, 100 µl 0.1 M EDTA, 50 µl 100 mM deoxypyridoxine, 150 µl 1 M sodium fluoride, 0.7 µl 2-mercaptoethanol, 50 µl 0.2 M sodium orthovanadate, 10 µl 10 mg/ml leupeptin and aprotinin, 10 µl 10 mg/ml trypsin inhibitor, 86.4 mg β-glycerolphosphate, 20 µl 0.2 M PMSF, 4 ml 50% glycerol, and 5.36 ml of water as described [12]. The homogenate was centrifuged at 100,000 x g for 40 min and the supernatant collected as the cytosolic fraction. 1% Triton-100 was added to the pellet, extracted with a 1 ml syringe and put on the ice for 30 min, then centrifuged at 100,000 x g for 40 min. The supernatant was collected as membrane fraction. The remaining pellet was the Triton-insoluble fraction.

2.5. Sphingosine kinase (SphK) activity assay
Cytosolic and membrane fractions were used for the SphK assay as described [22]. The reaction mixture (200 µl) for the SphK assay contained 16.7 µM [3H] sphingosine (PerkinElmer, Wellesley, MA)(0.2 µCi), 80 mM Tris–HCl, 20 mM MgCl2, 1 mM ATP, 4 mg/ml BSA and 200 µg heart tissue protein. The mixture was incubated for 1 h at 37 °C. The Eppendorff tube containing the reaction mixture was then placed on ice and 20 µl of 1 M HCl was added to stop the reaction. The [3H] S1P formed was extracted by chloroform/methanol/HCl (100:200:1, v/v). Resultant lower chloroform phase samples were analyzed for [3H] S1P formation from [3H] sphingosine by thin layer chromatography separation with liquid scintillation counting (LS 6500 Multi-purpose Scintillation Counter, Beckman).

2.6. PKC{varepsilon}-null mice
PKC{varepsilon}-null mice were obtained from Dr. Robert Messing (Gallo Research Center, Emeryville, CA) as previously described in our laboratory [12]. These mice along with their wild-type littermates were used for studies of cardiac function and SphK activity measurement in both cytosolic and membrane fractions. Only male mice were used for all studies. Genotyping using PCR to confirm the absence of PKC{varepsilon} DNA was routinely performed on tail samples.

2.7. Western blot analysis
Measurements of Akt, phospho-Akt, and PKC{varepsilon} protein were performed using standard SDS-PAGE Western blot techniques. Briefly, 50 µg of protein derived from either the cytosolic fraction or the membrane fraction of each homogenate was electrophoresed on a 10.0% denaturing gel at 30 mA per lane for 2 h. Proteins were electrotransferred onto a nitrocellulose membrane (Bio-Rad) at 200 mA for 1.5 h. The transfer efficiency was checked by Ponceau S (Sigma-Aldrich). Adequate background blocking was accomplished by incubating the nitrocellulose membrane with 5% nonfat dry milk in phosphate buffer solution (pH 7.4). Primary antibodies (Cell Signaling Technology, Inc, Beverly, MA and Upstate USA, Inc., Lake Placid, NY) were used to measure the expression of Akt, phospho-Akt, PKC{alpha}, {delta}, and {varepsilon}. The 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.

2.8. Co-immunoprecipitation
For these experiments, a homogeneous suspension of protein G agarose beads was added to the cytosolic and membrane samples prepared by homogenization and centrifugation at 100,000 x g for 40 min. The mixture was incubated overnight at 4 °C on a rocking platform. Beads were pelleted by centrifugation at 12,000 x g for 20 s and the supernatants transferred to fresh tubes. SphK1 (Novus Biologicals, Inc., Littleton, CO) antibody was added to the supernatant and the mixture gently rocked for 1 h at 4 °C. Protein G suspension was added to the mixture and incubated overnight at 4 °C. Complexes were collected by centrifugation at 12,000 x g for 20 s. Beads were then washed 3 times with wash buffer. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes and incubated with primary antibodies for PKC{alpha}, {delta}, {varepsilon}, and phospho-PKC{varepsilon} (Ser729). Nitrocellulose membranes were washed and incubated with secondary antibodies (Amersham Bioscience, Piscataway, NJ) for 1 h at room temperature. Protein was detected by ECL and quantitated by densitometric analysis of digitized autoradiograms with NIH Image 1.61 software.

2.9. Statistical analysis
The data are presented as mean±S.E.M. The significance of the differences in mean values for the LVDP, ±dP/dtmax and infarction size was evaluated by one-way ANOVA, followed by post-hoc testing (Newman–Keuls). Sphingosine kinase activity between two groups was evaluated by Student's t-test. P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Cardioprotection induced by DMS was abolished in PKC{varepsilon}-null mouse hearts
To determine if a low concentration of DMS can improve cardiac contractile function, we perfused mouse hearts with 0.3 µM and 1 µM DMS for 10 min and washed the preparation for 7 min before global ischemia and reperfusion. As shown in Fig. 1, there were no significant differences among all groups at baseline. After ischemia/reperfusion, LVDP, +dP/dtmax, and – dP/dtmax were decreased in the control group. DMS pretreatment improved cardiac function: LVDP at the end of reperfusion increased from 43.8±5.1 mm Hg in the vehicle-treated control group to 74.7±7.5 mm Hg in the DMS pretreated group (P<0.05, vs control group) (Fig. 1A). +dP/dtmax and – dP/dtmax were similarly increased (Fig. 1B and C). DMS also reduced infarction size to 24.2±2.4% of risk area compared with 41.5±3.3 in the control group (Fig. 2, P<0.05). Both 0.3 µM and 1 µM DMS exhibited cardioprotection (Fig. 3A). These beneficial effects induced by DMS were abrogated in PKC{varepsilon}-null mouse hearts (Figs. 1 and 2Go).


Figure 1
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  		Fig. 1 LVDP (A), +dP/dtmax (B), and – dP/dtmax (C) changes in 1 µM N, N-dimethyl-sphingosine (DMS) pretreated C57BL/6J, PKC epsilon knockout (KO), and wild-type (WT) littermate mouse hearts. Black: baseline; Gray: at the end of 30 min reperfusion. *P<0.05, compared with control group. #P<0.05, compared with DMS-WT group. n=3–6/group.

 

Figure 2
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  		Fig. 2 Infarction size measurement in N, N-dimethylsphingosine (DMS)-pretreated C57BL/6J PKC epsilon knockout (KO) and wild-type (WT) littermate mouse hearts. *P<0.05, compared with control group. n=3–6/group.

 

Figure 3
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  		Fig. 3 Dose–response effect of N, N-dimethylsphingosine (DMS) (A) and effect of EGF receptor inhibitor (AG1478) on cardiac function change induced by DMS (B). Black: baseline; Gray: at the end of 40 min reperfusion. *P<0.05, compared with control group. DMS 0.3, 1, and 3 refer to 0.3 µM, 1 µM, and 3 µM DMS correspondingly. AG: AG1478. n=3/group.

 
3.2. DMS-induced cardioprotection involves the epidermal grown-factor (EGF) receptor
Previous work has shown that DMS enhances EGF receptor autophosphorylatioon [14] and that the EGF receptor is involved in Akt phosphorylation [20]. We found that AG1478, an EGF receptor inhibitor, abolished the beneficial hemodynamic effects of DMS (Fig. 3B), thereby implicating the EGF receptor in the mechanism of DMS-induced cardioprotection.

3.3. DMS translocated and phosphorylated Akt in murine heart
As shown in Figs. 4 and 5Go, Akt distributed in 3 different fractions: cytosol, membrane, and a Triton-insoluble fracion. Pretreatment with 1 µM DMS for 10 min significantly increased Akt translocation from the Triton-insoluble fraction to the cytosol (P<0.05, vs control group). DMS infusion at 1 µM for 10 min also increased pAkt (Ser 473) expression in the cytosolic fraction (Fig. 5, P<0.05 vs control group). In our previous study, 10 µM DMS abolished cardioprotection induced by ischemic preconditioning and reduced PKC{varepsilon} phosphorylation (Ser 729) [12]. Here we found that 10 µM DMS slightly increased Akt translocation from the Triton-insoluble fraction to the cytosolic fraction, but had no effect on Akt phosphorylation (Ser 473) (Figs. 4 and 5Go, P=N.S.).


Figure 4
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  		Fig. 4 Akt translocation in N, N-dimethylsphingosine (DMS)-pretreated mouse heart (A) and Akt distribution in cytosol, membrane, and Triton-insoluble fractions after DMS pretreatment (B). NC: normal control; DMS: N, N-dimethylsphingosine. *P<0.05, compared with control (NC) group. NC: control; D1: DMS 1 µM; D10: DMS 10 µM. n=4/group.

 

Figure 5
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  		Fig. 5 Phospho-Akt (Ser 473) expression in N, N-dimethylsphingosine (DMS)-pretreated mouse heart. A) A characteristic western blot of Akt 473 phosphorylation and PKC{alpha} and PKC{varepsilon} responses in mouse heart tissue. C: cytosol; M: membrane fraction; T-I: Triton-insoluble fraction. NC: normal control; D1: DMS 1 µM; D10: DMS 10 µM. The PKC{alpha} and PKC{varepsilon} data were replicated 3 additional times in conjunction with the DMS data shown in panel B. B) Bar graphs showing phospho-Akt (473) expression in cytosol, membrane, and Triton-insoluble fractions after DMS pretreatment. *P<0.05, compared with the NC group. n=4/group.

 
3.4. DMS translocated and activated PKC{varepsilon} in murine heart
As shown in Fig. 5, 1 µM DMS increased PKC{varepsilon} expression in the cytosolic fraction, but had no effect on PKC{alpha} expression.

3.5. DMS pretreatment increased SphK activity in the cytosolic fraction
As shown in Fig. 6, SphK activity in the cytosolic fraction was increased after 1 µM DMS pretreatment for 10 min. S1P formation also was increased, whereas sphingosine content was reduced. The ratio of S1P to sphingosine was enhanced from 6.1±1.1% in the control group to 10.3±2.7% after pretreatment with 1 µM DMS (P<0.05, compared with the control group). In contrast to the results in the cytosolic fraction, DMS decreased SphK activity in the membrane fraction. The ratio of S1P to sphingosine was reduced from 1.01±0.12% to 0.66±0.026% (P<0.05, compared with the control group).


Figure 6
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  		Fig. 6 Sphingosine kinase (SphK) activity in the cytosolic fraction from control and 1 µM N, N-dimethyl-sphingosine (DMS)-pretreated mouse hearts. SphK activity is expressed as the ratio of [3H] S1P to [3H] Sphingosine. *P<0.05, compared with control group. n=5/group.

 
3.6. DMS did not increase SphK activity in PKC{varepsilon}-null murine hearts
As shown in Fig. 7, 1 µM DMS failed to increase SphK activity in the cytosolic fraction of PKC{varepsilon}-null murine hearts. The ratio of S1P to sphingosine was 6.7±0.9% in PKC{varepsilon}-null group. However, 1 µM DMS still increased SphK activity in wild-type littermate mouse hearts. The ratio of S1P to sphingosine was 11.4±1.7% in the wild-type littermate group.


Figure 7
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  		Fig. 7 Sphingosine kinase (SphK) activity in the cytosolic fraction from N, N-dimethylsphingosine (DMS)-pretreated PKC epsilon knockout (KO) and wildtype littermate (WT) mouse hearts. SphK activity is expressed as the ratio of [3H] S1P to [3H] Sphingosine. *P<0.05, compared with WT group. n=5/group.

 
3.7. SphK1 interacted with PKC{varepsilon}
SphK1 antibody was used to precipitate proteins in both cytosolic and membrane fractions of murine heart. PKC{alpha}, {delta}, and {varepsilon} antibodies were used to investigate the interaction between SphK1 and PKC isoforms. Only phospho-PKC{varepsilon} (Ser 729) and PKC{varepsilon} were detected in the cytosolic fraction (Fig. 8).


Figure 8
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  		Fig. 8 Interaction between SphK1 and PKC isoforms in cytosol and membrane fractions of mouse heart. C: Cytosol; M: Membrane. IP: Immunoprecipitation; WB: Western blotting. Mouse heart was separated into cytosol and membrane fractions. SphK1 antibody was added to both cytosol and membrane fractions for immunoprecipitation. The immunoprecipitation complex was separated by SDS-polyacrylamide gel electrophoresis. Protein was transferred to nitrocellulose membrane and incubated with PKC isoforms ({alpha}, {delta}, {varepsilon}) and Phospho-PKC{varepsilon} (Ser729) antibodies. The experiment was repeated a total of 3 times with identical results.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
4.1. DMS, PKC{varepsilon} and cardioprotection
N, N-Dimethylsphingosine (DMS) is an endogenous sphingolipid derived from the metabolism of ceramide within the cell [14]. Activation of neutral sphingomyelinase and accumulation of ceramide have been proposed as mediators of reperfusion injury [23]. Although ceramide induced cardiac apoptosis and decreased cardiac function [24,25], it has also been reported to be cardioprotective [9]. Both DMS and N, N, N-trimethylsphingosine (TMS) are methylation products of sphingosine that inhibited superoxide anion production in neutrophils and reduced neutrophil trans-endothelial migration [26]. Indeed, TMS, a synthetic sphingosine analogue, protected isolated rat heart against ischemia/reperfusion injury [27].

It is well-recognized that PKC{varepsilon} is involved in cardioprotection and ischemic preconditioning [28,29]. Phosphorylation (Ser 729) of PKC{varepsilon} has been shown to be associated directly with activation of PKC{varepsilon} [30]. Cytosloic phosphorylation (Ser 729) of PKC{varepsilon} was increased by IPC and pharmacological preconditioning [31,32]. Thus, PKC activators increased SphK activity and PKC inhibition had the opposite effect [33]. In HEK 293 cells, PKC-mediated activation of SphK was accompanied by a 4-fold increase in S1P in the media and SphK activation was not associated with its translocation [34,35]. In a previous study, we reported that IPC increased SphK activity in wild-type mouse hearts, but this enhanced SphK activity was abolished in hearts from PKC{varepsilon}-null mice [12].

Although DMS is present in mammalian brain [15], the role of endogenous DMS in response to injury in the cardiovascular system is unknown. DMS has been recognized as a selective competitive SphK inhibitor [16,17] and has been widely employed to measure the effect of SphK [36,37]. The IC50 is 5 µM in vitro. Our previous report showed that 10 µM DMS inhibited cardiac protection induced by ischemic preconditioning [12]. In this study, which was initially undertaken to determine whether a lower concentration of DMS would behave similarly, we were surprised to find that a low dose of DMS (0.3 µM and 1 µM) for 10 min had the opposite effect. Thus, these novel results are the first to show that DMS given at a low concentration protected isolated murine hearts against ischemia/reperfusion injury.

We also found that DMS-induced cardioprotection was abolished in PKC{varepsilon}-null mice. The failure of DMS to increase cardiac SphK activity in these genetically altered mice suggested that enzyme activation was indirect and indicated that DMS-induced SphK activation required PKC{varepsilon}. Indeed co-immunoprecipitation experiments revealed that SphK1 interacted with PKC{varepsilon} and phospho-PKC{varepsilon} in mouse heart. These results provide further evidence supporting the critical role of PKC{varepsilon} in the activation of SphK, and emphasize the importance of combined PKC{varepsilon}/SphK signaling in cardiac protection.

4.2. SphK, S1P, Akt, EGF receptor and cell survival
Activation of SphK, the key enzyme in the formation of S1P, has been implicated in enhanced cell survival, proliferation, and inhibition of apoptosis and oncogenesis. SphK activation increased intracellular S1P content and promoted cell growth and survival [38]. Overexpression of SphK induced endothelial cell survival primarily through the activation of PI-3K/Akt pathway and an associated up-regulation of Bcl-2 and down-regulation of Bim [39]. SphK1 expression also counteracted ceramide-induced cell death related to Bcl-2 [40].

In the heart, S1P, the metabolic product of SphK, achieved cardioprotection through a PKC{varepsilon}-independent pathway, whereas ganglioside GM1, a SphK activator, protected hearts against ischemia/reperfusion injury via a PKC{varepsilon} dependent pathway [8]. In separate preliminary experiments, we have found that myocardial ischemia/reperfusion injury was exaggerated in SphK1-null mice, while ischemic preconditioning was abolished [41]. In a previous study, we found that ex vivo ischemic preconditioning enhanced and 10 µM DMS reduced SphK activity and also abolished ischemic preconditioning [12].

Several studies have shown that S1P, the product of SphK, increased Akt activation in many cell types [42–44], including adult cardiac myocytes [45]. Akt is a pro-survival kinase that is activated by ischemic preconditioning [46]. In isolated adult murine cardiac myocytes we found S1P induced cardioprotection via a Gi/PI-3 kinase/Akt pathway that resulted in phosphorylation of BAD and GSK-3β. Wortmannin, the PI3 kinase inhibitor, abolished the beneficial response induced by S1P [45]. In the present study, 1 µM DMS increased Akt translocation from the Triton-insoluble fraction to the cytosol, where its activity was increased, and enhanced cytosolic phosphorylation of Akt at Ser 473. Conversely, DMS at 10 µM had no effect on phosphorylation of Akt (Ser 473) and lost its ability to induce cardioprotection at this concentration, as previously shown in our laboratory [12].

EGF receptor activation was involved in the cardioprotection induced by low dose of DMS. AG 1478, an EGF receptor inhibitor, abolished the DMS-induced beneficial effect on hymodynamics. Recently, Sukocheva et al. reported that an autocrine or paracrine S1P signaling loop, triggered by SphK1 activation, plays a critical role in transactivating the EGF receptor via the S1P3 receptor subtype [47].


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
Exogenously administrated DMS elicited a biphasic response on activation of SphK and hence on cardioprotection. At higher concentrations (such as 10 µM), it inhibited SphK activity, and prevented cardioprotection associated with ischemic preconditioning [10]. Conversely, at a lower concentration, such as the 1 µM dose used in the present study, it activated SphK via PKC{varepsilon}, and was cardioprotective (Fig. 9). Fig. 9 also illustrates that ceramide can be metabolized both to sphingosine and to DMS and thus under some circumstances could be cardioprotective [9]. Our observations also suggest the possibility, which deserves further study, that endogenous low dose DMS could play a role in preconditioning, and be a therapeutic target for enhancing cardioprotection.


Figure 9
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  		Fig. 9 Proposed pathway for N, N-dimethylsphingosine (DMS)-induced cardioprotection. DMS exerts dual effects on sphingosine kinase activity. At low concentration (1 µM), DMS enhances cytosolic SphK activity by activating PKC{varepsilon}. At high concentration (>5 µM), DMS inhibits both cytosol and membrane SphK activity by a direct competitive mechanism [16].

 


   Acknowledgement
 
This work was supported by NIH grant P01 HL068738 (JSK).


   Notes
 
Time for primary review 14 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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