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Deletion of the Sphingosine Kinase-1 gene influences cell fate during hypoxia and glucose deprivation in adult mouse cardiomyocytes

Rong Tao, Jianqing Zhang, Donald A. Vessey, Norman Honbo, Joel S. Karliner
DOI: http://dx.doi.org/10.1016/j.cardiores.2007.01.015 56-63 First published online: 1 April 2007

Abstract

Objectives: Activation of sphingosine kinase (SphK), which has two known isoforms, is responsible for the synthesis of sphingosine 1-phosphate (S1P), a cell survival factor. We tested the following hypotheses: 1] cardiac myocytes null for the SphK1 gene are more vulnerable to the stress of hypoxia+glucose deprivation; 2] the monoganglioside GM-1, which activates SphK via protein kinase C ε, is ineffective in SphK1-null myocytes; 3] S1P generated by SphK activation requires cellular export to be cardioprotective.

Methods: We cultured adult mouse cardiac myocytes from wildtype and SphK1-null mice (deletion of exons 3–6) and measured cell viability by trypan blue exclusion.

Results: In wildtype adult mouse cardiomyocytes subjected to 4 h of hypoxic stress+glucose deprivation, cell viability was significantly higher than in SphK1-null cardiomyocytes. SphK1-null cells also displayed more mitochondrial cytochrome C release. Cell death induced by hypoxia+glucose deprivation was substantially prevented by pretreatment with exogenous S1P in both wildtype and SphK1-null myocytes, but S1P was effective at a lower concentration in wildtype cells. Hence, the absence of the Sphk1 gene did not affect receptor coupling or downstream signal transduction. Pretreatment for 1 h with 1 μM of the monoganglioside GM-1 increased survival in wildtype cells, but not in SphK1-null myocytes. Thus, activation of SphK1 by GM-1 leads to cell survival. In wildtype cells, enhanced survival produced by GM-1 was abrogated by pretreatment either with 300 nM of the S1P1 receptor-selective antagonist VPC23019 or with 100 ng/ml of pertussis toxin for 16 h before exposure to hypoxia+glucose deprivation.

Conclusion: As the effect of GM-1 is blocked both at the receptor and the G-protein (Gi) levels, we conclude that S1P generated by GM-1 treatment must be exported from the cell and acts in a paracrine or autocrine manner to couple with its cognate receptor.

Keywords
  • Sphingosine kinase
  • Sphingosine-1-phosphate
  • Cytochrome C
  • Hypoxia

This article is referred to in the Editorial by Q. Yang (pages 4–5) in this issue.

1 Introduction

Sphingosine-1-phosphate (S1P), a product of sphingosine kinase (SphK) activation, is an intracellular and extracellular signaling molecule that regulates many important cellular processes including growth, survival, differentiation, cytoskeletal rearrangements, motility, angiogenesis, and calcium mobilization [1,2]. In contrast, ceramide is a growth-inhibiting lipid implicated in differentiation and apoptosis [3]. These findings have led to the so-called “sphingolipid rheostat” hypothesis which proposes that the relative levels of these lipids are important determinants of cell fate [4]. It is now accepted that many of the actions of S1P are mediated by a family of G protein-coupled S1P receptor isoforms, termed S1P1–5 [5,6]. We previously identified S1P as a cardioprotective agent in cultured cardiac myocytes [7,8] and also in isolated hearts [9].

In many cell types the normal intracellular amount of S1P is very low, due to degradation by sphingosine phosphate lyase (SPL) and dephosphorylation by S1P phosphohydrolases [10]. Activation of SphK is the final and rate-limiting step in the synthesis of S1P, and thus this enzyme serves the dual function of modulating both ceramide and S1P levels [11]. Two isozymes of mammalian SphK, SphK1 and SphK2, have been cloned and characterized [12,13]. SphK1 and SphK2 have different cellular functions. SphK1 is the isoform implicated in cell growth, proliferation, anti-apoptosis, and inflammatory responses [14,15]. SphK2, on the other hand, has been described as a growth inhibitor, and is considered to be pro-apoptotic [16]. A recent study showed that SphK1 and SphK2 have opposing roles in the regulation of cell responses and suggested that the location of S1P production dictates its functions [17]. S1P produced by translocation of SphK1 to the plasma membrane has been implicated in transactivation of cell surface S1P receptors [18,19].

SphK1 inhibitors [20], dominant negative SphK1 [21] and small interfering RNA [14] have been used to study the function of endogenous SphK1. Limitations to these studies include the lack of a specific SphK1 inhibitor and the absence of an effect on basal enzyme activity. To circumvent these limitations, we used SphK1-null mice to ask the following questions: 1] Are cardiac myocytes null for the Sphk1 gene more vulnerable to hypoxic stress? 2] If so, is exogenous S1P capable of preventing hypoxic cell death in SphK1-null cells? 3] Does intracellular S1P generated by SphK activation require cellular export to exert cardioprotection?

2 Materials and methods

2.1 Materials

Sphingosine 1-phosphate and pertussis toxin were from BIOMOL International (Plymouth Meeting, Pennsylvania). VPC23019 was from Avanti Polar Lipids, inc. (Alabaster, Alabama). GM1 was from Sigma (St. Louis, Missouri). Antibodies directed against cytochrome C and Cox4 were from Cell Signaling Technology (Danvers, Massachusetts). Antibody directed against SphK-1 was from ECM Biosciences (Versailles, Kentucky). Antibody directed against SphK-2 was from Santa Cruz Biotechnology (Santa Cruz, California).

2.2 Animals

SphK1-null mice were provided by Drs. Shaun Coughlin and Rajita Pappu (Cardiovascular Research Institute, University of California, San Francisco). Only male mice were used for all studies. Genotyping using PCR to confirm the absence of exons 3–6 of SphK1 DNA was routinely performed on tail samples as described [22]. The background of these mice is C57BL6. All studies were approved by the Institutional Animal Care and Use Committee of the San Francisco Veterans Affairs Medical Center. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.3 Adult mouse cardiac myocyte isolation and culture

Adult mouse cardiac myocytes were isolated and cultured using a modification of the collagenase dissociation method described by Zhou et al. [23]. Mice were treated with heparin (50 U) and anesthetized by intraperitoneal injection with sodium pentobarbital (200 mg/kg). The heart was quickly excised and the aorta cannulated for retrograde perfusion in a Langendorff apparatus at a constant flow rate of 3 ml/min at 37 °C. The heart was perfused for 9–10 min with isolation buffer (120 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 5.6 mM glucose, 5 mM NaHCO3, 10 mM HEPES, 50 μM CaCl2, 10 mM 2, 3-butanedione monoxime (BDM) and 5 mM taurine followed by digestion for 9 min with collagenase II (1.5 mg/ml, Worthington, Lakewood, New Jersey) in isolation buffer. After digestion, the soft and flaccid heart was removed, and myocytes were suspended in isolation buffer. A series of four centrifugations (40 ×g, 1 min) and resuspensions were used for stepwise Ca2+ reintroduction from 50 μM to 1.0 mM, which was the final medium Ca2+ concentration.

Isolated cardiac myocytes were plated for 2 h on 35 mm and 60 mm tissue culture dishes coated with 10 μg/ml laminin. The cells were suspended in minimum essential medium (MEM) with Hanks Buffered Salt Solution (HBSS), 10 μg/ml penicillin, 1.5 μM vitamin B12, and 10 mM BDM. After this period of attachment, the medium was changed to MEM/HBSS containing 10 μg/ml penicillin, 1.5 μM vitamin B12, and 1 mM BDM, and incubated overnight at 37 °C in a humidified atmosphere of 1% CO2 and air. The culture protocol yielded an average of 80% rod-shaped myocytes at a plating density of 50 cells/mm2 that were viable at pH 7.2 for 48 h. Experiments were performed the day following isolation and culture.

2.4 Hypoxia protocol

On the day after isolation and culture, cardiac myocytes were treated with pharmacological agonists and antagonists under normoxic conditions for the indicated times. The cells were rinsed and subsequently incubated in a Bactron I anaerobic chamber containing a humidified atmosphere of 1% CO2 and 99% N2 for 4–5 h. Experimental medium was changed to serum-free, glucose-free MEM with HBSS that did not contain BDM. This medium was pre-equilibrated overnight in the anaerobic chamber containing 1% CO2 and 99% N2. Normoxic experimental medium was pre-equilibrated overnight in water-jacketed incubators in a humidified atmosphere of 1% CO2 and air.

2.5 Measurement of cell survival

Cardiac myocyte survival was measured by staining cells in tissue culture dishes with trypan blue solution (Sigma Chemical, St. Louis, Missouri) diluted to a final concentration of 0.04% (w/v). Myocytes were visualized using brightfield microscopy at 100× magnification. The number of viable (unstained) and non-viable (blue stained) cardiac myocytes in ten random microscopic fields was recorded, and at least 300 cells were counted in each dish. Percent survival was defined as the number of unstained myocytes counted in each hypoxic treatment dish divided by the number of unstained myocytes counted in each corresponding normoxic control dish. This calculation accounted for the detachment and loss of non-viable cells during the experimental hypoxia protocols.

2.6 Assay for sphingosine kinase activity

On the day after isolation and culture, dishes were scraped and cells decanted into a buffer containing 0.13 M KCl, 20 mM HEPES pH 7.4, 1 mM EGTA, 1 μg/L leupeptin, and 0.25 μg/L each of chymostatin and pepstain A. Cell lysates were prepared by sonication, followed by centrifugation at 45,000 ×g for 30 min. SphK activity was assayed as previously described in our laboratory [24].

2.7 Western blot analysis

For whole cell extraction, cells were lysed in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 0.1% protease inhibitor mixture (Roche) and phosphatase inhibitor cocktails (Sigma). For cellular fractionation extraction, cells were lysed in buffer (0.25 M sucrose, 5 mM HEPES buffer, 1 mM EDTA, pH 7.2) for 10 min on ice. The lysate was homogenized with a grinder and centrifuged at 700 ×g for 10 min. The supernatant was transferred to a fresh tube and centrifuged at 10,000 ×g for 25 min. Protein concentration was determined using the Bradford method. Equal amounts of protein were resuspended in 4× Laemmli sample buffer, boiled for 5 min, and subjected to sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis. After transfer to polyvinylidene fluoride membrane, the extract was blocked in 5% nonfat milk in Tris-buffered saline (TBS)+0.1% Tween-20 for 1 h. The membranes were probed overnight with primary antibodies, washed three times with TBS/Tween-20 for 5 min, and probed with secondary antibodies for 1 h. The membranes were rinsed three times, and the signal was detected using enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer's instructions.

2.8 Statistical analysis

Data are expressed as the mean±SEM. Mean values were compared by one-way analysis of variance (ANOVA) and post hoc testing (Newman–Keuls). Differences in SphK activity, cytochrome C expression and SphK2 expression were evaluated by Student's t-test. P<0.05 was considered statistically significant.

3 Results

3.1 Effect of SphK1 gene deletion on cardiac myocyte survival

As shown in Fig. 1, SphK1 is expressed only in the wildtype cardiomyocytes, confirming the absence of this isozyme in the SphK1-null mouse heart. In initial experiments, we exposed adult mouse cardiomyocytes from wildtype (WT) and SphK1-null mice to hypoxia+glucose deprivation for 4 h. Cell viability was quantified using the trypan blue exclusion assay. As shown in Fig. 2, after 4 h of hypoxia, cell viability in WT cardiomyocytes was significantly higher than in SphK1-null cardiomyocytes. This result suggested that reduced SphK1 activity is associated with impaired survival in adult mouse cardiomyocytes subjected to the stress of hypoxia+glucose deprivation. In additional experiments we found that SphK1-null cardiomyocyte viability was not different from wildtype cardiomyocytes under normoxic culture conditions (data not shown).

Fig. 2

Cell viability after exposure to hypoxia+glucose deprivation. (A) Isolated adult mouse cardiomyocytes were exposed to hypoxia+glucose deprivation for 4 h. Cell viability was quantified by staining cells with trypan blue solution and counting the number of cells that excluded this agent. The left panel shows four viable rod-shaped cardiac myocytes and three contracted myocytes that have taken up trypan blue. The arrow points to a contracted myocyte that continues to exclude trypan blue. The right panel shows two rod-shaped myocytes and four contracted myocytes that have taken up trypan blue (arrow). Scale=50 μM. (B) Percent survival is shown. Data are expressed as the percentage of the normoxic control values, which are set at 100%. Data represent the mean±SEM of at least five independent experiments. WT: wildtype cells; SphK1 KO: SphK1-null cells. *p<0.05 vs WT.

Fig. 1

SphK1 expression in WT and SphK1-null mouse hearts. SphK1 protein expression was determined by Western blotting in cell lysates. SphK1 migrated at the expected molecular mass. This experiment was repeated 3 times with identical results. WT: wild type cells; SphK1 KO: SphK1-null cells.

3.2 Cytochrome C release

Because the mitochondrion is a key integrator of apoptotic signals, the effects of deleting SphK1 on mitochondrial function were assessed. Fig. 3 illustrates the effects of hypoxia+glucose deprivation for 4 h on the intracellular localization of cytochrome C in cardiomyocytes. Compared with WT cardiomyocytes, the absence of SphK1 caused a decline in cytochrome C immunoreactivity in the mitochondrial fraction, with a concomitant increase in the cytosolic fraction. This result suggested that the absence of SphK1 was associated with the release of pro-apoptotic cytochrome C from the intermembrane space into the cytosol under the stress of hypoxia+glucose deprivation.

Fig. 3

Cytochrome C release in wild type and SphK1-null adult cardiomyocytes. Cardiac myocytes were exposed to hypoxia+glucose deprivation for 4 h. Upper panel: The expression of cytochrome C protein and its intracellular distribution were determined by Western blotting in cell lysates. Cox4 and GAPDH were used to confirm equal loading of the mitochondrial and cytosolic extracts, respectively. Lower Panel: Quantification of western blotting data. Results are mean±SEM from five independent experiments. WT: wildtype cells; SphK1 KO: SphK1-null cells. *p<0.05 vs WT cytosol. #p<0.05 vs WT mitochondria.

3.3 Effects of SphK1 deletion on sphingosine kinase activity

As shown in Fig. 4A, SphK activity was detected in both cytosolic and membrane fractions in WT and SphK1-null cardiac myocytes, with the majority of enzyme activity detected in cytosol. Total SphK activity was higher in WT cardiac myocytes than SphK1-null cardiac myocytes. As shown in Fig. 4B, there was no difference in SphK2 protein expression between WT and SphK1-null mouse myocytes.

Fig. 4

SphK activity in wild type and SphK1-null mice. (A) Total SphK activity was determined in adult cardiac myocytes. SphK activity is expressed as pmol of S1P formed per min per mg of cell protein. Data represent the mean±SEM of five independent experiments. WT: wildtype cells; SphK1 KO: SphK1-null cells. *p<0.05 vs WT. (B). SphK2 protein expression in myocytes was determined by Western blotting. GAPDH was used to confirm equal loading of the whole cell extracts. Data are expressed as mean±SEM of three independent experiments.

3.4 Effects of treatment with sphingosine 1-phosphate (S1P) on hypoxia-induced adult cardiomyocyte viability of WT and SphK1-null mice

Cardiomyocytes were pretreated for 1 h with vehicle or S1P, washed and transferred to a hypoxia chamber for 4 h in 99% N2/1% CO2 in glucose-free medium. The effect of S1P on cardiomyocyte viability after hypoxia+glucose deprivation and exposure is shown in Fig. 5. Preincubation of WT adult mouse cardiomyocytes with 100 nM or 1 μM S1P for 1 h reduced cell death induced by hypoxia+glucose deprivation compared with control. After 4 h of hypoxia+glucose deprivation, the viability of SphK1-null cardiomyocytes pretreated only at 1 μM S1P was significantly higher than control myocytes treated with vehicle alone. These data suggest that cell death induced by hypoxia was substantially prevented by pretreatment with exogenous S1P in both WT and SphK1-null cardiac myocytes. However, as shown in Fig. 5, S1P also was effective at a lower concentration in WT cells.

Fig. 5

Effects of treatment with sphingosine 1-phosphate (S1P) on SphK1 null and wildtype adult cardiac myocytes subjected to hypoxia+glucose deprivation. Cardiomyocytes were treated for 1 h with vehicle, S1P 100 nM or 1 μM before exposure to 4 h of hypoxia+glucose deprivation. The effect of S1P on cardiomyocyte viability as measured by trypan blue exclusion after hypoxia+glucose deprivation is shown. Data are expressed as the percentage of viable normoxic control cells, which was set as 100%. The mean±SEM of at least five independent experiments is shown. WT: wildtype cells; SphK1 KO: SphK1-null cells. *p<0.05 vs WT vehicle, #p<0.05 vs SphK1 KO vehicle.

3.5 Effects of treatment with GM1 and a S1P receptor antagonist or pertussis toxin (PTX) on hypoxia-induced adult cardiomyocyte viability

As shown in Fig. 6, preincubation of WT adult cardiac myocytes with 1 μM of the monoganglioside GM1 for 1 h reduced cell death associated with hypoxia+glucose deprivation. The increased cell viability induced by GM1 was abolished after overnight treatment with 100 ng/ml of pertussis toxin (PTX). It is known that PTX ADP-ribosylates and thereby inactivates Gi through which S1P transduces signals after binding to its cognate receptors [25]. Pretreatment of WT myocytes with 300 nM VPC23019, an inhibitor of S1P1 and S1P3 receptors at this concentration [26], abrogated cell survival stimulated by GM1. The viability of GM1-pretreated SphK1-null adult cardiac myocytes was not significantly different from that of control myocytes incubated under conditions of hypoxia+glucose deprivation. The viability of GM1+VPC23019 or GM1+PTX pretreated SphK1-null adult cardiac myocytes was not different from that of control myocytes. These data indicate that GM1, which increases intracellular S1P levels by activating SphK [27], can prevent only WT myocyte death induced by hypoxia, thus indicating that GM1 activates only the SphK1 isoform. Abrogation of the GM1 effect by VPC23019 and pertussis toxin also suggests that intracellular S1P must be exported to act extracellularly in a paracrine or autocrine manner (Fig. 7).

Fig. 7

Proposed model for SphK1-mediated cell survival. Our results are consistent with a prosurvival model of cellular export of S1P. We used the monoganglioside GM1 to stimulate SphK1 activity resulting in the formation of intracellular S1P which our evidence suggests is then exported from the cell. Extracellular S1P then binds to cognate receptors, particularly the S1P1 receptor, to initiate a prosurvival signaling cascade as previously described in our laboratory (8). Cell viability is thereby enhanced, and mitochondrial cytochrome C release is diminished.

Fig. 6

Effect of treatment with GM1 and a S1P receptor antagonist or pertussis toxin (PTX) on SphK1 null and wildtype adult cardiac myocytes subjected to hypoxia+glucose deprivation. Cardiomyocytes were treated for 1 h with 1 μM of the monoganglioside GM1, for 30 min with 300 nM of the S1P1 and 3 receptor antagonist VPC23019, or for 16 h with 100 ng/ml of the Gi/o inhibitor pertussis toxin (PTX) before exposure to 4 h hypoxia. Data are expressed as the percentage of the normoxic control cells which was set at 100%. The mean±SEM of the following number of experiments is shown, vehicle and GM1, 11–19 experiments; GM1+VPC23019 and GM1+PTX, 6 experiments. *, #p<0.05 vs WT vehicle.

4 Discussion

The major findings of this study, which is the first to use SphK1-null cardiac myocytes to examine responses to in vitro stress due to hypoxia+glucose deprivation are as follows: 1]. Cardiac myocytes null for the Sphk-1 gene are more vulnerable to hypoxic stress. 2]. Exogenous S1P is capable of preventing hypoxic cell death in SphK-1-null cells. 3]. Intracellular S1P generated by SphK activation requires cellular export to exert cardioprotection.

Mammals carry two known SphK genes, which in mice are encoded by SphK1 and SphK2. Several additional isoform variants of SphK 1 and 2 have also been postulated [28]. The tissue distribution and developmental expression pattern differ between these two major isoforms [10,13]. Diverse external stimuli, particularly growth and survival factors, stimulate SphK1, and intracellularly generated S1P has been implicated in their mitogenic and anti-apoptotic effects [16]. In contrast, SphK2, a BH3-only protein, enhances apoptosis in diverse cell types induced by a variety of stressful stimuli [16]. Our results show that endogenous SphK1 is necessary to maintain the viability of adult mouse cardiac myocytes during stress due to hypoxia+glucose deprivation. Deletion of the SphK1 gene activates the intrinsic mitochondrial cell death pathway compared with WT cardiomyocytes. Thus, the absence of SphK1 augmented adult cardiomyocyte death during the stress of hypoxia+glucose deprivation, in association with cytochrome C release, a known marker and instigator of cell demise. Consistent with these observations is a recent report which showed that knockdown of SphK1 by small interfering RNA caused MCF-7 cell cycle arrest and induced apoptosis [14]. Endogenous SphK1 is an important regulator of ceramide levels in the cell, and its downregulation results in enhanced ceramide synthesis and its accumulation in the mitochondrion, which may be key in initiating the mitochondrial events leading to cell death [14]. Pchejeski et al. found the generation of ROS resulted in SphK1 inhibition and caused apoptosis in H9c2 cardiomyoblasts [29]. Moreover, the balance between the intracellular levels of ceramide and S1P and their regulatory effects on different family members of prosurvival and pro-apoptotic protein kinases has been proposed as a major determinant of cell fate [30]. In this connection it should be noted that both isolated and intact hearts null for the SphK1 gene exhibit a higher susceptibility to ischemic damage [22,31].

Allende et al. have also reported the generation of SphK1-null mice [32]. These mice differed from our model in which all of exons 3–6 of the SphK1 gene were targeted while Allende et al. deleted exons 3–5 and the 5′ end of exon 6 [32]. Like them, we also found these mice to be fertile, long lived, and without any obvious abnormalities. Although they performed no functional cardiac studies, Allende et al. reported a greater than 60% reduction in serum S1P levels, but surprisingly no significant decrease in tissue levels, including brain, heart, kidney, liver, spleen and testis. However, substantial reductions in SphK1 enzyme activity were observed in all of these organs, suggesting a compensatory increase in SphK2 activity. Of note, in their SphK1-null mice, Allende et al. reported no changes in the mRNA levels for the genes encoding enzymes that are known to regulate S1P levels, including S1P lyase, S1P phosphatase, or ceramidase [32].

We measured SphK activity of cultured adult mouse cardiac myocytes both in cytosolic and membrane fractions, whereas SphK activity reported by Allende et al. was prepared from a whole heart homogenate [32]. Like Allende et al., we found a significant reduction in enzyme activity in the cytosolic fraction isolated from SphK1-null cardiomyocytes, but in a membrane fraction there was no difference. In this connection, Igarashi et al. reported that SphK1 localizes mainly in the cytosol and that the enzyme stimulates DNA synthesis [33]. We did not detect a difference in SphK2 protein expression between SphK1-null and WT myocytes. The remaining SphK activity in Sphk1-null cardiomyocytes is likely attributable to SphK2, which has been found to be widely distributed among mouse tissues [32].

S1P is the ligand for a family of specific G-protein-coupled receptors that regulate a wide variety of important cellular functions, including growth, survival and motility [34]. In present study, we found exogenously applied S1P was effective in preventing cell death induced by stress of hypoxia+glucose deprivation both in WT and SphK1-null cardiomyocytes. We have previously shown that exogenous S1P initiates a pertussis toxin inhibitable survival cascade in adult mouse cardiac myocytes by binding to the S1P1 receptor [8]. Subsequent signals include activation of the phosphorylated forms of Akt, PI3 kinase, BAD, and GSK-3β, and a reduction in cytochrome C release [8]. Our finding that S1P was effective in SphK1-null mouse myocytes suggests that this prosurvival signal transduction pathway remains intact. However a lower concentration of S1P (100 nM) was totally effective in wildtype cells, while efficacy was reduced in SphK1 knockout myocytes (Fig. 5).

In this study we addressed another unresolved issue, which relates to the intracellular vs extracellular action of S1P. The monoganglioside GM1 increases intracellular S1P levels by activating SphK [27]. We reasoned that if intracellular S1P levels increased by incubation of cardiomyocytes with GM1, blockade of S1P-mediated responses by the S1P receptor antagonist VPC23019 [26] and by the guanine nucleotide binding protein Gi, which transduces signals from S1P bound to its cognate receptors [25], should not inhibit the intracellular action of S1P. However, if S1P requires export from the cell to act in an autocrine or paracrine manner, these maneuvers should abrogate S1P-dependent cell survival. We found GM1 was cardioprotective in WT myocytes but not in SphK1-null myocytes, indicating that GM1 stimulates only SphK1 activity. We also noted that VPC23019 and pertussis toxin reduced cell survival stimulated by GM1. These results are consistent with export of intracellular S1P to the cell surface and/or extracellular space and are in agreement with recent work in other cell types. For example, Anelli et al. also provided evidence for the extracellular release of S1P by primary cells from CNS, which supports a role of S1P as an autocrine/paracrine physiological messenger in the cerebellum [35]. An alternative or complementary explanation proposed by Venkataraman et al. [28] is that an isoform of SphK1 itself is exported from the cell and thereby could be responsible for substantial extracellular S1P synthesis.

In summary, this study has examined the role of endogenous SphK1 in the regulation of adult cardiac myocyte survival subjected to stress of hypoxia+glucose deprivation. We found that cardiac myocytes null for the Sphk-1 gene are more vulnerable to stress of hypoxia+glucose deprivation and that exogenous S1P is capable of preventing hypoxic cell death in SphK-1-null myocytes. Furthermore, as shown schematically in Fig. 7, our data are consistent with the hypothesis that S1P generated by GM-1 treatment must be exported from the cell and acts in a paracrine or autocrine manner to couple with its cognate receptor.

Acknowledgements

This work was supported by NIH grant 1P01 HL068738-01A1 (JSK).

The authors thank Drs. Shaun Coughlin and Rajita Pappu for supplying the SphK1-null mice.

Footnotes

  • Time for primary review 28 days

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View Abstract