Ischaemic postconditioning protects isolated mouse hearts against ischaemia/reperfusion injury via sphingosine kinase isoform-1 activation

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1 Cardiovascular Research (2008) 79, doi: /cvr/cvn065 Ischaemic postconditioning protects isolated mouse hearts against ischaemia/reperfusion injury via sphingosine kinase isoform-1 activation Zhu-Qiu Jin 1, Joel S. Karliner 1,2,3, and Donald A. Vessey 2,4 * 1 Cardiology Section, Veterans Affairs Medical Center, San Francisco, CA 94121, USA; 2 Department of Medicine, University of California, San Francisco, CA 94143, USA; 3 Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA; and 4 Liver Study Unit (151-K), Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA Received 9 November 2007; revised 28 February 2008; accepted 6 March 2008; online publish-ahead-of-print 11 March 2008 Time for primary review: 30 days KEYWORDS Ischaemia; Preconditioning; Postconditioning; Sphingosine kinase; Signal transduction Aims Sphingosine-1-phosphate (S1P) plays a vital role in cytoskeletal rearrangement, development, and apoptosis. Sphingosine kinase-1 (SphK1), the key enzyme catalyzing the formation of S1P, mediates ischaemic preconditioning. Ischaemic postconditioning (POST) has been shown to protect hearts against ischaemia/reperfusion injury (IR). To date, no studies have examined the role of SphK1 in POST. Methods and results Wild-type (WT) and SphK1 null (KO) mouse hearts were subjected to IR (45 min of global ischaemia and 45 min of reperfusion) in a Langendorff apparatus. Left ventricular developed pressure (LVDP), maximum velocity of increase or decrease of LV pressure (+dp/dt max ), and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by 1% triphenyltetrazolium chloride staining. POST, consisting of 5 s of ischaemia and 5 s of reperfusion for three cycles after the index ischaemia, protected hearts against IR: recovery of LVDP and +dp/dt max were elevated; LVEDP was decreased; infarction size (% of risk area) was reduced from % in the control group to % of the risk area in the POST group (P, 0.05, n ¼ 4 per group). Phosphorylation of Akt and extracellular signal-regulated kinases detected by Western blotting was increased at 10 min of reperfusion. The protection induced by POST was abolished in KO hearts. Infarction size in KO hearts (57 + 5%) was not different from the KO control group (53 + 5% of risk area, n ¼ 4, P ¼ NS). Conclusions A short period of ischaemic POST protected WT mouse hearts against IR. The cardiac protection induced by POST was abrogated in SphK1 KO mouse hearts. Thus, SphK1 is critical for successful ischaemic POST. 1. Introduction Short periods of ischaemia and reperfusion subsequent to a long period of myocardial ischaemia protect hearts against ischaemia/reperfusion damage. This is referred to as ischaemic postconditioning (POST). POST reduces infarction size, improves cardiac function, and decreases arrhythmias. 1 Adenosine, K ATP channel opening, phosphorylation of Akt, and PKC activation mediate this protection. 2,3 POST has been observed in different species (dog, rabbit, mouse, rat, and human). The mouse heart model is ideal for genetic manipulation and useful for recording of cardiac function. Sphingosine kinase (SphK) is a bioactive lipid kinase which is responsible for the formation of sphingosine-1-phosphate (S1P) within the cell. 4 S1P plays a vital role in cellular mitosis, apoptosis, cytoskeletal rearrangement, and survival. * Corresponding author. Tel: þ ; fax: þ address: donald.vessey@va.gov Recent studies indicate that S1P protects hearts against ischaemia/reperfusion injury (IR) when administered prior to the index ischaemia (preconditioning) and mediates myocardial ischaemic preconditioning (IPC) in isolated mouse hearts. 5 7 IPC-induced cardioprotection is abolished in SphK1-null mouse hearts. Phosphorylation of Akt (Ser437) remains unchanged in SphK1-null mouse hearts after IPC. 8 There are two isoforms of sphingosine kinase: isoforms 1 and 2. SphK1 exerts anti-apoptotic effects and SphK2 appears to be pro-apoptotic. 9 In the present study, we used a genetic approach to address the hypothesis that blocking the activity of SphK isoform 1 abrogates the beneficial effects provided by ischaemic POST. 2. Methods This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academic Press, Published on behalf of the European Society of Cardiology. All rights reserved. & The Author For permissions please journals.permissions@oxfordjournals.org.

2 Sphingosine kinase-1 in ischaemia/reperfusion and postconditioning 135 Washington, DC, 1996), and all procedures were approved by the Animal Care Subcommittee of the San Francisco Department of Veterans Affairs Medical Center. 2.1 SphK1-null mice SphK1-null 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, CA, USA). Male homozygous null (SphK12/2) and wild-type (WT) mice were generated by breeding heterozygous (SphK1þ/2) mice. Genotyping using PCR to confirm the absence of exons 3 6 of SphK1 DNA was routinely performed on tail biopsies of 3-week old mice. Details have been reported previously s of reperfusion for three cycles. POST III was 20 s of ischaemia and 20 s of reperfusion for three cycles. IPC consisted of 2 min of global ischaemia and 2 min of reperfusion, followed immediately by prolonged IR as described above. Haemodynamics [left ventricular developed pressure (LVDP), LV end-diastolic pressure (LVEDP), LV + dp/dt max, and coronary flow] were recorded as previously described. 5,6 2.2 Langendorff-isolated perfused heart preparation WT and SphK1 null mice (males, weighing g) were heparinized (500 U/kg, IP) and anaesthetized with sodium pentobarbital (60 mg/ kg, i.p.). 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 mmhg on a modified Langendorff apparatus using Krebs Henseleit solution containing (mmol/l) NaCl 118, KCl 4.7, CaCl 2 2.5, MgSO 4 1.2, KH 2 PO 4 1.2, NaHCO 3 24, glucose 5.5, and sodium pyruvate 5.0 bubbled with 95% O 2 /5% CO 2 at 378C as previously described in our laboratory. 5 Platinum electrodes connected to a stimulus generator (Grass Instruments, West Warwick, RI, USA) were used to pace hearts at 360 b.p.m. An isovolumic balloon filled with degassed distilled water was inserted into the left ventricle (LV) to record haemodynamics. 2.3 Ischaemic postconditioning protocols and ischaemia-reperfusion protocol For ischaemia-reperfusion experiments, the protocol (IR) consisted of a 20 min equilibration period, followed by 45 min of global ischaemia and 45 min of reperfusion. For POST, the protocol was identical except that it was performed at the onset of reperfusion (Figure 1). POST I was composed of 5 s of ischaemia and 5 s of reperfusion for three cycles. POST II consisted of 10 s of ischaemia and Figure 1 Protocols for studies of ischaemic preconditioning and postconditioning in isolated mouse hearts. Hearts were subjected to 20 min of equilibration, 45 min of global ischaemia, and 45 min of reperfusion. POST I consisted of 5 s of reperfusion and 5 s of ischaemia for three cycles immediately after the cessation of the 45 min ischaemic period. Similarly, following prolonged ischaemia, POST II consisted of 10 s of reperfusion and 10 s of ischaemia for three cycles. POST III was 20 s of reperfusion and 20 s of ischaemia for three cycles following prolonged ischaemia. Figure 2 Left ventricular developed pressure (LVDP) (A), its maximum increase (B) and decrease (C) of velocity (+dp/dt max ) in ischaemic preconditioning- and postconditioning-treated mouse hearts. Data are expressed as mean + SEM n ¼ 4 10 per group. *P, 0.05 vs. control.

3 136 Z.-Q. Jin et al. 2.4 Infarct size measurement After 45 min of global ischaemia and 45 min of reperfusion, a subset of hearts in each group was infused with 15 ml of 1% triphenyltetrazolium chloride (Sigma) in phosphate-buffed saline at a rate of 1.5 ml/min as described previously. 5 Hearts were then removed from the cannula, weighed, and fixed overnight in 10% formalin. Hearts were removed from formalin and stored frozen at 2208C until sectioning for analysis of LV infarct size as previously described in our laboratory. 5,6 The infarct size of each section was expressed as a fraction of the area at risk defined as the total area of the LV in this global ischaemia model. 2.5 Western blot Measurement of phospho-akt and phospho-erk protein expressions after POSTwas performed using standard SDS-PAGE Western blotting as previously described in our laboratory. 8 Primary antibodies for phospho-akt, phospho-erk, total Akt, and total-erk (Cell Signaling Technology, Inc. Beverly, MA, USA and Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used to measure the expression of phosphorylation of Akt (Ser 473), total Akt, phosphorylation of ERK and total ERK. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham Bioscience, Piscataway, NJ, USA) and quantified by densitometric analysis of digitized autoradiograms with NIH Image 1.61 software. 2.6 Sphingosine kinase measurement Mice were anaesthetized by i.p. injection of pentobarbital sodium (60 mg/ml) and the hearts and spleen then rapidly excised and washed in ice-cold buffer A (0.13 M KCl, 20 mm HEPES, ph 7.4, 1 mm EGTA, 1 mg/l leupeptin, 0.25 mg/l each of chymostatin and pepstatin A). The tissue was minced and homogenized in a homogenizer with a loose-fitting Teflon pestle. The homogenate was centrifuged for 6 min at 350 g in a TOMY microcentrifuge to remove cell debris and nuclei. The supernatant was centrifuged for 50 min at g. The supernatant was decanted and designated as the cytosolic fraction. Both cytosolic and membrane fractions were used separately for assay of SphK. The assay uses a chloroform/methanol/aqueous trisodium EDTA extraction system to separate reactant ([ 3 H]sphingosine) from product ([ 3 H]S1P) as previously described in our laboratory. 10 A standard assay contains Triton X-100 (0.05%), 250 mm KCl, 1 mm [ 3 H]sphingosine ( c.p.m./pmol), 5 mm ATP, 10 mm Mg 2þ, 100 mm Tris, ph 8.0 (308C), 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 in a Beckman LS6500 Scintillation Counter. 2.7 Statistics Data are mean + SEM. The significance of the differences between groups was evaluated by one-way ANOVA, followed by post hoc testing (Student Newman Keuls). P-values of,0.05 were considered significant. 3. Results 3.1 Cardiac protection induced by ischaemic postconditioning in isolated mouse hearts A 45 min period of ischaemia followed by 45 min of reperfusion substantially impaired cardiac function (Figure 2). However, postconditioning the heart with short ischaemia/ reperfusion cycles instituted immediately after 45 min of index ischaemia enhanced recovery (Figure 2). POST regimen I (POST I) consisting of 5 s of ischaemia and 5 s of reperfusion significantly improved LVDP recovery (control hearts recovered to % of baseline whereas POST I treated hearts recovered to % of baseline, Figure 2A, P, 0.05). The peak levels of (þ)dp/dt max increased with POST I from % of baseline in the control group to % of baseline (Figure 2B, P, 0.05). Cardiac diastolic function was also improved. The recovery of (2)dP/dt max was enhanced to % compared with % in the control group (P, 0.05, Figure 2C). At the end of reperfusion, LVEDP in the POST I group was decreased by 33.9% to mmhg (P, 0.05, compared with mmhg in the control group). In contrast, POST II and POST III (10 and 20 s cycles, respectively) were not effective POST regimens (Figure 2A C). As shown in Table 1, there was no difference at baseline (prior to ischaemia) among the different groups. Different cycles of short ischaemia/reperfusion after ischaemia also exerted variable effects on infarction area. Only POST I (5 s of ischaemia/reperfusion for three cycles) markedly reduced the infarction area ( % in the POST I group compared with % of the risk area in the control group, P, 0.05, Figure 3). Longer periods of ischaemia/reperfusion (10 or 20 s) with three cycles caused no reduction of infarction size (Figure 3). Table 1 Cardiac functional parameters at the baseline of isolated mouse hearts Group n LVEDP (mmhg) LVDP (mmhg) þdp/dt max (mmhg/s 10 3 ) 2dP/dt max (mmhg/s 10 3 ) CF (ml/min) Control Post I Post II Post III IPC KOCN KOPOS I Data are expressed as mean + SEM of 4210 animals per group. No significant difference was observed in above groups. Post I, II, and III, ischeamic postconditioning with different reperfusion/ischaemia periods; IPC, ischaemic postconditioning; KOCN, control in SphK1-null hearts; KOPOS I, postconditioning in SphK1-null hearts; LVEDP, left ventricular end-diastolic pressure; LVDP, LV developed pressure; CF, coronary flow.

4 Sphingosine kinase-1 in ischaemia/reperfusion and postconditioning 137 As expected, IPC reduced infarction size and improved cardiac function (Figure 2A C, P, 0.05 vs. the control group). The cardioprotective effect induced by POST I was similar to IPC. Figure 3 Effects of ischaemic preconditioning and postconditioning on infarction size expressed as percentage of risk area (RA) in both wild-type and Sphk1 KO hearts. Data are expressed as mean + SEM n ¼ 4 10 per group. *P, 0.05 vs. control. # P, 0.05 vs. POST I. POST I: postconditioning immediately following prolonged ischaemia with 5 s of reperfusion and 5 s of ischaemia for three cycles, then followed by prolonged reperfusion. POST II: postconditioning with 10 s of reperfusion and 10 s of ischaemia. POST III: postconditioning with 20 s of reperfusion and 20 s of ischaemia. IPC, ischaemic preconditioning; KOCN, control SphK1-null hearts with ischaemia/reperfusion injury; KOPOS I, postconditioning in SphK1-null hearts. 3.2 Sphingosine kinase isoform 1 mutation abrogates the cardioprotection induced by ischaemic POST In contrast to the results in WT hearts, SphK1 knockout (2/2, KO) mouse hearts did not exhibit an improvement in cardiac systolic and diastolic functions in response to POST relative to non-conditioned KO controls (Figure 4). The recovery of LVDP was only % in the KOPOST I group and only % in the control group (P. 0.05). The recovery of (þ)dp/dt max in the KOPOST I group was % vs % in control group, P. 0.05). (2)dP/dt max recovery in the KOPOST I group was % vs % in the control group (P. 0.05). LVEDP in KOPOST I group was mmhg vs mmhg in the control group (P. 0.05). Ischaemic POST also did not result in any reduction of infarction size in SphK1 (2/2) mouse hearts ( % of risk area in the POST I group, vs % of risk area in SphK1 (2/2) control group, P. 0.05, Figure 3). It is of interest to note that the infarct sizes are larger in SphK1 (2/2) hearts, both with and without POST, than in WT hearts. 3.3 Phosphorylation of Akt and ERK is enhanced after POST I but abolished in sphingosine kinase 1-null hearts Five seconds of ischaemia and 5 s of reperfusion for three cycles significantly increased the phosphorylation of Akt Figure 4 Left ventricular developed pressure (A), its maximum increase (B) and decrease (C) of velocity (+dp/dt max ) and left ventricular end-diastolic pressure (D) in wild-type and Sphk1-KO mouse hearts subjected to ischaemic postconditioning. Data are expressed as mean + SEM n ¼ 4 10 per group. *P, 0.05 vs. control. CN, control hearts with ischaemia/reperfusion injury; POS I, postconditioning with 5 s reperfusion and 5 s ischaemia for three cycles, followed by reperfusion injury; KOCN, SphK1-null hearts subjected to ischaemia/reperfusion injury; KOPOS I, postconditioning in SphK1-null hearts.

5 138 Z.-Q. Jin et al. Figure 5 Time course of phosphorylation of Akt and ERK in wild-type mouse hearts subjected to ischaemic postconditioning. Upper panel: phosphorylation of ERK1/2 and phosphorylation of AKT. Bottom panel: total ERK and total Akt expression. Total and phospho-erk and AKT were measured after equilibration (NC), and after 30 s, 5, and 10 min of reperfusion following postconditioning. A similar result was observed in one additional experiment. and ERK (Figures 5 and 6). The time of maximal expression was 10 min after reperfusion (Figure 5). In contrast, POST failed to increase the phosphorylation of Akt and ERK in SphK1-null mouse hearts (Figure 6). 3.4 Sphingosine kinase activity in postconditioned mouse hearts SphK activity in cytosol and particulate fractions was measured. As shown in Figure 7A, for WT hearts cytosolic SphK activity was reduced from pmol/g tissue to pmol/g tissue after ischaemia/ir (n ¼ 5, P, 0.05). POST I-enhanced SphK activity to pmol/g tissue (n ¼ 5, P, 0.05 vs. control and IR). IR injury also decreased SphK activity in the particulate fraction (n ¼ 5, P, 0.05 vs. control group). There was no significant difference in the change between the IR and POST I groups for the particulate fraction. For SphK1 (2/2) KO hearts (Figure 7B), the SphK activity, which is attributable to SphK2, was not significantly affected by ischaemia/ir ( pmol/g tissue before ischaemia and pmol/g tissue after ischaemia/ir), nor was it affected by POST ( pmol/g tissue). We also measured the response of SphK activity to POST protocols that were less than optimal. POST I, which gave optimal POST, consisted of 5 s of ischaemia and 5 s of reperfusion for three cycles. Unsuccessful regimens consisted of 10 s of ischaemia and 10 s of reperfusion for three cycles (POST II) and 20 s of ischaemia and 20 s of reperfusion for three cycles (POST III). POST II and POST III did not successfully postcondition hearts and, as shown in Fig. 8, did not result in recovery of SphK activity or phosphorylation of Akt upon reperfusion. 4. Discussion Our previous studies have shown that sphingosine kinase activity and S1P content during reperfusion are enhanced by IPC 7 and that inhibition of this enzyme abolishes the cardioprotection induced by IPC. 7,8 IPC also retards formation of the pro-apoptotic molecule ceramide. 17 When sphingosine, the precursor of S1P, is not phosphorylated by sphingosine kinase, mitochondrial permeability transition is increased leading to uncoupled respiration and cardiac Figure 6 Effect of ischaemic postconditioning on phosphorylation of Akt and ERK in wild-type and Sphk1 KO mouse hearts. Data are expressed as mean + SEM n ¼ 4. *P, 0.05 vs. control. WTCN, control (ischaemia/reperfusion) in WT hearts; WTPOS I, postconditioning in wild-type hearts; KOCN, control (ischaemia/reperfusion) in SphK1-null hearts; KOPOS I, postconditioning in SphK1-null hearts. contractile dysfunction. 18 Recent studies have identified the sphingosine kinase-1 isoform as the critical mediator of cytoprotection. Thus, in vitro knockdown of SphK-1 by sirna caused cell-cycle arrest and induced apoptosis via effector caspase activation, cytochrome C release and Bax oligomerization in the mitochondrial membrane. 19 We recently reported that the infarct-size-lowering effect of IPC is abolished in hearts null for SphK-1. 8 In the present study, we asked whether SphK-1 plays a role in ischaemic POST. We found that ischaemic POST improved cardiac function and decreased infarct size in isolated mouse hearts. Phosphorylation of Akt and ERK and recovery of SphK activity were enhanced after POST. Conversely, cardiac protection induced by POST was abrogated in SphK-1 null mouse hearts. Moreover, POST failed to increase phosphorylation of Akt and ERK in these hearts. Thus, short

6 Sphingosine kinase-1 in ischaemia/reperfusion and postconditioning 139 Figure 7 Effect of ischaemic postconditioning (POST I) on sphingosine kinase (SphK) activity in cytosol and particulate fractions of isolated wild-type (A) and Sphk1 KO (B) mouse hearts. Data are expressed as mean + SEM n ¼ 5. *P, 0.05 vs. ischaemia/reperfusion group. NC, normal control (no ischaemia); CN, ischaemia/reperfusion; POST, postconditioning. cycles of ischaemia/reperfusion, whether applied before or after the index ischaemia, rely on similar signalling mechanisms involving activation of SphK-1, Akt, and ERK. Further, the infarct sizes are larger in SphK1 (2/2) hearts, both with and without POST, than for WT hearts (Figure 3). This is also suggestive of a cardioprotective effect for SphK1. Using SphK-1 null mouse hearts, the present study is the first to show that to be effective POST, like preconditioning, 8 depends on the SphK-1/S1P axis for myocyte survival. As reported previously, 8 and shown in Fig. 7, the SphK-1-KO hearts still have considerable SphK activity resulting from SphK-2. The level of activity is about half of that in the WT hearts. The data for the KO hearts indicate that this SphK-2 activity is not capable of supporting cardioprotective mechanisms. The data in Fig. 7 also suggest that SphK-2 activity is not sensitive to ischaemia or responsive to POST. If true, this would mean that the variation seen in the WT is due predominantly to variation in the activity of SphK-1 and that SphK-2 and SphK-1 activities are differentially regulated. Postconditioning has also been observed in different species: mouse, rat, rabbit, dog, and human However, the duration and cycle number of short bouts of ischaemia/reperfusion required to induce POST vary in different animal models. In the mouse heart we found that effective POST was produced by 5 s of reperfusion and 5 s of ischaemia for three cycles. We also found that extension of the reperfusion and ischaemia times to 10 or 20 s was less effective. Our data and previous studies of the mechanism of POST by others have revealed many additional similarities to IPC. Both adenosine receptor antagonists and bradykinin receptor antagonists block the beneficial effects of POST. 11,12 In addition to ERK 14 and Akt, 2 PKCe and K ATP channel opening also mediate POST. 3,13 Other pivotal factors in POST include mitochondrial permeability transition and ph changes induced by acidosis. 15,20 Recent studies of preconditioning have shown that S1P activates the PI3K/Akt pathway in both cardiac myocytes and endothelial cells through S1P receptor-dependent pathways We have previously demonstrated in adult mouse cardiac myocytes that S1P synthesized intracellularly by activation of SphK must be exported to the extracellular space where it binds to S1P receptors on the cell surface to initiate survival signals. 24 In the present study of POST, phospho-akt and phospho-erk1/2 were increased after 5 and 10 min of reperfusion following POST I, an effect that was abolished in SphK-1 null mouse hearts. These observations in an ex vivo heart model suggest that SphK-1 is necessary for the activation of Akt and ERK1/2 in POST by rapidly synthesising S1P that acts in a paracrine and/or autocrine manner to induce cytoprotection. This is consistent with our studies of preconditioning in a cell culture model. 24 Thus, it appears that preconditioning and POST utilize similar pathways to induce cardioprotection. In summary, our previous work 5 8 and the present study indicate that short bouts of ischaemia either before or after ischaemia/reperfusion share a common mechanism of protection against IR injury. Both require SphK-1 and likely depend on exported S1P to initiate a cell survival signalling cascade.

7 140 Z.-Q. Jin et al. Figure 8 Effect of suboptimal ischaemic postconditioning (POST) regimens on SphK activity in cytosol (A) and expression of phosphorylation of Akt (B). Data are expressed as mean + SEM n ¼ 5. NC, normal control (no ischaemia); CN, ischaemia/reperfusion; POST, postconditioning. *P, 0.05 vs. NC group. Acknowledgements The authors thank Drs Shaun Coughlin and Rajita Pappu for supplying the SphK1 Null mice, Mr Michael Kelley for technical assistance, and Dr Jianqing Zhang and Mrs Mei-Fei Xu for breeding mice and genotyping PCR. Conflict of interest: none declared. Funding This work was supported by grants from the NIH grant 1P01 HL A1 (J.S.K.) and the Medical Service of the Department of Veterans Affairs (D.A.V.). References 1. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. 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