High-density lipoprotein protects cardiomyocytes against necrosis induced by oxygen and glucose deprivation through SR-B1, PI3K, and AKT1 and 2
The cardioprotective lipoprotein HDL (high-density lipoprotein) prevents myocardial infarction and cardiomyocyte death due to ischemia/reperfusion injury. The scavenger receptor class B, type 1 (SR-B1) is a high-affinity HDL receptor and has been shown to mediate HDL-dependent lipid transport as well as signaling in a variety of different cell types. The contribution of SR-B1 in cardiomyocytes to the protective effects of HDL on cardiomyocyte survival following ischemia has not yet been studied. Here, we use a model of simulated ischemia (oxygen and glucose deprivation, OGD) to assess the mechanistic involvement of SR-B1, PI3K (phosphatidylinositol-3-kinase), and AKT in HDL-mediated protection of cardiomyocytes from cell death. Neonatal mouse cardiomyocytes and immortalized human ventricular cardiomyocytes, subjected to OGD for 4 h, underwent substantial cell death due to necrosis but not necroptosis or apoptosis. Pretreatment of cells with HDL, but not low-density lipoprotein, protected them against OGD-induced necrosis. HDL-mediated protection was lost in cardiomyocytes from SR-B1−/− mice or when SR-B1 was knocked down in human immortalized ventricular cardiomyocytes. HDL treatment induced the phosphorylation of AKT in cardiomyocytes in an SR-B1-dependent manner. Finally, chemical inhibition of PI3K or AKT or silencing of either AKT1 or AKT2 gene expression abolished HDL-mediated protection against OGD-induced necrosis of cardiomyocytes. These results are the first to identify a role of SR-B1 in mediating the protective effects of HDL against necrosis in cardiomyocytes, and to identify AKT activation downstream of SR-B1 in cardiomyocytes.
Introduction<!>Mice<!>Culture of neonatal mouse cardiomyocytes and human immortalized ventricular cardiomyocytes<!>Oxygen and glucose deprivation<!>Measurement of cell viability<!>Measurement of necrosis<!>Measurement of apoptosis<!>Microscopy and quantification of necrotic and apoptotic cells<!>Immunoblotting<!>Statistics<!>HDL protects HIVCMs against OGD-induced necrosis.<!>Nec-1s, an inhibitor of necroptosis, does not prevent OGD-induced death of HIVCM.<!>SR-B1 is required for HDL-mediated protection of NMCM against OGD-induced necrosis.<!>siRNA-mediated knock down of SR-B1 impairs HDL-mediated protection of HIVCM against OGD-induced necrosis.<!>siRNA-mediated knock down of SR-B1 impairs HDL-stimulated AKT phosphorylation in HIVCMs.<!>PI3K, AKT1, and AKT2 are required for HDL-mediated protection of HIVCM against OGD-induced necrosis.<!>Proposed pathway for HDL-mediated protection against OGD-induced cardiomyocyte necrosis.<!>Discussion<!><!>Author Contribution<!>Funding<!>Competing Interests
<p>Cardiovascular disease is a leading cause of death and a major burden to the healthcare systems of developed societies [1]. About half of all cardiovascular disease-related deaths are due to coronary heart disease [1]. Partial or complete occlusion in one or more areas of the coronary arteries renders an ischemic myocardial environment, which, if left unperfused, results in infarction due to irreversible cardiomyocyte death and myocardial infarction. Over time, the increased demand on the non-infarcted tissue may lead to detrimental functional changes and heart failure; therefore, it is of utmost importance to prevent or limit the amount of infarcted tissue.</p><p>Low plasma HDL (high-density lipoprotein) cholesterol is associated with increased risk of mortality in patients recovering from myocardial infarction [2], and HDL cholesterol levels are inversely correlated with cardiovascular disease risk [3]. The clinical data are supported by ex vivo experimental evidence, where administration of HDL provides protection against ischemia–reperfusion injury in rodent hearts [4,5], and in vivo experimental evidence demonstrates that overexpression of apolipoprotein (apo) A1 (the major protein component of HDL) protects low-density lipoprotein (LDL) receptor (LDLR−/−) knockout mice from coronary artery ligation-induced myocardial infarction, cardiac dysfunction, and death [6]. Conversely, apoA1-deficiency, and a resultant reduction in HDL, impairs cardiomyocyte mitochondrial function and results in larger infarctions following coronary ligation in mice [7].</p><p>Although the physiological effects of HDL against myocardial ischemia are well documented in both humans and rodents, the receptor(s) and pathways through which HDL induces protection at the cardiomyocyte are not completely defined. The scavenger receptor class B type 1 (SR-B1) is a high-affinity HDL receptor that is present in heart tissue, and has been implicated in mediating HDL-dependent cytoprotective intracellular signaling, including activation of the PI3K (phosphatidylinositol-3-kinase)/AKT signaling pathway, in diverse cell types including endothelial cells, macrophages, and Chinese hamster ovary cells [8–14]. PI3K and AKT are well-characterized mediators of cardiomyocyte survival [15–18]. The requirement for SR-B1 in HDL-mediated induction of PI3K/AKT signaling in cardiomyocytes or its role in protection of cardiomyocytes against ischemic injury has not been tested.</p><p>Coronary artery atherosclerosis and non-lethal myocardial infarcts can be induced in LDLR/Apo E double knockout (dKO) mice by feeding them a high fat diet for extended periods of time [19]. On the other hand, coronary artery atherosclerosis and lethal myocardial infarcts develop spontaneously in mice lacking SR-B1 and ApoE (SR-B1/ApoE dKO) [20] and in a high fat/high cholesterol diet-dependent manner in SR-B1/LDLR dKO mice [21] or SR-B1−/− mice with a hypomorphic mutation in apoE [22]. In SR-B1-deficient models, spontaneous or diet-induced coronary artery atherosclerosis and myocardial infarction are accompanied by cardiac conductance abnormalities, reduced heart function, and early death [20–22]. The striking phenotype of extensive myocardial infarction when SR-B1 is knocked out in atherosclerosis-susceptible strains of mice led us to hypothesize that the effects of SR-B1 extend beyond its role in atherosclerosis, and that SR-B1 may play a more direct role in protecting cardiomyocytes against myocardial infarction.</p><p>In the present study, we directly tested the role of SR-B1 in HDL-activated PI3K/AKT signaling in cardiomyocytes and protection against oxygen and glucose deprivation (OGD)-induced cardiomyocyte death.</p><!><p>All procedures involving animals were approved by the McMaster University Animal Research Ethics Board and were in accordance with guidelines from the Canadian Council on Animal Care. Mice were bred and housed in ventilated cages with automatic watering and free access to food in the Division of Comparative Medicine Facility of the Thrombosis and Atherosclerosis Research Institute. SR-B1−/− mice (backcrossed >10 generations on C57BL/6 genetic background) were bred as homozygous mutants and breeders were fed a diet of chow containing 0.5% probucol [23]. Founders were originally obtained from Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA, U.S.A.). Wild-type C57BL/6 (SR-B1+/+) mice were bred from founders originally obtained from the Jackson Laboratories (Barr Harbor, ME, U.S.A.).</p><!><p>Neonatal mouse cardiomyocytes (NMCMs) were isolated from hearts of 1- to 3-day-old mice. Briefly, neonatal mice were killed by decapitation and hearts were excised and washed with Hank's balanced saline solution (HBSS, Life Technologies, Burlington, ON, Canada). The ventricles were serially digested at 37°C using 0.2 mg/ml collagenase type II (Worthington Biochemical Corp., Lakewood, NJ, U.S.A.) and 0.6 mg/ml pancreatin (Sigma–Aldrich Canada Co., Oakville, ON, Canada) in HBSS. The cell suspension was centrifuged at 300×g for 10 min at 4°C, the supernatant was removed, and the cell pellet was resuspended in 37°C DMEM (Dulbecco's modified Eagle's medium)/M199 (4 : 1) medium supplemented with 10% horse serum, 5% fetal bovine serum (FBS), 100 µM bromo-deoxyuridine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 mM l-glutamine (Life Technologies, Burlington, ON, Canada). Non-cardiomyocytes were removed by plating the cell suspension on an uncoated plate for 1 h. The myocyte-enriched suspension was collected and then plated at 1 × 105 cells/cm2 on collagen type I (Life Technologies, Burlington, ON, Canada)-coated dishes and incubated in a humid 5% CO2 incubator at 37°C for 48 h at which point they were used for experiments. SV40 large T-antigen immortalized human ventricular CMs (HIVCMs) were obtained from Applied Biological Materials, Inc. (Richmond, BC, Canada). HIVCMs were cultured in Prigrow I media (Applied Biological Materials, Inc., Richmond, BC, Canada) supplemented with 10% FBS and 100 U/ml penicillin.</p><!><p>Forty-eight hours after plating, cell culture media were replaced with media containing 3% newborn calf lipoprotein-deficient serum, and cells were cultured for a further 24 h; all experiments were carried out in media supplemented with lipoprotein-depleted serum [14]. Cells were incubated with or without 100 µg (protein)/ml human HDL or LDL (Alfa Aesar, Ward Hill, MA, U.S.A.) for 30 min, then media were changed to OGD media (DMEM with no glucose and 1% lipoprotein-deficient serum), and cells were incubated in a humidified chamber with 100% N2 at 37°C for 4 h. Alternatively, normoxic control cells received DMEM containing glucose and 1% lipoprotein-deficient serum, and were incubated in 95% air, 5% CO2 at 37°C. In some experiments, HIVCMs were transfected with 30 nM siRNA in the presence of Lipofectamine RNAi Max (Life Technologies, Burlington, ON, Canada), 24 h prior to lipoprotein starvation. SiRNA targeting human SR-B1 (siSR-B1: Hs_SCARB1_6), AKT1 (siAKT1: Hs_AKT1_10) and AKT2 (siAKT2: Hs_AKT2_7), and AllStars Negative Control siRNA (siCntrl) were purchased from Qiagen, Inc. (Toronto, ON, Canada). In other experiments, cells were treated with 10 µM LY294002 (PI3K inhibitor; Cell Signaling Technologies, Inc., Danvers, MA), 3 µM AKT inhibitor V (pan-AKT inhibitor; Millipore Canada Ltd, Etobicoke, ON, Canada), 10 µM Nec-1s (necroptosis inhibitor; BioVision, Inc., Milpitas, CA), or vehicle (DMSO, Sigma–Aldrich Canada Co., Oakville, ON, Canada). All inhibitors were applied to cells 10 min prior to other treatments (including HDL) and for the duration of OGD treatment.</p><!><p>Cells were cultured and treated in a 96-well plate. Following OGD, cells were incubated with the Cell Titer Blue (Promega Corp., Madison, WI, U.S.A.) assay reagent according to the manufacturer's instructions to assess cell viability using a spectrophotometer. Cell viability was expressed as a percentage of the viability of parallel cultures of cells not exposed to OGD.</p><!><p>Following OGD, cells were stained with propidium iodide (PI), washed extensively, fixed with 4% paraformaldehyde, and counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) to identify nuclei. Necrotic cells were identified as having PI-stained nuclei. Additionally, NMCMs were co-stained with a mouse monoclonal anti-cardiac troponin T antibody (cTnT, MA5-12960, Fisher Scientific, Inc., Waltham, MA, U.S.A.) using a Mouse on Mouse Kit (Vector Laboratories, Burlington, ON, Canada), biotinylated secondary, and tertiary labeling with streptavidin conjugated with AlexaFluor 488 (Life Technologies, Burlington, ON, Canada) to identify cardiomyocytes.</p><!><p>Following treatment, apoptosis was detected by staining fixed cells by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) using the ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (Millipore (Canada) Ltd, Etobicoke, ON, Canada) or by staining unfixed cells with fluorescein isothiocyanate (FITC)-conjugated Annexin V (AxV, Life Technologies, Burlington, ON, Canada) and PI according to the manufacturer's instructions. All cells were then fixed and counterstained with DAPI as above.</p><!><p>Fluorescent images were captured using a Zeiss Axiovert 200M inverted fluorescence microscope (Carl Zeiss Canada Ltd, Toronto, ON, Canada) or an Olympus BX41TF fluorescence microscope (Olympus Canada, Inc., Richmond Hill, ON, Canada). A minimum of 4 and maximum of 10 fields of view were imaged per well. Necrotic nuclei were identified as having PI nuclear staining, and apoptotic cells were identified as having AxV staining. Necrotic and apoptotic cell numbers were counted across 4–10 fields of view for each well and were averaged across triplicate wells. Percentage ratios of the numbers of PI/total (PI + DAPI)-stained nuclei were taken as measures of the proportions of necrotic cells and percentage ratios of the numbers of AxV-stained cells/DAPI-stained nuclei or TUNEL-stained/DAPI-stained nuclei were taken as measures of the proportions of apoptotic cells.</p><!><p>Cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM Na4P2O7, 1 mM β-glycerophosphate, 1 mM Na3VO3, with 1 µg/ml pepstatin A, 1 mg/ml leupeptin 2 µg/ml aprotinin and 50 µM p-amidinophenylmethylsulfonylfluoride (all from Sigma–Aldrich Canada Co., Oakville, ON, Canada). Samples (20–25 µg of total protein) prepared from lysates were subjected to SDS–polyacrylamide gel electrophoresis, transferred onto a PVDF membrane, and analyzed by immunoblotting as previously described [14]. The antibodies used (and catalog numbers) were: rabbit antibodies for horseradish peroxidase (HRP)-conjugated β-actin (12620), total AKT (9272), AKT1 (2938), AKT2 (3063), and phosphorylated AKT at Ser 473 (4060) (Cell Signaling Technology, Danvers, MA, U.S.A.); rabbit anti-SR-B1 (NB400-104; Novus Biologicals, Oakville, ON, Canada); and HRP-conjugated donkey anti-rabbit IgG (711-035-152; Jackson ImmunoResearch, West Grove, PA, U.S.A.). HRP was detected by enhanced chemiluminescence (GE Healthcare Life Sciences, Baie d'Urfe, QC, Canada) and quantified using a Gel Doc instrument (Bio-Rad Laboratories, Hercules, CA, U.S.A.). For analysis of AKT phosphorylation, blots were first probed with the anti-phospho-AKT primary and secondary HRP-conjugated anti-rabbit antibody, stripped, and then reprobed with the total AKT primary and secondary HRP-conjugated anti-rabbit antibody. All blots were stripped and reprobed using the HRP-conjugated anti-β-actin antibody.</p><!><p>All data are expressed as means ± SEM. Data were graphed and statistically analyzed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Data comparing two groups were tested for significance by Student's t-test, and data comparing multiple groups were tested using one-way ANOVA and Tukey's multiple comparisons test. A value of P < 0.05 was considered statistically significant.</p><!><p>(A) Cell viability measured by the Cell Titer Blue viability assay for HIVCM treated for 30 min without or with human HDL (100 μg protein/ml) and then exposed to OGD for 4 h. The dashed line indicates 100 % viability, determined by the measurement of cell viability in control cells not exposed to OGD. n = 10 wells/group. ***P < 0.001. (B) Quantification and (C, top row) representative images of TUNEL- (green) and DAPI- (blue) stained HIVCM. n = 3 wells/group. (C, bottom row) Representative images of FITC-AxV- (green), PI- (red), and DAPI- (blue) stained HIVCM treated with or without HDL (30 min) and exposed to 4 h of OGD or control normoxic conditions (as indicated). All scale bars = 50 µm. Quantification of OGD-induced cell death by FITC-AxV staining (D) or PI staining (E) in HIVCM treated as above. (F) Quantification of OGD-induced cell death by PI staining in HIVCM pretreated for 30 min with human LDL (100 μg protein/ml) and then exposed to OGD for 4 h. n = 3 wells/group. (G) HIVCM cells were pretreated with the indicated concentrations of human HDL and then either maintained under normoxic conditions (OGD−) or exposed to OGD for 4 h (OGD+) and stained with PI to detect necrosis. n = 3 wells/group. Data represents means ± SEM. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns, not statistically significant (P > 0.14). Statistical analysis was carried out by unpaired Student's t-test (A) or one-way ANOVA (B, D–G).</p><!><p>HIVCM cells were treated with or without 10 μM Nec-1s, an inhibitor of necroptosis, 10 min prior to and during exposure to OGD for 4 h (A and B) or (C) 24 h treatment with TNF-α (100 ng/ml). Control cells were treated with an equivalent volume of DMSO solvent (final concentration of 0.1%). (A) Quantification of the proportion of PI-positive cells (A) or AxV-positive cells (B) after 4 h of OGD. (C) Quantification of the proportion of PI-positive cells after 24 h of exposure to TNF-α. Data represent means ± SEM of n = 3 replicates. **P < 0.004; *P = 0.02; ns, not statistically significant (P > 0.99), analyzed by one-way ANOVA.</p><!><p>(A) Representative images and (B) quantification of PI staining in SR-B1+/+ and SR-B1−/− NMCM treated for 30 min with or without HDL (100 μg/ml) prior to exposure to OGD for 4 h. Control cells were maintained in normal media under normoxic conditions. In (A), cells were stained prior to fixation with PI (red) or, after fixation, immunostained for cTnT (green) and stained with DAPI (blue). Scale bars = 50 μm. (B) Data represent means ± SEM for n = 3 replicates. ****P < 0.0001; **P < 0.01; ns, not statistically significant (P > 0.99) by one-way ANOVA.</p><!><p>(A) Representative immunoblot and (B) quantification (n = 3) of SR-B1 knockdown by siRNA in HIVCM. UT: untransfected cells; siCntrl: transfected with control siRNA; siSR-B1: transfected with siRNA targeting SR-B1. (C) Quantification of the percentage of OGD-induced cell death in siCntrl- or siSR-B1-transfected HIVCMs treated with or without HDL (100 μg/ml) prior to 4 h of OGD. Cell viability was measured using the Cell Titer Blue assay and the reduction in viability in siCntrl-transfected cells treated without HDL was set as 100% OGD-induced cell death (n = 9). (D) Proportion of PI-stained cells when HIVCMs were transfected with siCntrl or siSR-B1, treated for 30 min with or without HDL, and exposed to OGD for 4 h as above (n = 3). (E) Representative immunoblots of SR-B1, AKT, and β-actin and quantification of (F) SR-B1/β-actin (n = 5, 6, 6) and (G) AKT/β-actin (n = 3), expressed as fold change in arbitrary relative units. ***P = 0.0003; **P < 0.005; *P < 0.03; ns, not statistically significant (P > 0.3) by one-way ANOVA.</p><!><p>(A) Representative immunoblots of phospho-AKT (pAKT), total AKT (tAKT), and β-actin (loading control) and (B) quantification of the ratio of pAKT/tAKT (n = 3) in HIVCMs incubated with HDL (100 μg protein/ml) for different times. (C) Representative immunoblots of pAKT and tAKT and (D) quantification of the ratio of pAKT/tAKT (n = 3) in HIVCMs incubated with HDL (100 μg protein/ml) for 30 min prior to and during exposure to OGD or maintenance under normal conditions ('Normoxia'). (E) Representative immunoblots of pAKT, tAKT, SR-B1, and β-actin (loading control) in siCntrl and siSR-B1-transfected HIVCMs incubated with or without HDL for 30 min. (F) Quantification of the ratio of pAKT/tAKT (expressed as fold change relative to untreated; n = 4). Data are means ± SEM. **P < 0.004; *P < 0.05; ns, not statistically significant (P > 0.9) by one-way ANOVA. §P < 0.02 by unpaired Student's t-test, relative to 0 h time point.</p><!><p>(A) Quantification of OGD-induced PI staining in HIVCM treated with or without HDL (100 μg/ml) and inhibitors of PI3K (10 µM LY294002) or AKT (3 µM AKT Inh. V) prior to 4 h of OGD. Control cells were treated with an equal volume of DMSO (0.1%). (B) Representative immunoblots of AKT1 and β-actin (loading control) and (C) quantification of fold change in AKT1/β-actin protein levels in HIVCMs transfected with the indicated siRNAs. (D) Representative immunoblots of AKT2 and β-actin (loading control) and (E) quantification of fold change in AKT2/β-actin protein levels in HIVCMs transfected with the indicated siRNAs. (F) Quantification of OGD-induced necrosis (% PI-positive cells) in HIVCMs transfected with the indicated siRNAs and treated with or without HDL (100 μg protein/ml) for 30 min prior to 4 h of OGD, as indicated. Data in all panels represent means ± SEM (n = 3 replicates). ****P < 0.0001; **P = 0.009; *P < 0.04; ns, not statistically significant (P > 0.33) by one-way ANOVA.</p><!><p>HDL binding to SR-B1 leads to activation of AKT1 and AKT2 signaling and induction of pathways protecting cardiomyocytes from necrosis induced by OGD. Activation of signaling pathways may be indirect, as indicated by broken arrows. Pharmacological or genetic inhibition of this pathway (shown by the boxed text) prevents protection by HDL.</p><!><p>Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is primarily carried by HDL [29,30] and, when applied to cardiomyocytes alone, or as part of reconstituted HDL, can protect cardiomyocytes against various stresses [28,31,32]. S1P receptors (S1PRs) have been implicated in mediating S1P-dependent protection against cardiomyocyte cell death following hypoxia and rexoygenation, although different receptors have been implicated in HDL-dependent protection under different conditions; S1PR3 is reportedly required for HDL-mediated protection against myocardial infarction in mice in vivo [32], whereas mouse cardiomyocytes subjected to hypoxia/reoxygenation required both S1PR1 and S1PR3 [31], and doxorubicin-treated cultured rat cardiomyocytes required S1PR2 but not S1PR1 or S1PR3 [28] for HDL/S1P-mediated cytoprotection. Here, we conclude that SR-B1 is also a critical receptor for HDL-mediated protection of cardiomyocytes. It is possible that SR-B1 could function to anchor HDL in close proximity to S1PRs. Recently, it has been reported that SR-B1 and S1PR1 form complexes in the presence of S1P [33]. Alternatively, since SR-B1 is known to mediate the cellular uptake of HDL-bound lipids [34], and since HDL is known to be a major carrier of S1P in blood [29,30], SR-B1 may mediate the uptake of HDL-bound S1P, making it accessible to S1PRs. It remains to be determined if SR-B1 and S1P receptors participate with each other in HDL-mediated signaling in cardiomyocytes and by what mechanism this may occur. Regardless, our data indicate a role of SR-B1 in facilitating HDL-mediated protection against OGD-induced cardiomyocyte necrosis (Figure 7).</p><p>OGD can be applied to cardiomyocytes in vitro in order to mimic the in vivo ischemic environment and assists in defining mechanistic and direct effects of cardioprotective agents specifically on the cardiomyocyte. Necrosis is believed to be an important pathway contributing to the death of cardiomyocytes in vivo following myocardial infarction [35]. Other pathways of cell death also contribute. For example, apoptosis appears to be an important pathway of cardiomyocyte cell death in the infarct border zone and during reperfusion after ischemia [36]. HDL protects cardiomyocytes against apoptosis induced by the cardiotoxic agent, doxorubicin, and we have found that this protection involves SR-B1 and PI3K/AKT signaling [27]. Evidence is also emerging implicating necroptosis in cardiomyocyte cell death during infarct generation and remodeling [37]. The effect of HDL on necroptosis has not yet been examined.</p><p>Given the extensive and lethal myocardial infarctions that occur spontaneously at a young age in SR-B1/ApoE dKO mice [20], and in a high fat/high cholesterol diet-dependent manner in SR-B1−/− mice either lacking LDLR or with a hypomorphic mutation in apoE [21,22], we hypothesized that increased susceptibility of cardiomyocytes lacking SR-B1 to cell death may contribute along with the occlusive coronary artery atherosclerosis that develops in these mice to the development of lethal myocardial infarction. Here, we have demonstrated that cardiomyocytes (human and mouse) depend on SR-B1 to facilitate the HDL-dependent activation of AKT and protection against cardiomyocyte necrosis triggered by deprivation of oxygen and glucose.</p><p>This research will have important implications for our understanding of how HDL protects against cardiovascular disease, beyond its roles in reverse cholesterol transport and attenuating atherosclerosis, by providing insights into the mechanisms by which it exerts direct cytoprotective effects on cardiomyocytes.</p><!><p>apolipoprotein</p><p>annexin V</p><p>cardiac troponin T</p><p>4′,6-diamidine-2′-phenylindole dihydrochloride</p><p>double knockout</p><p>Dulbecco's modified Eagle's medium</p><p>fetal bovine serum</p><p>fluorescein isothiocyanate</p><p>Hank's balanced salt solution</p><p>high-density lipoprotein</p><p>immortalized human ventricular cardiomyocytes</p><p>horseradish peroxidase</p><p>low-density lipoprotein</p><p>LDL receptor</p><p>neonatal mouse cardiomyocyte</p><p>oxygen and glucose deprivation</p><p>propidium iodide</p><p>phosphatidylinositol-3-kinase</p><p>siRNA against AKT1 and 2</p><p>scrambled siRNA</p><p>sphingosine-1-phosphate</p><p>S1P receptors</p><p>scavenger receptor, class B type 1</p><p>terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling</p><!><p>B.L.T. conceived of and supervised the study. K.K.D. designed the experiments. K.K.D. and K.M.C. performed the experiments, and collected and analyzed the data. B.L.T. and K.K.D. interpreted the data and wrote the manuscript. All authors contributed to editing the manuscript.</p><!><p>These studies were supported by funding from the Canadian Institutes of Health Research [MOP-74753] to B.L.T.</p><!><p>The Authors declare that there are no competing interests associated with the manuscript.</p>