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DHA and 19,20-EDP induce lysosomal-proteolytic-dependent cytotoxicity through de novo ceramide production in H9c2 cells with a glycolytic profile

DHA and 19,20-EDP induce lysosomal-proteolytic-dependent cytotoxicity through de novo ceramide... Docosahexaenoic acid (DHA) and their CYP-derived metabolites, epoxydocosapentaenoic acids (EDPs), are important fatty acids obtained from dietary sources. While it is known that they have significant biological effects, which can differ between cell type and disease state, our understanding of how they work remains limited. Previously, we demonstrated that DHA and 19,20-EDP triggered pronounced cytotoxicity in H9c2 cells correlating with increased ceramide production. In this study, we examine whether DHA- and 19,20-EDP-induced cell death depends on the type of metabolism (glycolysis or OXPHOS). We cultivated H9c2 cells in distinct conditions that result in either glycolytic or oxidative metabolism. Our major findings suggest that DHA and its epoxy metabolite, 19,20-EDP, trigger cytotoxic effects toward H9c2 cells with a glycolytic metabolic profile. Cell death occurred through a mechanism involving activation of a lysosomal-proteolytic degradation pathway. Importantly, accumulation of ceramide played a critical role in the susceptibility of glycolytic H9c2 cells to cytotoxicity. Furthermore, our data suggest that an alteration in the cellular metabolic profile is a major factor determining the type and magnitude of cellular toxic response. Together, the novelty of this study demonstrates that DHA and 19,20-EDP induce cell death in H9c2 cells with a glycolytic metabolicwct 2 profile through a lysosomal-proteolytic mechanism. Introduction cardiovascular system, suggesting they reduce the risk of Long chain n-3 polyunsaturated fatty acids (PUFAs) cardiovascular disease by protecting the heart and vas- such as docosahexaenoic acid (DHA, C22:6n-3) and culature against injury, such as limiting cardiac arrhyth- 1,2 eicosapentaenoic acids (C20:5n-3) are important fatty mias, myocardial infarction and hypertension . Overall, acids obtained from dietary sources. These essential fatty there is a growing body of evidence demonstrating that n- acids are required components of phospholipid mem- 3 PUFA have significant biological effects depending upon branes and serve as precursors to numerous lipid med- the cell and disease; however, understanding exactly how iators with various biological properties. Numerous n-3 PUFAs work remains unknown. In recent years, evi- studies report a positive effect of n-3 PUFAs toward the dence indicate that there is a biological role for cyto- 3,4 chrome P450 (CYP) epoxygenase metabolites of DHA . CYP epoxygenases metabolize DHA into 6 regioisomeric Correspondence: John M. Seubert (jseubert@ualberta.ca) epoxydocosapentaenoic acids (4,5-, 7,8-, 10,11-, 13,14-, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada 16,17- and 19,20-EDP), which may then undergo further Department of Pharmacological Sciences, School of Pharmaceutical Sciences, metabolism by epoxide hydrolase enzymes to corre- Health Sciences University of Hokkaido, Hokkaido, Japan sponding diols, dihydroxydocosapentaenoic acids . EDPs Full list of author information is available at the end of the article. Edited by A. Rufini © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Endo et al. Cell Death Discovery (2019) 5:29 Page 2 of 11 have been suggested as the active mediators impacting cells grown with 25 mM glucose was less than one-fifth of cellular responses to injury and disease such as cytotoxi- the oxygen consumed when cells were grown in 5.5 mM 5–9 city, cardiovascular disease and cancer . Among the glucose condition (Fig. 1a). These data suggest changing regioisomeric metabolites of DHA, 19,20-EDP gained the the cell culture conditions from 25 mM to 5.5 mM glu- most interest due to its pronounced biological effects in cose shifted the energy metabolism in the undifferentiated numerous aspects of cell biology . cells from glycolysis to OXPHOS, which was reflected in the significant change in ATP production and increased The reported biological effects of DHA and its meta- bolites, EDPs, appear to be dependent upon the specific Nicotinamide adenine dinucleotide/Nicotinamide ade- cellular phenotype being studied. H9c2 myoblast cells are nine dinucleotide hydrogen NAD/NADH ratio (Fig. 1b, c). an immortalized cell line derived from ventricular tissue Also, we detected a significantly higher lactate level in 11,12 of the BDIX rat heart . H9c2 cells are not fully dif- media with 25 mM glucose, which further reflects higher ferentiated until cultured in media with low serum, which glycolytic activity (Fig. 1d). Next, we assessed mitochon- triggers differentiating from mono-nucleated myoblasts to drial respiration in permeabilized cells to determine the a skeletal muscle phenotype or the addition of all-trans respiration control ratio (RCR), which is the ratio between retinoic acid to 1% serum media that induces adult car- basal and adenosine diphosphate (ADP)-stimulated 13,14 diac muscle phenotype . In the undifferentiated state, respiration. H9c2 cells grown in 25 mM glucose media H9c2 cells tend to be highly proliferative relying on gly- had an RCR of 1.44 ± 0.18, while cells cultivated in 5.5 colysis rather than mitochondrial oxidative phosphoryla- mM glucose media had RCR up to 6.9 ± 0.87, thus, tion . Such aerobically poised cells can demonstrate demonstrating that there was a shift from glycolysis to resistance to toxic agents that target mitochondria . OXPHOS in H9c2 cells grown in 5.5 mM glucose media. Ceramide is a central lipid component of sphingolipid Adding retinoic acid (RA, 10 nM) treatment to H9c2 structure that is biosynthesized by three pathways, which cells cultured in Dulbecco's modified Eagle's medium include de novo synthesis from palmitoyl-CoA and serine, (DMEM) with 1% fetal bovine serum (FBS) serum for hydrolysis of sphingomyelin or a salvage pathway .Itis 2 weeks has been shown to stimulate differentiation to a an important lipid mediator regulating various cellular cardiac-specific phenotype . We compared our cell responses like cell death, and recent evidence also sug- model in non-differentiated cells of shifting metabolism gests a role in various metabolic pathways influencing from a primarily glycolytic metabolism to OXPHOS to mitochondrial function . In vitro data indicate produc- differentiated cells. Neither 25 mM nor 5.5 mM cultured cells expressed troponin T, indicating our cells remained tion of ceramide increases in glycolytic cells but decreases in cells with developed OXPHOS . Previous data indicate in the non-differentiated state as compared to cells treated undifferentiated H9c2 cells are susceptible to DHA- with RA for 14 days in 1% FBS (Fig. 1e). We observed a induced cell death in a concentration-dependent man- marked difference in oxygen consumption, ADP/ATP, ner which does not occur in primary neonatal cardio- NAD/NADH and lactate levels between 25 mM, 5.5 mM myocytes . Furthermore, 19,20-EDP was demonstrated and differentiated cells (Fig. 1a–d). Our primary goal was to cause cytotoxicity in undifferentiated H9c2 cells cor- to investigate the impact of altered metabolism in non- relating with de novo synthesis of intracellular ceramide . differentiated cells, and hence we utilized these cells for While ceramide is known to induce cell death in tumor the remaining experiments. cells, the mechanisms involved in DHA-mediated events associated with a metabolic state remain unclear. In the DHA- and 19,20-EDP-induced cell cytotoxicity in non- present study, we investigated the effects of DHA differentiated H9c2 cells and 19,20-EDP in undifferentiated H9c2 cells cultured Our previous data demonstrated that H9c2 cells cul- under conditions triggering glycolytic or oxidative tured in 25 mM glucose were susceptible DHA-mediated 20,21 phosphorylation-mediated metabolism. cell death . In the current study, we investigated the effect of DHA (100 µM) and its CYP epoxygenase meta- Results bolite, 19,20-EDP (1 µM), on viability of H9c2 cells grown Culturing non-differentiated H9c2 cells in low glucose in either 25 mM (NG) or 5.5 mM (LG) glucose-containing media shifts cellular metabolism toward OXPHOS media (Fig. 2a, b). Consistently, both DHA and 19,20-EDP H9c2 cells are normally cultured in media containing treatment markedly reduced cell viability in cells cultured 25 mM glucose, and as such they primarily utilize glyco- in 25 mM glucose. Co-treatment of DHA with a CYP lysis for adenosine triphosphate (ATP) generation. In epoxygenase inhibitor (MSPPOH) prevented cell death contrast, H9c2 cells grown in galactose or low glucose suggesting it is the metabolite 19,20-EDP (Fig. 2a). (5.5 mM) rely on mitochondrial oxidative phosphoryla- Interestingly, neither DHA nor 19,20-EDP had any sig- tion (OXPHOS) to meet their energy requirements. We nificant effect on the cell viability under low (5.5 mM) first demonstrated that total oxygen consumption of H9c2 glucose conditions (Fig. 2b). Treatment with myriocin Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 3 of 11 (1 µM), a potent inhibitor of serine palmitoyl transferase with an OXPHOS metabolic profile. Both cell cultures that inhibits the de novo synthesis of ceramide, com- were susceptible to a non-metabolic toxicant saponin pletely blocked both DHA- and 19,20-EDP-induced cell (Fig. 2b). death under 25 mM glucose conditions (Fig. 2a). Potas- The proteasome is a large complex type protease that sium cyanide (KCN), an inhibitor of mitochondrial elec- degrades ubiquitinated proteins, consisting of a catalytic tron transport chain complex IV, had no effect on the cell 20S core and two 20S regulatory subunits. Proteasomes viability under 25 mM glucose conditions, consistent with are necessary for maintaining cell proliferation and a glycolytic metabolic phenotype. Conversely, KCN homeostasis by regulating proteolysis of ubiquitinated treatment to cells cultured under 5.5 mM resulted in an and damaged proteins . We measured 20S total protea- almost complete loss of viability, which is expected in cells some activity following DHA or 19,20-EDP treatment Fig. 1 Characterization of H9c2 cells. H9c2 cells were cultured in DMEM media supplemented with 10% FBS, 25 mM (NG) or 5.5 mM (LG) glucose or cultured in DMEM supplemented with 1% FBS and 10 nM retinoic acid (RA) for 2 weeks. Alterations in culture media conditions impacted a oxygen consumption; b ADP/ATP ratios; c NAD/NADH ratios; and d lactate levels. e Representative immunoblot demonstrating increased expression of Troponin T in differentiated H9c2 cells incubated with 1%FBS and RA. Values are expressed as mean ± SEM (n = 3 repeat experiments). *P< 0.05 vs. NG Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 4 of 11 Fig. 2 DHA and 19,20-EDP-induced cytotoxicity is attenuated by an inhibitor of de novo ceramide synthesis under glycolytic conditions. H9c2 cells were cultured in either 25 mM (NG) or 5.5 mM (LG) glucose-containing media. Differential effects of DHA and 19,20-EDP on a, b cell viability; c, d 20S proteasomal activity; and e, f MTT activity. g, h Alterations in ATP levels following incubation with DHA or 19,20-EDP. Cells were treated with or without DHA (100 μM), 19,20-EDP (1 μM), MSPPOH (50 μM), myriocin (1 μM), saponin (10 mg/mL) and/or KCN (10 μM) for 24 h. Values are expressed as mean ± SEM (n = 3). *P < 0.05 vs. untreated control, P < 0.05 vs. 19,20-EDP alone Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 5 of 11 under 25 mM glucose conditions to estimate the total mitochondrial fractions. Furthermore, the 19,20-EDP- protein degradation/turnover rate as an indication of induced ceramide accumulation in membrane and lyso- cellular injury. We observed a significant increase in 20S somal/mitochondrial fractions was completely inhibited total protease activity in both DHA- and 19,20-EDP- by myriocin. Cells cultured under low glucose conditions treated cells (Fig. 2c). Conversely, neither DHA nor 19,20- demonstrated lower basal ceramide content in both EDP affected total proteasome activity under 5.5 mM fractions. In contrast, DHA and 19,20-EDP only caused a marginal shift in ceramide levels. These results suggest glucose conditions (Fig. 2d). The MTT (3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction DHA- and 19,20-EDP-induced cytotoxicity correlated assay can provide insight into impairment of mitochon- with marked increases in movement of ceramide accu- drial oxidative activity. Our data demonstrated DHA and mulation to lysosomal/mitochondrial fractions when cells 19,20-EDP caused a marked decrease in activity, sug- were cultured under 25 mM glucose conditions. gesting impaired mitochondrial function, (Fig. 2e), which was inhibited with myriocin. Interestingly, 19,20-EDP DHA and 19,20-EDP suppress mitochondrial function in caused a significant increase in MTT reduction in H9c2 H9c2 cells cultured under 25 mM glucose conditions cells when treated under low glucose conditions (Fig. 2f). To define the impact of DHA and 19,20-EDP on The presence of MG-132, a cell-permeable potent pro- mitochondria, we first evaluated the expression and teasome inhibitor, in both normal and low glucose con- activity of key enzymes. Under both normal and low ditions resulted in a significant increase in MTT activity; glucose cell culture conditions, neither DHA nor 19,20- moreover, MG-132 blocked the effect of DHA and 19,20- EDP had any effect on citrate synthase and cytochrome c EDP (Fig. 2e, f). oxidase protein expressions (Fig. 3a–d). However, they significantly inhibited the catalytic activities when cul- DHA and 19,20-EDP cause ceramide accumulation under tured with 25 mM glucose conditions (Fig. 3e, g) and high glucose conditions myriocin treatment significantly reversed the DHA- and Since myriocin completely inhibited both DHA- and 19,20-EDP-induced inhibitory effect. There is no effect on 19,20-EDP-induced cell death under normal glucose enzyme activities under low glucose condition (Fig. 3f, h). conditions (Fig. 2a), we next examined the effect of DHA and 19,20-EDP treatment toward cellular ceramide levels. DHA and 19,20-EDP cause lysosome-dependent cell death The basal ceramide content in subcellular lysosomal/ under high glucose conditions mitochondrial fractions isolated from cells cultured under Lysosomes are unique membrane-bound organelles 25 mM glucose conditions was much lower than mem- found in the cytosol that contain degradative hydrolytic brane fractions (Table 1). Under 25 mM glucose condi- enzymes involved in digestion of many biomolecules. tions, both DHA and 19,20-EDP significantly increased Lysosomal-dependent cell death has been classified as a the amount of ceramide found in membrane fractions. pattern initiated by perturbations of intracellular home- However, both DHA and 19,20-EDP treatment resulted in ostasis and the permeabilization of lysosomal membranes significant increases of ceramide content in lysosomal and releasing proteases. In order to investigate the role of lysosomes we used a fluorescent LysoTracker Green dye to visualize the organelles under an epifluorescent Table. 1 De novo ceramide synthesis in H9c2 cells microscope (Fig. 4A, B), as well as quantify the intensity with a fluorescent-based plate reader assay (Fig. 4C, D). Crude membrane Lysosome/mitochondria Treating H9c2 cells cultured in either 25 mM or 5.5 mM glucose media with bafilomycin, as a positive control NG LG NG LG promoting acidification and maturation of lysosomes, resulted in marked increase lysosome formation (Fig. 4Ab, Control 32.81 ± 1.9 14.27 ± 0.62 1.45 ± 1.27 1.14 ± 0.79 Bb, c). Interestingly, both DHA and 19,20-EDP increased DHA 54.26 ± 1.73* 20.85 ± 0.87 470.73 ± 15.94* 0.25 ± 0.09 the intensity of LysoTracker Green fluorescence (Fig. 4Ac, 19,20-EDP 50.50 ± 4.63* 16.44 ± 0.52 601.73 ± 23.9* 0.40 ± 0.21 Ad) compared with control (Fig. 4Aa, C) under normal † † 19,20-EDP 18.23 ± 7.57 11.78 ± 0.24 64.20 ± 63.85 0.37 ± 0.22 glucose conditions, while inhibiting ceramide synthesis +myriocin with myriocin inhibited 19,20-EDP-increased intensity (Fig. 4Ag, C). DHA- and 19,20-EDP-induced intensity was LC/MS analysis was employed to measure ceramide levels in H9c2 cells cultured in either normal (25 mM) or low (5.5 mM) glucose-containing media reduced by MG-132 treatment (Fig. 4Ai, Aj, C). Con- Ceramide content in crude membrane and lysosomal/mitochondria fractions versely, addition of DHA or 19,20-EDP to H9c2 cells were assessed. Cells were treated with or without DHA (100 μΜ), 19,20-EDP incubated in low glucose conditions had the opposite (1 μΜ) and myriocin (1 μΜ) for 24 h Values are expressed as mean ± SEM (n = 4) effect with a significantly decreased level of lysosomal *P < 0.05 vs. control P < 0.05 vs. 19.20-EDP (NG) intensity (Fig. 4B, D). Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 6 of 11 Fig. 3 DHA and 19,20-EDP inhibit mitochondrial enzyme activities under glycolytic conditions. H9c2 cells were cultured in either 25 mM (NG) or 5.5 mM (LG) glucose-containing media. H9c2 cells were treated with or without DHA (100 μM), 19,20-EDP (1 μM), MSPPOH (50 μM) and/or myriocin (1 μM) for 24 h. Representative immunoblots and histograms of relative densities of a, b citrate synthase and c, d cytochrome c oxidase protein expression. Enzyme activities of e, f citrate synthase and g, h cytochrome c oxidase. Values are expressed as mean ± SEM (n = 3). *P < 0.05 vs. untreated control, P < 0.05 vs. 19,20-EDP alone Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 7 of 11 Fig. 4 DHA and 19,20-EDP induced lysosomal formation and activity under glycolytic conditions. H9c2 cells were cultured in either 25 mM (NG) or 5.5 mM (LG) glucose-containing media. H9c2 cells were treated with or without DHA (100 μM), 19,20-EDP (1 μM), MSPPOH (50 μM), bafilomycin (1 μM), myriocin (1 μM) and/or MG1-32 (1 μM) for 24 h and then stained with mitochondrial dye, TMRE (100 nm, red), lysosomal dye, LysoTracker (10 nm, green) and nuclear dye, Hoechest33342 (1 mM, blue). Representative images of mitochondrial and lysosomal organelles in H9c2 cells cultured in a 25 mM (NG) or b 5.5 mM (LG) glucose media. Histograms representing the relative quantification of lysosomal expression in H9c2 cells cultured in c 25 mM (NG) or d 5.5 mM (LG) glucose media. Values are expressed as mean ± SEM (n = 3). *P < 0.05 vs. untreated control, P < 0.05 vs. respective group untreated MG132 Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 8 of 11 Discussion sphingolipid, ceramide, has important roles in cellular In the current study we demonstrate that DHA and membrane structure and is an integral component of the 19,20-EDP selectively induce cell death in non- sphingomyelin cycle acting as an essential second mes- differentiated H9c2 cells with a developed glycolytic senger in the intracellular propagation of physiological, profile. It is well documented that CYP epoxygenases pharmacological and environmental signals in normal and catalyze the enzymatic transformation of PUFA into var- cancerous cells . Ceramide elicits many cell-stress ious potent epoxylipid including omega-3 epoxides of responses including apoptosis, senescence, inflammation, DHA referred as EDPs. Recent evidence suggests that mitochondrial dysfunction and altered cellular metabo- 17,26,27 EDPs are involved in regulating cellular processes such as lism . The hydrophobic nature of ceramide limits its inflammation, angiogenesis and cell death; however, our accumulation to membranous regions within the cell understanding of how EDPs regulate these effects remains where it may influence protein distribution and function. significantly limited. Previously, we have demonstrated In this regard, cytosolic concentrations of ceramide are immortalized H9c2 cells were prone to cytotoxicity negligible. While the majority of studies evaluating the caused by DHA and 19,20-EDP. The current study effects of an elevation in cellular ceramide have focused demonstrated the cytotoxicity of EDPs was dependent on its ability to induce apoptosis , several other studies upon the metabolic profile of the cells. have also described ceramide to be necessary for the Interestingly, an increased de novo synthesis of cer- induction of senescence, cell–cell interactions, death 25,28–33 amide occurs following treatment DHA and 19,20-EDP, receptor clustering and autophagy . Collectively, with a corresponding accumulation in intracellular these studies emphasize that ceramide is capable of 17,25,28–34 membrane fractions including mitochondria and lyso- modulating several biochemical pathways . Stra- somes, while H9c2 cells driven to demonstrate an tegies that pharmacologically or genetically decrease cer- OXPHOS phenotype were not susceptible to DHA- or amide have beneficial effects in reversing insulin 19,20-EDP-mediated cell death, or changes in intracel- resistance, preventing apoptosis of pancreatic β-cells and 28–30 lular ceramide production. Altered levels of endogenous cardiomyocytes . lipids, such as ceramide, are well known to be involved in Accumulation of ceramide has been linked to major both cell survival and cell death processes. Together, our perturbations in cell metabolism resulting in apoptosis, data suggest a novel cell death mechanism for DHA and necroptosis and lethal autophagy including mito- 15,28,31,33 19,20-EDP lipids, which occurs in non-differentiated phagy . Ceramide accumulation in mitochondria glycolytic cells. Noteworthy, during the last decade, stu- can lead to stress-induced mitochondrial fragmentation dies dedicated to biological effects of DHA have largely and decreased ATP production through disruption of ignored the roles of CYP-derived metabolites. In this electron transport chain and induction of injury, resulting study, we demonstrated that DHA produced cytotoxic in a pool of dysfunctional mitochondria capable of pro- 31,32 effect in glycolytic H9c2 cells similar to what was moting cell death . We found that both DHA and observed with 19,20-EDP. Furthermore, formation of 19,20-EDP induced ceramide accumulation in mito- endogenous EDPs has an essential role in DHA- asso- chondrial and lysosomal fractions, which was diminished ciated injury of glycolytic H9c2 cells. Since cytotoxic in the presence of myriocin. Moreover, the increased de effects of DHA toward glycolytic H9c2 cells were strongly novo synthesis of ceramide and its accumulation in sub- decreased when MSPPOH, a CYP epoxygenase inhibitor, cellular membrane fractions occurred under glycolytic was added, we presume that observed effects were asso- conditions and requires increased NADH reduction, ciated with EDPs endogenously produced from DHA. which was markedly reduced under OXPHOS . The One of the main metabolic divergences between quiescent increased production of ceramide correlated with elevated and proliferating cells is the pathway responsible for ATP lysosomal activity and was reversed by the proteasome synthesis. In non-cancerous cells with hallmarks of inhibitor MG-132, which additionally support the role of primary lines, ATP is usually synthesized during ceramide formed in glycolytic conditions in various types OXPHOS, whereas cancerous and highly proliferative of cell death . Detection of lysosomal-mediated cell death cells preferentially develop glycolysis for ATP synthesis originally was difficult, as lysosomal ultrastructure often and employ mitochondria solely to sustain redox appears intact in apoptotic cells analyzed by electron potential required to reduce NADH for glycolysis. This is microscopy . Partial lysosomal membrane permeabiliza- known as the Warburg effect that results in formation of a tion can occur early in many death paradigms, which pool of aberrant mitochondria with poor functional triggers proteasomal cell death . Interestingly, increases 23,24 activity . in ceramide levels have been shown to lower the integrity While our previous research correlated DHA-mediated of lysosomal membranes and hinders their fusion with cell death in H9c2 cells with ceramide production, the other intracellular vesicles including mitochondria .In current data demonstrated unique mechanisms . The the current study, accumulation of ceramide in lysosomal Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 9 of 11 fractions following treatment with DHA or 19,20-EDP LC/MS measurement of different pools of intracellular may have contributed to destabilization of lysosomal ceramide membranes and the subsequent increase in proteasomal The extraction of ceramide from H9c2 cells was per- and lysosomal activities. Early studies using compounds formed in accordance to a published protocol . Cells directly targeting the integrity of lysosomal membranes were cultured in 175 cm flasks and then harvested and provided evidence for their role in programmed cell subjected to subcellular fractionation. Cells were cen- 39–41 death . Moreover, inhibitors of lysosomal function trifuged at 700 × g for 10 min and then rinsed with cold would be more toxic to cancer and transformed cells than phosphate-buffered saline (PBS) and centrifuged again at normal cell lines . A quantitative relationship between 700 × g for 10 min, 4 °C. The resulting pellet was termed the amount of lysosomal rupture and the mode of cell and used as a crude membrane fraction. For the enrich- death has been suggested to explain the widely different ment of heavy membrane fraction containing light and morphological outcomes following lysosomal membrane heavy mitochondria as well as lysosomes, the remaining permeabilization . According to this model, low stress supernatant was centrifuged at 10,000 × g for 20 min, 4 °C. intensities trigger a limited release of lysosomal contents Then, 300 mL of a 0.4% NaCl solution and 1 mL of a to the cytoplasm followed by cell death, while high chloroform–methanol–HCl 1 N (100:100:1, v/v/v) mix- intensity stress factors lead to a generalized lysosomal ture were added to the samples. Following this, the rupture and rapid cellular necrosis. It is entirely possible samples were vortexed at 1000 rpm (room temperature) that DHA and 19,20-EDP induced accumulation of cer- for 20 min. Ceramide content in the samples was analyzed amide in H9c2 cells with glycolytic profile, which insti- using Waters ZQ 4000 Mass Spectrometer coupled to a gated cell death. Remarkably, co-treatment with MG-132, Water 2795 Separations Module. A previously described an inhibitor of lysosomal proteases, decreased toxicity of liquid chromatography/mass spectrometry (LC/MS) DHA and 19,20-EDP. method was utilized with modifications . Briefly, cer- In conclusion, we report a novel cell death mechanism amide as well as the internal standard, 4-methox- for DHA and its epoxylipid, 19,20-EDP, in non- ybenzophenone, were resolved using a reverse-phase C18 differentiated glycolytic H9c2 cells. Our study suggests column (Alltima HP, 150 × 2.1 mm) at 35 °C by the iso- these lipid mediators induce a ceramide-mediated process cratic elution of methanol–tetrahydrofuran–water–acetic that increases lysosomal-proteasome activity impacting acid (80:10:10:0.2, v/v/v/v) as the mobile phase. Ceramide mitochondria function leading to cell death. An increased and 4-methoxybenzophenone were detected using a sin- production of ceramide coupled with increased lysosomal gle ion recording under positive-ion mode at m/z = 342.4 membrane permeability in glycolytic cells causes a and 213.1, respectively. The cone voltage was 25 V and destabilization of mitochondria resulting in cell death. 15 V for ceramide and 4-methoxybenzophenone, respec- The shift in cellular metabolic profile from glycolytic to tively, and capillary voltage of 4.0 kV. During the acqui- OXPHOS highlights a novel toxicity of DHA and 19, 20- sition of the data, the mass spectrometer was maintained EDP toward H9c2 cells with a glycolytic profile. at a source and desolation temperature of 120 °C and 275 °C, respectively. The resulting values were then cal- Materials and methods culated using a ceramide standard curve and expressed as Cell culture and viability assay mg weight per mg of cellular mass, and then further H9c2 cardiac cells were obtained from ATCC. The cells normalized to account for the fold change between wet were cultured in DMEM medium containing either 25 and dry cellular mass for quantification purposes. mM (normal glucose) or 5.5 mM glucose (low glucose) and supplemented with 10% FBS and 1% penicillin/ Assessment of mitochondrial function streptomycin. Differentiation of H9c2 myoblasts into Cellular ATP was determined using a kit based on myotubes was performed in DMEM supplemented with fluorescent assay (Abcam or Sigma-Aldrich). Citrate 1% FBS, 1% penicillin/streptomycin and 10 nM retinoic synthase (CS) and cytochrome c oxidase enzymatic acid for 2 weeks. Cell viability was assessed using a activities were assayed spectrophotometrically in cell 46,43 commercially available kit from Promega based on lumi- lysates as previously described . Rapid assessment of nescent assay of intracellular proteases. Another approach oxygen consumption was performed using a kit based on to measure metabolically active cells was MTT test per- detection of oxygen with fluorescent trap (Abcam). NAD/ formed as previously described . The intensity of NADH ratio was assessed using a kit from Promega. reduction of MTT to formazan crystals by mitochondrial Mitochondrial respiration was measured in saponin per- dehydrogenases positively correlates with the overall meabilized HL-1 cells using Clark oxygen electrode con- activity of oxidative metabolism. Optical density of nected to Oxygraph Plus recorder (Hansatech dimethyl sulfoxide extracted from formazan was mea- Instruments Ltd, Norfolk, England). Respiration rates sured spectrophotometrically at 595 nm. were measured at 30 °C before and after addition of 2 mM Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 10 of 11 ADP with 5 mM malate and 10 mM glutamate as sub- Author details Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, strates. RCR was calculated as the ratio between basal and Edmonton, AB, Canada. Department of Pharmacological Sciences, School of ADP-stimulated respiration rates . Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido, Japan. Department of Clinical Pharmacy, Faculty of Pharmacy, Tanta University, 8130 Tanta, Egypt. Department of Pharmacology, Faculty of Immunoblotting Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Samples were subjected to western blot analysis as 7,20 previously described . Briefly, 25 μg of protein from Conflict of interest The authors declare that they have no conflict of interest. H9c2 lysates was probed on a 12% SDS-polyacrylamide gel. Membranes were washed four times with Tris- buffered saline with Tween-20 buffer and then incu- Publisher's note bated with primary antibodies, CS (1:000, Abcam., Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cambridge, UK, Cat No: ab129095), cytochrome c oxidase (COX IV) (1:1,000, Cell Signaling Tech., Inc., Whitby, ON, Cat No: 4850) and GAPDH (1:5000, Cell Signaling Received: 17 July 2018 Accepted: 24 July 2018 Tech., Inc., Whitby, ON, Cat No: 5174S), secondary antibodies were used as 1:5000 dilution. Relative band intensity to control was measured using Image J software References (NIH, USA). 1. Wang, C. et al. n-3 Fatty acids from fish or fish-oil supplements, but not alpha- linolenic acid, benefit cardiovascular disease outcomes in primary- and Assessment of lysosomal activity secondary-prevention studies: a systematic review. Am. J. Clin. Nutr. 84,5–17 (2006). Lysosomal activity was measured and quantitated using 2. Mori, T. A. Dietary n-3 PUFA and CVD: a review of the evidence. Proc. Nutr. Soc. LysoTracker Green DND-26 which specifically stains 73,57–64 (2014). acidic structures in live cells. H9c2 cells were treated with 3. Samokhvalov, V. et al. PPARgamma signaling is required for mediating EETs protective effects in neonatal cardiomyocytes exposed to LPS. Front. Phar- LysoTracker Green DND-26 added to a final concentra- macol. 5, 242 (2014). tion of 10 nM for 1 h. The cells were rinsed with PBS and 4. Arnold, C. et al. Arachidonic acid-metabolizing cytochrome P450 fluorescence was measured with excitation/emission 504/ enzymes are targets of {omega}-3 fatty acids. J. Biol. Chem. 285, 32720–32733 (2010). 511 nm. The intensity of fluorescence reflected the active 5. Zhang, G., Kodani, S. & Hammock, B. D. Stabilized epoxygenated fatty acids pool of lysosomes. MG-132, which acts as a potent cell- regulate inflammation, pain, angiogenesis and cancer. Prog. Lipid Res. 53, permeable inhibitor of lysosomal activity, was added to a 108–123 (2014). 6. Jung, F. et al. Effect of cytochrome P450-dependent epoxyeicosanoids on final concentration of 1 µM where indicated. Ristocetin-induced thrombocyte aggregation. Clin. Hemorheol. Microcirc. 52, 403–416 (2012). Microscopy 7. Samokhvalov, V.,Jamieson, K. L.,Vriend, J.,Quan, S.,& Seubert, J. M. CYP- epoxygenase metabolites of docosahexaenoic acid protect HL-1 cardiac cells H9c2 cells were grown on glass-bottom 35 mm dishes against LPS-induced cytotoxicity through SIRT1.Cell Death Discov. 1, 15054 suitable for fluorescent microscopy (MatTek) and treated (2015). with indicated reagents. Mitochondria were stained with 8. Fasano, E. et al. DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines. Biochim. Biophys. Acta 1822, TMRE (100 nM) and nucleus were stained with Hoech- 1762–1772 (2012). est33342 (1 mM). Acidic organelles were stained with 9. Kim, H. K., Della-Fera, M.,Lin,J.& Baile, C. A. Docosahexaenoic acid inhibits LysoTracker Green DND-26 (10 nM). The dishes were adipocyte differentiation and induces apoptosis in 3T3-L1 preadipocytes. J. Nutr. 136, 2965–2969 (2006). placed in a micro incubator installed on the objective stage 10. Schunck, W. H., Konkel, A., Fischer, R. & Weylandt, K. H. Therapeutic potential of of a Zeiss Axio Observer epifluorescence microscope. The omega-3 fatty acid-derived epoxyeicosanoids in cardiovascular and inflam- image system consisted of Zeiss Axio Observer Z1 inverted matory diseases. Pharmacol. Ther. 183,177–204 (2018). 11. Kimes,B. W.& Brandt,B.L.Propertiesofa clonalmusclecelllinefromrat heart. epifluorescence microscope equipped with a LED lamp, 63× Exp. Cell Res. 98,367–381 (1976). oil immersion objective lens and cooled CCD camera. 12. Hescheler, J. et al. Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ. Res. 69, 1476–1486 (1991). Statistical analysis 13. Sardao, V. A., Oliveira, P. J., Holy, J., Oliveira, C. R. & Wallace, K. B. Vital imaging of Values were expressed as mean ± standard error of mean. H9c2 myoblasts exposed to tert-butylhydroperoxide--characterization of Statistical significance was determined using one-way ana- morphological features of cell death. BMC Cell. Biol. 8,11 (2007). 14. Menard, C. et al. Modulation of L-type calcium channel expression during lysis of variance. To determine whether significant differ- retinoic acid-induced differentiation of H9C2 cardiac cells. J. Biol. Chem. 274, ences exist between the groups, Bonferonni post-hoc test was 29063–29070 (1999). performed. Values were considered significant as p < 0.05. 15. Pereira, S. L. et al. Metabolic remodeling during H9c2 myoblast differentiation: relevance for in vitro toxicity studies. Cardiovasc. Toxicol. 11,180–190 (2011). 16. Marroquin, L. D., Hynes, J., Dykens, J. A., Jamieson, J. D. & Will, Y. Circumventing Acknowledgements the Crabtree effect: replacing media glucose with galactose increases sus- This work was partially supported by an operating grant from the Canadian ceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97,539–547 Institutes of Health Research (to J.M.S.) and the Taiho Fund from the Faculty of (2007). Pharmacy and Pharmaceutical Sciences, University of Alberta (to T.E. and J.M.S.). Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 11 of 11 17. Bikman, B. T. & Summers, S. A. Ceramides as modulators of cellular and whole- 33. Scarlatti, F. et al. Ceramide-mediated macroautophagy involves inhibition of body metabolism. J. Clin. Invest. 121,4222–42300 (2011). protein kinase B and up-regulation of beclin 1. J. Biol. Chem. 279,18384–18391 18. He, X. &Schuchman,E. H.Ceramideand ischemia/reperfusion injury. J. Lipids (2004). 2018, 3646725 (2018). 34. Lu, C. Y. et al. Docosahexaenoic acid down-regulates phenobarbital-induced 19. Zhang, T., Barclay, L., Walensky, L. D. & Saghatelian, A. Regulation of mito- cytochrome P450 2B1 gene expression in rat primary hepatocytes via the chondrial ceramide distribution by members of the BCL-2 family. J. Lipid Res. sphingomyelinase/ceramide pathway. J. Nutr. Biochem. 21,338–344 (2010). 56,1501–1510 (2015). 35. Arboleda, G. et al. Insulin-like growth factor-1-dependent maintenance of 20. Qadhi, R. et al. Differential responses to docosahexaenoic acid in primary and neuronal metabolism through the phosphatidylinositol 3-kinase-Akt pathway immortalized cardiac cells. Toxicol. Lett. 219, 288–297 (2013). is inhibited by C2-ceramide in CAD cells. Eur. J. Neurosci. 25,3030–3038 21. Samokhvalov, V. et al. PPARdelta signaling mediates the cytotoxicity of DHA in (2007). H9c2 cells. Toxicol. Lett. 232,10–20 (2015). 36. Okazaki, T. & Okazaki, I. M. [Regulation of autoimmunity and tumor immunity 22. Coux, O., Tanaka, K. & Goldberg, A. L. Structure and functions of the 20S and by immunoinhibitory co-receptor, PD-1]. Seikagaku 87,693–704 (2015). 26S proteasomes. Annu. Rev. Biochem. 65, 801–847 (1996). 37. de Duve, C. Lysosomes revisited. Eur. J. Biochem. 137,391–397 (1983). 23. Schwartz, L.,Buhler, L.,Icard,P., Lincet,H.&Steyaert,J.M.Metabolic treatment 38. Appelqvist, H., Waster, P., Kagedal, K. & Ollinger, K. The lysosome: from waste of cancer: intermediate results of a prospective case series. Anticancer Res. 34, bag to potential therapeutic target. J. Mol. Cell Biol. 5, 214–226 (2013). 973–980 (2014). 39. Chiong, M. et al. Cardiomyocyte death: mechanisms and translational impli- 24. Zhang, S. et al. Homeostasis of redox status derived from glucose metabolic cations. Cell Death Dis. 2, e244 (2011). pathway could be the key to understanding the Warburg effect. Am.J.Cancer 40. Terman, A., Kurz, T., Gustafsson, B. & Brunk, U. T. The involvement of lysosomes Res. 5,1265–1280 (2015). in myocardial aging and disease. Curr. Cardiol. Rev. 4,107–115 (2008). 25. Ogretmen, B. & Hannun, Y. A. Biologically active sphingolipids in cancer 41. Tsubone,T.M.etal. Enhanced efficiency of cell death by lysosome-specific pathogenesis and treatment. Nat. Rev. Cancer 4, 604–616 (2004). photodamage. Sci. Rep. 7, 6734 (2017). 26. Stathem, M. et al. Glucose availability and glycolytic metabolism dictate gly- 42. Boya, P. & Kroemer, G. Lysosomal membrane permeabilization in cell death. cosphingolipid levels. J. Cell. Biochem. 116,67–80 (2015). Oncogene 27,6434–6451 (2008). 27. Morad, S. A. & Cabot, M. C. Ceramide-orchestrated signalling in cancer cells. 43. Samokhvalov, V. et al. Epoxyeicosatrienoic acids protect cardiac cells during Nat. Rev. Cancer 13,51–65 (2013). starvation by modulating an autophagic response. Cell Death Dis. 4,e885 28. Jiang, W. & Ogretmen, B. Ceramide stress in survival versus lethal autophagy (2013). paradox: ceramide targets autophagosomes to mitochondria and induces 44. Bose, R. & Kolesnick, R. Measurement of ceramide levels by the diacylglycerol lethal mitophagy. Autophagy 9,258–259 (2013). kinase reaction and by high-performance liquid chromatography- 29. Young,M.M., Kester,M. & Wang, H. G.Sphingolipids: regulators of crosstalk fluorescence spectrometry. Methods Enzymol. 322,373–378 (2000). between apoptosis and autophagy. J. Lipid Res. 54,5–19 (2013). 45. Sahle,F. F., Lange, S., Dobner,B., Wohlrab, J. & Neubert,R.H. Development and 30. Park, T. S. et al. Ceramide is a cardiotoxin in lipotoxic cardiomyopathy. J. Lipid validation of LC/ESI-MS method for the detection and quantification of exo- Res. 49, 2101–2112 (2008). genous ceramide NP in stratum corneum and other layers of the skin. J. 31. Sentelle, R. D. et al. Ceramide targets autophagosomes to mitochondria and Pharm. Biomed. Anal. 60,7–13 (2012). induces lethal mitophagy. Nat. Chem. Biol. 8, 831–838 (2012). 46. Spinazzi,M., Casarin, A.,Pertegato,V., Salviati,L.&Angelini, C. Assessment of 32. Dany, M. & Ogretmen, B. Ceramide induced mitophagy and tumor suppres- mitochondrial respiratory chain enzymatic activities on tissues and cultured sion. Biochim. Biophys. Acta 1853,2834–2845 (2015). cells. Nat. Protoc. 7,1235–1246 (2012). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

DHA and 19,20-EDP induce lysosomal-proteolytic-dependent cytotoxicity through de novo ceramide production in H9c2 cells with a glycolytic profile

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Copyright © 2018 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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Abstract

Docosahexaenoic acid (DHA) and their CYP-derived metabolites, epoxydocosapentaenoic acids (EDPs), are important fatty acids obtained from dietary sources. While it is known that they have significant biological effects, which can differ between cell type and disease state, our understanding of how they work remains limited. Previously, we demonstrated that DHA and 19,20-EDP triggered pronounced cytotoxicity in H9c2 cells correlating with increased ceramide production. In this study, we examine whether DHA- and 19,20-EDP-induced cell death depends on the type of metabolism (glycolysis or OXPHOS). We cultivated H9c2 cells in distinct conditions that result in either glycolytic or oxidative metabolism. Our major findings suggest that DHA and its epoxy metabolite, 19,20-EDP, trigger cytotoxic effects toward H9c2 cells with a glycolytic metabolic profile. Cell death occurred through a mechanism involving activation of a lysosomal-proteolytic degradation pathway. Importantly, accumulation of ceramide played a critical role in the susceptibility of glycolytic H9c2 cells to cytotoxicity. Furthermore, our data suggest that an alteration in the cellular metabolic profile is a major factor determining the type and magnitude of cellular toxic response. Together, the novelty of this study demonstrates that DHA and 19,20-EDP induce cell death in H9c2 cells with a glycolytic metabolicwct 2 profile through a lysosomal-proteolytic mechanism. Introduction cardiovascular system, suggesting they reduce the risk of Long chain n-3 polyunsaturated fatty acids (PUFAs) cardiovascular disease by protecting the heart and vas- such as docosahexaenoic acid (DHA, C22:6n-3) and culature against injury, such as limiting cardiac arrhyth- 1,2 eicosapentaenoic acids (C20:5n-3) are important fatty mias, myocardial infarction and hypertension . Overall, acids obtained from dietary sources. These essential fatty there is a growing body of evidence demonstrating that n- acids are required components of phospholipid mem- 3 PUFA have significant biological effects depending upon branes and serve as precursors to numerous lipid med- the cell and disease; however, understanding exactly how iators with various biological properties. Numerous n-3 PUFAs work remains unknown. In recent years, evi- studies report a positive effect of n-3 PUFAs toward the dence indicate that there is a biological role for cyto- 3,4 chrome P450 (CYP) epoxygenase metabolites of DHA . CYP epoxygenases metabolize DHA into 6 regioisomeric Correspondence: John M. Seubert (jseubert@ualberta.ca) epoxydocosapentaenoic acids (4,5-, 7,8-, 10,11-, 13,14-, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada 16,17- and 19,20-EDP), which may then undergo further Department of Pharmacological Sciences, School of Pharmaceutical Sciences, metabolism by epoxide hydrolase enzymes to corre- Health Sciences University of Hokkaido, Hokkaido, Japan sponding diols, dihydroxydocosapentaenoic acids . EDPs Full list of author information is available at the end of the article. Edited by A. Rufini © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Endo et al. Cell Death Discovery (2019) 5:29 Page 2 of 11 have been suggested as the active mediators impacting cells grown with 25 mM glucose was less than one-fifth of cellular responses to injury and disease such as cytotoxi- the oxygen consumed when cells were grown in 5.5 mM 5–9 city, cardiovascular disease and cancer . Among the glucose condition (Fig. 1a). These data suggest changing regioisomeric metabolites of DHA, 19,20-EDP gained the the cell culture conditions from 25 mM to 5.5 mM glu- most interest due to its pronounced biological effects in cose shifted the energy metabolism in the undifferentiated numerous aspects of cell biology . cells from glycolysis to OXPHOS, which was reflected in the significant change in ATP production and increased The reported biological effects of DHA and its meta- bolites, EDPs, appear to be dependent upon the specific Nicotinamide adenine dinucleotide/Nicotinamide ade- cellular phenotype being studied. H9c2 myoblast cells are nine dinucleotide hydrogen NAD/NADH ratio (Fig. 1b, c). an immortalized cell line derived from ventricular tissue Also, we detected a significantly higher lactate level in 11,12 of the BDIX rat heart . H9c2 cells are not fully dif- media with 25 mM glucose, which further reflects higher ferentiated until cultured in media with low serum, which glycolytic activity (Fig. 1d). Next, we assessed mitochon- triggers differentiating from mono-nucleated myoblasts to drial respiration in permeabilized cells to determine the a skeletal muscle phenotype or the addition of all-trans respiration control ratio (RCR), which is the ratio between retinoic acid to 1% serum media that induces adult car- basal and adenosine diphosphate (ADP)-stimulated 13,14 diac muscle phenotype . In the undifferentiated state, respiration. H9c2 cells grown in 25 mM glucose media H9c2 cells tend to be highly proliferative relying on gly- had an RCR of 1.44 ± 0.18, while cells cultivated in 5.5 colysis rather than mitochondrial oxidative phosphoryla- mM glucose media had RCR up to 6.9 ± 0.87, thus, tion . Such aerobically poised cells can demonstrate demonstrating that there was a shift from glycolysis to resistance to toxic agents that target mitochondria . OXPHOS in H9c2 cells grown in 5.5 mM glucose media. Ceramide is a central lipid component of sphingolipid Adding retinoic acid (RA, 10 nM) treatment to H9c2 structure that is biosynthesized by three pathways, which cells cultured in Dulbecco's modified Eagle's medium include de novo synthesis from palmitoyl-CoA and serine, (DMEM) with 1% fetal bovine serum (FBS) serum for hydrolysis of sphingomyelin or a salvage pathway .Itis 2 weeks has been shown to stimulate differentiation to a an important lipid mediator regulating various cellular cardiac-specific phenotype . We compared our cell responses like cell death, and recent evidence also sug- model in non-differentiated cells of shifting metabolism gests a role in various metabolic pathways influencing from a primarily glycolytic metabolism to OXPHOS to mitochondrial function . In vitro data indicate produc- differentiated cells. Neither 25 mM nor 5.5 mM cultured cells expressed troponin T, indicating our cells remained tion of ceramide increases in glycolytic cells but decreases in cells with developed OXPHOS . Previous data indicate in the non-differentiated state as compared to cells treated undifferentiated H9c2 cells are susceptible to DHA- with RA for 14 days in 1% FBS (Fig. 1e). We observed a induced cell death in a concentration-dependent man- marked difference in oxygen consumption, ADP/ATP, ner which does not occur in primary neonatal cardio- NAD/NADH and lactate levels between 25 mM, 5.5 mM myocytes . Furthermore, 19,20-EDP was demonstrated and differentiated cells (Fig. 1a–d). Our primary goal was to cause cytotoxicity in undifferentiated H9c2 cells cor- to investigate the impact of altered metabolism in non- relating with de novo synthesis of intracellular ceramide . differentiated cells, and hence we utilized these cells for While ceramide is known to induce cell death in tumor the remaining experiments. cells, the mechanisms involved in DHA-mediated events associated with a metabolic state remain unclear. In the DHA- and 19,20-EDP-induced cell cytotoxicity in non- present study, we investigated the effects of DHA differentiated H9c2 cells and 19,20-EDP in undifferentiated H9c2 cells cultured Our previous data demonstrated that H9c2 cells cul- under conditions triggering glycolytic or oxidative tured in 25 mM glucose were susceptible DHA-mediated 20,21 phosphorylation-mediated metabolism. cell death . In the current study, we investigated the effect of DHA (100 µM) and its CYP epoxygenase meta- Results bolite, 19,20-EDP (1 µM), on viability of H9c2 cells grown Culturing non-differentiated H9c2 cells in low glucose in either 25 mM (NG) or 5.5 mM (LG) glucose-containing media shifts cellular metabolism toward OXPHOS media (Fig. 2a, b). Consistently, both DHA and 19,20-EDP H9c2 cells are normally cultured in media containing treatment markedly reduced cell viability in cells cultured 25 mM glucose, and as such they primarily utilize glyco- in 25 mM glucose. Co-treatment of DHA with a CYP lysis for adenosine triphosphate (ATP) generation. In epoxygenase inhibitor (MSPPOH) prevented cell death contrast, H9c2 cells grown in galactose or low glucose suggesting it is the metabolite 19,20-EDP (Fig. 2a). (5.5 mM) rely on mitochondrial oxidative phosphoryla- Interestingly, neither DHA nor 19,20-EDP had any sig- tion (OXPHOS) to meet their energy requirements. We nificant effect on the cell viability under low (5.5 mM) first demonstrated that total oxygen consumption of H9c2 glucose conditions (Fig. 2b). Treatment with myriocin Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 3 of 11 (1 µM), a potent inhibitor of serine palmitoyl transferase with an OXPHOS metabolic profile. Both cell cultures that inhibits the de novo synthesis of ceramide, com- were susceptible to a non-metabolic toxicant saponin pletely blocked both DHA- and 19,20-EDP-induced cell (Fig. 2b). death under 25 mM glucose conditions (Fig. 2a). Potas- The proteasome is a large complex type protease that sium cyanide (KCN), an inhibitor of mitochondrial elec- degrades ubiquitinated proteins, consisting of a catalytic tron transport chain complex IV, had no effect on the cell 20S core and two 20S regulatory subunits. Proteasomes viability under 25 mM glucose conditions, consistent with are necessary for maintaining cell proliferation and a glycolytic metabolic phenotype. Conversely, KCN homeostasis by regulating proteolysis of ubiquitinated treatment to cells cultured under 5.5 mM resulted in an and damaged proteins . We measured 20S total protea- almost complete loss of viability, which is expected in cells some activity following DHA or 19,20-EDP treatment Fig. 1 Characterization of H9c2 cells. H9c2 cells were cultured in DMEM media supplemented with 10% FBS, 25 mM (NG) or 5.5 mM (LG) glucose or cultured in DMEM supplemented with 1% FBS and 10 nM retinoic acid (RA) for 2 weeks. Alterations in culture media conditions impacted a oxygen consumption; b ADP/ATP ratios; c NAD/NADH ratios; and d lactate levels. e Representative immunoblot demonstrating increased expression of Troponin T in differentiated H9c2 cells incubated with 1%FBS and RA. Values are expressed as mean ± SEM (n = 3 repeat experiments). *P< 0.05 vs. NG Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 4 of 11 Fig. 2 DHA and 19,20-EDP-induced cytotoxicity is attenuated by an inhibitor of de novo ceramide synthesis under glycolytic conditions. H9c2 cells were cultured in either 25 mM (NG) or 5.5 mM (LG) glucose-containing media. Differential effects of DHA and 19,20-EDP on a, b cell viability; c, d 20S proteasomal activity; and e, f MTT activity. g, h Alterations in ATP levels following incubation with DHA or 19,20-EDP. Cells were treated with or without DHA (100 μM), 19,20-EDP (1 μM), MSPPOH (50 μM), myriocin (1 μM), saponin (10 mg/mL) and/or KCN (10 μM) for 24 h. Values are expressed as mean ± SEM (n = 3). *P < 0.05 vs. untreated control, P < 0.05 vs. 19,20-EDP alone Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 5 of 11 under 25 mM glucose conditions to estimate the total mitochondrial fractions. Furthermore, the 19,20-EDP- protein degradation/turnover rate as an indication of induced ceramide accumulation in membrane and lyso- cellular injury. We observed a significant increase in 20S somal/mitochondrial fractions was completely inhibited total protease activity in both DHA- and 19,20-EDP- by myriocin. Cells cultured under low glucose conditions treated cells (Fig. 2c). Conversely, neither DHA nor 19,20- demonstrated lower basal ceramide content in both EDP affected total proteasome activity under 5.5 mM fractions. In contrast, DHA and 19,20-EDP only caused a marginal shift in ceramide levels. These results suggest glucose conditions (Fig. 2d). The MTT (3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction DHA- and 19,20-EDP-induced cytotoxicity correlated assay can provide insight into impairment of mitochon- with marked increases in movement of ceramide accu- drial oxidative activity. Our data demonstrated DHA and mulation to lysosomal/mitochondrial fractions when cells 19,20-EDP caused a marked decrease in activity, sug- were cultured under 25 mM glucose conditions. gesting impaired mitochondrial function, (Fig. 2e), which was inhibited with myriocin. Interestingly, 19,20-EDP DHA and 19,20-EDP suppress mitochondrial function in caused a significant increase in MTT reduction in H9c2 H9c2 cells cultured under 25 mM glucose conditions cells when treated under low glucose conditions (Fig. 2f). To define the impact of DHA and 19,20-EDP on The presence of MG-132, a cell-permeable potent pro- mitochondria, we first evaluated the expression and teasome inhibitor, in both normal and low glucose con- activity of key enzymes. Under both normal and low ditions resulted in a significant increase in MTT activity; glucose cell culture conditions, neither DHA nor 19,20- moreover, MG-132 blocked the effect of DHA and 19,20- EDP had any effect on citrate synthase and cytochrome c EDP (Fig. 2e, f). oxidase protein expressions (Fig. 3a–d). However, they significantly inhibited the catalytic activities when cul- DHA and 19,20-EDP cause ceramide accumulation under tured with 25 mM glucose conditions (Fig. 3e, g) and high glucose conditions myriocin treatment significantly reversed the DHA- and Since myriocin completely inhibited both DHA- and 19,20-EDP-induced inhibitory effect. There is no effect on 19,20-EDP-induced cell death under normal glucose enzyme activities under low glucose condition (Fig. 3f, h). conditions (Fig. 2a), we next examined the effect of DHA and 19,20-EDP treatment toward cellular ceramide levels. DHA and 19,20-EDP cause lysosome-dependent cell death The basal ceramide content in subcellular lysosomal/ under high glucose conditions mitochondrial fractions isolated from cells cultured under Lysosomes are unique membrane-bound organelles 25 mM glucose conditions was much lower than mem- found in the cytosol that contain degradative hydrolytic brane fractions (Table 1). Under 25 mM glucose condi- enzymes involved in digestion of many biomolecules. tions, both DHA and 19,20-EDP significantly increased Lysosomal-dependent cell death has been classified as a the amount of ceramide found in membrane fractions. pattern initiated by perturbations of intracellular home- However, both DHA and 19,20-EDP treatment resulted in ostasis and the permeabilization of lysosomal membranes significant increases of ceramide content in lysosomal and releasing proteases. In order to investigate the role of lysosomes we used a fluorescent LysoTracker Green dye to visualize the organelles under an epifluorescent Table. 1 De novo ceramide synthesis in H9c2 cells microscope (Fig. 4A, B), as well as quantify the intensity with a fluorescent-based plate reader assay (Fig. 4C, D). Crude membrane Lysosome/mitochondria Treating H9c2 cells cultured in either 25 mM or 5.5 mM glucose media with bafilomycin, as a positive control NG LG NG LG promoting acidification and maturation of lysosomes, resulted in marked increase lysosome formation (Fig. 4Ab, Control 32.81 ± 1.9 14.27 ± 0.62 1.45 ± 1.27 1.14 ± 0.79 Bb, c). Interestingly, both DHA and 19,20-EDP increased DHA 54.26 ± 1.73* 20.85 ± 0.87 470.73 ± 15.94* 0.25 ± 0.09 the intensity of LysoTracker Green fluorescence (Fig. 4Ac, 19,20-EDP 50.50 ± 4.63* 16.44 ± 0.52 601.73 ± 23.9* 0.40 ± 0.21 Ad) compared with control (Fig. 4Aa, C) under normal † † 19,20-EDP 18.23 ± 7.57 11.78 ± 0.24 64.20 ± 63.85 0.37 ± 0.22 glucose conditions, while inhibiting ceramide synthesis +myriocin with myriocin inhibited 19,20-EDP-increased intensity (Fig. 4Ag, C). DHA- and 19,20-EDP-induced intensity was LC/MS analysis was employed to measure ceramide levels in H9c2 cells cultured in either normal (25 mM) or low (5.5 mM) glucose-containing media reduced by MG-132 treatment (Fig. 4Ai, Aj, C). Con- Ceramide content in crude membrane and lysosomal/mitochondria fractions versely, addition of DHA or 19,20-EDP to H9c2 cells were assessed. Cells were treated with or without DHA (100 μΜ), 19,20-EDP incubated in low glucose conditions had the opposite (1 μΜ) and myriocin (1 μΜ) for 24 h Values are expressed as mean ± SEM (n = 4) effect with a significantly decreased level of lysosomal *P < 0.05 vs. control P < 0.05 vs. 19.20-EDP (NG) intensity (Fig. 4B, D). Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 6 of 11 Fig. 3 DHA and 19,20-EDP inhibit mitochondrial enzyme activities under glycolytic conditions. H9c2 cells were cultured in either 25 mM (NG) or 5.5 mM (LG) glucose-containing media. H9c2 cells were treated with or without DHA (100 μM), 19,20-EDP (1 μM), MSPPOH (50 μM) and/or myriocin (1 μM) for 24 h. Representative immunoblots and histograms of relative densities of a, b citrate synthase and c, d cytochrome c oxidase protein expression. Enzyme activities of e, f citrate synthase and g, h cytochrome c oxidase. Values are expressed as mean ± SEM (n = 3). *P < 0.05 vs. untreated control, P < 0.05 vs. 19,20-EDP alone Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 7 of 11 Fig. 4 DHA and 19,20-EDP induced lysosomal formation and activity under glycolytic conditions. H9c2 cells were cultured in either 25 mM (NG) or 5.5 mM (LG) glucose-containing media. H9c2 cells were treated with or without DHA (100 μM), 19,20-EDP (1 μM), MSPPOH (50 μM), bafilomycin (1 μM), myriocin (1 μM) and/or MG1-32 (1 μM) for 24 h and then stained with mitochondrial dye, TMRE (100 nm, red), lysosomal dye, LysoTracker (10 nm, green) and nuclear dye, Hoechest33342 (1 mM, blue). Representative images of mitochondrial and lysosomal organelles in H9c2 cells cultured in a 25 mM (NG) or b 5.5 mM (LG) glucose media. Histograms representing the relative quantification of lysosomal expression in H9c2 cells cultured in c 25 mM (NG) or d 5.5 mM (LG) glucose media. Values are expressed as mean ± SEM (n = 3). *P < 0.05 vs. untreated control, P < 0.05 vs. respective group untreated MG132 Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 8 of 11 Discussion sphingolipid, ceramide, has important roles in cellular In the current study we demonstrate that DHA and membrane structure and is an integral component of the 19,20-EDP selectively induce cell death in non- sphingomyelin cycle acting as an essential second mes- differentiated H9c2 cells with a developed glycolytic senger in the intracellular propagation of physiological, profile. It is well documented that CYP epoxygenases pharmacological and environmental signals in normal and catalyze the enzymatic transformation of PUFA into var- cancerous cells . Ceramide elicits many cell-stress ious potent epoxylipid including omega-3 epoxides of responses including apoptosis, senescence, inflammation, DHA referred as EDPs. Recent evidence suggests that mitochondrial dysfunction and altered cellular metabo- 17,26,27 EDPs are involved in regulating cellular processes such as lism . The hydrophobic nature of ceramide limits its inflammation, angiogenesis and cell death; however, our accumulation to membranous regions within the cell understanding of how EDPs regulate these effects remains where it may influence protein distribution and function. significantly limited. Previously, we have demonstrated In this regard, cytosolic concentrations of ceramide are immortalized H9c2 cells were prone to cytotoxicity negligible. While the majority of studies evaluating the caused by DHA and 19,20-EDP. The current study effects of an elevation in cellular ceramide have focused demonstrated the cytotoxicity of EDPs was dependent on its ability to induce apoptosis , several other studies upon the metabolic profile of the cells. have also described ceramide to be necessary for the Interestingly, an increased de novo synthesis of cer- induction of senescence, cell–cell interactions, death 25,28–33 amide occurs following treatment DHA and 19,20-EDP, receptor clustering and autophagy . Collectively, with a corresponding accumulation in intracellular these studies emphasize that ceramide is capable of 17,25,28–34 membrane fractions including mitochondria and lyso- modulating several biochemical pathways . Stra- somes, while H9c2 cells driven to demonstrate an tegies that pharmacologically or genetically decrease cer- OXPHOS phenotype were not susceptible to DHA- or amide have beneficial effects in reversing insulin 19,20-EDP-mediated cell death, or changes in intracel- resistance, preventing apoptosis of pancreatic β-cells and 28–30 lular ceramide production. Altered levels of endogenous cardiomyocytes . lipids, such as ceramide, are well known to be involved in Accumulation of ceramide has been linked to major both cell survival and cell death processes. Together, our perturbations in cell metabolism resulting in apoptosis, data suggest a novel cell death mechanism for DHA and necroptosis and lethal autophagy including mito- 15,28,31,33 19,20-EDP lipids, which occurs in non-differentiated phagy . Ceramide accumulation in mitochondria glycolytic cells. Noteworthy, during the last decade, stu- can lead to stress-induced mitochondrial fragmentation dies dedicated to biological effects of DHA have largely and decreased ATP production through disruption of ignored the roles of CYP-derived metabolites. In this electron transport chain and induction of injury, resulting study, we demonstrated that DHA produced cytotoxic in a pool of dysfunctional mitochondria capable of pro- 31,32 effect in glycolytic H9c2 cells similar to what was moting cell death . We found that both DHA and observed with 19,20-EDP. Furthermore, formation of 19,20-EDP induced ceramide accumulation in mito- endogenous EDPs has an essential role in DHA- asso- chondrial and lysosomal fractions, which was diminished ciated injury of glycolytic H9c2 cells. Since cytotoxic in the presence of myriocin. Moreover, the increased de effects of DHA toward glycolytic H9c2 cells were strongly novo synthesis of ceramide and its accumulation in sub- decreased when MSPPOH, a CYP epoxygenase inhibitor, cellular membrane fractions occurred under glycolytic was added, we presume that observed effects were asso- conditions and requires increased NADH reduction, ciated with EDPs endogenously produced from DHA. which was markedly reduced under OXPHOS . The One of the main metabolic divergences between quiescent increased production of ceramide correlated with elevated and proliferating cells is the pathway responsible for ATP lysosomal activity and was reversed by the proteasome synthesis. In non-cancerous cells with hallmarks of inhibitor MG-132, which additionally support the role of primary lines, ATP is usually synthesized during ceramide formed in glycolytic conditions in various types OXPHOS, whereas cancerous and highly proliferative of cell death . Detection of lysosomal-mediated cell death cells preferentially develop glycolysis for ATP synthesis originally was difficult, as lysosomal ultrastructure often and employ mitochondria solely to sustain redox appears intact in apoptotic cells analyzed by electron potential required to reduce NADH for glycolysis. This is microscopy . Partial lysosomal membrane permeabiliza- known as the Warburg effect that results in formation of a tion can occur early in many death paradigms, which pool of aberrant mitochondria with poor functional triggers proteasomal cell death . Interestingly, increases 23,24 activity . in ceramide levels have been shown to lower the integrity While our previous research correlated DHA-mediated of lysosomal membranes and hinders their fusion with cell death in H9c2 cells with ceramide production, the other intracellular vesicles including mitochondria .In current data demonstrated unique mechanisms . The the current study, accumulation of ceramide in lysosomal Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 9 of 11 fractions following treatment with DHA or 19,20-EDP LC/MS measurement of different pools of intracellular may have contributed to destabilization of lysosomal ceramide membranes and the subsequent increase in proteasomal The extraction of ceramide from H9c2 cells was per- and lysosomal activities. Early studies using compounds formed in accordance to a published protocol . Cells directly targeting the integrity of lysosomal membranes were cultured in 175 cm flasks and then harvested and provided evidence for their role in programmed cell subjected to subcellular fractionation. Cells were cen- 39–41 death . Moreover, inhibitors of lysosomal function trifuged at 700 × g for 10 min and then rinsed with cold would be more toxic to cancer and transformed cells than phosphate-buffered saline (PBS) and centrifuged again at normal cell lines . A quantitative relationship between 700 × g for 10 min, 4 °C. The resulting pellet was termed the amount of lysosomal rupture and the mode of cell and used as a crude membrane fraction. For the enrich- death has been suggested to explain the widely different ment of heavy membrane fraction containing light and morphological outcomes following lysosomal membrane heavy mitochondria as well as lysosomes, the remaining permeabilization . According to this model, low stress supernatant was centrifuged at 10,000 × g for 20 min, 4 °C. intensities trigger a limited release of lysosomal contents Then, 300 mL of a 0.4% NaCl solution and 1 mL of a to the cytoplasm followed by cell death, while high chloroform–methanol–HCl 1 N (100:100:1, v/v/v) mix- intensity stress factors lead to a generalized lysosomal ture were added to the samples. Following this, the rupture and rapid cellular necrosis. It is entirely possible samples were vortexed at 1000 rpm (room temperature) that DHA and 19,20-EDP induced accumulation of cer- for 20 min. Ceramide content in the samples was analyzed amide in H9c2 cells with glycolytic profile, which insti- using Waters ZQ 4000 Mass Spectrometer coupled to a gated cell death. Remarkably, co-treatment with MG-132, Water 2795 Separations Module. A previously described an inhibitor of lysosomal proteases, decreased toxicity of liquid chromatography/mass spectrometry (LC/MS) DHA and 19,20-EDP. method was utilized with modifications . Briefly, cer- In conclusion, we report a novel cell death mechanism amide as well as the internal standard, 4-methox- for DHA and its epoxylipid, 19,20-EDP, in non- ybenzophenone, were resolved using a reverse-phase C18 differentiated glycolytic H9c2 cells. Our study suggests column (Alltima HP, 150 × 2.1 mm) at 35 °C by the iso- these lipid mediators induce a ceramide-mediated process cratic elution of methanol–tetrahydrofuran–water–acetic that increases lysosomal-proteasome activity impacting acid (80:10:10:0.2, v/v/v/v) as the mobile phase. Ceramide mitochondria function leading to cell death. An increased and 4-methoxybenzophenone were detected using a sin- production of ceramide coupled with increased lysosomal gle ion recording under positive-ion mode at m/z = 342.4 membrane permeability in glycolytic cells causes a and 213.1, respectively. The cone voltage was 25 V and destabilization of mitochondria resulting in cell death. 15 V for ceramide and 4-methoxybenzophenone, respec- The shift in cellular metabolic profile from glycolytic to tively, and capillary voltage of 4.0 kV. During the acqui- OXPHOS highlights a novel toxicity of DHA and 19, 20- sition of the data, the mass spectrometer was maintained EDP toward H9c2 cells with a glycolytic profile. at a source and desolation temperature of 120 °C and 275 °C, respectively. The resulting values were then cal- Materials and methods culated using a ceramide standard curve and expressed as Cell culture and viability assay mg weight per mg of cellular mass, and then further H9c2 cardiac cells were obtained from ATCC. The cells normalized to account for the fold change between wet were cultured in DMEM medium containing either 25 and dry cellular mass for quantification purposes. mM (normal glucose) or 5.5 mM glucose (low glucose) and supplemented with 10% FBS and 1% penicillin/ Assessment of mitochondrial function streptomycin. Differentiation of H9c2 myoblasts into Cellular ATP was determined using a kit based on myotubes was performed in DMEM supplemented with fluorescent assay (Abcam or Sigma-Aldrich). Citrate 1% FBS, 1% penicillin/streptomycin and 10 nM retinoic synthase (CS) and cytochrome c oxidase enzymatic acid for 2 weeks. Cell viability was assessed using a activities were assayed spectrophotometrically in cell 46,43 commercially available kit from Promega based on lumi- lysates as previously described . Rapid assessment of nescent assay of intracellular proteases. Another approach oxygen consumption was performed using a kit based on to measure metabolically active cells was MTT test per- detection of oxygen with fluorescent trap (Abcam). NAD/ formed as previously described . The intensity of NADH ratio was assessed using a kit from Promega. reduction of MTT to formazan crystals by mitochondrial Mitochondrial respiration was measured in saponin per- dehydrogenases positively correlates with the overall meabilized HL-1 cells using Clark oxygen electrode con- activity of oxidative metabolism. Optical density of nected to Oxygraph Plus recorder (Hansatech dimethyl sulfoxide extracted from formazan was mea- Instruments Ltd, Norfolk, England). Respiration rates sured spectrophotometrically at 595 nm. were measured at 30 °C before and after addition of 2 mM Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 10 of 11 ADP with 5 mM malate and 10 mM glutamate as sub- Author details Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, strates. RCR was calculated as the ratio between basal and Edmonton, AB, Canada. Department of Pharmacological Sciences, School of ADP-stimulated respiration rates . Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido, Japan. Department of Clinical Pharmacy, Faculty of Pharmacy, Tanta University, 8130 Tanta, Egypt. Department of Pharmacology, Faculty of Immunoblotting Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Samples were subjected to western blot analysis as 7,20 previously described . Briefly, 25 μg of protein from Conflict of interest The authors declare that they have no conflict of interest. H9c2 lysates was probed on a 12% SDS-polyacrylamide gel. Membranes were washed four times with Tris- buffered saline with Tween-20 buffer and then incu- Publisher's note bated with primary antibodies, CS (1:000, Abcam., Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cambridge, UK, Cat No: ab129095), cytochrome c oxidase (COX IV) (1:1,000, Cell Signaling Tech., Inc., Whitby, ON, Cat No: 4850) and GAPDH (1:5000, Cell Signaling Received: 17 July 2018 Accepted: 24 July 2018 Tech., Inc., Whitby, ON, Cat No: 5174S), secondary antibodies were used as 1:5000 dilution. Relative band intensity to control was measured using Image J software References (NIH, USA). 1. Wang, C. et al. n-3 Fatty acids from fish or fish-oil supplements, but not alpha- linolenic acid, benefit cardiovascular disease outcomes in primary- and Assessment of lysosomal activity secondary-prevention studies: a systematic review. Am. J. Clin. Nutr. 84,5–17 (2006). Lysosomal activity was measured and quantitated using 2. Mori, T. A. Dietary n-3 PUFA and CVD: a review of the evidence. Proc. Nutr. Soc. LysoTracker Green DND-26 which specifically stains 73,57–64 (2014). acidic structures in live cells. H9c2 cells were treated with 3. Samokhvalov, V. et al. PPARgamma signaling is required for mediating EETs protective effects in neonatal cardiomyocytes exposed to LPS. Front. Phar- LysoTracker Green DND-26 added to a final concentra- macol. 5, 242 (2014). tion of 10 nM for 1 h. The cells were rinsed with PBS and 4. Arnold, C. et al. Arachidonic acid-metabolizing cytochrome P450 fluorescence was measured with excitation/emission 504/ enzymes are targets of {omega}-3 fatty acids. J. Biol. Chem. 285, 32720–32733 (2010). 511 nm. The intensity of fluorescence reflected the active 5. Zhang, G., Kodani, S. & Hammock, B. D. Stabilized epoxygenated fatty acids pool of lysosomes. MG-132, which acts as a potent cell- regulate inflammation, pain, angiogenesis and cancer. Prog. Lipid Res. 53, permeable inhibitor of lysosomal activity, was added to a 108–123 (2014). 6. Jung, F. et al. Effect of cytochrome P450-dependent epoxyeicosanoids on final concentration of 1 µM where indicated. Ristocetin-induced thrombocyte aggregation. Clin. Hemorheol. Microcirc. 52, 403–416 (2012). Microscopy 7. Samokhvalov, V.,Jamieson, K. L.,Vriend, J.,Quan, S.,& Seubert, J. M. CYP- epoxygenase metabolites of docosahexaenoic acid protect HL-1 cardiac cells H9c2 cells were grown on glass-bottom 35 mm dishes against LPS-induced cytotoxicity through SIRT1.Cell Death Discov. 1, 15054 suitable for fluorescent microscopy (MatTek) and treated (2015). with indicated reagents. Mitochondria were stained with 8. Fasano, E. et al. DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines. Biochim. Biophys. Acta 1822, TMRE (100 nM) and nucleus were stained with Hoech- 1762–1772 (2012). est33342 (1 mM). Acidic organelles were stained with 9. Kim, H. K., Della-Fera, M.,Lin,J.& Baile, C. A. Docosahexaenoic acid inhibits LysoTracker Green DND-26 (10 nM). The dishes were adipocyte differentiation and induces apoptosis in 3T3-L1 preadipocytes. J. Nutr. 136, 2965–2969 (2006). placed in a micro incubator installed on the objective stage 10. Schunck, W. H., Konkel, A., Fischer, R. & Weylandt, K. H. Therapeutic potential of of a Zeiss Axio Observer epifluorescence microscope. The omega-3 fatty acid-derived epoxyeicosanoids in cardiovascular and inflam- image system consisted of Zeiss Axio Observer Z1 inverted matory diseases. Pharmacol. Ther. 183,177–204 (2018). 11. Kimes,B. W.& Brandt,B.L.Propertiesofa clonalmusclecelllinefromrat heart. epifluorescence microscope equipped with a LED lamp, 63× Exp. Cell Res. 98,367–381 (1976). oil immersion objective lens and cooled CCD camera. 12. Hescheler, J. et al. Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ. Res. 69, 1476–1486 (1991). Statistical analysis 13. Sardao, V. A., Oliveira, P. J., Holy, J., Oliveira, C. R. & Wallace, K. B. Vital imaging of Values were expressed as mean ± standard error of mean. H9c2 myoblasts exposed to tert-butylhydroperoxide--characterization of Statistical significance was determined using one-way ana- morphological features of cell death. BMC Cell. Biol. 8,11 (2007). 14. Menard, C. et al. Modulation of L-type calcium channel expression during lysis of variance. To determine whether significant differ- retinoic acid-induced differentiation of H9C2 cardiac cells. J. Biol. Chem. 274, ences exist between the groups, Bonferonni post-hoc test was 29063–29070 (1999). performed. Values were considered significant as p < 0.05. 15. Pereira, S. L. et al. Metabolic remodeling during H9c2 myoblast differentiation: relevance for in vitro toxicity studies. Cardiovasc. Toxicol. 11,180–190 (2011). 16. Marroquin, L. D., Hynes, J., Dykens, J. A., Jamieson, J. D. & Will, Y. Circumventing Acknowledgements the Crabtree effect: replacing media glucose with galactose increases sus- This work was partially supported by an operating grant from the Canadian ceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97,539–547 Institutes of Health Research (to J.M.S.) and the Taiho Fund from the Faculty of (2007). Pharmacy and Pharmaceutical Sciences, University of Alberta (to T.E. and J.M.S.). Official journal of the Cell Death Differentiation Association Endo et al. Cell Death Discovery (2019) 5:29 Page 11 of 11 17. Bikman, B. T. & Summers, S. A. Ceramides as modulators of cellular and whole- 33. Scarlatti, F. et al. Ceramide-mediated macroautophagy involves inhibition of body metabolism. J. Clin. Invest. 121,4222–42300 (2011). protein kinase B and up-regulation of beclin 1. J. Biol. Chem. 279,18384–18391 18. He, X. &Schuchman,E. H.Ceramideand ischemia/reperfusion injury. J. Lipids (2004). 2018, 3646725 (2018). 34. Lu, C. Y. et al. Docosahexaenoic acid down-regulates phenobarbital-induced 19. Zhang, T., Barclay, L., Walensky, L. D. & Saghatelian, A. Regulation of mito- cytochrome P450 2B1 gene expression in rat primary hepatocytes via the chondrial ceramide distribution by members of the BCL-2 family. J. Lipid Res. sphingomyelinase/ceramide pathway. J. Nutr. Biochem. 21,338–344 (2010). 56,1501–1510 (2015). 35. Arboleda, G. et al. Insulin-like growth factor-1-dependent maintenance of 20. Qadhi, R. et al. Differential responses to docosahexaenoic acid in primary and neuronal metabolism through the phosphatidylinositol 3-kinase-Akt pathway immortalized cardiac cells. Toxicol. Lett. 219, 288–297 (2013). is inhibited by C2-ceramide in CAD cells. Eur. J. Neurosci. 25,3030–3038 21. Samokhvalov, V. et al. PPARdelta signaling mediates the cytotoxicity of DHA in (2007). H9c2 cells. Toxicol. Lett. 232,10–20 (2015). 36. Okazaki, T. & Okazaki, I. M. [Regulation of autoimmunity and tumor immunity 22. Coux, O., Tanaka, K. & Goldberg, A. L. Structure and functions of the 20S and by immunoinhibitory co-receptor, PD-1]. Seikagaku 87,693–704 (2015). 26S proteasomes. Annu. Rev. Biochem. 65, 801–847 (1996). 37. de Duve, C. Lysosomes revisited. Eur. J. Biochem. 137,391–397 (1983). 23. Schwartz, L.,Buhler, L.,Icard,P., Lincet,H.&Steyaert,J.M.Metabolic treatment 38. Appelqvist, H., Waster, P., Kagedal, K. & Ollinger, K. The lysosome: from waste of cancer: intermediate results of a prospective case series. Anticancer Res. 34, bag to potential therapeutic target. J. Mol. Cell Biol. 5, 214–226 (2013). 973–980 (2014). 39. Chiong, M. et al. Cardiomyocyte death: mechanisms and translational impli- 24. Zhang, S. et al. Homeostasis of redox status derived from glucose metabolic cations. Cell Death Dis. 2, e244 (2011). pathway could be the key to understanding the Warburg effect. Am.J.Cancer 40. Terman, A., Kurz, T., Gustafsson, B. & Brunk, U. T. The involvement of lysosomes Res. 5,1265–1280 (2015). in myocardial aging and disease. Curr. Cardiol. Rev. 4,107–115 (2008). 25. Ogretmen, B. & Hannun, Y. A. Biologically active sphingolipids in cancer 41. Tsubone,T.M.etal. Enhanced efficiency of cell death by lysosome-specific pathogenesis and treatment. Nat. Rev. Cancer 4, 604–616 (2004). photodamage. Sci. Rep. 7, 6734 (2017). 26. Stathem, M. et al. Glucose availability and glycolytic metabolism dictate gly- 42. Boya, P. & Kroemer, G. Lysosomal membrane permeabilization in cell death. cosphingolipid levels. J. Cell. Biochem. 116,67–80 (2015). Oncogene 27,6434–6451 (2008). 27. Morad, S. A. & Cabot, M. C. Ceramide-orchestrated signalling in cancer cells. 43. Samokhvalov, V. et al. Epoxyeicosatrienoic acids protect cardiac cells during Nat. Rev. Cancer 13,51–65 (2013). starvation by modulating an autophagic response. Cell Death Dis. 4,e885 28. Jiang, W. & Ogretmen, B. Ceramide stress in survival versus lethal autophagy (2013). paradox: ceramide targets autophagosomes to mitochondria and induces 44. Bose, R. & Kolesnick, R. Measurement of ceramide levels by the diacylglycerol lethal mitophagy. Autophagy 9,258–259 (2013). kinase reaction and by high-performance liquid chromatography- 29. Young,M.M., Kester,M. & Wang, H. G.Sphingolipids: regulators of crosstalk fluorescence spectrometry. Methods Enzymol. 322,373–378 (2000). between apoptosis and autophagy. J. Lipid Res. 54,5–19 (2013). 45. Sahle,F. F., Lange, S., Dobner,B., Wohlrab, J. & Neubert,R.H. Development and 30. Park, T. S. et al. Ceramide is a cardiotoxin in lipotoxic cardiomyopathy. J. Lipid validation of LC/ESI-MS method for the detection and quantification of exo- Res. 49, 2101–2112 (2008). genous ceramide NP in stratum corneum and other layers of the skin. J. 31. Sentelle, R. D. et al. Ceramide targets autophagosomes to mitochondria and Pharm. Biomed. Anal. 60,7–13 (2012). induces lethal mitophagy. Nat. Chem. Biol. 8, 831–838 (2012). 46. Spinazzi,M., Casarin, A.,Pertegato,V., Salviati,L.&Angelini, C. Assessment of 32. Dany, M. & Ogretmen, B. Ceramide induced mitophagy and tumor suppres- mitochondrial respiratory chain enzymatic activities on tissues and cultured sion. Biochim. Biophys. Acta 1853,2834–2845 (2015). cells. Nat. Protoc. 7,1235–1246 (2012). Official journal of the Cell Death Differentiation Association

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