Role of sphingomyelinase in mitochondrial ceramide accumulation during reperfusion

Biochimica et Biophysica Acta 1862 (2016) 1955–1963

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Role of sphingomyelinase in mitochondrial ceramide accumulation during reperfusion I. Ramírez-Camacho a,1, R. Bautista-Pérez b,1, F. Correa a, M. Buelna-Chontal a, N.G. Román-Anguiano a, M. Medel-Franco a, O.N. Medina-Campos c, J. Pedraza-Chaverri c, A. Cano-Martínez d, C. Zazueta a,⁎ a

Department of Cardiovascular Biomedicine, National Institute of Cardiology, I. Ch. 14080, Mexico City, Mexico Department of Molecular Biology, National Institute of Cardiology, I. Ch. 14080, Mexico City, Mexico c Department of Biology, Faculty of Chemistry, National Autonomous University of Mexico (UNAM), University City, 04510 Mexico City, DF, Mexico d Department of Physiology, National Institute of Cardiology, I. Ch., 14080, Mexico City, Mexico b

a r t i c l e

i n f o

Article history: Received 19 April 2016 Received in revised form 25 July 2016 Accepted 27 July 2016 Available online 30 July 2016 Keywords: Ceramide Mitochondria Sphingomyelinase

a b s t r a c t Ceramide accumulation in mitochondria has been associated with reperfusion damage, but the underlying mechanisms are not clearly elucidated. In this work we investigate the role of sphingomyelinases in mitochondrial ceramide accumulation, its effect on reactive oxygen species production, as well as on mitochondrial function by using the sphingomyelinase inhibitor, tricyclodecan-9-yl-xanthogenate (D609). Correlation between neutral sphingomyelinase (nSMase) activity and changes in ceramide content were performed in whole tissue and in isolated mitochondria from reperfused hearts. Overall results demonstrated that D609 treatment attenuates cardiac dysfuncion, mitochondrial injury and oxidative stress. Ceramide was accumulated in mitochondria, but not in the microsomal fraction of the ischemic-reperfused (I/R) group. In close association, the activity of nSMase increased, whereas glutathione (GSH) levels diminished in mitochondria after reperfusion. On the other hand, reduction of ceramide levels in mitochondria from I/R + D609 hearts correlated with diminished nSMase activity, coupling of oxidative phosphorylation and with mitochondrial integrity maintenance. These results suggest that mitochondrial nSMase activity contributes to compartmentation and further accumulation of ceramide in mitochondria, deregulating their function during reperfusion. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Ceramide is a key sphingolipid messenger that regulates cellular responses to stress including changes in cell cycle, differentiation, senescence and apoptosis by modifying the physical properties of the cellular membranes and by interacting with signal transduction molecules [1]. In heart, increased cell death via ceramide-induced apoptosis is a common feature between heart failure, lipotoxicityinduced dilated cardiomyopathy and ischemia–reperfusion (I/R) [2–4]. There are also evidences that the accumulation of ceramide depresses cardiac contractility through activation of protein kinase C (PKC) and

Abbreviations: D609, tricyclodecan-9-yl-xanthogenate; nSMase, neutral sphingomyelinase; DP, double product; 4′,6-Diamidino-2-phenylindole, DAPI; 4-HNE, 4hydroxynonenal; DNPH, dinitrophenyl hydrazine; mCB, mono-chloro-bimane; TMB, 3,3′,5,5′-tetramethylbenzidine; TPP, tetraphenylphosphonium; CCCP, carbonyl cyanide m-chlorophenylhydrazone; ANT, adenine nucleotide translocator; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; RC, respiratory control; Δψm, mitochondrial transmembrane electric potential; cyt c, cytochrome c. ⁎ Corresponding author at: Department of Cardiovascular Biomedicine, National Institute of Cardiology, I. Ch., Juan Badiano No. 1, Col. Sección XVI, Tlalpan, Mexico, D.F. 14080, Mexico. E-mail address: [email protected] (C. Zazueta). 1 These authors contribute equally to this work.

http://dx.doi.org/10.1016/j.bbadis.2016.07.021 0925-4439/© 2016 Elsevier B.V. All rights reserved.

subsequent phosphorylation of myofilament proteins in isolated cardiomyocytes [5]. Ceramide, is generated by de novo synthesis pathway in the endoplasmic reticulum (ER). The first reaction, catalyzed by serine palmitoyltransferase (SPT) produces 3-ketosphinganine from L-serine and palmitoyl CoA. The product is first reduced to sphinganine and then acylated at the amide group by dihydroceramide synthase (CerS) forming dihydroceramide. Most dihydroceramides are immediately converted to ceramides by the introduction of a characteristic 4,5-double bond in the sphingoid base of the molecule. Once ceramide is synthesized in the ER, it is carried to the Golgi apparatus and converted to sphingomyelin (SM) by sphingomyelin synthase (SMS) [6]. Golgisynthesized SM is directed to the plasma membrane through vesicular trafficking and hydrolyzed to ceramide and phosphorylcholin by sphingomyelinases (SMases), which are regulated by anionic phospholipids [7] and oxidative stress [8]. SMase localization is a highly dynamic and regulated process, that might be important for compartmentalized ceramide production [9]. The participation of SMases in stress-induced ceramide production is well established [10]; in particular, it has been reported that the neutral isoform regulates ceramide increase in cardiomyocytes subjected to hypoxia and reoxygenation [11]. Accordingly, we and others have shown that the SMase inhibitors tricyclodecan-9-yl-xanthogenate (D609) [12] and disipramine confer

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I. Ramírez-Camacho et al. / Biochimica et Biophysica Acta 1862 (2016) 1955–1963

cardioprotection against reperfusion injury [13]. Ceramide accumulation in mitochondria has been associated with reperfusion damage mainly due to the observation that the addition of this sphingolipid to isolated mitochondria, mimetizes some of the events observed in these organelles after I/R, e.g. increased outer membrane permeability [14] and electron transport chain inhibition [15]. In this regard, it is well established that both short and long chain ceramides diminish oxygen consumption in heart mitochondria by inhibiting complex III activity in vitro [15,16] and increase reactive oxygen species (ROS) production [17]. Although enhanced ROS generation is a hallmark in reperfusion damage, it is not clear which of the two-way interaction between ceramide and oxidant production perpetuates mitochondrial dysfunction. On one hand, inhibition of electron transport chain by ceramide increases ROS production; whereas in turn, ROS might modulate the enzymatic activities of SMases and ceramidase [18,19] stimulating the generation of sphingolipid metabolites and producing a “vicious cycle”, that magnifies cardiac damage. Besides the “in vitro” evidence, we have previously shown that ceramide is accumulated in mitochondrial membrane microenvironments in association with increased Bax docking into mitochondria from reperfused hearts [12]. Following the observation that the insertion of this pro-apoptotic protein to the mitochondrial membranes was diminished by D609 pretreatment, in this work we have focused on the role of the SM pathway in mitochondrial ceramide accumulation, and its effect on ROS production and on mitochondrial function in reperfused hearts. 2. Material and methods This investigation was approved by the Ethics Committee of the National Institute of Cardiology, “Ignacio Chávez” and the experimental protocols followed the guidelines of Norma Oficial Mexicana for the use and care of laboratory animals (NOM-062-ZOO-1999) and for disposal of biological residues (NOM-087-SEMARNAT-SSA1-2002). Chemicals were of reagent or higher grade from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. 2.1. Ischemia and reperfusion in isolated hearts Rats (400–450 g) were anaesthetized with sodium pentobarbital (60 mg/kg) and complete lack of pain response was assessed by determining pedal withdrawal reflex. Then, sodium heparin was injected (1000 U/kg) and five min later a midsternal thoracotomy was performed. The heart was rapidly excised and placed in frozen KrebsHenseleit buffer solution at pH 7.4, containing 118 mM NaCl, 4.75 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4·7H2O, 2.5 mM CaCl2, 25 mM NaHCO3, 4.3 mM glucose, and 0.1 mM sodium octanoate for few seconds, then it was mounted onto a Langendorff heart perfusion system. Hearts (average weight = 1 g) were perfused retrogradely via the aorta at a constant flow rate of 12 mL/min with Krebs-Henseleit solution, which was continuously bubbled with 95% O2 and 5% CO2 at 37 °C. All hearts were stabilized during 20 min. Control hearts were continuously perfused during 110 min; ischemia and reperfusion (I/R) consisted in 30 min of ischemia, followed by 60 min of reperfusion. A group of rats were injected with an intravenous bolus of the sphingomyelinase inhibitor, tricyclodecan-9-yl-xanthogenate (D609, 0.1 mg/kg) 10 min before the I/R protocol, as previously reported [12]. This xanthate compound has been used to inhibit sphingomyelinases (SMase) and other enzymes that regulate ceramide levels [20–22]. Cardiac performance was measured at left ventricular end-diastolic pressure (LVEDP) of 10 mm Hg using a latex balloon inserted into the left ventricle and connected to a pressure transducer as previously reported [23]. Throughout the experiment, left ventricular developed pressure (LVDP) was continuously recorded using a computer acquisition data system designed by the Instrumentation and Technical Development Department of the National Institute of Cardiology (Mexico, D.F., Mexico). Heart rate (HR) expresses beat number per min and

cardiac contractile function was calculated by subtracting LVEDP from left ventricular peak systolic pressure (LVSP), yielding LVDP. The double product (DP) was calculated by multiplying HR by LVDP. 2.2. Infarct size measurement The hearts to be used for infarct size calculations were frozen at − 20 °C for 3 h. Later, the hearts were cut into approximately 3-mm slices visually and immersed in 1% triphenyltetrazolium chloride solution in phosphate buffer, pH 7.4, for 20 min at 37 °C. The samples were incubated in a solution of formalin for 5 min, placed between two glasses separated by a fixed 2-mm distance and scanned on a Hewlett-Packard Scanjet 3800 scanner (Hewlett-Packard). The infarcted area was displayed as the area unstained by triphenyltetrazolium chloride. Infarct size was expressed as a percentage of the area at risk (IS/AAR%). 2.3. Active caspase-3 analysis At the end of the protocols, some hearts were frozen and stored in liquid nitrogen. Cardiac tissue was powdered with a pre-chilled pestle in a frozen mortar and dissolved in ice-cold lysis buffer containing 10 mM Tris-HCl, 0.1 M EDTA and 0.5% sodium dodecyl sulfate (SDS), pH 8.0 plus protease inhibitors cocktail (SIGMAFAST™) and centrifuged at 4000 g for 10 min. The proteins in the homogenates (50 μg of protein) were loaded under reducing conditions onto 15% resolving SDSpolyacrylamide gels, separated by polyacrylamide gel electrophoresis (PAGE) at 140 V for 3.5 h and equilibrated for 15 min in 25 mM Tris– HCl, pH 8.3, containing 192 mM glycine and 20% (v/v) methanol. Electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) and incubated at room temperature for 1 h in Tris-based saline + Tween 20 (TBS-T) blocking buffer containing 5% defat milk. Antibodies against caspase-3 p20 subunit and full length procaspase-3 (sc-1226; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted to 1:500 and incubated overnight. The immunoblots were washed three times in TBS-T buffer for 15 min and then incubated with 1:12,500 donkey anti-goat IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The immunoblots were then washed in TBS-T during 15 min, three times. The immunoblotted proteins were visualized using an enhanced chemiluminescence (ECL) western blotting luminal reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 2.4. Activity of neutral and acid sphingomyelinases in reperfused hearts At the end of the protocols, some hearts were frozen and stored in liquid nitrogen. Cardiac tissue was powdered with a pre-chilled pestle in a frozen mortar and dissolved in ice-cold buffer containing 50 mM Tris-HCl, 120 mM NaCl, 0.5% IGEPAL, 100 μM NaF, 200 μM NaVO3, pH 8.0, and centrifuged at 4000 g for 10 min. Heart homogenates (20 μg protein) were incubated in 100 mM Tris, pH 7.4 buffer, containing 100 mM MgCl2, 100 μM Amplex red, 2 U/mL horseradish peroxidase, 0.2 U/mL choline oxidase, 8 U/mL alkaline phosphatase and 500 μM sphingomyelin in a final volume of 200 μL at 37 °C, as previously described [24]. After 30 min, the increase in fluorescence was measured using a microplate reader (Laurier Research Instrumentation Inc. Otego, NY, USA) at λex = 545 nm and λem = 590 nm. Acid sphingomyelinase activity was measured at pH 5.0. 2.5. Activity of phospholipase C (PC-PLC) in reperfused hearts The activity of PC-PLC was measured using the amplex Red phosphatidylcholine-specific phospholipase C assay (Molecular Probes). Each reaction mixture contained 200 μM Amplex Red reagent, 1 U/mL HRP, 4 U/mL alkaline phosphatase, 0.1 U/mL choline oxidase, 0.5 mM

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phosphatidylcholine (lecithin) and 20 μg of protein in reaction buffer (50 mM Tris-HCl, pH 7.4, 140 mM NaCl, 10 mM dimethylglutarate, 2 mM CaCl2). Reactions were incubated at 37 °C for 1 h. Fluorescence was measured with a microplate reader at λ ex = 530 nm and λ em = 590 nm (Laurier Research Instrumentation Inc, Otego, NY, USA). The activity of unknowns was compared to that of the standard enzyme to calculate microunits per microgram of total protein. 2.6. ROS levels and oxidative stress markers in heart tissue Evaluation of ROS levels was performed incubating the heart homogenates with 10 μM 2,7-dichlorofluorescein-diacetate (DCFH-DA) during 15 min at 37 °C in darkness and with constant agitation. Fluorescence increase was assessed using a Shimadzu Spectrofluorometer RF5000U at λex = 488 nm and λem = 530 nm. Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were determined as previously described [25]. Protein carbonyl content, was measured by their reactivity with dinitrophenyl hydrazine (DNPH) to form protein hydrazones [26]. 2.7. Glutathione (GSH) determination In a reaction catalyzed by glutathion S transferase (GST), the compound mono-chloro-bimane (mCB) forms an stable fluorescent adduct with GSH that can be assessed fluorometrically. The reaction mix contained Krebs-Henseleit buffer pH 7.4, 1 mM mCB, 1 U/mL GST and samples obtained from ventricle homogenates and mitochondria. After 30 min of incubation at 37 °C, changes in fluorescence were measured at λex = 385 nm and λem = 478 nm in a Synergy® HT multimode microplate reader. Readings were performed each 15 min and the values obtained were compared with a GSH standard curve [27]. 2.8. Isolation of mitochondrial, cytosolic and microsomal fractions from reperfused hearts At the end of the protocols, some hearts were placed in cold buffer solution containing 250 mM sucrose, 10 mM Tris-HCl and 1 mM EDTA, pH 7.4. The hearts were minced and incubated for 10 min with the same buffer, plus 2 mg/mL subtilisin A in an ice bath. Thereafter, the tissue was washed and resuspended in the same buffer without the enzyme. Heart tissue was homogenized and centrifuged at 9000 g for 10 min; the pellet contained the mitochondrial fraction, whereas the supernatant containing the cytosol and the membrane fraction was further spun down at 100,000 g for 45 min. The resulting pellet was the enriched microsome fraction, which is mostly composed of endoplasmic reticulum membranes. Protein concentration was determined by the Lowry method [28]. 2.9. Lipid extraction from homogenates, mitochondria and microsomes Lipids were extracted according the modified method of Bligh and Dyer [29]. In brief, 100 μg of protein from homogenates, mitochondria and/or microsomes were homogenized with ice-cold chloroformmethanol-1 M NaCl [1:2:0.4 (v/v/v)] for 2 min. Additional chloroform and NaCl were added to separate the organic and aqueous layers. After centrifugation, the aqueous phase was removed, and the chloroform layer was decanted and evaporated at 70 °C. The residue was dissolved in 0.1 mL of methanol. 2.10. Enzyme-linked immunosorbent assay (ELISA) for ceramide determination Ceramides were measured using the modified method of Pellieux et al. [30]. Microplate wells were coated with lipids from homogenates, mitochondria or microsomes at 4 °C overnight. Remaining binding sites on the wells were blocked with 3% bovine serum albumin (BSA) in PBS

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at pH 7.4 for 2 h at room temperature and washed three times with PBS - 0.05% Tween 20. Mouse monoclonal antibody anti-ceramide IgM (1:50; Sigma Aldrich, C8104) was added to the wells and the plate was incubated overnight at 4 °C. After the primary antibody was removed, the wells were washed again and incubated with goat anti-mouse IgG-HRP (1:1000; Sigma) during 4 h. Following extensive washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added. The reaction was stopped after 10 min with 4 N H2SO4 and the developed color was measured at 450 nm. Ceramide concentration was determined by interpolating the data within standard curves. 2.11. Mitochondrial integrity measurements in reperfused hearts [3H]Tetraphenylphosphonium (TPP+) distribution in mitochondria was measured to determine mitochondrial membrane potential (ΔΨm). Mitochondria (2 mg) were suspended in 0.5 mL of basic medium supplemented with 10 mM succinate, 1 μM rotenone, and 0.8 μM [ 3H]TPP+ (sp. act. 1000 cpm/nmol). ADP (200 μM) was added to the medium and, where indicated, 50 μM CaCl2 or 0.05 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The samples were incubated for 2 min and immediately centrifuged at 14,000 g for 10 min at 4 °C. [3 H]TPP+ content was measured in both mitochondrial and supernatant fractions and ΔΨm values were calculated using the Nernst equation. Mitochondrial oxygen consumption was determined using a Clark-type oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH, USA). The experiments were carried out in 1.5 mL of basic medium, containing 125 mM KCl, 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and 3 mM NaH2PO4, pH 7.3. State 4 respiration was evaluated in the presence of 5 mM sodium glutamate and 5 mM sodium malate. State 3 respiration was measured after adding 200 μM adenosine diphosphate (ADP). Respiratory control index (RC) was calculated as the ratio between the State 3 and the State 4 rates [31]. 2.12. Cytochrome c content in heart mitochondria from reperfused hearts Cytochrome c content in mitochondria and in cytosol was evaluated by western blot, using a primary monoclonal antibody against cytochrome c (1:1000, 7H8·2C12; Abcam, Cambridge, UK). HRP-conjugated secondary antibodies were used, followed by enhanced chemiluminescence system detection. To assess protein loading, the membranes were “stripped” in a buffer containing 62.5 mM Tris/HCl, 100 mM β-mercaptoethanol, 2% SDS, pH 6.7, for 20 min at 50 °C; then the membranes were incubated against anti-adenine nucleotide translocator (ANT) polyclonal antibodies (1:500, sc-9299; Santa Cruz Biotechnology, Santa Cruz, CA, USA). 2.13. Statistical analysis Values are given as means ± SD and were assessed by simple t-Student test. A p ≤ 0.05 was considered the threshold for statistical significance between the indicated groups. 3. Results 3.1. Heart function and cell death in I/R + D609 hearts. Heart rate-pressure double product (DP), an indicator of myocardial work performance, was 65% lower in I/R than in the Control group; whereas the I/R + D609 group regained 78% of control hearts values at the end of the experiments (Fig. 1A). Infarct size decreased from 37% in I/R hearts to 14% in I/R + D609 hearts (Fig. 1B) and the ratio

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between active caspase 3/pro-caspase 3 increased significantly in the I/ R heart in comparison with both Control and I/R + D609 hearts (Fig. 1C).

3.3. ROS levels and oxidative damage in heart tissue from reperfused hearts treated with D609 It has been reported that ROS regulates the enzymatic activities of sphingomyelinases [19,32], therefore we measured ROS levels, oxidative damage markers and GSH in heart tissue from all groups. Increased ROS (Fig. 3A), MDA (Fig. 3B) and high protein carbonylation levels (Fig. 3C) were observed in the I/R group, along with GSH diminution (Fig. 3D). Conversely, ROS production and protein oxidative damage diminished in the I/R + D609 group in correlation with preserved GSH levels. On the other hand, 4-HNE content (Fig. 3B) was the same in all the experimental groups. As in our study, other reports described that 4-HNE levels remained unchanged in models associated to oxidative stress and increased MDA levels [32,33]. These data has been explained by the enchaned metabolism of 4-HNE by conjugative pathways [34] or by the presence of aldehyde dehydrogenase 2 [32] that metabolizes 4-HNE.

3.2. Ceramide accumulation in heart tissue and sphingomyelinases activity Ceramide levels in heart tissue from the different groups were evaluated by enzyme-linked immunosorbent assay. Ceramide increased in I/R hearts, whereas the I/R + D609 group showed significantly lower levels of this sphingolipid (Fig. 2A). To get insight into the role of SMase as possible mediator of ceramide production in reperfused hearts, we determined neutral (nSMase) and acid sphingomyelinase (aSMase) activities in heart homogenates. The activity of nSMase increased significantly in reperfused hearts by 25% as compared with the activity observed in the control group, although no significant diminution was detected in hearts treated with D609 (Fig. 2B). On the other hand, the acid isoform activity remained constant in all the experimental groups (Fig. 2C). As it is known that D609 also inhibits phosphatidylcholinephospholipase C (PC-PCL) and furthermore, that diacylglycerol (DAG) produced after breakdown of phosphatydylcholine is an activator of aSMase, we measured possible changes in the activity of this enzyme. PC-PLC activity was similar in the different groups in correlation with results of aSMase activity (Fig. 2D).

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3.4. Ceramide content and neutral sphingomyelinase activity in mitochondrial and microsome fractions from I/R + D609 hearts The demonstration that ceramide induces cell death specifically when is generated in mitochondria and the recent characterization of

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Fig. 1. Heart function and myocardial cell death in ischemic-reperfused (I/R) hearts treated with D609. (A) Double product of Control, I/R and I/R + D609 hearts. Control hearts were continuously perfused during 110 min. I/R hearts were subjected to 30 min of ischemia and to 60 min of reperfusion, after a 20 min period of stabilization. D609 was administered intravenously to the rats 10 min before the I/R protocol. Data represent the mean of at least six different experiments ± SD. (B) Final myocardial infarct size (IS) related with area at risk (AAR). Data represent the mean of at least four different experiments ± SD. *p b 0.05 vs. Control and I/R + D609. (C) Representative western blot of zymogen (pro-caspase 3) and cleaved caspase-3 content (upper image). Bars show the mean ± SD of the ratio of cleaved caspase-3 and procaspase-3 of three independent experiments (lower graph). AUF = Arbitrary units of fluorescence. *p b 0.05 vs. Control and I/R + D609.

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Fig. 2. (A) Ceramide content, B) neutral sphingomyelinase (nSMase), (C) acid sphingomyelinase (aSMase) and D) phosphatydylcholine-phospholipase C (PC-PLC) activities in homogenates from ischemic-reperfused (I/R) hearts. Data are expressed as the mean ± SD of at least 7 independent experiments. *p b 0.05 vs. Control and I/R + D609; **p b 0.05 vs. Control. Samples were obtained at the end of the protocols described in the Material and Methods section.

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Fig. 3. (A) Reactive oxygen species (ROS) quantification; (B) lipoperoxidation, evaluated by the measurement of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels; (C) protein carbonylation and (d) glutathione content (GSH) in heart homogenates. Data represent the mean ± SD of at least 3 different experiments. AUF = Arbitrary units of fluorescence; DPNH = dinitrophenylhydrazine. *p b 0.05 vs. Control and I/R + D609; **p b 0.05 vs. Control. Samples were obtained at the end of the protocols described in the Material and Methods section.

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a novel mitochondria-associated nSMase [35], led us to determined if nSMase compartmentalization in mitochondria might be related with ceramide accumulation in these organelles in reperfused hearts. Additionally, we measured nSMase activation and ceramide accumulation in hearts subjected only to 30 min of ischemia. We found that mitochondrial nSMase activity (16.9 ± 0.7 nmol/mg protein) and ceramide levels (63.2 ± 5.3 ng/100 μg protein) after ischemia were similar to control values (16.9 ± 3.4 nmol/mg protein and 56.2 ± 21.5 ng/100 μg protein, respectively), demonstrating that ceramide increases during reperfusion and not during ischemia. Next, we compared nSMase activity and ceramide content between mitochondrial and microsomal fractions. nSMase activity increased in mitochondria from I/R hearts and diminished in the I/R + D609 group, whereas no changes were detected in the microsomal fraction of any of the experimental groups (Fig. 4A). Accordingly, ceramide levels increased in mitochondria from I/R group, whereas the effect of the nSMase inhibitor was evident only in these organelles (Fig. 4B). On the other hand, nSMase activity did not change in the microsomal fraction from none of the experimental groups, although ceramide content was lower in the I/R group than in control and I/R + D609 microsomes (Fig. 4A and 4B). To evaluate the purity of these subcellular fractions, we analyzed the presence of ANT and of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Mitochondria contained a negligible amount of SERCA, whereas ANT was not detected in the microsomal fraction (Fig. 4C). Studies in vitro and in vivo indicate that ceramide directly triggers and expand the oxidant response in mitochondria by interacting with Complex III [36], thus we measured GSH levels (as a marker of the redox state) in these organelles. It was

observed that GSH content diminished significantly in I/R mitochondria as compared with Control and I/R + D609 mitochondria (Fig. 4D). The parallelism found between these parameters, suggested that nSMase activity is relevant to cellular compartmentation of ceramide in mitochondria. 3.5. Mitochondrial function in reperfused hearts treated with D609 To demonstrate that ceramide accumulation contributes to deregulate mitochondrial function, we performed oxygen consumption experiments using NADH-linked substrates in isolated mitochondria. Increased basal oxygen consumption (state 4) in the I/R group suggested membrane damage and increased proton leak, whereas loss of ADP-stimulated respiration (state 3) could represent inhibition of substrate oxidation and/or ATP dysfunction (Fig. 5A). Accordingly, respiratory control (RC), the single most useful general measure of function in isolated mitochondria, diminished from 5.7 ± 1.2 to 1.2 ± 0.6. ADP/O, which represents the maximum number of ATP molecules made as an electron pair passes down the respiratory chain from substrate to oxygen, also decreased from 3.4 ± 0.6 to 0.7 ± 0.6. D609 treatment diminished state 4 respiration, increased RC (2.4 ± 0.4) and ADP/O values (1.4 ± 0.2) (Fig. 5B), reflecting recovered mitochondrial function. We also quantified the mitochondrial transmembrane electric potential (Δψm) to further identify subtle changes in proton leak, as well as the efficiency of coupling among mitochondria from each group. Transmembrane potential in mitochondria isolated from I/R hearts was significantly lower than in mitochondria from control hearts

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Fig. 4. Ceramide content and neutral sphingomyelinase (nSMase) activity in mitochondria and in microsomes from ischemic-reperfused (I/R) hearts treated with D609. (A) nSMase activity and (B) ceramide levels in mitochondrial and microsomal fractions. Data are the mean ± SD of at least 6 mitochondrial and microsomal independent preparations from each group. *p b 0.05 vs. Control and I/R + D609. (C) Inmunodetection of sarcoplasmic reticulum Ca2+ ATPase (SERCA) and adenine nucleotide translocase (ANT) as marker proteins of microsomal and mitochondrial membranes, respectively. (D) Glutathione (GSH) in mitochondrial fractions. Data are the mean ± SD of four mitochondrial independent preparations from each group. *p b 0.05 vs. Control and I/R + D609. Hearts for mitochondria isolation were obtained at the end of the protocols as described in the Material and Methods section.

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Fig. 5. Oxygen consumption, transmembrane potential and cytochrome c (cyt c) content in mitochondria and cytosol from ischemic-reperfused (I/R) hearts treated with D609. (A) Oxygen consumption in state 4 (basal respiration) and in state 3 (ADP-stimulated respiration); (B) Respiratory control (RC) and ADP/O values; (C) transmembrane potential values (ΔΨm) and (D) cytochrome c (Cyt c) released from and retained in mitochondria. Data are the mean ± SD of at least 4 different preparations from each group. *p b 0.05 vs. Control and I/R + D609. Cytochrome c immunodetection from three independent preparations of each experimental protocol. Adenine nucleotide translocator (ANT) was used as control loading. nAtO = nanoatoms of oxygen, ADP = adenosine diphosphate; CCCP = carbonyl cyanide m-chlorophenylhydrazone. Hearts for mitochondria isolation were obtained at the end of the protocols.

(−95.4 ± −19 vs. −112.3 ± −8.9 mV), and was further collapsed in the presence of 50 μM CaCl2, (−86.3 ± 6.8 vs. −57 ± −8.18 mV) indicating deregulation in calcium handling; on the other hand, Δψm was recovered in the IR + D609 group with and without calcium. Complete depolarization of the membrane was achieved after addition of the uncoupler CCCP in mitochondria from all groups (Fig. 5C). Mechanical injury to the mitochondrial outer membrane related with unespecific increase in permeability has been associated with cyt c release and apoptosis triggering. We measured cyt c content in mitochondria from the experimental groups and found that the content of this protein diminished in correlation with augmented release in the I/R group, as compared with heart mitochondria from Control and I/R + D609 rats (Fig. 5D). 4. Discussion Besides the established notion that ceramide accumulation in heart is a critical element in tissue injury [14,37], emerging experimental data indicate that ceramide accumulation also affects mitochondrial functions in I/R hearts [3,12,38]. Evidences on the direct regulation of ceramide and other sphingolipids on mitochondrial function include the studies of mitochondria from ceramide synthase 2(CerS2) null mouse, in which the accumulation of C-16 chain ceramide was related with Complex IV activity inhibition [39] and, also the demonstration of the occurrence of ceramide and sphingomyelin in purified mitochondria [40]. However, the mechanism underlying mitochondrial ceramide

accumulation remains controversial, as ceramide synthase [39], neutral ceramidases [41] and neutral sphingomyelinases [42] has been characterized in these organelles. In this regard, the finding that the sphingomyelinase inhibitor D609, restores cardiac function and diminishes BAX insertion into mitochondrial membranes [12], led us to investigate the relevance of the SM pathway for ceramide accumulation in mitochondria and its effect on the function of these organelles. Our results showed that cardiac dysfunction, myocardial cell death and augmented levels of active caspase-3 correlate with increased nSMase activity during reperfusion. The observed cardioprotection in the I/R + D609 group was reflected in ceramide diminution in whole tissue (Fig. 2A), but neither nSMase nor aSMase activity in homogenates showed susceptibility to this compound. These data contrast with those from Argaud et al. [43], who reported myocardial ceramide reduction after blockade of sphingomyelinase during the first min of ischemia, using the same dose of D609. In such work, ceramide content was not evaluated at the end of reperfusion, on the basis that enhancement of ceramide production occurs rapidly in response to the fast and transient activation of neutral and acid sphingomyelinases by diverse exogenous stimuli during ischemia [44]. Therefore, ceramide increase in whole tissue during reperfusion might result on the combined action of several pathways, as the activation of the de novo synthesis and reduction in ceramidase activity have been related with high ceramide levels after left coronary artery occlusion/reperfusion in cardiac tissue [37]. In this sense and while this work was in preparation, ceramide accumulation and augmented inflammatory response was associated with enhanced

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expression of the rate-limiting enzyme of de novo pathway, serine palmitoyltransferase [45]. To get insight into the mechanisms that provide cardioprotection in the I/R + D609 group, we evaluated the antioxidant properties attributed to this compound [46]. The observed reduction in oxidative stress, partially explained the effect of this xanthate; however, as it has been proposed that ceramide induces cell death specifically when is generated in mitochondria after selective targeting of bacterial SMase to these organelles [47], we also determined if the compartmentation of ceramide in mitochondria sustained by SMase activity might be relevant for cardiomyocyte fate and mitochondrial function. We found that ceramide is accumulated in mitochondria from I/R hearts, but not in the microsomal fraction of this group. In close association, the activity of mitochondrial nSMase increased and GSH levels diminished. There are no reports on the mechanism of activation of the mitochondrial nSMase, but it is reasonable to assume that as with other isoforms, GSH exerts regulatory mechanisms. Our finding is supported by Monette et al. [48], who demonstrated that lipoic acid administration to aged rats, restores mitochondrial GSH levels, decreases nSMase activity and improves Complex IV activity. It is tempting to speculate that mitochondrial nSMase activity contributes to compartmentation of ceramide in these organelles. Relevant to this information, is the demonstration of the existence of a mitochondrial SM pool in cells [49]. Our data show that reduction of ceramide levels in mitochondria from I/R + D609 hearts, correlated with diminished nSMase activity, coupling of oxidative phosphorylation and mitochondrial integrity maintenance, reflected in the ADP/O ratio and RC, respectively. Results of membrane potential and basal respiration (state 4) indicate that ceramide accumulation induces unspecific increase in mitochondrial membrane permeability, providing a release pathway to pro-apoptotic proteins contained in the intermembranal space. However, as current evidence points out to a close association between ceramide and proteins of the Bcl-2 family, it is possible that channel forming proteins like Bax, might act synergically with ceramide to permeabilize the mitochondrial outer membrane. In accordance with this assumption, some reports indicate that BAK regulates ceramide generation [50] and that the anti-apoptotic protein Bcl-xL regulates the formation of Bax channels and also of ceramide channels [51]. We cannot discard that other mechanisms beside nSMAse activity participate in ceramide biosynthesis in reperfused hearts. Our observation that nSMase activity is not inhibited by D609 in heart homogenates, but it does changed in mitochondria, might fit in a temporal sequence of events that occurs at different cellular locations. We think that the acute local production of ceramide by mitochondrial nSMase activity might exert damage to these organelles increasing ROS production; therefore preventing local ceramide increase in mitochondria results in cardioprotection. In conclusion, results from this work indicate that compartmentalization of nSMase activity in mitochondria is linked to the generation of ceramide in these organelles, disrupting its function and adversely affecting cardiac performance during reperfusion.

Transparency document The Transparency document associated with this article can be found, in the online version.

Acknowledgments This work was partially supported by Grant 177527 to CZ and 181593 to FC from Consejo Nacional de Ciencia y Tecnología.

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