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• W2012897199 abstract "Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)4CL]. A decrease in (18:2)4CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)4CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A2 (iPLA2) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)4CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA2 inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)4CL in the failing rat heart, and that mitochondrial iPLA2 plays a role in the remodeling of CL's acyl composition. Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)4CL]. A decrease in (18:2)4CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)4CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A2 (iPLA2) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)4CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA2 inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)4CL in the failing rat heart, and that mitochondrial iPLA2 plays a role in the remodeling of CL's acyl composition. Cardiolipin (CL, 1,3-bis[1′,2′-diacyl-3′-phosphoryl-sn-glycerol]sn-glycerol) is a unique tetra-acyl phospholipid found in energy-transducing biological membranes (1Mileykovskaya E. Zhang M. Dowhan W. Cardiolipin in energy transducing membranes.Biochemistry. 2005; 70: 154-158PubMed Google Scholar). In mammals, CL accounts for approximately 15% of mitochondrial phospholipid mass and localizes largely to the inner mitochondrial membrane (IMM), although it has also been identified in the outer mitochondrial membrane (2Hatch G.M. Cell biology of cardiac mitochondrial phospholipids.Biochem. Cell Biol. 2004; 82: 99-112Crossref PubMed Scopus (92) Google Scholar, 3Van Q. Liu J. Lu B. Feingold K.R. Shi Y. Lee R.M. Hatch G.M. Phospholipid scramblase-3 regulates cardiolipin de novo biosynthesis and its resynthesis in growing HeLa cells.Biochem. J. 2007; 401: 103-109Crossref PubMed Scopus (50) Google Scholar, 4Hovius R. Thijssen J. van der Linden P. Nicolay K. de Kruijff B. Phospholipid asymmetry of the outer membrane of rat liver mitochondria. Evidence for the presence of cardiolipin on the outside of the outer membrane.FEBS Lett. 1993; 330: 71-76Crossref PubMed Scopus (84) Google Scholar). Within the IMM, CL physically associates with a number of proteins involved in mitochondrial energetics, including cytochrome c oxidase and the F1F0 ATPase (5Musatov A. Contribution of peroxidized cardiolipin to inactivation of bovine heart cytochrome c oxidase.Free Radic. Biol. Med. 2006; 41: 238-246Crossref PubMed Scopus (47) Google Scholar, 6Chicco A.J. Sparagna G.C. Role of cardiolipin alterations in mitochondrial dysfunction and disease.Am. J. Physiol. Cell Physiol. 2007; 292: C33-C44Crossref PubMed Scopus (478) Google Scholar, 7Zhang M. Mileykovskaya E. Dowhan W. Gluing the respiratory chain together. Cardiolipin is required for supercomplex formation in the inner mitochondrial membrane.J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar).A substantial body of evidence supports the presence of CL as essential for mitochondrial respiratory function. Paradies et al. (8Paradies G. Petrosillo G. Pistolese M. Di Venosa N. Serena D. Ruggiero F.M. Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.Free Radic. Biol. Med. 1999; 27: 42-50Crossref PubMed Scopus (200) Google Scholar) reported that cytochrome c oxidase activity was restored only when delipidated mitochondrial membranes were reconstituted in the presence of CL, and Sedlak and Robinson (9Sedlak E. Robinson N.C. Phospholipase A2 digestion of cardiolipin bound to bovine cytochrome c oxidase alters both activity and quaternary structure.Biochemistry. 1999; 38: 14966-14972Crossref PubMed Scopus (131) Google Scholar) have shown that a loss of CL destabilizes the noncovalent connections between cytochrome c oxidase subunits VIa and VIb. In addition to its role in IMM protein activity, CL also serves as a proton trap (10Haines T.H. Dencher N.A. Cardiolipin: a proton trap for oxidative phosphorylation.FEBS Lett. 2002; 528: 35-39Crossref PubMed Scopus (306) Google Scholar), cytochrome c anchor (11Brown L.R. Wuthrich K. NMR and ESR studies of the interactions of cytochrome c with mixed cardiolipin-phosphatidylcholine vesicles.Biochim. Biophys. Acta. 1977; 468: 389-410Crossref PubMed Scopus (115) Google Scholar, 12Petrosillo G. Ruggiero F.M. Paradies G. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria.FASEB J. 2003; 17: 2202-2208Crossref PubMed Scopus (301) Google Scholar), is involved in protein import (13Ardail D. Lerme F. Louisot P. Phospholipid import into mitochondria: possible regulation mediated through lipid polymorphism.Biochem. Biophys. Res. Commun. 1992; 186: 1384-1390Crossref PubMed Scopus (7) Google Scholar, 14Eilers M. Endo T. Schatz G. Adriamycin, a drug interacting with acidic phospholipids, blocks import of precursor proteins by isolated yeast mitochondria.J. Biol. Chem. 1989; 264: 2945-2950Abstract Full Text PDF PubMed Google Scholar), and is important for imparting a specific three-dimensional structure on the IMM (15Mannella C.A. Structure and dynamics of the mitochondrial inner membrane cristae.Biochim. Biophys. Acta. 2006; 1763: 542-548Crossref PubMed Scopus (281) Google Scholar).The acyl composition and molecular symmetry of CL have received attention for their importance in proper CL function (6Chicco A.J. Sparagna G.C. Role of cardiolipin alterations in mitochondrial dysfunction and disease.Am. J. Physiol. Cell Physiol. 2007; 292: C33-C44Crossref PubMed Scopus (478) Google Scholar, 16Sparagna G.C. Chicco A.J. Murphy R.C. Bristow M.R. Johnson C.A. Rees M.L. Maxey M.L. McCune S.A. Moore R.L. Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure.J. Lipid Res. 2007; 48: 1559-1570Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 17Schlame M. Kelley R.I. Feigenbaum A. Towbin J.A. Heerdt P.M. Schieble T. Wanders R.J.A. DiMauro S. Blanck T.J.J. Phospholipid abnormalities in children with Barth syndrome.J. Am. Coll. Cardiol. 2006; 42: 1994-1999Crossref Scopus (156) Google Scholar). In mammals, the majority of cardiac CL is enriched with the essential ω-6 fatty acid, linoleic acid (18:2) (2Hatch G.M. Cell biology of cardiac mitochondrial phospholipids.Biochem. Cell Biol. 2004; 82: 99-112Crossref PubMed Scopus (92) Google Scholar, 6Chicco A.J. Sparagna G.C. Role of cardiolipin alterations in mitochondrial dysfunction and disease.Am. J. Physiol. Cell Physiol. 2007; 292: C33-C44Crossref PubMed Scopus (478) Google Scholar, 16Sparagna G.C. Chicco A.J. Murphy R.C. Bristow M.R. Johnson C.A. Rees M.L. Maxey M.L. McCune S.A. Moore R.L. Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure.J. Lipid Res. 2007; 48: 1559-1570Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Tetralinoleoyl CL [(18:2)4CL] accounts for 75–80% of total CL content in both rat and human cardiac mitochondria (2Hatch G.M. Cell biology of cardiac mitochondrial phospholipids.Biochem. Cell Biol. 2004; 82: 99-112Crossref PubMed Scopus (92) Google Scholar, 16Sparagna G.C. Chicco A.J. Murphy R.C. Bristow M.R. Johnson C.A. Rees M.L. Maxey M.L. McCune S.A. Moore R.L. Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure.J. Lipid Res. 2007; 48: 1559-1570Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The notion that 18:2 is essential for CL's function in the mammalian heart is based on three independent observations. First, the vast majority of CL is remodeled from its de novo form to (18:2)4CL subsequent to its biosynthesis. Second, the high prevalence of (18:2)4CL over other CL species is unlikely if one considers the remodeling process to be random with respect to acyl selection, which suggests that the loading of CL with 18:2 is purposeful. Lastly and most importantly, a loss of (18:2)4CL, along with an increase in 18:2-deficient CL species, occurs in a number of cardiac disease states (6Chicco A.J. Sparagna G.C. Role of cardiolipin alterations in mitochondrial dysfunction and disease.Am. J. Physiol. Cell Physiol. 2007; 292: C33-C44Crossref PubMed Scopus (478) Google Scholar, 18Sparagna G.C. Lesnefsky E.J. Cardiolipin remodeling in the heart.J. Cardiovasc. Pharmacol. 2008; (In press.)Google Scholar). The disease most directly associated with a loss of (18:2)4CL is Barth syndrome, caused by an X-linked mutation in the tafazzin gene (19Hauff K.D. Hatch G.M. Cardiolipin metabolism and Barth syndrome.Prog. Lipid Res. 2006; 45: 91-101Crossref PubMed Scopus (122) Google Scholar, 20Schlame M. Ren M. Barth syndrome, a human disorder of cardiolipin metabolism.FEBS Lett. 2006; 580: 5450-5455Crossref PubMed Scopus (239) Google Scholar, 21Barth P.G. Valianpour F. Bowen V.M. Lam J. Duran M. Vaz F.M. Wanders R.J. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): an update.Am. J. Med. Genet. A. 2004; 126A: 349-354Crossref PubMed Scopus (224) Google Scholar, 22Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J.A. Barth P.G. Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome.Biochem. Biophys. Res. Commun. 2000; 279: 378-382Crossref PubMed Scopus (300) Google Scholar). Levels of 18:2 in cardiac CL also decline in congestive heart failure (HF) (16Sparagna G.C. Chicco A.J. Murphy R.C. Bristow M.R. Johnson C.A. Rees M.L. Maxey M.L. McCune S.A. Moore R.L. Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure.J. Lipid Res. 2007; 48: 1559-1570Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), ischemic HF (23Heerdt P.M. Schlame M. Jehle R. Barbone A. Burkhoff D. Blanck T.J.J. Disease-specific remodeling of cardiac mitochondria after a left ventricular assist device.Ann. Thorac. Surg. 2002; 73: 1216-1221Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24Nasa Y. Sakamoto Y. Sanbe A. Sasaki H. Yamaguchi F. Takeo S. Changes in fatty acid compositions of myocardial lipids in rats with heart failure following myocardial infarction.Mol. Cell. Biochem. 1997; 176: 179-189Crossref PubMed Scopus (16) Google Scholar), and diabetes (25Han X. Yang J. Yang K. Zhongdan Z. Abendschein D.R. Gross R.W. Alterations in myocardial cardiolipin content and composition occur at the very earliest stages of diabetes: a shotgun lipidomics study.Biochemistry. 2007; 46: 6417-6428Crossref PubMed Scopus (217) Google Scholar). Because (18:2)4CL seems to be important for myocardial energy homeostasis, a complete understanding of the CL remodeling process is essential in designing future treatments for patients with HF and other mitochondrial diseases.The formation of (18:2)4CL is dependent on the coupling of CL biosynthesis and remodeling. The biosynthesis of CL occurs within the IMM (26Hatch G.M. Cardiolipin biosynthesis in the isolated heart.Biochem. J. 1994; 297: 201-208Crossref PubMed Scopus (65) Google Scholar, 27Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblasts by cytidine 5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28Hostetler K.Y. Van Den Bosch H. Van Deenen L.L.M. The mechanism of cardiolipin biosynthesis in liver mitochondria.Biochim. Biophys. Acta. 1972; 260: 507-513Crossref PubMed Scopus (80) Google Scholar, 29Schlame M. Hostetler K.Y. Cardiolipin synthase from mammalian mitochondria.Biochim. Biophys. Acta. 1997; 1348: 207-213Crossref PubMed Scopus (66) Google Scholar), where nascent CL is formed from the condensation of phosphatidylglycerol (PG) and cytidinediphosphate-diacylglycerol (CDP-DAG) in a reaction catalyzed by CL synthase (CLS) (for review, see 30Hatch G.M. Cardiolipin: biosynthesis, remodeling and trafficking in the heart and mammalian cells.Int. J. Mol. Med. 1998; 1: 33-41PubMed Google Scholar, 31Hatch G.M. Regulation of cardiolipin biosynthesis in the heart.Mol. Cell. Biochem. 1996; 159: 139-148Crossref PubMed Scopus (45) Google Scholar). Neither the acyl composition of PG and CDP-DAG nor the acyl specificity of CLS results in an enrichment of CL with 18:2 de novo; thus, nascent CL must be converted to (18:2)4CL through an acyl remodeling cycle. Presumably, the remodeling of CL occurs through a series of deacylation-reacylation reactions, though the details of CL remodeling in vivo remain in question (18Sparagna G.C. Lesnefsky E.J. Cardiolipin remodeling in the heart.J. Cardiovasc. Pharmacol. 2008; (In press.)Google Scholar, 30Hatch G.M. Cardiolipin: biosynthesis, remodeling and trafficking in the heart and mammalian cells.Int. J. Mol. Med. 1998; 1: 33-41PubMed Google Scholar). To date, three enzymes have been identified that are capable of adding 18:2 to a monolysoCL (MLCL): tafazzin (32Xu Y. Malhotra A. Ren M. Schlame M. The enzymatic function of tafazzin.J. Biol. Chem. 2006; 281: 39217-39224Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), MLCL-acyltransferase (MLCL-AT, 33Taylor W.A. Hatch G.M. Purification and characterization of monolysocardiolipin acyltransferase from pig liver mitochondria.J. Biol. Chem. 2003; 278: 12716-12721Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and acylCoA-lysocardiolipin acyltransferase (ALCAT-1, 34Cao J. Liu Y. Lockwood J. Burn P. Shi Y. A novel cardiolipin-remodeling pathway revealed by a gene encoding an endoplasmic reticulum-associated acyl-CoA:lysocardiolipin acyltransferase (ALCAT-1) in mouse.J. Biol. Chem. 2004; 279: 31727-31734Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). None of these enzymes, however, possess phospholipase activity. In fact, very little research has examined the role of endogenous phospholipases in CL remodeling.To the best of our knowledge, there are only two reports examining the role of mitochondrial phospholipases in CL remodeling. Mancuso et al. (35Mancuso D.J. Sims H.F. Han X. Jenkins C.M. Guan S.P. Yang K. Moon S.H. Pietka T. Abumrad N.A. Schlesinger P.H. et al.Genetic ablation of calcium-independent phospholipase A2 gamma leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype.J. Biol. Chem. 2007; 282: 34611-34622Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) created a murine model deficient in the functional form of calcium-independent phospholipase A2 (iPLA2) γ (iPLA2γ, also PLA2GVIB), and a decrease in (18:2)4CL in these animals occurred concomitantly with symptoms of myocardial energetic disequilibrium. More recently, Malhotra et al. (36Malhotra A. Edelman-Novemsky I. Xu Y. Plesken H. Ma J. Schlame M. Ren M. Role of calcium-independent phospholipase A2 in the pathogenesis of Barth syndrome.Proc. Natl. Acad. Sci. USA. 2009; 106: 2337-2341Crossref PubMed Scopus (107) Google Scholar) examined the role of iPLA2β (also PLA2GVIA), reporting that iPLA2β is not necessary for CL remodeling, but does increase the MLCL/CL ratio in tafazzin-deficient Drosophila melanogaster. Because these two reports are the first of their kind, much is still unknown about iPLA2 in CL remodeling. As such, the purpose of this study was to first develop a method to examine CL remodeling at the level of the isolated rat cardiomyocyte, and thereafter, use this method to investigate potential alterations in CL remodeling in the Spontaneously Hypertensive HF (SHHF) rat as well as the potential remodeling role of iPLA2 in this model of cardiac stress. We report that the incorporation of 18:2 into (18:2)4CL in SHHF cardiomyocytes: a) occurs singly over time, b) is attenuated with the development of HF, and c) is partially sensitive to inhibition of iPLA2.MATERIALS AND METHODSMaterialsAll materials used in this study were purchased from the Sigma Chemical Co. with the following exceptions: Type-2 collagenase was purchased from Worthington, racemic bromoenol lactone (BEL) and the iPLA2γ-specific enantiomer R-BEL were purchased from Cayman Chemical Co., and carbon stable isotope linoleic acid (13C18-18:2, >98% isotope enrichment, abbreviated in the text as 13C-18:2) was purchased from Spectra Stable Isotopes (Cambridge Isotope Laboratories).AnimalsThe SHHF rat is a model of human HF that is genetically predisposed to death from hypertrophic followed by dilated cardiomyopathy, the etiology of which has been described by McCune et al. (37McCune S.A. Park S. Radin M.J. Jurin J.J. The SHHF/Mcc-facp: a genetic model of congestive heart failure.in: Singal P.K. Dixon I.M.C. Kluwer M.A. Dhalla N.S. Mechanisms of Heart Failure. Kluwer Academic Publishers, Boston1995: 91-106Crossref Google Scholar). Briefly, female SHHF rats become hypertensive by approximately 3 months of age and this development progresses to overt hypertension by 5 months. Secondary to this hypertension, myocardial hypertrophy begins around 17 months of age and progresses to dilated cardiomyopathy between 23–25 months (38Haas G.J. McCune S.A. Brown D.M. Cody R.J. Echocardiographic characterization of left ventricular adaptation in a genetically determined heart failure rat model.Am. Heart J. 1995; 130: 806-811Crossref PubMed Scopus (47) Google Scholar, 39Gomez A.M. Valdiva H.H. Cheng H. Lederer M.R. Santana L.F. Cannell M.B. McCune S.A. Altschuld R.A. Lederer W.J. Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure.Science. 1997; 276: 800-806Crossref PubMed Scopus (641) Google Scholar). Female SHHF rats (colony kept at the University of Colorado by S.A.M.) were designated as nonHF or HF based on age (2–3 months and 21–23 months, respectively), left ventricular (LV) function as assessed by echocardiography, and the absence or presence, respectively, of at least one of the following symptoms: labored breathing, piloerection, or orthopnea. Aged-matched (3 months and ≥22 months) female Fisher Brown Norway (Fischer 344 x Brown Norway F1, FBN) rats (Harlan) were used in select experiments as an aging control. The FBN rat was chosen as an aging control, rather than the Fischer 344, Wistar, or Sprague Dawley rat, because the documented incidence of cardiovascular dysfunction and disease is milder and of later onset in FBN rats relative to the other strains (40Li Y.M. Steffes M. Donnelly T. Liu C. Fuh H. Basgen J. Bucala R. Vlassara H. Prevention of cardiovascular and renal pathology of aging by the advanced glycation inhibitor aminoguanidine.Proc. Natl. Acad. Sci. USA. 1996; 93: 3902-3907Crossref PubMed Scopus (164) Google Scholar, 41Sample J. Cleland J.G. Seymour A.M. Metabolic remodeling in the aging heart.J. Mol. Cell. Cardiol. 2006; 40: 56-63Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 42Lipman R.D. Chrisp C.E. Hazzard D.G. Bronson R.T. Pathologic characterization of Brown Norway, Brown Norway x Fischer 344, and Fischer 344 x Brown Norway rats with relation to age.J. Gerontol. A Biol. Sci. Med. Sci. 1996; 51: B54-B59Crossref PubMed Scopus (205) Google Scholar). All animals were housed in groups of 2–3 on a 12:12 h light:dark cycle with ad libitum access to chow and water. The n values for each experiment are located within figure legends. All animal treatment was conducted in conformity with the Public Health Service Policy on Humane Use and Care of Laboratory Animals and in accordance with guidelines set forth on animal care at the University of Colorado, Boulder.Echocardiographic analysisTransthoracic echocardiography was performed on all rats 2–5 days prior to euthanasia under inhaled isoflurane anesthesia (5% initial, 2% maintenance) using a 12 MHz pediatric transducer connected to a Hewlett Packard Sonos 5500 Ultrasound system. Short axis M-mode echocardiograms on the LV were obtained for measurement of LV internal diameters at diastole (LVIDd) and systole (LVIDs), fractional shortening (FS), ejection fraction (EF), anterior wall thickness in diastole (AWTd), and posterior wall thickness in diastole (PWTd) as previously described (43Chicco A.J. McCune S.A. Emter C.A. Sparagna G.C. Rees M.L. Bolden D.A. Marshall K.D. Murphy R.C. Moore R.L. Low-intensity exercise training delays heart failure and improves survival in female spontaneously hypertensive heart failure rats.Hypertension. 2008; 51: 1096-1102Crossref PubMed Scopus (58) Google Scholar).Cardiomyocyte isolationCardiomyocytes were isolated from whole hearts with modifications to methods previously described (44Emter C.A. McCune S.A. Sparagna G.C. Radin M.J. Moore R.L. Low-intensity exercise training delays onset of decompensated heart failure in spontaneously hypertensive heart failure rats.Am. J. Physiol. Heart Circ. Physiol. 2005; 289: H2030-H2038Crossref PubMed Scopus (76) Google Scholar, 45Cheung J.Y. Thompson I.G. Bonventre J.V. Effects of extracellular calcium removal and anoxia on isolated rat myocytes.Am. J. Physiol. Cell Physiol. 1982; 243: C184-C190Crossref PubMed Google Scholar). Animals received 2,000 units/kg body mass heparin and after 12 min were deeply anesthetized with 35 mg/kg sodium pentabarbitol, both through intraperitoneal injection. Hearts were rapidly excised, immersed in ice-cold saline, and cannulated by the aorta on a modified Langendorff perfusion apparatus. Hearts were perfused in a retrograde manner for 5 min with “buffer B” (described in refs. 44Emter C.A. McCune S.A. Sparagna G.C. Radin M.J. Moore R.L. Low-intensity exercise training delays onset of decompensated heart failure in spontaneously hypertensive heart failure rats.Am. J. Physiol. Heart Circ. Physiol. 2005; 289: H2030-H2038Crossref PubMed Scopus (76) Google Scholar, 45Cheung J.Y. Thompson I.G. Bonventre J.V. Effects of extracellular calcium removal and anoxia on isolated rat myocytes.Am. J. Physiol. Cell Physiol. 1982; 243: C184-C190Crossref PubMed Google Scholar), after which the perfusate was changed to a digestion buffer identical to the first, except containing 1.30 or 1.50 mg/ml type-2 collagenase (for nonHF and HF hearts, respectively), 1.30 mg/ml hyaluronidase, 100 μg/ml dialyzed albumin, and 55 μM CaCl2. When sufficiently digested (as determined by increases in coronary flow and softness to touch), hearts were cut from the cannula and the right ventricular free wall was removed. Remaining LV and septal tissue was cut into smaller pieces and teased apart with blunted glass pipette tips. Cells were washed once with a buffer identical to buffer B, except containing 100 μM CaCl2, and twice with medium 199 (37°C, pH 7.4, with 100 units/ml penicillin and 5 μg/ml gentamycin). After the final wash, cells were seeded on laminin-coated glass microscope coverslips in Springhorn medium (medium 199 with the addition of 2 mg/ml BSA, 100 nM insulin and (in mM): 2 carnitine, 5 creatine, 5 taurine, 1.3 glutamine, 2.5 sodium pyruvate, 10 2,3- butane dione monoxime; pH 7.70 before equilibration with 5% CO2) and incubated at 37°C for 2–3 h.Cardiomyocyte treatmentFor each experimental group, three laminin-coated glass coverslips plated with cardiomyocytes were bathed in Springhorn medium in 100 × 15 mm sterile Petri dishes (final volume 12 ml). The first group served as a control and was incubated with 0.17 mM fatty acid-free BSA and 0.1% DMSO vehicle. The second group of cells was incubated with BSA-bound 13C-18:2 such that the final concentrations were 1 mM 13C-18:2 and 0.17 mM BSA (a 6:1 18:2:BSA ratio), with 0.1% DMSO vehicle. The third group of cells was treated in a manner similar to the second group, but was incubated for 30 min with 10 μM BEL in 0.1% DMSO prior to the addition of BSA-bound 13C-18:2 (13C-18:2 + BEL). The final group was treated exactly the same as the third group, except 5 μM of the iPLA2γ-specific BEL enantiomer, R-BEL, was used instead of the racemic BEL mixture (13C-18:2 + R-BEL). Data was not shown for a fifth, “BEL control” group (10 μM BEL in 0.1% DMSO, 0.17 mM BSA), because this treatment did not result in any measurable effects. In the event that incubations lasted longer than 24 h, Springhorn medium and all necessary chemicals were replaced every 24 h. Myocytes were photographed preceding and subsequent to each treatment period using a Sony Cybershot DSC-S75 digital camera with a VAD-S70 adaptor ring under an inverted light microscope at 100× magnification.Cardiomyocyte harvest and lipid extractionFollowing treatment, myocytes were scraped from coverslips and centrifuged at 1600 g for 5 min at room temperature. Pelleted myocytes were resuspended in HPLC-grade methanol and stored at −20°C until lipid extraction. Lipids were extracted according to Bligh and Dyer (46Sparagna G.C. Johnson C.A. McCune S.A. Moore R.L. Murphy R.C. Quantitation of cardiolipin molecular species in spontaneously hypertensive heart failure rats using electrospray ionization mass spectrometry.J. Lipid Res. 2005; 46: 1196-1204Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and subject to ESI-MS for phospholipid content analysis.Phospholipid analysisSingly-ionized CL and PG species were quantified by ESI-MS as described by Sparagna et al. (46Sparagna G.C. Johnson C.A. McCune S.A. Moore R.L. Murphy R.C. Quantitation of cardiolipin molecular species in spontaneously hypertensive heart failure rats using electrospray ionization mass spectrometry.J. Lipid Res. 2005; 46: 1196-1204Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Tetramyristoyl CL [(14:0)4CL] was included as an internal standard to verify the quality of the spectra. Differences in cell yield between groups were controlled for by expressing each analyte as a fraction of its total respective phospholipid content. The specific acyl compositions, mass to charge ratios (m/z), and text abbreviations for all phospholipids presented can be found in Table 1.TABLE 1Phosphatidylglycerol and cardiolipin species measured using ESI mass spectrometryPhospholipid SpeciesMass to Charge Ratio (m/z)Abbreviation in TextPhosphatidylglycerol1-, 2-di-13C-linoleoyl phosphatidylglycerolaIn all phospholipids presented, acyl composition is arbitrary with respect to sn- position.805.5(13C-18:2)2PG1-13C-linoleoyl-2-oleoyl phosphatidylglycerol789.5(13C-18:2)(18:1)PG1-13C-linoleoyl-2-palmitoyl phosphatidylglycerol763.5(13C-18:2)(16:0)PG1-palmitoyl-2-oleoyl phosphatidylglycerol747.5(16:0)(18:1)PG1-13C-linoleoyl-2-linoleoyl phosphatidylglycerol787.5(13C-18:2)(18:2)PGCardiolipin1-, 2-, 3-, 4-tetralinoleoyl cardiolipin1448.0(18:2)4CL1-13C-linoleoyl, 2-, 3-, 4-trilinoleoyl cardiolipin1466.0(13C-18:2)(18:2)3CL1-, 2-di-13C-linoleoyl, 3-, 4-dilinoleoyl cardiolipin1484.0(13C-18:2)2(18:2)2CL1-, 2-, 3-tri-13C-linoleoyl, 4-linoleoyl cardiolipin1502.0(13C-18:2)3(18:2)CL1-, 2-, 3-, 4-tetra-13C-linoleoyl cardiolipinbThe atomic mass of this phospholipid is shared with phospholipids containing alternate acyl-compositions (see ref. 46). The acyl composition presented here represents the most common species.1520.0(13C-18:2)4CL1-, 2-, 3-trilinoleoyl, 4-docosahexaenoyl cardiolipinbThe atomic mass of this phospholipid is shared with phospholipids containing alternate acyl-compositions (see ref. 46). The acyl composition presented here represents the most common species.1496.0(18:2)3(22:6)CLSingly-ionized CL and PG species were quantified using ESI mass spectrometry. 16:0, palmitoyl; 18:1, oleoyl; 18:2, linoleoyl; 13C-18:2, carbon stable isotope linoleoyl; 22:6, docosahexaenoyl.a In all phospholipids presented, acyl composition is arbitrary with respect to sn- position.b The atomic mass of this phospholipid is shared with phospholipids containing alternate acyl-compositions (see ref. 46Sparagna G.C. Johnson C.A. McCune S.A. Moore R.L. Murphy R.C. Quantitation of cardiolipin molecular species in spontaneously hypertensive heart failure rats using electrospray ionization mass spectrometry.J. Lipid Res. 2005; 46: 1196-1204Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The acyl composition presented here represents the most common species. Open table in a new tab CLIn the first experiment, cardiomyocytes were incubated under control conditions for 48 h to verify that the CL composition in cell culture matched typical cardiac CL in the intact SHHF" @default.
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