Quantitation of cardiolipin molecular species in spontaneously hypertensive heart failure rats using electrospray ionization mass spectrometry

Abstract

Electrospray ionization mass spectrometry has previously been used to probe qualitative changes in the phospholipid cardiolipin (CL), but it has rarely been used in a quantitative manner. We assessed changes in the amount of individual molecular species of cardiac CL present in a model of congestive heart failure using 1,1′,2,2′-tetramyristoyl cardiolipin as an internal standard. There was a linear relationship between the ratio of the negative molecular ion ([M-H]−) current from four different CL reference standards and the [M-H]− from the internal standard, as a function of the concentration of CL molecular species. Therefore, this internal standard can be used to quantitate many naturally occurring CL molecular species over a wide range of CL concentrations. Using this method, changes to individual molecular species of CL in failing hearts from male spontaneously hypertensive heart failure rats were examined. CL isolated from cardiac mitochondria was compared with left ventricular tissue to demonstrate the feasibility of extracting and quantitating CL from either mitochondrial or tissue samples.The acyl chain composition of individual CL molecular species was identified using tandem mass spectrometry. In animals with heart failure, the major cardiac CL species (tetralinoloyl) decreased significantly, whereas other minor CL species were significantly increased. Electrospray ionization mass spectrometry has previously been used to probe qualitative changes in the phospholipid cardiolipin (CL), but it has rarely been used in a quantitative manner. We assessed changes in the amount of individual molecular species of cardiac CL present in a model of congestive heart failure using 1,1′,2,2′-tetramyristoyl cardiolipin as an internal standard. There was a linear relationship between the ratio of the negative molecular ion ([M-H]−) current from four different CL reference standards and the [M-H]− from the internal standard, as a function of the concentration of CL molecular species. Therefore, this internal standard can be used to quantitate many naturally occurring CL molecular species over a wide range of CL concentrations. Using this method, changes to individual molecular species of CL in failing hearts from male spontaneously hypertensive heart failure rats were examined. CL isolated from cardiac mitochondria was compared with left ventricular tissue to demonstrate the feasibility of extracting and quantitating CL from either mitochondrial or tissue samples. The acyl chain composition of individual CL molecular species was identified using tandem mass spectrometry. In animals with heart failure, the major cardiac CL species (tetralinoloyl) decreased significantly, whereas other minor CL species were significantly increased. Cardiolipin [(1′,2′-diacyl-3′-phosphoryl-sn-glycerol)2-sn-glycerol] (CL) is a unique mitochondrial phospholipid containing four acyl groups and is required for normal mitochondrial function (1Hatch G.M. Cardiolipin: biosynthesis, remodeling, and trafficking in the heart and mammalian cells.Int. J. Mol. Med. 1998; 1: 33-41PubMed Google Scholar, 2Hatch G.M. Cell biology of cardiac mitochondrial phospholipids.Biochem. Cell Biol. 2004; 82: 99-112Crossref PubMed Scopus (94) Google Scholar). Numerous enzymes involved in electron transport and oxidative phosphorylation have been shown to require CL for normal function (3Hoch F.L. Cardiolipins and biomembrane function.Biochim. Biophys. Acta. 1992; 1113: 71-133Crossref PubMed Scopus (546) Google Scholar). In addition, a decrease in CL levels has been implicated in mitochondrial membrane protein activity abnormalities during aging (4McMillin J. Taffet G. Taegtmeyer H.T. Hudson E.K. Tate C.A. Mitochondrial metabolism and substrate competition in the aging Fischer rat heart.Cardiovasc. Res. 1993; 27: 2222-2228Crossref PubMed Scopus (90) Google Scholar, 5Pepe S. Tsuchiya N. Lakatta E.G. Hansford R.G. PUFA and aging modulate cardiac mitochondrial membrane lipid composition and Ca2+ activation of PDH.Am. J. Physiol. 1999; 276: H149-H158PubMed Google Scholar, 6Paradies G. Ruggiero F.M. Petrosillo G. Quagliariello E. Age-dependent decline in the cytochrome c oxidase activity in rat heart mitochondria: role of cardiolipin.FEBS Lett. 1997; 406: 136-138Crossref PubMed Scopus (146) Google Scholar), ischemia (7Lesnefsky E.J. Slabe T.J. Stoll M.S. Minkler P.E. Hoppel C.L. Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria.Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H2770-H2778Crossref PubMed Google Scholar), and, most recently, Barth syndrome, a genetic disease characterized by heart abnormalities (8Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Placko 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 (310) Google Scholar). In addition to the quantitative changes in mitochondrial CL levels that have been implicated in various pathologies of the heart, there is now evidence that alterations in the acyl side chain composition of CL can contribute to aberrant mitochondrial function (9Yamaoka-Koseki S. Urade R. Kito M. Cardiolipins from rats fed different dietary lipids affect bovine heart cytochrome c oxidase activity.J. Nutr. 1991; 121: 956-958Crossref PubMed Scopus (42) Google Scholar). Recently, alterations in CL have also been strongly implicated in several key events during programmed cell death (apoptosis) in the heart and other tissues (10Ostrander D.B. Sparagna G.C. Amoscato A.A. McMillin J.B. Dowhan W. Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis.J. Biol. Chem. 2001; 276: 38061-38067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Kirkland R.A. Adibhatla R.M. Hatcher J.F. Franklin J.L. Loss of cardiolipin and mitochondria during programmed neuronal death: evidence of a role for lipid peroxidation and autophagy.Neuroscience. 2002; 115: 587-602Crossref PubMed Scopus (157) Google Scholar, 12Ott M. Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. Cytochrome c release from mitochondria proceeds by a two-step process.Proc. Natl. Acad. Sci. USA. 2002; 99: 1259-1263Crossref PubMed Scopus (796) Google Scholar, 13Fernandez M.G. Troiano L. Moretti L. Nasi M. Pinti M. Salvioli S. Dobrucki J. Cossarizza A. Early changes in intramitochondrial cardiolipin distribution during apoptosis.Cell Growth Differ. 2002; 13: 449-455PubMed Google Scholar, 14McMillin J.B. Dowhan W. Cardiolipin and apoptosis.Biochim. Biophys. Acta. 2002; 1585: 97-107Crossref PubMed Scopus (240) Google Scholar, 15Degli Esposti M. The mitochondrial battlefield and membrane lipids during cell death signalling.Ital. J. Biochem. 2003; 52: 43-50PubMed Google Scholar, 16Iverson S.L. Orrenius S. The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis.Arch. Biochem. Biophys. 2004; 423: 37-46Crossref PubMed Scopus (206) Google Scholar, 17Wright M.M. Howe A.G. Zaremberg V. Cell membranes and apoptosis: role of cardiolipin, phosphatidylcholine, and anticancer lipid analogues.Biochem. Cell Biol. 2004; 82: 18-26Crossref PubMed Scopus (122) Google Scholar), although the extent to which quantitative versus compositional changes in the CL pool influence these processes is poorly understood. CL has most often been studied using HPLC, which is able to separate CL from other phospholipids to assess changes in the amount of total CL. To date, determinations of CL acyl side chain composition have been largely qualitative, technically arduous, or limited to only a few of the many CL molecular species known to exist (18Schlame M. Shanske S. Doty S. Konig T. Sculco T. DiMauro S. Blanck T.J. Microanalysis of cardiolipin in small biopsies including skeletal muscle from patients with mitochondrial disease.J. Lipid Res. 1999; 40: 1585-1592Abstract Full Text Full Text PDF PubMed Google Scholar). Valianpour et al. (19Valianpour F. Wanders R.J.A. Barth P.G. Overmars H. VanGennip A.H. Quantitative and compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients.Clin. Chem. 2002; 48: 1390-1397Crossref PubMed Scopus (104) Google Scholar) reported an important advance in the quantitation of CL molecular species in platelets of Barth syndrome patients, with a quantitation method based on the abundance of the doubly charged species ([M-2H]2−). An alternative approach for the quantitative determination of CL and the resolution of the CL pool on the basis of fatty acyl side chain composition is reported here. A more thorough understanding of CL biosynthesis and how CL biosynthesis is altered by a variety of pathophysiological and physiological states requires a method for measuring changes in molecular CL species. To this end, we have successfully developed a method using electrospray ionization (ESI) quadrupole mass spectrometry based on the singly charged negative molecular ion ([M-H]−) of CL using a commercially available CL internal standard, 1,1′,2,2′-tetramyristoyl cardiolipin (M4CL), and have assessed its use in both isolated heart mitochondria and left ventricular cardiac tissue during the progression toward heart failure in spontaneously hypertensive heart failure (SHHF) rats, a well-established model of congestive heart failure (20McCune S.A. Park S. Radin M.J. Jurin R.R. The SHHF/Mcc-facp: a genetic model of congestive heart failure.in: Singal P.K. Dixon I.M.C. Beamish R.E. Dhalla N.S. Mechanisms of Heart Failure. Kluwer Academic Publishers, Boston1995: 91-106Crossref Google Scholar, 21McCune S.A. Baker P.B. Stills H.F. SHHF/Mcc-cp rat: model of obesity, non-insulin-dependent diabetes, and congestive heart failure.Ilar News. 1990; 32: 23-27Crossref Google Scholar). The CL internal standard, M4CL, and the CL reference standard containing four oleic acid side chains (O4CL) were obtained from Avanti Polar Lipids (Alabaster, AL) and reported as >99% pure. Beef heart CL was also from Avanti Polar Lipids. Deuterium-labeled [2H4]linoleic acid (9Z,12Z-octadecadienoic-9,10,12,13-d4 acid) was obtained from Cayman Chemicals (Ann Arbor, MI). HPLC solvents and hydrochloric acid were purchased from Fisher Chemicals (Fairlawn, NJ). Remaining chemicals were purchased from Sigma (St. Louis, MO). Male SHHF rats were provided out of the breeding colony maintained by S. McCune (University of Colorado at Boulder). All animals were treated according to the guidelines set forth by the University of Colorado Institutional Animal Care and Use Committee. The CL reference standards with four linoleate side chains (L4CL), three linoleates with one oleate (L3OCL), and two linoleates and two oleates (L2O2CL) were obtained from beef heart CL and purified by preparative reverse-phase HPLC. Briefly, a Chromolith Performance RP-18e (4.6 × 100 mm) column (Merck KGaA, Darmstadt, Germany) was used to separate the beef heart CL molecular species (1 mg repeated for four runs) using a mobile phase of isopropanol-20 mM ammonium acetate in water (2:1, v/v) for solvent A and hexane-isopropanol-20 mM ammonium acetate in for solvent B. was The was for and to by to by and was at which the solvents to for a total were at with an quadrupole mass 2000; was to a separate and for using the mass and the CL species of were CL molecular species with commercially available in as the reference standard to the standard The concentration of the CL species in reference standard was by as molecular species used as reference standards for the quantitative of on the CL in linoleate oleate mass of molecular species from to as a of the of in these but as the in this and the from bovine heart as in from bovine heart as in from bovine heart as in linoleate oleate The mass of molecular species from to as a of the of in these but as the in this and the from bovine heart as in in a The fatty acyl side chains of the four reference standards and the internal standard were from the CL by A fatty acid internal standard of acid was to was out by the addition of previously with to fatty acid a the was by the addition of previously with The fatty were two in (1 and were in a were as previously A. negative ion ionization of PubMed Scopus Google Scholar). Briefly, in and in were to were and at for to a of the were in for A standard containing [2H4]linoleic acid and known of and oleic was also and by The was a quadrupole mass which used a and a × × column and as the The column was programmed from to at The mass was in the negative ion ionization with used as the were in the ion were for of and from the column at of and The [2H4]linoleic acid was also at but at the of The for fatty was by the [2H4]linoleic acid as the quantitative of fatty acid in the A standard using known of and oleic was used to the of fatty over the range of quantitation of CL in reference standard of CL reference standard were with a amount of internal standard, and the were in v/v) on the mass the the for the singly charged was for both reference and internal the ion mobile and used the singly charged was in these with singly charged [M-H]− and doubly charged the solvent of CL using the quadrupole a of the doubly charged species Using a of mass the the of doubly charged was that the has a influence on the of singly charged CL Male SHHF rats and heart were using of hearts were in and left and and of these three were used for subsarcolemmal mitochondrial and the of the tissue was in for of CL from were isolated by the heart with containing 100 mM mM mM mM and mM and with The tissue was and using a for at The was at for the was and the was and The were and at for and the mitochondrial was in a containing mM mM mM mM mM and mM The mitochondrial were at were were isolated using a method of and E.G. A method of total lipid and J. Biochem. Physiol. PubMed Scopus Google Scholar). a protein determination was of mitochondrial protein and of was and as with the tissue previously heart mg of previously left ventricular tissue was using a in and its protein concentration was of tissue protein and of were of the were to either tissue or mitochondria: and of containing mM ammonium were for and at and the was to a and a of phospholipid from heart tissue or mitochondria were in 100 of for CL quantitation were a HPLC column 100 × to a mass in negative ion ion was out at with of of and of V. resolution was used with a of in the range of over a of The mobile phase was The solvent of hexane-isopropanol-20 mM ammonium acetate in in a ratio of for solvent A and v/v) for solvent B. were for from to from to and at which it to the of B. CL molecular species the column between and The of individual molecular species of CL were by the ratio of the of the CL molecular species of to the internal standard. with the and of the standard was used to of CL using the for a is is is and is of of CL by of protein the in of protein or was To the fatty acyl composition of CL species of mitochondrial from rats in heart failure were as with mg of mitochondrial protein and the internal standard. of the was by a quadrupole mass in the negative ion with a of of and as the was also using the quadrupole mass spectrometry in the negative ion with a of and as the Using either CL species of were in the of by the of acyl side chains from the CL fatty were as the molecular species identification M. Electrospray mass spectrometry of 2003; PubMed Scopus Google Scholar). All were using of in individual is known the changes that in individual molecular species of cardiac CL during To changes to CL during heart failure, negative ion mass spectrometry was used to study CL in mitochondria from failing rat The mass and heart SHHF rat heart mitochondrial molecular species of CL. changes in CL species were in the failing heart, most an in the of the CL pool of CL molecular species other as in The molecular species distribution in was with a decrease in an in minor or a of To the of the changes to CL during heart failure, a method was developed to CL using the addition of a commercially available internal standard, The of of the CL molecular species present in the failing hearts were using activation and tandem mass spectrometry to assess fatty acyl A of these shown in activation of the most CL species a ion with a at to The of the ion abundance at was most as the of acyl groups CL molecular species were activation of major to and A and In fatty acyl groups from to were used to molecular species of there be more species an [M-H]− at with fatty acyl groups that to the fatty by used to the species the most that this were with acyl side chains of and the molecular CL an of but was limited to a species by the of only two major at and for this species. the of this species was a CL with the acyl side chains this of it was to The to the CL species of molecular were in of the of different molecular species activation of at [M-H]− ion to species total acyl and of only the most the species present the side chains is and but many more minor species were present at this fatty were in these CL species. A of the major CL molecular species in the failing rat heart in with molecular as A of these in the rat were also studied using and to the composition of CL molecular species in the rats in heart acid side chain composition of various CL molecular species in failing rat heart mitochondria as by of on CL composition of the molecular ion based on tandem mass spectrometry shown in mass of molecular species from to as a of the of in these but as the in this and the of this is of the of the CL molecular ion was in either a tandem quadrupole or quadrupole mass and of fatty acyl groups in molecular species was by the of fatty acyl composition of the molecular ion based on tandem mass spectrometry shown in The mass of molecular species from to as a of the of in these but as the in this and the The of this is of the of the in a CL molecular ion was in either a tandem quadrupole or quadrupole mass and of fatty acyl groups in molecular species was by the of fatty acyl To mass spectrometry ion to a quantitative of it was to a internal standard most quantitative mass have used internal the of molecular species and the of CL on an with a molecular from the naturally occurring molecular species internal standard method was with to the ratio between the and the internal standard linear with to the concentration of the range of in the cardiac and of CL molecular species. The internal standard, was with four different CL molecular species for side chain and mass over a wide range of The CL of these were using and the ratio was from the for the reference standard to that of the internal standard. In the range the relationship of the ratio of the of CL reference standard and internal standard versus of reference standard was linear the on the in on the of side chain the feasibility of using this to quantitate CL molecular species of acyl chain was A of and a of were for the linear in The standard in was to heart mitochondrial with internal standard to assess changes in CL molecular species during heart Mitochondrial from SHHF rat hearts of three different heart failure were the of and internal as these individual molecular species from the HPLC in the The were as a of the of the internal standard. Using the and from the ratio of of CL molecular species to the internal standard the of which was for the amount of mitochondria heart protein used in the heart mitochondrial were in this changes in CL molecular species with heart failure were There was a in with of heart failure There were also many CL minor species that during heart failure The acyl chain of these in of CL molecular species in SHHF rat heart CL was using the standard in in with heart failure by species of CL that during heart failure and which identified in for these and by An was to separate out molecular species that by a for the CL at linoleate and one and the species at one and one The of the abundance that was to the abundance was using a previously and for Use in Scholar) with the for and of and P. H. of the Chem. 2003; Scopus Google Scholar). The of the of that was to the of to the species was of the In the rat heart, CL is studied using isolated this a more CL mitochondrial as in of tissue biopsies from patients, the tissue is and mitochondria require tissue for to be CL is to mitochondrial membranes and, in the heart, the of mitochondria is in other of CL in cardiac tissue was To the feasibility of using previously heart tissue lipids were isolated from left ventricular of the rats from which the mitochondrial were of the unique of the singly CL the ratio of other and the of CL to be from other phospholipid using there was by other shown in the quantitative of in left ventricular heart tissue was to the quantitative from isolated mitochondria CL is as an in the regulation of mitochondrial and as a that altered be the of mitochondrial An of on CL out with HPLC a that the total pool of CL in a it can be but this the to separate out different CL molecular species from is that the composition of in addition to its a role in its Valianpour et al. (19Valianpour F. Wanders R.J.A. Barth P.G. Overmars H. VanGennip A.H. Quantitative and compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients.Clin. Chem. 2002; 48: 1390-1397Crossref PubMed Scopus (104) Google Scholar) have used mass spectrometry to study CL in platelets of patients with Barth also used as an internal standard, there several between the doubly charged CL it more to the naturally occurring by and of the singly charged ion the resolution of these and a more for molecular species abundance CL species by only or We have also that the standard was of changes in CL acyl side chain composition over the range of In the the and of several CL molecular species present in mitochondrial or tissue were The rat heart CL from SHHF rats heart failure were to have a decrease in the amount of the major CL molecular species (L4CL), whereas the of several minor species with the of heart In the heart, of is in two separate CL is CL from phosphatidylglycerol containing fatty acyl The CL fatty acyl groups and a remodeling either G.M. and of from Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and or R. Blanck M. of cardiolipin by phospholipid Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) to these fatty acyl groups with the progression to heart failure, and CL containing other fatty acyl either a remodeling of the that is or a in the remodeling that is to which of these during heart In a of mitochondrial and tissue CL molecular a amount and in was to mitochondrial or tissue but mitochondrial protein of total heart tissue protein A and distribution in normal and Biol. Chem. Google Scholar), the of in tissue is is most to between the tissue and mitochondrial samples. although from the rats, were different for two mitochondria were isolated from the heart, whereas the tissue in were from the left and isolated mitochondria were only from the subsarcolemmal which whereas tissue We have that left ventricular which of the heart by has the CL of ventricular or tissue has been reported previously that mitochondria B. Hoppel C.L. of subsarcolemmal and mitochondria isolated from rat cardiac Biol. Chem. Full Text PDF PubMed Google Scholar) of a in total CL the subsarcolemmal mitochondria with S. Stoll Minkler P.E. R.G. Hoppel C.L. E.J. of cardiolipin during aging in rat heart 2002; Scopus Google Scholar) and with ischemia (7Lesnefsky E.J. Slabe T.J. Stoll M.S. Minkler P.E. Hoppel C.L. Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria.Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H2770-H2778Crossref PubMed Google Scholar). The and in to the of CL in heart In this for the of changes that in CL during various pathologies and it the of CL to both the quantitative and compositional alterations that in a variety of pathophysiological approach be in the processes that alterations in CL biosynthesis and remodeling and the of these alterations to normal mitochondrial The for with the was by Heart and and of and