CARDIOLIPIN (DIPHOSPHATIDYLGLYCEROL )

STRUCTURE, OCCURRENCE, BIOLOGY AND ANALYSIS

1. Structure and Composition

Cardiolipin is the trivial but universally used name for a lipid that should be correctly termed 'diphosphatidylglycerol' or more precisely 1,3-bis(sn-3'-phosphatidyl)-sn-glycerol. It is a unique phospholipid with in essence a dimeric structure, having four acyl groups and potentially carrying two negative charges. The tetra-linoleoyl molecular species, important in heart mitochondria, is illustrated.

Structure of cardiolipin

It is found almost exclusively in membranes of bacteria and of mitochondria, i.e. those whose function is to generate an electrochemical potential for substrate transport and ATP synthesis. The trivial name 'cardiolipin' is derived from the fact that it was first found in animal hearts, where it is especially abundant, but it can be found in mitochondria of all animal tissues and indeed of the eukaryotic kingdom. For example, it amounts to about 10% of the phospholipids of bovine heart muscle, and 20% of the phospholipids of the mitochondrial membrane in this organ. It is a minor component of human plasma lipoproteins, although it is the most abundant anionic lipid.

Scottish thistle Even with four identical acyl residues, cardiolipin has two chemically distinct phosphatidyl moieties, as two chiral centers exist, one in each outer glycerol group. These could give rise to diastereomers, although natural cardiolipin has the R/R configuration. In consequence, the two phosphate groups have different chemical environments, and they produce distinct ³¹P-NMR resonances. They are designated 1'-phosphate and 3'-phosphate with respect to the central glycerol. Each of them contains one acidic proton, but they have very different levels of acidity, i.e. pK₁ = 2.8 and pK₂ > 7.5. The weak acidity of the second phosphate is believed to be a result of formation of a stable intramolecular hydrogen bond with the central 2'-hydroxyl group. In fact, under normal physiological conditions, the molecule may carry only one negative charge, and the above figure may be inaccurate in displaying two. Molecular models of cardiolipin show that in aqueous dispersions its phosphates can form a tight bicyclic resonance structure with the central hydroxyl group, producing an acid-anion and giving an especially compact structure in which one of the protons is trapped.

Because of this unique structure, cardiolipin is able to form micellar, lamellar, and hexagonal states in aqueous dispersions, depending on pH and ionic strength. It is believed to exist mainly in a bilayer state in natural membranes, but with a tendency to form transient non-bilayer domains, which may have a profound influence on its function in vital cellular processes.

As there are four distinct fatty acyl groups in cardiolipin, the potential for complexity in the distribution within molecular species is enormous. However, the compositions can be remarkably simple, very different from those of other phospholipids, and in animals they are resistant to dietary manipulation. For example, in most animal tissues, cardiolipin contains almost exclusively 18 carbon fatty acids, and 80% of this is typically linoleic acid (18:2(n-6)). This appears to be true of higher plants also. Amongst animal tissues, testis cardiolipin is the exception in that it contains mainly palmitic acid. Yeast cardiolipin can differ in having mainly 16:1 and 18:1 fatty acids, while the bacterial lipid contains saturated and monoenoic fatty acids with 14 to18 carbons. In some marine species, cardiolipin contains only docosa- or tetracosahexaenoic acids (22:6 or 24:6), while lymphoblast cardiolipin contains only monoenoic fatty acids. Indeed, a common feature of this lipid in a variety of very different organisms is a relatively simple fatty acid composition, leading to a high degree of structural symmetry.

Analysis of the molecular species of cardiolipin, including the positional distributions on the various glycerol moieties, has been a technically daunting task, but it has been accomplished for many species and data for bovine heart and rat liver are listed in Table 1.

Source	Molecular species*				Amount (Mol%)

Table 1. The main molecular species of mammalian cardiolipin
	Fatty acid 1A	Fatty acid 2A	Fatty acid 1B	Fatty acid 2B

Bovine heart	18:2	18:2	18:2	18:2	48
	18:3	18:2	18:2	18:2 ]	\| 21 \|
	18:2	18:3	18:2	18:2 ]
	18:2	18:2	18:2	18:3 ]
	18:2	18:2	18:3	18:2 ]
	18:2	18:1	18:2	18:2 }	15
	18:2	18:2	18:2	18:1 }	15
Rat liver	18:2	18:2	18:2	18:2	57
	18:2	18:1	18:2	18:2 ]	35
	18:2	18:2	18:2	18:1 ]	35

* Fatty acyl residues are designated as shown in the formula above.
From Schlame, M., Brody,S. and Hostetler, K.Y. Eur. J. Biochem., 212, 727-735 (1993).

Until relatively recently, it was thought that cardiolipin was associated exclusively with the mitochondrial inner membrane, where it represents about 25% of the total phospholipids in bovine heart mitochondria, for example. It is now know to occur in the mitochondrial outer membrane also if only at a level of about 4%. However, this may be significant as it appears to predominate at sites connecting the outer membrane with the inner, where its unique physical properties may be important.

The highly specific location of cardiolipin is used as an argument in favour of the hypothesis that mitochondria are derived from prokaryotes, which lived inside a eukaryotic progenitor cell in symbiosis. If this did indeed occur, the function of cardiolipin has changed during evolution, as mitochondria require a constant level of cardiolipin to function correctly, while prokaryotes only appear to require it in specific circumstances.

2. Biosynthesis and Metabolism

The biosynthetic pathway to cardiolipin is similar to that of some other phospholipids in that it passes through the common intermediates, phosphatidic acid and phosphatidyl-CDP (see our web pages on phosphatidylglycerol). However, the final step is a unique reaction, which is different in prokaryotes and eukaryotes. In prokaryotes such as bacteria, diphosphatidylglycerol synthase catalyses a transfer of the phosphatidyl moiety of one phosphatidylglycerol to the free 3'-hydroxyl group of another, with the elimination of one molecule of glycerol, via the action of an enzyme related to phospholipase D. The enzyme can operate in reverse under some physiological conditions. Cardiolipin biosynthesis is regulated via that of phosphatidylglycerol.

formula

With eukaryotes, the first committed step in step in the biosynthesis of cardiolipin is the formation phosphatidylglycerolphosphate, a key intermediate in the biosynthesis of phosphatidylglycerol (as described in the web page on phosphatidylglycerol). The diphosphatidylglycerol synthase (a phosphatidyl transferase) then links phosphatidylglycerol to diacylglycerol phosphate from the activated phosphatidyl moiety cytidine diphosphate diacylglycerol, with elimination of cytidine monophosphate (CMP). In rat liver, the cardiolipin synthase resides in the inner mitochondrial membrane, while in yeast it is part of a large protein complex. The catalytic centre of cardiolipin synthase is exposed to the matrix side of the inner membrane.

formula

As eukaryotic cardiolipin synthase is a mitochondrial enzyme and mitochondria are believed to be phylogenetic derivatives of ancient prokaryotes, it appears strange that there has been such a change in mechanism. In eukaryotes, cardiolipin is the only phospholipid synthesised in the mitochondrion, and it remains there for the life of the cell.

The ultimate fatty acid composition of cardiolipin in eukaryotes is attained by a cycle of acylation-deacylation or re-modelling in which an enzyme termed ‘tafazzin’ plays a major part. This is necessary as the precursor phospholipids are very different in composition from that which is apparently required in the final product if it is to function correctly. In particular, tafazzin is known to transfer linoleate groups highly selectively from phosphatidylcholine to monolysocardiolipin thus promoting molecular symmetry among the molecular species. The reaction does not require a coenzyme A ester as an intermediate, and it is reversible. This is believed to be the first CoA-independent phospholipid transacylase to have been identified, and it may be involved in re-modelling of other phospholipid classes. The reaction is thus quite different from the cycle of acylation and deacylation involved in the remodelling of other phospholipids. It must be assumed that the monolysocardiolipin intermediate is produced by the action of a phospholipase A₂, various forms of which have been identified in mitochondria.

In yeast, tafazzin is located in outer leaflet of the inner membrane so cardiolipin must be translocated by means of a scramblase or other transporter from the inner leaflet of this membrane for remodelling to occur. Similarly, the final product must be transported by appropriate mechanisms to its final membrane destination.

Catabolism of cardiolipin may occur by the action of phospholipase A₂ to remove fatty acyl groups, possibly after oxidation as part of the process of apoptosis (see below). There is also a specific mitochondrial phospholipase D, which hydrolyses cardiolipin to phosphatidic acid (and phosphatidylglycerol) and in so doing promotes the fusion of mitochondria. The rate of hydrolysis by all phospholipases is significantly higher in the 3'-phosphatidyl moiety.

3. Function

As cardiolipin is the specific lipid component of mitochondria, its biological function in this organelle is clearly crucial. It is located mainly on the inner membrane of mitochondria, where it interacts with a large number of mitochondrial proteins. This interaction effects functional activation of certain enzymes, especially those involved in oxidative phosphorylation and photo-phosphorylation. In animals, the respiratory chain consists of four enzymes ((NADH dehydrogenase, succinate dehydrogenase, cytochrome bc1 complex, and cytochrome oxidase), which are now believed to be organized in large complexes, designated complexes I to IV and constituting a supramolecular network. Cardiolipin has been identified as an integral component in crystals of mitochondrial complex III, complex IV and the ADP-ATP-carrier. Similarly in yeast, the related enzymes appear to from part of a single functional unit, components of which contain tightly bound cardiolipin. This is an essential component of the interface between the complex and its membrane environment or between subunits within the complex. Removal of cardiolipin leads to break-up of the complex and loss of functionality.

thistle The physical chemistry of the interaction between cardiolipin and enzymes is the key to understanding this function. Cardiolipin has a strong binding capacity for many structurally unrelated proteins, so its structure must be adapted to differing protein surfaces. This interaction has been studied intensively for the cytochrome bc1 complex of the respiratory chain, which couples electron transfer between ubiquinol and cytochrome c to the translocation of protons across the lipid bilayer. One cardiolipin molecule is bound close to the site of ubiquinone reduction and is believed to ensure the stability of the catalytic site as well as being involved in proton uptake. In general, the head group of cardiolipin and certain amino acid residues interact strongly via electrostatic forces, hydrogen bonds, and water molecules, while the acyl chains retain their flexibility and interact through van-der-Waals forces with the protein surface at a number of sites. However, we do not yet know why the highly specific fatty acid and molecular species compositions are necessary for these functions.

Cardiolipin has been implicated in the process of apoptosis (programmed cell death) in some animal cells through an interaction with cytochrome c. The enzyme is believed to act as a peroxidase to generate oxidized cardiolipin, which can no longer bind to it. The consequence is that the cytochrome c is released into the inter-membrane space, while the oxidized cardiolipin is translocated to the outer mitochondrial membrane and participates in the formation of the mitochondrial permeability transition pore that facilitates release of cytochrome c into the cytosol where it triggers apoptosis. Specific non-typical molecular species of cardiolipin with a high content of arachidonic and docosahexaenoic acid may be involved in the process. As a result, the cellular concentration of cardiolipin decreases rapidly while some monolyso-cardiolipin may accumulate.

thistle Cardiolipin is believed to be an important cofactor for cholesterol translocation from the outer to the inner mitochondrial membrane, and in steroidogenic tissues, it activates mitochondrial cholesterol side-chain cleavage and is a potent stimulator of steroidogenesis. Cardiolipin may also have a specific role in the import of proteins into mitochondria, and it can behave as a molecular chaperone to promote folding of mitochondrial proteins. It binds in a highly specific way to the DNA in eukaryotic chromatin (the material of which chomosomes are composed), and indeed all of this lipid in chromatin is bound to DNA, where both have a common 'interphosphate' structural motive. Thus, cardiolipin appears to have a functional role in the regulation of gene expression. As a component of the plasma lipoproteins, it is believed to have an anti-coagulant function.

In higher plants, cardiolipin is an integral constituent of the photosystem II complexes, which are also involved in oxidative processes, where it may be required for the maintenance of structural and functional properties. In eubacteria, it also has a role in oxidative phosphorylation, but can be replaced by other phospholipids in selected mutants at least. Bacterial membranes are believed to contain regions of micro-domains of cardiolipin (and of phosphatidylethanolamine), which assemble spontaneously because of the intrinsic physical properties of the lipid. These micro-domains seem be located where there is intense phospholipid biosynthesis and may be relevant to other cellular processes, including cell division and sporulation.

4. Cardiolipin in Disease

Barth syndrome, a human disease state (an infantile cardiomyopathy) linked to the X-chromosome, is associated with marked abnormalities in the composition of cardiolipin, i.e. a decrease in tetra-linoleoyl molecular species, and an accumulation of monolysocardiolipin. There is evidence that the metabolic defect involves a phospholipid acyltransferase (‘tafazzin’) that under normal circumstances remodels cardiolipin by a specific transference of linoleate (see above). The consequence may be a reduction in the efficiency of oxidative phosphorylation in mitochondria or an increase in the permeability of the mitochondrial membranes. Similar phenomena have been observed in yeast mutants that lack the corresponding acyltransferase.

In addition, reductions in the concentrations of cardiolipin or changes in its composition in heart mitochondria have been implicated in many different human diseases states, including heart failure and diabetes, although it is not clear whether these effects are symptoms or the cause. Although oxidation of cardiolipin is part of the normal process of apoptosis, there is evidence that the proximity of this lipid to highly reactive oxygen species can lead to excessive peroxidation and oxidative stress, for example in the ischemic heart and skeletal muscle or during aging. Malfunctions of cardiolipin metabolism in brain mitochondria have been implicated in Alzheimer’s disease and Parkinson’s disease.

The bacteria responsible for syphilis produce antibodies to cardiolipin. Although the reasons for this are not properly understood, cardiolipin is widely used an antigen in tests for the disease. Antibodies to cardiolipin are used in diagnostic tests after unexplained venous or arterial thrombotic episodes or recurrent miscarriages.

5. Related Lipids

Animals and higher plants appear to contain only cardiolipin per se, but structural analogues of diphosphatidylglycerol, such as phosphatidylglycerophosphoglycerol, D-glucopyranosylcardiolipin, D-alanylcardiolipin, L-lysylcardiopin and phosphatidylglycerol acetal of plasmenyl diphosphatidylglycerol, have been found in bacteria. In these lipids, the alanyl, lysyl and glucosyl residues are linked to the hydroxyl-2' on the central glycerol moiety. The amino-acid containing lipids are obviously related to the complex lipoamino acids derived from phosphatidylglycerol. In the glucosylcardiolipin from Geobacillus stearothermophilus, the main fatty acids present were iso-and anteiso-methyl branched 15:0 to 17:0 fatty acids.

lysyl-cardiolipin

In Halobacterium salinarum (Archaea), osmotic shock induces formation of an even more complex lipid consisting of sulfo-triglycosyl-diether esterified to the phosphate group of phosphatidic acid, which has been termed 'glycocardiolipin'. However, use of the term ‘cardiolipin’ in this instance seems something of a misnomer as there is no central glycerol-phosphate unit.

6. Analysis

Cardiolipin elutes close to phosphatidylglycerol and phosphatidic acid in many chromatographic systems, but it can be resolved with care. Modern liquid chromatography-mass spectrometry and 'shotgun lipidomics' techniques are proving to be sensitive and specific. However, such methods do not distinguish between the two chiral glycerol moieties, so detailed analyses of molecular species and positional distributions are still difficult technical problems. The papers by Schlame et al. cited below are invaluable guides.

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