Functional role of cardiolipin in mitochondrial bioenergetics

Cardiolipin is a unique phospholipid which is almost exclusively located in the inner mitochondrial membrane where it is biosynthesized. Considerable progress has recently been made in understanding the role of cardiolipin in mitochondrial function and bioenergetics. This phospholipid is associated with membranes designed to generate an electrochemical gradient that is used to produce ATP, such as bacterial plasma membranes and inner mitochondrial membrane. This ubiquitous and intimate association between cardiolipin and energy transducing membranes indicates an important role for cardiolipin in mitochondrial bioenergetic processes. Cardiolipin has been shown to interact with a number of proteins, including the respiratory chain complexes and substrate carrier proteins. Over the past decade, the significance of cardiolipin in the organization of components of the electron transport chain into higher order assemblies, termed respiratory supercomplexes, has been established. Moreover, cardiolipin is involved in different stages of the mitochondrial apoptotic process, as well as in mitochondrial membrane stability and dynamics. This review discusses the current understanding of the functional role that cardiolipin plays in several reactions and processes involved in mitochondrial bioenergetics. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Role of cardiolipin peroxidation and Ca in mitochondrial dysfunction and disease

Cardiolipin is a unique phospholipid which is almost exclusively located at the level of the inner mitochondrial membrane where it is biosynthesized. This phospholipid is known to be intimately involved in several mitochondrial bioenergetic processes. In addition, cardiolipin also has active roles in several of the mitochondrial-dependent steps of apoptosis and in mitochondrial membrane dynamics. Alterations in cardiolipin structure, content and acyl chains composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions, including ischemia/reperfusion, different thyroid states, diabetes, aging and heart failure. Cardiolipin is particularly susceptible to ROS attack due to its high content of unsaturated fatty acids. Oxidative damage to cardiolipin would negatively impact the biochemical function of the mitochondrial membranes altering membrane fluidity, ion permeability, structure and function of components of the mitochondrial electron transport chain, resulting in reduced mitochondrial oxidative phosphorylation efficiency and apoptosis. Diseases in which mitochondrial dysfunction has been linked to cardiolipin peroxidation are described. Ca²⁺, particularly at high concentrations, appears to have several negative effects on mitochondrial function, some of these effects being linked to CL peroxidation. Cardiolipin peroxidation has been shown to participate, together with Ca²⁺, in mitochondrial permeability transition. In this review, we provide an overview of the role of CL peroxidation and Ca²⁺ in mitochondrial dysfunction and disease.     

In yeast,cardiolipin unsaturation level plays a key role in mitochondrial function and inner membrane integrity

Cardiolipin is the signature phospholipid of the mitochondrial inner membrane. It participates in shaping the inner membrane as well as in modulating the activity of many membrane-bound proteins. The acyl chain composition of cardiolipin is finely tuned post-biosynthesis depending on the surrounding phospholipids to produce mature or unsaturated cardiolipin. However, experimental evidence showing that immature and mature cardiolipin are functionally equivalents for mitochondria poses doubts on the relevance of cardiolipin remodeling. In this work, we studied the role of cardiolipin acyl chain composition in mitochondrial bioenergetics, including a detailed bioenergetic profile of yeast mitochondria. Cardiolipin acyl chains were modified by genetic and nutritional manipulation. We found that both the bioenergetic efficiency and osmotic stability of mitochondria are dependent on the unsaturation level of cardiolipin acyl chains. It is proposed that cardiolipin remodeling and, consequently, mature cardiolipins play an important role in mitochondrial inner membrane integrity and functionality.     

Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues.

Non-immune activation of the first component of complement (C1) by the heart mitochondrial inner membrane has been investigated. Cardiolipin, the only strong activator of C1 among phospholipids, is present in large amounts in the heart mitochondrial inner membrane. We therefore studied its contribution to C1 activation by mitochondria. The proteins of the mitochondrial inner membrane were found to activate C1 only weakly, in contrast with the phospholipid fraction which induces strong C1 activation. Furthermore, the digestion of mitochondrial inner membranes with proteolytic enzymes did not affect C1 activation. Additional support in favour of cardiolipin being the responsible activator came from competition experiments with mitochondrial creatine kinase (mt-CPK) and adriamycin, known to bind to cardiolipin. Both mt-CPK and adriamycin displaced C1q from the mitochondrial inner membrane. In addition, C1q displaced mt-CPK bound to mitoplasts.     

Lipid packing variations induced by pH in cardiolipin-containing bilayers: the driving force for the cristae-like shape instability

Cardiolipin is a four-tailed acidic lipid found predominantly within the inner membrane of mitochondria, and is thought to be a key component in determining inner membrane properties and potential. Thus, cardiolipin may be involved in the dynamics of the inner membrane characteristic invaginations (named cristae) that protrude into the matrix space. In previous studies, we showed the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid model membrane mimicking the inner mitochondrial membrane. In addition, we reported a theoretical model describing the dynamics of a chemically driven membrane shape instability caused by a modification of the plane-shape equilibrium density of the lipids in the membrane. In the present work, we focus on the lipid-packing modifications observed in a model cardiolipin-containing lipid membrane submitted to pH decrease because this is the driving force of the instability. Laurdan fluorescence and ζ-potential measurements show that under pH decrease, membrane surface charge decreases, but that significant modification of the lipid packing is observed only for CL-containing membranes. Our giant unilamellar vesicle experiments also indicate that cristae-like morphologies are only observed for CL-containing lipid membranes. Taken together, these results highlight the fact that only a strong modulation of the lipid packing of the exposed monolayer leads to membrane shape instability and suggest that mitochondrial lipids, in particular the cardiolipin, play a specific role under pH modulation in inner mitochondrial membrane morphology and dynamics.     

Cardiolipin in energy transducing membranes

Cardiolipin is a phospholipid located exclusively in energy transducing membranes such as the bacterial cytoplasmic membrane and the inner membrane of mitochondria. It plays both a structural and a functional role in many multimeric complexes associated with these membranes. The role of cardiolipin in higher order organization of components of the mitochondrial respiratory chain revealed by a combined molecular genetic and biochemical approach is described.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 191–196.Original Russian Text Copyright © 2005 by Mileykovskaya, Zhang, Dowhan.This revised version was published online in April 2005 with corrections to the post codes.     

PTPMT1: connecting cardiolipin biosynthesis to mitochondrial function

Cardiolipin, a phospholipid component of the inner mitochondrial membrane, is required for mitochondrial metabolism. In this issue, Zhang et?al. (2011) highlight a critical role for PTPMT1, a mitochondrial phosphatase, in cardiolipin biogenesis and possibly in cardiolipin deficiency diseases. Their findings also unveil a yet uncharacterized pathway affecting cell growth.     

Cardiolipin content in mitochondria from cultured skin fibroblasts harboring mutations in the mitochondrial <Emphasis Type="Italic">ATP6</Emphasis> gene

The role of phospholipids in normal assembly and organization of the membrane proteins has been well documented. Cardiolipin, a unique tetra-acyl phospholipid localized in the inner mitochondrial membrane, is implicated in the stability of many inner-membrane protein complexes. Loss of cardiolipin content, alterations in its acyl chain composition and/or cardiolipin peroxidation have been associated with dysfunction in multiple tissues in a variety of pathological conditions. The aim of this study was to analyze the phospholipid composition of the mitochondrial membrane in the four most frequent mutations in the ATP6 gene: L156R, L217R, L156P and L217P but, more importantly, to investigate the possible changes in the cardiolipin profile. Mitochondrial membranes from fibroblasts with mutations at codon 217 of the ATP6 gene, showed a different cardiolipin content compared to controls. Conversely, results similar to controls were obtained for mutations at codon 156. These findings may be attributed to differences in the biosynthesis and remodeling of cardiolipin at the level of the inner mitochondrial transmembrane related to some mutations of the ATP6 gene.     

Cardiolipin dynamics and binding to conserved residues in the mitochondrial ADP/ATP carrier

Cardiolipin in eukaryotes is found in the mitochondrial inner membrane, where it interacts with membrane proteins and, although not essential, is necessary for the optimal activity of a number of proteins. One of them is the mitochondrial ADP/ATP carrier, which imports ADP into the mitochondrion and exports ATP. In the crystal structures, cardiolipin is bound to three equivalent sites of the ADP/ATP carrier, but its role is unresolved. Conservation of residues at these cardiolipin binding sites across other members of the mitochondrial carrier superfamily indicates cardiolipin binding is likely to be important for the function of all mitochondrial carriers. Multiscale simulations were performed in a cardiolipin-containing membrane to investigate the dynamics of cardiolipin around the yeast and bovine ADP/ATP carriers in a lipid bilayer and the properties of the cardiolipin-binding sites. In coarse-grain simulations, cardiolipin molecules bound to the carriers for longer periods of time than phosphatidylcholine and phosphatidylethanolamine lipids—with timescales in the tens of microseconds. Three long-lived cardiolipin binding sites overlapped with those in the crystal structures of the carriers. Other shorter-lived cardiolipin interaction sites were identified in both membrane leaflets. However, the timescales of the interactions were of the same order as phosphatidylcholine and phosphatidylethanolamine, suggesting that these sites are not specific for cardiolipin binding. The calculation of lipid binding times and the overlap of the cardiolipin binding sites between the structures and simulations demonstrate the potential of multiscale simulations to investigate the dynamics and behavior of lipids interacting with membrane proteins.     

Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass

Cardiolipin, a polyunsaturated acidic phospholipid, is found exclusively in bacterial and mitochondrial membranes where it is intimately associated with the enzyme complexes of the respiratory chain. Cardiolipin structure and concentration are central to the function of these enzyme complexes and damage to the phospholipid may have consequences for mitochondrial function. The fluorescent dye, 10 nonyl acridine orange (NAO), has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. Additionally, NAO staining has been used to measure the mitochondrial content of cells as dye binding to mitochondria is reportedly independent of the membrane potential. We used confocal microscopy to examine the properties of NAO in cortical astrocytes, neonatal cardiomyocytes and in isolated brain mitochondria. We show that NAO, a lipophilic cation, stained mitochondria selectively. However, the accumulation of the dye was clearly dependent upon the mitochondrial membrane potential and depolarisation of mitochondria induced a redistribution of dye. Moreover, depolarisation of mitochondria prior to NAO staining also resulted in a reduced NAO signal. These observations demonstrate that loading and retention of NAO is dependant upon membrane potential, and that the dye cannot be used as an assay of either cardiolipin or mitochondrial mass in living cells.     

Cardiolipin remodeling and the function of tafazzin

Cardiolipin, the specific phospholipid of mitochondria, is involved in the biogenesis, the dynamics, and the supramolecular organization of mitochondrial membranes. Cardiolipin acquires a characteristic composition of fatty acids by post-synthetic remodeling, a process that is crucial for cardiolipin homeostasis and function. The remodeling of cardiolipin depends on the activity of tafazzin, a non-specific phospholipid–lysophospholipid transacylase. This review article discusses recent findings that suggest a novel function of tafazzin in mitochondrial membranes. By shuffling fatty acids between molecular species, tafazzin transforms the lipid composition and by doing so supports changes in the membrane conformation, specifically the generation of membrane curvature. Tafazzin activity is critical for the differentiation of cardiomyocytes, in which the characteristic cristae-rich morphology of cardiac mitochondria evolves. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Interaction of the Intermembrane Space Domain of Tim23 Protein with Mitochondrial Membranes

Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23^IMS is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import.     

Cardiolipin mediates curcumin interactions with mitochondrial membranes

Curcumin, the main molecular ingredient of the turmeric spice, has been reported to exhibit therapeutic properties for varied diseases and pathological conditions. While curcumin appears to trigger multiple signaling pathways, the precise mechanisms accounting for its therapeutic activity have not been deciphered. Here we show that curcumin exhibits significant interactions with cardiolipin (CL), a lipid exclusively residing in the mitochondrial membrane. Specifically, we found that curcumin affected the structures and dynamics of CL-containing biomimetic and biological mitochondrial membranes. Application of several biophysical techniques reveals the CL-promoted association and internalization of curcumin into lipid bilayers. In parallel, curcumin association with CL containing bilayers increased their fluidity and reduced lipid ordering. These findings suggest that membrane modifications mediated by CL interactions may play a role in the therapeutic functions of curcumin, and that the inner mitochondrial membrane in general might constitute a potential drug target.     

Cardiolipin clusters and membrane domain formation induced by mitochondrial proteins

We show in this study that mitochondrial creatine kinase promotes segregation and clustering of cardiolipin in mixed membranes, a phenomenon that has been proposed to occur at contact sites in the mitochondria. This property of mitochondrial creatine kinase is dependent on the native octameric structure of the protein and does not occur after heat-denaturation or with the native dimeric form of the protein. Cardiolipin segregation was demonstrated by differential scanning calorimetry using membranes containing cardiolipin and either dipalmitoylphosphatidylethanolamine or 1-palmitoyl-2-oleoylphosphatidylethanolamine. Addition of the ubiquitous form of mitochondrial creatine kinase leads to the formation of a phosphatidylethanolamine-rich domain as a result of the protein binding preferentially to the cardiolipin. Such phase separation does not occur if cardiolipin is replaced with dioleoyl phosphatidylglycerol. Lipid phase separation is observed with other cardiolipin-binding proteins, including cytochrome c and, to a very small extent, with truncated Bid (t-Bid), as well as with the cationic polypeptide poly-L-lysine, but among these proteins the octameric form of mitochondrial creatine kinase is by far the most effective in causing segregation and clustering of cardiolipin. The proteins included in this study are found at mitochondrial contact sites where they are known to associate with cardiolipin. Domains in mitochondria enriched in cardiolipin play an important role in apoptosis and in energy flux processes.     

Solubilization, purification, and characterization of cardiolipin synthase from rat liver mitochondria. Demonstration of its phospholipid requirement.

Cardiolipin is a specific and functionally important phospholipid of mitochondria, and its biosynthesis is considered to be crucial for the assembly of this organelle. However, little information is available about the enzyme cardiolipin synthase, largely because it has not yet been isolated. We solubilized cardiolipin synthase from rat liver mitochondrial membranes with Zwittergent 3-14 and purified it by Mono Q anion exchange chromatography, Superose 12 gel filtration, and Mono P chromatofocusing. Cardiolipin synthase is one of the most acidic mitochondrial proteins (isoelectric point, pH 4-5) and appears as a 50-kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme requires CO2+ for activity, has an alkaline pH optimum (pH 8-9), and exhibits Km values of 45 and 1.6 microM for phosphatidylglycerol and CDP-diacylglycerol, respectively. Cardiolipin synthase loses activity during purification, and the activity can be partially reconstituted by the addition of phospholipids. The most effective phospholipid is phosphatidylethanolamine which reactivates in a cooperative manner. Cardiolipin reactivates hyperbolically at low concentrations but inhibits the enzyme at higher concentrations. In addition, cardiolipin shifts the sigmoidal reactivation curve of phosphatidylethanolamine toward lower concentrations. It is suggested that cardiolipin synthase requires interaction with several molecules of phosphatidylethanolamine and at least one molecule of cardiolipin for full enzymatic activity.     

The effect of hexadecaprenol on molecular organisation and transport properties of model membranes

The Langmuir monolayer technique and voltammetric analysis were used to investigate the properties of model lipid membranes prepared from dioleoylphosphatidylcholine (DOPC), hexadecaprenol (C80), and their mixtures. Surface pressure-molecular area isotherms, current-voltage characteristics, and membrane conductance-temperature were measured. Molecular area isobars, specific molecular areas, excess free energy of mixing, collapse pressure and collapse area were determined for lipid monolayers. Membrane conductance, activation energy of ion migration across the membrane, and membrane permeability coefficient for chloride ions were determined for lipid bilayers. Hexadecaprenol decreases the activation energy and increases membrane conductance and membrane permeability coefficient. The results of monolayer and bilayer investigations show that some electrical, transport and packing properties of lipid membranes change under the influence of hexadecaprenol. The results indicate that hexadecaprenol modulates the molecular organisation of the membrane and that the specific molecular area of polyprenol molecules depends on the relative concentration of polyprenols in membranes. We suggest that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols.     

Cardiolipin synthesis during the cell cycle of the yeastSaccharomyces cerevisiae

Cardiolipin synthesis was studied during the aerobio synchronous growth of aerobically grown yeastSaccharomyces cerevisiae. The time course of the synthesis was stepwise and the rise in cardiolipin level in cells coincided in time with the increase in cytochrome oxidase activity. This finding supports the notion of discontinuous completion of the inner mitochondrial membrane and hints at a close relation between cardiolipin and cytochrome oxidase activity.     

Identification of a Mammalian-type Phosphatidylglycerophosphate Phosphatase in the Eubacterium Rhodopirellula baltica

Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs.     

Membrane microenvironment regulation of carnitine palmitoyltranferases I and II

CPT (carnitine palmitoyltransferase) 1 and CPT2 regulate fatty acid oxidation. Recombinant rat CPT2 was isolated from the soluble fractions of bacterial extracts and expressed in Escherichia coli. The acyl-CoA chain-length-specificity of the recombinant CPT2 was identical with that of the purified enzyme from rat liver mitochondrial inner membranes. The Km for carnitine for both the mitochondrial preparation and the recombinant enzyme was identical. In isolated mitochondrial outer membranes, cardiolipin (diphosphatidylglycerol) increased CPT1 activity 4-fold and the Km for carnitine 6-fold. It decreased the Ki for malonyl-CoA inhibition 60-fold, but had no effect on the apparent Km for myristoyl-CoA. Cardiolipin also activated recombinant CPT2 almost 4-fold, whereas phosphatidylglycerol, phosphatidylserine and phosphatidylcholine activated the enzyme 3-, 2- and 2-fold respectively. Most of the recombinant CPT2 was found to have substantial interaction with cardiolipin. A model is proposed whereby cardiolipin may hold the fatty-acid-oxidizing enzymes in the active functional conformation between the mitochondrial inner and outer membranes in conjunction with the translocase and the acyl-CoA synthetase, thus combining all four enzymes into a functional unit.     

Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria

Mitochondria contribute to myocyte injury during ischemia. After 30 and 45 min of ischemia in the isolated perfused rabbit heart, subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, sustain a decrease in oxidative phosphorylation through cytochrome oxidase. In contrast, oxidation through cytochrome oxidase in interfibrillar mitochondria (IFM), located between the myofibrils, remains unaffected. Cytochrome oxidase activity in the intact membrane requires an inner mitochondrial membrane lipid environment enriched in cardiolipin. During ischemia, the content of cardiolipin decreased only in SSM, whereas the content of other phospholipids was preserved. Ischemia did not alter the composition of the cardiolipin that remained in SSM. Cardiolipin content was preserved in IFM during ischemia. Thus cardiolipin is a relatively early target of ischemic mitochondrial damage, leading to loss of oxidative phosphorylation through cytochrome oxidase in SSM.