Effect Of Cardiolipin Research Articles

Effect of cardiolipin on the lamellarity and elongation of liposomes hydrated in PBS

Lamellarity and shape are important factors in the formation of vesicles and determine their role in biological systems and pharmaceutical applications. Cardiolipin (CL) is a major lipid in many biological membranes and exerts a great influence on their structural organization due to its particular structure and physico-chemical properties. Here, we used small-angle X-ray and neutron scattering to study the effects of CL with different acyl chain lengths and saturations (CL14:0, CL18:1, CL18:2) on vesicle morphology and lamellarity in membrane models containing mixtures of phosphatidylcholine and phosphatidylethanolamine with different acyl chain lengths and saturations (C14:0 and C 18:1). Measurements were performed in the presence of Phosphate Buffer Saline (PBS), at 37°C, to better reflect physiological conditions, which resulted in strong effects on vesicle morphology, depending on the type and amount of CL used. The presence of small quantities of CL (from 2.5%) reduced inter-membrane correlations and increased perturbation of the membrane, an effect which is enhanced in the presence of matched shorter saturated acyl chains, and mainly unilamellar vesicles (ULV) are formed. In extruded vesicles, employed for SANS experiments, flattened vesicles are observed partly due to the hypertonic effect of PBS, but also influenced by the type of CL added.Our experimental data from SAXS and SANS revealed a strong dependence on CL content in shaping the membrane microstructure, with an apparent optimum in the PC:CL mixture in terms of promoting reduced correlations, preferred curvature and elongation. However, the use of PBS caused distinct differences from previously published studies in water in terms of vesicle shape, and highlights the need to investigate vesicle formation under physiological conditions in order to be able to draw conclusions about membrane formation in biological systems.

The effects of cardiolipin on the structural dynamics of the mitochondrial ADP/ATP carrier in its cytosol-open state

Cardiolipin (CL) has been shown to play a crucial role in regulating the function of proteins in the inner mitochondrial membrane. As the most abundant protein of the inner mitochondrial membrane, the ADP/ATP carrier (AAC) has long been the model of choice to study CL-protein interactions, and specifically bound CLs have been identified in a variety of crystal structures of AAC. However, how CL binding affects the structural dynamics of AAC in atomic detail remains largely elusive. Here we compared all-atom molecular dynamics simulations on bovine AAC1 in lipid bilayers with and without CLs. Our results show that on the current microsecond simulation time scale: 1) CL binding does not significantly affect overall stability of the carrier or structural symmetry at the matrix-gate level; 2) pocket volumes of the carrier and interactions involved in the matrix-gate network become more heterogeneous in parallel simulations with membranes containing CLs; 3) CL binding consistently strengthens backbone hydrogen bonds within helix H2 near the matrix side; and 4) CLs play a consistent stabilizing role on the domain 1-2 interface through binding with the R30:R71:R151 stacking structure and fixing the M2 loop in a defined conformation. CL is necessary for the formation of this stacking structure, and this structure in turn forms a very stable CL binding site. Such a delicate equilibrium suggests the strictly conserved R30:R71:R151stacking structure of AACs could function as a switch under regulation of CLs. Taken together, these results shed new light on the CL-mediated modulation of AAC function.

Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.

The Effects of Cardiolipin on Vascular Smooth Muscle Cell Dedifferentiation and Migration

PURPOSEThe initial phases of atherosclerosis involve the transition of smooth muscle cells (SMCs) to a synthetic, dedifferentiated phenotype, which is associated with alterations in receptor, ion channel and contractile filament composition. The late, irreversible atherosclerosis stages are characterized by SMC migration and subsequent formation of a ‘neointima.’ This leads to blood flow obstruction which, when destined to the cardiomyocytes, induces hypoxic conditions and myocardial infarction. Apoptotic cardiomyocytes release a phospholipid called cardiolipin (CL) into the systemic circulation. Previously, we have shown that physiological concentrations of CL inhibit endothelial cell migration. The objective of this project was to investigate the impact of CL on SMCs, specifically addressing cellular dedifferentiation and migration.METHODSFor this study, we used adult, male C57Bl/6 mice. Aortas from these mice were extracted and incubated in Dulbecco's modification of Eagle medium containing physiological CL concentrations (1 μM and 10 μM) at 37°C for 48 hours. We subsequently quantified contractile proteins (such as smooth muscle α‐actin and calponin) using immunoblotting to determine whether CL affects SMC dedifferentiation. These concentrations were also used to investigate the migration of SMCs using a migration assay. For this, we mimicked wounds by scraping off the cell culture plates' diameters to create cell‐free gaps. The plates were then incubated at 37°C for six days, during which the width of the gaps was measured every 48 hours under a light microscope.RESULTSThe immunoblotting results demonstrated a significant drop in calponin in a dose‐dependent manner with increasing CL concentrations, while the other de/differentiation markers remained unchanged. The migration assay revealed that the gap width was significantly narrower in control plates compared to CL‐treated plates.CONCLUSIONSOur results suggest that the effect of physiological concentrations of CL on SMCs is calponin‐specific and does not initiate dedifferentiation within 48 hours. The significantly wider gaps observed in the CL‐treated plates compared to the control plates indicate the potent effect CL has on inhibiting SMC migration. Since SMC migration is considered the key step in the atherogenic progression, our migration assay data suggest that, in a physiological context, CL has the potential to arrest the progression of the disease into its irreversible stages. Thus, we hypothesize the potential involvement of CL in developing anti‐atherosclerosis therapeutics.Support or Funding InformationCanadian Institute of Health Research (CIHR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of cardiolipin on the antimicrobial activity of a new amphiphilic aminoglycoside derivative on Pseudomonas aeruginosa

Amphiphilic aminoglycoside derivatives are promising new antibacterials active against Gram-negative bacteria such as Pseudomonas aeruginosa, including colistin resistant strains. In this study, we demonstrated that addition of cardiolipin to the culture medium delayed growth of P. aeruginosa, favored asymmetrical growth and enhanced the efficiency of a new amphiphilic aminoglycoside derivative, the 3’,6-dinonylneamine. By using membrane models mimicking P. aeruginosa plasma membrane composition (POPE:POPG:CL), we demonstrated the ability of 3’6-dinonylneamine to induce changes in the biophysical properties of membrane model lipid systems in a cardiolipin dependent manner. These changes include an increased membrane permeability associated with a reduced hydration and a decreased ability of membrane to mix and fuse as shown by monitoring calcein release, Generalized Polarization of Laurdan and fluorescence dequenching of octadecyl rhodamine B, respectively. Altogether, results shed light on how cardiolipin may be critical for improving antibacterial action of new amphiphilic aminoglycoside derivatives.

The Effect of Cardiolipin on Microglial Activation and Microglia‐Mediated Neuronal Death

Microglia, the immune cells of the brain, become activated in response to a variety of stimuli. Under normal physiological conditions, microglial activation is advantageous, as it produces an acute inflammatory response that helps rid the brain of the noxious stimuli through the release of pro‐inflammatory mediators and cytotoxins, including reactive oxygen species (ROS) and reactive nitrogen species (RNS). When microglial activation is sustained, however, the persistent and non‐specific release of these cytotoxic molecules results in damage to surrounding cells, including neurons and other microglia. Such state of chronic neuroinflammation is a characteristic feature of Alzheimer's disease (AD). Cardiolipin (CL), a phospholipid located in the inner mitochondrial membrane, is significantly reduced in AD brains. The remaining CL is exposed to the high levels of ROS present in AD brains, which modifies the structure of CL and hinders its ability to regulate mitochondrial processes. This results in the further production of ROS and extensive neuron death. The intracellular role of CL is well defined; however, the effects of extracellular CL on microglial activation, and subsequently on neuronal viability, have not been described.We utilized cell culture techniques to study the effects of extracellular CL on the activation of microglia, and their subsequent toxicity towards neurons. BV‐2 murine microglia, as well as human HL‐60 promyelocytic and THP‐1 monocytic cells (microglia models), were exposed to CL in the presence of noxious stimuli for 24 or 48 hours. Pro‐inflammatory mediators including tumor necrosis factor (TNF)‐alpha, monocyte chemoattractant protein (MCP)‐1, ROS and RNS in supernatant were quantified to determine the effect CL has on the secretory profile of activated microglia. Additionally, THP‐1 cell supernatants were transferred onto human SH‐SY5Y neuroblastoma cells (neuron model) to determine whether CL regulates microglia‐mediated neurotoxicity.We discovered that CL decreased the secretion of TNF‐alpha, MCP‐1, ROS and RNS from activated microglial cells. Additionally, CL reduced the cytotoxicity of activated THP‐1 cells towards SH‐SY5Y neuronal cells.These findings indicate that CL may act as a novel intercellular signaling molecule that is capable of reducing microglial activation, as well as neuron death. Further studies will explore glial signaling pathways engaged by CL and reveal its role in neuron‐microglia communication.

The nitrite reductase activity of horse heart carboxymethylated-cytochrome c is modulated by cardiolipin.

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0°C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k on=(7.3±0.7)×10(-2)M(-1)s(-1); at pH 7.4], whereas the value of k on for NO2 (-) reduction by CM-cytc-Fe(II) is 1.1±0.2M(-1)s(-1) (at pH 7.4). CL facilitates the NO2 (-)-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k on for the NO2 (-)-mediated conversion of CL-CM-cytc-Fe(II) to CL-CM-cytc-Fe(II)-NO (5.6±0.6M(-1)s(-1); at pH 7.4) being slightly higher than that for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO (2.6±0.3M(-1)s(-1); at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3±0.3)×10(-6)M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k on versus pH are -1.05±0.07 and -1.03±0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH(-). These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL-CM-cytc.

Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis

Cardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2 ) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c. Interactions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria. SS-31 interacted only with liposomes and bicelles containing cardiolipin in about 1:1 ratio. NMR studies demonstrated that the aromatic residues of SS-31 penetrated deep into cardiolipin-containing bilayers. SS-31 restored cytochrome c reduction and mitochondrial oxygen consumption in the presence of added cardiolipin. In fresh mitochondria, SS-31 increased state 3 respiration and efficiency of ATP synthesis. SS-31 selectively targeted cardiolipin and modulated its interaction with cytochrome c. SS-31 inhibited the cytochrome c/cardiolipin complex peroxidase activity while protecting its ability to serve as an electron carrier, thus optimizing mitochondrial electron transport and ATP synthesis. This novel class of cardiolipin therapeutics has the potential to restore mitochondrial bioenergetics for treatment of numerous age-related diseases.

Enhancing the peroxidase activity of cytochrome c by mutation of residue 41: implications for the peroxidase mechanism and cytochrome c release

The peroxidase activity of cytochrome c may play a key role in the release of cytochrome c from the mitochondrial intermembrane space in the intrinsic apoptosis pathway. Induction of the peroxidase activity of cytochrome c is ascribed to partial unfolding and loss of axial co-ordination between the haem Fe and Met80, and is thought to be triggered by interaction of cytochrome c with cardiolipin (diphosphatidylglycerol) in vivo. However, the reaction mechanism for the peroxidase activity of either native or cardiolipin-bound cytochrome c is uncertain. In the present study we analyse the peroxidase activity of human and mouse cytochrome c residue 41 variants and demonstrate that stimulation of peroxidase activity can occur without prior loss of Fe-Met80 co-ordination or partial unfolding. The effects of cardiolipin and mutation of residue 41 are not additive, suggesting that cardiolipin stimulates peroxidase activity by the same mechanism as residue 41 mutation. Consistent with this, mutation of residue 41 did not enhance apoptotic release of cytochrome c from mitochondria. We propose that mutation of residue 41, and interaction with cardiolipin, increase peroxidase activity by altering the 40-57 Ω loop and its hydrogen bond network with the propionate of haem ring A. These changes enhance access of hydrogen peroxide and substrate to the haem.

Impairment of brain mitochondrial functions by β-hemolytic Group B Streptococcus. Effect of cardiolipin and phosphatidylcholine

Group B Streptococcus (GBS) causes severe infection in the central nervous system. In this study, brain mitochondrial function was investigated by simulating infection of isolated mitochondria with GBS, which resulted in loss of mitochondrial activity. The β-hemolysin expressing strains GBS-III-NEM316 and GBS-III-COH31, but not the gGBS-III-COH31 that does not express β-hemolysin, caused dissipation of preformed mitochondrial membrane potential (Δψm). This indicates that β-hemolysin is responsible for decreasing of the reducing power of mitochondria. GBS-III-COH31 interacted with mitochondria causing increase of oxygen consumption, due to uncoupling of respiration, blocking of ATP synthesis, and cytochrome c release outside mitochondria. Moreover, the mitochondrial systems contributing to the control of cellular Ca(2+) uptake were lost. In spite of these alterations, mitochondrial phospholipid content and composition did not change significantly, as evaluated by MALDI-TOF mass spectrometry. However, exogenous cardiolipin (CL) and dipalmitoylphosphatidylcholine (DPPC) attenuated the uncoupling effect of GBS-III-COH31, although with different mechanisms. CL was effective only when fused to the inner mitochondrial membrane, probably reducing the extent of GBS-induced proton leakage. DPPC, which is not able to fuse with mitochondrial membranes, exerted its effect outside mitochondria, likely by shielding mitochondria against GBS β-hemolysin attack.

Relationship between cardiolipin metabolism and oxygen availability in Bacillus subtilis

We report changes of the content of anionic phospholipids in Bacillus subtilis in response to hypoxic conditions and inhibition of terminal respiration. Cardiolipin accumulates rapidly when bacteria are suspended in non-growth medium under reduced aeration or exposed to the inhibitor cyanide; the increase of cardiolipin occurs at the expense of its precursor phosphatidylglycerol and is temperature-dependent. Depending on the extent of hypoxic stress, membranes containing different levels of cardiolipin can be isolated from B. subtilis cells. The NADH oxidase activity in cardiolipin-enriched membranes is cyanide-resistant; furthermore O2 consumption measurements indicated that cardiolipin-enriched cells are resistant to cyanide. Results point out a possible interdependence between the effect of cyanide on cardiolipin metabolism and the effect of cardiolipin on the effectiveness of cyanide inhibition.

Peroxynitrite detoxification by horse heart carboxymethylated cytochrome c is allosterically modulated by cardiolipin

Carboxymethylation of equine heart cytochrome c (cyt c) changes its tertiary structure by disrupting the heme-Fe-Met80 distal bond, such that carboxymethylated cyt c (CM-cyt c) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) on peroxynitrite isomerization by ferric CM-cyt c (CM-cyt c-Fe(III)) is reported. Unlike native ferric cyt c (cyt c-Fe(III)), CM-cyt c-Fe(III) catalyzes peroxynitrite isomerization, the value of the second order rate constant ( k on) is 6.8 × 10 4 M −1 s −1. However, CM-cyt c-Fe(III) is less effective in peroxynitrite isomerization than CL-bound cyt c-Fe(III) (CL-cyt c-Fe(III); k on = 3.2 × 10 5 M −1 s −1). Moreover, CL binding to CM-cyt c-Fe(III) facilitates peroxynitrite isomerization ( k on = 5.3 × 10 5 M −1 s −1). Furthermore, the value of the dissociation equilibrium constant for CL binding to CM-cyt c-Fe(III) ( K = 1.8 × 10 −5 M) is lower than that reported for CL-cyt c-Fe(III) complex formation ( K = 5.1 × 10 −5 M). Although CM-cyt c-Fe(III) and CL-cyt c-Fe(III) display a different heme distal geometry and heme-Fe(III) reactivity, the heme pocket and the CL cleft are allosterically linked.

Effect of cardiolipin on the iron uptake of F1FOATP synthase in heart mitochondria

Effect of cardiolipin on the iron uptake of F1FOATP synthase in heart mitochondria

Mitochondrial dysfunction in rat brain with aging: Involvement of complex I, reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in brain aging process. Mitochondrial respiration is an important site of ROS production and hence a potential contributor to brain functional changes with aging. In this study we examined the effect of aging on complex I activity, oxygen consumption, ROS production and phospholipid composition in rat brain mitochondria. The activity of complex I was reduced by 30% in brain mitochondria from 24 months aged rats relative to young animals. These changes in complex I activity were associated with parallel changes in state 3 respiration. H 2O 2 generation was significantly increased in mitochondria isolated from aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 31% as function of aging, while there was a significant increase in the level of peroxidized cardiolipin. The age-related decrease in complex I activity in brain mitochondria could be reversed by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids. It is proposed that aging causes brain mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with brain aging.

Dose‐dependent protective effect of propofol against mitochondrial dysfunction in ischaemic/reperfused rat heart: role of cardiolipin

Ischaemia damages to the cardiac mitochondria by increasing generation of reactive oxygen species (ROS) and peroxidation of cardiolipin. The inhibited mitochondrial function leads to the cardiac injury during reperfusion. Propofol (2, 6-diisopropylphenol), an intravenous anaesthetic, has been shown to decrease cardiac ischaemia and reperfusion injury. In the present study, we propose that propofol protects mitochondrial function and decreases cardiac injury by prevention of cardiolipin peroxidation during ischaemia and reperfusion. After isolation of mitochondria from isolated rat heart perfused on a Langendorff model, various mitochondrial bioenergetic parameters were evaluated such as rates of mitochondrial oxygen consumption, H(2)O(2) production, complex I and III activity as well as the degree of lipid peroxidation and cardiolipin content. The action of propofol was also explored in isolated mitochondria. And the effect of cardiolipin was evaluated by fusing cardiolipin liposome with mitochondria. Propofol treatment had strong dose-dependent protection attenuating these parameters alterations in reperfused rat heart and isolated mitochondria. Additionally, cardiolipin treatment had the same protective effect, compared with propofol treatment at high concentration. The protective effect of propofol appears to be due, at least in part, as a chemical uncoupler, to the interruption of the vicious circle of ROS-cardiolipin-complexes of the respiratory chain-ROS through preserving the content and integrity of cardiolipin molecules by ROS attack. These findings may provide an explanation for some of the factors responsible for cardioprotection and one approach exploring an available antioxidant.

Mitochondrial dysfunction in rat with nonalcoholic fatty liver: Involvement of complex I, reactive oxygen species and cardiolipin

Mitochondrial dysfunction and oxidative stress play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). This study aimed to elucidate the mechanism(s) responsible for mitochondrial dysfunction in nonalcoholic fatty liver. Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days. We examined the effect of CD diet on various parameters related to mitochondrial function such as complex I activity, oxygen consumption, reactive oxygen species (ROS) generation and cardiolipin content and oxidation. The activity of complex I was reduced by 35% in mitochondria isolated from CD livers compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. Hydrogen peroxide (H 2O 2) generation was significantly increased in mitochondria isolated from CD livers. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 38% as function of CD diet, while there was a significantly increase in the level of peroxidized cardiolipin. The lower complex I activity in mitochondria from CD livers could be completely restored to the level of control livers by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD.

2P250 ウシラクトフェリシンと酸性リン脂質二重膜との相互作用およびその抗菌作用に及ぼすカルジオリピンの影響(生体膜・人工膜,口頭発表,第45回日本生物物理学会年会)

2P250 ウシラクトフェリシンと酸性リン脂質二重膜との相互作用およびその抗菌作用に及ぼすカルジオリピンの影響(生体膜・人工膜,口頭発表,第45回日本生物物理学会年会)

Effects of cardiolipin on carnitine palmitoyltransferase kinetics

We previously reported [Biochem. J. (1994) 299, 761-767] that certain phospholipids added to intact rat liver mitochondria increased sensitivity of carnitine palmitoyltransferase-I (CPT1A) to inhibition by malonyl-CoA. The most effective phospholipid was cardiolipin, which enhanced inhibition so that it mimicked the normal change in malonyl-CoA sensitivity occurring in the fasting to fed transition. Since cardiolipin is a normal component of eukaryotic mitochondrial membranes, we used mitochondria from Saccharomyces cerevisiae to test the hypothesis that the absence of cardiolipin in mitochondria in vivo would change the enzyme kinetics of CPT1. Yeast do not oxidize fatty acids in mitochondria. They do not express any of the components of the CPT system for facilitating transport of fatty acids into mitochondria. From the Greenberg lab we obtained a cardiolipin synthase knock-out strain of S. cerevisiae lacking cardiolipin and having mitochondrial function deficiencies [J. Biol. Chem. (2000) 275, 22387-94], and we obtained its parent strain for use as controls. We transformed both strains with an expression vector for CPT1A, and harvested mitochondria from the cells after breaking cells with glass beads. CPT1A in mitochondria from the parent wild-type yeast had a Km for carnitine of 0.65 ± 0.05 mM, while mitochondria from the cardiolipin synthase knockout yeast had a Km of 0.31 ± 0.03 mM. We also tested malonyl-CoA sensitivity and found mitochondria from wild-type yeast to have an I-50 (concentration of malonyl-CoA producing 50% inhibition) = 0.50 ± 0.07 microM, while mitochondria from cardiolipin synthase knockout yeast had an I-50 = 2.0 ± 0.3 microM. These results suggest that membrane cardiolipin in vivo alters CPT1A to increase its sensitivity to inhibition by malonyl-CoA, and this may be a physiologically significant mechanism for regulating fatty acid oxidation. (Supported by NIH Grant HL-66924)

Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin.

Reactive oxygen species (ROS) are considered an important factor in ischemia/reperfusion injury to cardiac myocytes. Mitochondrial respiration is an important source of ROS production and hence a potential contributor to cardiac reperfusion injury. In this study, we have examined the effect of ischemia and ischemia followed by reperfusion of rat hearts on various parameters related to mitochondrial function, such as complex I activity, oxygen consumption, ROS production, and cardiolipin content. The activity of complex I was reduced by 25% and 48% in mitochondria isolated from ischemic and reperfused rat heart, respectively, compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. The capacity of mitochondria to produce H2O2 increased on reperfusion. The mitochondrial content of cardiolipin, which is required for optimal activity of complex I, decreased by 28% and 50% as function of ischemia and reperfusion, respectively. The lower complex I activity in mitochondria from reperfused rat heart could be completely restored to the level of normal heart by exogenous added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is proposed that the defect in complex I activity in ischemic/reperfused rat heart could be ascribed to a ROS-induced cardiolipin damage. These findings may provide an explanation for some of the factors responsible for myocardial reperfusion injury.

A simple method to discriminate between β2-glycoprotein I- and prothrombin-dependent lupus anticoagulants

Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against beta2-glycoprotein I (beta2GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against beta2GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT-based clotting assay (PTT-LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between beta2GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-beta2GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti-beta2GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on beta2GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of beta2GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-beta2GPI antibodies of the CL-induced prolongation of the PTT-LA.