Unsaturated Cardiolipin Research Articles

Loss of stearoyl-CoA desaturase 2 disrupts inflammatory response in macrophages.

Macrophages are innate immune cells that patrol tissues and are the first responders to detect infection. They orchestrate the host immune response in eliminating invading pathogens and the subsequent transition from inflammation to tissue repair. Macrophage dysfunction contributes to age-related pathologies, including low-grade inflammation in advanced age that is termed "inflammaging." Our laboratory has previously identified that macrophage expression of a fatty acid desaturase, stearoyl-CoA desaturase 2 (SCD2), declines with age. Herein, we delineate the precise cellular effects of SCD2 deficiency in murine macrophages. We found that deletion of Scd2 from macrophages dysregulated basal and bacterial lipopolysaccharide (LPS)-stimulated transcription of numerous inflammation-associated genes. Specifically, deletion of Scd2 from macrophages decreased basal and LPS-induced expression of Il1b transcript that corresponded to decreased production of precursor IL1B protein and release of mature IL1B. Furthermore, we identified disruptions in autophagy and depletion of unsaturated cardiolipins in SCD2-deficient macrophages. To assess the functional relevance of SCD2 in the macrophage response to infection, we challenged SCD2-deficient macrophages with uropathogenic Escherichia coli and found that there was impaired clearance of intracellular bacteria. This increased burden of intracellular bacteria was accompanied by increased release of pro-inflammatory cytokines IL6 and TNF but decreased IL1B. Taken together, these results indicate that macrophage expression of Scd2 is necessary for maintaining the macrophage response to inflammatory stimuli. This link between fatty acid metabolism and fundamental macrophage effector functions may potentially be relevant to diverse age-related pathologies. IMPORTANCE Macrophages are immune cells that respond to infection, but their dysfunction is implicated in many age-related diseases. Recent evidence showed that macrophage expression of a fatty acid enzyme, stearoyl-CoA desaturase 2, declines in aged organisms. In this work, we characterize the effects when stearoyl-CoA desaturase 2 is deficient in macrophages. We identify aspects of the macrophage inflammatory response to infection that may be affected when expression of a key fatty acid enzyme is decreased, and these findings may provide cellular insight into how macrophages contribute to age-related diseases.

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.

Current Knowledge on the Role of Cardiolipin Remodeling in the Context of Lipid Oxidation and Barth Syndrome.

Barth syndrome (BTHS, OMIM 302060) is a genetic disorder caused by variants of the TAFAZZIN gene (G 4.5, OMIM 300394). This debilitating disorder is characterized by cardio- and skeletal myopathy, exercise intolerance, and neutropenia. TAFAZZIN is a transacylase that catalyzes the second step in the cardiolipin (CL) remodeling pathway, preferentially converting saturated CL species into unsaturated CLs that are susceptible to oxidation. As a hallmark mitochondrial membrane lipid, CL has been shown to be essential in a myriad of pathways, including oxidative phosphorylation, the electron transport chain, intermediary metabolism, and intrinsic apoptosis. The pathological severity of BTHS varies substantially from one patient to another, even in individuals bearing the same TAFAZZIN variant. The physiological modifier(s) leading to this disparity, along with the exact molecular mechanism linking CL to the various pathologies, remain largely unknown. Elevated levels of reactive oxygen species (ROS) have been identified in numerous BTHS models, ranging from yeast to human cell lines, suggesting that cellular ROS accumulation may participate in the pathogenesis of BTHS. Although the exact mechanism of how oxidative stress leads to pathogenesis is unknown, it is likely that CL oxidation plays an important role. In this review, we outline what is known about CL oxidation and provide a new perspective linking the functional relevance of CL remodeling and oxidation to ROS mitigation in the context of BTHS.

Unsaturation in the Fatty Acids of Phospholipids Drastically Alters the Structure and Toxicity of Insulin Aggregates Grown in Their Presence.

Lipid bilayers play an important role in the pathological assembly of amyloidogenic proteins and peptides. This assembly yields oligomers and fibrils, which are highly toxic protein aggregates. In this study, we investigated the role of saturation in fatty acids of two phospholipids that are present in cell membranes. We found that unsaturated cardiolipin (CL) drastically shortened the lag phase of insulin aggregation. Furthermore, structurally and morphologically different aggregates were formed in the presence of unsaturated CL vs saturated CL. These aggregates exerted drastically different cell toxicity. Both saturated and unsaturated phosphatidylcholine (PC) were able to inhibit insulin aggregation equally efficiently. Similar to CL, structurally different aggregates were formed in the presence of saturated and unsaturated PC. These aggregates exerted different cell toxicities. These results show that unsaturated phospholipids catalyze the formation of more toxic amyloid aggregates comparing to those formed in the presence of saturated lipids.

Investigation of cardiolipin oxidation products as a new endpoint for oxidative stress in C. elegans by means of online two-dimensional liquid chromatography and high-resolution mass spectrometry

The investigation of neurodegenerative and age-related diseases is a highly relevant topic in current research. Especially oxidative stress is thought to be the common underlying mechanism in diseases such as Parkinson’s or Alzheimer’s disease. The nematode Caenorhabditis elegans (C. elegans) is a prominent model organism, which is often used for such investigations and has gained extensive recognition in research regarding the linkage of reactive oxygen species (ROS) and neurodegeneration. Not only studies regarding genomics and proteomics have been increasingly conducted, also the number of studies based on the lipidome is rising. The phospholipid class of cardiolipin (CL) is a unique lipid class, which is exclusively located in mitochondria and is therefore of great relevance regarding oxidative stress and associated diseases. CL oxidation products have become a prominent marker for oxidative stress in various organisms. However, the CL distribution in the nematode C. elegans is still scarcely known on the molecular level and oxidation products have not yet been identified. In this work, we demonstrate the importance of CL distribution and the applicability of CL oxidation products as a sensitive marker for oxidative stress in C. elegans. For this reason, the CL distribution was determined by means of online two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry (2D-LC/HRMS). Subsequently, worms were treated with tert-butyl hydroperoxide (tBOOH) in order to provoke oxidative stress and induce the artificial formation of oxidized CL. We were able to detect increasing amounts of CL oxidation products of highly unsaturated CL species in a concentration-dependent manner. This finding emphasizes the great potential of CL oxidation products as a sensitive marker substance of oxidative stress in C. elegans, which is not only directly linked to mitochondria function but also favourable to other oxidative stress markers in terms of the needed sample material, relative substance stability and specificity of the oxidation site.

Cardiolipin in Immune Signaling and Cell Death.

Cardiolipin (CL) is a tetra-acylated diphosphatidylglycerol lipid. In physiological conditions, CL presents unsaturated chains and is located in the inner mitochondria membrane (IMM). Dead signals, infection, or disease may change the level of CL saturation and oxidation and cause its translocation to the cytosolic side of the outer mitochondrial membrane (OMM), affecting mitochondrial function and the inflammatory response. In this review, we summarize the emerging proapoptotic, pro-, and anti-inflammatory functions of cytosolic-exposed CL and how they are regulated by CL chain saturation and oxidation. We underline how the unique dimeric phospholipid structure confers peculiar properties on CL in the regulation of cell death and immune system proteins, such as the Nucleotide-binding domain and leucine-rich repeat-containing pyrin protein 3 (NLRP3), caspases (Casp), and Toll-like receptor 4 (TLR4). We also provide an overview of the human diseases in which CL deficiency or modification are implicated and of the use of exogenous unsaturated CL (uCL) as a novel therapeutic approach.

What Links Neutropenia to Immature Cardiolipin in Patients with Barth Syndrome (tafazzin-deficiency)?

Barth syndrome is an inherited X-linked disorder characterized by cardiomyopathy, skeletal muscle myopathy, and neutropenia. The syndrome arises because of inherited mutations in the gene TAZ, resulting in a loss of function of the protein tafazzin. Of note, a group of investigators recently described how tafazzin can regulate 'stemness' in models of acute myeloid leukemia (Cell Stem Cell, 2019). Tafazzin is an enzyme that processes the final step of cardiolipin maturation, replacing saturated with unsaturated acyl chains. Cardiolipin is a 4-tailed phospholipid that is almost-exclusively found in the inner membrane of the mitochondria. The lack of tafazzin activity results in a cardiolipin pool that contains more highly saturated lipid tails and it is this lack of unsaturated cardiolipins that contributes to a disorganized inner mitochondrial membrane. The link between tafazzin-deficiency and myopathy is generally explained by the dependence of muscle cells on mitochondrial function as well as oxidative respiration. The components of the electron transport chain are co-localized with cardiolipin in the inner mitochondrial membrane, and it is felt that their appropriate organization within the membrane lipid bilayer is dependent on the presence of mature cardiolipin which is lacking in those individuals with Barth syndrome. The link between tafazzin-deficiency and neutropenia is less clear. Neutrophils are terminally-differentiated effector cells of the innate immune system. They are critical for protection against bacterial and fungal pathogens and patients without sufficient neutrophils are among the most immunocompromised and at risk of lethal infection. Neutrophils have few mitochondria at baseline and are generally believed to rely primarily on glycolysis for energy production. It is not known if the mechanism of neutropenia in Barth syndrome is due to a lack of production or due to increased clearance (e.g. more prone to apoptosis). We undertook the study of tafazzin-deficient neutrophils to try to elucidate the mechanism of neutropenia in patients with Barth syndrome. We took advantage of an existing tafazzin-knockout mouse and a system of conditional immortalization of granulocyte-monocyte progenitors (GMP) using the ER-Hoxb8 system pioneered in our laboratory. This ER-Hoxb8 system allows for the unlimited ex vivo expansion of myeloid progenitors in the presence of estradiol and active Hoxb8. Once estradiol is removed from culture media, the Hoxb8 protein is inactive and the cells undergo normal, synchronous and terminal neutrophilic differentiation. In this manner, we were able to generate tafazzin-wild-type and knockout GMP lines from murine fetal liver cells. Analysis of the myeloid progenitor compartment in fetal liver cells (d14.5-d16.5) showed no difference between wild-type and knockout mice, arguing against a developmental defect (E15 results shown in PANEL A). Furthermore, the tafazzin-deficient ER-Hoxb8 GMPs and neutrophils were remarkably normal when tested across a variety of assays including phagocytosis, cytokine production and ROS generation (ROS by H2DCFDA shown in PANEL B). We hypothesized that the unpredictable neutropenia in patients with Barth Syndrome might be due to an increased proclivity to apoptosis because of the mitochondrial membrane defect. Indeed, the tafazzin-deficient GMPs showed an increased sensitivity to Bcl2-inhibition following treatment with ABT199 (PANEL C). Two lines of evidence have suggested that the increased tendency towards apoptosis may be due to endoplasmic-reticulum (ER) stress. (1) Transmission electron microscopy demonstrated 'swollen' ER in the tafazzin-deficient cells (not shown) and (2) a comparison of gene expression patterns demonstrated an increased expression of ATF4 and CHOP (DDIT3) in the tafazzin-deficient cells (PANEL D). We are now focused on validating these findings and in establishing models to confirm the ER-stress phenotype in vivo in the TAZ-knockout mouse model as well as primary samples from patients with Barth Syndrome. We hope that this line of work will confirm the mechanism of neutropenia and shed light on potential targets for therapeutic intervention. In addition, this very rare disorder has provided insight into a previously-unexpected link between neutrophil survival and the membrane integrity of the inner mitochondrial membrane. Figure Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-Founder.

Saturation of acyl chains converts cardiolipin from an antagonist to an activator of Toll-like receptor-4

Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1β release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases.Graphical abstract

Genetic re-engineering of polyunsaturated phospholipid profile of Saccharomyces cerevisiae identifies a novel role for Cld1 in mitigating the effects of cardiolipin peroxidation

Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL-specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CLOX, has been implicated in the cellular signaling events that initiate apoptosis. CLOX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ12-desaturase gene in yeast, which then synthesized poly unsaturated fatty acids(PUFAs) that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; it also resulted in increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CLOX, and treatment of cells with H2O2 increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.

Imaging mass spectrometry reveals loss of polyunsaturated cardiolipins in the cortical contusion, hippocampus, and thalamus after traumatic brain injury.

Traumatic brain injury (TBI) leads to changes in ion fluxes, alterations in mitochondrial function, and increased generation of reactive oxygen species, resulting in secondary tissue damage. Mitochondria play important signaling rolesincoordination of multiple metabolic platforms in addition to their well-known role in bioenergetics. Mitochondrial signaling strongly depends on cardiolipin (CL), a mitochondria-specific structurally unusual anionic phospholipid containing four fatty acyl chains. While our previous reports indicated that CL is selectively oxidized and presents itself as a target for the redox therapy following TBI, the topography of changes of CL in the injured brain remained to be defined. Here, we present a matrix-assisted laser desorption/ionization imaging study which reports regio-specific changes in CL, in a controlled cortical impact model of TBI in rats. Matrix-assisted laser desorption/ionization imaging revealed that TBI caused early decreases in CL in the contusional cortex, ipsilateral hippocampus, and thalamus with the most highly unsaturated CL species being most susceptible to loss. Phosphatidylinositol was the only other lipid species that exhibited a significant decrease, albeit to a lesser extent than CL. Signals for other lipids remained unchanged. Thisis the first study evaluating the spatial distribution of CL loss after acute brain injury. We propose that the CL loss may constitute an upstream mechanism for CL-driven signaling in different brain regions as an early response mechanism and may also underlie the bioenergetic changes that occur in hippocampal, cortical, and thalamic mitochondria after TBI.

Molecular species composition of plant cardiolipin determined by liquid chromatography mass spectrometry

Cardiolipin (CL), an anionic phospholipid of the inner mitochondrial membrane, provides essential functions for stabilizing respiratory complexes and is involved in mitochondrial morphogenesis and programmed cell death in animals. The role of CL and its metabolism in plants are less well understood. The measurement of CL in plants, including its molecular species composition, is hampered by the fact that CL is of extremely low abundance, and that plants contain large amounts of interfering compounds including galactolipids, neutral lipids, and pigments. We used solid phase extraction by anion exchange chromatography to purify CL from crude plant lipid extracts. LC/MS was used to determine the content and molecular species composition of CL. Thus, up to 23 different molecular species of CL were detected in different plant species, including Arabidopsis, mung bean, spinach, barley, and tobacco. Similar to animals, plant CL is dominated by highly unsaturated species, mostly containing linoleic and linolenic acid. During phosphate deprivation or exposure to an extended dark period, the amount of CL decreased in Arabidopsis, accompanied with an increased degree in unsaturation. The mechanism of CL remodeling during stress, and the function of highly unsaturated CL molecular species, remains to be defined.

Dietary fatty acids specifically modulate phospholipid pattern in colon cells with distinct differentiation capacities.

Although beneficial effects of the dietary n-3 docosahexaenoic acid (DHA) or butyrate in colon carcinogenesis have been implicated, the mechanisms of their action are not fully clear. Here, we investigated modulations of composition of individual phospholipid (PL) classes, with a particular emphasis on cardiolipins (CLs), in colon cells treated with DHA, sodium butyrate (NaBt), or their combination (DHA/NaBt), and we evaluated possible associations between lipid changes and cell fate after fatty acid treatment. In two distinct human colon cell models, foetal colon (FHC) and adenocarcinoma (HCT-116) cells, we compared patterns and composition of individual PL classes following the fatty acid treatment by HPLC-MS/MS. In parallel, we measured the parameters reflecting cell proliferation, differentiation and death. In FHC cells, NaBt induced primarily differentiation, while co-treatment with DHA shifted their response towards cell death. In contrast, NaBt induced apoptosis in HCT-116 cells, which was not further affected by DHA. DHA was incorporated in all main PL types, increasing their unsaturation, while NaBt did not additionally modulate these effects in either cell model. Nevertheless, we identified an unusually wide range of CL species to be highly increased by NaBt and particularly by DHA/NaBt, and these effects were more pronounced in HCT-116 cells. DHA and DHA/NaBt enhanced levels of high molecular weight and more unsaturated CL species, containing DHA, which was specific for either differentiation or apoptotic responses. We identified a wide range of CL species in the colon cells which composition was significantly modified after DHA and NaBt treatment. These specific CL modulations might contribute to distinct cellular differentiation or apoptotic responses.

Polyunsaturated fatty acids incorporation into cardiolipin in H9c2 cardiac myoblast

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), known as ω-3 polyunsaturated fatty acid (PUFA), are common nutrients in daily food intake and have been shown to prevent cardiovascular disease and improve cardiac functions. Cardiolipin is a mitochondrial phospholipid necessary for maintaining physiological function of mitochondria. Several studies have indicated that the cardiolipin acyl chain compositions affect the function of cardiolipin and mitochondria. Here, we investigated the structural changes of cardiolipin after DHA and EPA supplementation and compared them to arachidonic acid (AA) treatment. H9c2 cardiac myoblast was used as a cell model, and cardiolipin species was monitored and identified via LC-MS and MS/MS. Our results showed distinct mass envelopes of cardiolipin with the same carbon number but different double bonds in mass spectrum. There were 116 cardiolipin species with 36 distinct mass in 6 mass envelopes identified by MS/MS. Three days of PUFA treatment resulted in decreases of low-molecular-weight cardiolipin and increases of high-molecular-weight cardiolipin, suggesting the incorporation of exogenous DHA, EPA and AA into mitochondrial cardiolipin. PUFA incorporation was further verified by MS/MS analysis. More importantly, we found that DHA supplementation elevated the percent content of less unsaturated cardiolipin species and highly unsaturated cardiolipin species, containing ω-3 fatty acyl chains, indicating a ω-3 fatty acid incorporation mechanism with peroxidation protection. Our results indicate that PUFA supplementation differentially perturbed the fatty acyl chain compositions in the mitochondrial cardiolipin in the H9c2 cardiac myoblast, suggesting that mitochondrial membrane and the function of mitochondria are susceptible to exogenous lipid species.

Deletion of the cardiolipin‐specific phospholipase Cld1 rescues growth and lifespan defects in the tafazzin mutant: implications for Barth syndrome (605.4)

Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects and, in humans, results in the life‐threatening disorder Barth syndrome (BTHS). Tafazzin deficiency causes a decrease in the CL/MLCL ratio and decreased unsaturated CL species. Which of these biochemical outcomes contributes to the physiological defects is not known. Yeast cells have a single CL‐specific phospholipase, Cld1, which can be exploited to distinguish between these outcomes. The cld1Δ mutant has decreased unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ mutant. This suggests that defective growth and respiration in tafazzin‐deficient cells is caused by the decreased CL/MLCL ratio, not by a deficiency in unsaturated CL. CLD1 expression is increased during respiratory growth and regulated by the HAP transcriptional activation complex. Overexpression of CLD1 leads to decreased mitochondrial respiration and growth, and instability of mitochondrial DNA. However, ATP concentrations are maintained by increasing glycolysis. We conclude that transcriptional regulation of Cld1‐mediated deacylation of CL influences energy metabolism by modulating the relative contribution of glycolysis and respiration.Grant Funding Source: the Barth Syndrome Foundation

Deletion of the Cardiolipin-specific Phospholipase Cld1 Rescues Growth and Life Span Defects in the Tafazzin Mutant: IMPLICATIONS FOR BARTH SYNDROME

Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects, and in humans, this results in the life-threatening disorder Barth syndrome. Tafazzin deficiency causes a decrease in the CL/MLCL ratio and decreased unsaturated CL species. Which of these biochemical outcomes contributes to the physiological defects is not known. Yeast cells have a single CL-specific phospholipase, Cld1, that can be exploited to distinguish between these outcomes. The cld1Δ mutant has decreased unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ mutant. This suggests that defective growth and respiration in tafazzin-deficient cells are caused by the decreased CL/MLCL ratio and not by a deficiency in unsaturated CL. CLD1 expression is increased during respiratory growth and regulated by the heme activator protein transcriptional activation complex. Overexpression of CLD1 leads to decreased mitochondrial respiration and growth and instability of mitochondrial DNA. However, ATP concentrations are maintained by increasing glycolysis. We conclude that transcriptional regulation of Cld1-mediated deacylation of CL influences energy metabolism by modulating the relative contribution of glycolysis and respiration.

Acyl chain composition determines cardiolipin clustering induced by mitochondrial creatine kinase binding to monolayers

It has been recently shown that mitochondrial creatine kinase (mtCK) organizes mitochondrial model membrane by modulating the state and fluidity of lipids and by promoting the formation of protein–cardiolipin clusters. This report shows, using Brewster angle microscopy, that such clustering is largely dependent on the acyl chain composition of phospholipids. Indeed, mtCK-cardiolipin domains were observed not only with unsaturated cardiolipins, but also with the cardiolipin precursor phosphatidylglycerol. On the other hand, in the case of saturated dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin, mtCK was homogeneously distributed underneath the monolayer. However, an overall decrease in membrane fluidity was indicated by infrared spectroscopy as well as by extrinsic fluorescence spectroscopy using Laurdan as a fluorescent probe, both for tetramyristoylcardiolipin and bovine heart cardiolipin containing liposomes. The binding mechanism implicated the insertion of protein segments into monolayers, as evidenced from alternative current polarography, regardless of the chain unsaturation for the phosphatidylglycerols and cardiolipins tested.

Role of mitochondrial cardiolipin peroxidation in apoptotic photokilling of 5-aminolevulinate-treated tumor cells

In 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), ALA taken up by tumor cells is metabolized to protoporphyrin IX (PpIX), which sensitizes photodamage leading to apoptotic or necrotic cell death. Since lipophilic PpIX originates in mitochondria, we postulated that photoperoxidation of highly unsaturated cardiolipin (CL), which anchors cytochrome c (cyt c) to the inner membrane, is an early proapoptotic event. As initial evidence, PpIX-sensitized photooxidation of liposomal CL to hydroperoxide (CLOOH) species precluded cyt c binding, but this could be reinstated by GSH/selenoperoxidase (GPX4) treatment. Further support derived from site-specific effects observed using (i) a mitochondrial GPX4-overexpressing clone (7G4) of COH-BR1 tumor cells, and (ii) an ALA treatment protocol in which most cellular PpIX is either inside (Pr-1) or outside (Pr-2) mitochondria. Sensitized cells were exposed to a lethal light dose, and then analyzed for death mechanism and lipid hydroperoxide (LOOH) levels. Irradiated Pr-1 vector control (VC) cells died apoptotically following cyt c release and caspase-3 activation, whereas 7G4 cells were highly resistant. Irradiated Pr-2 VC and 7G4 cells showed negligible cyt c release or caspase-3 activation, and both types died via necrosis. CLOOH (detected long before cyt c release) accumulated ∼70% slower in Pr-1 7G4 cells than in Pr-1 VC, and this slowdown exceeded that of all other LOOHs. These and related findings support the hypothesis that CL is a key upstream target in mitochondria-dependent ALA-PDT-induced apoptosis.

Intramolecular hydrogen bonding in cardiolipin

Fourier transform infrared (FT-IR) spectroscopy was used to determine whether intramolecular hydrogen bonding between the C-OH and P-OH groups exists in beef heart cardiolipin (CL) or in hydrogenated beef heart cardiolipin (18:0-CL) as compared to the synthetic 2′-deoxy analogue of cardiolipin (16:0-dCL). Such intramolecular hydrogen bonding would provide a structural basis for proton conduction on the molecular level. In aqueous dispersions at 20°C, both 18:0-CL and 16:0-dCL exist in the gel phase as bilayers with gel to liquid-crystalline transitions ( T m) at 61 and 56°C, respectively, whereas the unsaturated CL exists in the non-bilayer (hexagonal II) state. Evidence for intramolecular hydrogen bonding of the C-OH group in aqueous dispersions of 18:0-CL is provided by the large increase in T m observed on changing the aqueous medium from H 2O to D 2O but specific hydrogen-bonded C-OH... PO 2 − species cannot be identified because water molecules also compete for the PO 2 − binding sites. However, C-OH... PO 2 − hydrogen bonds can be identified in dry films of the sodium salt of 18:0-CL or in CCl 4 solution. In contrast, such hydrogen bonds cannot be formed in the deoxy analogue (16:0-dCL) indicating that the central C-OH group in 18:0-CL could provide a structural basis for proton conduction, involving the phosphate groups.