Mitochondrial phospholipid composition and microviscosity in myocardial ischaemia

Normothermic ischaemic arrest of the isolated perfused rat heart causes profound changes in mitochondrial ultrastructure. Since the mitochondrial membranes contain a high percentage of phospholipids, an evaluation of the effect of different periods of ischaemia on mitochondrial phospholipid content and fatty acid composition was made. The results showed that ischaemia had no effect on the content of the different phospholipid classes and no correlation was observed between ultrastructural changes and mitochondrial phospholipid content. However, the phospholipid fatty acid composition of several phospholipids showed marked changes. For example, with lysophosphatidylcholine a progressive increase in the percentage saturated fatty acids was observed with increasing periods of ischaemia, while a reduction occurred in lysophosphatidylethanolamine. To determine whether the ischaemia-induced changes in mitochondrial phospholipid fatty acid composition had an effect on the physical properties of the membrane, the microviscosity of mitochondrial preparations was studied, using the lipophilic probe, 1,6-diphenyl-1,3,5-hexatrine. Mitochondria isolated from ischaemic hearts showed a progressive increase in fluorescence polarization with longer periods of ischaemia, indicating an overall increase in microviscosity. This phenomenon may be responsible for the increased mitochondrial fragility which is characteristic of ischaemic damage.     

Changes in muscle mitochondrial lipid composition resulting from training and exhaustive exercise.

This study was undertaken to determine if the changes in mitochondrial structure and function that occur in muscle with exhaustive exercise could be caused by alterations in lipid composition of mitochondrial membranes. Further, the effect of training on lipid composition was studied to ascertain if lipid changes accompany the adaptation in the level of mitochondrial protein. Training decreased free fatty acids and triglycerides. Exhaustion of untrained animals resulted in increases of total phospholipid and phosphatidyl choline while exhaustion of trained rats caused a lowering of total phospholipid and phosphatidyl choline. Alterations in membrane lipid composition are most likely not the cause of changes in mitochondrial structure and function after exhaustive exercise since mitochondrial yield and lipid levels did not change in concert; i.e. muscle mitochondrial yield was decreased in both untrained and trained rats while total phospholipids were increased in untrained rats and decreased in trained rats as a result of exhaustive exercise. Although the physiological significance of the effects observed remains to be determined, this study does demonstrate that the lipid composition of mitochondria is not a constant parameter but can change in response to a chronic (training) or acute (exhaustive exercise) physiological condition.     

Interaction of mitochondrial malate dehydrogenase monomer with phospholipid vesicles.

The association between bovine and porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) and phospholipid vesicles was investigated. At concentrations at which malate dehydrogenase exists as a dimer, entrapment within the aqueous compartment but not binding of the 14C-labelled enzyme was observed. The dissociated enzyme was labile to moderate heat and to p-chloromercuribenzoate, but in both cases inactivation was decreased by incubation with suspensions of charged phospholipid vesicles. This suggested an interaction between enzyme subunits and phospholipid, and this was confirmed by direct binding measurements and by studies that followed changes in the fluorescein-labelled enzyme. The circular-dichroism spectra of the enzyme indicated a high alpha-helix content, and suggested that a small conformational change occurred when the enzyme dissociated. Fluorescence data also suggested less-rigid molecules after dissociation. A possible mechanism, based on the flexibility of enzyme monomer and its interaction with phospholipids, by which mitochondrial matrix enzymes are specifically localized in cells, is discussed.     

Effect of different levels of thyroid hormones on the fatty acid composition of lipids from rat liver mitochondria

The abundance or deficiency of thyroid hormones in rat organism influence the unsaturation and desaturation indices of total lipid fatty acids and phospholipids in liver mitochondria. The most conspicuous changes were observed in the fatty acid composition of the phospholipid fraction. The changes in the structure and function of rat liver mitochondria are considered to be due to alterations in the fatty acid composition of mitochondrial phospholipids.     

Modulation of the lipid composition of boar sperm plasma membranes during an acrosome reaction in vitro

An in vitro acrosome-like reaction was induced in spermatozoa from the boar cauda epididymis by incubation in Tyrode's solution containing 1 mg/ml fatty acid-free bovine serum albumin. Plasma membranes were isolated from the spermatozoa at different times during the incubation and analyzed for their lipid composition. The total lipid, phospholipid, and glycolipid content of the membranes did not change during the acrosome-like reaction, whereas the amount of diacylglycerols and free fatty acids increased. Within the phospholipid class, a decrease of the inositol phospholipid and and sphingomyelin content was observed, whereas the other phospholipids of the plasma membranes did not decrease significantly after 2 h of incubation. Changes in the sterol composition of the membranes were also observed. The onset of the lipid changes was correlated with the uptake of extracellular calcium by the spermatozoa. These results for the lipid changes in isolated sperm plasma membranes during an in vitro acrosome reaction provide the first direct evidence that a modulation of the plasma membrane lipid composition is involved in an acrosome-like reaction of mammalian spermatozoa.     

Dietary fatty acids modulate liver mitochondrial cardiolipin content and its fatty acid composition in rats with non alcoholic fatty liver disease

No data are reported on changes in mitochondrial membrane phospholipids in non-alcoholic fatty liver disease. We determined the content of mitochondrial membrane phospholipids from rats with non alcoholic liver steatosis, with a particular attention for cardiolipin (CL) content and its fatty acid composition, and their relation with the activity of the mitochondrial respiratory chain complexes. Different dietary fatty acid patterns leading to steatosis were explored. With high-fat diet, moderate macrosteatosis was observed and the liver mitochondrial phospholipid class distribution and CL fatty acids composition were modified. Indeed, both CL content and its C18:2n-6 content were increased with liver steatosis. Moreover, mitochondrial ATP synthase activity was positively correlated to the total CL content in liver phospholipid and to CL C18:2n-6 content while other complexes activity were negatively correlated to total CL content and/or CL C18:2n-6 content of liver mitochondria. The lard-rich diet increased liver CL synthase gene expression while the fish oil-rich diet increased the (n-3) polyunsaturated fatty acids content in CL. Thus, the diet may be a significant determinant of both the phospholipid class content and the fatty acid composition of liver mitochondrial membrane, and the activities of some of the respiratory chain complex enzymes may be influenced by dietary lipid amount in particular via modification of the CL content and fatty acid composition in phospholipid.     

Effect of heat inactivation and freezing on fatty acid composition of plasma and red blood cells.

The effect of heat inactivation and freezing on fatty acid composition of plasma and red blood cells was investigated. Analysis was completed at baseline; after freezing; after incubation; after incubation and subsequent freezing; after incubation, freezing and a second incubation; and after freezing and subsequent incubation. There were changes in fatty acid levels observed in all groups with the phospholipid fractions showing the greatest changes. Those bloods that had been incubated, frozen and incubated again, and those which had been frozen initially followed by incubation showed the greatest change when compared to baseline samples. Even though there were changes in fatty acid levels seen in all groups, the changes were small except in those two groups. Treatment of blood with either of those two treatment regimens changes the fatty acid values so that they do not accurately reflect the composition of fatty acids in the blood.     

Changes in fatty acid composition of phospholipids and triglycerides of Musca domestica resulting from choline deficiency

The fatty acid composition of the phospholipids and triglycerides extracted from housefly larvae reared on diets containing no added fatty acids but containing differing concentrations of choline has been determined. Reducing the choline content of the diet resulted in a graded reduction of the percentage of phosphatidylcholine present in the phospholipids of the larvae. This was accompanied by changes in the fatty acid composition, choline deficiency causing an increased utilization of 16-C rather than 18-C acids by the phospholipids. Changes in the fatty acid composition of the triglyceride fraction were also observed but these were associated with insects containing very low levels of phosphatidylcholine. Examination of the fatty acids in the different classes of phospholipids showed that the major change resulting from choline deficiency was in the fatty acids of the phosphatidylethanolamine fraction—the phospholipid which increased as the phosphatidylcholine decreased.Although the fatty acid composition of the different classes of phospholipids was not completely fixed, some preferential utilization of certain fatty acids by certain classes was observed, in both larval and adult insects. The fatty acid composition of the phospholipids extracted from larval gut, muscle, fat body, cuticle, trachea, nervous tissue, and haemolymph was determined. Changes resulting from choline deficiency similar to those seen in the whole larva were observed in all tissues except the nervous tissue. The effect of rearing larvae at temperatures between 24 and 35°C resulted in only minor changes in the fatty acid composition of both phospholipid and triglyceride fractions but the difference due to choline deficiency was observed at all temperatures. The possibility that the observed changes in the fatty acids of the phospholipids are compensatory to the changes in the proportion of the choline to the ethanolamine phospholipids is discussed.     

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.     

Growth of the mitochondrial inner membrane in synchronous cultures of Tetrahymena pyriformis: an examination of phospholipid accumulation

Based on morphological evidence, mitochondrial inner membrane growth has been reported to be discontinuous in heat shock-synchronized Tetrahymena pyriformis. As a biochemical measure of membrane growth under these conditions, we have examined phospholipid accumulation in the cell. No marked modulation of the accumulation of any of the major phospholipids could be detected through the cell cycle. At least 89% of the cardiolipin in the cells is restricted to the mitochondria, and we have used it as a marker for the growth of the mitochondrial inner membrane. During the heat shock synchrony, cardiolipin accumulates uniformly in parallel with the exponential rate of increase of total cellular phospholipids. These results suggest that at least the phospholipid component of all membrane systems in the cell grow continuously and uniformly. Additionally, we have shown that the total phospholipid content of Tetrahymena increases by a factor of 2.4 per generation following a series of heat shocks. No such net overaccumulation is observed for protein content.     

Murine diet-induced obesity remodels cardiac and liver mitochondrial phospholipid acyl chains with differential effects on respiratory enzyme activity

Cardiac phospholipids, notably cardiolipin, undergo acyl chain remodeling and/or loss of content in aging and cardiovascular diseases, which is postulated to mechanistically impair mitochondrial function. Less is known about how diet-induced obesity influences cardiac phospholipid acyl chain composition and thus mitochondrial responses. Here we first tested if a high fat diet remodeled murine cardiac mitochondrial phospholipid acyl chain composition and consequently disrupted membrane packing, supercomplex formation and respiratory enzyme activity. Mass spectrometry analyses revealed that mice consuming a high fat diet displayed 0.8–3.3 fold changes in cardiac acyl chain remodeling of cardiolipin, phosphatidylcholine, and phosphatidylethanolamine. Biophysical analysis of monolayers constructed from mitochondrial phospholipids of obese mice showed impairment in the packing properties of the membrane compared to lean mice. However, the high fat diet, relative to the lean controls, had no influence on cardiac mitochondrial supercomplex formation, respiratory enzyme activity, and even respiration. To determine if the effects were tissue specific, we subsequently conducted select studies with liver tissue. Compared to the control diet, the high fat diet remodeled liver mitochondrial phospholipid acyl chain composition by 0.6–5.3-fold with notable increases in n-6 and n-3 polyunsaturation. The remodeling in the liver was accompanied by diminished complex I to III respiratory enzyme activity by 3.5-fold. Finally, qRT-PCR analyses demonstrated an upregulation of liver mRNA levels of tafazzin, which contributes to cardiolipin remodeling. Altogether, these results demonstrate that diet-induced obesity remodels acyl chains in the mitochondrial phospholipidome and exerts tissue specific impairments of respiratory enzyme activity.     

Cell biology of cardiac mitochondrial phospholipids.

Phospholipids are important structural and functional components of all biological membranes and define the compartmentation of organelles. Mitochondrial phospholipids comprise a significant proportion of the entire phospholipid content of most eukaroytic cells. In the heart, a tissue rich in mitochondria, the mitochondrial phospholipids provide for diverse roles in the regulation of various mitochondrial processes including apoptosis, electron transport, and mitochondrial lipid and protein import. It is well documented that alteration in the content and fatty acid composition of phospholipids within the heart is linked to alterations in myocardial electrical activity. In addition, reduction in the specific mitochondrial phospholipid cardiolipin is an underlying biochemical cause of Barth Syndrome, a rare and often fatal X-linked genetic disease that is associated with cardiomyopathy. Thus, maintenance of both the content and molecular composition of phospholipids synthesized within the mitochondria is essential for normal cardiac function. This review will focus on the function and regulation of the biosynthesis and resynthesis of mitochondrial phospholipids in the mammalian heart.     

The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormones.

Three possible mechanisms by which different levels of thyroid hormones in rats might cause the observed sevenfold change in the apparent proton permeability of the inner membrane of isolated liver mitochondria were investigated. (a) Cytochrome c oxidase was isolated from the livers of hypothyroid, euthyroid and hyperthyroid rats and incorporated into liposomes made with soya phospholipids. There was no difference between the proton current/voltage curves of the three types of vesicles. The hormonal effects, therefore, were not an inherent property of the enzymes, and were not due to different coupling of electron flow through the enzyme to proton transport. (b) The surface area of the mitochondrial inner membrane was shown by three different assays to be greater by a factor of between two and three in mitochondria from hyperthyroid animals than in mitochondria from hypothyroid animals; euthyroid controls were intermediate. This difference in surface area of the inner membrane explains less than half of the difference in apparent proton permeability. (c) The proton permeability of liposomes prepared from phospholipids extracted from mitochondrial inner membranes of hyperthyroid rats was three times greater than the proton permeability of those from hypothyroid rats; euthyroid controls were intermediate. This suggests, first, that the proton permeability of the phospholipid bilayer is an important component of the proton permeability in intact mitochondria and, second, thyroid hormone-induced changes in the bilayer are a major part of the mechanism of increased proton permeability. Such changes may be due to the known differences in fatty acid composition of mitochondrial phospholipids in different thyroid states. Thus we have identified two mechanisms by which thyroid hormone levels in rats change proton flux/mass protein in isolated liver mitochondria: a change in the area of the inner membrane/mass protein and a change in the intrinsic permeability of the phospholipid bilayer.     

Binding of cardiolipin/lecithin and monoamine oxidase to lipid-depleted mitochondrial membrane structure.

Lipid-depleted pig liver mitochondrial residues were incubated with different proportions of the acidic phospholipid cardiolipin and the zwitterionic phospholipid lecithin in either separate or mixed liposomes. When cardiolipin and lecithin were present in separate liposomes all of the cardiolipin but no lecithin bound to the residues. When present in the same liposomes, cardiolipin also caused binding of lecithin to the mitochondrial residues. When monoamine oxidase solubilized from pig liver mitochondria by extraction of the phospholipids was included in the incubation, binding of the enzyme to the residues occurred in the presence of cardiolipin. The percentage of enzyme bound followed the same trend as the binding of phospholipids to the mitochondrial residues.     

Decreased cytochrome oxidase activity in hepatic mitochondria after chronic ethanol consumption and the possible role of decreased cytochrome aa3 content and changes in phospholipids

In ethanol-fed baboons, hepatic mitochondrial cytochrome oxidase activity and cytochrome aa₃ content were significantly decreased by 58.3 and 50.5%, respectively, compared to their pair-fed controls. However, there was no significant correlation between the two, suggesting that other factors in addition to cytochrome aa₃ may be responsible for the depression in cytochrome oxidase activity. The total phospholipid content of the mitochondrial membranes was significantly decreased (0.24 ± 0.03 μmol of phospholipid phosphorus/mg of protein vs. 0.32 ± 0.04 in controls). This change was accounted for, in part, by the significant decrease in the levels of phosphatidylcholine and cardiolipin. In addition, the fatty acid pattern of the phospholipids was changed. There was a marked increase in the relative amounts of oleic and linoleic acid and a decrease in arachidonic acid. These changes were associated with an increase in the activity of phospholipase A₂. The reactivation rate of phospholipid-depleted cytochrome oxidase by endogenous phospholipids from ethanol-fed baboons was significantly lower than that by phospholipid from pair-fed controls, when measured at an optimal phospholipid to protein ratio. Thus, it appears that alterations in the phospholipid composition of the mitochondrial membranes are responsible, at least in part, for the depression of cytochrome oxidase activity produced by chronic ethanol consumption.     

Degradation of Mitochondrial Phospholipids During Experimental Cerebral Ischemia in Rats

Changes in content of brain mitochondrial phospholipids were examined in rats after 30 and 60 min of decapitation ischemia compared with controls, to explore the degradation of the mitochondrial membrane and its relation to dysfunction of mitochondria. Activities of respiratory functions and respiratory enzymes (cytochrome c oxidase; F0F1-ATPase) decreased significantly during ischemia. Considerable decreases in cardiolipin and phosphatidylinositol content were observed after 60 min, and other phospholipids showed similar but nonsignificant decreases in content. The amount of polyunsaturated fatty acids chains, such as arachidonic and docosahexaenoic acids, was reduced in each phospholipid, in some cases significantly, after 30 and 60 min of ischemia in time-dependent manners. Degradation of mitochondrial phospholipids during ischemia associated with the deterioration of mitochondrial respiratory functions suggested the significance of such changes in phospholipid content in disintegration of cellular energy metabolism during cerebral ischemia.     

Manipulation of phospholipid polar headgroup composition in primary cultures of rat hepatocytes

Isolated hepatocytes in suspension or in primary culture were incubated with different phospholipid bases and the effects on the synthesis and composition of phospholipids were studied. After incubation in the presence of 1 mM diethylethanolamine for three days, an unnatural phospholipid, phosphatidyl-diethylethanolamine, constituted more than 20% of total phospholipids. Its fatty acid composition differed from that of other phospholipids. Incubation of hepatocytes with ethanolamine gave smaller effects and in this case the increased synthesis of phosphatidylethanolamine was compensated for by methylation to phosphatidylcholine. This system can be used for studies on the functional significance of phospholipid polar headgroups in a specialized type of cell.     

Phospholipid composition and fatty acid patterns of isolated phospholipids from rabbit reticulocyte mitochondria

The phospholipid composition and fatty acid patterns of individual phospholipid classes were determined in mitochondria from rabbit reticulocytes. Compared to mitochondria from rat liver reticulocyte, mitochondria exhibit about twice the amount of phospholipids. The phospholipid pattern of reticulocyte mitochondria (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) is comparable with other mitochondrial species. Mitochondrial fractions from reticulocytes are characterized, however, by an additional content of sphingomyelin. This sphingomyelin differs in its fatty acid composition from the sphingomyelin of the plasma membrane. The fatty acid patterns of all other phospholipids essentially correspond to those of mitochondria from other sources and to those of plasma membranes as well.     

Phospholipid composition of Mycobacterium smegmatis susceptible and resistant to antitubercular drugs

The phospholipid composition, distribution and metabolism in mono drug resistant mutants towards antitubercular drugs, viz, streptomycin, ethambutol and isoniazid, were investigated. Though their total phospholipid content was not altered significantly, changes were observed in their individual phospholipid content. Reduced biosynthesis and degradation of phospholipids (monitored by pulse and chase technique using [32P]orthophosphoric acid as a precursor) was observed in all the mutants studied. The subcellular distribution of phospholipids revealed accumulation of phospholipids in the cell walls and reduction in cell membranes of the drug-resistant mutants. Similar alterations were seen in individual phospholipids of these subcellular fractions.     

Changes in the level of tightly-bound mitochondrial phospholipids induced by exogenous factors

The effects of some parameters of the incubation medium (tonicity, H+ and Pi concentration) on the phospholipid content in rat mitochondrial extracts prepared by normal extraction and by extraction with strongly acidified solvent mixtures, were studied. It was found that after normal extraction with a chloroform-methanol mixture, 8% to 25% of mitochondrial phospholipids can be additionally extracted by acidified solvent mixtures. The concentration of weakly extractable lipids depends on the tonicity and ionic composition of the incubation medium. It was suggested that there exist in mitochondria two lipid pools, a loosely and a tightly-bound ones; the equilibrium between these pools is effectively regulated by medium tonicity and other external factors.