Fas-independent mitochondrial damage triggers cardiomyocyte death after ischemia-reperfusion.

The Fas/Fas ligand and mitochondria pathways have been involved in cell death in several cell types. We combined the genetic inactivation of the Fas receptor (lpr mice), on the one hand, to the pharmacological inhibition of the mitochondrial permeability transition pore (mPTP), on the other hand, to investigate which of these pathways is predominantly activated during prolonged ischemia-reperfusion. Anesthetized C57BL/6JICO (control) and C57BL/6-lpr mice were pretreated with either saline or cyclosporin A (CsA; 40 mg/kg, 3 times a day), an inhibitor of the mPTP, and underwent 25 min of ischemia and 24 h of reperfusion. After 24 h of reperfusion, hearts were harvested: infarct size was assessed by 2,3,5-triphenyltetrazolium chloride staining, myocardial apoptosis by caspase 3 activity, and mitochondrial permeability transition by Ca2+-induced mPTP opening using a potentiometric approach. Infarct size was comparable in untreated control and lpr mice, ranging from 77 +/- 5% to 83 +/- 3% of the area at risk. CsA significantly reduced infarct size in control and lpr hearts. Control and lpr hearts exhibited comparable increase in caspase 3 activity that averaged 57 +/- 18 and 49 +/- 5 pmol x min(-1) x mg(-1), respectively. CsA treatment significantly reduced caspase 3 activity in control and lpr hearts. The Ca2+ overload required to open the mPTP was decreased to a similar extent in lpr and controls. CsA significantly attenuated Ca2+-induced mPTP opening in both groups. Our results suggest that the Fas pathway likely plays a minor role, whereas mitochondria are preferentially involved in mice cardiomyocyte death after a lethal ischemia-reperfusion injury.

The 8 kD product of the putative oncogene MTCP-1 is a mitochondrial protein.

An unusually small (8 kD) protein (p8MTCP-1) is coded by the putative oncogene MTCP-1 (also called c6.1B), involved in the translocation t(X;14)(q28;q11) associated with some mature T-cell proliferations. Here, we show by subcellular fractionation and by confocal microscopy that this protein is located in the mitochondria. This localization orientates toward a role of p8MTCP-1 in the mitochondrial metabolism which may be relevant for the oncogenic process.

Involvement of mitochondrial contact sites in the subcellular compartmentalization of phospholipid biosynthetic enzymes.

The concerted synthesis of phospholipids derived from serine involving two microsomal enzymes (phosphatidylserine synthase and phosphatidylethanolamine N-methyltransferase) and a mitochondrial one (phosphatidylserine decarboxylase) occurs in reconstituted cell-free systems. Subfractionation of crude mitochondria after swelling and separating on a sucrose density gradient resulted in the isolation of two contact site-enriched fractions from total outer membranes and inner membranes, respectively. Estimation of marker enzyme activities shows a high recovery of glucose-6-phosphate phosphatase (a marker for the endoplasmic reticulum) associated with contact site-enriched fractions. Accordingly, the linked synthesis of phosphatidylserine, phosphatidylethanolamine, and at a lesser extent phosphatidylcholine can occur. This biosynthetic pathway was absent from purified contact site-enriched fractions correlative with the absence of glucose-6-phosphate phosphatase activity. Reconstitution experiments, including contact site-enriched fractions incubated with endoplasmic reticulum-rich fraction, led to the restoration of the linked synthesis of phospholipids, thereby demonstrating that a reversible association between these two fractions can occur. These functional interactions between the endoplasmic reticulum and mitochondria are confirmed at the ultrastructural level using either chemical or physical fixation before resin embedding. These results show that the interorganelle trafficking of lipids may involve only highly specialized microdomains of both membranes, thereby allowing the maintenance of a specific lipid composition and distribution within membranes.

Use of Percoll gradients for isolation of human placenta mitochondria suitable for investigating outer membrane proteins.

Human mitochondria were isolated from placenta by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation as assessed by marker enzyme analysis and electron microscopy. The advantages over previous methods are the rapidity of the procedure and the excellent resolution of mitochondria and lysosomes. Moreover, the high extent of intactness of the mitochondria so obtained made them particularly well suited for investigating outer membrane proteins. Taking advantage of this method, we have purified human mitochondrial porin. The purified protein consists of a single unglycosylated polypeptide of molecular mass 33 kDa.

Mitochondrial dolichyl-phosphate mannose synthase. Purification and immunogold localization by electron microscopy.

Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.

Investigation of glycosylation processes in mitochondria and microsomal membranes from human skeletal muscle.

Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined.

Glycosyltransferase activities in normal and leukaemic monocytic cells.

Glycosyltransferase activities were measured in normal monocytes and in leukaemic monoblasts. Biosynthesis of glycosylated derivatives of dolichyl-monophosphate, which act as intermediates in glycosylation, was measured. Transfer of mannose from GDP-mannose was greatly increased in leukaemic monoblasts. Galactosyltransferase activities, using endogenous protein acceptors, were increased in leukaemic cells of the monocytic lineage compared to normal cells. No significant difference was observed on specific exogenous glycoprotein acceptors. These selective increases of some glycosyltransferase activities in normal and leukaemic monocytic cells can be correlated either with different expression of specific carbohydrate structures or with changes in glycosylation regulation.