Monolysocardiolipins accumulate in Barth syndrome but do not lead to enhanced apoptosis

Barth syndrome (BTHS) is an X-linked recessive disorder that is biochemically characterized by low cellular levels of the mitochondrial phospholipid cardiolipin (CL). Previously, we discovered that the yeast disruptant of the TAZ ortholog in Saccharomyces cerevisiae not only displays CL deficiency but also accumulates monolysocardiolipins (MLCLs), which are intermediates in CL remodeling. Therefore, we set out to investigate whether MLCL accumulation also occurs in BTHS. Indeed, we observed MLCL accumulation in heart, muscle, lymphocytes, and cultured lymphoblasts of BTHS patients; however, only very low levels of these lysophospholipids were found in platelets and fibroblasts of these patients. Although the fatty acid composition of the MLCLs was different depending on the tissue source, it did parallel the fatty acid composition of the (remaining) CLs. The possible implications of these findings for the two reported CL remodeling mechanisms, transacylation and deacylation/reacylation, are discussed. Because MLCLs have been proposed to be involved in the initiation of apoptosome-mediated cell death by the sequestration of the proapoptotic protein (t)BH3-interacting domain death agonist (Bid) to the mitochondrial membrane, we used control and BTHS lymphoblasts to investigate whether the accumulation of MLCLs results in higher levels of apoptosis.We found no differences in susceptibility to death receptor-mediated apoptosis or in cellular distribution of Bid, cytochrome c, and other parameters, implying that MLCL accumulation does not lead to enhanced apoptosis in cultured BTHS lymphoblasts. Barth syndrome (BTHS) is an X-linked recessive disorder that is biochemically characterized by low cellular levels of the mitochondrial phospholipid cardiolipin (CL). Previously, we discovered that the yeast disruptant of the TAZ ortholog in Saccharomyces cerevisiae not only displays CL deficiency but also accumulates monolysocardiolipins (MLCLs), which are intermediates in CL remodeling. Therefore, we set out to investigate whether MLCL accumulation also occurs in BTHS. Indeed, we observed MLCL accumulation in heart, muscle, lymphocytes, and cultured lymphoblasts of BTHS patients; however, only very low levels of these lysophospholipids were found in platelets and fibroblasts of these patients. Although the fatty acid composition of the MLCLs was different depending on the tissue source, it did parallel the fatty acid composition of the (remaining) CLs. The possible implications of these findings for the two reported CL remodeling mechanisms, transacylation and deacylation/reacylation, are discussed. Because MLCLs have been proposed to be involved in the initiation of apoptosome-mediated cell death by the sequestration of the proapoptotic protein (t)BH3-interacting domain death agonist (Bid) to the mitochondrial membrane, we used control and BTHS lymphoblasts to investigate whether the accumulation of MLCLs results in higher levels of apoptosis. We found no differences in susceptibility to death receptor-mediated apoptosis or in cellular distribution of Bid, cytochrome c, and other parameters, implying that MLCL accumulation does not lead to enhanced apoptosis in cultured BTHS lymphoblasts. Barth syndrome BTHS; Mendelian Inheritance in Man (MIM) 302060 is an X-linked recessive disorder that clinically is characterized by cardiomyopathy, skeletal myopathy, growth retardation, and neutropenia (1Barth P.G. Scholte H.R. Berden J.A. Van der Klei-Van Moorsel J.M. Luyt-Houwen I.E. Van't Veer-Korthof E.T. Van der Harten J.J. Sobotka-Plojhar M.A. An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leucocytes.J. Neurol. Sci. 1983; 62: 327-355Abstract Full Text PDF PubMed Scopus (568) Google Scholar). Additional laboratory findings include intermittent lactic acidemia, low blood cholesterol, and increased urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid, and 2-ethylhydracrylic acid (2Kelley R.I. Cheatham J.P. Clark B.J. Nigro M.A. Powell B.R. Sherwood G.W. Sladky J.T. Swisher W.P. X-linked dilated cardiomyopathy with neutropenia, growth retardation, and 3-methylglutaconic aciduria.J. Pediatr. 1991; 119: 738-747Abstract Full Text PDF PubMed Scopus (227) Google Scholar). Moreover, mitochondria of BTHS patients have an abnormal ultrastructure, and several different respiratory chain defects in muscle and fibroblasts have been reported (3Barth P.G. Valianpour F. Bowen V.M. Lam J. Duran M. Vaz F.M. Wanders R.J. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): an update.Am. J. Med. Genet. 2004; 126A: 349-354Crossref PubMed Scopus (231) Google Scholar, 4Barth P.G. Wanders R.J. Vreken P. Janssen E.A. Lam J. Baas F. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome) (MIM 302060).J. Inherit. Metab. Dis. 1999; 22: 555-567Crossref PubMed Scopus (114) Google Scholar). The disease can be fatal in childhood, as a result of cardiac failure or sepsis. The clinical expression of the disease, however, is quite variable in severity and may show profound intrafamilial variability. Mutations in the TAZ gene, which is located at Xq28, are responsible for BTHS (5Bione S. D'Adamo P. Maestrini E. Gedeon A.K. Bolhuis P.A. Toniolo D. A novel X-linked gene, G4.5, is responsible for Barth syndrome.Nat. Genet. 1996; 12: 385-389Crossref PubMed Scopus (625) Google Scholar). Because of the homology of the predicted TAZ gene product(s) with acyltransferases involved in phospholipid metabolism, it is suggested that TAZ is involved in the remodeling of phospholipids (6Neuwald A.F. Barth syndrome may be due to an acyltransferase deficiency.Curr. Biol. 1997; 7: R465-R466Abstract Full Text Full Text PDF PubMed Google Scholar). This suggestion was supported by our finding that the incorporation of linoleic acid into cardiolipin (CL) was defective in patients with BTHS, even though their biosynthetic capacity to synthesize CL was entirely normal (7Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J. Barth P.G. Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome.Biochem. Biophys. Res. Commun. 2000; 279: 378-382Crossref PubMed Scopus (310) Google Scholar). Additionally, the CL levels have been shown to be markedly decreased in platelets (8Valianpour F. Wanders R.J. Barth P.G. Overmars H. van Gennip A.H. 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The and of Res. 2000; PubMed Scopus Google Scholar, remodeling and in the and J. Med. Google Scholar, M. S. cardiolipin in of biosynthetic and J. PubMed Scopus Google Scholar). The proposed for the remodeling of CL is a that the of the by the of and the of by or and of acyltransferase Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. B. and in J. PubMed Scopus (104) Google Scholar). Therefore, it has been suggested that the TAZ gene an acyltransferase involved in the remodeling of that the intermediates of remodeling in BTHS. we and have reported that a of Saccharomyces cerevisiae in which the yeast ortholog of the TAZ gene, has been not only displays CL deficiency but also accumulates MLCLs Valianpour F. S. Vaz F.M. Wanders R.J. cardiolipin in the yeast a for Barth 2004; 51: PubMed Scopus Google Scholar, F.M. Valianpour F. Barth P.G. Wanders R.J. of the TAZ gene a protein with a in cardiolipin Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the levels of CL and MLCL of the disruptant with F.M. Valianpour F. Barth P.G. Wanders R.J. of the TAZ gene a protein with a in cardiolipin Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), the of TAZ in the remodeling of however, MLCL accumulation has not been observed in or tissues BTHS patients. investigate whether MLCLs also in BTHS, we our for the of CL (8Valianpour F. Wanders R.J. Barth P.G. Overmars H. van Gennip A.H. Quantitative and compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients.Clin. Chem. 2002; 48: 1390-1397Crossref PubMed Scopus (104) Google Scholar) to and other phospholipids in and We also TAZ deficiency other phospholipids in these results show that in to CL deficiency in BTHS are also abnormalities in other phospholipid Additionally, is a accumulation of MLCLs in muscle, heart, and lymphocytes, but only of these are observed in fibroblasts and Because that MLCLs are proapoptotic we whether the MLCL accumulation to enhanced apoptosis in BTHS. that MLCLs not lead to enhanced apoptosis in cultured BTHS were of and were The and MLCL were An was and were cultured in and were by at for and with were cultured as by Valianpour (9Valianpour F. Wanders R.J. Overmars H. Vreken P. Van Gennip A.H. Baas F. Plecko B. Santer R. Becker K. Barth P.G. Cardiolipin deficiency in X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060): a study in cultured skin fibroblasts.J. Pediatr. 2002; 141: 729-733Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). were used for or were at were to for of of the involved or their were in of and on at a and cell was used for protein was to the of a of in by of This was and on for which it was at for The was into and the protein was with of The were a of at The was in of and of was into the of cardiac or muscle tissue was in a and was a The the of a of in which been a of at The tissue were in a The tissues were on and to an of was to the tissue and was for at of was and was on for and for at The was into and the protein was with of The were a of at The was in of and of was into the The of an a and a and a The was at The was a The phospholipids were by a and A of of The was as A to A to and with A. were and the the was A the and the mass was and was into the mass A mass was used in the electrospray ionization was used as was used as at a of The used was The was set at for MLCLs and was of CL and MLCL were by to with a of in a of which of the of CL and MLCL were of as (8Valianpour F. Wanders R.J. Barth P.G. Overmars H. van Gennip A.H. Quantitative and compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients.Clin. Chem. 2002; 48: 1390-1397Crossref PubMed Scopus (104) Google Scholar). were a to for in the were in the a to of the mass a the was used to a of MLCLs and CLs. The of and that of the was the were for the of the was also for the protein in of cell or tissue for tissue Because the is not for the different and we did not whether the of the was to that of the we show these as of tissue or protein The and for were by of with different of and a of as the The for the and were by their in the with in the with the The of the was by a and a with at low and The was by and with and The of the was by different and with a of We by of the of and in the with of with and BTHS were by at and with were in and and by a cell The was the for at at and protein was The was for at to the the The was and the was with the was in an as that of the of the and to of the were by and a a and was as F.M. R. K. Wanders R.J. and of the of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of of lymphoblasts were by with and by in The protein of the was and of protein was on a the of domain death agonist (Bid) mitochondrial control and BTHS were with at for in and at for mitochondrial were in and of protein was on a The were used for and the cell was as P.G. levels for mitochondrial in death cell 2004; PubMed Scopus Google Scholar), as was with an and were in of to different and for of cell by was by in a cell on and an of were in of and in of and an to was to and in was by of and by as J. T. P.G. M. and of of apoptosis and Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). were to in Because MLCLs are in the yeast for BTHS Valianpour F. S. Vaz F.M. Wanders R.J. cardiolipin in the yeast a for Barth 2004; 51: PubMed Scopus Google Scholar, F.M. Valianpour F. Barth P.G. Wanders R.J. of the TAZ gene a protein with a in cardiolipin Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), we set out to investigate whether these lysophospholipids also in and tissues of BTHS patients. We our to CL to the of MLCLs and The in our is the of a to the of This did not the of CL and that of other MLCL were by by The of the was to of at to protein The of the was at and at The of at low and levels of were and The at low and levels of were and We used to the levels of and MLCLs in lymphocytes, muscle, and of and BTHS patients. the MLCL cultured lymphoblasts of a control and a BTHS The in are of observed in control and BTHS cell This that the levels of and are increased in BTHS lymphoblasts with control that the levels of MLCL in BTHS were higher in the CL in the of to different CL are in the control The of of the fatty with higher a The mass to with fatty of different to a to the CL are and the CL are in and which and as fatty also fatty in to and with the CL in control the in the with the of the are decreased in BTHS the levels of in as are in normal mass of CL in cultured control and BTHS lymphoblasts used in The of cellular protein was for control and BTHS The at to the the control different CL are BTHS lymphoblasts CL with a of the a CL with low and A of the levels of their fatty acid is in are different control and BTHS cell Because of the of MLCLs in BTHS we the of BTHS and control to whether these BTHS also an accumulation of these CL the CL of control lymphoblasts and were very We found that is the CL in control lymphoblasts CL and a of chain and in BTHS we did a CL deficiency and a accumulation of MLCLs in BTHS This is in to BTHS platelets and fibroblasts in which MLCL accumulation was very or even not Because and muscle of CL and BTHS patients with to these tissues we the and MLCLs in these tissues of two control and two BTHS patients. control heart, is the CL This CL is in BTHS heart, other and are and their levels even to be increased with control The CL of skeletal muscle in control and BTHS was very to that of not control as muscle not we MLCL with the fatty acid composition which is in low with BTHS and muscle, however, is a accumulation of different and of these MLCL were in control The levels of these MLCL were higher in BTHS in control and were in as the CLs. The levels of in BTHS to in the as in the we the composition and of other and in the BTHS and control tissues used for the abnormalities in tissue of a control and a BTHS was to higher in BTHS with control The fatty acid composition of was by The abnormalities were found in the but were with levels to be at two to higher in BTHS with This to fatty acid was not Additionally, in which a to be increased in BTHS and acid by in to be decreased in BTHS We did not abnormalities in other phospholipid and of in control and BTHS lymphoblasts. of control and BTHS lymphoblasts and a control and is no of in BTHS and not of the protein is distribution of domain death agonist (Bid) and cytochrome in control and BTHS lymphoblasts by and and is no the distribution of and cytochrome mitochondria and in BTHS and control lymphoblasts. The of the mitochondria is by the distribution of the mitochondrial the and was in of the it was shown that CL and MLCL to Bid, a proapoptotic which is involved in death receptor-mediated apoptosis in cell in and 2004; PubMed Google Scholar, to a mitochondrial and cell 2003; PubMed Scopus Google Scholar). an is to the mitochondrial membrane, in with other it the of as cytochrome c, apoptosome-mediated apoptosis. is by in a protein which is the and to a mitochondrial and cell 2003; PubMed Scopus Google Scholar) have shown that CL and MLCL in with MLCL with higher and also is of the cytochrome capacity of The in MLCL levels apoptosis that MLCL in mitochondria be involved in the initiation of apoptosis by the of to Therefore, we that the accumulation of MLCLs in BTHS and tissues result in enhanced which may a in the in BTHS. we used BTHS which were found to and several we several in of is by which as in control is no of in BTHS lymphoblasts and that not of the protein is we the distribution of and cytochrome in BTHS and control cell show that is no the distribution of and cytochrome in mitochondria and in BTHS and control lymphoblasts. is as and only in the cytochrome is found only in mitochondria and is not in the The distribution of the mitochondrial that the mitochondria were and the we whether BTHS cell differences in susceptibility to death receptor-mediated apoptosis. BTHS patients and were for with of A cell was used as a as these levels of A. S. and to J. PubMed Scopus Google Scholar). a of cell death even at of and BTHS patients were also to cell levels were to cell death for with cell death of at BTHS and control cell no differences in of to the is to result in the and of which in Bid, to to the with the results of the of and were in control and BTHS lymphoblasts in cell of BTHS and control lymphoblasts of as by the of the higher mass and the of the and of in BTHS and control as by the of the we were to accumulation of in not or or in cell cell Because we not a of the of to the in control and BTHS we the of to control and BTHS were with for at and mitochondrial were by with was no in the of to control and BTHS mitochondria not on the finding that the yeast disruptant of the TAZ ortholog in Saccharomyces cerevisiae not only displays CL deficiency but also accumulates MLCLs Valianpour F. S. Vaz F.M. Wanders R.J. cardiolipin in the yeast a for Barth 2004; 51: PubMed Scopus Google Scholar, F.M. Valianpour F. Barth P.G. Wanders R.J. of the TAZ gene a protein with a in cardiolipin Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), we set out to investigate whether MLCL accumulation also occurs in BTHS. Therefore, we our to the of MLCLs in to CL and other observed in the yeast for BTHS, our show an accumulation of MLCL in BTHS cell and We found that MLCLs in BTHS heart, muscle, and no MLCL accumulation was observed in cultured fibroblasts and platelets of BTHS patients. The accumulation of a of CL remodeling in BTHS the suggestion that the decreased levels of CL in BTHS are to a in the remodeling that the BTHS even in the it is an that the of MLCL accumulation in different with the to investigate are The that cell show accumulation of MLCLs and show no accumulation of these is and that remodeling of may be different in different cell or has been for that CL is the M. B. and in J. PubMed Scopus (104) Google Scholar), with MLCL as an The responsible for the of MLCL has been mitochondria by and and of acyltransferase Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), but the gene has not been a gene has been that an acyltransferase J. J. P. A novel by a gene an acyltransferase in Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). This MLCL and as with a for and as to be in the as to the mitochondrial reported by and and of acyltransferase Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the by or acyltransferase that can to be Kelley R.I. Blanck T.J. M. of cardiolipin by phospholipid Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) reported that in CL and MLCL can also be and as that the in mitochondria was for in to in which also be used as an The that MLCLs in however, does not the of and transacylation mechanisms, MLCL occurs in of the fatty acid composition of the MLCL to our of the CL remodeling to be a the fatty acid composition of the MLCLs and that of the of a tissue or cell control and muscle we only found MLCL the of MLCL in BTHS tissues and were of and fatty which also the composition of the CLs. This was also for the MLCLs in lymphoblasts and lymphocytes, which the CL fatty acid Kelley R.I. Blanck T.J. M. of cardiolipin by phospholipid Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) observed that the for was in lymphoblasts patients with BTHS, which to that CL is by phospholipid which is MLCL acyltransferase is by a different gene, which is supported by the identification of The that not cell MLCL accumulation the expression of that of an with This that tissues with low or no expression of MLCL acyltransferase are or of which be the of the different which are for the of Because the fatty acid composition of the MLCLs that of the it is that the of MLCLs is not a This in is in with the observed of the MLCL acyltransferase by and and of acyltransferase Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), which is of and even This of MLCL be by a transacylation in the of the The of a is supported by the that and linoleic acid, which are reported to as in the transacylation of in BTHS The results of Kelley R.I. Blanck T.J. M. of cardiolipin by phospholipid Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) that the transacylation has a for or in and or were in the transacylation is however, that in the transacylation was very for in lymphoblasts transacylation also be with Kelley R.I. Blanck T.J. M. of cardiolipin by phospholipid Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This that may be a for the transacylation or that with different the CL yeast K. H. Cardiolipin respiratory chain Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, F. cardiolipin and is for growth of Saccharomyces PubMed Scopus Google Scholar, V.M. of cardiolipin results in respiratory and mitochondrial of Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) and the yeast disruptant Valianpour F. S. Vaz F.M. Wanders R.J. cardiolipin in the yeast a for Barth 2004; 51: PubMed Scopus Google Scholar) show increased on is a The structure is in with a of the as the of the mitochondrial M.A. of of and PubMed Scopus Google Scholar). parallel to was proposed for V.M. of cardiolipin results in respiratory and mitochondrial of Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), an for the accumulation of in BTHS also be that these for the decreased mitochondrial CL levels and abnormal CL Because MLCLs were suggested to apoptosis by the proapoptotic protein and to the mitochondrial to a mitochondrial and cell 2003; PubMed Scopus Google Scholar), we whether BTHS lymphoblasts a higher of or apoptosis. of and the distribution of and cytochrome were in BTHS and control and BTHS lymphoblasts also of cell death by and of and We were to the of to mitochondria in BTHS patients and we not accumulation of in the This is the result of the of which has been reported to in other cell by and with a of K. S. of the proapoptotic of A on apoptosis Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). mitochondria BTHS and control lymphoblasts no differences in these that BTHS and control lymphoblasts show of cell Although these results are in with the findings of and Becker K. Plecko B. Valianpour F. Wanders R.J. R. J. Van in Barth syndrome (BTHS) in the of 2004; PubMed Scopus Google Scholar), that BTHS are not even though are MLCL have different in other cell in that the lymphoblasts used for these are in BTHS blood are The that MLCLs in also has an on the BTHS is Previously, we a to BTHS patients by CL levels in platelets (8Valianpour F. Wanders R.J. Barth P.G. Overmars H. van Gennip A.H. Quantitative and compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients.Clin. Chem. 2002; 48: 1390-1397Crossref PubMed Scopus (104) Google Scholar). Although has been and is very it is on a of CL which has the accumulation of to a We are whether the of MLCL and CL levels a of these in is a for BTHS. This was supported by a the The to the Barth to the and of to and the of and to The M. for the BTHS T. and A. for the and cytochrome and for the and cell