Faculty Bibliography

The dynamical behavior of mitochondria has attracted much attention, but little is known about the dynamics of mitochondrial lipids, specifically cardiolipin (CL). Here, we estimated the turnover of select molecular species of CL in mammalian cell cultures and compared it to the turnover of other lipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol. Cells were labeled with myristic acid, 9,10-(2)H2-oleic acid, or d-[U-(13)C6]-glucose and analyzed by mass spectrometry at different time points of pulse-chase experiments. The turnover of glycerol groups was monitored by specific isotopologues that carried (13)C primarily in the glycerol carbons, whereas the turnover of acyl groups was monitored by molecular species that carried myristoyl or (2)H2-oleoyl groups. We found that the turnover of CL, but not of mitochondrial PC and PE, was substantially slower than the turnover of other cellular phospholipids. In dioleoyl-PC and dioleoyl-PE, the acyl turnover was faster than the glycerol turnover, indicating continuous deacylation and reacylation of the oleoyl residues. In contrast, the acyl turnover was similar to the glycerol turnover in tetraoleoyl-CL, suggesting that oleoyl remodeling did not take place continuously in endogenous CL. We conclude that CL, once assembled in mitochondrial membranes, remains largely inert to degradation and acyl remodeling.

The idea of a Cardiolipin workshop in Italy came to the meeting organizers in June 2011, during the mini-sabbatical of Angela Corcelli in New York City in the Laboratory of Michael Schlame. They thought to take advantage of the presence of the 54th International Conference on the Bioscience of Lipids (ICBL) at Bari in 2013 to organize the Cardiolipin workshop as a satellite event. The web page of the Cardiolipin Meeting was kindly supported by the Euro Fed Lipid organization. About 60 scientists attended the meeting focused on the multiple roles of cardiolipin in mitochondria in physiological and pathological states in various organisms as well as in bacterial membranes. In addition to ICBL participants, many students and colleagues of the Universities of Bari and Lecce attended the meeting, increasing the number of total participants to about 100. As defects in cardiolipin metabolism may cause Barth syndrome, the meeting also presented an occasion to establish contacts between the nascent Italian Barth Syndrome Foundation and scientists actively involved in cardiolipin research. C1 [Corcelli, Angela] Univ Bari A Moro, Dept Basic Med Sci Neurosci & Sensory Organs, Bari, Italy. [Schlame, Michael] NYU, Sch Med, New York, NY USA

Cardiolipin, the specific phospholipid of mitochondria, is involved in the biogenesis, the dynamics, and the supramolecular organization of mitochondrial membranes. Cardiolipin acquires a characteristic composition of fatty acids by post-synthetic remodeling, a process that is crucial for cardiolipin homeostasis and function. The remodeling of cardiolipin depends on the activity of tafazzin, a non-specific phospholipid-lysophospholipid transacylase. This review article discusses recent findings that suggest a novel function of tafazzin in mitochondrial membranes. By shuffling fatty acids between molecular species, tafazzin transforms the lipid composition and by doing so supports changes in the membrane conformation, specifically the generation of membrane curvature. Tafazzin activity is critical for the differentiation of cardiomyocytes, in which the characteristic cristae-rich morphology of cardiac mitochondria evolves. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Cardiolipin is a mitochondrial phospholipid with a characteristic acyl chain composition that depends on the function of tafazzin, a phospholipid-lysophospholipid transacylase, although the enzyme itself lacks acyl specificity. We incubated isolated tafazzin with various mixtures of phospholipids and lysophospholipids, characterized the lipid phase by (31)P-NMR and measured newly formed molecular species by MS. Substantial transacylation was observed only in nonbilayer lipid aggregates, and the substrate specificity was highly sensitive to the lipid phase. In particular, tetralinoleoyl-cardiolipin, a prototype molecular species, formed only under conditions that favor the inverted hexagonal phase. In isolated mitochondria, <1% of lipids participated in transacylations, suggesting that the action of tafazzin was limited to privileged lipid domains. We propose that tafazzin reacts with non-bilayer-type lipid domains that occur in curved or hemifused membrane zones and that acyl specificity is driven by the packing properties of these domains.

Cardiolipin is a dimeric phospholipid with a characteristic acyl composition that is generated by fatty acid remodeling after de novo synthesis. Several enzymes have been proposed to participate in acyl remodeling of cardiolipin. In order to compare the effect of these enzymes, we determined the pattern of cardiolipin molecular species in Drosophila strains with specific enzyme deletions, using MALDI-TOF mass spectrometry with internal standards. We established the linear range of the method for cardiolipin quantification, determined the relative signal intensities of several cardiolipin standards, and demonstrated satisfying signal-to-noise ratios in cardiolipin spectra from a single fly. Our data demonstrate changes in the cardiolipin composition during the Drosophila life cycle. Comparison of cardiolipin spectra, using vector algebra, showed that inactivation of tafazzin had a large effect on the molecular composition of cardiolipin, inactivation of calcium-independent phospholipase A(2) had a small effect, whereas inactivation of acyl-CoA:lysocardiolipin-acyltransferase and of the trifunctional enzyme did not affect the cardiolipin composition.

BACKGROUND: Barth syndrome is a rare, multisystem disorder caused by mutations in tafazzin that lead to cardiolipin deficiency and mitochondrial abnormalities. Patients most commonly develop an early-onset cardiomyopathy in infancy or fetal life. METHODS AND RESULTS: Knockdown of tafazzin (TAZKD) in a mouse model was induced from the start of gestation via a doxycycline-inducible shRNA transgenic approach. All liveborn TAZKD mice died within the neonatal period, and in vivo echocardiography revealed prenatal loss of TAZKD embryos at E12.5-14.5. TAZKD E13.5 embryos and newborn mice demonstrated significant tafazzin knockdown, and mass spectrometry analysis of hearts revealed abnormal cardiolipin profiles typical of Barth syndrome. Electron microscopy of TAZKD hearts demonstrated ultrastructural abnormalities in mitochondria at both E13.5 and newborn stages. Newborn TAZKD mice exhibited a significant reduction in total mitochondrial area, smaller size of individual mitochondria, reduced cristae density, and disruption of the normal parallel orientation between mitochondria and sarcomeres. Echocardiography of E13.5 and newborn TAZKD mice showed good systolic function, but early diastolic dysfunction was evident from an abnormal flow pattern in the dorsal aorta. Strikingly, histology of E13.5 and newborn TAZKD hearts showed myocardial thinning, hypertrabeculation and noncompaction, and defective ventricular septation. Altered cellular proliferation occurring within a narrow developmental window accompanied the myocardial hypertrabeculation-noncompaction. CONCLUSIONS: In this murine model, tafazzin deficiency leads to a unique developmental cardiomyopathy characterized by ventricular myocardial hypertrabeculation-noncompaction and early lethality. A central role of cardiolipin and mitochondrial functioning is strongly implicated in cardiomyocyte differentiation and myocardial patterning required for heart development. (J Am Heart Assoc. 2012;1:jah3-e000455 doi: 10.1161/JAHA.111.000455.).

Abstract Barth's syndrome (BTHS) is an X-linked mitochondrial disease that is due to a mutation in the Tafazzin (TAZ) gene. Based on sequence homology, TAZ has been characterized as an acyltransferase involved in the metabolism of cardiolipin (CL), a unique phospholipid almost exclusively located in the mitochondrial inner membrane. Yeast, Drosophila, and zebrafish models have been invaluable in elucidating the role of TAZ in BTHS, but until recently a mammalian model to study the disease has been lacking. Based on in vitro evidence of RNA-mediated TAZ depletion, an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model has been developed (TaconicArtemis GmbH, Cologne, Germany), and herein we describe the assessment of this mouse line as a model of BTHS. Upon induction of the TAZ-specific shRNA in vivo, transgenic mouse TAZ mRNA levels were reduced by >89% in cardiac and skeletal muscle. TAZ deficiency led to the absence of tetralineoyl-CL and accumulation of monolyso-CL in cardiac muscle. Furthermore, mitochondrial morphology from cardiac and skeletal muscle was altered. Skeletal muscle mitochondria demonstrated disrupted cristae, and cardiac mitochondria were significantly enlarged and displace neighboring myofibrils. Physiological measurements demonstrated a reduction in isometric contractile strength of the soleus and a reduction in cardiac left ventricular ejection fraction of TAZKD mice compared with control animals. Therefore, the inducible TAZ-deficient model exhibits some of the molecular and clinical characteristics of BTHS patients and may ultimately help to improve our understanding of BTHS-related cardioskeletal myopathy as well as serve as an important tool in developing therapeutic strategies for BTHS