Role of calcium-independent phospholipase A2 in the pathogenesis of Barth syndrome

Quantitative and qualitative alterations of mitochondrial cardiolipin have been implicated in the pathogenesis of Barth syndrome, an X-linked cardioskeletal myopathy caused by a deficiency in tafazzin, an enzyme in the cardiolipin remodeling pathway. We have generated and previously reported a tafazzin-deficient Drosophila model of Barth syndrome that is characterized by low cardiolipin concentration, abnormal cardiolipin fatty acyl composition, abnormal mitochondria, and poor motor function. Here, we first show that tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility. This phenotype can be genetically suppressed by inactivation of the gene encoding a calcium-independent phospholipase A₂, iPLA2-VIA, which also prevents cardiolipin depletion/monolysocardiolipin accumulation, although in wild-type flies inactivation of the iPLA2-VIA does not affect the molecular composition of cardiolipin. Furthermore, we show that treatment of Barth syndrome patients' lymphoblasts in tissue culture with the iPLA₂ inhibitor, bromoenol lactone, partially restores their cardiolipin homeostasis. Taken together, these findings establish a causal role of cardiolipin deficiency in the pathogenesis of Barth syndrome and identify iPLA2-VIA as an important enzyme in cardiolipin deacylation, and as a potential target for therapeutic intervention.     

Phospholipid abnormalities in children with Barth syndrome

OBJECTIVES: We sought to identify characteristic lipid abnormalities in patients with Barth syndrome (BTHS) and to correlate the lipid profile to phenotype and genotype. BACKGROUND: Barth syndrome typically includes cardiomyopathy, skeletal myopathy, neutropenia, growth retardation, and 3-methylglutaconic aciduria, and it is commonly associated with mutations in the tafazzin (TAZ) gene, whose products are homologous to phospholipid acyltransferases. However, clinical features of BTHS have also been found in patients with normal TAZ gene. METHODS: We analyzed molecular species of phospholipids in left and right ventricle, skeletal muscle, platelets, lymphoblasts, and fibroblasts from 19 children with BTHS (positive TAZ mutation), 6 children with BTHS-like syndromes (wild-type TAZ), 4 children with isolated cardiomyopathy (wild-type TAZ), and various controls. RESULTS: Cardiolipin, the specific lipid found only in mitochondria, was decreased in all tissues from BTHS patients, whereas concentrations of other phospholipids were normal. The molecular composition of cardiolipin was altered in all tissues from BTHS patients. The molecular compositions of phosphatidylcholine and phosphatidylethanolamine were altered in the heart. Cardiolipin abnormalities were only found in children with true BTHS, not in children with BTHS-like disease or with isolated cardiomyopathy. The degree of cardiolipin deficiency was tissue-specific but did not correlate with severity or specific phenotypic expression of BTHS. CONCLUSIONS: Abnormal cardiolipin is a specific diagnostic marker of cardiomyopathies caused by TAZ mutations. These mutations lead to alterations in the fatty acid composition of several phospholipids, supporting the idea that TAZ encodes a human acyltransferase.     

Characterization of a transgenic short hairpin RNA-induced murine model of Tafazzin deficiency

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.     

Structural basis for potassium transport in prokaryotes by KdpFABC

KdpFABC is an oligomeric K⁺ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K⁺ transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K⁺ or water and which define a pathway for K⁺ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K⁺ transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K⁺ to the cytoplasm.

KdpFABC is an ATP-dependent K⁺ pump in prokaryotes, essential for osmoregulation in K⁺-deficient environments. Expression of kdpFABC is induced when external K⁺ concentrations fall into the micromolar range, where constitutive K⁺-uptake systems, Trk and Kup, can no longer maintain intracellular K⁺ levels. Under these conditions, a high-affinity active transport system is required to maintain internal concentrations of K⁺, essential for regulating pH, membrane potential, and the turgor pressure that drives cell growth and division (1). As a molecular machine, the oligomeric KdpFABC complex represents a fascinating hybrid that couples a channel-like subunit (KdpA)—related to the superfamily of K⁺ transporters (SKT)—with a pump-like subunit (KdpB)—belonging to the superfamily of P-type ATPases (2). Early studies established a role for KdpA in the selectivity and transport of K⁺ (3). Furthermore, analysis of KdpA sequence and topology established the existence of four approximate repeats of the “MPM” fold that characterizes K⁺ channels and a close resemblance to bacterial potassium channels TrkH and KtrB (4). KdpB, on the other hand, harnesses the energy of ATP to drive transport of K⁺ against a concentration gradient. KdpB was shown to employ the Post–Albers reaction scheme (5) that features two main conformational states, E1 and E2, and an aspartyl phosphate intermediate (Fig. 1A). However, mechanisms for coupling between these two subunits have remained elusive, as has the specific transport pathway of K⁺ through the complex (6).Open in a separate windowFig. 1.Overview of cryo-EM structures. (A) Cryo-EM density maps of KdpFABC in four unique conformational states, corresponding to intermediates in the Post–Albers reaction cycle. KdpA is green; KdpB is brown, blue, yellow, and red; KdpC is purple; and KdpF is cyan. (B) Close-up of catalytic sites in which density is clearly visible for AMP-PCP and MgF₄; lower resolution of the E2-P map makes explicit placement of BeF₃ ambiguous. Mg²⁺ ions are green, and protein domains are colored as in A.Recent structural studies have generated renewed interest in KdpFABC and have led to new and conflicting ideas about its transport process. The first structure was determined by X-ray crystallography, defining the architecture of the complex (7). This structure revealed a sperical density modeled as water bound at the unwound part of the M4 transmembrane helix of KdpB (SI Appendix, Fig. S1A). This site, here denoted Bx, is conserved among P-type ATPases and plays a key role in binding and transporting their respective substrates. Structures of the well-studied sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) revealed ions at two sites, dubbed site Ca(I) and site Ca(II), the latter of which is congruent with the Bx site in KdpB. Structures of Na,K-ATPase show a similar ion binding pattern, with a third Na⁺ ion also accommodated near these two conserved sites. In KdpA, a K⁺ ion was bound within the selectivity filter (SF) and, like TrkH and KtrB, the third MPM repeat featured a kinked helix (D3M₂ in SI Appendix, Fig. S1A) with a loop that blocks this ion from traveling across the membrane. This loop was suggested to function as a gate that would move aside to open a pore for transport. In addition, a 40-Å–long tunnel was seen encapsulated within the membrane domain of KdpFABC. This intramembrane tunnel connects the SF in KdpA with the Bx site in KdpB, leading to a proposal for energy coupling based on a Grothuss mechanism for charge transfer between these sites. According to this mechanism, the tunnel is filled with water molecules acting as a water wire to shuttle protons between the subunits. K⁺ binding to the SF would initiate proton hopping through the tunnel. Arrival of this charge at the Bx site would stimulate the cycle of ATP hydrolysis, as documented in other P-type ATPases. A salt-bridge network connecting cytoplasmic loops of KdpA and KdpB would then tug on the kinked D3M₂ helix in KdpA, thus displacing the gating loop and allowing K⁺ to transit the membrane.Two additional structures of KdpFABC were subsequently determined by cryogenic electron microscopy (cryo-EM), revealing two additional conformations (8). Whereas the crystal structure appeared to represent an inhibited conformation due to the influence of serine phosphorylation (9), these cryo-EM structures were compatible with E1 and E2 states. The intramembrane tunnel was intact in the E1 state but was interrupted in the E2 state due to conformational changes in KdpB. These observations led to the proposal of an alternative mechanism in which K⁺ ions move through the tunnel, from the SF of KdpA to the Bx site of KdpB, where they are released to the cytoplasm at the appropriate step in the reaction cycle. The other two subunits, KdpC and KdpF, were also observed in all of these structures. KdpF consists of a single transmembrane helix at the interface of KdpA and KdpB, possibly serving to stabilize the complex. KdpC has a unique periplasmic domain anchored by a single TM helix; the location at the entrance to the SF suggested that this domain might act as a periplasmic filter or gate, though evidence for this role is currently lacking.To shed more light on the coupling between KdpA and KdpB and to resolve the role of the intramembrane tunnel, we determined structures of the KdpFABC complex in all of the major enzymatic states. We used inhibitors to trap the complex in various discrete states, in the presence of either K⁺ or Na⁺, and imaged these samples by cryo-EM. In this way, we produced 14 independent density maps at resolutions between 2.9 and 3.7 Å; four of these were selected to represent the primary intermediates from the reaction cycle. These resulting structures display significant conformational changes in KdpB as well as nonprotein spherical densities within the SF of KdpA, within the intramembrane tunnel, and in KdpB near the Bx site. In contrast, KdpA is static suggesting that it serves simply to select K⁺ ions from the periplasm and shuttle them to transport sites in KdpB. By providing high-resolution detail to structural changes in these sites during the reaction cycle, we provide evidence and a comprehensive model for the transport mechanism in which K⁺ from the periplasm enters the SF of KdpA, moves through the tunnel to the Bx site, and is released to the cytoplasm by KdpB.     

Inhibition by Volatile Anesthetics of Endogenous Glutamate Release from Synaptosomes by a Presynaptic Mechanism

Background: Synaptic transmission is more sensitive than axonal conduction to the effects of general anesthetics. Previous studies of the synaptic effects of general anesthetics have focused on postsynaptic sites of action. We now provide direct biochemical evidence for a presynaptic effect of volatile anesthetics on neurotransmitter release.

Methods: Rat cerebrocortical synaptosomes (isolated presynaptic nerve terminals) were used to determine the effects of general anesthetics on the release of endogenous L-glutamate, the major fast excitatory neurotransmitter. Basal and evoked (by 4-aminopyridine, veratridine, increased KCl, or ionomycin) glutamate release were measured by continuous enzyme-coupled fluorometry.

Results: Clinical concentrations of volatile halogenated anesthetics, but not of pentobarbital, inhibited 4-aminopyridine-evoked Calcium2+ -dependent glutamate release. Halothane also inhibited veratridine-evoked glutamate release but not basal, KCl-evoked, or ionomycin-evoked glutamate release. Halothane inhibited both the 4-aminopyridine-evoked and the KCl-evoked increase in free intrasynaptosomal [Calcium2+].     

Characterization of lymphoblast mitochondria from patients with Barth syndrome

Barth syndrome (BTHS) is a multisystem disorder of individuals who carry mutations in tafazzin, a putative phospholipid acyltransferase. We investigated the hypothesis that BTHS is caused by specific impairment of the mitochondrial lipid metabolism. The fatty acid composition of all major mitochondrial phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL), changed in lymphoblasts from BTHS patients. These changes were most extensive in CL and least extensive in PE. The complementary nature of the fatty acid alterations in CL and PC suggested that fatty acid transfer between these two lipids was inhibited in BTHS. Fluorescence staining and electron microscopy showed abnormal proliferation of mitochondria in BTHS lymphoblasts. The mitochondrial membrane potential, monitored with the fluorescence probe JC-1, was reduced in BTHS lymphoblasts. However, mitochondrial ATP formation of permeabilized lymphoblasts remained unaffected in BTHS. The data suggest that phospholipid abnormalities of BTHS mitochondria led to partial uncoupling of oxidative phosphorylation and that lymphoblasts compensated for this deficiency by expanding the mitochondrial compartment.     

Effect of cardiolipin oxidation on solid-phase immunoassay for antiphospholipid antibodies.

Diagnostic assays for antiphospholipid antibodies are routinely performed on microtitre plates coated with cardiolipin. Here we show that contact between cardiolipin and NUNC-Immuno plates leads to extensive oxidation, generating a series of peroxy-cardiolipins which were identified by electrospray ionization mass spectrometry. To investigate the impact of oxidation on the antibody assay. cardiolipin was resolved into 12 molecular species, including oxidized species and non-oxidized species with different degrees of unsaturation. All 12 species reacted under anaerobic conditions with serum from patients with primary antiphospholipid syndrome. Immune reactivity was similar for tetralinoleoyl-cardiolipin, trilinoleoyl-oleoyl-cardiolipin, and peroxycardiolipins, but somewhat lower for tristearoyl-oleoyl-cardiolipin. Oxidative treatment of cardiolipin with air, cytochrome c, or Cu2+/tert-butylhydroperoxide, either before or during the assay, did not enhance immune reactivity. Similar results were obtained with a monoclonal IgM from lupus-prone mice, that binds cardiolipin in the absence of protein cofactors. We conclude that the solid-phase assay for antiphospholipid antibodies can be supported by various oxidized and non-oxididized molecular species of cardiolipin.     

Heparin induces release of phospholipase A(2) into the splanchnic circulation

Cardiopulmonary bypass results in increased plasma activity of phospholipase A(2) (PLA(2)) that appears to be caused by the administration of heparin. High PLA(2) activity may be responsible for increased production of eicosanoids and, thus, may be implicated in various pathophysiologic events associated with cardiac surgery. To investigate the site of PLA(2) secretion, blood samples were simultaneously collected from the radial artery, the pulmonary artery, and the hepatic vein at 2, 4, 6, and 20 min after systemic heparinization (350 U/kg). Within 2 min of the heparin injection, plasma activity of PLA(2) increased 4- to 9-fold and remained so for at least 20 min. Two minutes after the heparin injection, PLA(2) was significantly higher in the hepatic vein than in the radial artery (P: < 0.01). No such difference was detected between pulmonary and radial arteries. When heparin was added to blood samples in vitro (5-100 U/mL), plasma activity of PLA(2) did not increase, which suggests that the enzyme was not secreted by blood cells. IMPLICATIONS: Heparin, given in the dosage required for cardiopulmonary bypass, caused release of phospholipase A(2) into the splanchnic circulation.     

Evaluation of Aromatherapy in Treating Postoperative Pain: Pilot Study

Abstract:   This study compared the analgesic efficacy of postoperative lavender oil aromatherapy in 50 patients undergoing breast biopsy surgery. Twenty-five patients received supplemental oxygen through a face mask with two drops of 2% lavender oil postoperatively. The remainder of the patients received supplemental oxygen through a face mask with no lavender oil. Outcome variables included pain scores (a numeric rating scale from 0 to 10) at 5, 30, and 60 minutes postoperatively, narcotic requirements in the postanesthesia care unit (PACU), patient satisfaction with pain control, as well as time to discharge from the PACU. There were no significant differences in narcotic requirements and recovery room discharge times between the two groups. Postoperative lavender oil aromatherapy did not significantly affect pain scores. However, patients in the lavender group reported a higher satisfaction rate with pain control than patients in the control group ( P  = 0.0001).