Substantial Decrease in Plasmalogen in the Heart Associated with

Article Cite This: Biochemistry 2018, 57, 2162−2175

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Substantial Decrease in Plasmalogen in the Heart Associated with Tafazzin Deficiency Tomohiro Kimura,*,† Atsuko K. Kimura,† Mindong Ren,§,∥ Bob Berno,‡ Yang Xu,∥ Michael Schlame,§,∥ and Richard M. Epand*,† †

Department of Biochemistry and Biomedical Sciences and ‡Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada § Department of Cell Biology and ∥Department of Anesthesiology, NYU Langone Medical Center, New York, New York 10016, United States S Supporting Information *

ABSTRACT: Tafazzin is the mitochondrial enzyme that catalyzes transacylation between a phospholipid and a lysophospholipid in remodeling. Mutations in tafazzin cause Barth syndrome, a potentially life-threatening disease with the major symptom being cardiomyopathy. In the tafazzin-deficient heart, cardiolipin (CL) acyl chains become abnormally heterogeneous unlike those in the normal heart with a single dominant linoleoyl species, tetralinoleoyl CL. In addition, the amount of CL decreases and monolysocardiolipin (MLCL) accumulates. Here we determine using highresolution 31P nuclear magnetic resonance with cryoprobe technology the fundamental phospholipid composition, including the major but oxidation-labile plasmalogens, in the tafazzinknockdown (TAZ-KD) mouse heart as a model of Barth syndrome. In addition to confirming a lower level of CL (6.4 ± 0.1 → 2.0 ± 0.4 mol % of the total phospholipid) and accumulation of MLCL (not detected → 3.3 ± 0.5 mol %) in the TAZKD, we found a substantial reduction in the level of plasmenylcholine (30.8 ± 2.8 → 18.1 ± 3.1 mol %), the most abundant phospholipid in the control wild type. A quantitative Western blot revealed that while the level of peroxisomes, where early steps of plasmalogen synthesis take place, was normal in the TAZ-KD model, expression of Far1 as a rate-determining enzyme in plasmalogen synthesis was dramatically upregulated by 8.3 (±1.6)-fold to accelerate the synthesis in response to the reduced level of plasmalogen. We confirmed lyso-plasmenylcholine or plasmenylcholine is a substrate of purified tafazzin for transacylation with CL or MLCL, respectively. Our results suggest that plasmenylcholine, abundant in linoleoyl species, is important in remodeling CL in the heart. Tafazzin deficiency thus has a major impact on the cardiac plasmenylcholine level and thereby its functions.

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respiratory supercomplexes.17 These CL alterations are (i) a decreased level, (ii) diversification of acyl chain species contrary to the normal control, e.g., with one dominant species in the heart, and (iii) accumulation of monolysocardiolipin (MLCL).10,11,18 Therefore, CL has been the major focus among phospholipids in unveiling the molecular mechanism leading to Barth syndrome.1,19 In this work, we report the highresolution 31P nuclear magnetic resonance (NMR) determination of the phospholipid composition of the heart of genetically engineered tafazzin-knockdown (TAZ-KD) mice.20 We found a substantial reduction in the choline plasmalogen level (from 30.8 ± 2.8 to 18.1 ± 3.1 mol % of the total phospholipid). The reduction was shown to be ∼3-fold larger than the reduction in the level of CL (from 6.4 ± 0.1 to 2.0 ± 0.4 mol %) that is known as the hallmark phenomenon of lipid

ipids play a tremendous variety of functional roles in biological systems that are performed according to the specific structure of the entire lipid molecule, including the hydrocarbon chains.1 In recent years, lipid−protein interactions therefore have been attracting more attention together with changes in our fundamental understanding, with regard to both specific binding and nonspecific regulation of the physical and chemical membrane environment.1−4 Barth syndrome is an Xlinked potentially life-threatening recessive disease caused by mutations of a G4.5 gene in distal Xq28,5−8 which encodes a transacylase named tafazzin;9−11 tafazzin catalyzes transfer of an acyl group between a phospholipid and a lysophospholipid in phospholipid remodeling.12−15 One of the major clinical symptoms due to the defect in the activity of tafazzin is cardiomyopathy.5,6 The deficiency in tafazzin function leads to abnormal mitochondrial ultrastructure and impaired respiratory function,5,6 where dramatic alterations have been elucidated in the state of cardiolipin (CL) as a key player with its cell type specific distribution of acyl chain species16 in stabilizing the © 2018 American Chemical Society

Received: January 12, 2018 Revised: March 16, 2018 Published: March 20, 2018 2162

DOI: 10.1021/acs.biochem.8b00042 Biochemistry 2018, 57, 2162−2175

Article

Biochemistry alteration in Barth syndrome. This finding was realized by the simultaneous quantification of all the phospholipids along with adoption of the cutting-edge cryoprobe technology in 31P NMR to enhance the detection sensitivity. A particular precaution was made in the material processing to maintain the plasmalogen structure that is known to be labile to oxidation. Plasmalogens constitute as much as 18% of the total phospholipid mass in humans,21 yet their physical, chemical, and biological roles are still largely unknown.21−23 The plasmalogen deficiency in relation to Barth syndrome has not been recognized previously. In general, situations that have been contributing to our poor understanding of plasmalogen functions are due largely to (i) the nature of the structure that is not defined and characterized only by the headgroup, (ii) instability against oxidation, (iii) instability against acid during lipid extraction and chromatographic separation, and (iv) fragmentation complexity in tandem mass spectrometry (MS). Plasmalogens are specifically characterized by the sn-1 vinyl ether linkage (Figure 1), in contrast to the counterpart ester

standard phospholipids of different headgroups and hydrocarbon species to correct for differences in their ionization efficiency and/or by minimization of such differences in the ionization efficiency by taking a lower limit of lipid concentration for sample infusion to avoid formation of lipid aggregation that causes nonlinear responses in detection.27,28 A phospholipid content of each class or subclass is determined by identification and summing up of contributions from various hydrocarbon chain species. The presence of differential fragmentation during collision-induced dissociation (CID) in tandem MS has to be taken into account. The tandem MS detection of plasmalogens that contain the vinyl ether linkage is known to be difficult and requires particular attention due to their unique behavior that is different from the typical neutral loss of the headgroup observed for diacyl and plasmanyl glycerophospholipids.29 31 P NMR with inverse-gated 1H decoupling has an advantage in accurately quantifying contents of phospholipid classes and subclasses, each of which gives a discrete signal as a sum of contributions from different hydrocarbon chain species. The signal separation is based on a unique chemical shift value of a phosphate group in each class or subclass that reflects its electronic structure influenced by specific types of nearby functional groups in the headgroup as well as the linkage with hydrocarbon chains. Plasmalogen 31P NMR signals can be distinctively observed for both the choline and ethanolamine classes.30−32 In high-resolution 31P NMR of phospholipid quantification, use of either (i) a solvent mixture or (ii) detergent micelles in water is common for solubilizing lipids to obtain sharp resonance lines.33 Here we use sodium dodecyl sulfate (SDS) detergent micelles to measure the detailed phospholipid compositions of the TAZ-KD and WT mouse heart. Accurate quantification of plasmalogens in a biological material requires precautions to avoid degradation of the structure due to oxidation during sample preparation by including an antioxidant and a metal ion chelating reagent. The characteristic sn-1 vinyl ether linkage of plasmalogens is readily cleaved by reaction with reactive oxygen species (ROS), such as radicals and singlet oxygen.34−38 This vinyl ether has often been considered as the most probable target site for the oxidative reaction in phospholipids, including other susceptible sites like polyunsaturation in the acyl chain.36 Such preferential oxidation at the vinyl ether is notable even in micelles36 or in a membrane with a large surface curvature like small unilamellar vesicles (SUVs),37,38 where molecular motions of lipids are much less restricted and the degree of hydration is enhanced compared with that of a flat bilayer.39 In fact, the high reactivity of the vinyl ether with ROS has suggested an antioxidant role of plasmalogens in the cell. Mutants of Chinese hamster ovary (CHO) cells that were deficient in plasmalogen biosynthesis were hypersensitive to photosensitized exposure to long wavelength ultraviolet (UV) light (>300 nm).35,40 An inverse correlation between the peroxide-producing potential of tissues and the longevity of mammalian species is well-known.41,42 Contrary to the inverse correlation for peroxide-producing potential as specified by the double-bond content of phospholipids, a positive correlation was observed with the maximum life span for the plasmalogen level, which supports the antioxidant function of plasmalogens.43 Our 31P NMR observation of the drastic decrease in the level of plasmenylcholine in the TAZ-KD mouse heart led us to investigate further the cause of the decrease as well as cellular

Figure 1. Structures of plasmenylcholine and diacyl phosphatidylcholine (PC). Structures are drawn with the most abundant acyl chain species for those lipids in the mammalian heart.

linkage in the diacyl glycerophospholipids and the alkyl ether linkage in the plasmanyl glycerophospholipids. There are only two major types of plasmalogens called plasmenylcholine and plasmenylethanolamine, which have choline and ethanolamine headgroups, respectively. The heart is a rare organ that has abundant plasmenylcholine (26−41 mol % of the total choline glycerophospholipids in humans; the differences come from different sources in the literature) (cf. 1−3 mol % in the brain and 0−1 mol % in the liver), whereas plasmalogens in other organs are primarily ethanolamine glycerophospholipids.1,24,25 In the wild type (WT) mouse heart, the level of plasmenylcholine was measured in this work to be 30.8 ± 2.8 mol % and that of plasmenylethanolamine to be 8.2 ± 0.4 mol % of the total phospholipid. The numbers compare with the rest of choline glycerophospholipids (diacyl plus plasmanyl) at 19.2 ± 1.9 mol % and ethanolamine glycerophospholipids (diacyl plus plasmanyl) at 26.2 ± 1.2 mol %. Therefore, the drastic reduction in the level of plasmenylcholine due to tafazzin deficiency is expected to have substantial effects on the integrity of the heart tissue (see Discussion). MS methods have an advantage in that they are more sensitive for signal detection than 31P NMR. MS is also capable of providing information about the hydrocarbon chain composition of various phospholipid classes and subclasses.26 Quantification of phospholipid species in a biological material using MS is enabled by inclusion of a mixture of internal 2163


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protected against oxidation by the inclusion of 50 μM BHT. Lipid was then extracted from the homogenate by the Folch method47 using a chloroform/methanol [2/1 (v/v)] solvent containing 250 μM BHT. Solvent was evaporated under a stream of nitrogen gas to form a lipid film. The film was vacuum-dried for 2 h (note here high volatility of BHT) and dissolved in 600 μL of degassed 10% (w/v) aqueous SDS [pH 6.0, 50 mM MES, 50 μM BHT, and 10% (v/v) D2O]. High-Resolution 31P NMR. High-resolution 31P NMR spectra of the samples placed in 5 mm diameter NMR tubes were recorded with temperature control at 25 °C on a Bruker AVANCE-III 700 MHz spectrometer (31P frequency, 283.4 MHz) that was equipped with a QNP cryoprobe. Spectra were acquired with an 80° excitation pulse on 31P nuclei and inversegated broadband 1H decoupling with the WALTZ-16 sequence at a decoupling power of 3.8 kHz. Free induction decays were acquired for 2048 scans over a 12 ppm (3.4 kHz) bandwidth with a 2.4 s acquisition time and a 1.0 s recycle delay. The chemical shift scale was referenced to an external 85% (w/v) phosphoric acid standard set to 0 ppm. With the total 3.4 s pulse interval time on the 80° excitation pulse, 31P resonances of the measured phospholipids undergo full relaxation to ensure accurate quantification of their composition. This was confirmed by measurements of 31P spin−lattice relaxation times (T1) of the component phospholipids, as well as by direct comparison of the composition as a function of the excitation pulse angle varied in the range of 30−90° in a 10° increment. Parameters for the inversion−recovery experiments (d1−180°− t−90°−acquire)n were as follows: a 5.0 s delay time (d1), a 12.9 μs 90° pulse on 31P nuclei, 256 scans n for each variable delay time t (0.10, 0.25, 0.50, 0.70, 1.25, 1.50, 1.75, 2.50, 3.00, and 5.00 s), and WALTZ-16 1H decoupling at 1.6 kHz during the d1−180°−t period and at 3.8 kHz during the 90°−acquire period. Quantitative Western Blot. Quantitative Western blotting experiments were conducted on a V3 Western Workflow system (Bio-Rad) with some modifications to the procedure. Sample Preparation. 48−51 All the steps of sample preparation were performed at 0−4 °C. The homogenization medium (HM) [0.25 M sucrose, 1 mM EDTA, 20 mM TrisHCl (pH 7.4), and protease inhibitor cocktail] and the equipment used in the homogenization, centrifugation, and fractionation were prechilled. The heart immediately after being taken from a freshly sacrificed mouse was washed three or four times with HM. The heart was minced coarsely in 4 volumes of HM and homogenized in a homogenizer with a pestle rotating at 500 rpm for eight up-and-down strokes at a rate of ∼10 s/ stroke. The homogenate obtained was centrifuged at 750g for 5 min. The supernatant was centrifuged one more time at 750g for 5 min. Thus, the obtained supernatant, i.e., postnuclear supernatant (PNS), was aliquoted and stored at −80 °C until it was used. A part of PNS was fractionated into a pellet fraction containing peroxisomes and a supernatant fraction by ultracentrifugation at 100000g for 30 min. The pellet was resuspended with a volume of HM equal to the supernatant volume. The protein concentration of PNS was determined by the Bradford assay (Bio-Rad). For Western blot experiments, the samples were heated at 95 °C for 5 min with SDS in the presence of a reducing agent (5% β-mercaptoethanol or 100 mM DTT) to denature proteins. The samples were briefly (∼2 min) centrifuged at 6000 rpm, and the supernatant was used for the Western blot experiment.

responses to it based on quantitative Western blotting of relevant proteins.

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MATERIALS AND METHODS Materials for the NMR Experiments. Most phospholipid standards used for signal assignment in the 31P NMR experiments, except sphingomyelin (SM) from Sigma (St. Louis, MO), were purchased from Avanti Polar Lipids (Alabaster, AL) as summarized in Appendix S1. SDS and sodium cholate hydrate used to dissolve lipids in water for 31P NMR measurements were from Bioshop Canada (Burlington, ON) and Sigma-Aldrich (St. Louis, MO), respectively. 2-(NMorpholino)ethanesulfonic acid (MES) hydrate used to prepare the buffered SDS or cholate micellar solution was from Sigma-Aldrich. Ethylenediaminetetraacetic acid (EDTA) and cesium hydroxide hydrate or the cesium hydroxide solution used to prepare the Cs-EDTA aqueous solution to homogenize the mouse hearts were from Sigma-Aldrich. An antioxidant, butylated hydroxytoluene (BHT), was from Sigma-Aldrich. Methanol (HPLC grade) and chloroform (HPLC grade) were from Sigma-Aldrich. NMR solvent deuterium oxide (99.9 atom % D) was from Cambridge Isotope Laboratories (Andover, MA). Doxycycline-Induced TAZ-KD in Transgenic Mice. All protocols were approved by the Institutional Animal Care and Use Committee of the NYU School of Medicine and Langone Medical Center and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH). The tafazzin-knockdown (TAZ-KD) transgenic mice (JAX stock 014648)44 were housed in temperaturecontrolled conditions under a 12 h light/dark cycle with free access to drinking water and food. The mice used in this study are the offspring of heterozygote (male)−wild type (WT) (female) C57BL/6N crosses. To knock down tafazzin expression, 3-month-old transgenic mice were treated with doxycycline in drinking water, as well as their WT littermates, for the next 8 months. Briefly, drinking water containing 2 mg/ mL doxycycline and 10% sucrose was prepared every 3−4 days. The use of 10% sucrose was necessary to improve the palatability of the doxycycline solution. The phospholipid composition of the WT mouse heart showed no significant difference with and without doxycycline induction. Preparation of the TAZ-KD and WT Mouse Heart Phospholipid Samples for 31P NMR. Cs-EDTA buffer was prepared by titration of free EDTA (at a final concentration of 0.2 M) in water with 50 wt % aqueous CsOH until the pH reached 6.0, followed by volume adjustment, addition of 50 μM BHT, readjustment of the pH to 6.0, and degassing by argon bubbling.31 Milli-Q purified water was used. The heart was taken from a freshly sacrificed TAZ-KD or WT mouse and placed immediately in the cold Cs-EDTA buffer in a beaker. The buffer with the heart in it was swirled gently, excess blood removed with the buffer by decantation, and the buffer readded (typically 1 mL for one heart). The heart tissue from two or three mice was used for one 31P NMR sample; for each sample, the hearts were minced and homogenized in the cold buffer using a motor-driven Teflon pestle and a glass vessel. Here EDTA not only captures paramagnetic ions to prevent NMR line broadening but also protects plasmalogens and polyunsaturated chains of phospholipids from ion-catalyzed oxidative degradation.34,45,46 Cs cation is used because of the absence of formation of a precipitate of the cation-EDTA salt upon mixing with organic solvent.31 Lipids were further 2164


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Figure 2. (A) 31P NMR spectra of phospholipids in the heart of the wild type (WT, blue trace) and tafazzin-knockdown (TAZ-KD, red trace) mice. A magnified region at the right shows inversion of the spectral asymmetry, indicating a drastic decrease in the level of plasmenylcholine (−0.163 ppm) and a counterbalancing increase in the level of diacyl PC (−0.174 ppm) due to the TAZ-KD. The signal from the minor component plasmanylcholine (see the text) overlaps with the signal from diacyl PC (Figure S6). A magnified region at the top left shows a large decrease in the level of CL (0.745 ppm) along with increases in levels of 2-MLCL (1.137 ppm) and 1-MLCL (1.009 ppm). An increase in the level of PG (0.845 ppm) is also seen. *A part of the CL decrease is masked by contributions from the resonance of a phosphate group in the diacyl half of MLCLs (Figure S5). (B) Spectrum of the standard plasmenylcholine (green) overlaid with that of the WT mouse heart phospholipid (blue).

SDS−Polyacrylamide Gel Electrophoresis. Precision Plus Protein All Blue Prestained Protein Standards (Bio-Rad) were used as molecular weight markers. Proteins were separated using a 4−15% Mini-PROTEIN TGX Stain-Free Protein Gel (Bio-Rad) at 250 V for 25 min. The amount of sample to be loaded per lane for the quantitative Western blot analysis of the target proteins was determined by evaluating their respective linear dynamic range as a function of the total protein content (Figures S1 and S2). This evaluation of the linear dynamic range was done with a serial dilution of a mixture of an equal amount (per total protein) of the WT and TAZ-KD PNS fractions. Introduction of a Fluorophore for Quantification of the Total Protein Content. UV irradiation of the gel after the electrophoretic separation of proteins initiates reaction between trihalo compounds in the gel and tryptophan residues in proteins to introduce a fluorophore. UV irradiation was conducted for 45 s on a ChemiDoc Imaging System (BioRad), after which ∼10% of tryptophan residues in proteins was fluorophore-labeled. This ∼10% labeling of tryptophan residues is sufficient to accurately quantify the total protein content, while having a negligible effect on immunodetection of target proteins downstream. The fluorescence from the introduced fluorophore was quantified after transfer to the polyvinylidene difluoride (PVDF) membrane to confirm the electrophoretic protein separation and to determine the lane-dependent transfer efficiency (Figure S2 legend). Protein Transfer from Gel to Membrane, and Correction for Lane-Dependent Transfer Efficiency. Proteins were

transferred from the gel onto an Immun-Blot Low Fluorescence PVDF Membrane (Bio-Rad) at 2.5 A and 25 V for 7 min using a Trans-Blot Turbo Blotting System (Bio-Rad). The transfer efficiency in each lane was determined by quantification of the total protein in the membrane on the ChemiDoc Imaging System (Bio-Rad) (Figure S2 legend). Incubation with Antibodies. After being blocked with 5% BSA in TBST, the membranes were incubated with the primary antibody at 4 °C overnight and then with the secondary antibody at room temperature for 1 h. A wash was made after each incubation. The antibodies that were used are summarized in Table S1. Quantification of Target Proteins. The membranes were incubated with ECL reagent,52 and chemiluminescence was used to quantify the target proteins with a ChemiDoc Imaging System (Bio-Rad).

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RESULTS High-Resolution 31P NMR Measurements of the Phospholipid Composition Including Oxidation-Labile Plasmalogens in the Heart of WT and TAZ-KD Mice. High-resolution 31P NMR in combination with the use of a solubilizing detergent is a powerful method for accurately determining the detailed phospholipid composition of a biological material in a single measurement, based on its ability to distinctively identify different phospholipid classes and subclasses according to the unique chemical shift values of the phosphate group. Sensitivity of the chemical shift to reflect the difference in the type of sn-1 linkage for plasmalogens is of 2165


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Biochemistry Table 1. Contents and Changes (mol %)a of the Heart Phospholipids of the WT versus TAZ-KD Mice content phospholipid diacyl PC (with plasmanylcholine)c plasmenylcholine PI PS lyso PC diacyl PE (with plasmanylethanolamine)c plasmenylethanolamine SM CL PG 1-MLCL 2-MLCL

chemical shiftb (ppm)

WT

TAZ-KD

−0.174

19.2 ± 1.9

27.0 ± 2.9

−0.163 0.039 0.128 0.208 0.295

30.8 ± 2.8 3.6 ± 0.1 1.8 ± 0.2 0.5 ± 0.1 26.2 ± 1.2

18.1 4.1 2.1 0.6 30.0

± ± ± ± ±

3.1 0.3 0.4 0.1 1.1

−12.7 +0.5 +0.3 +0.1 +3.8

± ± ± ± ±

4.2 0.3 0.5 0.1 1.6

−41.3 ± 13.6 +14.3 ± 9.1 +15.9 ± 26.8 +10.9 ± 23.3 +14.6 ± 6.2

8.3 3.1 2.0 1.4 0.9 2.4

± ± ± ± ± ±

0.4 0.5 0.4 0.1 0.4 0.2

+0.2 +0.7 −4.4 +0.6 +0.9 +2.4

± ± ± ± ± ±

0.5 0.5 0.4 0.2 0.4 0.2

+2.2 ± 6.5 +26.9 ± 21.1 −68.7 ± 6.3 +65.7 ± 20.0 NAe NAe

0.336 0.409 0.745 0.845 1.009 1.137

8.2 ± 2.5 ± 6.4 ± 0.9 ± NDd NDd

0.4 0.2 0.1 0.2

change in contribution to the total phospholipid (TAZ-KD − WT) +7.8 ± 3.5

change in terms of the individual content {[(TAZ-KD − WT)/WT] × 100} +40.4 ± 18.1

a

The average and error, shown as the standard deviation, are obtained from three independent biological samples (N = 3) for each of the WT and TAZ-KD mice. bValues are from observation at 25 °C in a 10% (w/v) SDS micellar solution at pH 6 [50 mM MES, 50 μM BHT, and 10% (v/v) D2O], in reference to an external standard of 85% (w/v) phosphoric acid in water set to 0 ppm. cThe signal of the plasmanyl glycerophospholipid overlaps with the signal of the diacyl counterpart (Figure S6 shows the case of plasmanylcholine and diacyl PC). dNot detected. eNot applicable.

was used, as described in Materials and Methods, to prevent oxidation of plasmalogens. Plasmalogens were confirmed to be fully protected and stable even in the micellar aqueous solution on the order of days according to 31P NMR. Extraction of lipid from the mouse heart homogenate was performed by the Folch method47 using a cold chloroform/methanol [2/1 (v/v)] solvent containing 250 μM BHT (see Materials and Methods); the method is one of the most recommended in lipidomics studies.57 We also confirmed that lysophospholipids of the heart tissue were efficiently extracted by this method (Figure S3). The Plasmenylcholine Level Drastically Decreases in the TAZ-KD Mouse Heart. Figure 2A shows high-resolution 31P NMR spectra of the TAZ-KD and WT mouse heart phospholipids in a SDS micellar solution. The phospholipid compositions were determined as summarized in Table 1 along with their changes from the WT to the TAZ-KD mice. Signal assignment was based on comparison with spectra of phospholipid standards, literature data, as well as use of a cholate micellar solution.30−32 Sensitive detection and accurate quantification of the phospholipids in the 31P NMR experiments with inverse-gated 1H decoupling were ensured by optimization of the experimental parameters according to the measured spin−lattice relaxation times (T1) (Table S2 and Figure S4). In addition to confirming the well-known changes in cardiolipin (CL) due to tafazzin deficiency, i.e., the decrease in the amount of CL by 4.4 ± 0.4 mol % (from 6.4 ± 0.1 to 2.0 ± 0.4 mol %) and accumulation of monolysocardiolipins (MLCLs) to a level of 3.3 ± 0.5 mol % (from not detected to 3.3 ± 0.5 mol %) in the heart of the TAZ-KD mice (Table 1 and Figure S5), we found a drastic decrease in the level of plasmenylcholine (at −0.163 ppm) by 12.7 ± 4.2 mol % (from 30.8 ± 2.8 to 18.1 ± 3.1 mol %) (Figure 2A,B and Table 1) and a counterbalancing increase in the level of diacyl phosphatidylcholine (PC) (at −0.174 ppm) of 7.8 ± 3.5 mol % (from 19.2 ± 1.9 to 27.0 ± 2.9 mol %) (Figure 2A and Table 1) that are seen as clear inversion of spectral asymmetry with the partially overlapping signals. The mechanism of the counterbalance is separately discussed in Appendix S2. The decrease in

particular importance in reliable quantification of those major but unique phospholipids.30−32 In 1H and 13C NMR of phospholipids, a proton or carbon signal from one of the sn-1 vinyl ether protons or carbons of plasmalogens, which is known to be distinct from all the other signals in the spectrum, may be used to quantify plasmalogens in a biological system.53−55 However, a procedure for identifying relative contributions to the 1H or 13C spectrum from various phospholipid classes and subclasses will be somewhat labor intensive also after assignment of heavily overlapping signals. Such quantification as well as cross-check for accuracy may be conducted by calculating a combination of a number of intensity ratios for uniquely identifiable and well-separated signals from the individual phospholipid class or subclass. Calculation can be done using only minor fractions of the entire dynamic range of detection even for the major classes because of heavy signal overlap for common structural moieties of phospholipids. In some cases, use of a detergent with an extremely small aggregation number (n) like cholate (n = 4) to dissolve lipids56 for measurements in 31P NMR not only results in resonance sharpening due to enhanced molecular motions but also causes specific detergent−lipid interactions that depend on hydrocarbon chain species and at least partially differentiate among them within a given class or subclass.33 In the work presented here, it turned out that use of SDS with a moderate aggregation number (n = 62−101)56 is suitable to determine the phospholipid composition of a biological material, where each class or subclass gives a distinct single resonance as the sum of contributions to the intensity from various hydrocarbon chain species. This simplifies determination of the phospholipid composition without the need to identify unevenly separated signals of diverse hydrocarbon chain species like in the case of using cholate, where some of them can overlap with or appear between signals of chain species of another class that is nearby in the spectrum. Nevertheless, we used cholate as well in this work to aid assignment of observed signals using SDS, according to a different profile of intensities versus chemical shifts for the phospholipid classes and subclasses as presented below. Throughout the process for preparation of the NMR samples of the mouse heart phospholipids, particular caution 2166


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in the whole heart tissue.62 Our preliminary data (not shown) in an ongoing subcellular fractionation study, using a single-cell type system of lymphoblasts derived from Barth syndrome patients that is ideal for studies of the mechanism, indicated that a decrease in the level of plasmalogen (in this case plasmenylethanolamine as the dominant form) takes place almost exclusively in mitochondria. The observed decrease in the level of plasmenylcholine in the TAZ-KD mouse heart may suggest an increase in the level of lyso-plasmenylcholine in the deficiency of tafazzin. Although the presence of only a trace amount of lysophospholipid is required to initiate the catalytic transacylation cycles by tafazzin according to the affinity that is orders of magnitude higher for a lysophospholipid than for a parent phospholipid,15 it is conceivable that the maintenance of the plasmenylcholine level is associated with the lyso-plasmenylcholine level. In fact, a decrease in the level of CL due to tafazzin deficiency is accompanied by an increase in the level of MLCL. In the 31P NMR spectra of both the WT and TAZ-KD mouse heart phospholipids (Figure 2A), we observed a signal at 0.208 ppm that can be unambiguously assigned to lyso PC as described below. Measurements on the standard lysoplasmenylcholine and lyso PC in a SDS micellar solution showed that the 31P NMR signal of lyso-plasmenylcholine appears slightly downfield of the signal of lyso PC, whereas the separation between the two signals was ∼4-fold smaller than that observed for plasmenylcholine and diacyl PC (Figure 3A).

the level of plasmenylcholine is drastic with regard to being both (i) the largest change in the content and (ii) for the most abundant phospholipid in the WT mouse heart (Table 1). The impact of such a drastic change in the most abundant phospholipid in the tissue is expected to be substantial as a part of not only the lipid biochemistry of the cell but also the membrane physical properties (see Discussion). The magnitude of the decrease in terms of a change in the individual content was also estimated to be large as 41.3 ± 13.6 mol % (Table 1). This value is quite comparable to the decrease in CL by 68.7 ± 6.3 mol %. As a minor component, a plasmanylcholine signal overlaps with the diacyl PC signal (Figure S6), whereas contents of plasmanylcholine in the mammalian heart tissues are commonly less than 1/10 of that of diacyl PC,25,58 and essentially the same applies to the ratio between plasmanylethanolamine and diacyl PE.25,58 It is noted that in sharp contrast to the dramatic decrease in the level of plasmenylcholine in the TAZ-KD heart, no significant change in plasmenylethanolamine content was detected (Figure 2A and Table 1). Lysoplasmalogen as a Counterpart of Plasmalogen in Transacylation by Tafazzin. The large decrease in the level of plasmenylcholine due to deficiency in transacylation by tafazzin poses the question of whether there is any change in the content of its lysophospholipid either as a precursor or as a product of transacylation in the control WT. Although tafazzin has been shown to be a nonspecific transacylase for any phospholipid class examined,12−15 its reactivity for plasmalogen phospholipids has not been probed specifically to date. In our matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS experiments, lysoplasmalogens indeed were confirmed to be a substrate of tafazzin for both lysoplasmenylcholine and lyso-plasmenylethanolamine, using purified recombinant yeast tafazzin59 (see Figure S7 for the reactivity). The reversible nature of the transacylation by tafazzin, as has been reported in experiments using reversed pairs with regard to the donor/acceptor roles in each pair of different phospholipid classes,14,15 indicates that lysoplasmalogens and plasmalogens are both substrates of tafazzin. The absence of the sn-1 unsaturation adjacent to the ether bond, corresponding to the structure of plasmanyl glycerophospholipids as minor components in mammalian organs, including the heart,25,58 showed in contrast a lack of reactivity with tafazzin.60 The schematic analogy in reversibility of biochemical reactions to regulate a compositional equilibrium may be familiarly presented by transaminase to transfer an amino group between a donor and an acceptor in the bidirectional reaction.61 The net direction of the reaction mutually depends on concentrations of the four substrates, i.e., two substrates (a donor and an acceptor) on each side of the equation that correspond to the two counterpart products (an acceptor and a donor in the opposite reaction, respectively). While we chose here CL as a transacylation partner to directly probe the reaction for CL remodeling, in biological systems the partner can also be other lipid classes, including the major PC and PE species as part of the catalytic reaction cycle. Another important feature of the transacylation cycles by tafazzin is that only a trace amount of lysophospholipid is required and actually present in the membrane for progression of remodeling and attainment of the equilibrium in acyl species distributions of the component phospholipids.15 Both choline and ethanolamine plasmalogens are major components of the heart mitochondria, and their fractions in this organelle are as abundant as they are

Figure 3. 31P NMR identification of lysophospholipids in the mouse heart, using different solubilizing detergent systems of SDS and cholate. (A) 31P NMR of lyso PC and lyso-plasmenylcholine (lysoplsCho) in SDS micelles yields similar chemical shift values. (B) Use of cholate micelles results in distinct signal separation for lyso PC and lyso-plsCho, thus unambiguously identifying the presence of lyso PC and the absence of a detectable amount of lyso-plsCho in the WT and TAZ-KD mouse heart. (C) The absence of a detectable amount of lyso PE in the WT and TAZ-KD mouse heart is evident from the measurements in SDS micelles. Lyso-plasmenylethanolamine (lysoplsEtn) shows a chemical shift close to that of CL and the diacyl half of MLCLs at 0.745 ppm in SDS micelles. (D) Use of cholate micelles enabled identification of the absence of a detectable amount of lysoplsEtn in the WT and TAZ-KD mouse heart. 2167


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Quantitative Western Blot for Monitoring Proteins for Biochemical Insights into the NMR Observation of the Decrease in the Level of Plasmalogen. In this section, we investigate levels of proteins in the mouse heart that may be considered to be relevant to the observation described above of the substantial loss of plasmenylcholine due to the TAZ-KD. Monitoring of the proteins has been performed by quantitative Western blotting. Quantification in a linear dynamic range of immunodetection was carefully assured for each target protein with regard to both the binding capacity with the PVDF membrane and integration of the chemiluminescence signal from the horseradish peroxidase (HRP) secondary antibody using a CCD camera (Figure S1). Lane-dependent unevenness in the efficiency of transfer from the gel to the membrane was also taken into account and corrected by normalization to the total protein content that was quantified in each lane using Image Lab Software (Bio-Rad) (Figure S2 legend). A loss of plasmalogen found in other diseases like Zellweger syndrome is associated with a significant loss of peroxisomes,24 where the early steps in biosynthesis of plasmalogen take place (Figure 4).21−23 In the work presented here, it is noteworthy that the steady state level of plasmenylethanolamine that is a precursor for plasmenylcholine synthesis showed no significant change due to the TAZ-KD in contrast to the dramatic loss of plasmenylcholine (Figure 2A and Table 1). In light of the wellcharacterized cases of the other diseases in which plasmalogen loss is associated with peroxisome deficiency, the clear difference observed for choline and ethanolamine plasmalogens in the TAZ-KD mouse heart was intriguing and led us to evaluate the amount of peroxisomes. Among the series of peroxins that are responsible for functions of peroxisomes, Pex19p, Pex16p, and Pex3p are known to be critical in de novo formation of the peroxisomal membrane structure.64−66 Pex19p is a water-soluble peroxin predominantly located in the cytosol, while Pex16p and Pex3p are membrane-associated. Pex19p functions as a cargo protein as well as a chaperone to incorporate peroxisomal membrane proteins (PMPs) into the peroxisomal membrane. In this work, we quantified Pex19p as well as PMP70, which is a frequently used marker of the peroxisome level,67,68 to investigate the amount of peroxisomes. Another reason to look into peroxisomes is related to a recent report that the de novo formation of peroxisomes is initiated by vesicular preperoxisomal structures budding off from mitochondria,68 which are damaged in Barth syndrome, as well as from the ER. We also measured the level of catalase, which has been used as a histological marker of peroxisomes.69 Catalase levels in the cytosol and in peroxisomes were monitored by paying attention to the previously reported correlation of tafazzin deficiency with impaired respiratory function70−72 and relevant enhancement of ROS formation.73,74 In examining a direct response to a plasmalogen decrease in the cell, we expected to find it insightful to look into the expression level of fatty acyl-CoA reductase 1 (Far1). Far1 functions as a rate-determining enzyme for plasmalogen synthesis,75 converting fatty acyl-CoA to fatty alcohol (Figure 4).76 Fatty alcohol is used to form a plasmalogen precursor 1O-alkyl-dihydroxyacetone phosphate (alkyl-DHAP) in peroxisomes (acyl-DHAP → alkyl-DHAP) (Figure 4). The level of Far1 expression is known to be regulated by a feedback mechanism in response to the amount of plasmalogen in the cell;75 in the case of a decrease in the level of plasmalogens, we would expect Far1 upregulation to increase the rate of plasmalogen synthesis.

The separation between those two lysophospholipids became more distinct in a cholate micellar solution with an altered chemical shift profile for phospholipids, giving a separation 10 times larger than that in a SDS micellar solution (Figure 3B). Measurements of the WT and TAZ-KD mouse heart phospholipids in a cholate micellar solution confirmed the absence of a detectable level of lyso-plasmenylcholine in those samples (Figure 3B). The absence of detectable levels of lyso PE and lyso-plasmenylethanolamine in either the WT or the TAZ-KD mouse heart was confirmed via similar experiments comparing those different detergent systems (Figure 3C,D). Plasmenylcholine versus Plasmenylethanolamine. Early steps in the biosynthesis of plasmalogens take place in peroxisomes, from which an intermediate precursor is transferred to the endoplasmic reticulum (ER), where further synthetic steps lead to the formation of plasmenylethanolamine (1-O-alk-1′-enyl-2-acyl-GPE) (Figure 4).21,22,63 Plasmenylcho-

Figure 4. Biosynthesis pathway for plasmalogens [1-O-alk-1′-enyl-2acyl-GPE (plasmenylethanolamine) and 1-O-alk-1′-enyl-2-acyl-GPC (plasmenylcholine)] in the cell.

line (1-O-alk-1′-enyl-2-acyl-GPC) is synthesized from plasmenylethanolamine via the cleavage in the headgroup either with or without steps including exchange of the acyl chain prior to headgroup cleavage (Figure 4).21,22,63 The observed decrease in the plasmalogen level in the heart of TAZ-KD mice was exclusive for plasmenylcholine (Figure 2A and Table 1). Other important pieces of experimental evidence in ongoing studies are that in other TAZ-KD mouse organs or human lymphoblast cells derived from Barth syndrome patients, there is a significant decrease in the level of plasmenylethanolamine, the dominant form of plasmalogen in these cells and tissues [e.g., in the lymphoblast, a decrease of 3.5 ± 2.2% (from 14.4 ± 1.9 to 10.8 ± 1.1%)] compared to control (unpublished results). A counterbalancing increase was observed, as well, in overlapping signals corresponding to the other ethanolamine phospholipids (diacyl and plasmanyl) that most likely reflect an increase in the level of diacyl PE [e.g., in the lymphoblast, 7.5 ± 1.8% (from 13.6 ± 1.1 to 21.1 ± 1.4%)]. 2168


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Figure 5. Quantitative Western blot of proteins in the mouse heart that are indicative of (i) the amount of peroxisomes (Pex19p, PMP70, and catalase), (ii) a degree of oxidative stress (catalase), (iii) a degree of feedback regulation of the plasmalogen level (Far1), and (iv) a degree of plasmalogen-selective lipid degradation (iPLA2β). (A) Ratios of expression levels (TAZ-KD/WT) in the TAZ-KD and WT mouse heart on cytosolic Pex19p (N = 3) and peroxisomal integral membrane protein PMP70 (N = 3). Measurements to determine the ratio were conducted on the postnuclear supernatant (PNS), while fractionation by ultracentrifugation (see Materials and Methods) results in detection of Pex19p exclusively in the supernatant fraction (S), and PMP70 exclusively in the membrane pellet fraction (P) that includes peroxisomes. (B) TAZ-KD/WT ratios on catalase determined for the PNS (N = 9). Furthermore, fractionation was performed to determine the TAZ-KD/WT ratio in each of the S (N = 3) and P (N = 3) fractions. The Western blotting image shows quantities of catalase in the PNS, S, and P fractions of the WT and TAZ-KD mouse heart homogenate. Distributions of cytosolic (in S) and peroxisomal (in P) catalase in the WT and TAZ-KD samples are illustrated as pie charts. (C) Western blotting image showing greatly enhanced expression of Far1 in the TAZ-KD mouse heart, where the large decrease in the level of plasmenylcholine was observed by 31P NMR. The fractionation experiment resulted in detection of Far1 dominantly in the supernatant. The TAZKD/WT ratio on the PNS is shown (N = 10). (D) Western blotting image showing quantities of iPLA2β. The fractionation experiment resulted in detection of the protein in the supernatant fraction. The TAZ-KD/WT ratio determined from the PNS is shown (N = 10).

conclusion of no significant reduction in the amount of peroxisomes is in accord with the NMR observation that the plasmenylethanolamine content remains essentially unchanged (Figure 2A and Table 1). That is, the state of peroxisomes is normal in their ability to synthesize the precursors of plasmalogens. In skin fibroblasts of Barth syndrome patients, the level of catalase was observed to be upregulated.74 Catalase is often used as a histological marker for peroxisomes by detecting peroxisomal catalase in comparison to the level of cytosolic catalase.69 The presence of catalase in mammalian heart mitochondria was also indicated,83 although its role in H2O2 removal in this organelle is likely to be minor compared to the role played by glutathione peroxidase.84 In the work presented here, we observed an increase in the level of catalase expression in the TAZ-KD mouse heart with a TAZ-KD/WT ratio of 1.47 ± 0.08 in measurements on the postnuclear supernatant (PNS) of the tissue (Figure 5B and Table S3). The distribution of catalase in the membrane pellet (P) and the supernatant (S) fractions of the PNS in the WT mouse heart was measured to be 45.4 ± 1.7 and 54.6 ± 1.7%, respectively (Figure 5B). The increased catalase level due to tafazzin deficiency was mostly attributed to the membrane fraction, i.e., peroxisomal catalase with a TAZ-KD/WT ratio of 2.15 ± 0.03, in contrast to the ratio of 1.10 ± 0.06 obtained for cytosolic catalase (Figure 5B).

In the mammalian heart, plasmenylcholine is degraded by consecutive enzymatic actions of (i) phospholipase A2 to first cleave off the acyl chain at the sn-2 position and (ii) lysoplasmalogenase to next cleave off the fatty aldehyde at the sn-1 vinyl ether bond on lysoplasmalogen.77 Note that plasmalogenase activity, which was observed in the heart to cleave the sn-1 vinyl linkage in plasmenylethanolamine,78 has been found to be absent for plasmenylcholine in this organ.77,79 In the work presented here, we measured expression levels of calcium-independent phospholipase A2 isoforms that are known to be plasmalogen-selective, iPLA2β and -γ.79−82 The Amount of Peroxisomes Is Normal in the TAZ-KD Mouse Heart, while the Catalase Expression Level Is Significantly Increased. The ratio of expression of Pex19p in the TAZ-KD to WT mouse heart (TAZ-KD/WT) was determined to be 1.12 ± 0.10 and that of PMP70 to be 0.97 ± 0.05 (Figure 5A and Table S3). The results indicate that the amount of peroxisomes is not reduced and thus do not ascribe the NMR observation of the decrease in the level of plasmenylcholine to a change in the amount of peroxisomes. Similar observations were made for the ratio for those proteins in human lymphoblast cells derived from Barth syndrome patients to a healthy control (BTHS/control) with a somewhat higher ratio for Pex19p than in the case of the mouse heart (1.29 ± 0.03 for Pex19p and 0.95 ± 0.01 for PMP70). The 2169


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other acyl transferases in remodeling.19,87 However, such responses can contribute to a complication to increase phenotypic severity. In fact, knockout of iPLA2β in tafazzin deficiency is known to decrease the MLCL/CL ratio as a molecular indicator of the inner mitochondrial membrane defect and to alleviate phenotypic severity in disease models (Appendix S4). We also measured the iPLA2γ levels in the TAZ-KD and WT mouse heart. However, we detected only a faint band corresponding to iPLA2γ (Figure S8). There was no consistent change in the TAZ-KD/WT ratio, ranging randomly between 0.5 and 3.1 in five sets of independent experiments (N = 12) (Figure S8).

The increase in the former is in accord as well with the observations that there is no loss of the normal level of peroxisomes in the TAZ-KD model according to quantification of Pex19p and PMP70 described above. The highly preferential increase in the peroxisomal catalase level results in the catalase distributions in the P and S fractions in the TAZ-KD mouse heart of 61.8 ± 0.2 and 38.2 ± 0.2%, respectively (Figure 5B). The conceivable mechanism leading to the catalase increase is described in Appendix S3. Our observation of an upregulated level of catalase in the TAZ-KD mouse heart likely indicates an enhanced defensive response to oxidation in the tissue. The more prominent increase for peroxisomal catalase than for cytosolic catalase may reflect the critical role of catalase in detoxifying H2O2 in peroxisomes in contrast to its minor contribution compared to that of glutathione peroxidase in the cytosol.85,86 The Rate of Plasmalogen Synthesis Is Enhanced in the TAZ-KD Mouse Heart by Substantial Upregulation of Far1 Expression. Far1 is a rate-determining enzyme of plasmalogen biosynthesis, the expression of which is regulated by a feedback mechanism in response to the amount of plasmalogen in the cell.75 We compared Far1 expression levels of the TAZ-KD and WT mouse heart. The abundance of Far1 was found to be substantially enhanced by 8.3 ± 1.6-fold in the TAZ-KD model compared to that of the control WT (Figure 5C and Table S3). The specificity of the immunodetection was further confirmed by use of a commercially available blocking peptide for Far1. The strong upregulation of the Far1 level is consistent with a positive feedback response to the decreased steady state level of plasmenylcholine that is caused by the tafazzin deficiency, yet the presence of the strong upregulation of Far1 expression does not fully compensate for the decrease in the level of plasmenylcholine. Another relevant observation is that there is no significant change in the content of plasmenylethanolamine (Figure 2A and Table 1). As plasmenylethanolamine is on the route to the production of plasmenylcholine (Figure 4), it is reasonable to consider that the observed lack of change in the steady state level of plasmenylethanolamine is merely a reflection of a balance between the accelerated plasmalogen synthesis caused by Far1 upregulation and a conceivable decrease in the level of plasmenylethanolamine by a mechanism analogous to that for the decrease in the level of plasmenylcholine. In other words, Far1 upregulation was sufficient to compensate for a decrease in the level of plasmenylethanolamine due to the tafazzin deficiency but not for a decrease in the level of plasmenylcholine. Plasmalogen-Selective iPLA2β Is Upregulated, Contributing to the Reduction of the Plasmalogen Level. The β isoform of calcium-independent phospholipase A2, iPLA2β, is known to be plasmalogen-selective especially in releasing its sn2 polyunsaturated fatty acid.79−81 It is therefore of interest to quantify the expression level of iPLA2β, when tafazzin is deficient compared with normal controls. We observed a TAZKD/WT expression ratio of 1.39 ± 0.14 (Figure 5D and Table S3). The significantly increased level of expression of iPLA2β in the absence of tafazzin likely contributes to the decrease in the plasmalogen level. No detectable amount of lyso-plasmenylcholine or lyso-plasmenylethanolamine in the NMR of the TAZKD (Figure 3) indicates that degradation of lysoplasmalogens is efficient to prevent their accumulation. The upregulation of iPLA2β in the tafazzin deficiency may be a part of spontaneous cellular responses to rescue the loss of transacylation, by an elevated level of formation of a substrate for reacylation by

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DISCUSSION Plasmalogen Species in the Mammalian Heart and Potential Influences of a Reduction of the Level of Plasmalogen in Tafazzin Deficiency. Tafazzin Function and CL Remodeling in the Context of the Membrane Environment. CL species, in a biological system particularly with a high energy turnover rate, are highly specific, typically containing one or two dominant unsaturated acyl chain species.16 The dominant acyl chain species and the distribution of acyl chain species vary depending on the type of cell, tissue, organ, and organism.16 In the case of the mammalian heart, including the mouse heart investigated in this work,16,88 the dominant CL species is tetralinoleoyl (18:2)4. Tafazzin is centrally responsible for the regulation of the unique distribution of CL species via transacylation cycles in remodeling. Deficiency in tafazzin function results in a highly diverse and heterogeneous distribution of CL species.16 The molecular mechanism that regulates the system-dependent dominant CL species in remodeling is currently being actively investigated. Differences in dominant CL species in different systems cannot be explained merely by confining our focus to an intrinsic specificity of tafazzin.1,2,19 Key observations in this regard are that expression of heterologous tafazzin yields formation of CL species characteristic to the expression system rather than CL species of the system, to which the expressed tafazzin orthologue originally belongs; e.g., human TAZ expressed in yeast yields yeast CL species,89 human TAZ in Drosophila yields Drosophila CL species,90 and human or Drosophila TAZ in Sf9 insect cells yields CL species of Sf9 insect cells.15 Therefore, the physical and chemical membrane environment of a system, including the composition of phospholipids and their acyl chain species, must play a role in determining the distribution of CL species typically with one or two dominant species. The observation of the substantially decreased steady state level of plasmenylcholine, but not of other lipids except CL, in the TAZ-KD mouse heart (Figure 2A and Table 1) suggests that plasmenylcholine plays an important role in transacylation cycles between alternating lysophospholipid and phospholipid substrates of tafazzin in remodeling. The decrease in the level of plasmenylcholine caused by tafazzin deficiency may be associated with degradation of plasmenylcholine by the activity of upregulated plasmalogen-selective iPLA2β as shown above (Figure 5D and Table S3). Tafazzin has been shown to have reactivity with any phospholipid class examined,12−15 and choline phospholipids showed a high reactivity with CL in experiments using purified mitochondria as the source of tafazzin. In addition to the high reactivity at a given concentration, we note that the WT mouse heart contains plasmenylcholine at 30.8 ± 2.8 mol % as the 2170


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Biochemistry single dominant phospholipid, followed by 19.2 ± 1.9 mol % diacyl PC and a minor fraction of plasmanylcholine (Table 1). When quantification was conducted for the transacylation reactivity by tafazzin between MLCL and a 14C-labeled phospholipid in the 2-acyl chain in sonicated liposomes14 >3fold faster reaction rates were observed for PC than for PE having the corresponding acyl chain species, with a marked preference in reactivity observed for a linoleoyl (18:2) chain among others. Note here that tafazzin preferentially transfers an sn-2 acyl chain of a phospholipid91,92 to either the sn-1 or the sn-2 site of a lysophospholipid,15,91 and the site of an acyl chain in a lysophospholipid readily shuffles between the sn-1 and sn-2 positions via acyl chain migration. Lysoplasmalogens are an exception in which the hydrocarbon chain is vinyl ether bonded in the sn-1 position and, therefore, does not migrate. An earlier work demonstrated the same trend in the reactivity preference (2-[14C]linoleoyl PC > 2-[14C]linoleoyl PE), although the difference was smaller.12 On both PC and PE, the reactivity of their sn-2 oleoyl (18:1) or arachidonoyl (20:4) chain species was PC(18:1−18:1) and PC(16:1−16:1)] using purified recombinant yeast tafazzin reconstituted in large unilamellar vesicles,91,92 while the effect of high concentrations of the residual detergent that was used in the protein purification in the work was discussed in detail later.3,59,92 The discussion was joined particularly because the tafazzin reactivity is known to be substantially accelerated in a highly disordered hydrocarbon chain environment, such as in micelles and inverted hexagonal phase membranes.2 A similar acceleration of the reactivity toward lipid substrates in those disordered environments is observed on another recombinant, human peripheral membrane protein, DGKε, by our group93 (Bozelli et al., unpublished results). Those results suggest that membrane morphology, including surface curvature (see Discussion), can have a large impact on the enzymatic reactivity and hence the phospholipid composition. In the mammalian heart in general, the dominant acyl chain species of plasmenylcholine is a linoleoyl chain, followed by an arachidonoyl or an oleoyl chain.58,62,94 The dominant sn-2 species of diacyl PC is also a linoleoyl chain, followed by an oleoyl chain. In the case of the mouse heart, the abundance of diacyl PC having an sn-2 linoleoyl chain is accompanied by the characteristic presence of diacyl PC having an sn-2 docosahexaenoyl or arachidonoyl chain.88 In contrast, the dominant acyl chain species of plasmenylethanolamine in the mammalian heart are often comparable amounts of arachidonoyl and linoleoyl chains, while that in the sn-2 in diacyl PE is an arachidonoyl chain.58,62,94 In the mouse heart, docosahexaenoyl and arachidonoyl chains are the dominant species of plasmenylethanolamine and the sn-2 chain of diacyl PE.88 The amounts of those phospholipids in the WT mouse heart were determined in this work to be 30.8 ± 2.8 mol % for plasmenylcholine, 19.2 ± 1.9 mol % for diacyl PC plus a minor fraction of plasmanylcholine, 8.2 ± 0.4 mol % for plasmenylethanolamine, and 26.2 ± 1.2 mol % for diacyl PE plus a minor fraction of plasmanylethanolamine. The high transacylation reactivity of choline phospholipid having an sn-2

linoleoyl chain with MLCL mentioned above, along with the measured phospholipid composition in the mouse heart, suggests that plasmenylcholine and diacyl PC are the highly preferred reaction partners of MLCL in CL remodeling. This is in accord with the well-known fact that the single dominant CL species in the mammalian heart, including the mouse heart, is tetralinoleoyl CL.16,44,88 The drastic reduction in the level of plasmenylcholine but not in diacyl PC due to tafazzin deficiency is suggestive of an important role of tafazzin in maintaining the plasmalogen level. The importance can be related to the regulation of the dominant CL species, i.e., tetralinoleoyl CL in the case of the mammalian heart. Plasmalogen and Membrane Morphology. The drastic loss of plasmenylcholine in the absence of functional tafazzin is likely to have a major impact on the structure, function, and integrity of mitochondria. While there are reports about correlations between the lack of peroxisome biogenesis as the site of synthesis of plasmalogen precursors and morphological and/or functional abnormalities of mitochondria, especially in cristae,95,96 little is known regarding physical and chemical roles of plasmalogens in mitochondria at the molecular level. From a biophysical perspective, plasmenylethanolamine has a tendency to stabilize the inverse hexagonal membrane phase (HII) above a transition temperature Th between the liquidcrystalline (Lα) and HII phases.97,98 This tendency is more notable than the one for diacyl PE and plasmanylethanolamine in the following order: plasmenylethanolamine ≫ plasmanylethanolamine > diacyl PE. Thus, plasmenylethanolamine is highly capable of stabilizing and being stable in a negative surface curvature. The sn-2 acyl chain of diacyl PE and PC in a bilayer is known to take the characteristic bent conformation near the ester bond, with which the uppermost carbon segments are almost parallel to the bilayer surface. In contrast, the presence of the sn-1 vinyl ether bond in ethanolamine and choline plasmalogens causes structural arrangement such that their sn-2 acyl chain takes its preferential conformation with its primary axis, throughout the chain, perpendicular to the surface,98−100 and as a consequence, plasmalogen molecules induce tighter lateral packing of lipids in a bilayer membrane compared to the diacyl counterparts.98,101,102 The tighter packing of the hydrocarbon chains brings the headgroups into closer contact, which favors HII phase formation in the case of the plasmenylethanolamine membrane. The mammalian heart mitochondria have percentages of plasmalogens of the total lipid at least comparable to those of the entire organ, according to a study using guinea pigs.62 This applies to both plasmenylcholine and plasmenylethanolamine. The membrane of cristae as the site of oxidative phosphorylation forms a tubular structure with a significant degree of curvature; the estimated outer diameter is ∼35 nm.103 Such curvature has been proposed to be stabilized by preferential localization of cone-shaped lipids such as CL and PE in the inner leaflet of the membrane.103,104 Indeed, PE deficiency in mammalian mitochondria impairs oxidative phosphorylation and alters mitochondrial morphology, including that of the cristae.105 CL deficiency has a similar impact (see, e.g., ref 19 for a review). Alterations of mitochondrial ultrastructures were also observed in the cells of Zellweger syndrome patients and mouse models, where plasmenylethanolamine as the dominant form of plasmalogen in the tissue was deficient due to deficiency in functional peroxisomes.95,96 In the work presented here, no significant change in the plasmenylethanolamine and diacyl PE contents was observed in the TAZ-KD mouse heart, 2171


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while common reduction of plasmenylethanolamine was confirmed in cells and tissues where ethanolamine is the dominant form of plasmalogens, such as in lymphoblast cells derived from Barth syndrome patients as well as in other organs of TAZ-KD mice (unpublished results). The reduction in the level of highly negative curvature prone plasmenylethanolamine is thought to decrease the stability of mitochondrial membranes of cristae even in the presence of the counterbalancing increase in the level of less negative curvature prone diacyl PE. Likewise, the decrease in the level of plasmenylcholine is considered to affect the stability of the membrane of cristae due to the reduction in the membrane packing ability despite the presence of the counterbalance by the diacyl PC. An indication that plasmenylcholine may play a critical role in maintaining the structure and function of mitochondrial membranes of cristae is that loss of functional SLC25A46, a mitochondrial metabolite carrier that is located in the outer membrane and associates with the “mitochondrial contact site and cristae organizing system” (MICOS), results in a prominent increase in plasmenylcholine content along with abnormal mitochondrial architecture with markedly shortened cristae.106 The dramatic reduction in the level of plasmalogen can be a useful diagnostic probe for Barth syndrome. A study to reverse the effects of the loss of tafazzin function associated with the plasmalogen level is in progress in our group using the tafazzindeficient human lymphoblast cell culture system. Findings in the study are expected to contribute to relief from the disease by recovering the reduced plasmalogen levels. The fundamental cellular mechanism of changes in lipid composition, not only in the mitochondria where tafazzin and CL are specifically located but also in other organelles, is being revealed as well by our group in view of the physical and chemical functions of those lipids.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The illustrated heart tissue images in the abstract graphic were based on electron micrographs of the WT and TAZ-KD mouse heart in ref 44.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00042. Appendices S1−S4, Figures S1−S9, and Tables S1−S3 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Richard M. Epand: 0000-0002-9602-9558 Author Contributions

T.K. and R.M.E. led the project development. T.K., B.B., and R.M.E. designed and conducted the NMR work. A.K.K. designed and conducted the quantitative Western blot work. M.R., Y.X., and M.S. designed and conducted the biological work to condition the TAZ-KD and WT mice and their heart samples and designed and conducted the MS work. T.K. wrote the manuscript. All authors contributed to completion of the manuscript. Funding

This work was supported by National Institute of General Medical Sciences (NIGMS) Grant R01GM115593-01 to M.S. and NIGMS Subaward 15-A0-00-003774-01 to R.M.E. 2172


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