Barth Syndrome: From Mitochondrial Dysfunctions Associated with Aberrant Production of Reactive Oxygen Species to Pluripotent Stem Cell Studies

Abstract

Mutations in the gene encoding the enzyme tafazzin, TAZ, cause Barth syndrome (BTHS). Individuals with this X-linked multisystem disorder present cardiomyopathy (CM) (often dilated), skeletal muscle weakness, neutropenia, growth retardation, and 3-methylglutaconic aciduria. Biopsies of the heart, liver and skeletal muscle of patients have revealed mitochondrial malformations and dysfunctions. It is the purpose of this review to summarize recent results of studies on various animal or cell models of Barth syndrome, which have characterized biochemically the strong cellular defects associated with TAZ mutations. Tafazzin is a mitochondrial phospholipidlysophospholipid transacylase that shuttles acyl groups between phospholipids and regulates the remodeling of cardiolipin (CL), a unique inner mitochondrial membrane phospholipid dimer consisting of two phosphatidyl residues linked by a glycerol bridge. After their biosynthesis, the acyl chains of CLs may be modified in remodeling processes involving up to three different enzymes. Their characteristic acyl chain composition depends on the function of tafazzin, although the enzyme itself surprisingly lacks acyl specificity. CLs are crucial for correct mitochondrial structure and function. In addition to their function in the basic mitochondrial function of ATP production, CLs play essential roles in cardiac function, apoptosis, autophagy, cell cycle regulation and Fe-S cluster biosynthesis. Recent developments in tafazzin research have provided strong insights into the link between mitochondrial dysfunction and the production of reactive oxygen species (ROS). An important tool has been the generation of BTHS-specific induced pluripotent stem cells (iPSCs) from BTHS patients. In a complementary approach, disease-specific mutations have been introduced into wild-type iPSC lines enabling direct comparison with isogenic controls. iPSC-derived cardiomyocytes were then characterized using biochemical and classical bioenergetic approaches. The cells are tested in a "heart-on-chip" assay to model the pathophysiology in vitro, to characterize the underlying mechanism of BTHS deriving from TAZ mutations, mitochondrial deficiencies and ROS production and leading to tissue defects, and to evaluate potential therapies with the use of mitochondrially targeted antioxidants.

Keywords: barth syndrome; cardiolipin; cellular models; mitochondria; mitochondrially targeted antioxidant; stem cells; tafazzin.

FIGURE 1

Left ventricular non-compaction (LVNC) associated with a dilated cardiomyopathy phenotype in Barth syndrome (BTHS) patient. (A) Example of a normal heart and a heart with dilated CM associated with LVNC. (B) BTHS patient with LVNC associated with dilated CM. Echocardiogram (apical 4-chamber view) of patient with BTHS depicting LVNC with associated DCM phenotype. The scale relates to Doppler effect velocity. Note the deep left ventricular trabeculations and dilated left ventricular chamber (Jefferies, 2013, with the permission of the editor, Wiley-Blackwell, John Wiley & Sons, USA).

FIGURE 2

Electron micrographs of mitochondria with an abnormal appearance in BTHS dilated cardiomyopathy and of lymphoblasts from a patient with Barth syndrome. (A) Higher power electron micrograph. The mitochondria are enlarged and crowded together, with many touching each another. The cristae are not parallel, but are stacked, and many are organized into abnormal circular arrays (white big arrows). The nucleus (N) is centrally placed, but the myofilaments, with their cross-striations (small white arrows), are displaced to the periphery of the cell. Lamellar bodies are also dispersed within the cytoplasm. One autophagolysosome is also detectable (large black arrow; AutoPh.) [reprinted with the permission of Sarah Clarke et al. (2013) and BiomedCentral]. (B) Control lymphoblasts. (C,D) Structure of mitochondria from control lymphoblasts. (E) Lymphoblasts from a patient. (F,G) Structure of mitochondria from the lymphoblasts of a patient. Size bars are ^∗Electron-translucent areas surrounded by the outer mitochondrial membrane (or a membrane derived from it). [from Dr. Patrice X. Petit, unpublished data and (Gonzalvez et al., 2013)].

FIGURE 3

Biosynthesis and remodeling of cardiolipin. Putative pathways of CL biosynthesis, remodeling, and degradation in mammalian cells. Note that the length of the acyl chains in the final phospholipids does not reflect their true length, which is typically 16C, 18C, or 22C, nor their degree of unsaturation. Abbreviations: ALCAT 1, Acyl-CoA:lysocardiolipin acyltransferase 1; CDP-DAG, cytidine diphosphate-diacylglycerol; CDS, CDP-DAG synthase; CS, CL synthase; CMP, cytidine monophosphate; CTP, cytidine triphosphate; DLCL, dilyso-CL; FFA, free fatty acid; G3P, glycerol-3-phosphate; LPC, lysophosphatidyl choline; LPE, lysophosphatidylethanolamine; MLCL, monolyso-CL; MLCLAT 1, MLCL acyltransferase 1; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PG-P phosphatidylglycerol phosphate; PGPP, phosphatidylglycerol phosphate phosphatase; PGPS, phosphatidylglycerol phosphate synthase; P_i, inorganic phosphate; PLA₂, phospholipase A₂; tafazzin, transacylase. [Reprinted, with permission of the authors and modified from (Chicco and Sparagna, 2007; Mejia et al., 2014b; Ren et al., 2014) and with information from Claypool and Koehler (Claypool and Koehler, 2012), and also from the American Journal of Cell Physiology, edited by the American Physiology Society, USA).

FIGURE 4

Cardiolipin as a “glue” optimizing electron transport within the electron transport chain supercomplex. (A) CL microlocalization on cristae membranes, “gluing” the respiratory complexes (Zhang et al., 2002) together to form supercomplexes, thereby decreasing the distance between redox partners. In addition to providing a platform for the aggregation of respiratory complexes, CL is also thought to serve as a proton trap (Haines and Dencher, 2002) on the outer leaflet of the IMM, mediating the rapid lateral diffusion of protons to the ATP synthase with minimal changes in bulk phase-pH. ADP, adenosine diphosphate; ATP, adenosine triphosphate; FADH₂, reduced flavin adenine dinucleotide; FAD⁺, oxidized flavin adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NAD⁺ oxidized nicotinamide adenine dinucleotide. (B) CL is a dimeric phospholipid with a small acidic head group and four acyl chains, resulting in a conical structure and a large surface area. As a result of its conical shape, CL exerts lateral pressure on a membrane containing other phospholipids, such as phosphatidylcholine (PC), resulting in curvature of the membrane and the proteins it contains.

FIGURE 5

Functions of cardiolipins, modifiers of Barth syndrome. CL is involved in a plethora of cellular events and signaling pathways. The tafazzin mutations affect the CL acyl chain length and its degree of saturation and the synthesized CLs stay saturated instead of being converted into mature CL with unsaturated acyl chains. This impacts all CL-related functions that are listed in the Figure. IRGM; Immunity-related GTPase family, M [(human)], OPA1; Dynamin-like 120 kDa protein, mitochondrial encoded by the gene OPA1.

FIGURE 6

Mitochondrial dysfunctions due to tafazzin mutations lead to ROS production, CL translocation and dysregulation of autophagic signaling. CL is synthesized in the inner leaflet of the IMM by cardiolipin synthase (CLS). This is also the localization where it can be remodeled by tafazzin and monolysocardiolipin acyltransferase (MLCLAT), in a balanced system with PLA₂. During processing or following stress signaling, CLs may also be translocated to the OMM and ER for remodeling, whereas in healthy mitochondria most of the CL remains at its site of synthesis in the IMM. The externalization of mature CL to the mitochondrial surface, through a mechanism involving phospholipid scramblase-3 (PLSR3), is a prerequisite for its recognition by light chain 3 protein (LC3) as a mitophagic “eat-me signal” targeting dysfunctional mitochondria to the autophagosomal machinery. The precise mechanism of scramblase regulation remains unclear, but its action may be physically facilitated by the proximity of nucleoside diphosphate kinase (NDPK-D). NDPK-D regulates the balance between di- and tri-phosphonucleotides, which provide GTP for the GTPase activity of a dynamin-like protein encoded by the gene OPA1 (for optic atrophy gene 1). By forming a hexamer in the intermembrane space, NDPK-D forms a physical bridge between the IMM and OMM. The switch from the phosphotransfer mode to a CL-translocating mode would thus facilitate the redistribution of CLs between the IMM and OMM. Note that immature CLs containing saturated acyl chains are gray while mature CLs with unsaturated acyl chains are blue. [Figure reproduced with modifications from Kagan et al. (2014), with the permission of the authors and of Elsevier Press].

FIGURE 7

Tafazzin knockdown in neonatal cardiac fibroblasts. Tafazzin gene knockdown results in mitochondrial dysfunction, with the production of lower levels of ATP and higher levels of superoxide anions and associated ROS. ROS activate MAPKs, induce gene expression factors and cell cycle regulators and lead to cell cycle progression. Alternatively, ATP deficiency may activate AMPK, leading to mitochondrial biogenesis and the cessation of cell proliferation. Tafazzin knockdown ultimately results in multinucleation, hypertrophy and enhanced collagen secretion [this scheme is derived from the work of He and collaborators (He et al., 2013) and has been freely interpreted with permission of the American Journal of Physiology: Heart and Circulatory Physiology from the American Physiology Society, USA].

FIGURE 8

Future role of iPSC-CMs in the clinical care of patients with hereditary cardiac disorders. iPSC-CMs can be profiled by molecular techniques, and electrical and mechanical measurements, and on the basis of their response to various pharmacological agents. Such studies can provide information about the predicted severity of disease, associated risks, and response to therapy, which can be used to support clinical decision making. In addition, the functional characterization of the tissue might help to identify candidate causal variants and to facilitate the screening of family members for the disease. Abbreviations: iPSCs, induced pluripotent stem cells; iPSC-CMs, induced pluripotent stem cell-derived cardiomyocytes; PMBCs, peripheral blood mononuclear cells.

FIGURE 9

Induced pluripotent stem cells generated from patients suffering from Barth syndrome. Disease-specific mutations are introduced into wild-type iPSC lines, facilitating direct comparison with isogenic controls. iPSC-CMs are subjected to biochemical analyses, such as phospholipid analysis by mass spectrometry and mitochondrial structure/function analysis. iPSC-CMs are also used in “heart-on-chip” assays, to model the pathophysiology of the disease in vitro and to investigate the underlying mechanism of BTHS (particularly as concerns binding capacity or force characteristics during contraction) and to evaluate potential therapies involving untargeted or targeted antioxidants. [This figure was developed from a publication of Wang et al. (2014b) and from comments recently published concerning the missing link between mitochondrial ROS production and the destabilization of sarcomere organization, raising questions about the relationship between ROS and dilated LVNC in BTHS patients (Raval and Kamp, 2014; Zweigerdt et al., 2014)].

FIGURE 10

Pathophysiology of Barth syndrome and iPSC-CMs. Healthy iPSC-CMs have organized myofilaments and mitochondria with active transacylase tafazzin linked to the outer membrane and facing the intermembrane space (Brandner et al., 2005), with tafazzin in an intermembrane position (Herndon et al., 2013), allowing CL processing. This results in the conversion of monolysocardiolipin (MLCL; immature CL) to L4-CL (mature CL) in the IMM and the formation of normal electron transport chain (ETC) complexes and normal ATP synthase function. However, the ETC normally produces few superoxide anions in a process homeostatically controlled by endogenous mitochondrial antioxidant (e.g., MnSOD). By contrast, in BTHS, iPSC-CMs with mutant TAZ lack L4-CL. CL cannot be processed correctly and respiratory complexes do not form normally (as they have a lower compaction) resulting in ETC complex malfunction and abnormally high levels of ROS production (red circle), contributing to myofilament disarray and contractile dysfunction. By contrast, when the defect is complemented, the respiratory supercomplexes are at the right distance, compacted and superoxide ion production is low (dashed black circle) (Claypool et al., 2008). modRNA encoding tafazzin can rescue the disease phenotype, as can buffering mitochondrial ROS with mitoTEMPO or providing a precursor for L4-CL, linoleic acid (redrawn from Raval and Kamp, 2014).

FIGURE 11

Rationale for the accumulation of MitoQ10 or SKQ1 in cells and mitochondria. The antioxidant component of MitoQ₁₀ is the same ubiquinone found in coenzyme Q₁₀ (Kelso et al., 2001), whereas the antioxidant of SKQ1 is a plastoquinone (Antonenko et al., 2008). Within mitochondria, the ubiquinone part of MitoQ is rapidly activated to yield the active ubiquinol antioxidant by the enzyme complex II (also called succinate dehydrogenase) in the mitochondrial respiratory chain (RC). As a result of detoxifying a free radical or other ROS, the ubiquinol part of MitoQ is converted to a ubiquinone, which is then recycled back to the active antioxidant by complex II within the mitochondria (James et al., 2005). It is this combination of a 1000-fold concentration within mitochondria, with conversion to the active antioxidant and recycling back to the active antioxidant after the detoxification of a free radical that makes MitoQ an effective mitochondrion-targeted antioxidant.

FIGURE 12

Cardiolipin and the main transduction pathways for apoptosis and autophagy. Collapse of cardiolipin content allows movements of CL from the inner mitochondrial membrane to the outer membrane required for both mitochondrially driven apoptosis and autophagic processes. If cardiolipin peroxidation is a prerequisite for the release of pro-apoptotic factors from the mitochondria into the cytosol, it could be problematic for mitophagy homeostasis. Moreover, changes in acyl chain composition associated with TAZ gene mutation and tafazzin dysfunction are directly involved in the inhibition of mitochondrially driven apoptosis (Gonzalvez et al., 2008, 2013) but has also be implicated in the possible inhibition of the mitophagic processes (Hsu et al., 2015). LC3-II is localized to preautophagosomes and autophagosomes, making this protein an autophagosomal marker (Kabeya et al., 2000) but also a marker of more specific mitophagosomes (that perform a specific type of autophagy called mitophagy). Normal and mutated tafazzin are represented by yellow and red stars, respectively, IMM, inner mitochondrial membrane; IMS, intermembrane space; OMM, outer mitochondrial membrane, LC3-II,microtubule-associated protein 1 light chain 3-II). A black cross marks the abolition of protein-to-protein interactions.