**9. Dilated Cardiomyopathy with Ataxia (DCMA)**

Mutations in the gene encoding for the mitochondrial protein DNAJC19 were found to be causative for Dilated Cardiomyopathy with Ataxia (DCMA) [135]. DCMA patients presenting with dilated cardiomyopathy and arrhythmias due to abnormalities in repolarization after a heartbeat (long QT syndrome, LQTS) [136]. Similar to BTHS and Sengers syndrome, 3-methylglutaconic aciduria is commonly described in DCMA (Figure 5). Other symptoms include cerebellar ataxia, growth retardation or genital anomalies in male patients [137]. Patient-derived iPSC-CM models of DCMA have been developed recently [138,139]. The function of the affected gene (DNAJC19) is unknown but it shares sequence similarities with the family of DnaJ proteins, which act as cofactors of Hsp70 chaperones. DNAJC19 binds to the mitochondrial protein prohibitin which is integrated in the inner membrane and oligomerizes into large ring-like structures, which restrict CL into specific membrane domains. The role of prohibitin appears to be to segregate specific membrane domains, which facilitate CL remodeling. The direct interaction of DNAJC19 with prohibitin suggests a participation of DNAJC19 in CL remodeling. In fact, deletion of DNAJC19 in a cell model resulted in changes in the CL species composition [140]. Interestingly, these alterations in the CL pool were not approved in an iPSC-CM model of DCMA [139]. The reason for these inconsistencies have not been resolved yet. A consistent finding, however, is that DNAJC19 deficiency causes highly fragmented and abnormally shaped mitochondria and changes in mitochondrial cristae morphology.

#### **10. Therapeutic Approaches**

Improvements in the therapy of BTHS cardiomyopathy and the associated symptoms such as neutropenia and skeletal myopathy have resulted in improved survival of the deadly disease. Standard therapy addressing the cardiac defects are angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers, in combination with beta-adrenergic receptor blockers [141]. More severe cases are treated with vasodilators or inotropes, left ventricular assist devices, and/or cardiac transplantation [142–144]. Neutropenia is treated with granulocyte colony-stimulating factor (G-CSF) and complemented with prophylactic antibiotics. Growth hormone (GH) supplementation is used to treat the growth delay and arginine supplementation acts as a complement for arginine depletion [141,145,146].

Gene therapy in inherited diseases allows to directly target affected genes and has been tested in mouse models of Barth syndrome. Adeno-associated virus (AAV)-mediated *TAZ* gene replacement ameliorated cardiac function in *Taz*-KD mice, indicating the reversibility of the clinical phenotype and the feasibility of the approach [147]. Additionally, pharmaceutical intervention in CL biosynthesis has been tested in experimental studies. The mitochondrial enzyme phospholipase A<sup>2</sup> is responsible for the deacylation of premature CL after its initial synthesis. The resulting monolysocardiolipin MLCL is then acetylated to mature CL by Tafazzin [148]. Inhibiting phospholipase A<sup>2</sup> would result in stabilizing the premature CL pool and prevents the accumulation of MLCL. This intervention has been tested in a *Taz* deletion Drosophila model of BTHS. *Taz* deletion causes male sterility in flies, which was prevented by inhibiting phospholipase A<sup>2</sup> [148]. However, as the human homolog of the *Drosophila* phospholipase A<sup>2</sup> has not been identified and its inhibition might have severe side effects, translation into medicine is difficult [149].

Therapeutic approaches are interesting with regard to the targeting of metabolic defects in BTHS. Due to a central role in energy metabolism and mitochondrial bioenergetics, peroxisome proliferator-activated receptors (PPARs) have been considered as potential therapeutic targets. Activation of the PPAR/PGC1α axis using the PPARα agonist bezafibrate has been a successful strategy in various mitochondrial disorders [150]. PPARα activation regulates the transcription of numerous genes involved in mitochondrial energy metabolism and fatty acid oxidation [151,152]. When tested in the BTHS mouse model, bezafibrate prevented the development of systolic dysfunction and improved exercise capacity when combined with voluntary exercise [153].

As the structural change in the respiratory chain in Barth syndrome is associated with a marked increase in ROS emission from mitochondria [36,154,155], an intriguing therapeutic strategy is the use of anitoxidants. The mitochondria-targeted ROS scavenger mitoTEMPO was tested in the human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes of BTHS patients [156,157]. ROS-induced changes in sarcomere assembly and the resulting deficits in contractility undergo an improvement with mitoTEMPO [158]. The translation into a mouse model was tested by expressing the ROS-scavenging catalase in the mitochondrial matrix to target mitochondrial hydrogen peroxide (H2O2) emission in the BTHS mouse. This approach efficiently reduced H2O<sup>2</sup> levels and lipid peroxidation, but did not eradicate cardiac dysfunction or skeletal muscle fatigue in *Taz*-knockdown (KD) mice [159]. The discrepancy of these contradictory results has not been resolved, yet.

Cytochrome c mediates electron transfer from complex III to complex IV in the respiratory chain and is also involved in the peroxidation of cardiolipin (CL), which has been observed in a variety of pathological conditions, including BTHS [160]. Peroxidized CL is associated with energy deficiency and plays a role in the opening of the permeability transition pore (PTP) [161]. The PTP is a large pore in the mitochondrial membranes and consists of the proapoptotic Bcl-2 members Bax/Bak in the outer membrane and the F1/F<sup>O</sup> ATPase in the inner membrane and cyclophilin D in the matrix [162]. PTP opening causes a depletion of the membrane potential and induces apoptosis. The Szeto-Schiller peptide (SS-31 or Elamipretide) is an aromatic-cationic mitochondria-targeting tetrapeptide that penetrates the plasma membrane and localizes to the inner mitochondrial membrane based on its direct interaction with CL. Elamipretide prevents the peroxidase activity of cytochrome c and normalizes the CL pool in models of BTHS and other models of heart failure [163,164]. Elamipretide also improves inner membrane cristae structures and re-establishes mitochondrial respiration and ATP production in models of heart failure including BTHS [165,166]. Unfortunately, a phase II clinical

trial (TAZPOWER trial, NCT03098797) treatment with Elamipretide in BTHS patients did not improve exercise capacity. Recently, Elamipretide was also tested in fibroblasts from DCMA patients and was found to rescue mitochondrial fragmentation and increased ROS production [167]. Heart tissue from a rat model of ischemia-reperfusion showed deterioration of mitochondrial complexes I, II, and IV. Application of Elamipretide significantly alleviated the structural changes in respirasomes, improved the fragmentation of mitochondria and enhanced the formation of cristae structures [168]. As changes in the CL pool were also found in the aging heart, Elamipretide was tested in aging mice. Here, it reduced mitochondrial ROS and normalized protein oxidation in old hearts, and even showed beneficial effects for aging-related diastolic defects. Interestingly, expression of the mitochondrial catalase presented similar beneficial effects, which were not further improved by Elamipretide application, indicating normalizing mitochondrial oxidative stress as the main mechanism for Elamipretide in aging hearts [67].

#### **11. Conclusions**

Mitochondria play a crucial role in energy metabolism, redox homeostasis and intermediate metabolism, not only having additional anabolic functions but also participating in signaling pathways. Many of these functions are membrane-associated and were shown to be dependent on CL. CL-deficient cells and animal models, including BTHS patient-derived lymphoblasts, *Drosophila*, *C.elegans*, *Trypanosoma* and mice, have been developed [35,169–173]. These model systems have helped to understand the role of CL in mitochondrial biogenesis and in shaping mitochondrial morphology. CL is an integral component of the respiratory chain since CL deficiency causes a decline in respiratory capacity and an increase in ROS production. A defect in MCU uncouples mitochondrial energy metabolism from myocardial Ca2<sup>+</sup> signaling, which mediates an important response during accelerated workload conditions. Deficiencies in the Krebs cycle require remodeling of intermediate metabolism for compensatory anaplerotic pathways. Finally, defects in the biogenesis of iron sulfur clusters and Coenzyme A might endanger fatty acid oxidation, one of the most prevalent energy sources for the heart. The tight interaction between CL and membrane proteins, such as respirasomes, may also have implications on the half-life of CL itself, although CL presents a longer half-life compared to other phospholipids [118,174,175]. As the slow turnover of CL is dependent on its interaction with the respiratory chain, respiratory chain remodeling as in BTHS may induce a vicious cycle when increased CL turnover may contribute to low CL levels in Barth syndrome [118]. The exact mechanisms of CL degradation remain unclear. The phospholipase HSD10 has been suggested to mediate rapid degradation, particularly of oxidized CL (CLOX) [176]. Patients with mutations in the gene encoding HSD10 suffer from progressive cardiomyopathy and neurodegenerative disease. The contribution of HSD10-mediated CLOX clearance for the maintenance of functional mitochondria has not been studied yet.

Barth syndrome patients suffer from a general metabolic remodeling including changes in serum amino acid levels, lactic acidosis during exercise and elevated urinary excretion of 3-MGA [113,114]. In the future it will be interesting to understand the exact mechanisms behind changes in the metabolism, how they relate to mitochondrial dysfunction and how dysfunctional mitochondria induce compensatory mechanisms such as anaplerotic pathways. Based on the integration of mitochondria in cellular signaling pathways, changes in mitochondrial function might be monitored and trigger a cellular response. ROS may be at the center of this mechanism since alteration in ROS according to CL deficiency has been widely documented. Being a part of a large number of cellular signaling pathways, ROS affect a wide variety of biological processes including responses to hypoxia, apoptosis, autophagy, cell proliferation and differentiation. Alterations in ROS signaling due to CL deficiency cause a defect in key cellular signaling pathways involved in the response to hypoxia [155]. Several signaling pathways are also directly dependent on CL. Kinases of the protein kinase C (PKC) family regulate diverse biological functions such as growth and differentiation and influence multiple physiological processes in the heart, including heart rate, contraction, and relaxation. Members of the PKC family locating to

the mitochondria and requiring CL for activation have been described previously [177,178]. As CL predominantly is located in the inner membrane, externalization of CL onto the outer membrane serves as a signaling platform in many signaling events, such as mitophagy and apoptosis. How these changes relate to the clinical picture in heart disease is not understood and remain a challenging task for the future.

**Funding:** The work in the laboratory of the authors is supported by the Deutsche Forschungsgemeinschaft (DFG; DU1839/2-1), the Bundesministerium für Bildung und Forschung (BMBF) and the Barth Syndrome Foundation.

**Conflicts of Interest:** The authors declare no conflict of interest.
