**4. Function of CL in Energy Metabolism**

In total, 95% of the cardiac energy demand is covered by oxidative phosphorylation of the respiratory chain in the inner membrane, consisting of five complexes (I–V). Four complexes (I–IV) transport electrons from reducing equivalents (NADH, FADH2) to oxygen, forming water. The resulting energy is stored in a membrane potential across the inner membrane and converted to ATP by the last complex in the chain (V). The structure of the respiratory chain has been resolved and specific interaction sites for CL were identified in all respiratory chain complexes [36,49]. Correspondingly, CL is a structural component of the respiratory chain and is essential for its integrity and full enzymatic activity [50–53]. Changes in the CL pool, including the accumulation of MLCL, may interfere with

the structure of respiratory chain complexes. MLCL, which strongly accumulates in Barth syndrome, binds to complex IV with a substantially reduced affinity and significantly lowers the enzymatic activity of this complex [54]. The structure of the dimeric ATP synthase from bovine mitochondria recently shed light upon the mechanism of proton uptake in the matrix. Interestingly, this study also described the incidence of CL in the integral membrane subunits of complex V [55]. Furthermore, CL is directly involved in the proton export and required for the organization of the F1F<sup>O</sup> ATPase into highly ordered structures [56,57].

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The respiratory chain complexes assemble into higher-ordered structures—the respirasomes. These supercomplexes include complex I, a dimer of complex III, and one or several complex IV units (Figure 3). CL molecules are not an only integral to individual complexes but are also associated with the mitochondrial supercomplex formation. It was found to support the structure of supercomplexes and mediate the interaction with the lipid phase of the inner membrane [58–60]. Due to its role in the structure of the respirasomes, CL deficiency causes a defect in respiratory function and a decrease in membrane potential and in ATP synthesis [36,37,61] (Table 1). — –

**Figure 3.** CL is an essential constituent of respirasomes: The mitochondrial respiratory chain assembles into large oligomeric structures called respirasomes. Respirasomes consist of complex I, a dimer of complex III, and several copies of complex IV. CL is required for respirasome formation and is essential for the activity of the complexes. CL molecules partially interacting with membrane protein complexes are shown in red. ANT, ADP/ATP carrier; Cyt c, Cytochrom c; IM inner membrane; IMS, intermembrane space.

In many forms of cardiac disease, reduced CL levels, alterations in the CL pool or Reactive Oxygen Species (ROS)-induced damage of CL have been observed. Given the important structural function of CL, these changes may have direct structural implications for the respiratory chain. Remodeling of the respiratory chain due to changes in CL has been described in aging, ischaemia/reperfusion and heart failure [62–65]. These findings are of particular importance as the architecture of respirasomes prevents ROS production. Structural alterations may induce increased ROS generation at the respirasomes, and increased oxidative stress may be an important contributor to the development of heart failure [66]. Therefore, a large number of studies have focused on preventing mitochondrial ROS in various forms of cardiac disease [67,68]. These studies, although somewhat inconsistent, showed that reducing ROS levels has the potential to ameliorate ROS-mediated cardiac abnormalities [69–71].

#### **5. Function of CL in Intermediate Metabolism**

In order to maintain the membrane potential, the inner membrane must be tightly sealed and transport processes across the membrane need to be vigorously controlled. This contrasts with the demands of energy metabolism, which requires an intensive exchange of metabolites between the cytosol and the matrix. This is ensured by the superfamily of carrier proteins in the inner membrane mediating the transport of metabolites across the inner membrane. The most abundant carrier protein is the ADP/ATP carrier (ANT), which exchanges ATP in the matrix with ADP in the cytosol. The recent resolution of the matrix-open state advances our understanding of the molecular mechanism of metabolite cycling, which most likely applies to the whole carrier family [72]. The ADP/ATP carrier possesses not only a tight binding to CL (Figure 3), but CL was also identified in its crystal structure from bovine heart mitochondria [73,74]. In addition, studies of bakers' yeast show that the conformation of the ADP/ATP carrier is controlled by CL acting as a link for dimer formation and the integration of ANT into a complex interaction network with the respiratory chain [75]. A requirement for CL has also been documented for other members of the carrier family including the Phosphate carrier (PiC), the monocarboxylate carrier (MCT1), carnitine/acylcarnitine translocase, pyruvate carrier and the tricarboxylate carrier [76–80]. Carrier proteins are integral proteins of the inner membrane, synthesized in the cytosol and then transported across the outer membrane to become subsequently integrated into the inner membrane. Their incorporation depends on the protein Translocase of Inner Membrane TIM22. TIM22 is a protein complex and is integrated in the inner membrane in a CL-dependent manner. Moreover, one structural component of the TIM22 complex is acylglycerol kinase (AGK). Besides its role in protein translocation, it also has a second function as a kinase in the biosynthesis of phosphatidic acid (PA), which serves as a precursor of CL biosynthesis [81,82].

The protein creatine kinase (CK) catalyzes the reversible conversion of creatine and adenosine triphosphate (ATP) into phosphocreatine (PCr) and adenosine diphosphate (ADP) and is expressed in the heart, skeletal muscle, brain and kidney. Phosphocreatine serves as a buffer for rapid regeneration of ATP in tissue with a high energy demand. Mitochondrial creatine kinase (mtCK) is located in the mitochondrial intermembrane space, where it uses the local ATP concentration to generate phosphocreatine. In heart and skeletal muscle, sarcomeric mtCK catalyzes the reverse reaction to regenerate ATP in close proximity of the site of high energy turnover. In-vitro experiments suggest CL as one of the main binding sites of the creatine kinase to the inner membrane [83,84]. mtCK was also found to form large oligomeric complexes in the intermembrane space [85]. By binding to CL, the creatine kinase was also suggested to mediate CL transfer between the inner and outer membrane [86].

Studies in CL-deficient yeast show reduced levels of acetyl-CoA due to a decreased activity of acetyl-CoA synthetase. Despite a compensatory upregulation of pyruvate dehydrogenase (PDH), the enzymatic activity was not increased, suggesting a defect in the specific PDH activity [75]. A C2C12 myoblast model of BTHS confirmed diminished PDH activity and proposed that an increased level of inhibitory phosphorylation was responsible for the defect. The authors suggested that CL is required to facilitate the binding of the pyruvate dehydrogenase phosphatase to the E2 subunit and reduced binding enhances inhibitory PDH phosphorylation [87]. To compensate for the deficiency in PDH activity, the pyruvate carboxylase is upregulated. A defect in PDH activity was not verified in an induced Pluripotent Stem Cell-derived Cardiomyocyte (iPSC-CM) model of BTHS, which even showed an increased flux of glucose into the Krebs cycle intermediate citrate. Accordingly, the anaplerotic supplementation by carboxylation of pyruvate was reduced in this cell model [88].

CL deficiency can also affect the mitochondrial Krebs cycle (TCA). The α-ketoglutarate dehydrogenase complex was found to be structurally affected in human BTHS patient fibroblasts; however, its enzymatic activity and the metabolic flux of glutamate into the Krebs cycle remained unaffected [38]. In addition, a CL-deficient yeast model showed alterations in the enzymatic activities of aconitase and succinate dehydrogenase [87]. Metabolic flux analyses in iPSC-CM displayed an increased level of the Krebs cycle intermediate citrates and decreased level of fumarate in BTHS, indicative of a reduced turnover of succinate into fumarate [88]. Aconitase and succinate dehydrogenase are strictly dependent on iron sulfur clusters as a cofactor. These data and an increase in mitochondrial iron amounts pointed to a defect in the biogenesis of iron sulfur clusters. CL is required for the correct processing of the mitochondrial protein frataxin, which is an important scaffold protein in

the iron sulfur cluster biogenesis [89,90]. Consistent with a defect in the TCA cycle, a recent report showed that anaplerotic pathways are required to ameliorate TCA cycle dysfunction in yeast cells [91]. Other cofactors may also be affected. A recent study assumed that lower levels of the cofactor Coenzyme A contribute to the respiratory deficiency in BTHS [92]. Coenzyme A is an important cofactor for fatty acid metabolism. Studies in yeast, however, described that increased fatty acid oxidation can compensate for the Krebs cycle defects in CL-deficient yeast [91]. Under conditions of a high-fat diet, Cole et al. were able to show intensified accumulation of triacylglycerides in cardiac tissue of BTHS mice compared to control animals. The authors found the upregulated synthesis of fatty acid synthase to be responsible for this effect. Increased triacylglycerides may enhance the susceptibility to lipotoxicity in BTHS under conditions of increased fat uptake [93].

Diseases, which are associated with defects in CL, such as DCMA, Sengers syndrome and Barth syndrome, commonly present with 3-methylglutaconic aciduria (3-MGA) accompanied by increased levels of lactic acid [94]. 3-Methylglutaconic and the related 3-methylglutaric acid are catabolic intermediates of the branched-chain amino acid (BCAA) leucine. Additionally, elevated levels of the intermediate of isoleucine metabolism, 2-ethylhydracrylic acid (2-EHA), occurred in the urine of BTHS patients [95]. A few studies allow for speculations of a defect in the metabolism of the branched-chain amino acids valine, leucine and isoleucine in BTHS. Transcriptome analysis of shTAZ mouse model revealed reduced gene expression of genes involved in BCAA breakdown [32]. A recent analysis of protein complexes in the mitochondria of BTHS patient skin fibroblasts revealed a profound destabilization of the branched-chain ketoacid dehydrogenase complex, which is involved in the initial oxidative degradation step of branched-chain amino acids [38]. Further investigations are required to elucidate the molecular mechanism of the observed secretion of organic acids in BTHS.
