**3. Function of CL in Mitochondrial Morphology**

Structural abnormalities of mitochondria, including fragmentation, disruption of mitochondrial membranes and loss of the electron-dense matrix have been observed in animal models of heart failure and in patients suffering from hypertrophic cardiomyopathy [24]. In a dog model of chronic heart failure, the mitochondrial ultrastructure changes from tightly packed to disorganized cristae [25]. Mitochondria possess two highly specialized membranes—the outer and the inner mitochondrial membrane. Invaginations in the inner membrane form the cristae structures, which are areas of intensive membrane curvature. CL plays a considerable role in shaping the morphology of mitochondria and prominently locates in the bended regions of the inner membrane [26]. CL adopts a cone-shaped structure due to the high content of unsaturated fatty acids, which is causative for the membrane bending at sites of high CL concentration. In addition, mitochondrial morphology is also shaped by a constant remodeling through the fission and fusion of individual mitochondria [27]. The opposing processes of fusion (merging of two mitochondria) and fission (segregation of two mitochondria) are shown to form a highly dynamic network of mitochondria in the cell. The continuing remodeling by fission and fusion is essential for normal mitochondrial function, downregulation of mitochondrial fusion promotes apoptosis and cardiomyocyte loss [28]. Mitochondrial fragmentation was elucidated to influence heart failure notably [29]. By its role in regulating mitochondrial fission and fusion, CL actively participates in shaping the mitochondrial network. Fission and fusion are regulated by a set of dynamin-related GTPases, whereby CL serves as a modulator of the activity of the fission protein Drp1 and the fusion protein OPA1 [30,31]. Morphological alterations of mitochondria are evident in human patients with defects in CL biosynthesis and remodeling as well as in animal models (Table 1).


The formation of cristae structures critically depends on the mitochondrial contact site and cristae organizing system (MICOS). These protein complexes locate at the cristae junctions and seal individual cristae to maintain individual membrane potentials [8]. The MICOS complex is a central component of a large interaction network which includes several complexes in the inner membrane and outer membrane [41]. MIC27 (APOOL) as a MICOS complex constituent directly interacts with CL and this interaction was found to be essential for MIC27 assembly in the MICOS complex [42,43] (Figure 2). MIC27 and the structurally related MIC26 also modulate the cardiolipin remodeling enzyme Tafazzin (see below) [44]. In fibroblasts from Barth syndrome patients, a compensatory increase in MICOS subunits was observed [38,45]. Interestingly, a slightly lower molecular mass was detected for MICOS subunits in Barth syndrome (BTHS) fibroblasts, indicative of structural changes due to CL deficiency. Application of the CL-interacting molecule SS31/Elamipretide normalized deregulated structural proteins, such as Drp1, Mfn2, Opa1 and Mic60 in human heart failure patients [46].

**Figure 2.** The role of CL in mitochondrial morphology: The mitochondrial contact site and cristae organizing system (MICOS) complex requires CL for optimal structural integrity. MICOS is a protein complex located in the inner mitochondrial membrane, playing an essential role in cristae junction formation. The resulting membrane invaginations harbor the respiratory chain complexes. By interactions with other proteins in the outer mitochondrial membrane such as the Translocase of the Outer membrane (TOM) and the sorting and Assembly Machinery (SAM), the mitochondrial intermembrane space (IMS) bridging complex (MIB) is formed. The MICOS subunits MIC27 and MIC26 influence the regulation of CL levels, while CL is remodeled by Tafazzin [47]. Not only the integration of MIC27 in MICOS, but also the function of other mitochondrial membrane anchored proteins such as the fission and fusion proteins Drp1 and OPA1 is affected by CL [48]. OM, outer membrane; IMS, intermembrane space; IM, inner membrane.
