Targeting High‐Density Aromatic Peptides to Cardiolipin Optimizes the Mitochondrial Membrane Potential and Inhibits Oxidative Stress

Cardiolipin (CL), a mitochondria-specific non-bilayer phospholipid, plays an essential role in the assembly and structural dynamics of the respiratory chain, affecting the membrane morphology and functional activity of inner mitochondria membrane (IMM)-embedded proteins. CL forms CL-rich domains on the IMM where negative curvature is required to increase the stability of cristae. However, CL constantly transitions between lamellar bilayer and non-bilayer phases, such as inverted CL hexagonal phases and inverted CL micelles. Non-bilayer phases of CL promote mitochondrial fission and fragmentation, transition of CL to the outer mitochondrial membrane (OMM), and mitophagy. In addition, non-bilayer phases of CL can increase proton leakage, which leads to mitochondrial depolarization and decreased mitochondrial ATP synthesis. Thus, therapeutic applications for minimizing non-bilayer CL phases should be able to optimize mitochondrial stability during various stresses. We have developed a novel, high-density aromatic peptide (HDAP2) that targets CL and enhances the stability of CL within the lipid core of bilayers in CL-POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes. We also demonstrated that HDAP2 interacts with inverted CL micelles, forming HDAP2-CL micelles. This suggests that HDAP2 interacts with the non-bilayer phase of CL, thereby stabilizing CL in the bilayer configuration. Scanning electron microscopy confirmed that HDAP2 assembles into spherical micelles approximately 1-3 μm in diameter. We have also demonstrated that this novel, water-soluble peptide is cell-permeable and targets mitochondria without causing cell toxicity. Furthermore, we used a well-known mitochondrial toxicity model of serum starvation to demonstrate that HDAP2 significantly promoted cell survival in a dose-dependent manner in mitochondria-dependent Madin-Darby bovine kidney (MDBK) cells. Importantly, HDAP2 preserved mitochondrial membrane potential and mitigated oxidative stress during serum deprivation. These protective effects suggest that, through its unique mechanism of action, HDAP2 can enhance cellular homeostasis, which would offer broad therapeutic potential for the prevention, recovery, and reversal of many acute and chronic disease conditions, including neurodegeneration, ischemia-reperfusion injury, and inflammation.