Targeting high-density aromatic peptides to cardiolipin optimizes the mitochondrial membrane potential

ABSTRACT The mitochondrial membrane potential (ΔΨm) is created by the accumulation of protons on an outer leaflet of the inner mitochondrial membrane and drives the synthesis of most cellular ATP, which is essential for cellular bioenergetics and survival. The ΔΨm also facilitates the electrogenic transport of cations, such as Ca 2+ , and regulates generation of reactive oxygen species, which serves as a powerful bioenergetic and stress-signaling regulator. Proton trapping on the outer leaflet of the inner mitochondrial membrane of mitochondrial cristae could be controlled by cardiolipin when the local pH is above 8. However, there is presently no technology that effectively targets strong bases to cardiolipin. We have developed a novel, high-density aromatic peptide (HDAP2) to preserve a proton gradient-driven potential in mitochondria by increasing proton trapping on cardiolipin (CL). HDAP2-induced formation of cardiolipin-HDAP2 complexes accumulated positive charges at the head of CL. The HDAP2-CL vesicles could accumulate the mitochondrial transmembrane potential probe, Tetramethylrhodamine (TMRM). This potential could be uncoupled with Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and Dinitrophenol (DNP), indicating that an interaction of HDAP2 with CL could support a proton gradient-driven transmembrane potential. We demonstrated that this novel, water-soluble peptide is cell-permeable, targets mitochondria without causing cell toxicity, and promotes cell survival during serum starvation. Importantly, the HDAP2-cardiolipin complex-mediated optimization of the proton gradient was supported by the ability of HDAP2 to prevent CCCP-mediated mitochondrial depolarization in ARPE-19 cells in a dose-dependent manner. Based on its mechanism of action, HDAP2 could promote cellular homeostasis, which would have broad clinical applicability for the prevention, recovery and reversal of many acute and chronic disease conditions, such as neurodegeneration, ischemia– reperfusion injury, and inflammation.