Modulation of the Protein Kinase Cδ Interaction with the “d” Subunit of F1F0-ATP Synthase in Neonatal Cardiac Myocytes: DEVELOPMENT OF CELL-PERMEABLE, MITOCHONDRIALLY TARGETED INHIBITOR AND FACILITATOR PEPTIDES

The F1F0-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F1F0 activity via an interaction with the “d” subunit of F1F0-ATP synthase (dF1F0) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831–29840). We have now identified a dF1F0-derived peptide (NH2-2AGRKLALKTIDWVSF16-COOH) that inhibits PKCδ binding to dF1F0 in overlay assays. We have also identified a second dF1F0-derived peptide (NH2-111RVREYEKQLEKIKNMI126-COOH) that facilitates PKCδ binding to dF1F0. Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCδ-dF1F0 interaction inhibitor, decreased 100 nm 4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF1F0 by 50 ± 15% and abolished the 30 nm 4β-PMA-induced inhibition of F1F0-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCδ-dF1F0 facilitator peptide by itself induced the PKCδ-dF1F0 co-immunoprecipitation and inhibited F1F0-ATPase activity. In in vitro PKC add-back experiments, the PKCδ-F1F0 inhibitor blocked PKCδ-mediated inhibition of F1F0-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCδ-dF1F0 interaction in NCMs. These novel peptides will improve our understanding of cardiac F1F0 regulation and may have potential as therapeutics to attenuate cardiac injury.