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 (NH ₂-²AGRKLALKTIDWVSF¹⁶-COOH) that inhibits PKCδ binding to dF₁F₀ in overlay assays. We have also identified a second dF₁F₀-derived peptide (NH ₂-¹¹¹RVREYEKQLEKIKNMI¹²⁶-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 dF ₁F₀ by 50 ± 15% and abolished the 30 nM 4β-PMA-induced inhibition of F₁F₀-AT Pase 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-F ₁F₀ inhibitor blocked PKCδ-mediated inhibition of F₁F₀-AT Pase 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 F ₁F₀ regulation and may have potential as therapeutics to attenuate cardiac injury.