Protein kinase A catalytic subunit alters cardiac mitochondrial redox state and membrane potential via the formation of reactive oxygen species

Abstract

Background: The identification of protein kinase A (PKA) anchoring proteins on mitochondria implies a direct effect of PKA on mitochondrial function. However, little is known about the relationship between PKA and mitochondrial metabolism. Methods and Results: The effects of PKA on the mitochondrial redox state (flavin adenine dinucleotide (FAD)), mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) production were investigated in saponin-permeabilized rat cardiomyocytes. The PKA catalytic subunit (PKAcat; 50 unit/ml) increased FAD intensities by 56.6±7.9% (p<0.01), 2′7′-dichlorofluorescin diacetate (DCF) intensities by 10.5±3.3 fold (p<0.01) and depolarized ΔΨm to 48.1±9.5% of the control (p<0.01). Trolox (a ROS scavenger; 100 μmol/L) inhibited PKAcat-induced ΔΨm, FAD and DCF alteration. PKAcat-induced ΔΨm depolarization was inhibited by an inhibitor of the inner membrane anion channel (IMAC), 4,4′-diisothiocyanatostilbene-2, 2′-disulfonic acid (DIDS: 1; μmol/L) but not by an inhibitor of mitochondrial permeability transition pore (mPTP), cyclosporine A (100 nmol/L). Conclusions: PKAcat alters FAD and ΔΨm via mitochodrial ROS generation, and PKAcat-induced ΔΨm depolarization was not caused by mPTP but rather by DIDS-sensitive mechanisms, which could be caused by opening of the IMAC. The effects of PKA on mitochondrial function could be related to myocardial function under the condition of extensive β-adrenergic stimulation.