Protein Kinase A-mediated Phosphorylation Modulates Cytochrome c Oxidase Function and Augments Hypoxia and Myocardial Ischemia-related Injury

Abstract

We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O 2 for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O 2 species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nM H89 or 50 nM myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions. Cytochrome c oxidase (CcO) 3 is the terminal oxidase of the mitochondrial electron transport chain, whose activity is modulated in response to O 2 tension and the work load of the tissue (1-6). This rate-limiting enzyme is an important site of regulation of mitochondrial respiration and oxidative phosphorylation (7). In the yeast, altered CcO activity in response to aerobic and anaerobic conditions is associated with the differential expression of the two isologs of the CcO Vb gene (8), although the precise mechanism by which the mammalian CcO modulates its activity remains unknown. Mitochondrial electron transport chain complexes are major sources of cellular ROS under both normoxic and hypoxic conditions (9, 10). Hypoxia-tolerant and hypoxia-sensitive human glioma cells exhibit distinct patterns of mitochondrial function in response to hypoxia (9, 11). Submitochondrial particles exposed to hypoxic conditions in vitro show reduced CcO activity (1,10,12). Some studies also suggest that the myocardial ischemia/reperfusion injury is manifested through altered CcO activity and reduced mitochondrial oxidative phosphorylation (13,14).Protein kinases have been suggested to play a role in the modulation of myocardial ischemia/reperfusion injury (15), although the roles of different cellular components in mediating this injury remain unclear. The presence of PKA and PKC activities in the mitochondrial inner membrane-matrix compartment and the role of PKC-mediated phosphorylation in the regulation of pyruvate dehydrogenase activity are well established (16). An 18-kDa subunit of the NADH dehydrogenase (complex I) (17)...

Cardioprotective Pathways During Reperfusion: Focus on Redox Signaling and Other Modalities of Cell Signaling

“…Yet, we have been unable to reproduce cardioprotection with ROS generation by purine=xanthine oxidase given at reperfusion (141). Since ROS scavengers, given at the beginning of reperfusion, abolished both pre-and postconditioning-induced protection (72,141,153,186), it is likely that the type, the concentration, and=or the compartmentalization of reactive species may play a pivotal role in triggering protection at reperfusion time. We can not exclude that a different ROS generator could trigger PostC protection (142).…”

Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction

“…The reason for this difference remains unclear. The membrane topology of proteins associated with the mitochondrial fraction was evaluated using the alkaline Na 2 CO 3 extraction method (26,49). Both full-length and CYP2C8 Var_3 in the mitochondrial fraction were nearly quantitatively extracted with the alkaline aqueous phase, whereas the microsomal WT CYP2C8 protein partitioned more into the insoluble fraction, suggesting a transmembrane topology.…”

“…The addition of 5 g of brain cytosolic fraction was used as the source of esterase for assaying mitochondrial fractions (49). Stable non-fluorescent 2Ј,7Ј-dichlorodihydrofluorescein diacetate (2.5 M in CH 3 OH) was used in 200 l of assay mixture containing 135 l of PBS, 20 g of mitochondrial protein, 5 g of brain cytosol, and 0.1 mM NADPH.…”

“…Measurement of ROS Production Using 2Ј,7Ј-Dichlorodihydrofluorescein Diacetate-Mitochondrial ROS was assayed using 2Ј,7Ј-dichlorodihydrofluorescein diacetate oxidation (Molecular Probes, Eugene, OR) (48) as modified by Prabu et al (49). The addition of 5 g of brain cytosolic fraction was used as the source of esterase for assaying mitochondrial fractions (49).…”

The role of protein kinase A in hypoxia-induced PC-12 cell death.

“…Furthermore, PKA has been shown to phosphorylate subunits I, IV, and Vb of complex IV of the mitochondrial electron transport chain. In vitro phosphorylation of COX IV by PKA in bovine heart and rat macro phages decreases its activity by 40-70% (17). Furthermore, the Ria regulatory subunit of PKA directly binds to the Vb subunit of COX IV and inhibits its activity (18).…”