Protein Kinase C activates glucose‐6‐phosphate dehydrogenase and NAD(P)H oxidase in failing human myocardium

Elevation of reactive oxygen species (ROS) in heart failure is well documented. However, sources of ROS in the failing myocardium are still unknown. In this study, we examined whether NADPH oxidase is a major source of ROS in failing human myocardium. Superoxide (O2−), a precursor of ROS, levels were detected by lucigenin (5 uM) chemiluminescence. The pre-incubation of human myocardium with NADPH (Nox) oxidase inhibitors, respectively, gp91dstat (50 uM), apocynin (100 uM) and diphenyleneiodonium (DPI; 10 uM), significantly inhibited O2− generation to 48.0, 21.4, and 16.4% of the untreated control O2− levels (378±102 Units/mg protein). Determination of NADPH oxidase activity indicates that O2− generation increased by 9.6- and 2.9-fold by NADPH (100 uM) and NADH (100 uM), respectively, in DPI- and gp91dstat-inhibitable manner. Interestingly, myocardial O2− was also inhibited by 6-aminonicotinamide (5 mM), a glucose-6-phosphate dehydrogenase (G6PD) inhibitor, to 53.5% of the untreated control. Furthermore, staurosporine (100 nM), a PKC inhibitor, and PP2 (0.010 mM), a Src inhibitor, attenuated G6PD to 2.2 and 49.1%, respectively, of the untreated control activity (0.29±0.04 nM/min/mg protein) and myocardial O2− to 62.1 and 58.7% of the control. Our data suggest that NADPH oxidase is a major source of ROS. G6PD-derived NADPH fuel NADPH oxidase activity, and both G6PD and NADPH oxidase are activated in failing cardiac muscle by PKC-Src signaling pathways. (Supported by AHA grant 0435070N and NIH grants HL 31069, HL43023, HL66331).