Mitochondrial Sources of H 2 O 2 Generation Play a Key Role in Flow-Mediated Dilation in Human Coronary Resistance Arteries

Abstract

We previously showed that hydrogen peroxide (H 2 O 2 ) contributes to flow-induced dilation in human coronary resistance arteries (HCRAs); however, the source of this H 2 O 2 is not known. We hypothesized that the H 2 O 2 is derived from superoxide (O 2 ·− ) generated by mitochondrial respiration. HCRAs were dissected from right atrial appendages obtained from patients during cardiac surgery and cannulated with micropipettes. H 2 O 2 -derived radicals and O 2 ·− were detected by electron spin resonance (ESR) using BMPO as the spin trap and by histofluorescence using hydroethidine (HE, 5 μmol/L) and dichlorodihydrofluorescein (DCFH, 5 μmol/L). Diameter changes to increases in pressure gradients (20 and 100 cm H 2 O) were examined in the absence and the presence of rotenone (1 μmol/L), myxothiazol (100 nmol/L), cyanide (1 μmol/L), mitochondrial complex I, III, and IV inhibitors, respectively, and apocynin (3 mmol/L), a NADPH oxidase inhibitor. At a pressure gradient of 100 cm H 2 O, ubisemiquinone and hydroxyl radicals were detected from effluents of vessels. Including superoxide dismutase and catalase in the perfusate reduced the ESR signals. Relative ethidium and DCFH fluorescence intensities in HCRAs exposed to flow were enhanced (1.45±0.15 and 1.57±0.12, respectively compared with no-flow) and were inhibited by rotenone (0.87±0.17 and 0.95±0.07). Videomicroscopic studies showed that rotenone and myxothiazol blocked flow-induced dilation (% max. dilation at 100 cm H 2 O: rotenone, 74±3% versus 3±13%; myxothiazol, 67±3% versus 28±4%; P <0.05). Neither cyanide nor apocynin altered flow-induced dilation. These results suggest that shear stress induced H 2 O 2 formation, and flow-induced dilation is derived from O 2 ·− originating from mitochondrial respiration.