Primary role of mitochondrial Rieske iron–sulfur protein in hypoxic ROS production in pulmonary artery myocytes

This study was designed to determine whether: (1) hypoxia could directly affect ROS production in isolated mitochondria and mitochondrial complex III from pulmonary artery smooth muscle cells (PASMCs) and (2) Rieske iron–sulfur protein in complex III might mediate hypoxic ROS production, leading to hypoxic pulmonary vasoconstriction (HPV). Our data, for the first time, demonstrate that hypoxia significantly enhances ROS production, measured by the standard ROS indicator dichlorodihydrofluorescein/diacetate, in isolated mitochondria from PASMCs. Studies using the newly developed, specific ROS biosensor pHyPer have found that hypoxia increases mitochondrial ROS generation in isolated PASMCs as well. Hypoxic ROS production has also been observed in isolated complex III. Rieske iron–sulfur protein silencing using siRNA abolishes the hypoxic ROS formation in isolated PASM complex III, mitochondria, and cells, whereas Rieske iron–sulfur protein overexpression produces the opposite effect. Rieske iron–sulfur protein silencing inhibits the hypoxic increase in [Ca2+]i in PASMCs and hypoxic vasoconstriction in isolated PAs. These findings together provide novel evidence that mitochondria are the direct hypoxic targets in PASMCs, in which Rieske iron–sulfur protein in complex III may serve as an essential, primary molecule that mediates the hypoxic ROS generation, leading to an increase in intracellular Ca2+ in PASMCs and HPV.