Prohibitin Overexpression Promotes Opa1 Cleavage, Reduces ROS and Improves Respiration Post Hypoxia

Abstract

Prohibitin is a multifunctional protein highly localized to mitochondria. Prohibitin 1 and 2 can make large oligomeric complexes that are associated with cristae formation and localization of mitochondrial signaling cascades including Opa1, an important mediator of mitochondrial inner membrane fusion, cristae formation, and respiration. These studies were performed to determine any beneficial effects of PHB2 on Opa1, ROS, and mitochondrial function following hypoxic injury. H9c2 cells were infected with adenoviral PHB2 or empty virus for 48 hours. ROS was measured using a mitochondrial matrix-targeted roGFP imaged intermittently in a gas and temperature-controlled imaging plate reader for 1 hour baseline, 3 hours hypoxia, 1 hour reoxygenation. Respiration was measured using a Seahorse Analyzer. PHB2 infection caused significant increase in both PHB2 and PHB1 expression. PHB2 overexpression did not significantly change total Opa1 levels, but did cause a significant increase in the short form of Opa1 as well as an increase in short:long ratio. In addition, PHB2 overexpression was associated with an increase in mitochondrial specific proteins HSP60 (matrix), VDAC (outer membrane) and electron transport chain protein subunits normalized to total cellular proteins indicating a general increase in mitochondrial protein expression and/or biogenesis. In vitro hypoxia (3h) and reperfusion (1 hour) caused a significant increase in ROS production during reoxygenation that was attenuated by PHB2 overexpression (32.8 +/- 1.8 vs 49.6 +/-2.4, % oxidized roGFP, P<.01) PHB2 overexpression also significantly increased mitochondrial basal, maximal and ATP-linked respiration following 3 hours hypoxia and reoxygenation. Overexpression of PHB2 is associated with increased Opa1 cleavage, increased ETC and mitochondrial proteins, but decreased ROS generation and improved respiration post-hypoxia. Therefore, strategies to increase prohibitin expression may be cytoprotective against hypoxic injury.