Glutathione S-Transferase Kappa 1 Knockdown Exacerbates Complex-III-Mediated ROS Production in H9c2 Cardiac Cells

Abstract

Background: Mitochondria produce reactive oxygen species (ROS) that are scavenged by local antioxidant enzymes. Glutathione (GSH) is key intermediate in many of these reactions and its availability determines the antioxidant capacity of mitochondria. Glutathione S-transferases (GSTs) are known to consume GSH during xenobiotic detoxification but their involvement in ROS scavenging is less clear. Gstκ1 was originally identified as a novel mitochondrial GST but its role there remains unknown.Objective: To examine whether loss of Gstκ1 affects the [GSSG/GSH] ratios in cytosol and mitochondria of the cardiac-derived cell line H9c2.Methods: Knockdown of Gstκ1 (Gstκ1-KD) in H9c2 cells was achieved by transfection with siRNA. Changes in [GSSG/GSH] were monitored using genetically-encoded ratiometric sensors that localize to cytosol or mitochondria. Global oxidative stress was induced with hydrogen peroxide (HP). Mitochondrially-targeted oxidative stress was induced by the superoxide generator di-methoxy-naphtho-quinone (DMNQ) and by antimycin-A (AA) which interacts specifically with mitochondrial complex III.Results: Treatment with HP elevatedcytosolic [GSSG/GSH] ratios equally in both control and Gstκ1-KD cells. Mitochondrial oxidative stress elicited by HP or DMNQ likewise increased mitochondrial [GSSG/GSH] and was unaffected by Gstκ1-KD. Inhibition of Complex III by AA decreased mitochondrial [GSSG/GSH] persistently in control cells. However, in Gstκ1-KD cells exposed to AA, the decrease was transient and was followed by a sustained increase in [GSSG/GSH].Conclusions: Here we examine for the first time the role of Gstκ1 in ROS scavenging in cardiac cells. While our data suggest that Gstκ1 does not participate in the general response against exogenous ROS, they indicate that low levels of this enzyme associate with increased superoxide production from mitochondrial complex III. Our findings may provide insights for experimental models of cardiac disease where Gstκ1 expression is downregulated.