1 2 Mitochondrial Reactive Oxygen Species — Which ROS Signals Cardioprotection ? 3 4

30 Mitochondria are the major effectors of cardioprotection by procedures that open the 31 mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and 32 pharmacological preconditioning. MitoKATP opening leads to increased reactive oxygen 33 species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates 34 mitoKATP and leaves it in a persistent open state (Costa, ADT and Garlid, KD. Am J 35 Physiol 295, H874-82, 2008). The ROS responsible for this effect is not known. The 36 present study focuses on superoxide (O2 ), hydrogen peroxide (H2O2), and hydroxyl 37 radical (HO), each of which has been proposed as the signaling ROS. Feedback 38 activation of mitoKATP provides an ideal setting for studying endogenous ROS signaling. 39 Respiring rat heart mitochondria were preincubated with ATP and diazoxide, together 40 with an agent being tested for interference with this process, either by scavenging ROS 41 or by blocking ROS transformations. The mitochondria were then assayed to determine 42 whether or not the persistent phosphorylated state was achieved. Dimethylsulfoxide 43 (DMSO), dimethylformamide (DMF), deferoxamine, Trolox, and bromoenol lactone 44 (BEL) each interfered with formation of the ROS-dependent open state. Catalase did 45 not interfere with this step. We also found that DMF blocked cardioprotection by both 46 ischemic preconditioning and diazoxide. The lack of a catalase effect and the inhibitory 47 effects of agents acting downstream of HO excludes H2O2 as the endogenous signaling 48 ROS. Taken together, the results support the conclusion that the ROS message is 49 carried by a downstream product of HO and that it is probably a product of phospholipid 50 oxidation. 51 52 53 54 55 56 57 58