Abstract
Ischaemia reperfusion (IR) injury occurs when blood flow is restored to a previously ischaemic tissue, resulting in increased cellular and tissue dysfunction. During ischaemia, myocardial cellular oxygen and nutrient supply are stopped resulting in damage to the electron transport chain and increased reactive oxygen species (ROS) production. Upon reperfusion, a large influx of molecular oxygen enters the ischaemic tissue resulting in production of pathological levels of ROS, triggering mitochondrial permeability transition pore opening and cell death. Current ischaemic preconditioning techniques utilise periods of sublethal IR to protect the tissue prior to a predicted ischaemic event. By triggering small bursts of ROS, preconditioning protocols initiate downstream protective mechanisms, such as the activation and translocation of protein kinase C ε (PKCε) to the mitochondria to prevent mitochondrial permeability transition pore opening and cell death. Carbon monoxide (CO) is an endogenous signalling molecule with a high affinity for haem and binds to electron transport chain complexes to cause small increases in ROS. oCOm-21 is an organic CO releasing molecule which releases CO under physiological conditions. In ex vivo perfused hearts, oCOm-21 has produced preconditioning effects which have shown to be inhibited by the PKC inhibitor chelerythrine.
This project aimed to determine if CO released from oCOm-21 triggers a preconditioning response by increasing mitochondrial ROS production to trigger the translocation of PKCε to the mitochondria. This mechanism was investigated in vitro in human cardiomyocyte AC16 cells. MitoSOX Red fluorescence (assessed at excitation of 488 ± 15 nm and emission of 575 ± 25 nm) for mitochondrial ROS showed no significant difference between cells treated for 1 hour with 0.1 µM, 1 µM, 3 µM and 10 µM oCOm-21 and the acidified water vehicle control (P > 0.05). PKCε mitochondrial colocalisation assessment with MitoTracker Red and PKCε immunofluorescence showed a significant difference between 0.1 µM and 1 µM oCOm-21 and between 0.1 µM and 10 µM oCOm-21 at 5 minutes after treatment of cells (P < 0.05). This evidence is preliminary due to inadequate control groups and further experiments must be carried out to confirm whether CO derived from oCOm-21 has a preconditioning effect through PKCε translocation to the mitochondria.