Ischemic preconditioning and diazoxide limit mitochondrial Ca2+ overload during ischemia/reperfusion: Role of reactive oxygen species

BACKGROUND:

Generation of reactive oxygen species (ROS) is associated with cardioprotection imparted by ischemic preconditioning (IPC) and pharmacological PC (PPC). The authors have previously shown that IPC or PPC, using the mitochondrial ATP-sensitive K⁺ channel opener diazoxide (DZ), reduce mitochondrial Ca²⁺ ([Ca²⁺]_m) during ischemia and reperfusion.

OBJECTIVES:

To test the hypothesis that both IPC and PPC (using DZ) lead to reduced [Ca²⁺]_m and improved functional recovery via a ROS-dependent mechanism.

METHODS:

Intracellular Ca²⁺ ([Ca²⁺]_i) and [Ca²⁺]_m were measured in isolated perfused rat hearts loaded with the fluorescent indicator indo-1 acetoxymethyl ester. [Ca²⁺]_m was determined by quenching the cytosolic indo-1 signal using manganese before ischemia (25 min). IPC and DZ (100 μM) group hearts were studied with and without the ROS scavenger N-2-mercaptopropionyl glycine (400 μM) (2-MPG).

RESULTS:

Both IPC and DZ significantly reduced [Ca²⁺]_i and [Ca²⁺]_m on reperfusion compared with the control. Administration of 2-MPG with washout before ischemia significantly attenuated the reduction in [Ca²⁺]_m observed on reperfusion in both the IPC and DZ groups. Additionally, the myocardial functional protection imparted by IPC or DZ was lost with the administration of 2-MPG.

CONCLUSIONS:

The [Ca²⁺]_m-reducing effect of IPC and DZ was attenuated with the administration of 2-MPG, resulting in decreased myocardial functional performance and increased release of creatine kinase, a marker of cellular injury. It can be concluded that IPC and DZ impart their protective effect via a mechanism involving ROS generation before the ischemic episode.