Differential Activation of Protein Kinase C between Ischemic and Pharmacological Preconditioning in the Rabbit Heart.

Abstract

The objective of the present study was to investigate the differential activation of protein kinase C between ischemic (IPC) and pharmacological preconditioning (PPC) in the rabbit heart. Control, IPC, diazoxide (Diaz), and chelerythrine (Chel)+IPC groups underwent prolonged coronary artery occlusion (CAO) for 30 minutes followed by 180 minutes' reperfusion (protocol I). In protocol II, sham, IPC-only, Diaz-only, and Chel+IPC-only groups did not undergo prolonged CAO. IPC was induced with 4 cycles of 5-min regional ischemia and 10-min reperfusion before prolonged CAO. Diaz (5 mg/kg) was administered 30 min before prolonged CAO. Chel (5 mg/kg) was administered 5 min before the IPC procedure. Infarct size was determined by tetrazolium staining. Assessment of protein kinase C (PKC) isoforms from a left ventricular (LV) sample was conducted by western blotting. Apoptosis in situ was determined by TUNEL assay. The infarction area in the IPC (11.6 ± 1.0%) and Diaz (19.5 ± 3.8%) groups was reduced significantly (plt;0.01, plt;0.05) relative to the control group (40.0 ± 3.8%). The reduction by IPC was abolished by pretreatment with Chel. Apoptosis was significantly decreased (plt;0.01) in the IPC and diazoxide groups compared with the control and Chel+IPC groups (control: 4.78 ± 0.56% vs. IPC: 2.00 ± 0.38% vs. Diaz: 2.20 ± 0.32% vs. Chel+IPC: 4.32 ± 0.41%) and DNA laddering was attenuated in the IPC and Diaz groups. Membrane PKC-ε levels in the IPC and Diaz groups increased significantly relative to the control and Chel+IPC groups. Membrane PKC-ε levels in the IPC-only group showed greater increases than the Diaz-only and Chel+IPC-only groups. These findings suggest that whereas PPC suppresses apoptosis when diazoxide opens mitochondrial K_ATP channels and then activates PKC-ε through ischemia-reperfusion, IPC activates PKC-ε in the particulate fraction prior to continuous ischemia-reperfusion. We concluded that the difference between IPC and PPC appears to consist in the difference in the timing of PKC-ε activation, though both IPC and PPC provide the cardioprotection in ischemia-reperfusion injury.