Molecular adaptation of cellular defences following preconditioning of the heart by repeated ischaemia

OBJECTIVE: The molecular mechanism of preconditioning the heart by repeated ischaemia was investigated by examining the expression of stress related and antioxidative genes and proteins. METHODS: Isolated buffer-perfused rat hearts were made ischaemic for 5 min followed by 10 min of reperfusion (1 x PC). Another group of experiments was performed by repeating the ischaemia/reperfusion episode four times (4 x PC). Both 1 x PC and 4 x PC groups were reperfused for 60 min. Control experiments were performed by perfusing the hearts with buffer using the same time frame, but without ischaemia/reperfusion. The induction of the expression of two oncogenes (c-fos and c-myc), three heat shock protein (HSP) genes (HSP 27, HSP 70, and HSP 89 mRNAs) and two antioxidative enzyme genes (Mn-superoxide dismutase (SOD) and catalase mRNAs) by northern hybridisation, as well as the activities of four major antioxidative enzymes (SOD, catalase, glutathione peroxidase, and glutathione reductase) were examined in control and 1 x PC and 4 x PC hearts. In addition, 2-D gel electrophoresis of the proteins in all groups was performed to examine the induction of any new protein. Myocardial preservation was studied by examining left ventricular functions. RESULTS: Northern hybridisation detected enhanced level of c-fos 2.2 kb mRNA and c-myc 2.4 kb mRNA in 4 x PC hearts after 60 min of reperfusion, induction being stronger for c-fos. 1 x PC hearts showed minimal expression of c-fos mRNA, but not c-myc mRNA. 4 x PC hearts also showed the induction of HSP 27, HSP 70, and HSP 89 mRNAs as well as catalase and Mn-SOD mRNAs, whereas 1 x PC hearts showed some induction of HSP 70 mRNA and catalase mRNA only. 2-D gel electrophoresis revealed the expression of 15-20 new proteins in 4 x PC hearts only. The activities of three major antioxidative enzymes, Mn-SOD, peroxisomal catalase, and glutathione peroxidase, but not Cu/Zn-SOD, cytosolic catalase, and glutathione reductase, were enhanced after 60 min of reperfusion in 4 x PC hearts only compared to both 1 x PC and baseline values. 4 x PC hearts, but not 1 x PC hearts, showed reduction of subsequent myocardial ischaemic and reperfusion injury. CONCLUSIONS: Repeated ischaemia and reperfusion (4 x PC), and not 1 x PC, caused the induction of several stress related and antioxidant genes including HSP 27, 70, and 89 genes, Mn-SOD and catalase genes, and proto-oncogenes (c-fos and c-myc), as well as the expression of several stress inducible proteins and stimulation of three antioxidative enzymes. Mn-SOD, peroxisomal catalase, and glutathione peroxidase, simultaneously protecting the heart from subsequent ischaemia-reperfusion injury. The expression of stress inducible and antioxidant genes and stimulation of antioxidant enzyme activities may reflect the heart's response enabling it to survive against ischaemic stress by eliminating the oxidative assault.