Multiple signaling pathways, including the c-Jun N-terminal kinase (JNK) pathway, are activated in myocardial ischemia and reperfusion (MI/R) and correlatewith cell death. However, the role of the JNK pathway with respect to protection ordestruction in MI/R-induced cell death is poorly understood. In a rabbit model, we foundthat ischemia followed by reperfusion resulted in JNK activation which could be detectedin cytosol as well as in mitochondria. To address the functional role of the JNKactivation, we examined the consequences of blockade of JNK activation in isolatedcardiomyocytes under conditions of simulated ischemia. The JNK activity was stimulated ～6-foldby simulated ischemia and reperfusion (simulated MI). When a dominant negative mutant ofJNK kinase-2(dnJNKK2), an upstream regulator of JNK, and JNK-interacting protein-1 (JIP-1)were expressed in myocytes by recombinant adenovirus, the activation of JNK by simulatedMI was reduced 53%. Furthermore, the TNFα-activated JNK activity in H9c2 cells wascompletely abolished by dnJNKK2 and JIP-1. In correlation, when dnJNKK2 and JIP-1 wereexpressed in cardiomyocytes, both constructs significantly reduced cell death aftersimulated MI compared to vector controls.

To further delineate the mechanism of JNK-induced cell death, weattempted to identify targets of JNK, especially those in mitochondria. Mitochondria wereprepared from untreated cardiomyocytes or myocardium and incubated with cytosols derivedfrom different conditions, using [³² P]-γ-ATP to indicate phosphorylation.A 46kD protein (p46) from mitochondrial inner membrane was specifically phosphorylated byMI cytosol. In addition, the ischemia-induced phosphorylation of p46 was reduced byischemic preconditioning. We further characterized the mitochondrial p46 and its kinase.We show that JNK depletion from cytosol failed to abolish phosphorylation,indicating thatthe kinase activity is not dependent on JNK. To identify the kinase responsible for the phosphorylation, we have examined the effect of a number of kinase inhibitors. Preliminarydata show that the phosphorylation of p46 was exclusively dependent on tyrosine kinaseactivity. When the purified p46 was subjected to mass spectrophotometry analysis, thefinger print of the tryptic peptides indicated that p46 is a novel protein.
In conclusion, we demonstrate that JNK pathway is important for MI/R inducedcardiomyocyte death. We further show that ischemia induces phosphorylation ofmitochondrial p46 while ischemic preconditioning suppresses phosphorylation. Finally, thekinase responsible for phosphorylation of p46 is present in mitochondria and appears to bea tyrosine kinase. (HE Hua-ping Roberta A.Gottlieb)