Inhibition of c-Jun N-terminal kinase 1, but not c-Jun N-terminal kinase 2, suppresses apoptosis induced by ischemia/reoxygenation in rat cardiac myocytes

In the present study, rat cardiac myocytes were used as an in vitro ischemi a/reperfusion injury model to delineate the role of c-Jun N-terminal kinase (JNK) 1 and JNK2 isoforms in ischemia/reoxygenation-induced apoptosis usin g an antisense approach. Exposure of rat cardiac myocytes to ischemia did n ot induce apoptosis as detected by staining with either acridine orange/eth idium bromide or annexin-V-fluorescein/propidium iodide. In contrast, a tim e-dependent increase in the number of apoptotic cells was noted after reoxy genation of ischemic myocytes, whereas the level of necrotic cells remained unaltered. Reoxygenation, but not ischemia alone, also caused a time-depen dent increase in JNK activation that preceded apoptosis induction. Treatmen t of cardiac myocytes with antisense (AS) oligonucleotides that specificall y targeted either JNK1 or JNK2 significantly reduced both mRNA and protein expression of the target isoform but had no effect on the expression of the alternate isoform. Pretreatment of cardiac myocytes with JNK1 AS, but not JNK2 AS, resulted in almost complete attenuation of reoxygenation-induced a poptosis. Furthermore, control oligonucleotides for JNK1 AS or JNK2 AS had no effect on JNK mRNA or protein expression or reoxygenation-induced apopto sis, indicating a sequence-specific mode of action. Additional studies reve aled that apoptosis induced by other JNK-activating stimuli, including cera mide, heat shock, and UV irradiation, was partly suppressed after treatment with JNK1 AS but not JNK2 AS. These findings demonstrate that the JNK1 iso form plays a preferential role in apoptosis induced by ischemia/reoxygenati on as well as diverse JNK-activating cellular stresses.