In the present study, rat cardiac myocytes were used as an in vitro
ischemia/reperfusion injury model to delineate the role of
c-Jun N-terminal kinase (JNK) 1 and JNK2
isoforms in
ischemia/reoxygenation-induced apoptosis using an antisense approach. Exposure of rat cardiac myocytes to
ischemia did not induce apoptosis as detected by staining with either
acridine orange/
ethidium bromide or
annexin-V-
fluorescein/
propidium iodide. In contrast, a time-dependent increase in the number of apoptotic cells was noted after reoxygenation of ischemic myocytes, whereas the level of necrotic cells remained unaltered. Reoxygenation, but not
ischemia alone, also caused a time-dependent increase in JNK activation that preceded apoptosis induction. Treatment of cardiac myocytes with antisense (AS)
oligonucleotides that specifically 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 apoptosis. Furthermore, control
oligonucleotides for JNK1 AS or JNK2 AS had no effect on JNK
mRNA or
protein expression or reoxygenation-induced apoptosis, indicating a sequence-specific mode of action. Additional studies revealed that apoptosis induced by other JNK-activating stimuli, including
ceramide, heat shock, and UV irradiation, was partly suppressed
after treatment with JNK1 AS but not JNK2 AS. These findings demonstrate that the JNK1
isoform plays a preferential role in apoptosis induced by
ischemia/reoxygenation as well as diverse JNK-activating cellular stresses.