Reperfusion injury occurs when ischaemic tissue is reperfused. It involves the generation and release of reactive oxygen that activates numerous signalling pathways and initiates cell death. Exposure of isolated cardiac myocytes to chronic hypoxia followed by reoxygenation results in the early activation of c-Jun N-terminal kinase (JNK) and death by apoptosis of approx. 30% of the myocytes. Although JNK activation has been described in a number of models of ischaemia/reperfusion, the contribution of JNK activation to cell fate has not been established. Here we report that the activation of JNK by reoxygenation correlates with myocyte survival. Transfection of myocytes with JNK pathway interfering plasmid vectors or infection with adenoviral vectors support the hypothesis that JNK is protective. Transfection or infection with JNK inhibitory mutants increased the rates of apoptosis by almost 2-fold compared with control cultures grown aerobically or subjected to hypoxia and reoxygenation. Caspase 9 activity, measured by LEHD cleavage, increased > 3-fold during reoxygenation and this activity was enhanced significantly at all times in cultures infected with dominant negative JNK adenovirus. Hypoxia—reoxygenation mediated a biphasic (2.6- and 2.9-fold) activation of p38 mitogen-activated protein kinase, as well as a small increase of tumour necrosis factor α (TNFα) secretion, but treatments with the p38 MAPK-specific inhibitor SB203580 or saturating levels of a TNFα-1 blocking antibody provided only partial protection against apoptosis. The results suggest that JNK activation is protective and that the pathway is largely independent of p38 MAPK or secreted TNFα.
Abbreviations used: JNK, c-Jun N-terminal kinase; TNFα, tumour necrosis factor α; MAPK, mitogen-activated protein kinase; ERK, extracellular-signal-regulated protein kinase; MEKK1, MAPK/ERK kinase kinase 1; MKK4, MAPK kinase 4; MAPKAP kinase 2, MAPK-activated protein kinase 2; HA, haemagglutinin antigen; Hsp27, heat-shock protein 27; TUNEL, terminal transferase deoxytidyl uridine end labelling; dn, dominant negative; ca, constitutively active; GFP, green fluorescent protein; PI, propidium iodide.