Cobalt chloride induces delayed cardiac preconditioning in mice through selective activation of HIF-1α and AP-1 and iNOS signaling

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Abstract
Acute systemic hypoxia induces delayed cardioprotection against ischemia (I)-reperfusion (R) injury via inducible nitric oxide synthase (iNOS)-dependent mechanism. Because CoCl2 is known to elicit hypoxia-like responses, we hypothesized that this chemical would mimic the delayed preconditioning effect in the heart. Adult male mice were pretreated with CoCl2 or saline. The hearts were isolated 24 h later and subjected to 20 min of global I and 30 min of R in Langendorff mode. Myocardial infarct size (% of risk area; mean ± SE, n = 6–8/group) was reduced in mice pretreated with 30 mg/kg CoCl2 (16.1 ± 3.1% vs. 27.6 ± 3.3% with saline control; P < 0.05) without compromising postischemic cardiac function. Higher doses of CoCl2 failed to induce similar protection. Electrophoretic mobility gel shift assay demonstrated significant enhancement in DNA binding activity of hypoxia-inducible factor 1α (HIF-1α) and activator protein 1 (AP-1) in nuclear extracts from CoCl2-treated hearts. Activation of HIF-1α and AP-1 was evident at 30 min and sustained for the next 4 h after CoCl2 injection. In contrast, CoCl2-induced protection was independent of NF-κB activation because no DNA binding or p65 translocation was observed in nuclear extracts. Also, CoCl2-induced cardioprotection was preserved in p50 subunit NF-κB-knockout (KO) mice (11.1 ± 3.0% vs. 25.1 ± 5.0% in saline-treated p50-KO mice; P < 0.05). The infarct-limiting effect of CoCl2 was absent in iNOS-KO mice (20.9 ± 3.0%). We conclude that in vivo administration of CoCl2 preconditions the heart against I/R injury. The delayed protective effect of CoCl2 is achieved through a distinctive signaling mechanism involving HIF-1α, AP-1, and iNOS but independent of NF-κB activation.