Abstract 2280: Intra-ischemic Cooling Protects Cardiomyocytes by Nitric Oxide Signaling
Background: Optimal timing for therapeutic hypothermia in ischemia/reperfusion (I/R) injury is not yet clear. Prior work suggests that rapid reperfusion can accelerate cell death, while transient extended ischemia is not as damaging. Also, nitric oxide (NO) is associated in this model with cardioprotection. Thus, we hypothesized that hypothermia is most protective if induced before reperfusion– even at the cost of delayed reperfusion and mediated by NO signaling.
Methods: In an established chick cardiomyocyte model of I/R, hypothermia (25oC) was induced either concurrently with reperfusion (RC), 15 min after reperfusion (DC), or before reperfusion using an extended 10 min of ischemia (IC). Hypothermia was maintained for 1 h during reperfusion. The cell death was assessed by propidium iodide at 3 and 24 h of reperfusion. Intracellular nitric oxide (NO) generation was continuously monitored using 4,5-diaminofluorescin diacetate. Also, caspase-3 activity was measured at 3 h and DNA fragmentation at 24 h reperfusion.
Results: RC protected against I/R injury by reducing cell death from 49.7 ± 3.4% in I/R control to 22.4 ± 2.9% (P<0.001). If hypothermia was delayed by 15 min into reperfusion (DC), the protective effect was lost (45.0 ± 8.2% death, P=NS). IC was most protective despite delaying reperfusion by 10 min (14.3 ± 0.6%, P<0.05 compared to RC). IC was associated with a significant increase in NO generation sustained throughout reperfusion, an effect not seen in DC, and was associated with decreased caspase-3 activation at 3 h and decreased DNA fragmentation at 24 h. IC protection and NO generation were abrogated by L-NAME (200 μM) and PKCϵ inhibiting peptide (5 μM).
Conclusions: I/R injury in this cardiomyocyte model is significantly determined by events occurring within the first 15 minutes of reperfusion, and can be abrogated substantially by extending the time of ischemia transiently in order to cool prior to reperfusion. Such protection is mediated by NO signaling pathways, leading to decreased caspase-3 activation and apoptosis.