Abstract 30: Hypothermia Protects against Reperfusion Injury in Murine Cardiomyocytes
Background: We have previously demonstrated lethal cardiac post-resuscitation injury in 2 models: chick cardiomyocyte and mouse whole body (i.e. cardiac arrest and resuscitation) ischemia/reperfusion (I/R). Hypothermia is protective in both models when initiated for 1h after ischemia. We sought to investigate whether a similar I/R injury pattern and protection occurs in neonatal mouse cardiomyocytes.
Methods: Murine ventricular cardiomyocytes isolated from neonatal C57/BL 6 mice (aged 1 day) were cultured for 6–9 days until they displayed synchronous spontaneous contractions. Cells were perfused in a Sykes-Moor chamber and subjected to 90min I/3h R. Cell viability in the same cell field was monitored over time by propidium iodide exclusion and LDH release. Affymetrix mouse 430 2.0 arrays were used to characterize reperfusion-induced expression profiles from in vivo mouse hearts before and after 8 min of cardiac arrest, and in vitro murine cardiomyocytes before and after ischemia. Intra-ischemic cooling (IC) in cells was initiated during the last 20 min of ischemia at 32°C through 1 h reperfusion, and then re-warmed for 2 h.
Results: Reperfusion accelerated cell death and LDH release compared to prolonged 4.5 h ischemia (cell death 44.7 ± 2.5% vs. 13.1 ± 1.9%, P < 0.001; LDH release 26.5 ± 1.0% vs. 11.8 ± 1.1%, P < 0.001). Similarly, mice exposed to 8min cardiac arrest and 2 h resuscitation as before demonstrate lethal cardiovascular collapse with approximate 50% mortality. Pathway analysis identified a number of statistically enriched signaling pathways including IL-6 signaling, chemokine signaling and NF-κB signaling–during reperfusion of both cardiomyocytes and whole hearts after cardiac arrest. Compared to I/R control, the IC group demonstrated protection against post-resuscitation injury (cell death reduced to 19.3 ± 2.1% vs. 44.7 ± 2.5%, P < 0.001).
Conclusions: We conclude that lethal post-resuscitation cardiac injury seen in mammalian cardiomyocytes resembles the injury pattern seen in our previously published models of chick cardiomyocyte I/R and mouse cardiac arrest. In addition, these cells may be useful for studying molecular mechanisms of hypothermia protection.