Cardiomyocytes: The Heart of DAMPs Release and Sterile Inflammation in Ischemia/Reperfusion Injury

Objectives: Targeted temperature management (TTM), also referred to as therapeutic hypothermia (TH), is the recommended standard of care for adult out-of-hospital cardiac arrest patients but fever is often observed in this cohort upon rewarming from cooling. Acute myocardial ischemia/reperfusion (I/R) injury results in the release of damage-associated molecular patterns (DAMPs), which can induce a sterile inflammatory response in the myocardial penumbra. However, the effect of cooling on cardiomyocytes in acute I/R-induced sterile inflammation remains to be elucidated. Therefore, we investigated the effect of cooling on cell viability, oxidative stress, and DAMPs release during oxygen-glucose deprivation/reperfusion (OGD/R) in a murine primary cardiomyocyte model.

Methods: Primary cardiomyocytes from P1 to 3 mice were exposed to 2, 4, or 6 hours of OGD (0.2% oxygen in medium without glucose and serum) followed by 6 hours simulated reperfusion (21% oxygen and complete medium). Cooling to 33.5°C was initiated intra-OGD (1-hour post-OGD) and a control group was maintained at 37°C normoxia. Necrotic cell death was assessed by LDH release and apoptosis by caspase-3 activation. OGD-induced DAMPs (cold-inducible RNA-binding protein [CIRBP], heat-shock protein 70 [Hsp70] and high-mobility group box-1 [HMGB-1]) secretions were analyzed by Western Blotting. Oxidative stress mediator, iNOS, and antiapoptotic RBM3 and CIRBP gene expression were assessed by RT-qPCR.

Result: Prolonged exposure to OGD resulted in a transition from apoptotic programmed cell death to necrosis, which was attenuated by cooling as observed in changes in caspase-3 cleavage and LDH release. Interestingly, necrotic cell death correlated with increased iNOS expression and DAMPs release, resulting in a significant increase in proinflammatory IL-6 expression, which was also attenuated by cooling. Moreover, cooling induced gene expressions of cytoprotective RBM3 and CIRBP.

Conclusion: Our findings show that TTM not only protects the myocardium by attenuating cardiomyocyte death and oxidative stress response during OGD but also diminishes the sterile inflammatory response, a clinically relevant adverse event often observed after acute myocardial I/R injury. Moreover, cooling induces cytoprotective RBM3 and CIRBP gene expressions and is therefore, an effective therapy for both the acute ischemic injury and following reperfusion phase.

 

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