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.

No conflict of interest has been declared by the author(s).