Moderate therapeutic hypothermia induces multimodal protective effects in oxygen-glucose deprivation/reperfusion injured cardiomyocytes

Abstract

Objective: Therapeutic hypothermia has been shown to attenuate myocardial cell death due to ischemia/reperfusion injury. However, cellular mechanisms of cooling remain to be elucidated. Especially during reperfusion, mitochondrial dysfunction contributes to cell death by releasing apoptosis inductors. The aim of the present study was to investigate the effects of moderate therapeutic hypothermia (33.5°C) on mitochondrial mediated apoptosis in ischemia/reperfusion-injured cardiomyocytes.

Methods: Ischemic injury was simulated by oxygen-glucose deprivation for 6h in glucose/serum-free medium at 0.2% O₂ in mouse atrial HL-1 cardiomyocytes. Simulation of reperfusion was achieved by restoration of nutrients in complete supplemented medium and incubation at 21% O₂. Early application of therapeutic hypothermia, cooling during the oxygen-glucose deprivation phase, was initiated after 3h of oxygen-glucose deprivation and maintained for 24h. Mitochondrial membrane integrity was assessed by cytochrome c and AIF protein releases. Furthermore, mitochondria were stained with MitoTracker Red and intra-cellular cytochrome c localization was visualized by immunofluorescence staining. Moreover, anti-apoptotic Bcl-2 and Hsp70 as well as phagophore promoting LC3-II protein expressions were analyzed by Western-blot analysis.

Results: Therapeutic hypothermia initiated during oxygen-glucose deprivation significantly reduced mitochondrial release of cytochrome c and AIF in cardiomyocytes during reperfusion. Secondly, anti-apoptotic Bcl-2/Bax ratio and Hsp70 protein expressions were significantly upregulated due to hypothermia, indicating an inhibition of both caspase-dependent and -independent apoptosis. Furthermore, cardiomyocytes treated with therapeutic hypothermia showed increased LC3-II protein levels associated with the mitochondria during the first 3h of reperfusion, indicating the initiation of phagophores formation and sequestration of presumably damaged mitochondrion.

Conclusion: Early application of therapeutic hypothermia effectively inhibited cardiomyocyte cell death due to oxygen-glucose deprivation/reperfusion-induced injury via multiple pathways. As hypothermia preserved mitochondrial membrane integrity, which resulted in reduced cytochrome c and AIF releases, induction of both caspase-dependent and -independent apoptosis was minimized. Secondly, cooling attenuated intrinsic apoptosis via Hsp70 upregulation and increasing anti-apoptotic Bcl-2/Bax ratio. Moreover, therapeutic hypothermia promoted mitochondrial associated LC3-II during the early phase of reperfusion, possibly leading to the sequestration and degradation of damaged mitochondrion to attenuate the activation of cell death.

Keywords: Cardioprotection; Mitochondrial dysfunction; Reperfusion injury; Targeted temperature management; Therapeutic hypothermia.