How does hypothermia protect cardiomyocytes during cardioplegic ischemia?

OBJECTIVE: Insufficient myocardial protection is still a considerable cause for in-hospital mortality in children. The purpose of our study was to investigate underlying the basic mechanisms of cardioplegic cardioprotection during hypothermic and normothermic ischemia in a cardiomyocyte cell culture model. METHODS: We cooled cardiomyocytes to 20°C for 20min; during this time, cardiac arrest was simulated by oxidative damage with 2mM H2O2 and cardioplegic solution, followed by rewarming to 37°C. Later on, we analyzed cardiomyocyte cell morphology (phase-contrast-microscopy), viability (trypan blue staining), inflammation (cyclooxygenase-2 (Cox-2) and phosphorylated-extracellular signal-regulated kinase (pERK) 1/2 expression in Western blot analysis), and expression of Akt survival protein (Western blot technique). RESULTS: Hypothermia increases cell survival of cardiomyocytes after cardioplegic ischemia, as demonstrated in significantly higher cell viability and less cell death in these cells compared with normothermic H2O2-damaged cardiomyocytes. As a possible underlying cellular mechanism, we found that, during cold cardioplegic ischemia, ERK 1/2 enzyme is less phosphorylated than under conditions of normothermic cardioplegic ischemia. This is in line with significantly diminished Cox-2 expression during cold cardioplegic ischemia. Moreover, hypothermic cardioplegia preserved cell survival by upregulation of Akt transcription factor in cardiomyocytes. CONCLUSION: In the present cell culture study, we clearly demonstrated that hypothermia exerts additional protection for cardiomyocytes during cardioplegic ischemia. The understanding of underlying basic mechanisms is evident to improve current techniques of myocardial protection.

Hypothermia protects H9c2 cardiomyocytes from H2O2 induced apoptosis.

The purpose of our study was to investigate underlying basic mechanisms of hypothermia-induced cardioprotection during oxidative stress in a cardiomyocyte cell culture model. For hypothermic treatment we cooled H9c2 cardiomyocytes to 20°C, maintained 20min at 20°C during which short-term oxidative damage was inflicted with 2mM H(2)O(2,) followed by rewarming to 37°C. Later on, we analyzed lactate dehydrogenase (LDH), caspase-3 cleavage, reactive oxygen species (ROS), mitochondrial activity, intracellular ATP production, cytoprotective signal molecules as well as DNA damage. Hypothermia decreased H(2)O(2) damage in cardiomyocytes as demonstrated in a lower LDH release, less caspase-3 cleavage and less M30 CytoDeath staining. After rewarming H(2)O(2) damaged cells demonstrated a significantly higher reduction rate of intracellular ROS compared to normothermic H(2)O(2) damaged cardiomyocytes(.) This was in line with a significantly greater mitochondrial dehydrogenase activity and higher intracellular ATP content in cooled and rewarmed cells. Moreover, hypothermia preserved cell viability by up-regulation of the anti-apoptotic protein Bcl-2 and a reduction of p53 phosphorylation. DNA damage, proven by PARP-1 cleavage and H2AX phosphorylation, was significantly reduced by hypothermia. In conclusion, we could demonstrate that hypothermia protects cardiomyocytes during oxidative stress by preventing apoptosis via inhibiting mitochondrial dysfunction and DNA damage.

Specific p38 inhibition in stimulated endothelial cells: a possible new anti-inflammatory strategy after hypothermia and rewarming.

To protect immature organ systems during corrective cardiac surgery, patients are cooled to a minimal temperature of 17 degrees C during cardiopulmonary bypass (CPB). However hypothermic CPB triggers the whole body inflammatory response and results in unwanted prolonged inflammation. The present study was designed to clarify the hypothermia and rewarming induced mechanisms and examine interventional pharmacological strategies that could prevent prolonged inflammation. Stimulated primary human umbilical vein endothelial cells (HUVECs) were exposed to a dynamic temperature protocol analogous to clinical settings. Furthermore endothelial cells were pretreated with methylprednisolone and/or tacrolimus as well as with MAPK inhibitors (SB203580, U0126 and SP600125). Cell viability, expression of IL-6 and ERK 1/2, p38 and SAPK/JNK were investigated. Stimulated endothelial cells secreted significantly higher IL-6 protein 2h after rewarming in comparison to normothermic control cells. Moreover, dynamic temperature changes lead to increased MAPK phosphorylation. Only the combined pre-treatment with MP and TAC served to inhibit the IL-6 secretion. As intracellular signalling pathway we could demonstrate that SB203580 as specific p38 inhibitor most effectively down regulated the unwanted IL-6 release after cooling and rewarming. Therefore inhibition of p38 or components of the p38 pathway could be a promising and selective antiinflammatory therapeutic target after hypothermic CPB.