Intracellular ceramide accumulation associated with apoptosis and changes in energy metabolism in primary cultured myocytes exposed to hypoxia and re-oxygenation

本研究旨在观察心肌细胞缺氧-复氧过程中的细胞内事件。制备培养的肌细胞并暴露于缺氧90分钟，随后再充氧60分钟。主要检测葡萄糖提取、乳酸产生、细胞凋亡、细胞外调节蛋白激酶（ERK）磷酸化。还测试了特异性髓鞘酶抑制剂D-609，以观察是否对暴露于缺氧后再氧合损伤的肌细胞具有保护作用。在缺氧期间，对照组的乳酸产生显著高于D-609组，而对照组的葡萄糖摄取显著低于D-609组。D 609处理明显降低了细胞凋亡，同时显著降低了ERK的磷酸化水平.然而，细胞坏死的标志物之一，脂肪酸结合蛋白FABP在组间没有差异，表明清楚检测到的细胞凋亡不同于坏死性细胞死亡。用Western免疫印迹法测定D 699组神经酰胺蛋白质合成显著低于对照组.这可能意味着D 609有助于减少缺氧-复氧暴露的心肌细胞中神经酰胺的积累。基于我们的研究结果，我们得出结论，神经酰胺的积累可能与葡萄糖代谢的损害，可能与脂肪酸代谢以及显著增加的凋亡细胞死亡。抑制神经酰胺在心肌细胞中的积累可能有助于减少细胞凋亡和细胞外调节蛋白激酶磷酸化以及改善葡萄糖代谢。

This study has been designed to see the intracellular event in myocytes during hypoxia-reoxygenation process. Cultured myocytes were prepared and exposed to hypoxia for 90 minutes followed by 60 minutes of re-oxygenation. Glucose extraction, lactate production, apoptotic cell death, extracellular regulated protein kinase (ERKs) phophorylation were mainly measured. Specific sphyngomyelinase inhibitor, D-609 was also tested to see if there was protective effects on myocytes exposed to hypoxia followed by re-oxygenation insult. During hypoxia lactate production was significantly higher in control than D-609 group, whereas glucose uptake was significantly reduced in control group compared to D609 group. Apoptotic cell death was clearly decreased with D 609 treatment with significantly reduced phosphorylation of ERKs. However one of the marker for cell necrosis, fatty acid binding protein FABP was not different between the group, suggesting that clearly detected apoptosis is different from necrotic cell death. Ceramide Protein synthesis measured by Western immunoblotting was significantly lower in D 699 group than control. This may imply that D609 is contributed to a reduction of ceramide accumulation in hypoxia-reoxygenation exposed myocytes. Based on our findings, we conclude that accumulation of ceramide might be associated with impairement of glucose metabolism, possibly fatty acid metabolism as well as significantly increased apoptotic cell death. Suppression of ceramide accumulation in myocytes might be contributed to reduced apoptotic cell death and extracellular regulated protein kinase phosphorylation as well as improved glucose metabolism.