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在缺氧再氧化过程中的细胞内事件。制备培养的肌细胞，缺氧90分钟后再充氧60分钟。葡萄糖提取、乳酸生成、凋亡细胞死亡、细胞外调节蛋白激酶（ERKs）磷酸化主要测定。还测试了特异性脊髓鞘酶抑制剂D-609是否对暴露于缺氧后再氧化损伤的肌细胞有保护作用。缺氧时，对照组乳酸产量显著高于D-609组，而葡萄糖摄取显著低于D-609组。d609处理明显降低了凋亡细胞的死亡率，并显著降低了ERKs的磷酸化水平。而细胞坏死标志物之一脂肪酸结合蛋白FABP在两组间无明显差异，提示明显检测到的凋亡不同于坏死细胞死亡。Western免疫印迹法测定d699组神经酰胺蛋白合成显著低于对照组。这可能意味着D609有助于减少缺氧再氧化暴露的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有关，可能与脂肪酸代谢以及凋亡细胞死亡的显著增加有关。抑制神经酰胺在肌细胞中的积累可能有助于减少凋亡细胞死亡和细胞外调节的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.