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

细胞内神经酰胺积累与缺氧和复氧原代培养心肌细胞凋亡和能量代谢变化相关

• 批准号: 17591461
• 负责人: TAKEUCHI Koh
• 金额: $ 2.05万
• 依托单位: The University of Tokyo (2006)National Research Institute for Child Health and Development (2005)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591461/
• 关键词: Ischemic injury; Re-oxygenation injury; glucose metabolism; fatty acid metabolism; ceramide; apoptosis; 心筋細胞; 虚血-再酸素化

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.

本研究旨在观察心肌细胞在缺氧-复氧过程中的细胞内事件。将培养的心肌细胞置于低氧90分钟后复氧60分钟。主要检测葡萄糖提取、乳酸生成、细胞凋亡、细胞外调节蛋白激酶(ERKs)磷酸化。同时检测特殊的鞘磷脂酶抑制剂D-609对缺氧后复氧损伤的心肌细胞是否有保护作用。低氧时，对照组乳酸产量显著高于D-609组，而葡萄糖摄取量显著低于D609组。D609处理组细胞凋亡率明显降低，ERKs的磷酸化程度显著降低。但作为细胞坏死标志物之一的脂肪酸结合蛋白FABP在组间无差异，提示明确检测到的细胞凋亡不同于坏死性细胞死亡。免疫印迹法检测D699组神经酰胺蛋白合成明显低于对照组。这可能暗示D609可能与减少缺氧-复氧暴露的心肌细胞神经酰胺蓄积有关。根据我们的研究结果，我们得出结论，神经酰胺的积聚可能与葡萄糖代谢受损有关，可能与脂肪酸代谢有关，也可能与显著增加细胞凋亡有关。抑制神经酰胺在心肌细胞中的积聚可能与减少细胞凋亡性死亡和细胞外调节蛋白激酶磷酸化以及改善糖代谢有关。

项目成果

Surgical Outcome of Heterotaxy Syndrome in a Single Institution

单一机构异位综合征的手术结果

• 发表时间: 2006
• 期刊: Asian Cardiovasc Thorac Ann 14
• 作者: Koh Takeuchi;et al.
• 通讯作者: et al.

Fate of Equine Pericardial Roll Conduit for Rastelli Operation during Long-term Follow-up

长期随访期间用于 Rastelli 手术的马心包滚动导管的命运

• 发表时间: 2007
• 期刊: Congenital Heart Diseases 2
• 作者: Koh Takeuchi;et al.
• 通讯作者: et al.

Evaluation of Valved Saphenous Vein Homograft as Right Ventricle-Pulmonary Artery Conduit in Modified Stage I Norwood Operation.

改良诺伍德第一阶段手术中带瓣隐静脉同种移植物作为右心室-肺动脉导管的评估。

• 发表时间: 2006
• 期刊: Interact CardioVasc Thorac Surg 5
• 作者: Koh Takeuchi;et al.
• 通讯作者: et al.

Surgical Outcome of Heterotaxy Syndrome in a Single Institution.

单一机构异位综合征的手术结果。

• 发表时间: 2006
• 期刊: Asian Cardiovasc Thorac Ann 14
• 作者: Koh Takeuchi;et al.
• 通讯作者: et al.