School of Science and Technology, The Hong Kong Metropolitan University, Hong Kong, Hong Kong.
Department of Pharmacology and Pharmacy, University of Hong Kong, Hong Kong, Hong Kong.
Mol Biol Rep. 2022 Nov;49(11):11201-11208. doi: 10.1007/s11033-022-07902-3. Epub 2022 Sep 15.
Nucleoside transporters are crucial in regulating the functions of adenosine. This study investigated the contribution of equilibrative nucleoside transporter (ENT) type 4 to adenosine transport in cardiomyocytes under simulated ischemic conditions and whether the inhibition of ENT4 could protect cardiomyocytes against ischemia-reperfusion injury.
AC16 human cardiomyocytes were used to create a model to simulate ischemia/reperfusion injury. ENT4 activity was inhibited by decynium-22 or specific siRNA against ENT4. The protein expressions of nucleoside transporters were measured by western blot analysis. The transport activity was studied by [?H]adenosine uptake. The cell injury was studied by biochemical assays.
The [?H]adenosine uptake in AC16 cells was predominantly mediated by ENTs. ENT1 to ENT4 were present in AC16 cells and their protein expression levels were comparable in normal and ischemic conditions. Decynium-22 or siRNA against ENT4 did not affect the adenosine uptake in AC16 cells under normal conditions but could inhibit the adenosine uptake in AC16 cells by 28% under ischemic conditions. In addition, the cell viability and lactate dehydrogenase release of AC16 cells under ischemia conditions could be reduced by decynium-22 or siRNA against ENT4.
The cell culture model has suggested that ENT4 may play a role in adenosine transport in cardiomyocytes under ischemic conditions. Inhibition or downregulation of ENT4 may be a potential approach for cardioprotection but this notion should be further validated using animal model.
核苷转运体在调节腺苷的功能中起着至关重要的作用。本研究旨在探讨在模拟缺血条件下，核苷转运蛋白 4（ENT4）对心肌细胞中腺苷转运的贡献，以及抑制 ENT4 是否可以保护心肌细胞免受缺血再灌注损伤。
使用 AC16 人心肌细胞建立模拟缺血/再灌注损伤的模型。用 decynium-22 或针对 ENT4 的特异性 siRNA 抑制 ENT4 的活性。通过 Western blot 分析测定核苷转运蛋白的蛋白表达。通过[?H]腺苷摄取研究转运活性。通过生化测定研究细胞损伤。
AC16 细胞中的[?H]腺苷摄取主要由 ENTs 介导。ENT1 至 ENT4 存在于 AC16 细胞中，其蛋白表达水平在正常和缺血条件下相似。在正常条件下，decynium-22 或针对 ENT4 的 siRNA 并不影响 AC16 细胞中的腺苷摄取，但在缺血条件下可抑制 AC16 细胞中的腺苷摄取 28%。此外，decynium-22 或针对 ENT4 的 siRNA 可降低缺血条件下 AC16 细胞的细胞活力和乳酸脱氢酶释放。
细胞培养模型表明，在缺血条件下，ENT4 可能在心肌细胞中腺苷转运中发挥作用。抑制或下调 ENT4 可能是一种潜在的心脏保护方法，但这一观点需要进一步通过动物模型进行验证。
