Section of Animal and Human Physiology, Faculty of Biology, School of Science, National and Kapodistrian University of Athens, University Campus, Ilissia, Athens, Greece.
Eur Rev Med Pharmacol Sci. 2021 Jan;25(1):313-325. doi: 10.26355/eurrev_202101_24397.
Multiple pathophysiological conditions are associated with disturbance of myocardial osmotic equilibrium, exerting detrimental effects on cardiac performance. Cardiac myocytes may encounter hyperosmotic stress during hyperglycemia, ischemia/reperfusion injury, myocardial infarction, diabetes mellitus, severe dehydration, hypoxia or heat stress. Aquaporins (AQPs) constitute transmembrane channels that facilitate water transport in response to osmotic gradients. Therefore, the present study aimed at probing into AQPs mode of response and potential role as effector molecules and sensors, under hyperosmotic stress.
H9c2 cardiac myoblasts were left untreated (control) or were exposed to 0.5 M sorbitol so as to induce hyperosmotic stress conditions. After the experimental treatments, MTT assay was performed to assess cell viability. Endogenous mRNA levels of AQP1 and AQP7 were assessed by ratiometric RT-PCR. Their subcellular localization pattern was revealed by immunofluorescence microscopy. Protein levels of AQP1 and AQP7, as well as of apoptotic markers (cleaved caspase-3 and PARP), were detected by immunoblot analysis.
Hyperosmotic stress (0.5 M sorbitol) induced a time-dependent upregulation of AQP7 (but not of AQP1) mRNA in H9c2 cells. Of note, biochemical and immunocytochemical analyses revealed the increased membrane-associated protein expression of AQP1, under these conditions, while AQP7 respective levels remained unchanged. Interestingly, inhibition of AQP1 by HgCl2, aggravated the sorbitol-induced apoptosis in H9c2 cells, as evidenced by chromatin condensation and fragmentation of caspase-3 and PARP.
AQP1 and AQP7 are differentially regulated under hyperosmotic stress conditions in H9c2 cells. AQP1, acting as an osmotic stress sensor and response factor, exerts a beneficial effect against the sorbitol-induced apoptosis, potentially favoring preservation of cardiac function.
多种病理生理状况与心肌渗透平衡的紊乱有关，对心脏功能产生有害影响。心肌细胞在高血糖、缺血/再灌注损伤、心肌梗死、糖尿病、严重脱水、缺氧或热应激时可能会遇到高渗应激。水通道蛋白（AQP）构成跨膜通道，可响应渗透梯度促进水的运输。因此，本研究旨在探讨 AQP 在高渗应激下的反应模式和作为效应分子和传感器的潜在作用。
未处理（对照）或暴露于 0.5 M 山梨醇的 H9c2 心肌细胞用于诱导高渗应激条件。实验处理后，通过 MTT 测定法评估细胞活力。通过比率 RT-PCR 评估 AQP1 和 AQP7 的内源性 mRNA 水平。通过免疫荧光显微镜揭示其亚细胞定位模式。通过免疫印迹分析检测 AQP1 和 AQP7 以及凋亡标志物（裂解的 caspase-3 和 PARP）的蛋白水平。
高渗应激（0.5 M 山梨醇）诱导 H9c2 细胞中 AQP7（而非 AQP1）mRNA 的时间依赖性上调。值得注意的是，生化和免疫细胞化学分析显示，在这些条件下 AQP1 的膜相关蛋白表达增加，而 AQP7 的相应水平保持不变。有趣的是，HgCl2 抑制 AQP1 加重了山梨醇诱导的 H9c2 细胞凋亡，这表现在 caspase-3 和 PARP 的染色质浓缩和片段化。
AQP1 和 AQP7 在 H9c2 细胞的高渗应激条件下差异调节。AQP1 作为渗透应激传感器和反应因子，对山梨醇诱导的细胞凋亡具有有益作用，可能有利于心脏功能的维持。
