Department of Cardiology and Angiology, University Hospital Giessen and Marburg Klinikstr. 33, 35392 Giessen, Germany.
J Mol Cell Cardiol. 2013 May;58:209-16. doi: 10.1016/j.yjmcc.2013.01.003. Epub 2013 Jan 15.
Experimental and clinical studies demonstrated that postconditioning confers protection against myocardial ischemia/reperfusion injury. However the underlying cellular mechanisms responsible for the beneficial effect of postconditioning are still poorly understood. The aim of the present study was to examine the role of cytosolic and mitochondrial Ca(2+)-handling. For this purpose adult rat cardiomyocytes were subjected to simulated in vitro ischemia (glucose-free hypoxia at pH6.4) followed by simulated reperfusion with a normoxic buffer (pH7.4; 2.5 mmol/L glucose). Postconditioning, i.e., 2 repetitive cycles of normoxic (5s) and hypoxic (2.5 min) superfusion, was applied during the first 5 min of reoxygenation. Mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial Ca(2+) concentrations, cytosolic pH and necrosis were analysed applying JC-1, fura-2, fura-2/manganese, BCECF and propidium iodide, respectively. Mitochondrial permeability transition pore (MPTP) opening was detected by calcein release. Hypoxic treatment led to a reduction of ΔΨm, an increase in cytosolic and mitochondrial Ca(2+) concentration, and acidification of cardiomyocytes. During the first minutes of reoxygenation, ΔΨm transiently recovered, but irreversibly collapsed after 7 min of reoxygenation, which was accompanied by MPTP opening. Simultaneously, mitochondrial Ca(2+) increased during reperfusion and cardiomyocytes developed spontaneous cytosolic Ca(2+) oscillations and severe contracture followed by necrosis after 25 min of reoxygenation. In postconditioned cells, the collapse in ΔΨm as well as the leak of calcein, the increase in mitochondrial Ca(2+), cytosolic Ca(2+) oscillations, contracture and necrosis were significantly reduced. Furthermore postconditioning delayed cardiomyocyte pH recovery. Postconditioning by hypoxia/reoxygenation was as protective as treatment with cyclosporine A. Combining cyclosporine A and postconditioning had no additive effect. The data of the present study demonstrate that postconditioning by hypoxia/reoxygenation prevents reperfusion injury by limiting mitochondrial Ca(2+) load and thus opening of the MPTP in isolated cardiomyocytes. These effects seem to be supported by postconditioning-induced delay in pH recovery and suppression of Ca(2+) oscillations.
实验和临床研究表明，后处理对心肌缺血/再灌注损伤具有保护作用。然而，导致后处理有益效果的潜在细胞机制仍知之甚少。本研究旨在研究细胞质和线粒体 Ca(2+)处理的作用。为此，将成年大鼠心肌细胞进行体外模拟缺血（pH6.4 时无葡萄糖缺氧），然后用正常氧缓冲液（pH7.4；2.5mmol/L 葡萄糖）进行模拟再灌注。在后处理中，即在复氧的前 5 分钟内，应用 2 个重复的正常氧（5s）和低氧（2.5 分钟）灌注循环。线粒体膜电位（ΔΨm）、细胞质和线粒体 Ca(2+)浓度、细胞质 pH 和坏死分别用 JC-1、fura-2、fura-2/锰、BCECF 和碘化丙啶进行分析。通过钙黄绿素释放检测线粒体通透性转换孔（MPTP）的开放。低氧处理导致 ΔΨm 降低，细胞质和线粒体 Ca(2+)浓度增加，以及心肌细胞酸化。在复氧的最初几分钟内，ΔΨm 短暂恢复，但在复氧 7 分钟后不可逆地崩溃，这伴随着 MPTP 的开放。同时，线粒体 Ca(2+)在再灌注过程中增加，并且心肌细胞在再灌注 25 分钟后发展出自发性细胞质 Ca(2+)振荡和严重收缩，随后坏死。在后处理细胞中，ΔΨm 的崩溃以及钙黄绿素的渗漏、线粒体 Ca(2+)的增加、细胞质 Ca(2+)振荡、收缩和坏死均显著减少。此外，后处理延迟了心肌细胞 pH 的恢复。低氧/复氧的后处理与环孢素 A 的治疗一样具有保护作用。环孢素 A 与后处理联合使用没有叠加作用。本研究的数据表明，低氧/复氧的后处理通过限制线粒体 Ca(2+)负荷从而防止 MPTP 的开放，从而防止分离的心肌细胞再灌注损伤。这些作用似乎受到后处理诱导的 pH 恢复延迟和 Ca(2+)振荡抑制的支持。
