Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah, United States of America.
Department of Bioengineering, University of Utah, Salt Lake City, Utah, United States of America.
PLoS One. 2019 Feb 7;14(2):e0212076. doi: 10.1371/journal.pone.0212076. eCollection 2019.
While an ischemic insult poses a lethal danger to myocardial cells, a significant proportion of cardiac myocytes remain viable throughout the ischemic episode and die, paradoxically, only after the blood flow is reinstated. Despite decades of research, the actual chronology of critical events leading to cardiomyocyte death during the reperfusion phase remains poorly understood. Arguably, identification of the pivotal event in this setting is necessary to design effective strategies aimed at salvaging the myocardium after an ischemic attack. Here we used neonatal rat ventricular myocytes (NRVMs) subjected to 20-30 min of simulated ischemia followed by 1 hour of "reperfusion". Using different combinations of spectrally-compatible fluorescent indicators, we analyzed the relative timing of the following events: (1) abnormal increase in cytoplasmic [Ca2+] (TCaCy); (2) abnormal increase in mitochondrial [Ca2+] (TCaMi); (3) loss of mitochondrial inner membrane potential (ΔΨm) indicating mitochondrial permeability transitions (TMPT); (4) sacrolemmal permeabilization (SP) to the normally impermeable small fluorophore TO-PRO3 (TSP). In additional experiments we also analyzed the timing of abnormal uptake of Zn2+ into the cytoplasm (TZnCy) relative to TCaCy and TSP. We focused on those NRVMs which survived anoxia, as evidenced by at least 50% recovery of ΔΨm and the absence of detectable SP. In these cells, we found a consistent sequence of critical events in the order, from first to last, of TCaCy, TCaMi, TMPT, TSP. After detecting TCaCy and TCaMi, abrupt switches between 1.1 mM and nominally zero [Ca2+] in the perfusate quickly propagated to the cytoplasmic and mitochondrial [Ca2+]. Depletion of the sarcoplasmic reticulum with ryanodine (5 μM)/thapsigargin (1 μM) accelerated all events without changing their order. In the presence of ZnCl2 (10-30 μM) in the perfusate we found a consistent timing sequence TCaCy < TZn ≤ TSP. In some cells ZnCl2 interfered with Ca2+ uptake, causing "steps" or "gaps" in the [Ca2+]Cy curve, a phenomenon never observed in the absence of ZnCl2. Together, these findings suggest an evolving permeabilization of NRVM's sarcolemma during reoxygenation, in which the expansion of the pore size determines the timing of critical events, including TMPT.
虽然缺血性损伤对心肌细胞构成致命威胁，但在整个缺血期间，相当一部分心肌细胞仍然存活，只是在血流恢复后才死亡，这令人费解。尽管经过了几十年的研究，但在再灌注阶段导致心肌细胞死亡的关键事件的实际时间顺序仍知之甚少。可以说，在这种情况下，确定关键事件对于设计旨在在缺血攻击后挽救心肌的有效策略是必要的。在这里，我们使用 20-30 分钟模拟缺血后再灌注 1 小时的新生大鼠心室肌细胞（NRVM）。使用不同组合的光谱兼容荧光指示剂，我们分析了以下事件的相对时间顺序：（1）细胞质[Ca2+]（TCaCy）异常增加；（2）线粒体[Ca2+]（TCaMi）异常增加；（3）线粒体内膜电位（ΔΨm）丧失，表明线粒体通透性转换（TMPT）；（4）正常情况下不可渗透的小分子荧光探针 TO-PRO3（TSP）的质膜通透性。在额外的实验中，我们还分析了 Zn2+异常摄取到细胞质（TZnCy）相对于 TCaCy 和 TSP 的时间。我们专注于那些在缺氧条件下存活的 NRVM，证据是 ΔΨm 至少恢复了 50%，并且没有检测到可检测的 SP。在这些细胞中，我们发现了一个一致的关键事件序列，从 TCaCy 到 TCaMi 再到 TMPT 再到 TSP。在检测到 TCaCy 和 TCaMi 之后，灌流液中 1.1 mM 到名义上的零 [Ca2+] 之间的突然切换迅速传播到细胞质和线粒体 [Ca2+]。用 Ryanodine（5 μM）/ Thapsigargin（1 μM）耗竭肌浆网会加速所有事件的发生，而不会改变其顺序。在灌流液中存在 ZnCl2（10-30 μM）的情况下，我们发现了一个一致的时间顺序 TCaCy < TZn ≤ TSP。在一些细胞中，ZnCl2 干扰了 Ca2+摄取，导致 [Ca2+]Cy 曲线出现“台阶”或“间隙”，这在没有 ZnCl2 的情况下从未观察到。综上所述，这些发现表明，在再氧合过程中，NRVM 的质膜会逐渐发生通透性，其中孔大小的扩展决定了关键事件的时间顺序，包括 TMPT。
