How to give a cell a heart attack.
How to give a cell a heart attack.
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如何让细胞心脏病发作。
DOI：
10.1161/01.res.0000234908.21102.f9
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发表时间：
2006
影响因子：
20.1
通讯作者：
Stern,MichaelD
中科院分区：
文献类型：
--
作者：
Stern,MichaelD
心脏缺血是发达国家的头号杀手。它是一种复杂的病理，有无数的因素——电的、化学的、代谢的、机械的和免疫的——表现在从分子到整个生物体的尺度上。因此，尽管进行了数十年的研究，我们仍无法简洁地确定导致这种疾病的“核心”损伤，这并不奇怪。但是，最终，数百万人的命运取决于心肌细胞在血液供应被剥夺时会发生什么。它可以死于坏死、细胞凋亡或机械自我毁灭。决定它何时“死亡”并不总是那么容易。它可以失去它的收缩功能，它的松弛功能，和/或它的电功能。它可以冬眠，如果受到早期非致命性缺血的刺激，它可以在几分钟到几周的时间内“学会”保护自己。因此，在单细胞水平上研究心脏缺血是有意义的。事实证明，这是非常困难的。实验室中分离的心肌细胞通常浸泡在流动液体的海洋中。相比之下，即使在最好的条件下，肌细胞在其原生栖息地是与营养来源隔绝的。肌细胞表面的pO2通常不超过20torr。细胞外空间很小，细胞必须在其中处理其代谢废物。在心脏中，很容易使心肌细胞缺血。在培养皿中，这几乎是不可能的。当在膜片钳装置上研究心肌细胞时，习惯上是用氧气使灌注溶液起泡。如果你用氮代替，你不会看到生理上的变化！更糟糕的是，目前测量肌细胞电活动的方法需要用一根长微管从上面自由地接触到它。大约18年前，霍华德·西尔弗曼（Howard Silverman）和我为这个问题开发了一个粗略的解决方案。通过在锥形井中建立一种比空气重的惰性气体（氩气是唯一可行的选择）向上层流，可以排除井底培养皿中细胞中的氧气，即使允许上面的仪器开放。实际问题并没有就此结束。在培养皿中灌注缺氧缓冲液是必要的。因为所有的塑料都能渗透氧气（最好的是聚氯乙烯-萨兰，其次是聚氯乙烯；Tygon，紧随其后），所以盘子和系统中所有接触液体的部件都必须用玻璃或不锈钢制成。没有泵是不渗透氧气的，所以液体必须通过气体压力来移动。我们找不到足够密封的液体阀门，所以我们不得不临时制作了一个粗糙的浸入式阀门，由一根锥形不锈钢丝堵住不锈钢管组成，通过手动将金属丝从橡胶塞上的一个孔中上下拉出来操作。尽管做了这些努力，我们仍然不时地发现，微量的氧气在正压下泄漏到铜氩气管道中，这就需要重建所有的金属-金属连接点，以及它们的可压缩铜垫圈，尽管泄漏很少能直接定位。由于所有这些原因，这项技术很麻烦，只有少数勇敢的研究者采用了它。但它最大的问题是它是缺氧的模型，而不是缺血的模型。代谢物的积累不能在5毫米宽，1毫米深的培养皿的“巨大”体积中模拟。虽然可以通过同时进行溶液和气体转换（其尴尬可想而知）来研究外部施加的二氧化碳、酸中毒和钾含量升高的影响，但这仍然不是“真正的”缺血。在这一期的《循环研究》中，Ganitkevich等人……
Cardiac ischemia is the leading killer in the developed world. It is a complex pathology, with myriad factors—electrical, chemical, metabolic, mechanical, and immunological—manifesting on scales ranging from the molecular to the whole organism. So it is not surprising that, despite decades of study, we cannot succinctly identify “the” injury that is central to this disorder. But, ultimately, the fate of millions of people comes down to what happens to a cardiac myocyte when it is deprived of its blood supply. It can die—by necrosis, apoptosis, or mechanical self-destruction. It isn’t always easy to decide when it is “dead.” It can lose its contractile function, its relaxation function, and/or its electrical function. It can hibernate and, if provoked by earlier nonlethal episodes of ischemia, it can “learn” to protect itself on time scales from minutes to weeks. It would make sense, then, to study cardiac ischemia at the single-cell level. This turns out to be surprisingly difficult to do. The isolated cardiac myocyte in the laboratory is normally bathed in an ocean of moving fluid. In contrast, even under the best of conditions, the myocyte in its native habitat is secluded from its sources of nutrition. The pO2 at the myocyte surface is normally no more than 20 torr. The extracellular space, in which the cell must dispose of its metabolic waste, is tiny. In the heart, it is very easy to make a myocyte ischemic. In a petri dish, it is almost impossible. When studying cardiac myocytes on a patch-clamp apparatus, it is customary to bubble the perfusing solution with oxygen. If you use nitrogen instead, you will see no change in physiology! To make matters worse, present-day methods of measuring the electrical activity of a myocyte require that it be freely accessible from above by a long micropipette. Some 18 years ago, Howard Silverman and I developed a crude fix for this problem. 1 By establishing an upward laminar flow of a heavier-than-air inert gas (argon is the only practical choice) in a conical well, it was possible to exclude oxygen from cells in a dish at the bottom of the well, even while allowing open access above for instrumentation. The practical problems didn’t end there. It was necessary to perfuse the dish with anoxic buffer. Because all plastics are permeable to oxygen (the best is polyvinylidene chloride—Saran, with PVC; Tygon, a close second), it was necessary to make the dish and all parts of the system that contact the liquid out of glass or stainless steel. No pumps are impermeable to oxygen, so the liquid had to be moved by gas pressure. We were unable to find any liquid valves that were sufficiently air-tight, so we were forced to improvise a crude immersed valve consisting of a tapered stainless wire occluding stainless tubing, operated by pulling the wire manually up and down through a hole in a rubber stopper. Despite these efforts, we still found, from time to time, that traces of oxygen were leaking into the copper argon gas line against positive pressure, requiring all the metal–metal junctions, with their compressible copper gaskets, to be rebuilt, even though the leak could seldom be directly located. For all these reasons, the technique was cumbersome, and only a few other intrepid investigators adopted it. But its greatest problem was that it was a model of hypoxia, not ischemia. The accumulation of metabolites could not be emulated in the “vast” volume of the 5-mm-wide, 1-mm-deep dish. While it was possible to investigate the effects of externally applied CO2, acidosis and elevated potassium—by means of simultaneous solution and gas switching whose awkwardness can be imagined—this was still not “genuine” ischemia.In this issue of Circulation Research, Ganitkevich et …