Enzyme-dependent fluorescence recovery of NADH after photobleaching to assess dehydrogenase activity of isolated perfused hearts.
Enzyme-dependent fluorescence recovery of NADH after photobleaching to assess dehydrogenase activity of isolated perfused hearts.
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DOI：
10.1038/srep45744
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发表时间：
2017-03-31
影响因子：
4.6
通讯作者：
Kay MW
中科院分区：
文献类型：
--
作者：
Moreno A;Kuzmiak-Glancy S;Jaimes R Rd;Kay MW
通过脱氢酶还原NAD+以形成NADH是细胞代谢的关键组分。在细胞制剂和分离的线粒体悬浮液中，NADH的光漂白后的酶依赖性荧光恢复（ED-FRAP）已被证明是用于测量NADH产生速率以评估脱氢酶活性的有效方法。我们的目的是证明如何脱氢酶活性可以评估内的心肌灌注心脏使用NADH ED-FRAP。这是使用高强度UV脉冲的组合来光漂白心外膜NADH来实现的。然后使用低强度UV照射对心外膜NADH荧光的补充进行成像。优化了NADH ED-FRAP参数，以在6毫秒的脉冲宽度和50%的占空比下提供23.8 mJ的光漂白光能。这些参数提供了在多种代谢扰动过程中NADH产生速率的可重复测量，包括灌注液温度、机电解偶联和急性缺血/再灌注损伤的变化。在能量需求较高或未受影响的每个扰动中，NADH产生速率显著较高。我们还发现，在全脑缺血再灌注10分钟后，NADH的产生率仍然显着受损。总之，我们的研究结果表明，心肌NADH ED-FRAP是一个有用的光学非破坏性的方法来评估脱氢酶活性。
Reduction of NAD+ by dehydrogenase enzymes to form NADH is a key component of cellular metabolism. In cellular preparations and isolated mitochondria suspensions, enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH has been shown to be an effective approach for measuring the rate of NADH production to assess dehydrogenase enzyme activity. Our objective was to demonstrate how dehydrogenase activity could be assessed within the myocardium of perfused hearts using NADH ED-FRAP. This was accomplished using a combination of high intensity UV pulses to photobleach epicardial NADH. Replenishment of epicardial NADH fluorescence was then imaged using low intensity UV illumination. NADH ED-FRAP parameters were optimized to deliver 23.8 mJ of photobleaching light energy at a pulse width of 6 msec and a duty cycle of 50%. These parameters provided repeatable measurements of NADH production rate during multiple metabolic perturbations, including changes in perfusate temperature, electromechanical uncoupling, and acute ischemia/reperfusion injury. NADH production rate was significantly higher in every perturbation where the energy demand was either higher or uncompromised. We also found that NADH production rate remained significantly impaired after 10 min of reperfusion after global ischemia. Overall, our results indicate that myocardial NADH ED-FRAP is a useful optical non-destructive approach for assessing dehydrogenase activity.