Detection of mitochondrial depolarization/recovery during ischaemia--reperfusion using spectral properties of confocally recorded TMRM fluorescence.
Detection of mitochondrial depolarization/recovery during ischaemia--reperfusion using spectral properties of confocally recorded TMRM fluorescence.
复制标题
使用共聚焦记录的 TMRM 荧光的光谱特性检测缺血再灌注期间线粒体去极化/恢复。
DOI：
10.1113/jphysiol.2012.248153
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Zaitsev,AlexeyV
中科院分区：
文献类型：
--
作者：
Venable,PaulW;Taylor,TysonG;Sciuto,KatieJ;Zhao,Jerry;Shibayama,Junko;Warren,Mark;Spitzer,KennethW;Zaitsev,AlexeyV
要点•心肌缺血期间的线粒体内膜电位（ΔΨm）崩溃是决定缺血发作在缺血后心律失常和细胞存活方面的生理后果的关键事件之一。•缺血期间ΔΨm崩溃的时间和模式仍然存在争议，部分原因是难以解释常用于评估ΔΨmin细胞和多细胞实验模型的电位计阳离子探针的荧光。•本手稿提出一种基于心肌细胞中线粒体的规则排列来监测 ΔΨmin 全心的新方法，从而允许使用荧光光谱分析检测 ΔΨm 塌陷。•所提出的方法将有助于确定线粒体功能在急性心血管疾病中的作用，例如急性心肌梗死或心脏骤停。摘要无流量缺血再灌注 (I–R) 期间线粒体电位 (ΔΨm) 去极化的时间和模式仍然存在争议，至少在部分原因是难以解释 ΔΨm 敏感染料（例如 TMRM）的荧光变化。本研究的目的是开发一种新方法，基于心室心肌细胞线粒体包装的空间周期性来解释 I-R 期间的共焦 TMRM 信号。使用共焦显微镜或光学测绘系统记录固定有肌球蛋白抑制剂的 Langendorff 灌注兔心脏的 TMRM 荧光 (FTMRM)。在正常条件下、使用质子载体 FCCP 进行线粒体解偶联期间以及 I-R 期间对心脏进行了研究。 FTMRM 的共焦图像经过空间傅里叶变换，显示出空间频率为 ∼2 μm−1 的不同峰值。随着 FCCP 浓度 (0.3–20 μm) 的增加，峰下面积 (MPA) 逐渐减小，在 5–20 μmFCCP 下变得不可检测。在缺血期间，在缺血 27 至 69 分钟之间观察到 MPA 急剧下降，达到与 5-20 μmFCCP 诱导的低/不可检测水平相当的水平。再灌注后，观察到 MPA 异质恢复，但未观察到 MPA 新下降。在 5 μmFCCP 存在的情况下，共焦成像和宽视场成像均显示空间平均 FTMRMin 持续下降，但在 I-R 过程中该参数没有持续变化。我们得出的结论是，源自共焦图像的 MPA 提供了 I-R 期间显着线粒体去极化或恢复的敏感且特异的指标。相反，空间平均 FTMR 并不是 I-R 期间 ΔΨm 变化的可靠指标。
Key points•Mitochondrial inner membrane potential (ΔΨm) collapse during myocardial ischaemia is one of the key events determining the physiological consequences of ischaemic attack in terms of post‐ischaemic arrhythmias and cell survival.•Timing and pattern of ΔΨmcollapse during ischaemia remain controversial, in part due to difficulties in interpreting the fluorescence of potentiometric cationic probes commonly used for assessment of ΔΨmin cellular and multicellular experimental models.•This manuscript presents a new method for monitoring ΔΨmin whole hearts based on the regular arrangement of mitochondria in cardiac myocytes, thus permitting detection of ΔΨmcollapse using spectral analysis of fluorescence.•The proposed method will help to ascertain the role of mitochondrial function in acute cardiovascular conditions, such as acute myocardial infarction or sudden cardiac arrest.AbstractTiming and pattern of mitochondrial potential (ΔΨm) depolarization during no‐flow ischaemia–reperfusion (I–R) remain controversial, at least in part due to difficulties in interpreting the changes in the fluorescence of ΔΨm‐sensitive dyes such as TMRM. The objective of this study was to develop a new approach for interpreting confocal TMRM signals during I–R based on spatial periodicity of mitochondrial packaging in ventricular cardiomyocytes. TMRM fluorescence (FTMRM) was recorded from Langendorff‐perfused rabbit hearts immobilized with blebbistatin using either a confocal microscope or an optical mapping system. The hearts were studied under normal conditions, during mitochondrial uncoupling using the protonophore FCCP, and during I–R. Confocal images ofFTMRMwere subjected to spatial Fourier transform which revealed distinct peaks at a spatial frequency of ∼2 μm−1. The area under the peak (MPA) progressively decreased upon application of increasing concentrations of FCCP (0.3–20 μm), becoming undetectable at 5–20 μmFCCP. During ischaemia, a dramatic decrease in MPA, reaching the low/undetectable level comparable to that induced by 5–20 μmFCCP, was observed between 27 and 69 min of ischaemia. Upon reperfusion, a heterogeneous MPA recovery was observed, but not ade novoMPA decrease. Both confocal and wide‐field imaging registered a consistent decrease in spatially averagedFTMRMin the presence of 5 μmFCCP, but no consistent change in this parameter during I–R. We conclude that MPA derived from confocal images provides a sensitive and specific indicator of significant mitochondrial depolarization or recovery during I–R. In contrast, spatially averagedFTMRMis not a reliable indicator of ΔΨmchanges during I–R.