Two-photon laser scanning microscopy for the investigation of cardiomyocyte death during ischemia/reperfusion.

线粒体通透性转换孔的开放导致线粒体膜电位(ΔΨ_m)的丧失，是心肌缺血/再灌注过程中细胞死亡的关键步骤，活性氧(ROS)和钙离子被认为是这一过程的两个主要调节因子。我们的目的是利用实时双光子成像系统在细胞水平上显示缺血/再灌流心脏中ROS、Ca^&lt；2和Gt；与ΔΨ_m丢失之间的时空关系。在缺血/再灌流过程中，ROS主要发生在缺血早期，细胞间比速率不同，不可逆的ΔΨ_m丢失取决于细胞内ROS水平，并有一个明确的界值。细胞内Ca~(2+)水平在缺血过程中伴随升高，一旦ROS水平达到阈值水平，细胞内Ca~(2+)水平即显著升高。缺血预适应减轻ROS和Ca~(2+)升高，保护…更多的抗ΔΨ_m损失。抗氧化剂可以抑制ROS的积累，但不能抑制Ca^&lt；2&gt；的增加，对ΔΨ_m的损失没有保护作用。因此，仅减少ROS积聚不足以防止心肌细胞死亡；可能需要同时抑制ROS和钙。MPTP开放是导致细胞死亡的最早事件，这一过程可能是心肌缺血/再灌注治疗干预的主要靶点。应用双光子激光扫描显微镜观察亲环素D(CypD)RNAi介导的基因沉默对心肌缺血再灌注损伤的保护作用。我们创造了一种携带短干扰RNA(SiRNA)的腺病毒，它可以灭活CypD。为了研究其在体内的作用，我们用双光子成像技术监测了大鼠心脏缺血/再灌流后ΔΨ_m的时空变化。成年大鼠直接心肌内注射腺病毒。注射后2~3d，以朗宁多夫模式灌流大鼠心脏，监测单个细胞ΔΨ_m水平。与非转导细胞相比，转导CypD-siRNA的细胞中ΔΨ_m的进行性丢失受到显著抑制。此外，CypD-siRNA的保护作用具有剂量依赖性。因此，这种新的双光子成像技术为深入了解心肌细胞在缺血/再灌注过程中死亡的机制，为以线粒体为靶点的心脏保护治疗提供了更深入的了解。较少

An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨ_m), is a crucial step in the cell death during myocardial ischemia/reperfusion ; reactive oxygen species (ROS) and Ca^<2+> are considered as the two major regulators of this step. We aimed to visualize the spatio-temporal relationship between ROS, Ca^<2+> and ΔΨ_m loss at cellular level in the perfused heart subjected to ischemia/reperfusion using a real-time two-photon imaging system. During ischemia/ reperfusion, ROS were mainly generated during early phase of ischemia with variable cell-to-cell specific rates and irreversible ΔΨ_m loss occurred in an all-or-none manner depending on cellular ROS level with a clear cut-off value. Cellular Ca^<2+> level concomitantly increased during ischemia, and once ROS level reached a threshold level, Ca^<2+> level increased robustly. Ischemic preconditioning attenuated both the increase of ROS and Ca^<2+> and protected a … More gainst ΔΨ_m loss. An anti-oxidant attenuated the ROS accumulation but not the Ca^<2+> increase ; it did not protect against ΔΨ_m loss. Thus, reducing only ROS accumulation does not suffice to prevent cardiomyocyte death ; suppression of both ROS and Ca^<2+> may be required.An opening of the MPTP is the earliest event that commits cells to death, and this process is potentially a prime target for therapeutic intervention against myocardial ischemia/reperfusion. We aimed to investigate the protective effects of RNAi-mediated gene silencing of cyclophilin D (CypD), one of the putative components of the MPTP, against myocardial ischemia/reperfusion by using the two-photon laser scanning microscopy. We created an adenovirus carrying short interfering RNA (siRNA) which inactivates CypD. To investigate the effects in vivo, we monitored the spatio-temporal changes of ΔΨ_m in perfused rat hearts subjected to ischemia/reperfusion, using the two-photon imaging. Adult rats received direct intramyocardial injections of the adenovirus. Two to three days after injection, the rat hearts were perfused in Langendorff mode, and ΔΨ_m levels of individual cells were monitored. The progressive loss of ΔΨ_m during ischemia/reperfusion was significantly suppressed in CypD-siRNA-transduced cells, compared with non-transduced cells. Furthermore, the protective effect of CypD-siRNA was dose-dependent.Thus, this novel two-photon imaging provides deeper insights into the mechanisms of cardiomyocyte death during ischemia/reperfusion, into cardioprotective therapy that targets mitochondria.. Less