Department of Biomedical Engineering & Healthcare Industry Research Institute, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea.
Micron. 2013 Jan;44:167-73. doi: 10.1016/j.micron.2012.06.003. Epub 2012 Jun 13.
Mitochondrial dysfunction plays a central role in mediating both the necrotic and apoptotic components of reperfusion injury. Because mitochondrial swelling is one of the most important indicators of the beginning of mitochondrial permeability transition, quantification of morphological changes in mitochondria would be useful in evaluating the degree of IR injury, as well as the protective effects of various therapies. In this study, we characterized the morphological changes in heart mitochondria caused by the duration and severity of ischemia utilizing particle shape analysis on atomic force microscopy (AFM) topographic images. We also simultaneously investigated the nano-mechanical changes in rat heart mitochondria by injury using force-distance curve measurements. Rats were randomly divided into 3 groups: control group (n=3), myocardial ischemia without reperfusion (PI group, n=3), and myocardial ischemia with reperfusion (IR group, n=4). Normal mitochondria appeared ellipsoidal with a mean area of 3551±1559 nm(2) and mean perimeter of 217.54±52.09 nm (n=60). The mean area and perimeter of mitochondria in the IR groups increased to 28,181±21,248 nm(2) and 595.74±234.29 nm (n=40, p<0.0001 vs. control group, respectively), maintaining oval in shape. But, in the PI group, all parameters showed significant differences compared to parameters of the control group (n=35, p<0.0001). In particular, the mean axial ratio and roundness were significantly different from those in the IR group. Mitochondria in the PI group looked more spherical than those of control and IR groups. Adhesion force is the force before the last event on the retraction half of force-distance curve measurements, corresponding to the point where the tip and the surface loose contact. The adhesion forces of heart mitochondria in the IR and PI groups significantly decreased to 19.56±1.08 nN (n=30, p<0.0001) and 18.65±3.18 nN (n=30, p<0.0001), compared to normal mitochondria which had an adhesion force of 27.64±0.88 nN (n=30). Adhesion force is governed by the attractive portion of the interacting forces between the surface atoms of the contacts. From the morphological and nano-mechanical changes in heart mitochondria, we suggested that the outer membranes of mitochondria were broken by myocardial ischemic injury before they became swollen, and the swelling might be correlated with the ischemic injury. We inferred that the breakage of membranes leads to uptake of water and matrix swelling. As a result, shape measurement parameters for the quantitative analysis of mitochondrial swelling could be very effective for evaluating the myocardial injury.
线粒体功能障碍在介导再灌注损伤的坏死和凋亡成分中起着核心作用。由于线粒体肿胀是线粒体通透性转换开始的最重要指标之一，因此量化线粒体的形态变化将有助于评估 IR 损伤的程度以及各种治疗方法的保护作用。在这项研究中，我们利用原子力显微镜（AFM）形貌图像上的颗粒形状分析来描述心肌缺血持续时间和严重程度引起的心脏线粒体的形态变化。我们还通过损伤力-距离曲线测量同时研究了大鼠心脏线粒体的纳米力学变化。大鼠随机分为 3 组：对照组（n=3）、无再灌注心肌缺血组（PI 组，n=3）和再灌注心肌缺血组（IR 组，n=4）。正常线粒体呈椭圆形，平均面积为 3551±1559nm2，平均周长为 217.54±52.09nm（n=60）。IR 组的线粒体平均面积和周长分别增加到 28,181±21,248nm2 和 595.74±234.29nm（n=40，分别与对照组相比，p<0.0001），仍呈椭圆形。但是，PI 组的所有参数与对照组的参数相比均有显著差异（n=35，p<0.0001）。特别是，平均轴向比和圆形度与 IR 组有显著差异。PI 组的线粒体看起来比对照组和 IR 组的更球形。粘附力是力-距离曲线测量回缩半程上最后一个事件之前的力，对应于尖端和表面失去接触的点。IR 组和 PI 组心脏线粒体的粘附力分别显著降低至 19.56±1.08nN（n=30，p<0.0001）和 18.65±3.18nN（n=30，p<0.0001），而正常线粒体的粘附力为 27.64±0.88nN（n=30）。粘附力由接触表面原子之间相互作用力的吸引力部分决定。从心脏线粒体的形态和纳米力学变化来看，我们认为心肌缺血损伤前线粒体的外膜已经破裂，然后才发生肿胀，肿胀可能与缺血损伤有关。我们推断，膜的破裂导致水的摄取和基质肿胀。因此，用于量化线粒体肿胀的定量分析的形状测量参数对于评估心肌损伤可能非常有效。
