Quantitative and qualitative analysis of heart mitochondria for evaluating the degree of myocardial injury utilizing atomic force microscopy

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±1559nm² and mean perimeter of 217.54±52.09nm (n=60). The mean area and perimeter of mitochondria in the IR groups increased to 28,181±21,248nm² and 595.74±234.29nm (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±1.08nN (n=30, p<0.0001) and 18.65±3.18nN (n=30, p<0.0001), compared to normal mitochondria which had an adhesion force of 27.64±0.88nN (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.