Analysis of Mitochondrial Morphology in Cells Experiencing a "Heart Attack"
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Series/Report no.:2010 Edward F. Hayes Graduate Research Forum. 24th
Vascular endothelial cell (EC) dysfunction immediately following the recanalization of a blocked artery (known as reperfusion, RP) is attributed to the cellular generation of superoxide radicals and other reactive oxygen species (ROS). Nitric oxide (NO) is known to promote ROS production from the cell mitochondria. Our lab discovered that cultured EC exposure to fluid mechanical shear stress results in endogenous NO-mediated mitochondrial ROS production. Ischemia (I)/RP-induced EC injury occurs primarily due to mitochondrial ROS production, and the decline in bioavailable NO (due to the reaction of NO with superoxide) impairs the EC-dependent dilation in the heart coronary vessels. In general, mitochondrial ROS lead to activation of the mitochondrial apoptotic pathway. Recent literature suggests that induction of mitochondrial apoptosis correlates with mitochondrial fragmentation (fission), which may contribute to cell apoptosis/death. Our goals are to detect and quantify changes in mitochondrial morphology due to cultured EC exposure to simulated I/RP (with or without inhibitors of ROS sources, a NO synthase inhibitor or an inhibitor of the mitochondrial fission protein Drp1), and to determine whether these changes contribute to cell apoptosis/death. Following cultured EC exposure to treatments, such as fluid shear stress, hypoxia/reoxygenation (H/RO) or ischemia/reperfusion (I/RP; ischemia is simulated as hypoxia; reperfusion is simulated as reoxygenation with the addition of flow), ECs are stained with mitotracker green, which is used in conjunction with fluorescence microscopy to obtain digital images of the mitochondrial network. Morphological parameters are determined for each object in an image, and these are used for quantitative comparisons of the mitochondrial network between treatments. We found that static or sheared ECs maintain their mitochondrial network. H/RO-exposed ECs undergo changes in mitochondrial morphology, but fission is significantly less compared to that in ECs exposed to I/RP. The massive fission in I/RP-exposed ECs is inhibited by antioxidants, a NO synthase inhibitor or a Drp1 inhibitor. Hence, ROS, NO and Drp1 are all responsible for the I/RP-induced increase in mitochondrial fission. Mitochondrial morphology changes may be implicated in the induction of EC apoptosis after vessel recanalization following a heart attack suggesting that, by controlling mitochondrial morphology, we may be able to lessen the EC dysfunction and improve outcomes.
Poster Division 2: Science and Technology: 1st Place (The Ohio State University Edward F. Hayes Graduate Research Forum)
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