Emerging Functions of Mitochondrial Fission in Postischemic Endothelial Cells Endothelial cell (EC) dysfunction upon early reperfusion (RP) following ischemia (I) is thought to occur due to endogenous oxidative stress, and, specifically, due to mitochondrial superoxide (O27-)/reactive oxygen species (ROS) generation. The decline in bioavailable nitric oxide (NO) impairs the EC-dependent dilation in coronary vessels. NO, both by itself and via peroxynitrite formation, is known to promote mitochondrial O27- production. Cultured EC exposure to shear stress was also shown to result in NO-mediated mitochondrial O27- production. Oxidative stress is thought to induce the opening of the mitochondrial permeability transition pore (mtPTP) leading to activation of the mitochondrial pathway of apoptosis. However, recent literature suggests that induction of mitochondrial apoptosis correlates with mitochondrial fission, and the resultant depolarized mitochondria are degraded via autophagy, which can lead to cell survival, apoptosis or autophagic cell death. We found that static or sheared ECs maintain their mitochondrial network. Hypoxia (H)/reoxygenation (RO)- exposed ECs undergo mitochondrial morphology changes, but fission is significantly less compared to that in ECs exposed to in vitro I/RP (I is simulated as H;RP is simulated as RO with the addition of flow). Fission in I/RP-exposed ECs is inhibited by antioxidants or NO synthase inhibitors, and is accompanied by phosphorylation of the fission protein dynamin-related protein 1 (Drp1) and increased autophagy. In order to understand the I/RP-induced EC mitochondrial morphology changes and whether these dictate the cell fate, we propose to: (a) Assess the differential effects of H/RO and I/RP on cultured EC mitochondrial dynamics, and delineate the intracellular signaling pathways that lead to increased mitochondrial fission. Transfection with a lentivirus that expresses green fluorescent protein in mitochondria will be used to analyze their dynamic behavior during static, shear, H/RO or I/RP. We will examine if abnormal mitochondrial dynamics are accompanied by fusion/fission protein changes, with a focus on fission regulation by Drp1 levels/activity, and will also delineate the roles of ROS, NO, and mtPTP in Drp1 activation and mitochondrial fission. (b) Examine if blocking the extensive fission due to I/RP will preserve the endothelial mitochondrial function and suppress apoptosis, and if the effect of fission on cell function is, at least in part, mediated by autophagy. ECs, in the presence of a pharmacological Drp1 inhibitor or following overexpression of either a dominant negative Drp1 form or the fusion protein mitofusin 2, will be exposed to I/RP, and mitochondrial function, autophagy and apoptosis will be measured. The latter will also be measured in the presence of autophagy inhibitors. This study is based on the novel hypothesis that changes in mitochondrial network dynamics may be responsible for the EC dysfunction upon RP. Our goal is, via better understanding of the EC dysfunction (the first critical step in cardiac I/RP injury), to develop new therapeutic strategies that will target mitochondrial fission.
Although early coronary reperfusion following acute myocardial infarction is the primary clinical intervention for improving patient outcome, reperfusion itself, primarily via generation of free radicals, may cause further tissue damage. The endothelium in coronary arteries is an early critical target of ischemia/reperfusion injury. This proposal aims to better understand the role of mitochondrial fragmentation/fission and its repercussions on postischemic endothelial cell survival, and to propose new therapeutic strategies that target the endothelial mitochondrial dynamics.
|Scheitlin, Christopher G; Julian, Justin A; Shanmughapriya, Santhanam et al. (2016) Endothelial mitochondria regulate the intracellular Ca2+ response to fluid shear stress. Am J Physiol Cell Physiol 310:C479-90|
|Scheitlin, Christopher G; Nair, Devi M; Crestanello, Juan A et al. (2014) Fluid Mechanical Forces and Endothelial Mitochondria: A Bioengineering Perspective. Cell Mol Bioeng 7:483-496|
|Giedt, Randy J; Pfeiffer, Douglas R; Matzavinos, Anastasios et al. (2012) Mitochondrial dynamics and motility inside living vascular endothelial cells: role of bioenergetics. Ann Biomed Eng 40:1903-16|
|Giedt, Randy J; Yang, Changjun; Zweier, Jay L et al. (2012) Mitochondrial fission in endothelial cells after simulated ischemia/reperfusion: role of nitric oxide and reactive oxygen species. Free Radic Biol Med 52:348-56|