Mechanoregulation of Endothelial Mitochondrial Function It is known that nitric oxide (NO), through its interaction with electron transport chain (ETC) components, may function not only as a regulator of cell respiration, but also to augment the generation of reactive oxygen species (ROS) by mitochondria and thereby trigger mechanisms of cell survival or death. Following ischemia (I)/reperfusion (RP), the production of both NO and superoxide (O27 ) is increased and peroxynitrite (ONOO ) is formed. ONOO inactivates the ETC at several sites leading to enhanced ROS generation, protein nitration, lipid peroxidation and myocardial damage. Coronary endothelial cells (ECs) are injured following cardiac I/RP. Although some knowledge was gained from hypoxia (H)/reoxygenation (RO) models, the molecular me- chanisms leading to the EC dysfunction upon RP are not well understood. ECs regulate the vascular tone and thrombosis, and, hence, protection of coronary ECs from I/RP injury is an important goal. We recently showed that cultured EC exposure to steady laminar shear stress induces ONOO formation in the mitochondria leading to inhibition of ETC at multiple sites. Since, under flow conditions at atmos- pheric O2 concentrations (21% O2), the shear-induced NO is responsible for mitochondrial oxidative stress, we propose to characterize the mitochondrial (dys)function in cultured human coronary arterial ECs exposed to environments with controlled levels of shear stress and oxygen tension (PO2). More specifically, we propose to:
Aim 1 : Quantitatively characterize the mitochondrial function in coronary ECs exposed to: (a) shear stress under physiologic PO2 (10% O2) and (b) I (H, no glucose, acidotic pH)/RP (shear at 10% O2). In ECs sheared under the relatively `hyperoxic'21% O2, mitochondrial O27 formation may be increased lead- ing to enhanced ONOO formation and mitochondrial dysfunction. Mitochondrial function will be characteri- zed by measuring O27 levels, ETC complex activities, protein nitration and membrane potential;EC function by measuring NO production, O2 consumption, ATP levels, lipid peroxidation and apoptosis.
Aim 2 : Examine if mitochondria-targeted antioxidant peptides and ischemic postconditioning, treatments shown to protect the postischemic myocardium from RP injury, also protect the coronary ECs. The antioxidant peptides may scavenge mitochondrial ONOO leading to decreased protein nitration and lipid peroxidation upon RP. Ischemic postconditioning may reduce NO formation upon RP leading to decreased ge- neration of mitochondrial O27 and ONOO , and preservation of cell function. If time permits, coronary ECs flow-preconditioned overnight at 10% O2 prior to I/RP will also be examined. This proposal is novel (since it is based on a new discovery that EC mitochondrial function is modulated by shear-induced NO) and is exploratory (since we propose to explore the effect of PO2, prior I and potential therapeutic strategies on the mitochondrial function in sheared ECs), making it suitable for the R21 category.Relevance to Public Health This goal of this proposal is to better understand the molecular mechanisms that lead to mitochondrial and cell dysfunction of coronary endothelial cells (ECs) following cardiac ischemia (I)/reperfusion (RP). The coronary endothelium is a critical target of I/RP injury. Ischemic heart disease secondary to acute myocardial infarction is among the most prevalent health problems in the world and a major cause of morbidity and mortality.
| Chen, Jingfeng; Chen, Chwen-Lih; Alevriadou, B Rita et al. (2011) Excess no predisposes mitochondrial succinate-cytochrome c reductase to produce hydroxyl radical. Biochim Biophys Acta 1807:491-502 |
| Han, Zhaosheng; Varadharaj, Saradhadevi; Giedt, Randy J et al. (2009) Mitochondria-derived reactive oxygen species mediate heme oxygenase-1 expression in sheared endothelial cells. J Pharmacol Exp Ther 329:94-101 |
| Jones 3rd, Charles I; Han, Zhaosheng; Presley, Tennille et al. (2008) Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite. Am J Physiol Cell Physiol 295:C180-91 |
