Mitochondria as the major source of energy generation are essential for proper cellular function. There is considerable evidence supporting the key role of mitochondrial dysfunction in heart disease such as myocardial infarction and heart failure. At the myocardial level of the post-ischemic heart, a defect in energy metabolism associated with overproducing oxygen free radicals and NO in mitochondria was marked. Alterations of protein S-glutathionylation (PrSSG) and protein nitration have been detected in the mitochondrial complex I (NQR), complex II (SQR), and other ETC proteins during myocardial ischemia and reperfusion injury. Alterations of NQR/SQR-derived oxidative modifications are closely linked to oxygen free radical production, NO metabolism, and homeostasis of redox thiols in mitochondria. S-glutathionylation of the reactive and labile cysteine residues in the NQR or SQR is a reversible modification, whereas S-sulfonation of reactive cysteine residues is an irreversible modification. Our central hypotheses are that both cysteinyl modifications are highly regulated by the redox status in the mitochondria of the post-ischemic heart, and the mitochondrial redox status is controlled by ROS production, NO metabolism, and the homeostasis of the GSH pool. The long term objectives of this research are to elucidate the molecular mechanism of mitochondrial redox signals in the mediation of myocardial injury, to understand the pathogenesis, and to develop a treatment for cardiovascular diseases. The key hypotheses of the major signal pathway leading to protein S-glutathionylation/sulfonation in mitochondria will be tested by pursuing the following specific aims using novel animal models, EPR spectrometry, and mass spectrometry.
Specific aim 1 will determine whether irreversible protein S- sulfonation of NQR and SQR is induced in the mitochondria of the post-ischemic myocardium. The protein sulfonation marked in the NQR and SQR after myocardial infarction will be characterized by mass spectrometry. A sequence-specific antibody for sulfonation will be generated to detect this event in vitro and in vivo. EPR spectrometry with a spin probe and a spin trap will be used to measure the redox status and "O2- generation activity of mitochondria isolated from the post-ischemic heart.
Specific aim 2 will determine the role of glutathione reductase (GR) in the mechanism of glutathionylation of NQR/SQR and regulation of overall mitochondrial function in the post-ischemic heart. We will use a pharmacologic approach and mice deficient in GR (gsr-/-) to determine whether (i) enhancing GSSG in vivo will increase glutathionylation of NQR and SQR, and (ii) whether or not increasing NQR/SQR glutathionylation in vivo will be protective and reduce the susceptibility of mitochondria to post-ischemic injury.
Specific aim 3 will ascertain the role of eNOS in the mechanism of NQR/SQR glutathionylation and regulation of overall mitochondrial function in the post-ischemic heart. Mice with an eNOS-/- and a cardiac-specific eNOS-/- genotype will serve as an excellent in vivo model for studying the regulation of NQR/SQR glutathionylation, mitochondrial redox status, and its "O2- generation activity via NO metabolism. We will also create a novel mouse model by crossing cardiac-specific SOD2 transgenic mice with eNOS-/- mice in order to determine whether increased SOD2 signaling in mitochondria is sufficient to correct oxidative injury resulting from eNOS deficiency and post-ischemic injury.

Public Health Relevance

Mitochondria are the major sources of both energy and oxygen free radical production in the cell, a broad spectrum of cardiovascular diseases can thus have a mitochondrial etiology, and many of these diseases are prevalent in the US patient population, including myocardial infarction, heart failure, cardiomyopathy, atherosclerosis, and hypertension. Mitochondrial function can be altered by oxidative posttranslational modifications, such as glutathionylation and sulfonation of cysteine residues, which affect the functions of proteins and enzymes. The renewal application is designed to shed light on their roles in controlling mitochondrial function during periods of metabolic stress and how this contributes to disease, in which the information obtained will accelerate our knowledge of pathogenesis and aid design of treatments for cardiovascular diseases.

National Institute of Health (NIH)
Research Project (R01)
Project #
Application #
Study Section
Myocardial Ischemia and Metabolism Study Section (MIM)
Program Officer
Wong, Renee P
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Northeast Ohio Medical University
Other Basic Sciences
Schools of Medicine
United States
Zip Code
Kang, Patrick T; Chen, Chwen-Lih; Chen, Yeong-Renn (2015) Increased mitochondrial prooxidant activity mediates up-regulation of Complex I S-glutathionylation via protein thiyl radical in the murine heart of eNOS(-/-). Free Radic Biol Med 79:56-68
Chen, Yeong-Renn; Zweier, Jay L (2014) Cardiac mitochondria and reactive oxygen species generation. Circ Res 114:524-37
Kang, Patrick T; Chen, Chwen-Lih; Ren, Pei et al. (2014) BCNU-induced gR2 defect mediates S-glutathionylation of Complex I and respiratory uncoupling in myocardium. Biochem Pharmacol 89:490-502
Zhang, Liwen; Kang, Patrick T; Chen, Chwen-Lih et al. (2013) Oxidative modifications of mitochondria complex II. Methods Mol Biol 1005:143-56
Lee, Hsin-Ling; Chen, Chwen-Lih; Yeh, Steve T et al. (2012) Biphasic modulation of the mitochondrial electron transport chain in myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol 302:H1410-22
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
Cai, Ming; Li, Yuanjing; Xu, Yi et al. (2011) Endothelial NOS activity and myocardial oxygen metabolism define the salvageable ischemic time window for ischemic postconditioning. Am J Physiol Heart Circ Physiol 300:H1069-77
Chen, Chun-An; Lin, Cho-Hao; Druhan, Lawrence J et al. (2011) Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling. J Biol Chem 286:29098-107
Kang, Patrick T; Yun, June; Kaumaya, Pravin P T et al. (2011) Design and use of peptide-based antibodies decreasing superoxide production by mitochondrial complex I and complex II. Biopolymers 96:207-21
Chen, Weiguo; Druhan, Lawrence J; Chen, Chun-An et al. (2010) Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: transition from reversible to irreversible enzyme inhibition. Biochemistry 49:3129-37

Showing the most recent 10 out of 25 publications