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PI: Wang Wang, MD, PhD
Mitochondria play critical role in modulating cell function through energy metabolism, reactive oxygen species (ROS) generation, calcium (Ca2+) regulation, and cell fate determination. Mitochondria dysfunction often accompanies and underlies the pathogenesis of disease. Recently, we developed a genetically encoded superoxide indicator and discovered transient mPTP opening events coupled with bursting ROS production, named superoxide flashes, in intact cell of various tissues including cardiac myocyte (Figure 1 and online video: http://www.cell.com/supplemental/S0092-8674%2808%2900769-1). This breakthrough strongly supports the existence of physiologically relevant mPTP activities and highlights the regulation of microdomain ROS signaling. Based on these discoveries, the long-term goal of the lab is to understand how mitochondria integrate ROS, Ca2+ and permeability transition pore (mPTP) signaling to impact cell function under normal and disease conditions, especially in the cardiovascular system. Current research efforts are focused on the following three projects:
Figure 1. Discovery of superoxide flash in single mitochondria of intact adult cardiac myocyte. The superoxide indicator, cpYFP is targeted to mitochondrial matrix and fluorescence images taken by dual-wavelength excitation at 488 nm and 405 nm. A superoxide flash is highlighted in upper panel and its time course is shown in the lower traces (Wang et al, 2008, Cell 134:279-290).
Regulation and Significance of Physiological mPTP Opening and Superoxide Flash Activity in the Heart
Hypothesis: mitochondria accumulate Ca2+ and manipulate cardiac function through Ca2+ triggered physiological mPTP opening and quantal ROS generation (Figure 2). We will employ state-of-the-art techniques, including species specific ROS probes, compartmentalized Ca2+ indicators, high resolution in vivo confocal imaging and genetically engineered animal models, to determine the relationship of mitochondrial Ca2+ uptake, triggered mPTP opening and superoxide production in normal excitation-contraction coupling of intact cardiac myocyte and working heart.
Figure 2. Diagram shows the hypothesis of physiological mPTP regulation and function. Mitochondrial Ca2+ uptake ultimately triggers mPTP opening, which in turn releases ROS to modulate local signaling.
Molecular Mechanism of mPTP Triggered Superoxide Flash Production
This project will elucidate the specific mechanism that bridge mPTP opening, electron transport chain (ETC) activity and superoxide flash generation. The hypotheses to be tested are that critical mPTP component senses matrix Ca2+ or ROS to facilitate the pore opening, and the subsequent matrix acidosis and membrane depolarization, in turn, stimulate ETC activity to produce superoxide flash. We will combine genetic, biochemical and biophysical approaches together with functional assessment to identify the role of mPTP components in physiological mPTP activity, ETC activation and superoxide flash production.
Role of Mitochondrial ROS in Oxidative Stress
The main objective is to develop quantitative measurement of mitochondrial ROS production under physiological and pathological conditions by using superoxide flash as a biomarker. The working hypothesis is that mitochondrial superoxide flash represents fundamental ROS producing event and the major regulatory mechanism of intracellular ROS signaling. Since ROS can be either 'friends' or 'foes' to the cell function depending on the relative concentration and spatiotemporal distribution, we are developing methodology to quantify mitochondrial superoxide flash under various conditions, in different tissues and at different levels (Figure 3). The results will establish standards to differentiate normal and excessive mitochondrial ROS production. Information obtained in this project will advance our understanding on the role of mitochondria in oxidative stress related disorders including ischemic heart disease, metabolic syndrome, atherosclerosis, and neurodegenerative disease as well as identify novel mitochondrial targets for therapy.
Figure 3. Diagram showing multi-level analysis of mitochondrial superoxide flash in health and disease. Indicator bearing transgenic mice will facilitate the evaluation of superoxide flashes under different disease conditions and at different levels from individual cells to intact tissues and to living animals.
Nicolas Gutierrez PhD Senior Fellow
Shangcheng Xu PhD Senior Fellow
Huiliang Zhang PhD Senior Fellow
Mitochondria and Metabolism Center
850 Republican St., Room N113
Seattle, Wa 98109-8057
Email: Wang Wang