Grant: Research Tools and Instruments

<p>&nbsp; &nbsp; &nbsp; &nbsp;The mitochondrion is the power house of eukaryotic cells, where ATP is produced via oxidative phosphorylation. Vascular smooth muscle cells use L-cysteine or homocysteine as substrates to produce hydrogen sulfide (H₂S), catalyzed by cystathionine gamma-lyase (CSE) in the cytosol. Whether the mitochondrion can use H₂S to produce ATP in the absence of oxygen has been unknown and intriguing. We hypothesize that mitochondrial production of H₂S can be realized by translocation of CSE to mitochondria in response to different metabolic stresses, and that mitochondrial H₂S regulates mitochondrial functions. Our objectives are to examine 1) mitochondrial CSE translocation and H₂S production under different stress conditions, and 2) the role of H₂S in regulation of mitochondrial structure and function and the underlying mechanisms. This program has the potential to unmask the novel function of CSE/H₂S in mitochondria. Moreover, it may lead to a breakthrough in our understanding of the fundamental sulfur metabolism in mammalian cells, of the evolution and etiology of mitochondria, and of the regulation of mitochondrial biogenesis and function in the cardiovascular system of animals and humans.<br /> &nbsp; &nbsp; &nbsp; &nbsp; Over the last 3 years, this NSERC-funded program has proceeded well as planned. However, the advancement of the program is now challenged by the lack of essential instruments. To address this urgent challenge, two instruments are requested, which are not currently available at Laurentian University. In the current program, exogenous H₂S is used extensively. To reliably and accurately deliver exogenous H₂S to animals or cultured cells, pure H₂S gas must be used to bubble the physiological solutions to make physically saturated H₂S stock solutions, which can be later stepwise diluted. The H₂S-specific hypoxic Glove Box will enable us to accurately and safely handle H₂S gas and H₂S solutions in a tightly controlled environment. It is also essential for testing the integrated effect of hypoxia and H₂S on mitochondrial bioenergetics. A ProteinSimple Wes system (SimWes) is requested for the detection of protein abundance throughout the whole research program. The SimWes system is a fully automated western blot alternative, operating in gel-free and blot-free modes. It uses a capillary-based nanoimmunoassay size-based separation platform to detect all sizes of proteins. It is more sensitive and faster, and uses much smaller quantities of cell samples and antibodies than traditional Western blot. <br /> &nbsp; &nbsp; &nbsp; &nbsp; These instruments are essential for the successful completion of two PhD projects and for the progress of the research projects of two post-doctoral fellows. In short, this RTI application will ensure the successful accomplishment of all objectives of this NSERC Discovery program, the career development of HQP, and the continuation of our long-term research program.</p>