Revalesio Pilot Studies

Proposal to carry out the following experiments to explore the effects of RNS60 on mitochondrial dynamics, function, signaling and survival responses. The first study will examine its effects on mitochondria-dependent cell death responses, the second study will examine its effects on mitochondrial morphology/fission responses, and the third study will test its effects on the mitochondrial redox signaling responses to hypoxia: 1. Using the lung slice explant model, we will test whether cell survival during 72 hrs of culture under hyperoxic conditions is enhanced or degraded by RNS60. These primary tissue explants undergo progressive cell death under these conditions, as evidenced by increasing uptake of propidium iodide. Hyperoxia induces significant oxidant stress in the mitochondrial matrix, which may contribute to cell death by enhancing cytochrome c release from the intermembrane space to the cytosol. 2. Simulated ischemia triggers a mitochondrial fission response in primary cultured mouse cardiomyocytes. I will isolate and culture cardiomyocytes from neonatal transgenic mice expressing a mitochondria-targeted fluorescent (GFP) protein. We will study the changes in mitochondrial morphology in these cells in response to oxidant stress or simulated ischemia-reperfusion to determine whether RNS60 alters that response. 3. We will express the redox-sensitive roGFP protein in cultured primary vascular smooth muscle cells and assess the effects of RNS60 on the basal redox status of protein thiols, and determine whether it affects the increase in oxidant signaling observed during 1-2 hr of physiological hypoxia in these cells. We can use whatever interesting results we obtain in the above studies as a starting point for future endeavors.