OXYGEN CONCENTRATION & REDOX METABOLISM WITH TRITYL PROBES IN CARDIAC MYOCYTES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this collaborative project is to image O2 and redox metabolism in live cardiac myocytes or myocyte clusters from normal hearts and hearts with cardiomyopathy. Normal cardiac myocytes (from rats) are typically about 20 x 25 x 150 microns. With hypertrophic cardiomyopathy (CM) these cells can increase in size by over 50%. We will make use of recently developed ESRM technology at ACERT and recently developed intra-cellular trityl radical probes and trityl-nitroxide biradicals to provide subcellular and extra cellular imaging of oxygen and redox metabolism that will complement our present "tissue level" imaging on isolated and in vivo hearts, conducted with low field/lower resolution in-vivo ESR imaging. ESR microscopy will be employed with our recently developed novel trityl esters and trityl-nitroxide biradicals that enable measurements of O2 concentrations and redox metabolism in cells and tissues. This is an extension of our grants noted above. Specifically, we will: 1. Incubate and load cardiac myocytes with our recently developed trityl ester probes that we have shown to serve as intracellular spin labels and oximetry probes and then employ ESRM to measure their location and the localized intracellular O2 concentration. Similar measurements will be performed with the extracellular trityl CT-03. 2. Perform similar measurements with our new trityl nitroxide biradicals of intracellular redox metabolism. 3. Subject the cells to different Ca(II) levels and stimulation with beating at rates of 0.5 Hz to 5 Hz and measure the effects on O2 and redox metabolism within the normal and CM myocytes. Several important questions will be addressed including: Is intracellular O2 concentration and redox metabolism different than extracellular values? Are there spatial differences within the cell? Do these values change with myocyte contractile activity? Do these values change with Ca(II) concentration?