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TRUSTEES OF INDIANA UNIVERSITY

Documentation of cardiomyocyte cell cycle activity and myocardial tissue regeneration typically requires extensive histochemical processing and frequently relies on the use of subjective criteria for cell lineage determination. Although reporter transgenes can greatly assist such analyses, their use also requires considerable tissue processing, and current transgenic reporter systems do not readily permit monitoring of cumulative regenerative growth. The studies proposed in this R21 application will generate transgenic reporter models which can be used to quantitate cardiomyocyte cell cycle activity and cumulative myocardial regeneration with minimal tissue processing. Aim 1 will utilize a cardiomyocyte-restricted promoter to target expression of a fusion between proteins with intrinsic fluorescent activity and proteins which undergo cell cycle- dependent changes in sub-nuclear localization. Cardiomyocyte cell cycle status can be quantitated simply by monitoring the pattern of reporter protein epifluorescence within the nucleus. Aim 2 will develop a tertiary transgenic reporter system to quantitate cumulative de novo myocardial growth in adult hearts. The system will utilize an existing conditional Cre-recombinase transgenic model, in combination with two new conditional transgenes, to permanently activate a nuclear localized EGFP reporter protein in all adult cardiomyocytes undergoing de novo proliferation. Consequently, cumulative myocardial growth resulting from cardiomyocyte proliferation can be determined simply by scoring the number of cells with nuclear EGFP epifluorescence. We will utilize existing transgenic models that exhibit enhanced cardiomyocyte proliferation to validate both reporter gene systems. Once validated, we will develop automated data acquisition and analyses protocols. The reporter transgenes proposed in this application will have the distinct advantage over existing models in that data can be acquired with minimal sample processing (in essence, requiring only tissue fixation and sectioning). Moreover, the systems will permit quantitation of cumulative regenerative growth, which cannot easily be determined with existing models. The use of automated techniques for data acquisition and analysis should permit precise quantitation of low-frequency events, and the use of fluorescent reporters will permit analyses in living tissue.

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