The ability to reconstitute lost cardiac mass following myocardial infarction could thus be of considerable therapeutic value. One approach to accomplish this entails the direct transplantation of donor cardiomyocytes or cardiomyogenic stem cells into injured hearts. Recent studies have identified a population of Sca-1+ stem cells isolated from the adult heart that home to the infarct border zone following intravenous infusion after reperfusion injury. After homing, the Sca-1+ cells have at least two fates: some of the cells fuse to infarct border zone cardiomyocytes forming heterokaryons, while others undergo de novo cardiomyogenic differentiation. The net result is that approximately 5% of the cardiomyocytes at the infarct border zone are derived either totally or in part from Sca-1+ stem cells. In this application we will combine the use of Sca-1+ stem cell transplantation with several validated cardioproliferative and/or cardioprotective genetic pathways in an effort to increase the number of Sca-l+ derived cardiomyocytes that reconstitute the heart following myocardial injury.
Four Specific Aims are proposed.
In Aim 1, we will use cell cycle and apoptosis analyses to establish the replicative and survival characteristics of Sca-1+ derived cardiomyocytes. These somewhat descriptive studies will provide requisite base line parameters for the remaining aims of the application.
In Aim 2, we will test the hypothesis that lineage-restricted expression of cell cycle regulatory genes will enhance the ability of Sca-1+ stem cells to reconstitute damaged hearts. These experiments will utilize stem cells derived from mice expressing either the SV40 Large T Antigen oncoprotein, cyclin D2, or a dominant interfering pi93 mutant; all three proteins are able to drive cardiomyocyte cell cycle activity in transgenic animals.
In Aim 3, we will test the hypothesis that lineage-restricted expression of cell survival genes will enhance the ability of Sca-1+ stem cells to reconstitute damaged hearts. These studies will utilize transgenes expressing Bcl-2 or a dominant interfering version of p53, both of which bestow cardioprotective phenotypes when expressed in the hearts of transgenic mice. Finally, in Aim 4 we will test the hypothesis that border zone cardiomyocytes derived from Sca-1+ stem cells are able to participate in a functional syncytium with the host myocardium. These studies will utilize two photo molecular excitation laser scanning microscopy to simultaneously monitor intracellular calcium transients in host and Sca-1+ derived cardiomyocytes in intact hearts. The experiments proposed here will use a variety of transgenic mouse resources to determine if genetic modification can enhance the capacity of Sca-1+ derived cardiomyocytes to repopulate myocardial infarcts, and furthermore will determine if the nascent cells are able to participate in a functional syncytium with the surviving host myocardium. Ultimately these approaches might be useful to reconstitute myocardial mass following cardiac injury, as well as deliver beneficial genes to cardiomyocytes at the border zone. ? ?
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