Cardiomycocyte Regeneration and Multi-Isotope Imaging MS

Heart failure is a leading cause of mortality worldwide, and effective therapies to repair damaged cardiac tissue are badly needed given that myocardial regeneration is clearly inadequate in the setting of extensive injury. Recent studies indicate that mammalian myocardium does have some limited regenerative capability including resident cardiac stem cells capable of pluripotent differentiation. It is currently not clear what the relative rates of replenishment by stem cells versus division of pre-existing myocytes are, and much less is known regarding those relative rates post injury, when replacement of healthy tissue is far more crucial. In order to better assess the regenerative capacity of the heart, there is a need for more accurate, longer-term methods to quantitatively measure cell division and measure the rates of replacement. In this project I will address this need by combining two orthogonal techniques to quantitatively measure the rates of cell division and replenishment of damaged myocardium by stem cells versus pre-existing cardiomyocytes following injury. The first technique I will utilize is a mouse genetic cell fate-mapping approach using a bitransgenic labeling system based on inducible Cre/LoxP expression in cardiomyocytes. This method provides a means to identify and track pre-existing cardiomyocytes versus refreshed cells originating from a stem cell niche. However, this technique cannot demonstrate and quantify which cells have undergone division after injury. Therefore, I will utilize a second technique, Multi-Isotope Imaging Mass Spectrometry (MIMS), that can quantitatively assess cell division history. MIMS can simultaneously resolve multiple stable isotopes at subcellular resolution over a wide range of times. Thus, we can uniquely determine both new cell incorporation from a stem cell pool (via genetic fate-mapping using inducible Cre/Lox) and determine which cells have undergone division (via stable-isotope labeling and MIMS). Furthermore, in order to determine whether a precursor cell population exists within a niche in the bone-marrow that contributes to the replenishment of the cardiomyocyte pool, I will perform transplantation studies with genetically and isotopically labelled bone-marrow cells. I propose three specific aims. Aim 1: To test the hypothesis that stable isotope MIMS quantification combined with genetic fate mapping reveals a low rate of basal cardiomyocyte cell division with no measurable contribution of cell division from a precursor pool in adult mammalian myocardium. Combining MIMS with genetic fate-mapping in vivo, we will measure rates of turnover and division of cardiomyocytes in uninjured mice. Aim 2: To test the hypothesis that, following injury, cardiomyocytes are replenished primarily by stem cells and not by cell division of pre-existing cardiomyocytes. Combining MIMS with genetic fate-mapping in vivo, I will track and quantify cell division within the pre-existing cardiomyocyte pool versus the stem cell pool after pressure overload and after experimental myocardial infarction. Aim 3: To test the hypothesis that, following injury, mammalian cardiomyocytes are partly replenished by bone marrow-derived precursor cells. We will use a combination of genetic and stable isotope based labeling techniques, in concert with bone marrow transplantation, to track the fate of bone marrow-derived stem/progenitor populations after cardiac injury.