Nuclear Cardiac Troponins, Tropomyosin and Actin in Native Ventricular Cardiomyocytes

Changes in gene expression determine cellular differentiation and developmental programs as cells transform from a progenitor to a mature adult state. The difference in the organization of the nucleus between undifferentiated cells and their terminally differentiated counterparts, and the possible mechanisms that determine and alter gene expression in the nucleus is an area of intense investigation. Nuclear actin -- which is immunologically distinct from cytoplasmic actin -- has been documented in number of differentiated cell types (Bettinger et al., 2004; Dingová et al., 2009 and Gieni et al., 2009) and cardiac isoforms of troponinI (cTnI) and troponinT (cTnT ) have been detected in association with nuclei of adult human cardiacmyocytes (Bergman et al., 2009 and Kajstura et al., 2010). It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. Using an in vitro primary explant cell culture model, I investigated whether nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. I developed a cell permeabilization and extraction protocol that enabled an unambiguous determination of the distribution pattern of each of these proteins distinctly in the cell nucleus via immunocytochemistry and confocal microscopy. Adult stem cell cardiomyogenesis remains a precarious process. I developed a reproducible bone marrow stem cell isolation and differentiation protocol to further investigate the presence of these proteins in nuclei of multipotent, bone marrow-derived mesenchymal stem cells from adult rats. I investigated the temporal appearance of cardiac genes and structural proteins in bone marrow stem cells undergoing cardiomyogenesis. Using my permeabilization and extraction protocol, I investigated the presence of nuclear actin, cTnC, cTnI, cTnT and cTm in the nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs and in BM-MSCs treated to differentiate into cardiomyocytes. The efficacy of adult stem cells is known to be compromised as a function of age. This therefore raises questions about the effectiveness of autologous cell therapy in elderly patients. Using a rodent model, I investigated and showed that there are differences in BM-MSCs associated with the age of the animal from which the cells are isolated.