Molecular Regulation of Cardiac Stem Cell Growth and Differentiation by Extrinsic Factors and Novel Intracellular Signaling Pathways

T. J. Bartosh, Molecular regulation of cardiac stem cell growth and differentiation by extrinsic factors and novel intracellular signaling pathways. Doctors of Philosophy (Biomedical Sciences), May 2008, 293 pp., 4 tables, 59 illustrations, bibliography, 353 titles. Insufficient myocardial regeneration following ischemic injury provokes cardiac dysfunction, adverse tissue remodeling, and ultimately heart failure. Stem cell replacement therapy appears to be a promising strategy for improving cardiac function, however, challenges involving inadequate stem cell differentiation, engraftment, and survival following transplantation currently impede the efficacy of regeneration protocols. These limitations emphasize the significance of identifying extrinsic factors and corresponding molecular mechanisms regulating cardiac cell differentiation. Recent reports recognizing a stem cell component in the adult heart (i.e. cardiac stem cells, CSC) have provided additional targets and/or tools for myocardial repair. This investigation verified the presence of CSC in the adult dog heart, described methods to generate three dimensional (3D) cardiac microtissues (‘cardiospheres’) from CSC, and revealed features of cardiospheres potentially useful for identifying extrinsic procardiogenic factors, evaluating myocardial response to stress, and delivering functional CSC into the damaged heart. Specifically, cardiosphere formation was facilitated by culturing CSC in growth medium on a poly-L-ornithine substratum. Cardiospheres are comprised of interior cells that exhibited characteristics of CSC; differentiating cardiomyocytes at the periphery with organized contractile machinery; and/or vascular cells capable of forming vessel-like networks. Upon co-culture with neonatal cardiomycocytes, spheres developed foci of contracting regions. Furthermore, cardiospheres exhibited increased resistance to oxidative stress and survived subcutaneous injections without undergoing neoplastic transformation further supporting their ability to effectively promote myocardial regeneration. Retionic acid (RA), the active form of vitamin A, augmented expression of myocyte-specific proteins in cardiospheres, thus, RA may improve the success of cardiac regenerative therapies and provide an appropriate stimulus to model underlying mechanisms fundamental for CSC differentiation. The tendency for cultured stem cells to undergo various levels of multi-lineage commitment is, however, cumbersome for deciphering precise cues that direct cell fate decisions. In this study, the molecular pathways important for RA-induced cardiac differentiation were examined using h9c2 cardioblasts as a stable cell model. In h9c2 cells, RA treatment promoted transcriptional enhancement of the muscle-enriched gene regulatory protein MEF2C, morphological alterations indicative of differentiation, and a robust increase in expression of myocyte differentiation genes including cardiac myosin heavy chain (cMHC) and ventricular myosin light chain-2 (vMLC2). These changes were preceded by rapid events involving elevation of intracellular Ca^2+ and phosphorylation of p38 MAPK. The effects of RA were attenuated using CA^2+ buffering agents or chemical inhibitors of L-type Ca^2+ channels (LTCC) and the phosphatase calcinuerin, but not by RA receptor antagonists. Furthermore, overexpression of dominant negative (dn) MEF2C, dnp38 MAPK, or CAIN, a physiological calcineruin inhibitor, abrogated MEF2 activity and RA-induced differentiation. These results imply that RA promotes cardiomyocyte differentiation, independent of RA response element activation, via induction of CA^2+ -regulated signaling pathways by activating the LTCC-calcineurin/38MAPK-MEF2 axis. Taken together, these findings enhance our understanding of extrinsic factors and molecular mechanisms indispensable for myocyte differentiation and subsequently provide novel therapeutic targets and cellular tools for regeneration and repair of damaged myocardium.