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The possibility to induce myocardial regeneration by the activation of resident cardiac stem cells (CSCs) has raised great interest. However, to propose endogenous CSCs as therapeutic options, a better understanding of the complex mechanisms controlling heart morphogenesis is needed, including the cellular and molecular interactions that cardiomyocyte precursors establish with cells of the stromal compartment. In the present study, we co-cultured immature cardiomyocytes from neonatal mouse hearts with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte growth in vitro. We found that cardiomyocyte proliferation was enhanced by direct co-culture with MSCs compared with the single cultures. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch-1 receptor by its ligand Jagged-1 expressed by the adjacent MSCs. In fact, the cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notch-intracellular domain (Notch-ICD) as compared with those cultured alone and this response was significantly attenuated when MSCs were silenced for Jagged-1. The presence of various cardiotropic cytokines and growth factors in the conditioned medium of MSCs underscored the contribution of paracrine mechanisms to Notch-1 up-regulation by the cardiomyocytes. In conclusions these findings unveil a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation through Notch-1/Jagged-1 pathway and suggest that stromal-myocardial cell juxtacrine and paracrine interactions may contribute to the development of new and more efficient cell-based myocardial repair strategies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The possibility that resident myocardial progenitor cells may be re-activated by transplantation of exogenous stem cells into the post-infarcted heart has been suggested as a possible mechanism to explain the heart's functional improvement after stem cell therapy. Here we studied whether differentiation of mouse neonatal immature cardiomyocytes in vitro was influenced by mouse skeletal myoblasts C2C12, wild type or engineered to secrete the cardiotropic hormone relaxin. The cultured cardiomyocytes formed spontaneously beating clusters and temporally exhibited cardiac immunophenotypical (cKit, atrial natriuretic peptide, troponin T, connexin-43, HCN4) and electrical features (inward voltage-dependent Na(+), T- and L-type Ca(2+) currents, outward and inward K(+) currents, I(f) pacemaker current). These clusters were functionally connected through nanotubular structures and undifferentiated cardiac cells in the form of flattened stripes, bridging the clusters through connexin-43-containing gap junctions. These findings suggested the existence of long distance cell-to-cell communications among the cardiomyocyte aggregates involved in the intercellular transfer of Ca(2+) signals and organelles, likely required for coordination of myocardial differentiation. Co-presence of the myoblasts greatly increased cardiomyocyte differentiation and the amount of intercellular connections. In fact, these cells formed a structural support guiding elongation of nanotubules and stripe-like cells. The secretion of relaxin by the engineered myoblasts accelerated and enhanced the cardiomyogenic potential of the co-culture. These findings underscore the possibility that grafted myoblasts and cardiotropic factors, such as relaxin, may influence regeneration of resident immature cardiac cells, thus adding a tile to the mosaic of mechanisms involved in the functional benefits of cell transplantation for cardiac repair.

Unlabelled: The role of angiotensin II in pressure overload is still debated because notwithstanding its effects on myocyte contractility angiotensin II is not an obligatory factor for the development of hypertrophy. To define the role of angiotensin II in acute pressure overload we studied the effects of AT1 blockade (valsartan 80mg per day) on myocardial contractility, cardiac growth factor gene expression, and myocardial hypertrophy in aortic banded (60mmHg) pigs. Acute pressure overload caused an abrupt reduction of myocardial contractility, measured by the end-systolic stiffness constant, and a sharp increase in end-systolic stress which rapidly normalized (within 12h) in the placebo group. In AT1-blocked animals end-systolic stiffness constant remained significantly depressed up to 24h and end-systolic stress was still elevated up to 48h (both P<0.05 vs placebo). In both groups confocal microscopy revealed that granular staining of angiotensin II in cardiomyocyte cytoplasm disappeared after 30min of pressure overload. AT1 blockade abolished following cardiac overexpression of angiotensinogen and endothelin-1 genes as shown in RT-PCR studies and the consequent angiotensin II and endothelin-1 release in the coronary circulation. Conversely, insulin-like growth factor-I and ACE mRNA overexpression, as well as the onset of left ventricular mass increase, were not significantly affected by AT1 blockade., In Conclusion: (1) mechanical stress releases preformed angiotensin II from myocyte in vivo; (2) the AT1 blockade abolishes cardiac angiotensin II and endothelin-1 production with delayed recovery of myocardial contractility; whereas (3) the overexpression of insulin-like growth factor-I gene and the development of myocardial hypertrophy are not angiotensin II-mediated effects.