Investigating the role of Ca2+ signalling in the cell fate decision of cardiac myocyte precursor cells

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Heart failure is the leading cause of death in the developed world and heart transplantation and/or chronic drug administration are the only therapeutic means. Recently, the dogma that the heart is terminally differentiated has been overturned. There is an increasing body of evidence arguing for the continuous cardiac myocytes replacement. The mechanisms of myocyte turnover include replication of immature myocyte precursor cells, the so called small mononuclear myocytes (SMMs). The mechanisms that control SMM fate choices directing them to either proliferate or to differentiate are not established. The proposed project will investigate the signaling mechanisms that govern this fate choice of SMMs.Ca2+ signals mediated by Inositol triphosphate receptors are essential for development, differentiation and growth of cardiomyocytes. These Ca2+ signals are localized to nuclear microdomains, where they activate the calcineurin-NFAT pathway, which regulates gene expression. We hypothesize that the activation of NFAT also regulates the SMM cell fate decision, to either proliferate or differentiate. To test this hypothesis, I will first establish the Ca2+ phenotype of isolated SMMs and compare it to mature myocytes. Using adenoviruses I will express genetically encoded Ca2+ indicators (GCaMPs) in SMMs and investigate the Ca2+ signals using confocal laser scanning microscopy. Additionally, I will localize the proteins involved in regulating the Ca2+ homeostasis by immunofluorescence. Subsequently, I will determine the role of Ca2+ regulated pathways in the fate choice of SMMs. For this purpose, I will use pharmacological means and adenovirally expressed proteins to affect the signaling pathways and unravel the underlying mechanisms. Furthermore, I will develop techniques to image and manipulate Ca2+ signaling pathways in SMMs in vivo, in the intact heart - using adeno-associated viruses for SMM specific expression of reporter proteins (e.g. GCaMP) and two-photon microscopy.Understanding the underlying signaling mechanisms that govern the cell fate decision of SMMs offers the possibility to manipulate them - a novel therapeutic approach: helping the heart to repair itself.