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

在发达国家，心力衰竭是导致死亡的主要原因，心脏移植和/或慢性药物治疗是唯一的治疗手段。最近，心脏是最终分化的教条被推翻了。越来越多的证据支持心肌细胞的持续替换。肌细胞周转的机制包括未成熟肌细胞前体细胞的复制，即所谓的小单核肌细胞（SMMs）。控制SMM命运选择，引导它们增殖或分化的机制尚未建立。拟议的项目将研究控制这种命运选择的信号机制。肌醇三磷酸受体介导的Ca2+信号对心肌细胞的发育、分化和生长至关重要。这些Ca2+信号定位于核微域，在那里它们激活钙调磷酸酶- nfat通路，从而调节基因表达。我们假设NFAT的激活也调节SMM细胞的命运决定，无论是增殖还是分化。为了验证这一假设，我将首先建立分离的smm的Ca2+表型，并将其与成熟的肌细胞进行比较。我将使用腺病毒在SMMs中表达基因编码的Ca2+指示剂（GCaMPs），并使用共聚焦激光扫描显微镜研究Ca2+信号。此外，我将通过免疫荧光定位参与调节Ca2+稳态的蛋白质。随后，我将确定Ca2+调控途径在SMMs命运选择中的作用。为此，我将使用药理学手段和腺病毒表达蛋白来影响信号通路并揭示其潜在机制。此外，我将开发技术成像和操纵Ca2+信号通路的SMM在体内，在完整的心脏-使用腺相关病毒的SMM特异性表达报告蛋白（如GCaMP）和双光子显微镜。了解控制smm细胞命运决定的潜在信号机制，为操纵它们提供了可能——一种新的治疗方法：帮助心脏自我修复。

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.