RESUMO
The endocardium is a specialized endothelium that lines the inner surface of the heart. Functional studies in mice and zebrafish have established that the endocardium is a source of instructive signals for the development of cardiac structures, including the heart valves and chambers. Here, we characterized the NOTCH-dependent endocardial secretome by manipulating NOTCH activity in mouse embryonic endocardial cells (MEEC) followed by mass spectrometry-based proteomics. We profiled different sets of soluble factors whose secretion not only responds to NOTCH activation but also shows differential ligand specificity, suggesting that ligand-specific inputs may regulate the expression of secreted proteins involved in different cardiac development processes. NOTCH signaling activation correlates with a transforming growth factor-ß2 (TGFß2)-rich secretome and the delivery of paracrine signals involved in focal adhesion and extracellular matrix (ECM) deposition and remodeling. In contrast, NOTCH inhibition is accompanied by the up-regulation of specific semaphorins that may modulate cell migration. The secretome protein expression data showed a good correlation with gene profiling of RNA expression in embryonic endocardial cells. Additional characterization by in situ hybridization in mouse embryos revealed expression of various NOTCH candidate effector genes (Tgfß2, Loxl2, Ptx3, Timp3, Fbln2, and Dcn) in heart valve endocardium and/or mesenchyme. Validating these results, mice with conditional Dll4 or Jag1 loss-of-function mutations showed gene expression alterations similar to those observed at the protein level in vitro These results provide the first description of the NOTCH-dependent endocardial secretome and validate MEEC as a tool for assaying the endocardial secretome response to a variety of stimuli and the potential use of this system for drug screening.
Assuntos
Endocárdio/embriologia , Endocárdio/metabolismo , Valvas Cardíacas/embriologia , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Benzazepinas/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Endocárdio/citologia , Endocárdio/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Valvas Cardíacas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Camundongos Mutantes , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores Notch/genética , Reprodutibilidade dos TestesRESUMO
The zebrafish heart regenerates after ventricular damage through a process involving inflammation, fibrotic tissue deposition/removal and myocardial regeneration. Using 3D whole-mount imaging, we reveal a highly dynamic endocardium during cardiac regeneration, including changes in cell morphology, behaviour and gene expression. These events lay the foundation for an initial expansion of the endocardium that matures to form a coherent endocardial structure within the injury site. We studied two important endocardial molecules, Serpine1 and Notch, which are implicated in different aspects of endocardial regeneration. Notch signalling regulates developmental gene expression and features of endocardial maturation. Also, Notch manipulation interferes with attenuation of the inflammatory response and cardiomyocyte proliferation and dedifferentiation. serpine1 is strongly expressed very early in the wound endocardium, with decreasing expression at later time points. serpine1 expression persists in Notch-abrogated hearts, via what appears to be a conserved mechanism. Functional inhibition studies show that Serpine1 controls endocardial maturation and proliferation and cardiomyocyte proliferation. Thus, we describe a highly dynamic endocardium in the regenerating zebrafish heart, with two key endocardial players, Serpine1 and Notch signalling, regulating crucial regenerative processes.
Assuntos
Endocárdio/metabolismo , Proteínas de Homeodomínio/metabolismo , Inflamação/patologia , Proteínas do Tecido Nervoso/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptor Notch1/metabolismo , Regeneração , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Endocárdio/patologia , Células Endoteliais/metabolismo , Congelamento , Inflamação/metabolismo , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Sus scrofa , Regulação para Cima , CicatrizaçãoRESUMO
Mutations in the G proteincoupled receptor GPR126/ADGRG6 cause human diseases, including defective peripheral nervous system (PNS) myelination. To study GPR126 function, we generated new genetic mice and zebrafish models. Murine Gpr126 is expressed in developing heart endocardium, and global Gpr126 inactivation is embryonically lethal, with mutants having thin-walled ventricles but unaffected heart patterning or maturation. Endocardial-specific Gpr126 deletion does not affect heart development or function, and transgenic endocardial GPR126 expression fails to rescue lethality in Gpr126-null mice. Zebrafish gpr126 mutants display unaffected heart development. Gpr126 is also expressed in placental trophoblast giant cells. Gpr126-null mice with a heterozygous placenta survive but exhibit GPR126-defective PNS phenotype. In contrast, Gpr126-null embryos with homozygous mutant placenta die but are rescued by placental GPR126 expression. Gpr126-deficient placentas display down-regulation of preeclampsia markers Mmp9, Cts7, and Cts8. We propose that the placenta-heart axis accounts for heart abnormalities secondary to placental defects in Gpr126 mutants.