Bram van Wijk

Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart.

The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart, we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially derived fibroblasts eventually largely replace the endocardially derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development.

Epicardium and Myocardium Separate From a Common Precursor Pool by Crosstalk Between Bone Morphogenetic Protein– and Fibroblast Growth Factor–Signaling Pathways

Rationale: The epicardium contributes to the majority of nonmyocardial cells in the adult heart. Recent studies have reported that the epicardium is derived from Nkx2.5-positive progenitors and can differentiate into cardiomyocytes. Not much is known about the relation between the myocardial and epicardial lineage during development, whereas insights into these embryonic mechanisms could facilitate the design of future regenerative strategies. Objective: Acquiring insight into the signaling pathways involved in the lineage separation leading to the differentiation of myocardial and (pro)epicardial cells at the inflow of the developing heart. Methods and Results: We made 3D reconstructions of Tbx18 gene expression patterns to give insight into the developing epicardium in relation to the developing myocardium. Next, using DiI tracing, we show that the (pro)epicardium separates from the same precursor pool as the inflow myocardium. In vitro, we show that this lineage separation is regulated by a crosstalk between bone morphogenetic protein (BMP) signaling and fibroblast growth factor (FGF) signaling. BMP signaling via Smad drives differentiation toward the myocardial lineage, which is inhibited by FGF signaling via mitogen-activated protein kinase kinase (Mek)1/2. Embryos exposed to recombinant FGF2 in vivo show enhanced epicardium formation, whereas a misbalance between FGF and BMP by Mek1/2 inhibition and BMP stimulation causes a developmental arrest of the epicardium and enhances myocardium formation at the inflow of the heart. Conclusion: Our data show that FGF signaling via Mek1/2 is dominant over BMP signaling via Smad and is required to separate the epicardial lineage from precardiac mesoderm. Consequently, myocardial differentiation requires BMP signaling via Smad and inhibition of FGF signaling at the level of Mek1/2. These findings are of clinical interest for the development of regeneration-based therapies for heart disease.

Cardiac regeneration from activated epicardium.

In contrast to lower vertebrates, the mammalian heart has a very limited regenerative capacity. Cardiomyocytes, lost after ischemia, are replaced by fibroblasts. Although the human heart is able to form new cardiomyocytes throughout its lifespan, the efficiency of this phenomenon is not enough to substitute sufficient myocardial mass after an infarction. In contrast, zebrafish hearts regenerate through epicardial activation and initiation of myocardial proliferation. With this study we obtain insights into the activation and cellular contribution of the mammalian epicardium in response to ischemia. In a mouse myocardial infarction model we analyzed the spatio-temporal changes in expression of embryonic epicardial, EMT, and stem cell markers and the contribution of cells of the Wt1-lineage to the infarcted area. Though the integrity of the epicardial layer overlaying the infarct is lost immediately after the induction of the ischemia, it was found to be regenerated at three days post infarction. In this regenerated epicardium, the embryonic gene program is transiently re-expressed as well as proliferation. Concomitant with this activation, Wt1-lineage positive subepicardial mesenchyme is formed until two weeks post-infarction. These mesenchymal cells replace the cardiomyocytes lost due to the ischemia and contribute to the fibroblast population, myofibroblasts and coronary endothelium in the infarct, and later also to the cardiomyocyte population. We show that in mice, as in lower vertebrates, an endogenous, epicardium-dependent regenerative response to injury is induced. Although this regenerative response leads to the formation of new cardiomyocytes, their number is insufficient in mice but sufficient in lower vertebrates to replace lost cardiomyocytes. These molecular and cellular analyses provide basic knowledge essential for investigations on the regeneration of the mammalian heart aiming at epicardium-derived cells.

Cardiac myocyte follistatin-like 1 functions to attenuate hypertrophy following pressure overload

Factors secreted by the heart, referred to as “cardiokines,” have diverse actions in the maintenance of cardiac homeostasis and remodeling. Follistatin-like 1 (Fstl1) is a secreted glycoprotein expressed in the adult heart and is induced in response to injurious conditions that promote myocardial hypertrophy and heart failure. The aim of this study was to investigate the role of cardiac Fstl1 in the remodeling response to pressure overload. Cardiac myocyte-specific Fstl1-KO mice were constructed and subjected to pressure overload induced by transverse aortic constriction (TAC). Although Fstl1-KO mice displayed no detectable baseline phenotype, TAC led to enhanced cardiac hypertrophic growth and a pronounced loss in ventricular performance by 4 wk compared with control mice. Conversely, mice that acutely or chronically overexpressed Fstl1 were resistant to pressure overload-induced hypertrophy and cardiac failure. Fstl1-deficient mice displayed a reduction in TAC-induced AMP-activated protein kinase (AMPK) activation in heart, whereas Fstl1 overexpression led to increased myocardial AMPK activation under these conditions. In cultured neonatal cardiomyocytes, administration of Fstl1 promoted AMPK activation and antagonized phenylephrine-induced hypertrophy. Inhibition of AMPK attenuated the antihypertrophic effect of Fstl1 treatment. These results document that cardiac Fstl1 functions as an autocrine/paracrine regulatory factor that antagonizes myocyte hypertrophic growth and the loss of ventricular performance in response to pressure overload, possibly through a mechanism involving the activation of the AMPK signaling axis.

Wt1 and retinoic acid signaling in the subcoelomic mesenchyme control the development of the pleuropericardial membranes and the sinus horns.

Rationale: The cardiac venous pole is a common focus of congenital malformations and atrial arrhythmias, yet little is known about the cellular and molecular mechanisms that regulate its development. The systemic venous return myocardium (sinus node and sinus horns) forms only late in cardiogenesis from a pool of pericardial mesenchymal precursor cells. Objective: To analyze the cellular and molecular mechanisms directing the formation of the fetal sinus horns. Methods and Results: We analyzed embryos deficient for the Wt1 (Wilms tumor 1) gene and observed a failure to form myocardialized sinus horns. Instead, the cardinal veins become embedded laterally in the pleuropericardial membranes that remain tethered to the lateral body wall by the persisting subcoelomic mesenchyme, a finding that correlates with decreased apoptosis in this region. We show by expression analysis and lineage tracing studies that Wt1 is expressed in the subcoelomic mesenchyme surrounding the cardinal veins, but that this Wt1-positive mesenchyme does not contribute cells to the sinus horn myocardium. Expression of the Raldh2 (aldehyde dehydrogenase family 1, subfamily A2) gene was lost from this mesenchyme in Wt1−/− embryos. Phenotypic analysis of Raldh2 mutant mice rescued from early cardiac defects by retinoic acid food supply revealed defects of the venous pole and pericardium highly similar to those of Wt1−/− mice. Conclusions: Pericardium and sinus horn formation are coupled and depend on the expansion and correct temporal release of pleuropericardial membranes from the underlying subcoelomic mesenchyme. Wt1 and downstream Raldh2/retinoic acid signaling are crucial regulators of this process. Thus, our results provide novel insight into the genetic and cellular pathways regulating the posterior extension of the mammalian heart and the formation of its coelomic lining.

Comprehensive Gene-Expression Survey Identifies Wif1 as a Modulator of Cardiomyocyte Differentiation

During chicken cardiac development the proepicardium (PE) forms the epicardium (Epi), which contributes to several non-myocardial lineages within the heart. In contrast to Epi-explant cultures, PE explants can differentiate into a cardiomyocyte phenotype. By temporal microarray expression profiles of PE-explant cultures and maturing Epi cells, we identified genes specifically associated with differentiation towards either of these lineages and genes that are associated with the Epi-lineage restriction. We found a central role for Wnt signaling in the determination of the different cell lineages. Immunofluorescent staining after recombinant-protein incubation in PE-explant cultures indicated that the early upregulated Wnt inhibitory factor-1 (Wif1), stimulates cardiomyocyte differentiation in a similar manner as Wnt stimulation. Concordingly, in the mouse pluripotent embryogenic carcinoma cell line p19cl6, early and late Wif1 exposure enhances and attenuates differentiation, respectively. In ovo exposure of the HH12 chicken embryonic heart to Wif1 increases the Tbx18-positive cardiac progenitor pool. These data indicate that Wif1 enhances cardiomyogenesis.

Growth and Differentiation of the Developing Heart

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