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.

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.

Bone morphogenetic proteins (BMP) are involved in the regulation of a plethora of processes underlying cardiovascular development. This review summarizes the effects of BMP and the signaling pathways that regulate the differentiation of cardiomyocytes from mesoderm in the heart-forming region and at the distal borders of the heart tube from the second heart field. Subsequently, the role of BMPs in the formation of the ventricular chambers and septovalvulogenesis in the atrioventricular canal and outflow tract is described. Finally, the effects of BMPs in stem cell biology and cardiac regeneration are discussed.

Proepicardial cells give rise to epicardium, coronary vasculature and cardiac fibroblasts. The proepicardium is derived from the mesodermal lining of the prospective pericardial cavity that simultaneously contributes myocardium to the venous pole of the elongating primitive heart tube. Using proepicardial explant cultures, we show that proepicardial cells have the potential to differentiate into cardiac muscle cells, reflecting the multipotency of this pericardial mesoderm. The differentiation into the myocardial or epicardial lineage is mediated by the cooperative action of BMP and FGF signaling. BMP2 is expressed in the distal IFT myocardium and stimulates cardiomyocyte formation. FGF2 is expressed in the proepicardium and stimulates differentiation into the epicardial lineage. In the base of the proepicardium, coexpression of BMP2 and FGF2 inhibits both myocardial and epicardial differentiation. We conclude that the epicardial/myocardial lineage decisions are mediated by an extrinsic, inductive mechanism, which is determined by the position of the cells in the pericardial mesoderm.

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.TSC-36 | angiogenesis

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. (Circ Res. 2009;105:431-441.)Key Words: cardiovascular development Ⅲ proepicardium Ⅲ epicardium Ⅲ BMP Ⅲ FGF Ⅲ regeneration I n contrast to the adult heart, the embryonic heart tube is devoid of nonmyocardial cells and an epicardium, consisting of an outer myocardial and an inner endocardial layer separated by cardiac jelly. The heart tube expands by recruitment of progenitor cells from the splanchnic mesoderm at both poles of the heart. 1,2 The formation of the majority of nonmyocardial cells starts with the development of the proepicardium from splanchnic mesodermal cells at the inflow of the heart. Its villous outgrowths extend into the pericardial cavity, attach to the atrioventricular canal and gradually envelop the entire "naked" heart tube, deriving the epicardium. A subset of epicardial cells undergoes epithelial-to-mesenchymal transformation. The formed subepicardial mesenchymal cells contribute to the nonmyocardial component of the heart ie, the coronary vessels and the cardiac fibroblasts. In the adult heart, the nonmyocardial component occupies approximately 25% of the my...

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. T he systemic venous return of the mature mammalian heart, which terminates in the right atrium, consists of multiple anatomic components including the myocardial sleeves of the right superior and inferior caval veins, the sinoatrial node (SAN), the coronary sinus (persisting left caval vein in the mouse), and the sinus venarum. 1 The systemic venous return is a focus of congenital malformations and atrial arrhythmias, 2,3 necessitating insight into the cellular and molecular programs by which it arises during cardiac development. Most myocardial components of the heart are not represented in its initial anlage, but are continuously added by recruitment and differentiation of precursor cells. 4 The sinus horns, the myocardial parts of the common cardinal veins upstream of the venous valves that bulge into the pericardial cavity, form from pericardial precursors that differentiate into sinus venosus myocardium around the systemic venous connection to the atrium. 5 They form only after embryonic day (E)9.5, when outflow tract, left and right ventricle, and the common atrium have already been established. In adults, most of the right sinus horn myocardium is incorporated into the right atrium to form the sinus venarum. In humans, the left sinus horn will lose its connection to the body and form the coronary sinus, whereas in mouse it will persist as the left superior caval vein.Few genes regulating venous pole development have been characterized, including the Tbx18 (T-box transcription factor 18) that marks the sinus horn lineage. 14 which harbors a tetrameric repeat of the RAR␤2 RARE linked to the Hsp68 minimal promoter used to determine RA signaling, were all described before. For the Wt1 BAC-IRES-EGFPCre transgenic line, the BAC clone RP23-266M16 was modified by inserting an IRES/EGFP-Cre cassette 17bp downstream of the translation stop site of the Wt1 gene. (The generation and evaluation of this line will be described elsewhere.) In Wt1 tTA mice, the coding sequence of an improved tetracyclinedependent transactivator tTA2S 15 was introduced into the Wt1 locus by gene targeting (E Lausch, S Fees, C Steinwender, C Spangenberg, L Eshkind, E Bockamp, B Zabel, manuscript in preparation). All mouse lines were maintained on an outbred (NMRI or FvB) background.An expanded Materials and Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Results Defects in the Systemic Venous Return of Wt1-Deficient Hearts Wt1Ϫ/Ϫ mice maintained on an NMRI outbred background died during midgestation presenting defects reported on earlier, including lack of kidneys, diaphragmatic hernia, and defects in coronary vessel formation the latter of which may underlie embryonic lethality. Yet, histological inspection of surviving embryos at E14.5 revealed an undescribed variation in the systemic venous ...

IMPORTANCEIn children undergoing heart surgery, nitric oxide administered into the gas flow of the cardiopulmonary bypass oxygenator may reduce postoperative low cardiac output syndrome, leading to improved recovery and shorter duration of respiratory support. It remains uncertain whether nitric oxide administered into the cardiopulmonary bypass oxygenator improves ventilator-free days (days alive and free from mechanical ventilation).OBJECTIVE To determine the effect of nitric oxide applied into the cardiopulmonary bypass oxygenator vs standard care on ventilator-free days in children undergoing surgery for congenital heart disease.DESIGN, SETTING, AND PARTICIPANTS Double-blind, multicenter, randomized clinical trial in 6 pediatric cardiac surgical centers in Australia, New Zealand, and the Netherlands. A total of 1371 children younger than 2 years undergoing congenital heart surgery were randomized between July 2017 and April 2021, with 28-day follow-up of the last participant completed on May 24, 2021. INTERVENTIONS Patients were assigned to receive nitric oxide at 20 ppm delivered into the cardiopulmonary bypass oxygenator (n = 679) or standard care cardiopulmonary bypass without nitric oxide (n = 685). MAIN OUTCOMES AND MEASURESThe primary end point was the number of ventilator-free days from commencement of bypass until day 28. There were 4 secondary end points including a composite of low cardiac output syndrome, extracorporeal life support, or death; length of stay in the intensive care unit; length of stay in the hospital; and postoperative troponin levels. RESULTS Among 1371 patients who were randomized (mean [SD] age, 21.2 [23.5] weeks; 587 girls [42.8%]), 1364 (99.5%) completed the trial. The number of ventilator-free days did not differ significantly between the nitric oxide and standard care groups, with a median of 26.6 days (IQR, 24.4 to 27.4) vs 26.4 days (IQR, 24.0 to 27.2), respectively, for an absolute difference of −0.01 days (95% CI, −0.25 to 0.22; P = .92). A total of 22.5% of the nitric oxide group and 20.9% of the standard care group developed low cardiac output syndrome within 48 hours, needed extracorporeal support within 48 hours, or died by day 28, for an adjusted odds ratio of 1.12 (95% CI, 0.85 to 1.47). Other secondary outcomes were not significantly different between the groups. CONCLUSIONS AND RELEVANCEIn children younger than 2 years undergoing cardiopulmonary bypass surgery for congenital heart disease, the use of nitric oxide via cardiopulmonary bypass did not significantly affect the number of ventilator-free days. These findings do not support the use of nitric oxide delivered into the cardiopulmonary bypass oxygenator during heart surgery.