Disturbed myocardial connexin 43 and N-cadherin expressions in hypoplastic left heart syndrome and borderline left ventricle.

OBJECTIVES: Borderline left ventricle is the left ventricular morphology at the favorable end of the hypoplastic left heart syndrome. In contrast to the severe end, it is suitable for biventricular repair. Wondering whether it is possible to identify cases suitable for biventricular repair from a developmental viewpoint, we investigated the myocardial histology of borderline and severely hypoplastic left ventricles. METHODS: Postmortem specimens of neonatal, unoperated human hearts with severe hypoplastic left heart syndrome and borderline left ventricle were compared with normal specimens and hearts from patients with transposition of the great arteries. After tissue sampling of the lateral walls of both ventricles, immunohistochemical and immunofluorescence stainings against cardiac troponin I, N-cadherin, and connexin 43, important for proper cardiac differentiation, were done. RESULTS: All severely hypoplastic left hearts (7/7) and most borderline left ventricle hearts (4/6) showed reduced sarcomeric expressions of troponin I in left and right ventricles. N-cadherin and connexin 43 expressions were reduced in intercalated disks. The remaining borderline left ventricle hearts (2/6) were histologically closer to control hearts. CONCLUSIONS: Four of 6 borderline left ventricle hearts showed myocardial histopathology similar to the severely hypoplastic left hearts. The remainder were similar to normal hearts. Our results and knowledge regarding the role of epicardial-derived cells in myocardial differentiation lead us to postulate that an abnormal epicardial-myocardial interaction could explain the observed histopathology. Defining the histopathologic severity with preoperative myocardial biopsy samples of hearts with borderline left ventricle might provide a diagnostic tool for preoperative decision making.

Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes.

During heart development, cells from the proepicardial organ spread over the naked heart tube to form the epicardium. From here, epicardium-derived cells (EPDCs) migrate into the myocardium. EPDCs proved to be indispensable for the formation of the ventricular compact zone and myocardial maturation, by largely unknown mechanisms. In this study we investigated in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction, as well as the expression and cellular distribution of proteins involved in myocardial maturation. Embryonic quail EPDCs induced proliferation of neonatal mouse cardiomyocytes. This required cell-cell interactions, as proliferation was not observed in transwell cocultures. Western blot analysis showed elevated levels of electrical and mechanical junctions (connexin43, N-cadherin), sarcomeric proteins (Troponin-I, alpha-actinin), extracellular matrix (collagen I and periostin) in cocultures of EPDCs and cardiomyocytes. Immunohistochemistry indicated more membrane-bound expression of Cx43, N-cadherin, the mechanotransduction molecule focal adhesion kinase, and higher expression of the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a). Newly developed software for analysis of directionality in immunofluorescent stainings showed a quantitatively determined enhanced cellular alignment of cardiomyocytes. This was functionally related to increased contraction. The in vitro effects of EPDCs on cardiomyocytes were confirmed in three reciprocal in vivo models for EPDC-depletion (chicken and mice) in which downregulation of myocardial N-cadherin, Cx43, and FAK were observed. In conclusion, direct interaction of EPDCs with cardiomyocytes induced proliferation, correct mechanical and electrical coupling of cardiomyocytes, ECM-deposition and concurrent establishment of cellular array. These findings implicate that EPDCs are ideal candidates as adjuvant cells for cardiomyocyte integration during cardiac (stem) cell therapy.

Cardiac malformations in Pdgfralpha mutant embryos are associated with increased expression of WT1 and Nkx2.5 in the second heart field.

Platelet-derived growth factor receptor alpha (Pdgfralpha) identifies cardiac progenitor cells in the posterior part of the second heart field. We aim to elucidate the role of Pdgfralpha in this region. Hearts of Pdgfralpha-deficient mouse embryos (E9.5-E14.5) showed cardiac malformations consisting of atrial and sinus venosus myocardium hypoplasia, including venous valves and sinoatrial node. In vivo staining for Nkx2.5 showed increased myocardial expression in Pdgfralpha mutants, confirmed by Western blot analysis. Due to hypoplasia of the primary atrial septum, mesenchymal cap, and dorsal mesenchymal protrusion, the atrioventricular septal complex failed to fuse. Impaired epicardial development and severe blebbing coincided with diminished migration of epicardium-derived cells and myocardial thinning, which could be linked to increased WT1 and altered alpha4-integrin expression. Our data provide novel insight for a possible role for Pdgfralpha in transduction pathways that lead to repression of Nkx2.5 and WT1 during development of posterior heart field-derived cardiac structures.

Platelet-derived growth factor is involved in the differentiation of second heart field-derived cardiac structures in chicken embryos.

For the establishment of a fully functional septated heart, addition of myocardium from second heart field-derived structures is important. Platelet-derived growth factors (PDGFs) are known for their role in cardiovascular development. In this study, we aim to elucidate this role of PDGF-A, PDGF-C, and their receptor PDGFR-alpha. We analyzed the expression patterns of PDGF-A, -C, and their receptor PDGFR-alpha during avian heart development. A spatiotemporal pattern of ligands was seen with colocalization of the PDGFR-alpha. This was found in second heart field-derived myocardium as well as the proepicardial organ (PEO) and epicardium. Mechanical inhibition of epicardial outgrowth as well as chemical disturbance of PDGFR-alpha support a functional role of the ligands and the receptor in cardiac development.

Epicardium-derived cells in development of annulus fibrosis and persistence of accessory pathways.

BACKGROUND: The developmental mechanisms underlying the persistence of myocardial accessory atrioventricular pathways (APs) that bypass the annulus fibrosis are mainly unknown. In the present study, we investigated the role of epicardium-derived cells (EPDCs) in annulus fibrosis formation and the occurrence of APs. METHODS AND RESULTS: EPDC migration was mechanically inhibited by in ovo microsurgery in quail embryos. In ovo ECGs were recorded in wild-type (n=12) and EPDC-inhibited (n=12) hearts at Hamburger-Hamilton (HH) stages 38 to 42. Subsequently, in these EPDC-inhibited hearts (n=12) and in additional wild-type hearts (n=45; HH 38-42), ex ovo extracellular electrograms were recorded. Electrophysiological data were correlated with differentiation markers for cardiomyocytes (MLC2a) and fibroblasts (periostin). In ovo ECGs showed significantly shorter PR intervals in EPDC-inhibited hearts (45+/-10 ms) than in wild-type hearts (55+/-8 ms, 95% CI 50 to 60 ms, P=0.030), whereas the QRS durations were significantly longer in EPDC-inhibited hearts (29+/-14 versus 19+/-2 ms, 95% CI 18 to 21 ms, P=0.011). Furthermore, ex ovo extracellular electrograms (HH 38-42) displayed base-first ventricular activation in 44% (20/45) of wild-type hearts, whereas in all EPDC-inhibited hearts (100%, 12/12), the ventricular base was activated first (P<0.001). Small periostin- and MLC2a-positive APs were found mainly in the posteroseptal region of both wild-type and EPDC-inhibited hearts. Interestingly, in all (n=10) EPDC-inhibited hearts, additional large periostin-negative and MLC2a-positive APs were found in the right and left lateral free wall coursing through marked isolation defects in the annulus fibrosis until the last stages of embryonic development. CONCLUSIONS: EPDCs play an important role in annulus fibrosis formation. EPDC outgrowth inhibition may result in marked defects in the fibrous annulus with persistence of large APs, which results in ventricular preexcitation on ECG. These APs may provide a substrate for postnatally persistent reentrant arrhythmias.

Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos: Correlation with abnormal epicardial development.

Epicardium and epicardium-derived cells have been shown to be necessary for myocardial differentiation. To elucidate the function of podoplanin in epicardial development and myocardial differentiation, we analyzed podoplanin knockout mouse embryos between embryonic day (E) 9.5 and E15.5 using immunohistochemical differentiation markers, morphometry, and three-dimensional reconstructions. Podoplanin null mice have an increased embryonic lethality, possibly of cardiac origin. Our study reveals impairment in the development of the proepicardial organ, epicardial adhesion, and spreading and migration of the epicardium-derived cells. Mutant embryos show a hypoplastic and perforated compact and septal myocardium, hypoplastic atrioventricular cushions resulting in atrioventricular valve abnormalities, as well as coronary artery abnormalities. The epicardial pathology is correlated with reduced epithelial-mesenchymal transformation caused by up-regulation of E-cadherin, normally down-regulated by podoplanin. Our results demonstrate a role for podoplanin in normal cardiac development based on epicardial-myocardial interaction. Abnormal epicardial differentiation and reduced epithelial-mesenchymal transformation result in deficient epicardium-derived cells leading to myocardial pathology and cardiac anomalies.

Periostin expression by epicardium-derived cells is involved in the development of the atrioventricular valves and fibrous heart skeleton.

The epicardium is embryologically formed by outgrowth of proepicardial cells over the naked heart tube. Epicardium-derived cells (EPDCs) migrate into the myocardium, contributing to myocardial architecture, valve development, and the coronary vasculature. Defective EPDC formation causes valve malformations, myocardial thinning, and coronary defects. In the atrioventricular (AV) valves and the fibrous heart skeleton isolating atrial from ventricular myocardium, EPDCs colocalize with periostin, a matrix molecule involved in remodeling. We investigated whether proepicardial outgrowth inhibition affected periostin expression and how this related to development of the AV valves and fibrous heart skeleton. Periostin expression by epicardium and EPDCs was confirmed in vitro in primary cultures of human and quail EPDCs. Disturbing EPDC formation in quail embryos reduced periostin expression in the endocardial cushions and AV junction. Disturbed fibrous tissue development resulted in AV myocardial connections reflected by preexcitation electrocardiographic patterns. We conclude that EPDCs are local producers of periostin. Disturbance of EPDC formation results in decreased cardiac periostin levels and hampers the development of fibrous tissue in AV junction and the developing AV valves. The resulting cardiac anomalies might link to Wolff-Parkinson White syndrome with persistent AV myocardial connections.

PDGF-B signaling is important for murine cardiac development: its role in developing atrioventricular valves, coronaries, and cardiac innervation.

We hypothesized that PDGF-B/PDGFR-beta-signaling is important in the cardiac contribution of epicardium-derived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf-b-/- and Pdgfr-beta-/- mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for alphaSMA, periostin, ephrinB2, EphB4, VEGFR-2, Dll1, and NCAM was performed on wild-type and knockout embryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest-related innervation deficits. This implies a role for PDGF-B/PDGFR-beta-signaling specifically in the contribution of these cell lineages to cardiac development.

Origin, fate, and function of epicardium-derived cells (EPDCs) in normal and abnormal cardiac development.

During heart development, cells of the primary and secondary heart field give rise to the myocardial component of the heart. The neural crest and epicardium provide the heart with a considerable amount of nonmyocardial cells that are indispensable for correct heart development. During the past 2 decades, the importance of epicardium-derived cells (EPDCs) in heart formation became increasingly clear. The epicardium is embryologically formed by the outgrowth of proepicardial cells over the naked heart tube. Following epithelial-mesenchymal transformation, EPDCs form the subepicardial mesenchyme and subsequently migrate into the myocardium, and differentiate into smooth muscle cells and fibroblasts. They contribute to the media of the coronary arteries, to the atrioventricular valves, and the fibrous heart skeleton. Furthermore, they are important for the myocardial architecture of the ventricular walls and for the induction of Purkinje fiber formation. Whereas the exact signaling cascades in EPDC migration and function still need to be elucidated, recent research has revealed several factors that are involved in EPDC migration and specialization, and in the cross-talk between EPDCs and other cells during heart development. Among these factors are the Ets transcription factors Ets-1 and Ets-2. New data obtained with lentiviral antisense constructs targeting Ets-1 and Ets-2 specifically in the epicardium indicate that both factors are independently involved in the migratory behavior of EPDCs. Ets-2 seems to be especially important for the migration of EPDCs into the myocardial wall, and to subendocardial positions in the atrioventricular cushions and the trabeculae. With respect to the clinical importance of correct EPDC development, the relation with coronary arteriogenesis has been noted well before. In this review, we also propose a role for EPDCs in cardiac looping, and emphasize their contribution to the development of the valves and myocardial architecture. Lastly, we focus on the congenital heart anomalies that might be caused primarily by an epicardial developmental defect.

Epicardium-derived cells are important for correct development of the Purkinje fibers in the avian heart.

During embryonic development, the proepicardial organ (PEO) grows out over the heart surface to form the epicardium. Following epithelial-mesenchymal transformation, epicardium-derived cells (EPDCs) migrate into the heart and contribute to the developing coronary arteries, to the valves, and to the myocardium. The peripheral Purkinje fiber network develops from differentiating cardiomyocytes in the ventricular myocardium. Intrigued by the close spatial relationship between the final destinations of migrating EPDCs and Purkinje fiber differentiation in the avian heart, that is, surrounding the coronary arteries and at subendocardial sites, we investigated whether inhibition of epicardial outgrowth would disturb cardiomyocyte differentiation into Purkinje fibers. To this end, epicardial development was inhibited mechanically with a membrane, or genetically, by suppressing epicardial epithelial-to-mesenchymal transformation with antisense retroviral vectors affecting Ets transcription factor levels (n=4, HH39-41). In both epicardial inhibition models, we evaluated Purkinje fiber development by EAP-300 immunohistochemistry and found that restraints on EPDC development resulted in morphologically aberrant differentiation of Purkinje fibers. Purkinje fiber hypoplasia was observed both periarterially and at subendocardial positions. Furthermore, the cells were morphologically abnormal and not aligned in orderly Purkinje fibers. We conclude that EPDCs are instrumental in Purkinje fiber differentiation, and we hypothesize that they cooperate directly with endothelial and endocardial cells in the development of the peripheral conduction system.

Platelet-derived growth factors in the developing avian heart and maturating coronary vasculature.

Platelet-derived growth factors (PDGFs) are important in embryonic development. To elucidate their role in avian heart and coronary development, we investigated protein expression patterns of PDGF-A, PDGF-B, and the receptors PDGFR-alpha and PDGFR-beta using immunohistochemistry on sections of pro-epicardial quail-chicken chimeras of Hamburger and Hamilton (HH) 28-HH35. PDGF-A and PDGFR-alpha were expressed in the atrial septum, sinus venosus, and throughout the myocardium, with PDGFR-alpha retreating to the trabeculae at later stages. Additionally, PDGF-A and PDGFR-alpha were present in outflow tract cushion mesenchyme and myocardium, respectively. Small cardiac nerves and (sub)epicardial cells expressed PDGF-B and PDGFR-beta. Furthermore, endothelial cells expressed PDGF-B, while vascular smooth muscle cells and interstitial epicardium-derived cells expressed PDGFR-beta, indicating a role in coronary maturation. PDGF-B is also present in ventricular septal development, in the absence of any PDGFR. Epicardium-derived cells in the atrioventricular cushions expressed PDGFR-beta. We conclude that all four proteins are involved in myocardial development, whereas PDGF-B and PDGFR-beta are specifically important in coronary maturation.

Coronary artery and orifice development is associated with proper timing of epicardial outgrowth and correlated Fas-ligand-associated apoptosis patterns.

The proepicardial organ provides differentiated cell types to the myocardial wall and facilitates coronary development. Ingrowth of the coronary arteries into the aorta has recently been linked to apoptosis. This study was set up to examine the effect of an inhibition of epicardial outgrowth on apoptotic patterning and coronary development. Epicardial outgrowth was blocked at HH15-17 in quail embryos, which survived until HH25-35 (n=33). Embryos with complete inhibition of outgrowth did not survive after HH29. These embryos presented with thin compact myocardium, devoid of vessels. In embryos with delayed epicardial outgrowth the phenotype was less severe, and surviving embryos were studied up to HH35. In these embryos, myocardial vascularization was poor and apoptosis in the peritruncal region at HH30 was diminished. Embryos at HH35 displayed an abnormal coronary network and absent coronary orifices. In a further set of experiments (n=10), outgrowth was inhibited in chicken embryos at HH15, followed by transplantation of a quail proepicardial organ into the pericardial cavity to rescue cardiac phenotype. These chimeras were studied at HH29 and HH35. Myocardial development was restored; however, in 3 of 4 embryos (HH35), the coronary orifices were absent. Examination of double stainings of quail-chicken chimeras revealed that EPDCs produce Fas ligand as an apoptotic inductor at sites of coronary ingrowth. In the absence of proper timing of epicardial outgrowth, myocardial development and vascularization are disturbed. Also apoptosis in the peritruncal region is diminished. During later development, this leads to defective or absent connections of the coronary system to the systemic circulation.

Myocardial heterogeneity in permissiveness for epicardium-derived cells and endothelial precursor cells along the developing heart tube at the onset of coronary vascularization.

The coronary vasculature develops from mesothelial and endothelial precursor cells (EPCs) derived from the proepicardial organ (PEO), which migrate over the heart to form the epicardium. By epithelial-mesenchymal transition (EMT), the subepicardium and epicardium-derived cells (EPDCs) are formed. EPDCs migrate into the myocardium, where they differentiate into smooth muscle cells and fibroblasts that stabilize the developing coronary vasculature and contribute to myocardial architecture. Complete PEO ablation results in embryonic lethality due to cardiac defects, including a looping disorder with a too wide inner curvature. To investigate the behavior of early coronary contributors, we analyzed normal quail embryos and found lumenized endothelial vessels in the subepicardium already at stage HH19. Furthermore, EPCs had penetrated into the myocardium of the inner curvature. To confirm that the myocardium of the inner curvature is specifically permissive for EPCs and to study early EPDC migration in more detail, chimeric chicken embryos harboring a quail PEO were analyzed. Lateral epicardial outgrowth and EMT were observed throughout, but migration into the myocardium was restricted to the inner curvature between HH19 and 22. The permissive myocardial area expanded to the atrium, atrioventricular canal, and trabeculated ventricle at stage HH23-24. In contrast, outflow tract myocardium was never found to be permissive for EPDCs and EPCs until HH30, not even when the quail PEO was attached directly onto it. We conclude that early coronary formation starts in the inner curvature and hypothesize that the presence of PEO-derived cells is essential for the maturation of the inner curvature and subsequent looping of the heart tube.

Ets-1 and Ets-2 transcription factors are essential for normal coronary and myocardial development in chicken embryos.

In the development of a functional myocardium and formation of the coronary vasculature, epicardium-derived cells play an essential role. The proepicardial organ contributes to the developing coronary system by delivering mural cells to the endothelium-lined vessels. In search of genes that regulate the behavior of (pro)epicardial cells, the Ets-1 and Ets-2 transcription factors stand out as strong candidates. In the present study, the hypothesis that Ets transcription factors have a role in proper coronary and myocardial development was tested via antisense technology, by targeting Ets-1 and Ets-2 mRNAs to downregulate protein expression in chicken embryos. The results suggest that hereby the development of the coronary system is hampered, primarily by defects in the process of epithelial-mesenchymal transformation of the mesothelia of the primary and secondary heart fields. This was indicated by a lack of periarterial and epicardial mesenchyme, of peripheral coronary smooth muscle cells, and changes in myocardial morphology. A defect in myocardial perfusion caused by the absence of one or both coronary ostia seems to be "solved" by the development of numerous small fistulae connecting the ventricular lumen with the subepicardially located coronary vessels. The presence of coronary vascular aberrations in the antisense-Ets phenotype enabled us for the first time to study abnormal coronary development in a model that is not lethal to the embryo.

The role of the epicardium and neural crest as extracardiac contributors to coronary vascular development.

At species-specific times in embryonic development, the pro-epicardial organ appears as an outcropping of the mesothelial body wall, near the sinus venosus-liver region. The pro-epicardial vesicles attach to the myocardium, flatten, and join to form the epicardium. The epicardium shows epithelial-mesenchymal transformation: cells detach from the epithelium, fill the subepicardial space, and invade the heart tube. Epicardium-derived cells migrate as far as the core of the endocardial cushions, which differentiate into the atrioventricular valve leaflets. In the cardiac wall, other epicardium-derived cells differentiate into interstitial fibroblasts and adventitial and smooth muscle cells of the coronary arteries. Using neural crest tracings in mouse embryos (Wnt1-Cre-lacZ), we studied the patterning of cardiac neural crest cells during development. Participation of neural crest cells in the formation of the vascular media could not be excluded, although epicardium-derived cells have hitherto been considered responsible for formation of the coronary arterial smooth muscle cells. The endothelial cells of the coronary network derive mostly from the endothelium of the sinus venosus-liver region by vasculogenesis and angiogenesis. However, an epicardium-derived cell origin of some endothelial cells cannot be ruled out. The coronary vasculature is closely related to the differentiating Purkinje network, but isolated epicardium-derived cells are also associated with Purkinje cells. After ablating the pro-epicardial organ in quail embryos, we found severe malformations in the myocardial architecture, leading to the hypothesis that epicardium-derived cells give instructive signals to the myocardium for proper differentiation of the compact and the trabeculated compartments.