Extracellular matrix from embryonic myocardium elicits an early morphogenetic event in cardiac endothelial differentiation

A critical step in early cardiac morphogenesis can be faithfully duplicated in culture using a hydrated collagen substratum, and thereby serves as a useful model system for studying the molecular mechanisms of cell differentiation. Results from previous work suggested that the myocardium in the atrioventricular canal (AV) region of the developing chick heart secretes extracellular proteins into its associated basement membrane, which may function to promote an epithelial-mesenchymal transition of endothelium to form prevalvular fibroblasts (E. L. Krug, R. B. Runyan, and R. R. Markwald, 1985, Dev. Biol. 112, 414-426; C. H. Mjaatvedt, R. C. Lepera, and R. R. Markwald, 1987, Dev. Biol., in press). In the present study we show that an EDTA-soluble extract of embryonic chick hearts can substitute for the presence of myocardium, the presumptive stimulator tissue, in initiating mesenchyme formation from AV endothelium in culture. Ventricular endothelium was unresponsive to this material in keeping with observed in situ behavior. AV endothelial cells did not survive beyond 4-5 days when cultured in the absence of either the EDTA-soluble heart extract, myocardial conditioned medium, or the myocardium itself. Antibody prepared against a particulate fraction of the EDTA-solubilized heart extract immunohistochemically localized this material to the myocardial basement membrane. In addition, conditioned medium from embryonic myocardial cultures effectively induced mesenchyme formation. Neither a variety of growth factors nor a sarcoma basement membrane preparation were effective in promoting mesenchyme formation indicating a selectivity of the responding embryonic AV endothelial cells to myocardial basement membrane. These observations reflect a truly inductive phenomenon as there was an absolute dependence on the presence of the stimulating substance/tissue and retention, in culture, of both the temporal and regional characteristics observed in situ. This is in contrast to the results of others investigating the cytodifferentiation of committed cells whose phenotypic expression can be either accelerated or diminished but not obligatorily regulated by a specific agent, thus making the interpretation of data difficult, if not irrelevant, to the study of differentiation. The results of this study provide direct experimental support for the hypothesis that extracellular matrix can indeed serve as a direct stimulator or "secondary inducer" of cytodifferentiation.     

An antiserum (ES1) against a particulate form of extracellular matrix blocks the transition of cardiac endothelium into mesenchyme in culture

The epithelial-mesenchymal transition of cardiac endothelium is a critical developmental event in the formation of valvular and septal anlagen. We have demonstrated previously that this event can be mimicked in culture by treating atrioventricular canal (AV) endothelium with EDTA-soluble proteins extracted from embryonic heart tissue. This activity was fractionated by ultracentrifugation of the EDTA extract, indicating that the critical proteins existed as a multicomponent complex. Based on these results we propose that: (1) the in vitro particulates in EDTA extracts correspond to an observed particulate form of extracellular matrix within the myocardial basement membrane (MBM) of mesenchyme-forming regions and (2) one or more of the proteins in the MBM particulates function to elicit the epithelial-mesenchymal transition. To test these hypotheses we utilized an antiserum, termed ES1, prepared against EDTA-extractable particulates from embryonic chick hearts. Both ES1 and an anti-fibronectin monoclonal antibody (M3H) co-localized in situ to particles within the MBM; however, no ES1 reactivity towards fibronectin could be detected by ELISA or immunoblot analysis. The ES1-positive MBM particulates were removed by extraction with EDTA, but not with PBS, indicating a divalent cation-mediated association of the constituent proteins. ES1 antibodies recognized two major (28 and 46 kDa) and three minor (93, 109, and 180 kDa) proteins on immunoblots of EDTA-extractable proteins. When tested in culture, ES1 antiserum inhibited the formation of mesenchyme from AV endothelium in a dose-dependent manner, while M3H did not. These results are consistent with an active role for one or more of the ES1 antigens in initiating the formation of AV mesenchyme. The localization of ES1 antigens to the extracellular matrix at other dynamic interfaces, e.g., ectoderm/neural tube and limb bud ectoderm/mesoderm, point to a potentially general importance of ES1 antigens in mediating similar developmental interactions.     

Signal transduction of a tissue interaction during embryonic heart development.

During early cardiac development, progenitors of the valves and septa of the heart are formed by an epithelial-mesenchymal cell transformation of endothelial cells of the atrioventricular (AV) canal. We have previously shown that this event is due to an interaction between the endothelium and products of the myocardium found within the extracellular matrix. The present study examines signal transduction mechanisms governing this differentiation of AV canal endothelium. Activators of protein kinase C (PKC), phorbol myristate acetate (PMA) and mezerein, both produced an incomplete phenotypic transformation of endothelial cells in an in vitro bioassay for transformation. On the other hand, inhibitors of PKC (H-7 and staurosporine) and tyrosine kinase (genistein) blocked cellular transformation in response to the native myocardium or a myocardially-conditioned medium. Intracellular free calcium concentration ([Ca2+]i) was measured in single endothelial cells by microscopic digital analysis of fura 2 fluorescence. Addition of a myocardial conditioned medium containing the transforming stimulus produced a specific increase in [Ca2+]i in "competent" AV canal, but not ventricular, endothelial cells. Epithelial-mesenchymal cell transformation was inhibited by pertussis toxin but not cholera toxin. These data lead to the hypothesis that signal transduction of this tissue interaction is mediated by a G protein and one or more kinase activities. In response to receptor activation, competent AV canal endothelial cells demonstrate an increase in [Ca2+]i. Together, the data provide direct evidence for a regional and temporal regulation of signal transduction processes which mediate a specific extracellular matrix-mediated tissue interaction in the embryo.     

Induction of an epithelial-mesenchymal transition by an in vivo adheron-like complex

The embryonic vertebrate heart consists of two epithelia: the myocardium and endothelium, separated by the myocardial basement membrane (MBM). The myocardium has been shown to induce endothelial transformation into prevalvular mesenchyme in a temporally and site restricted manner. Previously, we hypothesized that the myocardial-endothelial interaction is mediated in vivo by aggregates of 30-nm particles in the MBM which can be removed by EDTA extraction. These MBM extracts contain fibronectin and other lower Mr proteins and can initiate an epithelial-mesenchymal transition in the AV (atrioventricular canal) endothelium of embryonic chick heart in collagen gel culture. These and other data suggested that the 30-nm multicomponent particles are similar, structurally and compositionally, to multimolecular complexes, termed adherons, secreted by L6 muscle cells in culture. The purpose of this study was to (1) test whether the removal of the 30-nm particles from MBM extracts of embryonic chick hearts would remove the in vitro biological activity and (2) determine if the fractionated MBM extracts can cause AV endothelial cells to follow the same differentiation pathway observed in vivo by monitoring immunohistochemically the cell surface expression of N-CAM. Results showed that centrifugation of extract at 100,000g for 1 hr produced a supernatant fraction that was unable to initiate mesenchyme formation from AV endothelium. However, the resuspended pellet fraction did initiate differentiation of endothelium into mesenchyme. Conditioned medium from L6 skeletal muscle cultures could not substitute for the EDTA extract of embryonic heart. Endothelial cells undergoing the transition to form mesenchyme, both in vivo and in vitro, showed a concomitant decrease in N-CAM staining. This suggested that the pellet-induced formation of migrating cells in the collagen gels is not the result a novel in vitro phenomenon.     

Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field

A hallmark of heart-valve development is the swelling and deposition of extracellular matrix in the heart-valve region. Only myocardium overlying this region can signal to underlying endothelium and cause it to lose cell-cell contacts, delaminate, and invade the extracellular space abutting myocardium and endocardium to form endocardial cushions (EC) in a process known as epithelial to mesenchymal transformation (EMT). The heart-valve myocardium expresses bone morphogenetic protein-2 (Bmp2) coincident with development of valve mesenchyme. BMPs belong to the transforming growth factor beta superfamily (TGF-beta) and play a wide variety of roles during development. We show that conditional ablation of Bmp2 in cardiac progenitors results in cell fate changes in which the heart-valve region adopts the identity of differentiated chamber myocardium. Moreover, Bmp2-deficient hearts fail to induce production and deposition of matrix at the heart-valve-forming region, resulting in the inability of the endothelium to swell and impairing the development of ECs. Furthermore, in collagen invasion assays, Bmp2 mutant endothelium is incapable of undergoing EMT, and addition of BMP2 protein to mutant heart explants rescues this phenotype. Our results demonstrate that Bmp2 is both necessary and sufficient to specify a field of cardiac progenitor cells as the heart-valve-inducing region amid developing atria and ventricles.     

Biosynthesis of glycosaminoglycans by the separated tissues of the embryonic chick cornea

Corneal tissues (epithelium, endothelium, and stroma) were isolated from chick embryos at 14, 17, and 20 days of incubation and immediately labeled in vitro with d-[6-³H]glucosamine and H₂³⁵SO₄. Amount of label incorporated into each type of glycosaminoglycan or into glycopeptides was determined by specific degradative techniques, in conjunction with gel filtration chromatography. Results suggested that corneal epithelium synthesized little, if any, corneal keratan sulfates, but that corneal endothelium may have synthesized small amounts of corneal keratan sulfates. Nearly all corneal keratan sulfates were derived from the stroma. Corneal heparan sulfates appeared to be derived predominantly from corneal epithelium at later stages of development. Corneal endothelium contributed large proportions of the hyaluronic acids of the cornea. Only epithelium produced a large proportion of sulfated glycoproteins. In addition, epithelium synthesized a large proportion of a sulfated, high molecular weight polysaccharide which was resistant to treatments degrading known types of glycosaminoglycans. Each corneal tissue may not only affect corneal morphogenesis directly by contributing a unique spectrum of glycosylated proteins to the extracellular matrix, but also may regulate the extracellular matrix composition indirectly by modulating the biosynthetic activities of the other corneal tissues.     

Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin

The early chick heart tube consists of myocardium and endothelium separated by a myocardially derived basement membrane (MBM). As development proceeds, the endothelium undergoes a transition into mesenchyme in a regionally specific manner; only the atrioventricular (AV) and outflow tract, but not the ventricular endothelium, is transformed into mesenchyme, the progenitor of heart septa and valves. Recent experiments have shown that an EDTA extract of MBM can initiate AV endothelium to form mesenchyme in an in vitro collagen gel culture system. Two-dimensional gel electrophoresis of AV region EDTA extracts showed potentially three isoelectric forms of fibronectin (Fn), while extracts from ventricle contained only two forms. The purpose of the present study was to further investigate the significance of these regional differences by testing of specific myocardial regions (AV vs ventricle) for their ability to induce endothelium to form mesenchyme in vitro, and to immunohistochemically determine if a regionally specific distribution of Fn exists in the MBM that can be correlated with previous electrophoretic data. Embryonic heart regions cultured on three-dimensional collagen gels showed that AV endothelium could only form mesenchyme if cocultured with AV myocardium. Coculture with ventricular myocardial explants did not initiate differentiation of AV endothelium. In contrast, ventricular endothelial cells did not form mesenchyme when cocultured with AV or ventricle myocardium. Immunohistochemical localization of Fn revealed three distinct morphological patterns of distribution in the AV-MBM, i.e., an intense lamina densa staining, diffuse staining in fibrils, and as particles. The Fn localized in particles (0.1 to 0.5 micron in diameter) appeared as a gradient of decreasing concentration extending from the myocardium toward the endothelium. In contrast, no particulate Fn staining was observed in the ventricular region. EDTA extraction selectively depleted the particulate form of Fn. Previous work has shown that this extract, which contains several lower Mr proteins in addition to Fn, is biologically active in initiating mesenchyme formation from AV endothelium in vitro. These results show that a regionally specific interaction of the myocardium with the endothelium is required to initiate the formation of prevalvular mesenchyme. This interaction may be mediated by a multicomponent complex involving Fn and other proteins which appear as a regionally distinct particulate only in areas of endothelial differentiation.     

Cardiac mast cell-mediated activation of gelatinase and alteration of ventricular diastolic function

Mast cells contain proteases capable of activating matrix metalloproteinases (MMPs). However, given the relatively low density of mast cells in the myocardium (i.e., 1.5-5.3 cells/mm(2)), it is unknown whether these enzymes are present in sufficient quantities in the normal heart to mediate MMP activation. Accordingly, this study sought to determine whether chemically induced degranulation of cardiac mast cells (with compound 48/80) would have an effect in isolated, blood-perfused, functioning rat hearts. Mast cell degranulation produced a 15% increase in histamine levels present in the coronary efflux, a significant increase in myocardial water (i.e., edema) relative to normal values (80.1 +/- 3.4% vs. 77.4 +/- 1.08%, P < or = 0.03), a substantial activation of MMP-2 (126% increase relative to controls, P < or = 0.02), and a marked decrease in myocardial collagen volume fraction (0.46 +/- 0.10% vs. 0.97 +/- 0.33%, P < or = 0.001). Furthermore, although an increase in ventricular stiffness was expected due to the extent of edema resulting from mast cell degranulation, modest ventricular dilatation was observed. These findings clearly demonstrate that the number of mast cells present in normal hearts is sufficient to mediate activation of MMPs and produce extracellular matrix degradation, thereby potentially causing subsequent ventricular dilatation.     

Sca-1+ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury

Background

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/Principal Finding

Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1⁺CD45^- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1⁺CD45^- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1⁺CD45^- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.

Conclusions/Significance

These studies demonstrate that cloned Sca-1⁺CD45^- cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1⁺) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.     

Intact calcium signaling in adrenergic-deficient embryonic mouse hearts

Mouse embryos that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to disruption of the dopamine beta-hydroxylase (Dbh^?/-) gene inevitably perish from heart failure during mid-gestation. Since adrenergic stimulation is well-known to enhance calcium signaling in developing as well as adult myocardium, and impairments in calcium signaling are typically associated with heart failure, we hypothesized that adrenergic-deficient embryonic hearts would display deficiencies in cardiac calcium signaling relative to adrenergic-competent controls at a developmental stage immediately preceding the onset of heart failure, which first appears beginning or shortly after mouse embryonic day 10.5 (E10.5). To test this hypothesis, we used ratiometric fluorescent calcium imaging techniques to measure cytosolic calcium transients, [Ca²⁺]_i in isolated E10.5 mouse hearts. Our results show that spontaneous [Ca²⁺]_i oscillations were intact and robustly responded to a variety of stimuli including extracellular calcium (5?mM), caffeine (5?mM), and NE (100?nM) in a manner that was indistinguishable from controls. Further, we show similar patterns of distribution (via immunofluorescent histochemical staining) and activity (via patch-clamp recording techniques) for the major voltage-gated plasma membrane calcium channel responsible for the L-type calcium current, I_Ca,L, in adrenergic-deficient and control embryonic cardiac cells. These results demonstrate that despite the absence of vital adrenergic hormones that consistently leads to embryonic lethality in vivo, intracellular and extracellular calcium signaling remain essentially intact and functional in embryonic mouse hearts through E10.5. These findings suggest that adrenergic stimulation is not required for the development of intracellular calcium oscillations or extracellular calcium signaling through I_Ca,L and that aberrant calcium signaling does not likely contribute to the onset of heart failure in this model.     

Human Myocardium Releases Heat Shock Protein 27 (HSP27) after Global Ischemia: The Proinflammatory Effect of Extracellular HSP27 through Toll-like Receptor (TLR)-2 and TLR4

The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27.     

Endothelial deletion of ADAM17 in mice results in defective remodeling of the semilunar valves and cardiac dysfunction in adults

Global inactivation of the metalloproteinase ADAM17 during mouse development results in perinatal lethality and abnormalities of the heart, including late embryonic cardiomegaly and thickened semilunar and atrioventricular valves. These defects have been attributed in part to a lack of ADAM17-mediated processing of HB-EGF, as absence of soluble HB-EGF results in similar phenotypes. Because valvular mesenchymal cells are largely derived from cardiac endothelial cells, we generated mice with a floxed Adam17 allele and crossed these animals with Tie2-Cre transgenics to focus on the role of endothelial ADAM17 in valvulogenesis. We find that although hearts from late-stage embryos with ablation of endothelial ADAM17 appear normal, an increase in valve size and cell number is evident, but only in the semilunar cusps. Unlike Hbegf^?/? valves, ADAM17-null semilunar valves do not differ from controls in acute cell proliferation at embryonic day 14.5 (E14.5), suggesting compensatory processing of HB-EGF. However, levels of the proteoglycan versican are significantly reduced in mutant hearts early in valve remodeling (E12.5). After birth, aortic valve cusps from mutants are not only hyperplastic but also show expansion of the glycosaminoglycan-rich component, with the majority of adults exhibiting aberrant compartmentalization of versican and increased deposition of collagen. The inability of mutant outflow valve precursors to transition into fully mature cusps is associated with decreased postnatal viability, progressive cardiomegaly, and systolic dysfunction. Together, our data indicate that ADAM17 is required in valvular endothelial cells for regulating cell content as well as extracellular matrix composition and organization in semilunar valve remodeling and homeostasis.     

Proteoglycan synthesis in the endothelium of embryonal vertebrate hearts: significance for the development of cardiovascular function

The endothelium of the embryonic heart is able to synthetize proteoglycans (PG) as it is the myocardium. In the extracellular matrix, PG form highly polymeric visco-elastic networks, which besides others act as shock absorber. That is apparently of evidence for the modulation of embryonic heart actions. Because during the embryonic period the large arteries are simple endothelial tubes without having an elastic-muscular wall. That means the typical "windkessel" function such as dumping of pulse waves or a continues pressure distribution is not existent. The embryonic vessels are perfused like rigid tubes. The continuous rhythmic flow pattern in the endothelial tubes, necessary for perfusion of the different organs, is apparently compensated by a high initial pressure level initiated by the heart. It is concluded that the continuity of the pressure profile is caused by intracardial PG. The endothelial synthesis of the PG of the heart decreases with increasing development of the muscular wall of the vessels and disappears completely post partum.     

Molecular Strategy to Reduce In Vivo Collagen Barrier Promotes Entry of NCX1 Positive Inducible Pluripotent Stem Cells (iPSCNCX1+) into Ischemic (or Injured) Myocardium

Objective

The purpose of this study was to assess the effect of collagen composition on engraftment of progenitor cells within infarcted myocardium.

Background

We previously reported that intramyocardial penetration of stem/progenitor cells in epicardial patches was enhanced when collagen was reduced in hearts overexpressing adenylyl cyclase-6 (AC6). In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium.

Methods

In vitro: A tri-cell patch (Tri-P) consisting of cardiac sodium-calcium exchanger-1 (NCX1) positive iPSC (iPSC^NCX1+), endothelial cells (EC), and mouse embryonic fibroblasts (MEF) was created, co-cultured, and seeded on isolated peritoneum. The expression of fibrosis-related genes was analyzed in cardiac fibroblasts (CFb) by qPCR and Western blot. In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI). The Tri-P was placed over the infarcted region 7 days later. Angiomyogenesis was analyzed by micro-CT imaging and immunofluorescent staining. Echocardiography was performed weekly.

Results

The number of green fluorescent protein positive (GFP⁺) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups. Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remodeling.

Conclusion

Overexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSC^NCX1+ penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI.     

Loss of connexin45 causes a cushion defect in early cardiogenesis

At around embryonic day 9, the primitive heart of a mouse embryo undergoes spectacular alterations within 24 hours. We created mice harboring an nls-lacZ gene in place of connexin45, which encodes the only known gap junction protein in the primitive heart before embryonic day 9, using the Cre-loxP system. Connexin45-deficient mice died of heart failure at around embryonic day 10. They initiated heart contractions, but conduction block appeared within 24 hours after the first contractions. Their cardiac walls displayed an endocardial cushion defect, while the cardiac jelly was present. These abnormalities were caused by impairment of the epithelial-mesenchymal transformation of the cardiac endothelium. Activation of the cardiac endothelium depended on the presence of the connexin45 gap junctions since signaling through Ca(2+)/calcineurin and NF-ATc1 (originally named NF-ATc) was disrupted in the mutant hearts. These results indicate a requirement for gap junction channels during early cardiogenesis and hence implicate connexin45 in congenital heart diseases. http://www. biologists.com/Development/movies/dev4369.html     

Analysis of the endocardium and cardiac jelly in truncal development in the cardiac lethal mutant axolotl Ambystoma mexicanum

Recessive mutant gene c in axolotls results in a failure of the heart to function because of abnormal embryonic induction processes. The myocardium in this mutant lacks organized sarcomeric myofibrils. The present study was undertaken to determine if developmental abnormalities were evident in other areas of the heart besides the myocardium. A detailed comparative survey of the structure of developing normal and mutant hearts, including the endocardium, its cellular derivatives, and the extracellular matrix, known as cardiac jelly, showed that in the mutant there are fewer than the normal number of endocardial cells lining the heart lumen, the number of mesenchyme cells is reduced, and the cardiac jelly area is greatly enlarged in the posterior part of the truncus adjacent to the ventricle.     

Heterogeneity in human cardiac troponin I standards

The LC-MS analysis of recombinant cardiac troponin I (cTnI) and cTnI extracted from human hearts showed a high degree of structural heterogeneity among all samples. The examined recombinant cTnI samples indicated posttranslational modifications, presumably due to their purification (i.e., 2-mercaptoethanol adducts and carbamylation) and related to their expression (i.e., an N-terminal expression tag). The extracted cTnI samples, while having a higher degree of structural heterogeneity, showed less structural variance between samples than the recombinant proteins. The LC-MS analysis of the extracted cTnI samples provided evidence of posttranslational modification by phosphorylation, acetylation, proteolytic cleavage, and intrachain disulfide bond formation.     

Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice

Transformation of endocardial endothelial cells into invasive mesenchyme is a critical antecedent of cardiac cushion tissue formation. The message for bone morphogenetic protein (BMP)-2 is known to be expressed in myocardial cells in a manner consistent with the segmental pattern of cushion formation [Development 109(1990) 833]. In the present work, we localized BMP-2 protein in atrioventricular (AV) myocardium in mice at embryonic day (ED) 8.5 (12 somite stage) before the onset of AV mesenchymal cell formation at ED 9.5. BMP-2 protein expression was absent from ventricular myocardium throughout the stages examined. After cellularization of the AV cushion at ED 10.5, myocardial BMP-2 protein expression was diminished in AV myocardium, whereas cushion mesenchymal cells started expressing BMP protein. Expression of BMP-2 in cushion mesenchyme persisted during later stages of development, ED 13.5-16, during valuvulogenesis. Intense expression of BMP-2 persisted in the valve tissue in adult mice. Based on the expression pattern, we performed a series of experiments to test the hypothesis that BMP-2 mediates myocardial regulation of cardiac cushion tissue formation in mice. When BMP-2 protein was added to the 16-18 somite stage (ED 9.25) AV endocardial endothelium in culture, cushion mesenchymal cells were formed in the absence of AV myocardium, which invaded into collagen gels and expressed the mesenchymal marker, smooth muscle (SM) alpha-actin; whereas the endothelial marker, PECAM-1, was lost from the invaded cells. In contrast, when noggin, a specific antagonist to BMPs, was applied together with BMP-2 to the culture medium, AV endothelial cells remained as an epithelial monolayer with little expression of SM alpha-actin, and expression of PECAM-1 was retained in the endocardial cells. When noggin was added to AV endothelial cells cocultured with associated myocardium, it blocked endothelial transformation to mesenchyme. AV endothelium treated with BMP-2 expressed elevated levels of TGFbeta-2 in the absence of myocardium, as observed in the endothelium cocultured with myocardium. BMP-2-supported elevation of TGFbeta-2 expression in endocardial cells was abolished by noggin treatment. These data indicated that BMP signaling is required in and BMP-2 is sufficient for myocardial segmental regulation of AV endocardial cushion mesenchymal cell formation in mice.     

Relationship between cardiac protein tyrosine phosphorylation and myofibrillogenesis during axolotl heart development

The axolotl, Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of protein tyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the state of protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomeric myofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosine phosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40-41 showed less tyrosine phosphorylated staining as compared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower content of phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstrated that P-Tyr staining gradually increased being localized primarily at cell-cell boundaries and cell-extracellular matrix boundaries. In contrast, mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell-cell and cell-matrix junctions. We also delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resulted in a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation is critical during myofibrillogenesis and embryonic heart development in axolotls.