Quail Heart Research Articles

The heart as a target for deltamethrin toxicity: Inhibition of Nrf2/HO-1 pathway induces oxidative stress and results in inflammation and apoptosis

As a synthetic pyrethroid pesticide, deltamethrin (DLM) is widely employed in veterinary medicine and farming, and DLM-triggered oxidative stress largely causes serious harm to the organism. It is well-known that nuclear factor erythroid-2-related factor 2/heme oxygenase-1 (Nrf2/HO-1), a pivotal endogenous anti-oxidative pathway, acts on inhibiting oxidative stress-induced cell injury under the activated state. The purpose of this research was to observe the impact and molecular mechanism of DLM on inflammation and apoptosis in quail cardiomyocytes based on the Nrf2/HO-1 signaling route. In this research, quails were established as a cardiac injury model through gastric infusion of various doses of DLM (0, 15, 30, and 45 mg/kg b. w.) for 12 weeks. Our results showed that DLM could induced cardiomyocyte injury in a dose-dependent manner though weakening antioxidant defense via down-regulating Nrf2 and its downstream protein HO-1. Furthermore, DLM stimulation induced apoptosis in quail heart by decreasing the protein expressions of B-cell lymphoma-extra large and B-cell lymphoma gene 2 (Bcl-2), as well as increasing P53, caspase 3, and Bcl-2-associated X protein levels. Meanwhile, relative levels of nuclear factor-kappa B and interleukin-1β in quail hearts were up-regulated under DLM intervention progressively. Collectively, our study demonstrates that chronic exposure to DLM can induce quail cardiomyocyte inflammation and apoptosis by mediating Nrf2/HO-1 signaling pathway-related oxidative stress.

Purification and characterization glutathione S-transferase enzyme from quail (Coturnix, coturnix japonica) heart and investigation the effect of some metal ions on enzyme activity

In this study glutathione S-transferase enzyme (EC: 2.5.1.18) from the heart of japonica quail was purified with 34.0 EU/mg specific activity, 10.44% purification yield and 78.29 purification folds and characterized. Purification processes are consist of three steps, firstly homogenate was prepared, and then ammonium sulfate precipitation was performed and finally glutathione-agarose gel affinity column chromatography was performed. To check the purity of GST enzyme used SDS-PAGE method. Then the M.W calculated at approximately 26.3 kDa by SDS-PAGE method. Enzymatic activity was determined spectrofotometrically according to Beutler`s method at 340 nm. Also characterizations study carry out, and the results obtained are stability-pH = 9.0 in Tris/HCL buffer, optimum pH = 8.0 in Tris/HCl buffer, optimum temperature 60 °C, optimum ionic strength was 1.2 M in Tris/HCl buffer. And kinetic studies performed for GST enzyme purified from quail heart by used both glutathione and 1-chloro 2,4-dinitrobenzen as substrate. KM and Vmax values are determined as 1.642 mM and 0.502 EU/mL respectively for GSH substrate and 3.880 mM and 0.588 EU/mL respectively for CDNB substrate. In addition, the effect of some metal ions (Cu2+, Cd2+, Fe2+, Fe3+ Zn2+, Ag+, Co2+, and Ti1+) were investigated on the GST enzyme activity in vitro.

Modulation of heat-shock response is associated with Di (2-ethylhexyl) phthalate (DEHP)-induced cardiotoxicity in quail (Coturnix japonica)

Di(2-ethylhexyl) phthalate (DEHP) is an omnipresent environmental pollutant with endocrine disrupting properties. As a plasticizer, DEHP can be leach from the plastic to transfer the external environment and thus enters the animal food chain, causing serious damage to the animal organs. The heat-shock response (HSR) comprising heat-shock protein (HSPs) and heat-shock transcription factor (HSFs) plays a pivotal role in various toxic stress conditions. For the sake of investigating the effects of DEHP exposure on cardiac toxicity and the regulation of HSR, male quail were fed the diet with 0, 250, 500 and 750 mg/kg DEHP by gavage administration for 45 days. Histopathological changes including cardiomyocyte swelling and muscle fiber dilatation were observed in the hearts exposed to DEHP. During the DEHP treatment, the mRNA expression of HSP60 and HSP70 were universally reduced, while the expression of other HSPs (HSP10, HSP25, HSP27, HSP40, HSP47, HSP90, HSP110) had different degrees of growth. In addition, the levels of HSF1, HSF2, and HSF3 were significantly increased. Given the facts above, DEHP exposure induced the toxic effects of quail heart. DEHP exposure did great harm to HSR via affecting the synthesis of HSFs to mediate the transcription of the HSPs. Ultimately, this study provided new evidence that DEHP-induced cardiotoxicity in quail was related to activation of HSR and playing a protective role.

Anatomy of the coronary artery and cardiac vein in the quail ventricle: patterns are distinct from those in mouse and human hearts.

Coronary vessel development has been investigated in avian and mouse embryonic hearts. Quail embryos are a useful tool to examine vascular development, particularly because the QH1 antibody and transgenic quail line, Tg (tie1:H2B-eYFP), are useful to trace endothelial cells. However, there are only a few descriptions of the quail coronary vessels. Using ink injection coronary angiography, we examined the course of coronary vessels in the fetal quail heart. The major coronary arteries were the right and left septal arteries, which, respectively, branched off from the right and left coronary stems. The right septal artery ran posteriorly (dorsally) and penetrated the ventricular free wall to distribute to the posterior surface of the ventricles. The left septal artery ran anteriorly (ventrally) and penetrated the ventricular free wall to distribute to the anterior surface of the ventricles. The right and left circumflex arteries were directed posteriorly along the atrioventricular sulci. The cardiac veins consisted of three major tributaries: the middle, great, and anterior cardiac veins. The middle cardiac vein ascended along the posterior interventricular sulcus and emptied into the right atrium. The great cardiac vein ran along the anterior interventricular sulcus, entered the space between the left atrium and conus arteriosus and emptied into the right atrium behind the aortic bulb. The anterior cardiac vein drained the anterior surface of the right ventricle and connected to the anterior base of the right atrium. The course of coronary vessels in the quail heart was basically the same as that observed in chick but was different from those of mouse and human.

Atrazine-xenobiotic nuclear receptor interactions induce cardiac inflammation and endoplasmic reticulum stress in quail (Coturnix coturnix coturnix)

Atrazine (ATR) is one of the most extensively used herbicide that eventually leaches into groundwater and surface water from agricultural areas. Exposure to ATR does harm to the health of human and animals, especially the heart. However, ATR exposure caused cardiotoxicity in bird remains unclear. To evaluate ATR-exerted potential cardiotoxicity in heart, quail were exposed with 0, 50, 250, and 500 mg/kg BW/day ATR by gavage treatment for 45 days. Cardiac histopathological alternation was observed in ATR-induced quail. ATR exposure increased the Cytochrome P450s and Cytochrome b5 contents, Cytochrome P450 (CYP) enzyme system (APND, ERND, AH, and NCR) activities and the expression of CYP isoforms (CYP1B1, CYP2C18, CYP2D6, CYP3A4, CYP3A7, and CYP4B1) in quail heart. The expression of nuclear xenobiotic receptors (NXRs) was also influenced in the heart by ATR exposure. ATR exposure significantly caused the up-regulation of pro-inflammatory cytokines (TNF-α, IL-6, NF-κB, and IL-8), down-regulation of anti-inflammatory cytokines (IL-10) expression levels and increased NO content and iNOS activity. The present research provides new insights into the mechanism that ATR-induced cardiotoxicity through up-regulating the expression levels of GRP78 and XBP-1s, triggering ER stress, activating the expression of IRE1α/TRAF2/NF-κB signaling pathway related factors (IRE1α, TRAF2, IKK, and NF-κB) and inducing an inflammatory response in quail hearts. In conclusion, ATR exposure could induce cardiac inflammatory injury via activating NXRs responses, disrupting CYP homeostasis and CYP isoforms transcription, altering NO metabolism and triggering ER stress and inflammatory response by activating IRE1α/TRAF2/NF-κB signaling pathway.

Abstract 19018: Histone 3 Lysine 4 Tri-Methylation is Necessary for Epicardial Epithelial-to-mesenchymal Transformation

Introduction: Recent whole exome sequencing carried out in patients with congenital heart disease revealed an over-representation of mutations in members of the Histone 3 Lysine 4 Methyltransferase Complex, indicating that the methylation of Histone 3 Lysine 4 (H3K4) is of particular importance during heart development. In the tri-methylated state (H3K4me3) this signature actively promotes gene transcription through the recruitment of the Pre-Initiation Complex to the promoter. There are currently no data regarding the role of the H3K4me3 modification in cardiogenesis. We investigated the hypothesis that the acquisition of H3K4me3 is necessary for Epicardial Epithelial-to-Mesenchymal Transformation (EMT). Methods: Global patterns of H3K4 tri-methylation were investigated in epicardial cells undergoing EMT in the embryonic quail heart in-vivo, via co-staining of H3K4me3 with the epicardial marker pan-cytokeratin, and the mesenchymal marker alpha-Smooth Muscle Actin. For the in-vitro investigation of H3K4me3 during Epicardial EMT, quail epicardial explants were induced to undergo mesenchymal transformation via treatment with Fetal Bovine Serum alone, and in the presence of an H3K4 methyltransferase inhibitor. Epicardial EMT was characterized by the induction of Snail expression, a loss of E-cadherin, the expression of alpha-Smooth Muscle Actin, and an increase in nuclear area. The acquisition of new H3K4me3 was investigated via Immunohistochemistry. Results: There is a global acquisiton of H3K4me3 in quail epicardial cells undergoing EMT in-vivo and in-vitro. Inhibition of H3K4 methyltransferase activity impaired Snail gene expression, rescued E-cadherin, prevented the expression of alpha-Smooth Muscle Actin, and prevented nuclear enlargement in epicardial cells in-vitro, while having no effect on cell proliferation. Conclusions: We conclude that the acquisition of tri-methylated H3K4 is featured in Epicardial EMT both in-vivo and in-vitro in embryonic quail epicardium. This acquisition is necessary for Epicardial Epithelial-to-Mesenchymal Transformation in-vitro. These data represent the first potential explanation as to how mutations in the H3K4 Methyltransferase Complex impair heart development.

Molecular and kinetic characterization of lactate dehydrogenase enzyme in the heart and breast muscle of Japanese quail (Coturnix japonica)

In the present investigation, attempts were made to characterize lactate dehydrogenase (LDH) isozyme in quail heart and breast muscle by using electrophoresis, kinetic, and molecular techniques. Polyacrylamide gel electrophoresis of the heart and breast muscle revealed a single isozyme. Kinetic studies were carried out for further functional characterization of LDH isozyme in the heart and breast muscle. Substrate inhibition, enzyme kinetics, and inhibitor (urea) studies indicated that the isozymes present in these tissues differed in their functional properties. LDH isozyme present in the heart and breast muscle exhibited properties of LDH-A and LDH-B isozyme, respectively. Furthermore, RT-PCR amplification of Ldh-a and Ldh-b mRNA in both tissues confirmed that these isozymes were formed by the association of both H and M subunits and may be of H3M1 and M3H1 in the heart and breast muscle, respectively.

Oedipus, King of the Falcons: Dissonant Fragments of a Work in Progress

Part 1 Five weeks ago today, Oedipus decided it was time for him to cut his way out of the egg. He pushed his feet against the side of the shell and pushed his head back fiercely, forcing the small hard notch on his beak to break through the shell above his head. A couple of small bits of shell fell onto his head as he pushed his head back a second time and etched another millimeter of shell. After a short pause, he pressed his feet again firmly against the shell, and swung his head back. Crunch. Another two millimeters of shell split apart. Crunch, crunch, crunch. After ten minutes, about one fifth of the circumference of the shell had been broken in a jagged line about his head. After a pause to rest for a further ten minutes, Oedipus started chopping again. Within half an hour, three quarters of the shell had been cut apart. Rocking from side to side, pressing his feet up against the wall of the shell, flinging his head further and further back, Oedipus was preparing his entry into the world beyond his shell. In ten more minutes, one more strong heave and the two sides of the shell fell apart. Panting a little from exertion, Oedipus stretched his legs and pushed himself free, leaving behind him the portable womb so kindly given to him by his mother. Inside the shell he left the membrane that had surrounded him while he had been growing, still containing just a little of the blood in the veins that had kept it alive. Until this moment it had been part of his body. Just before the final act of splitting the shell in half he had drawn most of the blood inside his body through a fragile umbilical cord. This now breaks as he crawls away from the egg's broken shell and nestles in a corner against a sterilized, cotton hippopotamus, lent to him by my daughter Robin. He is still wet so he rubs against the surrogate mother to dry himself, and in so doing ruffles up the smallest of small feathers that thinly cover him. But they soon fluff out and become white, so that after half an hour he resembles a ball of cotton wool, with a pair of claws at one end and a pointed beak at the other. I kill a quail and open it up, next to the electric brooder/incubator where Oedipus is being kept warm. I take off the top of the brooder and gently lift out Oedipus and place him in a small bowl lined with cotton wool. I bend over him and click my tongue a few times in imitation of an adult falcon. Oedipus, less than an hour old, balances himself upright between his two legs stretched out on each side of his body, sticks his head in the air, opens his huge mouth and chups back at me; so I place a hot, bloody morsel of quail heart into his mouth, and he promptly gobbles it down with obvious excitement. His eyes are open, gazing up at me, two translucent black suns that are bringing to this magical moment, one hundred million years of falcon consciousness. I feed him more small bloody morsels of meat, from muscles, the breast and some more from the heart. When I see that his small crop is almost full of food, I stop feeding him and place him back into the brooder, where he snuggles up to the hippopotamus and falls immediately to sleep. For the first five days he is fed every two hours or so, or whenever he calls to me for food. As he gets bigger, so his crop enlarges until after two weeks he can be fed every four hours, five times a day, with eight hours undisturbed at night when both of us can enjoy some sleep. When he hatched he weighed thirty grams. So when was Oedipus born? Was he born when he hacked his way out of his egg? But two days before that, he had pipped the egg, which means he had made the first break into the egg with the small notch on his beak, a hole that allows him to breathe air directly from the outside, and not having to pass through the pores of the egg shell. He had already been breathing air in his lungs for several days before this first punctured hole; maybe thinking about the ordeal ahead, of splitting the very very hard eggshell apart. …

High temporal resolution OCT using image-based retrospective gating

High temporal resolution OCT imaging is very advantageous for analyzing cardiac mechanics in the developing embryonic heart of small animals. An image-based retrospective gating technique is presented to increase the effective temporal resolution of an OCT system and to allow visualization of systolic dynamics in 3D. The gating technique employs image similarity measures for rearranging asynchronously acquired input data consisting of a time series of 2D images at each z position along the heart volume, to produce a time sequence of 3D volumes of the beating heart. The study includes a novel robust validation technique, which quantitatively evaluates the accuracy of the gating technique, in addition to visual evaluations by 2D multiplanar reformatting (MPR) and 3D volume rendering. The retrospective gating and validation is demonstrated on a stage 14 embryonic quail heart data set. Using the validation scheme, it is shown that the gating is accurate within a standard deviation of 4.7 ms, which is an order of magnitude shorter than the time interval during which systolic contraction (approximately 50 ms) occurs in the developing embryo. This gating method has allowed, for the first time, clear visualization of systolic dynamics of the looping embryonic heart in 3D.

Selective blockade of the vasoactive intestinal peptide (VIP) receptors affect formation and development of coronary arteries in quail embryonic heart

We have previously demonstrated that VIP receptors are morphogenic modulators of embryonic coronary vessel tube formation in vitro. This study tested the hypothesis that a selective blockade of VIP receptors will affect formation and development of the coronary vessels in vivo. At embryonic day (ED) 5, 6, 7, and 8, a selective VIP receptor antagonist, VIP (6‐28), was injected in ovo into the vitelline vein of the Japanese quail embryos at a final concentration of 10 µmol/L. Six embryos per each ED were sacrificed 4 days after the injection and the base of their hearts was fixed in 4% paraformaldehyde/PBS, and processed for frozen or paraffin‐embedded histological cross‐sections. The serial sections through the origin of the coronary vessels were studied by H&E staining or by immunostaining with the antibodies against α‐smooth muscle actin and quail endothelium (QH1). We found that the blockade of VIP receptors did not alter the formation of left and right coronary arteries in all experimental embryos. However, when the injection was done on ED8 (the time when multiple endothelial channels penetrate the aortic wall in all three aortic sinuses), an additional coronary artery originating from a non‐coronary sinus had been detected on ED12. Furthermore, the embryos from this experimental group showed a delay in the maturation of the tunica media of coronary arteries. Our findings demonstrate that VIP receptors are involved in morphogenic modulation of coronary artery development in the embryonic quail heart.Grant Funding SourceStart‐up funding

Imaging algorithm visualizes early heart abnormalities

Studying abnormalities that occur during early cardiac development is very important for understanding congenital heart defects. Optical coherence tomography (OCT) is a noninvasive imaging modality based on low-coherence interferometry that is capable of micrometer-scale resolution and 1–2mm of depth penetration in embryonic tissue. OCT can visualize and quantify real-time heart dynamics in 3D,making it a useful tool for assessing cardiac development. Real-time volume imaging performed with current systems, however, does not provide the necessary temporal resolution to analyze rapid cardiac dynamics in 3D. In the past, researchers used a physiological signal such as an electrocardiogram to trigger data acquisition, typically during multiple phases of the cardiac cycle.1–4 Alternatively, some recorded the physiological signal while imaging as fast as possible over several heartbeats at different spatial locations, and then used timing from the signal to create 3D volumes over time.1, 5–8 However, recording an additional signal is invasive and time-consuming. A possible remedy is retrospective cardiac gating,9–12 where one obtains data over multiple heartbeats without recording an external signal and rearranges the data after the fact to reconstruct 3D volumes over time. Our technique, called image-based retrospective gating, employs image similarity to rearrange out-of-order image data. The result is extremely high temporal resolution. Our image-based retrospective gating algorithm is influenced by the work of Liebling et al.10 However, due to the higher signal-to-noise ratio of our imaging setup, we work in the spatial rather than the wavelet transform domain. In addition, we created a novel quantitative validation technique to obtain a real-time reference scan of the same heart volume used for outof-order data acquisition. We then employ it to evaluate the accuracy of our algorithm. Figure 1. A 4D visualization of retrospective gated OCT data from a stage 14 embryonic quail heart. Movies of the data (90 volumes in a single cardiac cycle, or ∼270 volumes/s) are shown in (a) sagittal or en face (see video13), (b) transverse (see video14), and (c) coronal (see video15) views. In each case a cutaway view of the beating heart is shown with a moving orthogonal 2D slicer in the plane of interest.

Evidence of Endogenous Mono-ADP-Ribosylation of Cardiac Proteins Via Anti-ADP-Ribosylarginine Immunoreactivity

Abstract. Arginine-specific mono-ADP-ribosylation of proteins and arginine-specific mono-ADP-ribosyltransferase occur in heart. We developed a polyclonal antiserum, R-28, against ADP-ribosylpolyarginine that recognized mono-ADP-ribosylated proteins and identified the major mono-ADP-ribosylation products of quail heart. Treatment of Immobilon-bound ADP-ribosylated Gs protein with hydroxylamine under conditions that remove ADP-ribose from its arginines eliminated R-28 immunoreactivity to Gs. Also, R-28 immunoreactivity to quail heart proteins was removed by NaOH and phosphodiesterase I treatments. Similar treatment with mercuric chloride did not remove the immunoreactivity but did remove exogenously (via in vitro pertussis toxin treatment) added ADP-ribose from cysteine of cardiac Gi/Go proteins. The antiserum did not appear to react with ADP-ribosylasparagine of Rho (formed by C3 toxin), ADP-ribosyldiphthamide of elongation factor 2 (formed by diphtheria toxin) in quail heart preparations, or polyADP-ribosylated proteins of a neonate rat cardiac nuclear preparation. Thus, the R-28 antiserum appears to contain predominantly antibodies directed against ADP-ribosylarginine. To test the usefulness of R-28, immunoblotting of subcellular fractions of quail heart was performed. R-28 showed the greatest immunoreactivity in the sarcolemma with significant immunoreactivity in denser membrane fractions. The cytosol also contained an immunoreactive band distinct from those found in the membranes. Hydroxylamine treatment eliminated immunoreactivity in the sarcolemma and denser membrane fractions but not the cytosol, suggesting the membranous immunoreactive bands contain ADP-ribosylarginine. In conclusion, a polyclonal antiserum that recognizes ADP-ribosylarginine proteins has been raised. The usefulness of the antiserum is demonstrated by the characterization of endogenous arginine mono-ADP-ribosylation products in quail heart. The quail heart has several sarcolemmal and denser membrane fraction proteins that appear to be mono-ADP-ribosylated on arginines.

Temporally Expressed PDGF and FGF-2 Regulate Embryonic Coronary Artery Formation and Growth

PDGF and FGF-2 are important regulators of vascular wall assembly. We tested the hypothesis that their embryonic temporal expression facilitates 2 specific events: (1) the endothelial invasion of the aortic root to form the coronary artery stems and (2) the subsequent growth and development of the arterial tree. Addition of FGF-2 and PDGF-BB proteins to embryonic quail heart explants stimulated a 3- and 7-fold increase, respectively, in tubulogenesis, whereas neutralizing antibodies to these growth factors attenuated tubulogenesis by 40%. Anti-FGF-2 and anti-PDGF neutralizing antibodies were then introduced in ovo via the vitelline vein at various embryonic (E) days. When injections occurred before coronary ostial formation, the embryos usually developed only 1 coronary artery or lacked coronary arteries. When 1 or both major coronary arteries formed: (1) their branches had a thinner tunica media, and (2) smooth muscle investment did not progress as far distally as in shams. Other anomalies included smaller diameter coronary artery stems in some hearts. Inhibition of VEGF via injections of aflibercept (VEGF-Trap, a VEGFR-1 and -2 chimera), previously shown to be essential for coronary stem formation, limited development of the coronary arteries even though introduced after formation of coronary ostia (at E9 or EI0). Our data (1) document a role for FGF-2 and PDGF in the temporal regulation of coronary artery stem formation and growth of the coronary arterial tree and (2) reveal that VEGF expression is required for normal artery/arterial formation, even after coronary artery stem formation.

In vivo gated 4D imaging of the embryonic heart using optical coherence tomography

We demonstrate the first in vivo gated 4D images of avian embryonic hearts by use of optical coherence tomography (OCT). We present a gated 4D dataset of an in vivo beating quail heart consisting of approximately 864,000 A-scans accumulated over multiple heartbeats. Generation of a gating trigger from a laser Doppler velocimetry (LDV) signal, collected from an outlying vitelline vessel, enabled us to gate image acquisition to the cardiac cycle. To fully characterize the genesis and mechanisms of cardiac defects, a tool capable of assessing structure and function simultaneously at early stages of development is needed, and gated OCT has the capability to become such a tool.

Vascular patterning of the quail coronary system during development

Recent studies have provided insights into specific events that contribute to vasculogenesis and angiogenesis in the developing coronary vasculature. This study focused on the developmental progression of coronary vascularization beginning with tube formation and ending with the establishment of a coronary arterial tree. We used electron microscopy, histology of serial sections, and immunohistochemistry in order to provide a comprehensive view of coronary vessel formation during the embryonic and fetal periods of the quail heart, a species that has been used in a number of studies addressing myocardial vascularization. Our data reveal features of progenitor cells and blood islands, tubular formation, and the anatomical relationship of a transformed periarterial tubular network and sympathetic ganglia to the emergence and branching of the right and left coronary arteries. We have traced the pattern of coronary artery branching and documented its innervation. Finally, our data include the relationship of fibronectin, laminin, and apoptosis to coronary artery growth. Our findings bring together morphological events that occur over the embryonic and fetal periods and provide a baseline for studies into the mechanisms that regulate the various events that occur during these time periods.

Is the Fetal Heart a Hematopoietic Organ?

To the Editor: The article by Tomanek et al1 presents well the in vivo data on the requirement of VEGF family members for tubulogenesis and coronary artery formation. The authors observed that VEGF-Trap (a chimera of R1 and R2), apart from precluding formation of coronary arteries, causes a massive accumulation of erythrocytes in the subepicardium and in the interventricular septum. The important finding of this study was that erythrocytes that are components of blood islands derive from epicardial-derived precursors. The authors assume that proepicardial cells include a hemangioblast population. From this observation a conclusion can be drawn that either the proepicardium or the fetal heart exhibits a hematopoietic activity. Considering the topic of blood island derivation in the embryonic heart I would like to comment that although the derivation of the endothelial cell component in these structures has been proven, the origin of erythrocytes is …

Vasoactive intestinal polypeptide receptors (VIP1 and VIP2) are morphogenic modulators of embryonic coronary vessel tube formation

This study tested the hypothesis that selective activation or blockade of VIP1 and VIP2 receptors will affect embryonic coronary vessel tube formation. The in vitro quail heart explant model was us...

Transient expression of fast troponin C transcripts in embryonic quail heart

Most myofibrillar proteins, including troponin I and troponin T subunits of troponin complex, undergo developmental stage-specific isoform transitions in vertebrate heart before attaining adult contractile and regulatory characteristics. Only the cardiac/slow skeletal muscle type isoform of troponin C, however, has been shown to be expressed in both adult and developing heart. The changes in troponin C could be functionally important as the TnC isoforms vary in their affinities for Ca(2+). For example, fast troponin C has two Ca(2+) binding sites while slow/cardiac troponin C has a single regulatory site. This study demonstrates the co-expression of both fast and slow transcripts of troponin C in not only quail embryonic skeletal muscle but also embryonic heart using two different analytical techniques of polymerase chain reaction and in situ hybridisation procedure. Fast troponin C expression in the quail heart using in situ hybridisation procedure was first observed at embryonic day 3, with maximum expression at day 5 after which its level in the developing heart was gradually down regulated. In situ hybridisation staining of sections at these developmental stages demonstrated the expression of both fast and slow transcripts of troponin C in all cardiomyocytes.

TGF-β1, -β2 and -β3 Cooperate to Facilitate Tubulogenesis in the Explanted Quail Heart

Background: Transforming growth factor-β (TGF-β) isoforms have been implicated as both pro- and anti-angiogenic modulators. In this study we addressed the roles of TGF-β isoforms on coronary tubulogenesis. Methods: Embryonic (E6) quail ventricular specimens were explanted onto collagen gels allowing endothelial cells to migrate and form vascular tubes. Growth factors and/or neutralizing growth factor antibodies were added to the cultures. Endothelial cells were identified using a quail endothelial cell marker, QH1. Image analysis was used to quantify aggregate tube length. Results: Addition of any isoform (TGF-β₁, TGF-β₂ or TGF-β₃) virtually prevented tubulogenesis (>95% inhibition), while stimulation of tubulogenesis occurred by adding neutralizing antibodies to TGF-β₃, but not to TGF-β₁ or -β₂. When all three isoforms were added, tubulogenesis was enhanced, indicating the key role of TGF-β₃. Documentation of the inhibitory effect of TGF-β isoforms on tubulogenesis is further supported by our experiments in which the marked enhancement of tube formation by bFGF and VEGF was negated when exogenous TGF-β₁, -β₂, or -β₃ were added to the cultures. Conclusions: (1) TGF-β₁, -β₂ and -β₃ each inhibits angiogenesis; (2) cooperation between the three TGF-β isoforms and other angiogenic factors is essential for the regulation of normal tubulogenesis and (3) the stimulatory effect of VEGF or bFGF on tubulogenesis is negated by exogenous TGB-βs.

Role of VEGF family members and receptors in coronary vessel formation.

The specific roles of vascular endothelial growth factor (VEGF) family members and their receptors (VEGFRs) in coronary vessel formation were studied. By using the quail heart explant model, we found that neutralizing antibodies to VEGF-B or VEGF-C inhibited tube formation on the collagen gel more than anti-VEGF-A. Soluble VEGFR-1, a receptor for VEGF-A and -B, inhibited tube formation by 87%, a finding consistent with that of VEGF-B inhibition. In contrast, addition of soluble VEGFR-2, a receptor for VEGF family members A, C, D, and E, inhibited tube formation by only 43%. Acidic FGF-induced tube formation dependency on VEGF was demonstrated by the attenuating effect of a soluble VEGFR-1 and -2 chimera. The localization of VEGF R-2 and R-3 was demonstrated by in situ hybridization of serial sections, which documented marked accumulations of transcripts for both receptors at the base of the truncus arteriosus coinciding with the temporal and spatial formation of the coronary arteries by means of ingrowth of capillary plexuses. This finding suggests that both VEGFR-2 and R-3 may play a role in the formation of the coronary artery roots. In summary, these experiments document a role for multiple members of the VEGF family and their receptors in formation of the coronary vascular bed.