Virginio Garcia-Martinez

Complex I and cytochrome c are molecular targets of flavonoids that inhibit hydrogen peroxide production by mitochondria.

Flavonoids can protect cells from different insults that lead to mitochondria-mediated cell death, and epidemiological data show that some of these compounds attenuate the progression of diseases associated with oxidative stress and mitochondrial dysfunction. In this work, a screening of 5 flavonoids representing major subclasses showed that they display different effects on H₂O₂ production by mitochondria isolated from rat brain and heart. Quercetin, kaempferol and epicatechin are potent inhibitors of H₂O₂ production by mitochondria from both tissues (IC₅₀ approximately 1-2 μM), even when H₂O₂ production rate was stimulated by the mitochondrial inhibitors rotenone and antimycin A. Although the rate of oxygen consumption was unaffected by concentrations up to 10 μM of these flavonoids, quercetin, kaempferol and apigenin inhibited complex I activity, while up to 100 μM epicatechin produced less than 20% inhibition. The extent of this inhibition was found to be dependent on the concentration of coenzyme Q in the medium, suggesting competition between the flavonoids and ubiquinone for close binding sites in the complex. In contrast, these flavonoids did not significantly inhibit the activity of complexes II and III, and did not affect the redox state of complex IV. However, we have found that epicatechin, quercetin and kaempferol are able to stoichiometrically reduce purified cytochrome c. Our results reveal that mitochondria are a plausible main target of flavonoids mediating, at least in part, their reported preventive actions against oxidative stress and mitochondrial dysfunction-associated pathologies.

MORPHOLOGY AND SIGNIFICANCE OF PROGRAMMED CELL DEATH IN THE DEVELOPING LIMB BUD OF THE VERTEBRATE EMBRYO

Cell death constitutes a basic mechanism accounting for many morphogenetic and histogenetic events during normal and abnormal development of embryonic organs and tissues. This article focuses on the major areas of mesodermal cell death occurring during vertebrate limb development. In early stages of limb development, cell death appears to reduce the amount of mesodermal tissue destined to form the anlage of the autopodium. In later stages, cell death plays a role sculpturing the shape of the digits. The morphology of the dying cells corresponds with apoptosis, but internucleosomal DNA fragmentation by endonuclease activation does not appear to be a precocious feature. The cell death program can be inhibited in vivo and in vitro by changing the environmental conditions of the prospective dying cells up to 6–10 h before death. In this review, we survey possible factors controlling the establishment of the cell death program. Information concerning the biochemical basis of cell death in the developing limb is also revised. Finally, the possible role of genes whose pattern of expression is coincident with the dying processes is discussed.

Developmental regulation of a proinsulin messenger RNA generated by intron retention

Proinsulin gene expression regulation and function during early embryonic development differ remarkably from those found in postnatal organisms. The embryonic proinsulin protein content decreased from gastrulation to neurulation in contrast with the overall proinsulin messenger RNA increase. This is due to increasing levels of a proinsulin mRNA variant generated by intron 1 retention in the 5′ untranslated region. Inclusion of intron 1 inhibited proinsulin translation almost completely without affecting nuclear export or cytoplasmic decay. The novel proinsulin mRNA isoform expression was developmentally regulated and tissue specific. The proportion of intron retention increased from gastrulation to organogenesis, was highest in the heart tube and presomitic region, and could not be detected in the pancreas. Notably, proinsulin addition induced cardiac marker gene expression in the early embryonic stages when the translationally active transcript was expressed. We propose that regulated unproductive splicing and translation is a mechanism that regulates proinsulin expression in accordance with specific requirements in developing vertebrates.

Localization of Cells of the Prospective Neural Plate, Heart and Somites within the Primitive Streak and Epiblast of Avian Embryos at Intermediate Primitive-Streak Stages

By constructing avian transplantation chimeras using fluorescently-labeled grafts and antibodies specific for grafted cells, we have generated a prospective fate map of the primitive streak and epiblast of the avian blastoderm at intermediate primitive-streak stages (stages 3a/3b). This high-resolution map confirms our previous study on the origin of the cardiovascular system from the primitive streak at these stages and provides new information on the epiblast origin of the neural plate, heart and somites. In addition, the origin of the rostral endoderm is now documented in more detail. The map shows that the prospective neural plate arises from the epiblast in close association with the rostral end of the primitive streak and lies within an area extending 250 µm rostral to the streak, 250 µm lateral to the streak and 125 µm caudal to the rostral border of the streak. The future floor plate of the neural tube arises within the midline just rostral to the streak, confirming our earlier study, but unlike at the late-primitive streak stages when both Hensen’s node and the midline area rostral to Hensen’s node contribute to the floor plate, only the area rostral to the primitive streak contributes to the floor plate at intermediate primitive-streak stages. Instead of contributing to the floor plate of the neural tube, the rostral end of the primitive streak at intermediate primitive-streak stages forms the notochord as well as the rostromedial endoderm, which lies beneath the prechordal plate mesoderm and extends caudolaterally on each side toward the cardiogenic areas. The epiblast lateral to the primitive streak and caudal to the neural plate contributes to the heart and it does so in rostrocaudal sequence (i.e., rostral grafts contribute to rostral levels of the straight heart tube, whereas progressively more caudal grafts contribute to progressively more caudal levels of the straight heart tube), and individual epiblast grafts contribute cells to both the myocardium and endocardium. The prospective somites (i.e., paraxial mesoderm) lie within the epiblast just lateral to the prospective heart mesoderm. Comparing this map with that constructed at late primitive-streak stages reveals that by the late primitive-streak stages, prospective heart mesoderm has moved from the epiblast through the primitive streak and into the mesodermal mantle, and that some of the prospective somitic mesoderm has entered the primitive streak and is undergoing ingression.

Kaempferol protects against rat striatal degeneration induced by 3‐nitropropionic acid

3‐Nitropropionic acid (NPA) produces degeneration of striatum and some neurological disturbances characteristic of Huntington’s disease in rodents and primates. We have shown that the flavonoid kaempferol largely reduced striatal damage induced by cerebral ischaemia‐reperfusion in rats ( Lopez‐Sanchez et al. 2007 ). In this work, we report that intraperitoneal (i.p.) administration of kaempferol affords an efficient protection against NPA‐induced neurodegeneration in Wistar rats. We studied the effects of daily i.p. injections of 7, 14 and 21 mg of kaempferol/kg body weight during the NPA‐treatment (25 mg/kg body weight/12 h i.p., for 5 days) on the neurological deficits, degeneration of rat striatum and oxidative stress markers. Intraperitoneal injections of 14–21 mg of kaempferol/kg body weight largely attenuated motor deficit and delayed mortality. The higher dose of kaempferol prevented the appearance of NPA‐induced striatal lesions up to the end of treatment, as revealed by haematoxylin‐eosin and TUNEL staining, and also NPA‐induced oxidative stress, because it blocked the fall of reduced glutathione and the increase of protein nitrotyrosines in NPA‐treated rats. It was found that striatal degeneration was associated with calpains activation and a large inactivation of creatine kinase, which were also prevented when the higher doses of kaempferol were administered.

Induction of cardiogenesis by Hensen's node and fibroblast growth factors

Abstract. The earliest events underlying cardiac induction and morphogenesis remain largely unknown. In the present study, we show that Hensens node, the organizer of the avian embryo, induces cardiogenesis. Specifically, following heterotopic transplantation, Hensens node induces ectopic host tissue that expresses two early cardiac markers (cNkx-2.5 and cNkx-2.8), as well as a ventricular marker (VMHC1), but not an atrial marker (AMHC1). Moreover, we examine the potential roles of candidate growth factors known to be secreted by Hensens node. Our results show that fibroblast growth factors (FGF-2 and FGF-4) when ectopically expressed can initiate cardiac development, inducing host tissue to express the two cardiac transcription factors cNkx-2.5 and cNkx-2.8, as well as the cardiac-restricted structural gene VMHC1, but not AMHC1. In contrast to FGFs, TGFβ family members fail to induce ectopic tissue and expression of cardiac marker genes. We also examined the effects of growth factors on the morphogenesis of the host embryos heart. Both exogenous FGFs and TGFβ family members perturb normal morphogenesis of the early cardiac tube and alter patterns of ventricular and atria gene expression in characteristic ways. Namely, exogenous FGFs expand areas expressing the ventricular marker VMHC1 at the expense of areas expressing the atrial marker AMHC1. Conversely, exogenous TGFβ1 inhibits expression of VMHC1, expanding AMHC1 expression. We show here that Hensens node and FGFs induce ectopic expression of cardiac lineage markers, and that FGF and TGFβ family members can modulate early development of the heart. Collectively, these data suggest that the organizer plays a crucial role in cardiac induction and morphogenesis, mediated in part by endogenous members of the FGF and TGFβ families.

Myocardial Fiber Architecture in the Human Heart

Ventricular myocardial fiber architecture has been considered an important factor in heart dynamics. Most anatomical studies however have focussed on the analysis of normal hearts. The present study c

Molecular determinants of cardiac specification

In this review, we report and analyse the molecular factors involved in cardiogenesis from the earliest stages of development, using mainly the chick embryo as a model. The first part of the review demonstrates the areas where cardiogenic cells are located from gastrula stages, analysing a brief summary of the fate map of cardiogenic cells, from the epiblast through to the primitive heart tube. The next part analyses the commitment of pre-cardiac cells in cardiogenesis before, during, and after ingression through the primitive streak. Throughout the different journeys of the pre-cardiac cells, from the origin on the epiblast level up to the constitution of the tubular heart in the mid-line, the genes involved in the different stages of the process of cardiogenesis are very numerous. These have a greater or lesser importance depending on their specificity and the order in which they appear, bearing in mind that they become more valuable as the developmental process advances and the precursor cells start acquiring the commitment of pre-cardiac cells. Next, we show some box-filled diagrams to illustrate the dynamic gene expression pattern throughout the early stages of heart development, grouping the genes by their chronological significance. Finally, we discuss the implications that this temporal genomic expression could have in the induction and specification of the different types of cells and regions of the heart.

Immunofluorescent localization of tenascin during the morphogenesis of the outflow tract of the chick embryo heart

SummaryThe cono-truncus constitutes a complex segment of the developing heart that gives rise to the outflow tract of the ventricles and root of the pulmonary and aortic arteries. Numerous studies have revealed that the extracellular matrix plays a relevant role in most morphogenetic processes modulating cell behaviour. By means of immunofluorescence, we studied the distribution and possible involvement of tenascin during morphogenesis of the conus and truncus in chick embryo hearts between days 4.5–10 of incubation. Tenascin is an extracellular matrix glycoprotein with a significant role in morphogenesis and cell and tissue differentiation. Our results reveal a specific distribution of tenascin in the areas of the cono-truncus undergoing significant structural changes during morphogenesis of this cardiac segment, appearing mainly in the mesenchymal layer subjacent to the myocardial layer, the cono-truncal ridges and the aorto-pulmonary septum. The distribution of tenascin was compared and contrasted with that of collagen type I, which constitutes a further component of the extracellular matrix common to most developing connective tissues.

Morphological analysis of the fish heart ventricle: Myocardial and connective tissue architecture in teleost species

Light and scanning electron microscopy were used to study the structure of the heart ventricle in three species of marine teleost fishes: the hake (Merluccius merluccius), the angler fish (Lophius piscatorius) and the sea bream (Pagellus centrodontus). Our findings show the ventricle to be shaped differently in each species: tubular in the hake, saccular in the angler fish and pyramidal in the sea bream. From a structural viewpoint, interest was centered on two aspects: organization of the myocardial fibres and arrangement of connective tissue. In hake and angler fish ventricles, the myocardium was exclusively trabecular in nature, whereas the bream ventricle, in addition to trabecular myocardium, presented a thin compact layer. Muscle fibres showed precise patterns of organization at the level of the ventricular orifices. With the techniques used the intramyocardial connective tissue was detected in the following ventricular zones: i) at the level of subepicardial and subendocardial spaces, ii) surrounding the myocardial fascicles, and iii) surrounding individual myocardial cells. According to this structural study, the pyramidal ventricle of the fish should be considered as a ventricular pump with greater efficiency.