Vitamin A signals play critical roles during embryonic development. In particular, heart morphogenesis depends on vitamin A signals mediated by the retinoid X receptor ␣ (RXR␣), as the systemic mutation of this receptor results in thinning of the myocardium and embryonic lethality. However, the molecular and cellular mechanisms controlled by RXR␣ signaling in this process are unclear, because a myocardium-restricted RXR␣ mutation does not perturb heart morphogenesis. Here, we analyze a series of tissuerestricted mutations of the RXR␣ gene in the cardiac neural crest, endothelial, and epicardial lineages, and we show that RXR␣ signaling in the epicardium is required for proper cardiac morphogenesis. Moreover, we detect an additional phenotype of defective coronary arteriogenesis associated with RXR␣ deficiency and identify a retinoid-dependent Wnt signaling pathway that cooperates in epicardial epithelial-to-mesenchymal transformation.coronary vessels ͉ epicardium ͉ retinoids ͉ wnt ͉ FGF

We have previously identified several members of the Wnt/␤-catenin pathway that are differentially expressed in a mouse model with deficient coronary vessel formation. Systemic ablation of ␤-catenin expression affects mouse development at gastrulation with failure of both mesoderm development and axis formation. To circumvent this early embryonic lethality and study the specific role of ␤-catenin in coronary arteriogenesis, we have generated conditional ␤-catenin-deletion mutant animals in the proepicardium by interbreeding with a Cre-expressing mouse that targets coronary progenitor cells in the proepicardium and its derivatives. Ablation of ␤-catenin in the proepicardium results in lethality between embryonic day 15 and birth. Mutant mice display impaired coronary artery formation, whereas the venous system and microvasculature are normal. Analysis of proepicardial ␤-catenin mutant cells in the context of an epicardial tracer mouse reveals that the formation of the proepicardium, the migration of proepicardial cells to the heart, and the formation of the primitive epicardium are unaffected. However, subsequent processes of epicardial development are dramatically impaired in epicardial-␤-catenin mutant mice, including failed expansion of the subepicardial space, blunted invasion of the myocardium, and impaired differentiation of epicardium-derived mesenchymal cells into coronary smooth muscle cells. Our data demonstrate a functional role of the epicardial ␤-catenin pathway in coronary arteriogenesis. The embryonic epicardium originates from a primarily extracardiac primordium, the proepicardum, which is located at the septum transversum near the venous pole of the heart (2, 3). In mouse embryos, proepicardial cells reach the heart predominantly in the form of free-floating vesicles that traverse the pericardial cavity, adhere to the initially naked myocardial surface, and subsequently form the epicardial covering of the heart (reviewed in ref. 1). The recruitment of coronary vessel progenitor cells from the proepicardium and embryonic epicardium involves several steps of epithelial-mesenchymal transition (EMT). As a result of proepicardial EMT, the extracellular matrix of free-floating proepicardial vesicles becomes populated with mesenchymal cells (4-8). Subsequently, the primitive epicardium gives rise to the subepicardial mesenchyme, also by means of EMT.Recent data suggest that the primitive epicardium and epicardium-derived cells (EPDCs) modulate the maturation of other cardiac components, including the embryonic myocardium and the cardiac conduction system (9-14). We have previously shown that the embryonic epicardium is a key signaling tissue responsible for the transmission of the morphogenic signal derived from retinoic acid and identified several components of the Wnt/␤-catenin signaling pathway that are down-regulated upon retinoid signaling deficiency, in particular, ␤-catenin and its activator Wnt9b (15).However, it remains to be shown whether Wnt/␤-catenin signaling plays a specific role in the de...

Overexpression of adenine nucleotide translocase-1 (ANT1) is known to induce apoptosis (Bauer, M. K., Schubert, A., Rocks, O., and Grimm, S. (1999) J. Cell Biol. 147, 1493-1501), but the mechanisms involved remain unclear. In this study we show that ANT1 overexpression results in a recruitment of the IB␣-NF-B complex into mitochondria, with a coincident decrease in nuclear NF-B DNA binding activity. In this situation, NF-B transcriptionally regulated genes with antiapoptotic activity, such as Bcl-XL, MnSOD2, and c-IAP2, are down-regulated, and consequently, cells are sensitized to apoptosis. Accordingly, co-expression of p65 partially interferes with the proapoptotic effect of ANT1 overexpression. Despite the high identity of the two isoforms, overexpression of ANT2 does not exert an apoptotic effect; this lack of apoptotic activity is correlated with the absence of mitochondrial IB␣-NF-B recruitment or changes in NF-B activity. Thus, we propose that the mitochondrial recruitment of NF-B observed following ANT1 overexpression has an important role in ANT1 proapoptotic activity.Apoptosis is a form of cell death that plays a role in development, tissue homeostasis, and disease (1). The induction of apoptosis is governed by an elaborate array of checks and balances in the cell. Studies of apoptosis induction in "in vitro" systems have demonstrated that mitochondria are required for the apoptosis stimulated by a variety of different factors (2).The ANT 1 protein is localized in the inner mitochondrial membrane and exchanges cytosolic ADP for mitochondrial ATP (3). Three isoforms (ANT1, ANT2, and ANT3) with tissuespecific expression patterns have been described in humans (4). ANT interacts with several proteins of the outer mitochondrial membrane (peripheral benzodiazepine receptor, porin/VDAC, and Bax) as well as the matrix (cyclophilin D) to form the permeability transition pore (PTP) (5). The PTP appears to be an important regulator of the apoptotic process. Opening of the pore leads to a loss of mitochondrial transmembrane potential, ⌬⌿ m , which can ultimately culminate in matrix swelling and outer membrane rupture, allowing the release of apoptogenic proteins such as cytochrome c, apoptosis-inducing factor, and procaspases (6, 7). Proteins of the bcl-2 family essentially control the release of cytochrome c. Antiapoptotic members of the family (Bcl-2 and Bcl-XL) prevent cytochrome release, whereas the proapoptotic members Bax and Bak exert the opposite effect (8). Bax has been shown to interact with ANT to induce PTP opening and cytochrome c release (9). Several pharmacological compounds interfere with PTP. For instance, cyclosporin A, through its binding to cyclophilin D, prevents PTP opening, and bongkrekic acid and atractyloside are, respectively, a blocker and an inducer of apoptosis via binding of two different conformational states of ANT (10). In addition, alongside their modulation of pore formation by ANT, Bcl-2 and Bax also have been reported to influence ANT ADP/ATP antiporter activity (11). Although ...

Mitochondrial adenine nucleotide translocase 1 (ANT1), but not ANT2, can dominantly induce apoptosis [Bauer et al. (1999) J. Cell Biol. 439, 258^262]. Nothing is known, however, about the apoptotic activity of ANT3. We have transfected HeLa cells with the three human ANT isoforms to compare their potential as inducers of apoptosis. Transient overexpression of ANT3 resulted, like ANT1, in apoptosis as shown by an increase in the sub-G1 fraction, annexin V staining, low v v8 8 m , and activation of caspases 9 and 3. Moreover, the apoptosis produced by ANT3 was inhibited by bongkrekic acid and by cyclosporin A. The pro-apoptotic activities of the ANT1 and ANT3 isoforms contrast with the lack of apoptotic activity of ANT2. This ¢nding may help to identify the speci¢c factors associated with the pro-apoptotic activities of ANT isoforms. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Diabetic cardiomyopathy (DCM) has emerged as a relevant cause of heart failure among the diabetic population. Defined as a cardiac dysfunction that develops in diabetic patients independently of other major cardiovascular risks factors, such as high blood pressure and coronary artery disease, the underlying cause of DCMremains to be unveiled. Several pathogenic factors, including glucose and lipid toxicity, mitochondrial dysfunction, increased oxidative stress, sustained activation of the renin-angiotensin system (RAS) or altered calcium homeostasis, have been shown to contribute to the structural and functional alterations that characterize diabetic hearts. However, all these pathogenic mechanisms appear to stem from the metabolic inflexibility imposed by insulin resistance or lack of insulin signaling. This results in absolute reliance on fatty acids for the synthesis of ATP and impairment of glucose oxidation. Glucose is then rerouted to other metabolic pathways, with harmful effects on cardiomyocyte function. Here, we discuss the role that impaired cardiac insulin signaling in diabetic or insulin-resistant individuals plays in the onset and progression of DCM.

Abstract-The Drosophila pair-rule gene even skipped (eve) is required for embryonic segmentation and later in specific cell lineages in both the nervous system and the mesoderm. We previously generated eve mesoderm-specific mutants by combining an eve null mutant with a rescuing transgene that includes the entire locus, but with the mesodermal enhancer removed. This allowed us to analyze in detail the defects that result from a precisely targeted elimination of mesodermal eve expression in the context of an otherwise normal embryo. Absence of mesodermal eve causes a highly selective loss of the entire eve-expressing lineage in this germ layer, including those progeny that do not continue to express eve, suggesting that mesodermal eve precursor specification is not implemented. Despite the resulting absence of a subset of muscles and pericardial cells, mesoderm-specific eve mutants survive to fertile adulthood, providing an opportunity to examine the effects of these developmental abnormalities on adult fitness and heart function. We find that in these mutants, flying ability, myocardial performance under normal and stressed conditions, and lifespan are severely reduced. These data imply a nonautonomous role of the affected pericardial cells and body wall muscles in developing and/or maintaining cardiac performance and possibly other functions contributing to normal lifespan. Given the similarities of molecular-genetic control between Drosophila and vertebrates, these findings suggest that peri/epicardial influences may well be important for proper myocardial function. Key Words: cardiac development Ⅲ heart rate Ⅲ cardiac failure Ⅲ aging Ⅲ muscle A lthough much effort has gone into defining developmental pathways, many of the mechanistic aspects of regulatory processes occurring during tissue specialization and organogenesis remain to be elucidated. The homeoboxcontaining gene even skipped (eve) is involved in several such aspects of tissue specialization. It was first identified in Drosophila as a segmentation gene. 1 Later during development, eve is expressed in the nervous system and in dorsal muscle progenitors and pericardial cells. 2 Regulatory elements for each of these aspects of the pattern were localized, 3,4 and a transgenic copy of the eve gene was shown to fully supply eve function. 3 Specific repressor domains of Eve were identified in cultured cell assays 5,6 and subsequently shown to interact functionally with corepressors. [7][8][9] eve is expressed in, and required for the formation of, a subset of dorsal muscle and pericardial (PC) cells. 10 -12 In the developing mesoderm, early progenitors in the cardiogenic region express eve in segmentally repeated clusters that later differentiate into eve-positive pericardial cells (EPCs) and dorsal acute muscle 1 (DA1). 2 A transgene was generated that fully rescues the phenotype of eve null mutants in all other tissues, but gives no detectable expression or function in the mesoderm, and resulting embryos were shown to develop mesodermal-specific defec...

Cardiovascular diseases (CVDs) affect the myocardium and vasculature, inducing remodelling of the heart from cellular to whole organ level. To assess their impact at micro and macroscopic level, multi-resolution imaging techniques that provide high quality images without sample alteration and in 3D are necessary: requirements not fulfilled by most of current methods. In this paper, we take advantage of the non-destructive time-efficient 3D multiscale capabilities of synchrotron Propagation-based X-Ray Phase Contrast Imaging (PB-X-PCI) to study a wide range of cardiac tissue characteristics in one healthy and three different diseased rat models. With a dedicated image processing pipeline, PB-X-PCI images are analysed in order to show its capability to assess different cardiac tissue components at both macroscopic and microscopic levels. The presented technique evaluates in detail the overall cardiac morphology, myocyte aggregate orientation, vasculature changes, fibrosis formation and nearly single cell arrangement. Our results agree with conventional histology and literature. This study demonstrates that synchrotron PB-X-PCI, combined with image processing tools, is a powerful technique for multi-resolution structural investigation of the heart ex-vivo . Therefore, the proposed approach can improve the understanding of the multiscale remodelling processes occurring in CVDs, and the comprehensive and fast assessment of future interventional approaches.

-Cánoves (2008) Efficient adult skeletal muscle regeneration in mice deficient in p38β, p38γ and p38δ MAP kinases, Cell Cycle, 7:14, 2208-2214, DOI: 10.4161/cc.7.14.6273To link to this article: https://doi.org/10.4161/cc.7.14.6273 Adult skeletal muscle is a very stable tissue containing a small population of myofiber-associated quiescent satellite cells compared with late embryonic/neonatal skeletal muscle, which contains highly proliferating myoblasts and small actively growing myofibers, suggesting that specific regulatory pathways may control myogenesis at distinct developmental stages. The p38 MAPK signaling pathway is central for myogenesis, based on studies using immortalized and neonatal primary myoblasts in vitro. However, the contribution of this pathway to adult myogenesis has never been investigated. Four p38 isoforms (p38α, p38β, p38γ and p38δ) exist in mammalian cells, being p38α and p38γ the most abundantly expressed isoforms in adult skeletal muscle. Given the embryonic/neonatal lethality of p38α-deficient mice, here we investigate the relative contribution of p38β, p38γ and p38δ to adult myogenesis. Regeneration and myofiber growth of adult muscle proceeds with similar efficiency in mice lacking p38β, p38γ and p38δ as in wild-type control mice. In agreement with this, there is no difference in adult primary myoblasts behavior in vitro among the different genotypes. Importantly, the pattern of p38 activation (ascribed to p38α) remains unperturbed during satellite cell-mediated myogenesis in vitro and adult muscle regeneration in wild type and p38β-, p38γ-and p38δ-deficient mice, rendering p38α as the essential p38 isoform sustaining adult myogenesis. This study constitutes the first analysis addressing the functionality of p38β, p38γ and p38δ in satellite cell-dependent adult muscle regeneration and growth.