Honors & Awards
Fellow, American Heart Association (2011)
Faculty 1000, http://f1000.com/prime/717952921?bd=1 (2012)
Ph.D., University of Barcelona, Molecular Biology (1992)
Heart failure and coronary artery disease are leading clinical problems in the western countries with limited therapeutical options (www.americanheart.org). The heart, however, has endogenous mechanisms of repair that could potentially be enhanced pharmacologically and be used as novel approaches for treatment. Based on a genetic, stem cell and biochemical approaches, research in the Ruiz-Lozanos laboratory focuses on the discovery, analysis and applications of endogenous cardiac repair systems with particular emphasis on the role of epicardial progenitor cells.
Current projects in the laboratory include: 1. The determination of the fate of coronary progenitor cells from the epicardium and their response to growth signals. 2. The potential of epicardial cells to modulate cardiac morphogenesis. 3. Adaptation mechanisms of the heart to stress and aging.
Among the achievements of the laboratory is the generation of the first animal model targeting specific mutations to coronary progenitor cells and the identification of genetic pathways that regulate cardiac growth and coronary formation. A major effort is committed to the isolation of progenitor cells and tissue repair.
Nanoparticle-mediated sustained delivery of therapeutics is one of the highly effective and increasingly utilized applications of nanomedicine. Here, we report the development and application of a drug delivery system consisting of polyethylene glycol (PEG)-conjugated liposomal nanoparticles as an efficient in vivo delivery approach for [Pyr1]-apelin-13 polypeptide. Apelin is an adipokine that regulates a variety of biological functions including cardiac hypertrophy and hypertrophy-induced heart failure. The clinical use of apelin has been greatly impaired by its remarkably short half-life in circulation. Here, we investigate whether [Pyr1]-apelin-13 encapsulation in liposome nanocarriers, conjugated with PEG polymer on their surface, can prolong apelin stability in the blood stream and potentiate apelin beneficial effects in cardiac function. Atomic force microscopy and dynamic light scattering were used to assess the structure and size distribution of drug-laden nanoparticles. [Pyr1]-apelin-13 encapsulation in PEGylated liposomal nanocarriers resulted in sustained and extended drug release both in vitro and in vivo. Moreover, intraperitoneal injection of [Pyr1]-apelin-13 nanocarriers in a mouse model of pressure-overload induced heart failure demonstrated a sustainable long-term effect of [Pyr1]-apelin-13 in preventing cardiac dysfunction. We concluded that this engineered nanocarrier system can serve as a delivery platform for treating heart injuries through sustained bioavailability of cardioprotective therapeutics.
View details for DOI 10.1016/j.biomaterials.2014.08.045
View details for Web of Science ID 000346541100028
View details for PubMedID 25443792
Considering the impaired regenerative capacity of adult mammalian heart tissue, cardiovascular tissue engineering aims to create functional substitutes that can restore the structure and function of the damaged cardiac tissue. The success of cardiac regenerative therapies has been limited mainly due to poor control on the structure and properties of the tissue substitute, lack of vascularization, and immunogenicity. In this study we introduce a new approach to rapidly engineer dense biomimetic scaffolds consisting of type I collagen, to protect the heart against severe ischemic injury. Scaffold biomechanical properties are adjusted to mimic embryonic epicardium which is shown to be optimal to support cardiomyocyte contractile work. Moreover, the designed patch can serve as a delivery device for targeted, controlled release of cells or therapeutic macromolecules into the lesion area.
View details for DOI 10.1007/978-1-4939-1435-7_18
View details for PubMedID 25173173
Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction.
View details for DOI 10.1016/j.biomaterials.2013.08.017
View details for Web of Science ID 000326426500006
View details for PubMedID 23992980
View details for Web of Science ID 000326327100106
Upon incorporation of nanoparticles (NPs) into the body, they are exposed to biological fluids, and their interaction with the dissolved biomolecules leads to the formation of the so-called protein corona on the surface of the NPs. The composition of the corona plays a crucial role in the biological fate of the NPs. While the effects of various physico-chemical parameters on the composition of the corona have been explored in depth, the role of temperature upon its formation has received much less attention. In this work, we have probed the effect of temperature on the protein composition on the surface of a set of NPs with various surface chemistries and electric charges. Our results indicate that the degree of protein coverage and the composition of the adsorbed proteins on the NPs surface depend on the temperature at which the protein corona is formed. Also, the uptake of NPs is affected by the temperature. Temperature is, thus, an important parameter that needs to be carefully controlled in quantitative studies of bio-nano interactions.
View details for DOI 10.1021/nn305337c
View details for Web of Science ID 000323810600013
Anatomical congruence of peripheral nerves and blood vessels is well recognized in a variety of tissues. Their physical proximity and similar branching patterns suggest that the development of these networks might be a coordinated process. Here we show that large diameter coronary veins serve as an intermediate template for distal sympathetic axon extension in the subepicardial layer of the dorsal ventricular wall of the developing mouse heart. Vascular smooth muscle cells (VSMCs) associate with large diameter veins during angiogenesis. In vivo and in vitro experiments demonstrate that these cells mediate extension of sympathetic axons via nerve growth factor (NGF). This association enables topological targeting of axons to final targets such as large diameter coronary arteries in the deeper myocardial layer. As axons extend along veins, arterial VSMCs begin to secrete NGF, which allows axons to reach target cells. We propose a sequential mechanism in which initial axon extension in the subepicardium is governed by transient NGF expression by VSMCs as they are recruited to coronary veins; subsequently, VSMCs in the myocardium begin to express NGF as they are recruited by remodeling arteries, attracting axons toward their final targets. The proposed mechanism underlies a distinct, stereotypical pattern of autonomic innervation that is adapted to the complex tissue structure and physiology of the heart.
View details for DOI 10.1242/dev.087601
View details for Web of Science ID 000316096400011
View details for PubMedID 23462468
Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate G?i and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of ?-arrestins or by pharmacological doses of apelin acting through G?i. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.
View details for DOI 10.1038/nature11263
View details for Web of Science ID 000307501000045
View details for PubMedID 22810587
Bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly in humans. Despite recent advances, the molecular basis of BAV development is poorly understood. Previously it has been shown that mutations in the Notch1 gene lead to BAV and valve calcification both in human and mice, and mice deficient in Gata5 or its downstream target Nos3 have been shown to display BAVs. Here we show that tissue-specific deletion of the gene encoding Activin Receptor Type I (Alk2 or Acvr1) in the cushion mesenchyme results in formation of aortic valve defects including BAV. These defects are largely due to a failure of normal development of the embryonic aortic valve leaflet precursor cushions in the outflow tract resulting in either a fused right- and non-coronary leaflet, or the presence of only a very small, rudimentary non-coronary leaflet. The surviving adult mutant mice display aortic stenosis with high frequency and occasional aortic valve insufficiency. The thickened aortic valve leaflets in such animals do not show changes in Bmp signaling activity, while Map kinase pathways are activated. Although dysfunction correlated with some pro-osteogenic differences in gene expression, neither calcification nor inflammation were detected in aortic valves of Alk2 mutants with stenosis. We conclude that signaling via Alk2 is required for appropriate aortic valve development in utero, and that defects in this process lead to indirect secondary complications later in life.
View details for DOI 10.1371/journal.pone.0035539
View details for Web of Science ID 000305336200053
View details for PubMedID 22536403
The protein kinase product of the gene mutated in myotonic dystrophy 1 (DMPK) is reported to play a role in cardiac pathophysiology. To gain insight into the molecular mechanisms modulated by DMPK, we characterize the impact of DMPK ablation in the context of cardiac ?-adrenergic function. Our data demonstrate that DMPK knockout mice present altered ?-agonist-induced responses and suggest that this is due, at least in part, to a reduced density of ?(1)-adrenergic receptors in cardiac plasma membranes.
View details for DOI 10.1002/mus.22256
View details for Web of Science ID 000298477000023
View details for PubMedID 22190319
Blood vessel formation is important for many physiological and pathological processes and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumour or promoting 'normalization' of tumour vasculature in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows the investigation of cell-cell and cell-matrix interactions in a controlled environment and is therefore a crucial step in developing HCS (high content screening) and HTS (high throughput screening) assays to search for modulators of blood vessel formation. HUVECs (human umbilical-vein endothelial cells) exemplify primary cells used in angiogenesis assays. However, primary cells have significant limitations that include phenotypic decay and/or senescence by six to eight passages in culture, making stable integration of fluorescent markers and large-scale expansion for HTS problematic. To overcome these limitations for HTS, we developed a novel angiogenic model system that employs stable fluorescent endothelial cell lines based on immortalized HMECs (human microvascular endothelial cell). We then evaluated HMEC cultures, both alone and co-cultured with an EMC (epicardial mesothelial cell) line that contributes vascular smooth muscle cells, to determine the suitability for HTS or HCS.The endothelial and epicardial lines were engineered to express a panel of nuclear- and cytoplasm-localized fluorescent proteins to be mixed and matched to suit particular experimental goals. HMECs retained their angiogenic potential and stably expressed fluorescent proteins for at least 13 passages after transduction. Within 8 h after plating on Matrigel, the cells migrated and coalesced into networks of vessel-like structures. If co-cultured with EMCs, the branches formed cylindrical-shaped structures of HMECs surrounded by EMC derivatives reminiscent of vessels. Network formation measurements revealed responsiveness to media composition and control compounds.HMEC-based lines retain most of the angiogenic features of primary endothelial cells and yet possess long-term stability and ease of culture, making them intriguing candidates for large-scale primary HCS and HTS (of ~10000-1000000 molecules). Furthermore, inclusion of EMCs demonstrates the feasibility of using epicardial-derived cells, which normally contribute to smooth muscle, to model large vessel formation. In summary, the immortalized fluorescent HMEC and EMC lines and straightforward culture conditions will enable assay development for HCS of angiogenesis.
View details for DOI 10.1042/BC20100146
View details for Web of Science ID 000296483100002
View details for PubMedID 21732911
The adult human heart is an ideal target for regenerative intervention since it does not functionally restore itself after injury yet has a modest regenerative capacity that could be enhanced by innovative therapies. Adult cardiac cells with regenerative potential share gene expression signatures with early fetal progenitors that give rise to multiple cardiac cell types, suggesting that the evolutionarily conserved regulatory networks that drive embryonic heart development might also control aspects of regeneration. Here we discuss commonalities of development and regeneration, and the application of the rich developmental biology heritage to achieve therapeutic regeneration of the human heart.
View details for DOI 10.1101/gad.2018411
View details for Web of Science ID 000287365000003
View details for PubMedID 21325131
Epicardial signaling and Rxra are required for expansion of the ventricular myocardial compact zone. Here, we examine Raldh2(-/-) and Rxra(-/-) mouse embryos to investigate the role of retinoic acid (RA) signaling in this developmental process. The heart phenotypes of Raldh2 and Rxra mutants are very similar and are characterized by a prominent defect in ventricular compact zone growth. Although RA activity is completely lost in Raldh2(-/-) epicardium and the adjacent myocardium, RA activity is not lost in Rxra(-/-) hearts, suggesting that RA signaling in the epicardium/myocardium is not required for myocardial compact zone formation. We explored the possibility that RA-mediated target gene transcription in non-cardiac tissues is required for this process. We found that hepatic expression of erythropoietin (EPO), a secreted factor implicated in myocardial expansion, is dependent on both Raldh2 and Rxra. Chromatin immunoprecipitation studies support Epo as a direct target of RA signaling in embryonic liver. Treatment of an epicardial cell line with EPO, but not RA, upregulates Igf2. Furthermore, both Raldh2(-/-) and Rxra(-/-) hearts exhibit downregulation of Igf2 mRNA in the epicardium. EPO treatment of cultured Raldh2(-/-) hearts restores epicardial Igf2 expression and rescues ventricular cardiomyocyte proliferation. We propose a new model for the mechanism of RA-mediated myocardial expansion in which RA directly induces hepatic Epo resulting in activation of epicardial Igf2 that stimulates compact zone growth. This RA-EPO-IGF2 signaling axis coordinates liver hematopoiesis with heart development.
View details for DOI 10.1242/dev.054239
View details for Web of Science ID 000285046500015
View details for PubMedID 21138976
The circulating, adipocyte-secreted hormone adiponectin (APN) exerts protective effects on the heart under stress conditions. The receptors binding APN to cardiac tissue, however, have remained elusive. Here, we report that the glycosyl phosphatidylinositol–anchored cell surface glycoprotein T-cadherin (encoded by Cdh13) protects against cardiac stress through its association with APN in mice. We observed extensive colocalization of T-cadherin and APN on cardiomyocytes in vivo. In T-cadherin-deficient mice, APN failed to associate with cardiac tissue, and its levels dramatically increased in the circulation. Pressure overload stress resulted in exacerbated cardiac hypertrophy in T-cadherin-null mice and paralleled corresponding defects in mice lacking APN. During ischemia-reperfusion injury, the absence of T-cadherin increased infarct size similar to that in APN-null mice. Myocardial AMPK is a major downstream protective signaling target of APN. In both cardiac hypertrophy and ischemia-reperfusion models, T-cadherin was necessary for APN-dependent AMPK phosphorylation. In APN-null mice, recombinant adenovirus-expressed APN reduced exaggerated hypertrophy and infarct size and restored AMPK phosphorylation as previously reported. In contrast, rescue was ineffective in mice lacking T-cadherin in addition to APN. These data suggest that T-cadherin protects from stress-induced pathological cardiac remodeling by binding APN and activating its cardioprotective functions.
View details for DOI 10.1172/JCI43464
View details for Web of Science ID 000284971400020
View details for PubMedID 21041950
Frank-Ter Haar syndrome (FTHS), also known as Ter Haar syndrome, is an autosomal-recessive disorder characterized by skeletal, cardiovascular, and eye abnormalities, such as increased intraocular pressure, prominent eyes, and hypertelorism. We have conducted homozygosity mapping on patients representing 12 FTHS families. A locus on chromosome 5q35.1 was identified for which patients from nine families shared homozygosity. For one family, a homozygous deletion mapped exactly to the smallest region of overlapping homozygosity, which contains a single gene, SH3PXD2B. This gene encodes the TKS4 protein, a phox homology (PX) and Src homology 3 (SH3) domain-containing adaptor protein and Src substrate. This protein was recently shown to be involved in the formation of actin-rich membrane protrusions called podosomes or invadopodia, which coordinate pericellular proteolysis with cell migration. Mice lacking Tks4 also showed pronounced skeletal, eye, and cardiac abnormalities and phenocopied the majority of the defects associated with FTHS. These findings establish a role for TKS4 in FTHS and embryonic development. Mutation analysis revealed five different homozygous mutations in SH3PXD2B in seven FTHS families. No SH3PXD2B mutations were detected in six other FTHS families, demonstrating the genetic heterogeneity of this condition. Interestingly however, dermal fibroblasts from one of the individuals without an SH3PXD2B mutation nevertheless expressed lower levels of the TKS4 protein, suggesting a common mechanism underlying disease causation.
View details for DOI 10.1016/j.ajhg.2010.01.009
View details for Web of Science ID 000274637200015
View details for PubMedID 20137777
The outermost layer of the vertebrate heart originates from migratory mesothelial cells (epicardium) that give rise to coronary vascular smooth muscles and fibroblasts. The role of the epicardium in myocardial morphogenesis and establishment of normal heart function is still largely unknown. Here, we use Drosophila to investigate non-autonomous influences of epicardial-like tissue surrounding the heart tube on the structural and functional integrity of the myocardium. It has previously been shown that during Drosophila heart formation, mesodermal expression of the homeobox transcription factor even-skipped (eve) is required for specification of a subset of non-myocardial progenitors in the precardiac mesoderm. These progenitors may share some similarities with the vertebrate epicardium. To investigate a non-autonomous epicardial-like influence on myocardial physiology, we studied the consequences of reduced mesodermal Eve expression and epi/pericardial cell numbers on the maturation of the myocardial heart tube, its contractility, and acquisition of a normal heart rhythm in the Drosophila model. Targeting the eve repressor ladybird early (lbe) with the minimal eve mesodermal enhancer efficiently eliminates the mesodermal Eve lineages. These flies exhibit defects in heart structure, including a reduction in systolic and diastolic diameter (akin to 'restrictive cardiomyopathy'). They also exhibit an elevated incidence of arrhythmias and intermittent asystoles, as well as compromised performance under stress. These abnormalities are restored by eve reexpression or by lbe-RNAi co-overexpression. The data suggest that adult heart function in Drosophila is likely to be modulated non-autonomously, possibly by paracrine influences from neighboring cells, such as the epi/pericardium. Thus, Drosophila may serve as a model for finding genetic effectors of epicardial-myocardial interactions relevant to higher organisms.
View details for DOI 10.1016/j.ydbio.2009.02.013
View details for Web of Science ID 000265239100028
View details for PubMedID 19233157
The genetic basis of heart development is remarkably conserved from Drosophila to mammals, and insights from flies have greatly informed our understanding of vertebrate heart development. Recent evidence suggests that many aspects of heart function are also conserved and the genes involved in heart development also play roles in adult heart function. We have developed a Drosophila heart preparation and movement analysis algorithm that allows quantification of functional parameters. Our methodology combines high-speed optical recording of beating hearts with a robust, semi-automated analysis to accurately detect and quantify, on a beat-to-beat basis, not only heart rate but also diastolic and systolic intervals, systolic and diastolic diameters, percent fractional shortening, contraction wave velocity, and cardiac arrhythmicity. Here, we present a detailed analysis of hearts from adult Drosophila, 2-3-day-old zebrafish larva, and 8-day-old mouse embryos, indicating that our methodology is potentially applicable to an array of biological models. We detect progressive age-related changes in fly hearts as well as subtle but distinct cardiac deficits in Tbx5 heterozygote mutant zebrafish. Our methodology for quantifying cardiac function in these genetically tractable model systems should provide valuable insights into the genetics of heart function.
View details for DOI 10.2144/000113078
View details for Web of Science ID 000266095700006
View details for PubMedID 19317655
Trophic factors secreted both from the endocardium and epicardium regulate appropriate growth of the myocardium during cardiac development. Epicardially-derived cells play also a key role in development of the coronary vasculature. This process involves transformation of epithelial (epicardial) cells to mesenchymal cells (EMT). Similarly, a subset of endocardial cells undergoes EMT to form the mesenchyme of endocardial cushions, which function as primordia for developing valves and septa. While it has been suggested that transforming growth factor-betas (Tgf-beta) play an important role in induction of EMT in the avian epi- and endocardium, the function of Tgf-betas in corresponding mammalian tissues is still poorly understood. In this study, we have ablated the Tgf-beta type I receptor Alk5 in endo-, myo- and epicardial lineages using the Tie2-Cre, Nkx2.5-Cre, and Gata5-Cre driver lines, respectively. We show that while Alk5-mediated signaling does not play a major role in the myocardium during mouse cardiac development, it is critically important in the endocardium for induction of EMT both in vitro and in vivo. Moreover, loss of epicardial Alk5-mediated signaling leads to disruption of cell-cell interactions between the epicardium and myocardium resulting in a thinned myocardium. Furthermore, epicardial cells lacking Alk5 fail to undergo Tgf-beta-induced EMT in vitro. Late term mutant embryos lacking epicardial Alk5 display defective formation of a smooth muscle cell layer around coronary arteries, and aberrant formation of capillary vessels in the myocardium suggesting that Alk5 is controlling vascular homeostasis during cardiogenesis. To conclude, Tgf-beta signaling via Alk5 is not required in myocardial cells during mammalian cardiac development, but plays an irreplaceable cell-autonomous role regulating cellular communication, differentiation and proliferation in endocardial and epicardial cells.
View details for DOI 10.1016/j.ydbio.2008.07.038
View details for Web of Science ID 000259790000020
View details for PubMedID 18718461
Dissecting the molecular mechanisms that guide the proper development of epicardial cell lineages is critical for understanding the etiology of both congenital and adult forms of human cardiovascular disease. In this study, we describe the function of BAF180, a polybromo protein in ATP-dependent SWI/SNF chromatin remodeling complexes, in coronary development. Ablation of BAF180 leads to impaired epithelial-to-mesenchymal-transition (EMT) and arrested maturation of epicardium around E11.5. Three-dimensional collagen gel assays revealed that the BAF180 mutant epicardial cells indeed possess significantly compromised migrating and EMT potentials. Consequently, the mutant hearts form abnormal surface nodules and fail to develop the fine and continuous plexus of coronary vessels that cover the entire ventricle around E14. PECAM and *-SMA staining assays indicate that these nodules are defective structures resulting from the failure of endothelial and smooth muscle cells within them to form coronary vessels. PECAM staining also reveal that there are very few coronary vessels inside the myocardium of mutant hearts. Consistent with this, quantitative RT-PCR analysis indicate that the expression of genes involved in FGF, TGF, and VEGF pathways essential for coronary development are down-regulated in mutant hearts. Together, these data reveal for the first time that BAF180 is critical for coronary vessel formation.
View details for DOI 10.1016/j.ydbio.2008.04.020
View details for Web of Science ID 000257734600009
View details for PubMedID 18508041
Although the use of stem cells in cell-replacement therapies by transplantation is obvious, another equally important and interesting application of stem cells is to use them in disease modeling. Disease models serve as a platform to dissect the biochemical mechanisms of normal phenotypes and the processes which go awry during disease conditions. Particularly in complex, multigenic diseases, molecular studies lead to a greater understanding of the disease, and perhaps more targeted approaches for therapies. Stem cells provide an ideal in vitro system in which to study events related to development at the molecular and cellular level. Neural stem cells have been used as excellent models to study the mechanisms of differentiation of cells of the central nervous system. These studies may be particularly relevant to diseases of complex etiology such as psychiatric illnesses, neurodegenerative diseases and brain tumors. Stem cell-derived systems are also being developed to create models of cardiovascular disease. The application of stem cells to the study of cardiovascular illnesses, and vertebrate heart development, is discussed.
View details for PubMedID 18220912
We have previously identified several members of the Wnt/beta-catenin pathway that are differentially expressed in a mouse model with deficient coronary vessel formation. Systemic ablation of beta-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 beta-catenin in coronary arteriogenesis, we have generated conditional beta-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 beta-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 beta-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-beta-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 beta-catenin pathway in coronary arteriogenesis.
View details for DOI 10.1073/pnas.0702415104
View details for Web of Science ID 000251077000039
Myotonic dystrophy 1 (DM1) is caused by a CTG expansion in the 3'-unstranslated region of the DMPK gene, which encodes a serine/threonine protein kinase. One of the common clinical features of DM1 patients is insulin resistance, which has been associated with a pathogenic effect of the repeat expansions. Here we show that DMPK itself is a positive modulator of insulin action. DMPK-deficient (dmpk-/-) mice exhibit impaired insulin signaling in muscle tissues but not in adipocytes and liver, tissues in which DMPK is not expressed. Dmpk-/- mice display metabolic derangements such as abnormal glucose tolerance, reduced glucose uptake and impaired insulin-dependent GLUT4 trafficking in muscle. Using DMPK mutants, we show that DMPK is required for a correct intracellular trafficking of insulin and IGF-1 receptors, providing a mechanism to explain the molecular and metabolic phenotype of dmpk-/- mice. Taken together, these findings indicate that reduced DMPK expression may directly influence the onset of insulin-resistance in DM1 patients and point to dmpk as a new candidate gene for susceptibility to type 2-diabetes.
View details for DOI 10.1371/journal.pone.0001134
View details for Web of Science ID 000207459000018
View details for PubMedID 17987120
Growing evidence indicates that microRNAs (miRNAs or miRs) are involved in basic cell functions and oncogenesis. Here we report that miR-133 has a critical role in determining cardiomyocyte hypertrophy. We observed decreased expression of both miR-133 and miR-1, which belong to the same transcriptional unit, in mouse and human models of cardiac hypertrophy. In vitro overexpression of miR-133 or miR-1 inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by 'decoy' sequences induced hypertrophy, which was more pronounced than that after stimulation with conventional inducers of hypertrophy. In vivo inhibition of miR-133 by a single infusion of an antagomir caused marked and sustained cardiac hypertrophy. We identified specific targets of miR-133: RhoA, a GDP-GTP exchange protein regulating cardiac hypertrophy; Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis. Our data show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease.
View details for DOI 10.1038/nm1582
View details for Web of Science ID 000246302800029
View details for PubMedID 17468766
The adult heart displays a low proliferation capacity, compromising its function if exposed to distinct biological insults. Interestingly, the observation that an increasing number of cell types display an unpredicted cellular plasticity has opened new therapeutical avenues. In this review we will summarize the current knowledge of non-resident stem cells that can be putatively used for cardiac regeneration. At present, bone marrow stem cells have been extensively studied as a cellular source to heal the heart; however, their myocardial contribution is highly limited. Experimental studies have demonstrated that skeletal myoblasts can engraft into the heart, although, unfortunately, they lead to myocardial uncoupling. Embryonic stem cells can spontaneously generate cardiomyocytes that exhibit a variety of electrophysiological phenotypes. Several constrains should nonetheless be overcome before entering the clinical arena, such as the ability to direct and control the generation of cardiomyocytes into a single myocardial lineage.
View details for DOI 10.1007/s00018-007-6521-4
View details for Web of Science ID 000245131600005
View details for PubMedID 17380309
The heterogeneous cell types of the cardiac conduction system are responsible for coordinating and maintaining rhythmic contractions of the heart. While it has been shown that the cells of the conduction system are derived from myocytes, additional cell types, including neural crest cells, may play a role in the development and maturation of these specialized cell lineages. Previous work has shown that the expression of the hf-1b gene is required for specification of the cardiac conduction system. Using Cre-Lox technology, we conditionally mutated the hf-1b gene in the ventricular and the neural crest cell lineages. Cx40 immunohistochemistry on HF-1b tissue-restricted knockouts revealed a requirement for HF-1b in the cardiomyogenic lineage. Electrophysiological studies identified a second requirement for HF-1b in the neural crest-derived cells. Absence of HF-1b in the neural crest led to atrial and atrioventricular dysfunction resulting from deficiencies in the neurotrophin receptor trkC. Therefore, in this study, we document that a single transcription factor, HF-1b, acts through two separate cell types to direct distinct functions of the cardiac conduction system.
View details for DOI 10.1016/j.ydbio.2005.10.018
View details for Web of Science ID 000236410900003
View details for PubMedID 16430881
Laminin alpha4 chain is a component of extracellular matrix (ECM) laminin-8 and -9 and serves dual roles as a structure protein and as a signaling molecule. The abundance of laminin alpha4 chain transcripts in the heart suggests an important role of this protein in cardiovascular development and function. In this study, we demonstrate that laminin alpha4 deficient mice gradually develop cardiac hypertrophy with impaired function. We show that depletion of laminin alpha4 chain did not alter the levels of dystrophin-glycoprotein complex (DGC) components or affect cell membrane integrity. No alteration in integrin beta 1D protein was observed in terms of expression level or distribution pattern, indicating that the postnatal development of cardiac hypertrophy and cardiomyopathy in these mice is unlikely associated with the stability of sarcolemmal DGC and integrin complexes. Moreover, cardiomyocytes isolated from Lama4-/- mutant hearts maintained their contractility in vitro. In contrast, elevated levels of hypoxia-inducible factor 1alpha (Hif1alpha) and vascular endothelial growth factor A (Vegfa) transcripts, along with multiple foci of cardiomyocyte degeneration and fibrosis suggested sustained cardiac ischemia. Electron microscopy confirmed malformed blood vessels and wide pericapillary ECM spaces, suggesting the presence of microcirculation abnormalities in Lama4-/- mutant hearts. We thus conclude that mutation in the laminin alpha4 chain leads to abnormal cardiovascular ECM structure that cause insufficient oxygen supply to the heart and the subsequent ischemic cardiac phenotype observed. Our study links the genetic deficiency of an ECM protein to cardiomyopathy and implies a novel pathway of idiopathic cardiomyopathy in human.
View details for DOI 10.1074/jbc.M505061200
View details for Web of Science ID 000234307200029
View details for PubMedID 16204254
Vitamin A signals play critical roles during embryonic development. In particular, heart morphogenesis depends on vitamin A signals mediated by the retinoid X receptor alpha (RXRalpha), as the systemic mutation of this receptor results in thinning of the myocardium and embryonic lethality. However, the molecular and cellular mechanisms controlled by RXRalpha signaling in this process are unclear, because a myocardium-restricted RXRalpha mutation does not perturb heart morphogenesis. Here, we analyze a series of tissue-restricted mutations of the RXRalpha gene in the cardiac neural crest, endothelial, and epicardial lineages, and we show that RXRalpha signaling in the epicardium is required for proper cardiac morphogenesis. Moreover, we detect an additional phenotype of defective coronary arteriogenesis associated with RXRalpha deficiency and identify a retinoid-dependent Wnt signaling pathway that cooperates in epicardial epithelial-to-mesenchymal transformation.
View details for DOI 10.1073/pnas.0504343102
View details for Web of Science ID 000234174300039
View details for PubMedID 16352730
Here we address the capacity of bone marrow-derived cells (BMDCs) to trans-differentiate into mature myocytes under the physiological stimulus of exercise training.For this purpose, we have transplanted bone marrow from mice ubiquitously expressing enhanced green fluorescence protein (eGFP) into host mice that have been subjected to a prolonged program of exercise.In all successful bone marrow reconstitutions (greater than 80%), we observed rare but consistent events of bone marrow-derived cardiomyocytes, the frequency of which was unchanged upon exercise training. We have further determined whether these recruited myocytes are a product of trans-differentiation or fusion by the use of a genetic system that distinguishes cell fusion from trans-differentiation in a single-cell assay.We concluded that both in the unchallenged mouse and in the trained specimens, fusion is the most prominent mechanism by which bone marrow-derived cells are observed in the myocyte compartment.
View details for DOI 10.1016/j.cardiores.2005.09.016
View details for Web of Science ID 000233469400009
View details for PubMedID 16256964
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.
View details for DOI 10.1161/01.RES.0000191546.08532.B2
View details for Web of Science ID 000233460900008
View details for PubMedID 16239588
The development of the nervous system (NS) requires the coordinated migration of multiple waves of neurons and subsequent processes of neurite maturation, both involving selective guidance mechanisms. In Caenorhabditis elegans, unc-53 codes for a new multidomain protein involved in the directional migration of a subset of cells. We describe here the first functional characterization of the mouse homologue, mouse Neuron navigator 1 (mNAV1), whose expression is largely restricted to the NS during development. EGFP-mNAV1 associates with microtubules (MTs) plus ends present in the growth cone through a new microtubule-binding (MTB) domain. Moreover, its overexpression in transfected cells leads to MT bundling. The abolition of mNAV1 causes loss of directionality in the leading processes of pontine-migrating cells, providing evidence for a role of mNAV1 in mediating Netrin-1-induced directional migration.
View details for DOI 10.1016/j.mcn.2004.09.016
View details for Web of Science ID 000228352200001
View details for PubMedID 15797708
Myotonic dystrophy (DM) is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Cardiovascular disease is one of the most prevalent causes of death in DM patients. Electrophysiological studies in cardiac muscles from DM patients and from DMPK(-/-) mice suggested that DMPK is critical to the modulation of cardiac contractility and to the maintenance of proper cardiac conduction activity. However, there are no data regarding the molecular signaling pathways involved in DM heart failure. Here we show that DMPK expression in cardiac myocytes is highly enriched in the sarcoplasmic reticulum (SR) where it colocalizes with the ryanodine receptor and phospholamban (PLN), a muscle-specific SR Ca(2+)-ATPase (SERCA2a) inhibitor. Coimmunoprecipitation studies showed that DMPK and PLN can physically associate. Furthermore, purified wild-type DMPK, but not a kinase-deficient mutant (K110A DMPK), phosphorylates PLN in vitro. Subsequent studies using the DMPK(-/-) mice demonstrated that PLN is hypo-phosphorylated in SR vesicles from DMPK(-/-) mice compared with wild-type mice both in vitro and in vivo. Finally, we show that Ca(2+) uptake in SR is impaired in ventricular homogenates from DMPK(-/-) mice. Together, our data suggest the existence of a novel regulatory DMPK pathway for cardiac contractility and provide a molecular mechanism for DM heart pathology.
View details for DOI 10.1074/jbc.M412845200
View details for Web of Science ID 000227395700071
View details for PubMedID 15598648
The mammalian heart is known to contain only two isoformic myosin heavy chain (MHC) genes, alpha and beta. A previously uncharacterized MHC gene was isolated in Syrian hamster hearts (McCully et al., JMol Biol 1991). We identified the novel MHC gene as a hamster embryonic skeletal MHC gene based on the developmental stage- and tissue-specific expression pattern: the restricted expression ofmRNA to striated muscles was highest in embryonic skeletal muscle and was developmentally down-regulated. We confirmed that the embryonic skeletal MHC gene exhibited higher expression in cardiomyopathic than in normal hamster hearts, and was up-regulated during the development of cardiomyopathy. The sporadic expression was highly localized in the endocardium. The present study identified that a very small number of undifferentiated myogenic cells existed in adult hamster endocardium. Moreover, using RT-PCR, a homologue of embryonic skeletal MHC mRNA was also expressed in human embryonic, but not adult ventricles. Our data provide a new insight into the regulatory mechanisms of MHCs in the cardiomyopathic hamster heart.
View details for Web of Science ID 000186410900002
View details for PubMedID 14677642
The murine dishevelled 2 (Dvl2) gene is an ortholog of the Drosophila segment polarity gene Dishevelled, a member of the highly conserved Wingless/Wnt developmental pathway. Dvl2-deficient mice were produced to determine the role of Dvl2 in mammalian development. Mice containing null mutations in Dvl2 present with 50% lethality in both inbred 129S6 and in a hybrid 129S6-NIH Black Swiss background because of severe cardiovascular outflow tract defects, including double outlet right ventricle, transposition of the great arteries and persistent truncus arteriosis. The majority of the surviving Dvl2(-/-) mice were female, suggesting that penetrance was influenced by sex. Expression of Pitx2 and plexin A2 was attenuated in Dvl2 null mutants, suggesting a defect in cardiac neural crest development during outflow tract formation. In addition, approximately 90% of Dvl2(-/-) mice have vertebral and rib malformations that affect the proximal as well as the distal parts of the ribs. These skeletal abnormalities were more pronounced in mice deficient for both Dvl1 and Dvl2. Somite differentiation markers used to analyze Dvl2(-/-) and Dvl1(-/-);Dvl2(-/-) mutant embryos revealed mildly aberrant expression of Uncx4.1, delta 1 and myogenin, suggesting defects in somite segmentation. Finally, 2-3% of Dvl2(-/-) embryos displayed thoracic spina bifida, while virtually all Dvl1/2 double mutant embryos displayed craniorachishisis, a completely open neural tube from the midbrain to the tail. Thus, Dvl2 is essential for normal cardiac morphogenesis, somite segmentation and neural tube closure, and there is functional redundancy between Dvl1 and Dvl2 in some phenotypes.
View details for DOI 10.1242/dev.00164
View details for Web of Science ID 000180157100024
View details for PubMedID 12421720
Understanding the cell type-specific molecular mechanisms by which distinct signaling pathways combinatorially control proliferation during organogenesis is a central issue in development and disease. Here, we report that the bicoid-related transcription factor Pitx2 is rapidly induced by the Wnt/Dvl/beta-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Regulated exchange of HDAC1/beta-catenin converts Pitx2 from repressor to activator, analogous to control of TCF/LEF1. Pitx2 then serves as a competence factor required for the temporally ordered and growth factor-dependent recruitment of a series of specific coactivator complexes that prove necessary for Cyclin D2 gene induction. The molecular strategy underlying interactions between the Wnt and growth factor-dependent signaling pathways in cardiac outflow tract and pituitary proliferation is likely to be prototypic of cell-specific proliferation strategies in other tissues.
View details for Web of Science ID 000179594500009
View details for PubMedID 12464179
The identification of several peroxisomal proteins in the past decade has deepened our understanding of the biology of peroxisomes and their involvement in human disorders. We report the cloning and expression pattern during the mouse development of a cDNA encoding a novel protein, named PeP, and show that its product is imported specifically to the peroxisome matrix in a variety of cell types. We also demonstrate that PeP is imported to the organelle through the PEX5 receptor pathway, which indicates that the C-terminal tripeptide SKI behaves as a type 1 peroxisomal targeting signal (PTS1). PeP expression is tightly regulated, as shown by Northern and in situ hybridization experiments. Thus during embryonic development in the mouse, PeP mRNA is detected almost exclusively in the skeletal muscle, whereas in adult mice, strong expression is also found in the heart and brain. In addition, PeP mRNA accumulation is induced after myoblast differentiation in vitro, when myotube formation is promoted. Sequence analysis reveals that PeP has no significant homology to any known protein, except for a short stretch of amino acids containing the fingerprint of the fibronectin type III superfamily, a domain present in proteins often related to molecular and cellular recognition and binding processes. Thus our data suggest a connection between the function of PeP and murine cell differentiation and development.
View details for DOI 10.1002/dvdy.10099
View details for Web of Science ID 000175964000004
View details for PubMedID 12112469
The elastic fibre system has a principal role in the structure and function of various types of organs that require elasticity, such as large arteries, lung and skin. Although elastic fibres are known to be composed of microfibril proteins (for example, fibrillins and latent transforming growth factor (TGF)-beta-binding proteins) and polymerized elastin, the mechanism of their assembly and development is not well understood. Here we report that fibulin-5 (also known as DANCE), a recently discovered integrin ligand, is an essential determinant of elastic fibre organization. fibulin-5-/- mice generated by gene targeting exhibit a severely disorganized elastic fibre system throughout the body. fibulin-5-/- mice survive to adulthood, but have a tortuous aorta with loss of compliance, severe emphysema, and loose skin (cutis laxa). These tissues contain fragmented elastin without an increase of elastase activity, indicating defective development of elastic fibres. Fibulin-5 interacts directly with elastic fibres in vitro, and serves as a ligand for cell surface integrins alphavbeta3, alphavbeta5 and alpha9beta1 through its amino-terminal domain. Thus, fibulin-5 may provide anchorage of elastic fibres to cells, thereby acting to stabilize and organize elastic fibres in the skin, lung and vasculature.
View details for Web of Science ID 000173159300042
View details for PubMedID 11805835
The past three years can be considered in cardiology as critical for understanding the relevance of developmental genes in the adult cardiac physiology. Also, for the first time, endogenous control of programmed cell death has been demonstrated to mark the transition between normal adaptation and cardiac hypertrophy. Most of this work has been based on previous analysis using molecular markers of cardiac determination and differentiation, work that has served a double aim: First, the determination of the cellular process that contribute to the specification of the working heart and secondly, the characterization of key regulatory factors in cardiogenesis. These studies in conjunction with the recent availability of single gene mutation in transgenic mice have furnished a new perspective in the nature of cardiac defects either in shape or function. Here we review some of the key factors in cardiac morphogenesis from the perspective of the analysis of gene mutation.
View details for Web of Science ID 000172774700011
View details for PubMedID 11754790
Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK), IkappaB kinase (IKK)-alpha and -beta, and IkappaBalpha are common elements that signal NF-kappaB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-kappaB DNA-binding activity that correlated in time with the activation of IKKalpha, IKKbeta, and NIK. Moreover, the activation of IKKalpha, IKKbeta, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IkappaBalpha(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-kappaB activation and myoblast differentiation, indicating that phosphorylation of IkappaBalpha at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKalpha or IKKbeta revealed that IKKalpha is required for IGF-II-dependent myoblast differentiation, whereas IKKbeta is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II.
View details for Web of Science ID 000169135100076
View details for PubMedID 11279241
The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to beta-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.
View details for Web of Science ID 000168623300074
View details for PubMedID 11331753
HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.
View details for Web of Science ID 000089105200015
View details for PubMedID 11007485
Recent advances have given us new insights into the molecular basis of organ position. A gene cascade that determines left-right positioning of organ primordia has emerged. In here we present the current knowledge of the molecular determinants of organ positioning during vertebrate embryogenesis.
View details for Web of Science ID 000167797100005
View details for PubMedID 11282304
LIM domain containing proteins play critical roles in animal development and cellular differentiation. Here, we describe the cloning and expression patterns of three members of the four and a half LIM domain-only protein family, FHL1, 2, and 3, from mouse. A comparison of embryonic expression patterns of these three highly-related genes indicates that they are expressed in an overlapping pattern in the developing cardiovascular system, and skeletal muscle. In adult tissues, the three genes are expressed in a predominant and overlapping manner in cardiac and skeletal muscle. Of the three genes, FHL2 appears to have the most restricted expression pattern during development, in heart, blood vessels, and skeletal muscle. Expression in heart is highest in cardiac septa and in the region adjacent to the atrio-ventricular ring, suggesting a potential role in septation or conduction system development. In the heart, FHL1expression was observed strongly in developing outflow tract, and to a lesser extent in myocardium. FHL3 displays low and ubiquitous expression during mouse development. Cardiac ventricular expression of FHL1, but not FHL2 or FHL3, was upregulated in two mouse models of cardiac hypertrophic and dilated cardiomyopathy. Taken together, these data indicate the potential importance of this FHL family in the development and maintenance of the cardiovascular system and striated muscle, and suggest that FHL1 may play a role in the development of heart disease.
View details for Web of Science ID 000088607200029
View details for PubMedID 10906474
Recently the near complete cDNA of the regulatory atrial myosin light chain (MLC-2a) was cloned. The atrial specific isoform has been shown to be a useful molecular marker for cardiac chamber specification. Therefore, the regulatory sequence of the gene will provide clues on cardiomyocyte differentiation and atrial specific transcription regulation. Here we report the identification of the murine genomic sequence of the MLC-2a gene (Mylc2a). The entire 5' flanking region was identified and sequenced. In addition, the exon-intron boundaries and 3' flanking region were determined. Sequence comparison revealed the presence of the final exon (11) of the mouse glucokinase gene on chromosome 11, 2.0 kb upstream of the Mylc2a transcription start site. In addition, we compared the structure of the gene to other myosin light chains to show evolutionary conservation. The intron-exon boundaries turned out to be highly conserved and an increasing intron size in more complex mammalian species was found. At the amino acid level there is 95% homology between the human and mouse MLC-2a sequence.
View details for Web of Science ID 000166158200011
View details for PubMedID 11124527
Deletions or rearrangements of human chromosome 22q11 lead to a variety of related clinical syndromes such as DiGeorge syndrome (DGS) and velo--cardiofacial syndrome (VCFS). In addition, patients with 22q11 deletions have an increased incidence of schizophrenia and several studies have mapped susceptibility loci for schizophrenia to this region. Human molecular genetic studies have so far failed to identify the crucial genes or disruption mechanisms that result in these disorders. We have used gene targeting in the mouse to delete a defined region within the conserved DGS critical region (DGCR) on mouse chromosome 16 to prospectively investigate the role of the mouse DGCR in 22q11 syndromes. The deletion spans a conserved portion ( approximately 150 kb) of the proximal region of the DGCR, containing at least seven genes ( Znf74l, Idd, Tsk1, Tsk2, Es2, Gscl and Ctp ). Mice heterozygous for this deletion display no findings of DGS/VCFS in either inbred or mixed backgrounds. However, heterozygous mice display an increase in prepulse inhibition of the startle response, a manifestation of sensorimotor gating that is reduced in humans with schizophrenia. Homozygous deleted mice die soon after implantation, demonstrating that the deleted region contains genes essential for early post-implantation embryonic development. These results suggest that heterozygous deletion of this portion of the DGCR is sufficient for sensorimotor gating abnormalities, but not sufficient to produce the common features of DGS/VCFS in the mouse.
View details for Web of Science ID 000083658800012
View details for PubMedID 10545603
The nuclear hormone receptor PPAR gamma promotes adipogenesis and macrophage differentiation and is a primary pharmacological target in the treatment of type II diabetes. Here, we show that PPAR gamma gene knockout results in two independent lethal phases. Initially, PPAR gamma deficiency interferes with terminal differentiation of the trophoblast and placental vascularization, leading to severe myocardial thinning and death by E10.0. Supplementing PPAR gamma null embryos with wild-type placentas via aggregation with tetraploid embryos corrects the cardiac defect, implicating a previously unrecognized dependence of the developing heart on a functional placenta. A tetraploid-rescued mutant surviving to term exhibited another lethal combination of pathologies, including lipodystrophy and multiple hemorrhages. These findings both confirm and expand the current known spectrum of physiological functions regulated by PPAR gamma.
View details for Web of Science ID 000083356700013
View details for PubMedID 10549290
Although accumulating evidence suggests that the heart develops in a segmental fashion, the molecular mechanisms that control regional specification of cardiomyocytes in the developing heart remain largely unknown. In this study, we have used the mouse cardiac-restricted ankyrin repeat protein (CARP) gene as a model system to study these mechanisms. The CARP gene encodes a nuclear co-regulator for cardiac gene expression, which lies downstream of the cardiac homeobox gene, Nkx 2.5, and is an early marker of the cardiac muscle cell lineage. We have demonstrated that the expression of the gene is developmentally down regulated and dramatically induced as part of the embryonic gene program during cardiac hypertrophy. Using a lacZ/knock-in mouse and three lines of transgenic mouse harboring various CARP promoter/lacZ reporters, we have identified distinct 5' cis regulatory elements of the gene that can direct heart segment-specific transgene expression, such as atrial versus ventricular and left versus right. Most interestingly, a 213 base pair sequence element of the gene was found to confer conotruncal segment-specific transgene expression. Using the transgene as a conotruncal segment-specific marker, we were able to document the developmental fate of a subset of cardiomyocytes in the conotruncus during cardiogenesis. In addition, we have identified an essential GATA-4 binding site in the proximal upstream regulatory region of the gene and cooperative transcriptional regulation mediated by Nkx2.5 and GATA-4. We have shown that this cooperative regulation is dependent on binding of GATA-4 to its cognate DNA sequence in the promoter, which suggests that Nkx2.5 controls CARP expression, at least in part, through GATA-4.
View details for Web of Science ID 000083386600005
View details for PubMedID 10477291
Asymmetric expression of Sonic hedgehog (Shh) in Hensen's node of the chicken embryo plays a key role in the genetic cascade that controls left-right asymmetry, but its involvement in left-right specification in other vertebrates remains unclear. We show that mouse embryos lacking Shh display a variety of laterality defects, including pulmonary left isomerism, alterations of heart looping, and randomization of axial turning. Expression of the left-specific gene Lefty-1 is absent in Shh(-/-) embryos, suggesting that the observed laterality defects could be the result of the lack of Lefty-1. We also demonstrate that retinoic acid (RA) controls Lefty-1 expression in a pathway downstream or parallel to Shh. Further, we provide evidence that RA controls left-right development across vertebrate species. Thus, the roles of Shh and RA in left-right specification indeed are conserved among vertebrates, and the Shh and RA pathways converge in the control of Lefty-1.
View details for Web of Science ID 000082868500083
View details for PubMedID 10500184
We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries and neural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1-5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis by in situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling.
View details for Web of Science ID 000081868400053
View details for PubMedID 10428823
We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.
View details for Web of Science ID 000081377300044
View details for PubMedID 10391924
The role that the p53 tumor suppressor gene product plays in cellular differentiation remains controversial. However, recent evidence indicates that p53 is required for proper embryogenesis. We have studied the effect of p53 on the expression mediated by the promoter of the rat muscle-specific phosphoglycerate mutase gene (M-PGAM), a marker for cardiac and skeletal muscle differentiation. Experiments involving transient transfection, mobility shift assay, and site-directed mutagenesis demonstrated that p53 specifically binds and transactivates the M-PGAM promoter. The p53-related proteins p51A and p73L also transactivated M-PGAM. Moreover, stable expression of a p53 dominant mutant in C2C12 cells blocked the induction of M-PGAM expression during the myoblast to myotube transition and the ability of p53, p51A, and p73L to transactivate the M-PGAM promoter. In addition, impaired expression of M-PGAM was observed in a subset of p53-null animals in heart and muscle tissues of anterior-ventral location. These results demonstrate that p53 is a transcriptional activator of M-PGAM that contributes in vivo to the control of its cardiac expression. These data support previous findings indicating a role for p53 in cellular differentiation.
View details for Web of Science ID 000080501200002
View details for PubMedID 10359011
The embryonic cellular events that set the asymmetry of the genetic control circuit controlling left-right (L-R) axis determination in mammals are poorly understood. New insight into this problem was obtained by analyzing mouse mutants lacking the KIF3A motor subunit of the kinesin-II motor complex. Embryos lacking KIF3A die at 10 days postcoitum, exhibit randomized establishment of L-R asymmetry, and display numerous structural abnormalities. The earliest detectable abnormality in KIF3A mutant embryos is found at day 7.5, where scanning electron microscopy reveals loss of cilia ordinarily present on cells of the wild-type embryonic node, which is thought to play an important role in setting the initial L-R asymmetry. This cellular phenotype is observed before the earliest reported time of asymmetric expression of markers of the L-R signaling pathway. These observations demonstrate that the kinesin-based transport pathway needed for flagellar and ciliary morphogenesis is conserved from Chlamydomonas to mammals and support the view that embryonic cilia play a role in the earliest cellular determinative events establishing L-R asymmetry.
View details for Web of Science ID 000080130200057
View details for PubMedID 10220415
To investigate a potential role of protein-tyrosine phosphatases (PTPases) in myocardial growth and signaling, a degenerate primer-based reverse transcription-polymerase chain reaction approach was used to isolate cDNAs for proteins that contain a PTPase catalytic domain. Among the 16 cDNA clones isolated by reverse transcription-polymerase chain reaction from total neonatal rat cardiomyocyte RNA, one, designated PTP-TD14, was unique. Subsequent isolation and sequencing of a full-length PTP-TD14 cDNA confirmed that it encodes a novel 164-kDa protein, p164(PTP-TD14). The C-terminal region contains the PTP-like domain, whereas the N-terminal region shows no homology to any known mammalian protein. However, this region is homologous to a yeast protein, BRO1, that is involved in the mitogen-activated protein kinase signaling pathway. Like BRO1, p164(PTP-TD14) contains a proline-rich region with two putative SH3-domain binding sites. By Northern blot analysis, PTP-TD14 is expressed as a 5.3-kilobase pair transcript, not only in neonatal heart but also in many adult rat tissues. When expressed in either COS-7 or NIH-3T3 cells, p164(PTP-TD14) localizes to the cytoplasm in association with vesicle-like structures. Expression of p164(PTP-TD14) in NIH-3T3 cells inhibits Ha-ras-mediated transformation more than 3-fold. This inhibitory activity is localized to the C-terminal PTPase homology domain, since no inhibition of Ha-ras-mediated focus formation was observed with a PTP-TD14 mutant, in which the putative catalytic activity was presumably inactivated by a point mutation. These findings indicate that PTP-TD14 encodes a novel protein that may be critically involved in regulating Ha-ras-dependent cell growth.
View details for Web of Science ID 000075386100053
View details for PubMedID 9694860
Transcription regulation of genes active in the cardiovascular system is a complex process, involving DNA and RNA binding proteins. Nucleic acid binding proteins bind to the regulatory DNA and interact with other proteins, including RNA polymerase to initiate and control the level of transcription. The RNA binding proteins have a function in spliceosome formation and in stabilising mRNA. In this review the currently available molecular approaches to analyse regulatory DNA in relation to DNA binding proteins are discussed. Similar techniques that have been developed for RNA binding protein studies are included. In addition to an explanation of the various methods, examples are provided from DNA-protein interactions on genes active in the cardiovascular system, together with strategies for identification and characterisation of new nucleic acid binding proteins active in cardiac or vascular cell types.
View details for Web of Science ID 000074585800004
View details for PubMedID 9709391
RXRalpha null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRalpha-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRalpha-/- embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRalpha mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRalpha mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRalpha mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRalpha-/- phenotype and resembles an embryonic form of dilated cardiomyopathy.
View details for Web of Science ID 000072350300018
View details for PubMedID 9425147
Cortistatin is a 14-residue putative neuropeptide with strong structural similarity to somatostatin and is expressed predominantly in cortical GABAergic interneurons of rats. Administration of cortistatin into the brain ventricles specifically enhances slow-wave sleep, presumably by antagonizing the effects of acetylcholine on cortical excitability. Here we report the identification of cDNAs corresponding to mouse and human preprocortistatin and the mRNA distribution and gene mapping of mouse cortistatin. Analysis of the nucleotide and predicted amino acid sequences from rat and mouse reveals that the 14 C-terminal residues of preprocortistatin, which make up the sequence that is most similar to somatostatin, are conserved between species. Lack of conservation of other dibasic amino acid residues whose cleavage by prohormone convertases would give rise to additional peptides suggests that cortistatin-14 is the only active peptide derived from the precursor. As in the rat, mouse preprocortistatin mRNA is present in GABAergic interneurons in the cerebral cortex and hippocampus. The preprocortistatin gene maps to mouse chromosome 4, in a region showing conserved synteny with human 1p36. The human putative cortistatin peptide has an arginine for lysine substitution, compared to the rat and mouse products, and is N-terminally extended by 3 amino acids.
View details for Web of Science ID A1997XG51900018
View details for PubMedID 9205124
We have isolated the mouse homologue of human spliceosome-associated protein SAP49, mSAP49. mSAP49 contains two RNA recognition motifs (RRM) in the N terminus of the predicted amino acid sequence, and a highly basic C terminus rich in glycine/proline. mSAP49 displayed a plastic of expression in cardiac development. In the adult mouse, mSAP49 is widely distributed, although it was found at relatively lower levels in the heart. In situ hybridization analysis of mSAP49 mRNA distribution in staged mouse embryos showed that mSAP49 onset occurs later in the heart than in other embryonic tissues. While mSAP49 expression was found at day 10.0 postconception (pc) in the optic eminence, optic vesicle, hindbrain, and somites, it was not in cardiac structures. mSAP49 was detected in the ventricles at day 11.5, and at day 13.5 it was also detected in the atria. Northern analysis showed that mSAP49 mRNA displayed a peak of expression in the heart at days 14.0-15.0 pc, and its abundance decayed in the adult. This dynamic pattern of cardiac expression suggests that mSAP49 may be contributing to a change in the ratio of spliceosome components during cardiac growth and development, which may have consequences for tissue-specific splicing, RNA stabilization, or translation.
View details for Web of Science ID A1997WQ84200004
View details for PubMedID 9097020
The induction of the atrial natriuretic factor (ANF) gene during alpha 1-adrenergic stimulation of neonatal rat ventricular myocytes has served as a model for gene expression during cardiac muscle cell hypertrophy. This study describes and identifies a single regulatory element that mediates expression of the ANF gene. Deletional mutations were generated in a 639-bp fragment of the ANF promoter that confers alpha 1-adrenergic inducibility to a luciferase reporter gene in transient transfection assays in ventricular myocytes. The results of gel mobility shift and diethylpyrocarbonate (DEPC) interference studies with nuclear cardiac cell extracts identified the nucleotide contract points for a novel A/T-rich element (ANF-AT) at positions -582/-575 that partially mediates alpha 1-adrenergic inducibility. Mutations in the ANF-AT element reduced alpha-adrenergic inducibility of an ANF-TK-luciferase fusion gene in cardiac cells by 35% but had no effect on expression in other muscle and non-muscle cells tested. Gel mobility supershift assays with antibodies directed against the MEF-2 protein, the homeobox protein MHox, or the zinc finger protein HF-1b, document that these factors are not major components of the endogenous ANF-AT binding activity in cardiac muscle cells. The current study provides evidence for a role for a novel A/T-rich element in the regulation of ANF gene expression in cardiac ventricular myocytes.
View details for Web of Science ID A1997WU80800009
View details for PubMedID 9140811
The expression of the gene encoding the muscle-specific (M)-subunit of phosphoglycerate mutase (PGAM-M) is restricted to adult skeletal and cardiac muscle. In order to study its expression in muscle, the rat PGAM-M gene has been isolated and sequenced. Rat PGAM-M spans about 2.2 kb and is composed of three exons: 442, 181 and 186-bp long, and two introns of 97 bp and 1.3 bp. The analysis of the 5'-flanking region reveals a promoter which contains multiple DNA regulatory elements and constitutes an ideal model to study muscle gene transcriptional regulation. Thus, the elements responsible for rat PGAM-M muscle-specific expression have been identified by transient transfection in chicken embryo primary cultures, using chimeric constructs of the rat promoter linked to a cat reporter gene. Here, we report that in spite of the abundance of E-box motifs in the rat PGAM-M promoter known for their involvement in muscle gene expression, two DNA elements regulate the muscle-specific transcription of rat PGAM-M: an A/T motif, the putative MEF-2-binding site (myocyte-specific enhancer-binding factor 2), and a proximal 27-bp element which is conserved between the rat and human genes. These two elements define a small promoter (170 bp) sufficient to support potent and skeletal-muscle-specific expression. The conserved 27-bp region contains a transcriptional regulatory element able to confer muscle-specific expression when located upstream from a heterologous TATA box.
View details for Web of Science ID A1994PK88000015
View details for PubMedID 7926808
The isolation and characterization of cofactor-independent phosphoglycerate mutase gene from maize (Zea mays) is here reported. This gene sequence constitutes the first described from this enzyme. The gene spans 5.4 kilo base pairs and has nine exons in the translated region. Canonical TATA and CCAAT and Sp1-like boxes are present upstream of the gene. Comparison with cofactor-dependent phosphoglycerate mutase genes revealed no similarity between both groups of enzymes.
View details for Web of Science ID A1994PG80100066
View details for PubMedID 8093040
View details for Web of Science ID 000208702707019