Diego Franco

BMP10 is essential for maintaining cardiac growth during murine cardiogenesis.

During cardiogenesis, perturbation of a key transition at mid-gestation from cardiac patterning to cardiac growth and chamber maturation often leads to diverse types of congenital heart disease, such as ventricular septal defect (VSD), myocardium noncompaction, and ventricular hypertrabeculation. This transition, which occurs at embryonic day (E) 9.0-9.5 in murine embryos and E24-28 in human embryos, is crucial for the developing heart to maintain normal cardiac growth and function in response to an increasing hemodynamic load. Although, ventricular trabeculation and compaction are key morphogenetic events associated with this transition, the molecular and cellular mechanisms are currently unclear. Initially, cardiac restricted cytokine bone morphogenetic protein 10 (BMP10) was identified as being upregulated in hypertrabeculated hearts from mutant embryos deficient in FK506 binding protein 12 (FKBP12). To determine the biological function of BMP10 during cardiac development, we generated BMP10-deficient mice. Here we describe an essential role of BMP10 in regulating cardiac growth and chamber maturation. BMP10 null mice display ectopic and elevated expression of p57kip2 and a dramatic reduction in proliferative activity in cardiomyocytes at E9.0-E9.5. BMP10 is also required for maintaining normal expression levels of several key cardiogenic factors (e.g. NKX2.5 and MEF2C) in the developing myocardium at mid-gestation. Furthermore, BMP10-conditioned medium is able to rescue BMP10-deficient hearts in culture. Our data suggest an important pathway that involves a genetic interaction between BMP10, cell cycle regulatory proteins and several major cardiac transcription factors in orchestrating this transition in cardiogenesis at mid-gestation. This may provide an underlying mechanism for understanding the pathogenesis of both structural and functional congenital heart defects.

PITX2 Insufficiency Leads to Atrial Electrical and Structural Remodeling Linked to Arrhythmogenesis

Background— Pitx2 is a homeobox transcription factor that plays a pivotal role in early left/right determination during embryonic development. Pitx2 loss-of-function mouse mutants display early embryonic lethality with severe cardiac malformations, demonstrating the importance of Pitx2 during cardiogenesis. Recently, independent genome-wide association studies have provided new evidence for a putative role of PITX2 in the adult heart. These studies have independently reported several risk variants close to the PITX2 locus on chromosome 4q25 that are strongly associated with atrial fibrillation in humans. Methods and Results— We show for the first time that PITX2C expression is significantly decreased in human patients with sustained atrial fibrillation, thus providing a molecular link between PITX2 loss of function and atrial fibrillation. In addition, morphological, molecular, and electrophysiological characterization of chamber-specific Pitx2 conditional mouse mutants reveals that atrial but not ventricular chamber-specific deletion of Pitx2 results in differences in the action potential amplitude and resting membrane potential in the adult heart as well as ECG characteristics of atrioventricular block. Lack of Pitx2 in atrial myocardium impairs sodium channel and potassium channel expression, mediated in part by miRNA misexpression. Conclusions— This study thus identifies Pitx2 as an upstream transcriptional regulator of atrial electric function, the insufficiency of which results in cellular and molecular changes leading to atrial electric and structural remodeling linked to arrhythmogenesis.

The Role of Pitx2 during Cardiac Development: Linking Left–Right Signaling and Congenital Heart Diseases☆

Pitx2 is a bicoid-related homeodomain transcription factor that plays a critical role in directing cardiac asymmetric morphogenesis. Ectopic Pitx2c expression in the developing myocardium correlates with double outlet right ventricle (DORV) in laterality mutants. Pitx2 loss of function experiments cause severe cardiovascular defects, such as atrial isomerism (AI), double inlet left ventricle, transposition of the great arteries (TGA), persistent truncus arteriosus (PTA), and abnormal aortic arch (AAA) remodeling. Current studies suggest that Pitx2-mediated signaling during cardiogenesis is conducted within three different cell types: the myocardium, the cardiac neural crest (CNC) cells, and the pharyngeal arch mesenchyme. Impaired Pitx2 function in discrete myocardial regions seems to lead to DORV, AI, and possibly TGA. On the other hand, impaired Pitx2 expression in the CNC leads preferentially to PTA. AAA remodeling is likely to occur owing to impaired cross-talk of the CNC cells with the pharyngeal arch mesenchyme. Thus, Pitx2 appears to be directing left-right identity to the cardiac venous components (e.g., the atria), whereas it appears to be modeling the morphologic arrangement of distinct myocardial components in the arterial pole. These data suggest that altered left-right signaling underlies the etiology of several common congenital cardiac malformations.

Patterns of expression in the developing myocardium: towards a morphologically integrated transcriptional model

The heart is the first embryonic organ to function. Early in development, the heart shows autorhythmycity and peristaltoid contraction waves [1, 2]. Contraction requires the expression of a specific set of proteins that form the contractile apparatus, i.e. the sarcomere. The contraction–relaxation cycle of the sarcomeric apparatus is mediated by changing local concentrations of free calcium. This function is achieved by another set of specific proteins, located in the sarcoplasmic reticulum and in the sarcolemma. Fascinating questions that are still poorly understood are how the cardiogenic lineage becomes established to form the peristaltoid contracting tube without valves and how this tube becomes transformed into the synchronous-contracting four-chambered heart with unidirectional valves. It is well documented that the expression of the different isoforms of contractile proteins changes considerably during these stages (for a review see [3]). However, a detailed analysis of the changes in the patterns of gene expression in relation to cardiac morphogenesis is lacking. In the present review we try to fill this gap. We have centred our attention on gene products (mRNA and protein) expressed in the working myocardium of mammals and birds. No distinction has been made when mRNA and protein display the same pattern of expression, however we have highlighted those cases where the pattern of expression differs between mRNA and protein. The development and expression pattern of genes of the conduction system of the heart merits an independent review [4](Moorman et al., Circ. Res., in press). Data referring to other experimental models as Drosophila , Xenopus or zebrafish ( Danio rerio ) are included only if they are helpful for our general understanding. Often only gene expression in the presumptive atria and/or presumptive ventricles is mentioned, whereas understanding the functional significance of the patterns of gene expression requires knowledge of the entire pattern including the … * Corresponding author. Tel.: +31 (20) 5664928; fax: +31 (20) 6976177; e-mail: a.f.moorman@amc.uva.nl

Multiple Transcriptional Domains, With Distinct Left and Right Components, in the Atrial Chambers of the Developing Heart

During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and Pitx-2, and transgenic mouse lines expressing nlacZ under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.

MicroRNA profiling during mouse ventricular maturation: a role for miR-27 modulating Mef2c expression

AIMS non-coding RNA has been recently demonstrated to be a novel mechanism for modulation of gene expression at the post-transcriptional level. The importance of microRNAs in the cardiovascular system is now apparent. Mutations of distinct microRNAs have provided evidence for fundamental roles of microRNAs during cardiovascular development. However, there is limited information about global microRNA profiles during mouse heart development. In this study, we have gained insight from the expression profiles of microRNAs during mouse ventricular development by microarray and qRT-PCR analysis. METHODS AND RESULTS our microarray analysis reveals that relatively few microRNAs display either increasing or decreasing expression profiles during ventricular chamber formation. Interestingly, most of the differentially expressed microRNAs display a rather discrete peak of expression at particular developmental stages. Furthermore, we demonstrate that microRNA-27b (miR-27b) displays an overt myocardial expression during heart development and that the transcription factor-encoding gene Mef2c is an miR-27b target. CONCLUSION our data present a comprehensive profile of microRNA expression during ventricular maturation, providing an entry point for investigation of the functional roles of the most abundantly and differentially expressed microRNAs during cardiogenesis.

Divergent expression of delayed rectifier K+ channel subunits during mouse heart development

The repolarization phase of the cardiac action potential is dependent on transmembrane K(+) currents. The slow (I(Ks)) and fast (I(Kr)) components of the delayed-rectifier cardiac K(+) current are generated by pore-forming alpha subunits KCNQ1 and KCNH2, respectively, in association with regulatory beta-subunit KCNE1, KCNE2 and perphaps KCNE3. In the present study we have investigated the distribution of transcripts encoding these five potassium channel-forming subunits during mouse heart development as well as the protein distribution of KCNQ1 and KCNH2. KCNQ1 and KCNH2 mRNAs (and protein) are first expressed at embryonic day (E) 9.5, showing comparable levels of expression within the atrial and ventricular myocardium during the embryonic and fetal stages. In contrast, the beta-subunits display a more dynamic pattern of expression during development. KCNE1 expression is first observed at E9.5 throughout the entire myocardium and progressively is confined to the ventricular myocardium. With further development (E16.5), KCNE1 expression is mainly confined to the compact ventricular myocardium. KCNE2 is first expressed at E9.5 and it is restricted already to the atrial myocardium. KCNE3 is first expressed at E8.5 throughout the myocardium and with further development, it becomes restricted to the atrial myocardium. The fact that alpha subunits are homogeneously distributed within the myocardium, whereas the beta subunits display a regionalized expression profile during cardiac development, suggest that differences in the slow and fast component of the delayed-rectifier cardiac K(+) currents between the atrial and the ventricular cardiomyocytes are mainly determined by differential beta-subunit distribution.

Biphasic Development of the Mammalian Ventricular Conduction System

Rationale: The ventricular conduction system controls the propagation of electric activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes organized in defined structures including central components and a peripheral Purkinje fiber network. How the mammalian ventricular conduction system is established during development remains controversial. Objective: To define the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. Methods and Results: A retrospective clonal analysis using the &agr;-cardiac actinnlaacZ/+ mouse line was carried out in three week old hearts. Clusters of clonally related myocytes were screened for conductive cells using connexin40-driven enhanced green fluorescent protein expression. Two classes of clusters containing conductive cells were obtained. Mixed clusters, composed of conductive and working myocytes, reveal that both cell types develop from common progenitor cells, whereas smaller unmixed clusters, composed exclusively of conductive cells, show that proliferation continues after lineage restriction to the conduction system lineage. Differences in the working component of mixed clusters between the right and left ventricles reveal distinct progenitor cell histories in these cardiac compartments. These results are supported by genetic fate mapping using Cre recombinase revealing progressive restriction of connexin40-positive myocytes to a conductive fate. Conclusions: A biphasic mode of development, lineage restriction followed by limited outgrowth, underlies establishment of the mammalian ventricular conduction system.

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (Kd = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth.

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (αMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These αMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. αMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in αMHC-BMP10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the αMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies.