miRNAs in Heart Development and Disease.

Cardiovascular diseases (CVD) are a group of disorders that affect the heart and blood vessels. They include conditions such as myocardial infarction, coronary artery disease, heart failure, arrhythmia, and congenital heart defects. CVDs are the leading cause of death worldwide. Therefore, new medical interventions that aim to prevent, treat, or manage CVDs are of prime importance. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the posttranscriptional level and play important roles in various biological processes, including cardiac development, function, and disease. Moreover, miRNAs can also act as biomarkers and therapeutic targets. In order to identify and characterize miRNAs and their target genes, scientists take advantage of computational tools such as bioinformatic algorithms, which can also assist in analyzing miRNA expression profiles, functions, and interactions in different cardiac conditions. Indeed, the combination of miRNA research and bioinformatic algorithms has opened new avenues for understanding and treating CVDs. In this review, we summarize the current knowledge on the roles of miRNAs in cardiac development and CVDs, discuss the challenges and opportunities, and provide some examples of recent bioinformatics for miRNA research in cardiovascular biology and medicine.

CCBE1 Is Essential for Epicardial Function during Myocardium Development.

The epicardium is a single cell layer of mesothelial cells that plays a critical role during heart development contributing to different cardiac cell types of the developing heart through epithelial-to-mesenchymal transition (EMT). Moreover, the epicardium is a source of secreted growth factors that promote myocardial growth. CCBE1 is a secreted extracellular matrix protein expressed by epicardial cells that is required for the formation of the primitive coronary plexus. However, the role of CCBE1 during epicardial development was still unknown. Here, using a Ccbe1 knockout (KO) mouse model, we observed that loss of CCBE1 leads to congenital heart defects including thinner and hyper-trabeculated ventricular myocardium. In addition, Ccbe1 mutant hearts displayed reduced proliferation of cardiomyocyte and epicardial cells. Epicardial outgrowth culture assay to assess epicardial-derived cells (EPDC) migration showed reduced invasion of the collagen gel by EPDCs in Ccbe1 KO epicardial explants. Ccbe1 KO hearts also displayed fewer nonmyocyte/nonendothelial cells intramyocardially with a reduced proliferation rate. Additionally, RNA-seq data and experimental validation by qRT-PCR showed a marked deregulation of EMT-related genes in developing Ccbe1 mutant hearts. Together, these findings indicate that the myocardium defects in Ccbe1 KO mice arise from disruption of epicardial development and function.

DAND5 Inactivation Enhances Cardiac Differentiation in Mouse Embryonic Stem Cells.

Deciphering the clues of a regenerative mechanism for the mammalian adult heart would save millions of lives in the near future. Heart failure due to cardiomyocyte loss is still one of the significant health burdens worldwide. Here, we show the potential of a single molecule, DAND5, in mouse pluripotent stem cell-derived cardiomyocytes specification and proliferation. Dand5 loss-of-function generated the double of cardiac beating foci compared to the wild-type cells. The early formation of cardiac progenitor cells and the increased proliferative capacity of Dand5 KO mESC-derived cardiomyocytes contribute to the observed higher number of derived cardiac cells. Transcriptional profiling sequencing and quantitative RT-PCR assays showed an upregulation of early cardiac gene networks governing cardiomyocyte differentiation, cell cycling, and cardiac regenerative pathways but reduced levels of genes involved in cardiomyocyte maturation. These findings prompt DAND5 as a key driver for the generation and expansion of pluripotent stem cell-derived cardiomyocytes systems with further clinical application purposes.

Xenopus Pkdcc1 and Pkdcc2 Are Two New Tyrosine Kinases Involved in the Regulation of JNK Dependent Wnt/PCP Signaling Pathway.

Protein Kinase Domain Containing, Cytoplasmic (PKDCC) is a protein kinase which has been implicated in longitudinal bone growth through regulation of chondrocytes formation. Nevertheless, the mechanism by which this occurs remains unknown. Here, we identified two new members of the PKDCC family, Pkdcc1 and Pkdcc2 from Xenopus laevis. Interestingly, our knockdown experiments revealed that these two proteins are both involved on blastopore and neural tube closure during gastrula and neurula stages, respectively. In vertebrates, tissue polarity and cell movement observed during gastrulation and neural tube closure are controlled by Wnt/Planar Cell Polarity (PCP) molecular pathway. Our results showed that Pkdcc1 and Pkdcc2 promote the recruitment of Dvl to the plasma membrane. But surprisingly, they revealed different roles in the induction of a luciferase reporter under the control of Atf2 promoter. While Pkdcc1 induces Atf2 expression, Pkdcc2 does not, and furthermore inhibits its normal induction by Wnt11 and Wnt5a. Altogether our data show, for the first time, that members of the PKDCC family are involved in the regulation of JNK dependent Wnt/PCP signaling pathway.

Expression and function of Ccbe1 in the chick early cardiogenic regions are required for correct heart development.

During the course of a differential screen to identify transcripts specific for chick heart/hemangioblast precursor cells, we have identified Ccbe1 (Collagen and calcium-binding EGF-like domain 1). While the importance of Ccbe1 for the development of the lymphatic system is now well demonstrated, its role in cardiac formation remained unknown. Here we show by whole-mount in situ hybridization analysis that cCcbe1 mRNA is initially detected in early cardiac progenitors of the two bilateral cardiogenic fields (HH4), and at later stages on the second heart field (HH9-18). Furthermore, cCcbe1 is expressed in multipotent and highly proliferative cardiac progenitors. We characterized the role of cCcbe1 during early cardiogenesis by performing functional studies. Upon morpholino-induced cCcbe1 knockdown, the chick embryos displayed heart malformations, which include aberrant fusion of the heart fields, leading to incomplete terminal differentiation of the cardiomyocytes. cCcbe1 overexpression also resulted in severe heart defects, including cardia bifida. Altogether, our data demonstrate that although cardiac progenitors cells are specified in cCcbe1 morphants, the migration and proliferation of cardiac precursors cells are impaired, suggesting that cCcbe1 is a key gene during early heart development.

The dynamic right-to-left translocation of Cerl2 is involved in the regulation and termination of Nodal activity in the mouse node.

The determination of left-right body asymmetry in mouse embryos depends on the interplay of molecules in a highly sensitive structure, the node. Here, we show that the localization of Cerl2 protein does not correlate to its mRNA expression pattern, from 3-somite stage onwards. Instead, Cerl2 protein displays a nodal flow-dependent dynamic behavior that controls the activity of Nodal in the node, and the transmission of the laterality information to the left lateral plate mesoderm (LPM). Our results indicate that Cerl2 initially localizes and prevents the activation of Nodal genetic circuitry on the right side of the embryo, and later its right-to-left translocation shutdowns Nodal activity in the node. The consequent prolonged Nodal activity in the node by the absence of Cerl2 affects local Nodal expression and prolongs its expression in the LPM. Simultaneous genetic removal of both Nodal node inhibitors, Cerl2 and Lefty1, sustains even longer and bilateral this LPM expression.

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis.

Bacillus subtilis produces alpha-l-arabinofuranosidases (EC 3.2.1.55; AFs) capable of releasing arabinosyl oligomers and l-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (>250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 degrees C, with p-nitrophenyl-alpha-l-arabinofuranoside as substrate, gave an apparent K(m) of 0.498 mM and 0.421 mM, and V(max) of 317 U mg(-1) and 311 U mg(-1) for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 degrees C and 60 degrees C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1-->5) linkages of linear alpha-1,5-l-arabinan and alpha-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1-->2) and (1-->3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinose-containing polysaccharides by B. subtilis.

Overproduction, crystallization and preliminary X-ray characterization of Abn2, an endo-1,5-alpha-arabinanase from Bacillus subtilis.

Two Bacillus subtilis extracellular endo-1,5-alpha-L-arabinanases, AbnA and Abn2, belonging to glycoside hydrolase family 43 have been identified. The recently characterized Abn2 protein hydrolyzes arabinan and has low identity to other reported 1,5-alpha-L-arabinanases. Abn2 and its selenomethionine (SeMet) derivative have been purified and crystallized. Crystals appeared in two different space groups: P1, with unit-cell parameters a = 51.9, b = 57.6, c = 86.2 A, alpha = 82.3, beta = 87.9, gamma = 63.6 degrees , and P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 163.3, c = 202.0 A. X-ray data have been collected for the native and the SeMet derivative to 1.9 and 2.7 A resolution, respectively. An initial model of Abn2 is being built in the SeMet-phased map.

Characterization of abn2 (yxiA), encoding a Bacillus subtilis GH43 arabinanase, Abn2, and its role in arabino-polysaccharide degradation.

The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-alpha-1,5-l-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-alpha-1,5-l-arabinan as the preferred substrate, the enzyme exhibited an apparent K(m) of 2.0 mg ml(-1) and V(max) of 0.25 mmol min(-1) mg(-1) at pH 7.0 and 50 degrees C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a sigma(A)-dependent and a sigma(H)-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.

trans-Acting factors and cis elements involved in glucose repression of arabinan degradation in Bacillus subtilis.

In Bacillus subtilis, the synthesis of enzymes involved in the degradation of arabinose-containing polysaccharides is subject to carbon catabolite repression (CCR). Here we show that CcpA is the major regulator of repression of the arabinases genes in the presence of glucose. CcpA acts via binding to one cre each in the promoter regions of the abnA and xsa genes and to two cres in the araABDLMNPQ-abfA operon. The contributions of the coeffectors HPr and Crh to CCR differ according to growth phase. HPr dependency occurs during both exponential growth and the transitional phase, while Crh dependency is detected mainly at the transitional phase. Our results suggest that Crh synthesis may increase at the end of exponential growth and consequently contribute to this effect, together with other factors.

Transcriptional regulation of genes encoding arabinan-degrading enzymes in Bacillus subtilis.

Bacillus subtilis produces hemicellulases capable of releasing arabinosyl oligomers and arabinose from plant cell walls. In this work, we characterize the transcriptional regulation of three genes encoding arabinan-degrading enzymes that are clustered with genes encoding enzymes that further catabolize arabinose. The abfA gene comprised in the metabolic operon araABDLMNPQ-abfA and the xsa gene located 23 kb downstream most probably encode alpha-L-arabinofuranosidases (EC 3.2.1.55). Here, we show that the abnA gene, positioned immediately upstream from the metabolic operon, encodes an endo-alpha-1,5-arabinanase (EC 3.2.1.99). Furthermore, by in vivo RNA studies, we inferred that abnA and xsa are monocistronic and are transcribed from sigma(A)-like promoters. Transcriptional fusion analysis revealed that the expression of the three arabinases is induced by arabinose and arabinan and is repressed by glucose. The levels of induction by arabinose and arabinan are higher during early postexponential growth, suggesting a temporal regulation. Moreover, the induction mechanism of these genes is mediated through negative control by the key regulator of arabinose metabolism, AraR. Thus, we analyzed AraR-DNA interactions by in vitro quantitative DNase I footprinting and in vivo analysis of single-base-pair substitutions within the promoter regions of xsa and abnA. The results indicate that transcriptional repression of the abfA and xsa genes is achieved by a tightly controlled mechanism but that the regulation of abnA is more flexible. We suggest that the expression of genes encoding extracellular degrading enzymes of arabinose-containing polysaccharides, transport systems, and intracellular enzymes involved in further catabolism is regulated by a coordinate mechanism triggered by arabinose via AraR.

Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis.

The Bacillus subtilis proteins involved in the utilization of L-arabinose are encoded by the araABDLMNPQ-abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene and araR regulatory gene is induced by L-arabinose and negatively controlled by AraR. Additionally, expression of both the ara operon and the araE gene is regulated at the transcriptional level by glucose repression. Here, by transcriptional fusion analysis in different mutant backgrounds, it is shown that CcpA most probably complexed with HPr-Ser46-P plays the major role in carbon catabolite repression of the ara regulon by glucose and glycerol. Site-directed mutagenesis and deletion analysis indicate that two catabolite responsive elements (cres) present in the ara operon (cre araA and cre araB) and one cre in the araE gene (cre araE) are implicated in this mechanism. Furthermore, cre araA located between the promoter region of the ara operon and the araA gene, and cre araB placed 2 kb downstream within the araB gene are independently functional and both contribute to glucose repression. In Northern blot analysis, in the presence of glucose, a CcpA-dependent transcript consistent with a message stopping at cre araB was detected, suggesting that transcription 'roadblocking' of RNA polymerase elongation is the most likely mechanism operating in this system. Glucose exerts an additional repression of the ara regulon, which requires a functional araR.