Myocardial RNA Sequencing Reveals New Potential Therapeutic Targets in Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) represents a global health challenge, with limited therapies proven to enhance patient outcomes. This makes the elucidation of disease mechanisms and the identification of novel potential therapeutic targets a priority. Here, we performed RNA sequencing on ventricular myocardial biopsies from patients with HFpEF, prospecting to discover distinctive transcriptomic signatures. A total of 306 differentially expressed mRNAs (DEG) and 152 differentially expressed microRNAs (DEM) were identified and enriched in several biological processes involved in HF. Moreover, by integrating mRNA and microRNA expression data, we identified five potentially novel miRNA-mRNA relationships in HFpEF: the upregulated hsa-miR-25-3p, hsa-miR-26a-5p, and has-miR4429, targeting HAPLN1; and NPPB mRNA, targeted by hsa-miR-26a-5p and miR-140-3p. Exploring the predicted miRNA-mRNA interactions experimentally, we demonstrated that overexpression of the distinct miRNAs leads to the downregulation of their target genes. Interestingly, we also observed that microRNA signatures display a higher discriminative power to distinguish HFpEF sub-groups over mRNA signatures. Our results offer new mechanistic clues, which can potentially translate into new HFpEF therapies.

Depletion of DAND5 Hinders EMT in Mouse Embryonic Stem Cell Differentiation.

BACKGROUND: Dand5 encodes a protein that acts as an antagonist to Nodal/TGF-ß and Wnt pathways. A mouse knockout (KO) model has shown that this molecule is associated with left-right asymmetry and cardiac development, with its depletion causing heterotaxia and cardiac hyperplasia. OBJECTIVE: This study aimed to investigate the molecular mechanisms affected by the depletion of Dand5. METHODS: DAND5-KO and wild-type embryoid bodies (EBs) were used to assess genetic expression with RNA sequencing. To complement the expression results that pointed towards differences in epithelial to mesenchymal transition (EMT), we evaluated migration and cell attachment. Lastly, in vivo valve development was investigated, as it was an established model of EMT. RESULTS: DAND5-KO EBs progress faster through differentiation. The differences in expression will lead to differences in the expression of genes involved with Notch and Wnt signalling pathways, as well as changes in the expression of genes encoding membrane proteins. Such changes were accompanied by lower migratory rates in DAND5-KO EBs, as well as higher concentrations of focal adhesions. Within valve development, Dand5 is expressed in the myocardium underlying future valve sites, and its depletion compromises correct valve structure. CONCLUSION: The DAND5 range of action goes beyond early development. Its absence leads to significantly different expression patterns in vitro and defects in EMT and migration. These results have an in vivo translation in mouse heart valve development. Knowledge regarding the influence of DAND5 in EMT and cell transformation allows further understanding of its role in development, or even in some disease contexts, such as congenital heart defects.

Gene-Edited Human-Induced Pluripotent Stem Cell Lines to Elucidate DAND5 Function throughout Cardiac Differentiation.

(1) Background: The contribution of gene-specific variants for congenital heart disease, one of the most common congenital disabilities, is still far from our complete understanding. Here, we applied a disease model using human-induced pluripotent stem cells (hiPSCs) to evaluate the function of DAND5 on human cardiomyocyte (CM) differentiation and proliferation. (2) Methods: Taking advantage of our DAND5 patient-derived iPSC line, we used CRISPR-Cas9 gene-editing to generate a set of isogenic hiPSCs (DAND5-corrected and DAND5 full-mutant). The hiPSCs were differentiated into CMs, and RT-qPCR and immunofluorescence profiled the expression of cardiac markers. Cardiomyocyte proliferation was analysed by flow cytometry. Furthermore, we used a multi-electrode array (MEA) to study the functional electrophysiology of DAND5 hiPSC-CMs. (3) Results: The results indicated that hiPSC-CM proliferation is affected by DAND5 levels. Cardiomyocytes derived from a DAND5 full-mutant hiPSC line are more proliferative when compared with gene-corrected hiPSC-CMs. Moreover, parallel cardiac differentiations showed a differential cardiac gene expression profile, with upregulated cardiac progenitor markers in DAND5-KO hiPSC-CMs. Microelectrode array (MEA) measurements demonstrated that DAND5-KO hiPSC-CMs showed prolonged field potential duration and increased spontaneous beating rates. In addition, conduction velocity is reduced in the monolayers of hiPSC-CMs with full-mutant genotype. (4) Conclusions: The absence of DAND5 sustains the proliferation of hiPSC-CMs, which alters their electrophysiological maturation properties. These results using DAND5 hiPSC-CMs consolidate the findings of the in vitro and in vivo mouse models, now in a translational perspective. Altogether, the data will help elucidate the molecular mechanism underlying this human heart disease and potentiates new therapies for treating adult CHD.

A novel biallelic loss-of-function variant in DAND5 causes heterotaxy syndrome.

The majority of heterotaxy cases do not obtain a molecular diagnosis, although pathogenic variants in more than 50 genes are known to cause heterotaxy. A heterozygous missense variant in DAND5, a nodal inhibitor, which functions in early development for establishment of right-left patterning, has been implicated in heterotaxy. Recently, the first case was reported of a DAND5 biallelic loss-of-function (LoF) variant in an individual with heterotaxy. Here, we describe a second unrelated individual with heterotaxy syndrome and a homozygous frameshift variant in DAND5 (NM_152654.2:c.197del [p.Leu66ArgfsTer22]). Using an in vitro assay, we demonstrate that the DAND5 c.197del variant is unable to inhibit nodal signaling when compared with the wild-type expression construct. This work strengthens the genetic and functional evidence for biallelic LoF variants in DAND5 causing an autosomal recessive heterotaxy syndrome.

CCBE1 in Cardiac Development and Disease.

The collagen- and calcium-binding EGF-like domains 1 (CCBE1) is a secreted protein extensively described as indispensable for lymphangiogenesis during development enhancing VEGF-C signaling. In human patients, mutations in CCBE1 have been found to cause Hennekam syndrome, an inherited disease characterized by malformation of the lymphatic system that presents a wide variety of symptoms such as primary lymphedema, lymphangiectasia, and heart defects. Importantly, over the last decade, an essential role for CCBE1 during heart development is being uncovered. In mice, Ccbe1 expression was initially detected in distinct cardiac progenitors such as first and second heart field, and the proepicardium. More recently, Ccbe1 expression was identified in the epicardium and sinus venosus (SV) myocardium at E11.5-E13.5, the stage when SV endocardium-derived (VEGF-C dependent) coronary vessels start to form. Concordantly, CCBE1 is required for the correct formation of the coronary vessels and the coronary artery stem in the mouse. Additionally, Ccbe1 was found to be enriched in mouse embryonic stem cells (ESC) and revealed as a new essential gene for the differentiation of ESC-derived early cardiac precursor cell lineages. Here, we bring an up-to-date review on the role of CCBE1 in cardiac development, function, and human disease implications. Finally, we envisage the potential of this molecule's functions from a regenerative medicine perspective, particularly novel therapeutic strategies for heart disease.

Generation of a gene-corrected human induced pluripotent stem cell line derived from a patient with laterality defects and congenital heart anomalies with a c.455G > A alteration in DAND5.

Human induced pluripotent stem cells (hiPSCs) from individual patient basis are considered a powerful resource to model human diseases. However, to study complex multigenic diseases such as Congenital Heart Disease, it is crucial to generate perfect isogenic controls to understand gene singularity and contribution. Here, we report the engendering of an isogenic hiPSC line with homozygous correction of c.455G > A alteration in the DAND5 gene, using CRISPR/Cas9 technology. The characterization of a clone of this cell line demonstrates normal karyotype, pluripotent state, and potential to differentiate in vitro towards endoderm, mesoderm, and ectoderm.

Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells.

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.

CCBE1 is required for coronary vessel development and proper coronary artery stem formation in the mouse heart.

BACKGROUND: Proper coronary vasculature development is essential for late-embryonic and adult heart function. The developmental regulation of coronary embryogenesis is complex and includes the coordinated activity of multiple signaling pathways. CCBE1 plays an important role during lymphangiogenesis, enhancing VEGF-C signaling, which is also required for coronary vasculature formation. However, whether CCBE1 plays a similar role during coronary vasculature development is still unknown. Here, we investigate the coronary vasculature development in Ccbe1 mutant embryos. RESULTS: We show that Ccbe1 is expressed in the epicardium, like Vegf-c, and also in the sinus venosus (SV) at the stages of its contribution to coronary vasculature formation. We also report that absence of CCBE1 in cardiac tissue inhibited coronary growth that sprouts from the SV endocardium at the dorsal cardiac wall. This disruption of coronary formation correlates with abnormal processing of VEGF-C propeptides, suggesting VEGF-C-dependent signaling alteration. Moreover, Ccbe1 loss-of-function leads to the development of defective dorsal and ventral intramyocardial vessels. We also demonstrate that Ccbe1 mutants display delayed and mispatterned coronary artery (CA) stem formation. CONCLUSIONS: CCBE1 is essential for coronary vessel formation, independent of their embryonic origin, and is also necessary for peritruncal vessel growth and proper CA stem patterning. Developmental Dynamics 247:1135-1145, 2018. © 2018 Wiley Periodicals, Inc.

Full-length human CCBE1 production and purification: leveraging bioprocess development for high quality glycosylation attributes and functionality.

Collagen and calcium-binding EGF domain-1 (CCBE1) is a secreted protein critical for lymphatic/cardiac vascular development and regeneration. However, the low efficient production of the recombinant full-length CCBE1 (rCCBE1) has been a setback for functional studies and therapeutic applications using this protein. The main goal of this work was to implement a robust bioprocess for efficient production of glycosylated rCCBE1. Different bioprocess strategies were combined with proteomic tools for process/product characterization, evaluating the impact of process parameters on cell performance, rCCBE1 production and quality. We have shown that rCCBE1 volumetric yield was positively correlated with higher cell density at transfection (HDT), and under these conditions the secreted protein presented a mature glycosylated profile (complex N-glycans). Mild hypothermia was also applied to HDT condition that resulted in enhanced cell viability; however an enrichment of immature rCCBE1 variants was detected. Mass spectrometry-based tools allowed the identification of rCCBE1 peptides confirming protein identity in the affinity chromatography enriched product. rCCBE1 biological activity was validated by in vitro angiogenesis assay, where enhanced vessel formation was observed. Herein, we report a step forward in the production and characterization of human glycosylated rCCBE1, amenable for in vitro and in vivo studies to explore its regenerative therapeutic potential.

Generation and characterization of a human iPS cell line from a patient-related control to study disease mechanisms associated with DAND5 p.R152H alteration.

A DAND5-control human iPSC line was generated from the urinary cells of a phenotypically normal donor. Exfoliated renal epithelial (RE) cells were collected and reprogrammed into iPSCs using Sendai virus reprogramming system. The pluripotency, in vitro differentiation potential, karyotype stability, and the transgene-free status of generated iPSC line were analyzed and confirmed. This cell line can be exploited as a control iPSC line to better understand the mechanisms involved in DAND5-associated cardiac disease.

Generation of human iPSC line from a patient with laterality defects and associated congenital heart anomalies carrying a DAND5 missense alteration.

A human iPSC line was generated from exfoliated renal epithelial (ERE) cells of a patient affected with Congenital Heart Disease (CHD) and Laterality Defects carrying tshe variant p.R152H in the DAND5 gene. The transgene-free iPSCs were generated with the human OSKM transcription factor using the Sendai-virus reprogramming system. The established iPSC line had the specific heterozygous alteration, a stable karyotype, expressed pluripotency markers and generated embryoid bodies that can differentiate towards the three germ layers in vitro. This iPSC line offers a useful resource to study the molecular mechanisms of cardiomyocyte proliferation, as well as for drug testing.

Functional study of DAND5 variant in patients with Congenital Heart Disease and laterality defects.

BACKGROUND: Perturbations on the Left-Right axis establishment lead to laterality defects, with frequently associated Congenital Heart Diseases (CHDs). Indeed, in the last decade, it has been reported that the etiology of isolated cases of CHDs or cases of laterality defects with associated CHDs is linked with variants of genes involved in the Nodal signaling pathway. METHODS: With this in mind, we analyzed a cohort of 38 unrelated patients with Congenital Heart Defects that can arise from initial perturbations in the formation of the Left-Right axis and 40 unrelated ethnically matched healthy individuals as a control population. Genomic DNA was extracted from buccal epithelial cells, and variants screening was performed by PCR and direct sequencing. A Nodal-dependent luciferase assay was conducted in order to determine the functional effect of the variant found. RESULTS: In this work, we report two patients with a DAND5 heterozygous non-synonymous variant (c.455G > A) in the functional domain of the DAND5 protein (p.R152H), a master regulator of Nodal signaling. Patient 1 presents left isomerism, ventricular septal defect with overriding aorta and pulmonary atresia, while patient 2 presents ventricular septal defect with overriding aorta, right ventricular hypertrophy and pulmonary atresia (a case of extreme tetralogy of Fallot phenotype). The functional analysis assay showed a significant decrease in the activity of this variant protein when compared to its wild-type counterpart. CONCLUSION: Altogether, our results provide new insight into the molecular mechanism of the laterality defects and related CHDs, priming for the first time DAND5 as one of multiple candidate determinants for CHDs in humans.

The Role of Cerl2 in the Establishment of Left-Right Asymmetries during Axis Formation and Heart Development.

The formation of the asymmetric left-right (LR) body axis is one of the fundamental aspects of vertebrate embryonic development, and one still raising passionate discussions among scientists. Although the conserved role of nodal is unquestionable in this process, several of the details around this signaling cascade are still unanswered. To further understand this mechanism, we have been studying Cerberus-like 2 (Cerl2), an inhibitor of Nodal, and its role in the generation of asymmetries in the early vertebrate embryo. The absence of Cerl2 results in a wide spectrum of malformations commonly known as heterotaxia, which comprises defects in either global organ position (e.g., situs inversus totalis), reversed orientation of at least one organ (e.g., situs ambiguus), and mirror images of usually asymmetric paired organs (e.g., left or right isomerisms of the lungs). Moreover, these laterality defects are frequently associated with congenital heart diseases (e.g., transposition of the great arteries, or atrioventricular septal defects). Here, reviewing the knowledge on the establishment of LR asymmetry in mouse embryos, the emerging conclusion is that as necessary as is the activation of the Nodal signaling cascade, the tight control that Cerl2-mediates on Nodal signaling is equally important, and that generates a further regionalized LR genetic program in the proper time and space.

Expression pattern of zcchc24 during early Xenopus development.

We report the expression pattern of a novel Xenopus laevis gene, zcchc24, which encodes a protein containing two zinc finger domains from the zf-CCHC and zf-3CxxC superfamilies. This protein shares >84% amino acid identity with its vertebrate homologues. During X. laevis embryonic development, zcchc24 is expressed at gastrula stages in the dorsal mesoderm, including the cardiac precursors region. During neurula stages, zcchc24 is expressed as two stripes in the dorsal region, more precisely, in the somitogenic mesoderm until the cardiac mesoderm. At early tailbud stages, zcchc24 continues to be expressed in these regions, but starts to be expressed in the migrating neural crest. Later, this gene is expressed in the head, branchial arches, heart and somites. The zinc finger domains present in Zcchc24 protein and its dynamic gene expression pattern suggest that Zcchc24 might be involved in the regulation of heart, somites and of branchial arch formation/patterning, namely in the regulation of apoptosis.

New evidence for the role of calcium in the glycosidase reaction of GH43 arabinanases.

Endo-1,5-α-L-arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. Two extracellular endo-1,5-α-L-arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5-α-L-arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo-1,5-α-L-arabinanases, as it comprises two domains, an N-terminal catalytic domain, with a 3D fold similar to that observed for other endo-1,5-α-L-arabinanases, and an additional C-terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.