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.

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.

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.

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.

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.