Insertion of a FLAG-tag sequence at the end of exon 9 of the TBX5 gene in three induced pluripotent stem cell lines (DHMi004-A-4, DHMi004-A-5, DHMi004-A-6) by CRISPR/Cas9 technology.

The identification of TBX5-related regulatory sequences in genes essential for heart development is hampered by the absence of antibodies which allow precipitation of TBX5:DNA complexes. Employing CRISPR/Cas9 technology, we have inserted a FLAG-tag sequence at the end of exon 9 of the TBX5 gene prior to the stop codon by homologous recombination. The translated TBX5-FLAG fusion protein of the three iPSC lines can effectively be precipitated by anti-FLAG antibodies and, thus, allow the detection of specific TBX5-binding sites and their associated genes.

Correction of a deleterious TBX5 mutation in an induced pluripotent stem cell line (DHMi004-A-1) using a completely plasmid-free CRISPR/Cas 9 approach.

TBX5 is a transcription factor which plays an essential role at different checkpoints during cardiac differentiation. However, regulatory pathways affected by TBX5 still remain ill-defined. We have applied the CRISPR/Cas9 technology using a completely plasmid-free approach to correct a heterozygous causative "loss-of function" TBX5 mutation in an iPSC line (DHMi004-A), that has been established from a patient suffering from Holt-Oram syndrome (HOS). This isogenic iPSC line, DHMi004-A-1, represents a powerful in vitro tool to dissect the regulatory pathways affected by TBX5 in HOS.

Elucidation of the genetic causes of bicuspid aortic valve disease.

AIMS: The present study aims to characterize the genetic risk architecture of bicuspid aortic valve (BAV) disease, the most common congenital heart defect. METHODS AND RESULTS: We carried out a genome-wide association study (GWAS) including 2236 BAV patients and 11 604 controls. This led to the identification of a new risk locus for BAV on chromosome 3q29. The single nucleotide polymorphism rs2550262 was genome-wide significant BAV associated (P = 3.49 × 10-08) and was replicated in an independent case-control sample. The risk locus encodes a deleterious missense variant in MUC4 (p.Ala4821Ser), a gene that is involved in epithelial-to-mesenchymal transformation. Mechanistical studies in zebrafish revealed that loss of Muc4 led to a delay in cardiac valvular development suggesting that loss of MUC4 may also play a role in aortic valve malformation. The GWAS also confirmed previously reported BAV risk loci at PALMD (P = 3.97 × 10-16), GATA4 (P = 1.61 × 10-09), and TEX41 (P = 7.68 × 10-04). In addition, the genetic BAV architecture was examined beyond the single-marker level revealing that a substantial fraction of BAV heritability is polygenic and ∼20% of the observed heritability can be explained by our GWAS data. Furthermore, we used the largest human single-cell atlas for foetal gene expression and show that the transcriptome profile in endothelial cells is a major source contributing to BAV pathology. CONCLUSION: Our study provides a deeper understanding of the genetic risk architecture of BAV formation on the single marker and polygenic level.

Uncovering the molecular identity of cardiosphere-derived cells (CDCs) by single-cell RNA sequencing.

Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders.

Generation of a CRISPR/Cas edited human induced pluripotent stem cell line DHMi005-A-1 carrying a patient-specific disease-causing point mutation in the TBX5 gene.

A number of mutations in the human TBX5 gene have been described which cause Holt-Oram syndrome, a severe congenital disease associated with abnormalities in heart and upper limb development. We have used a prime-editing approach to introduce a patient-specific disease-causing TBX5 mutation (c.920_C > A) into an induced pluripotent stem cell (iPSC) line from a healthy donor. The resulting iPSC line provides a powerful tool to identify and analyze the biological and molecular impact of this specific TBX5 mutation in comparison to the isogenic control iPSC line during cardiac development.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Aggrecan: a new biomarker for acute type A aortic dissection.

Acute type A aortic dissection (ATAAD) constitutes a life-threatening aortic pathology with significant morbidity and mortality. Without surgical intervention the usual mortality rate averages between 1 and 2% per hour. Thus, an early diagnosis of ATAAD is of pivotal importance to direct the affected patients to the appropriate treatment. Preceding tests to find an appropriate biomarker showed among others an increased aggrecan (ACAN) mRNA expression in aortic tissue of ATAAD patients. As a consequence, we investigated whether ACAN is a potential biomarker for diagnosing ATAAD. Mean ACAN protein concentration showed a significantly higher plasma concentration in ATAAD patients (38.59 ng/mL, n = 33) compared to plasma of patients with thoracic aortic aneurysms (4.45 ng/mL, n = 13), patients with myocardial infarction (11.77 ng/mL, n = 18) and healthy volunteers (8.05 ng/mL, n = 12). Cardiac enzymes like creatine kinase MB and cardiac troponin T showed no correlation with ACAN levels in ATAAD patients. Receiver-operator characteristics (ROC) curve analysis for ATAAD patients versus control subjects an optimum discrimination limit of ACAN plasma levels at 14.3 ng/mL with a corresponding sensitivity of 97% and specificity of 81%. According to our findings ACAN is a reliable potential biomarker in plasma samples to detect ATAAD with high sensitivity and specificity.

Congenital heart disease risk loci identified by genome-wide association study in European patients.

Genetic factors undoubtedly affect the development of congenital heart disease (CHD) but still remain ill defined. We sought to identify genetic risk factors associated with CHD and to accomplish a functional analysis of SNP-carrying genes. We performed a genome-wide association study (GWAS) of 4034 White patients with CHD and 8486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified 4 highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence the expression of WNT3, and the variant rs870142 related to septal defects is proposed to influence the expression of MSX1. We analyzed the expression of all 4 genes during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNA-Seq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3, and MSX1 play an essential functional role in heart development at the embryonic and newborn stages.

miR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations.

MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent Nkx2.5 enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased Isl1, Sfrp5, and Hcn4 (cardiac transcription factors) but reduced Irx4 at the onset of cardiogenesis, (2) upregulated Isl1-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker Myl2 accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of Isl1, Sfrp5, Nkx2.5, and Mef2c, but increased Irx4, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (Tnnt2+, Myh6+, Shox2+) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.

Myosin binding protein H-like (MYBPHL): a promising biomarker to predict atrial damage.

Myosin binding protein H-like (MYBPHL) is a protein associated with myofilament structures in atrial tissue. The protein exists in two isoforms that share an identical amino acid sequence except for a deletion of 23 amino acids in isoform 2. In this study, MYBPHL was found to be expressed preferentially in atrial tissue. The expression of isoform 2 was almost exclusively restricted to the atria and barely detectable in the ventricle, arteria mammaria interna, and skeletal muscle. After atrial damage induced by cryo- or radiofrequency ablation, MYBPHL was rapidly and specifically released into the peripheral circulation in a time-dependent manner. The plasma MYBPHL concentration remained substantially elevated up to 24 hours after the arrival of patients at the intensive care unit. In addition, the recorded MYBPHL values were strongly correlated with those of the established biomarker CK-MB. In contrast, an increase in MYBPHL levels was not evident in patients undergoing aortic valve replacement or transcatheter aortic valve implantation. In these patients, the values remained virtually constant and never exceeded the concentration in the plasma of healthy controls. Our findings suggest that MYBPHL can be used as a precise and reliable biomarker to specifically predict atrial myocardial damage.

Bioprinting Approaches to Engineering Vascularized 3D Cardiac Tissues.

PURPOSE OF REVIEW: 3D bioprinting technologies hold significant promise for the generation of engineered cardiac tissue and translational applications in medicine. To generate a clinically relevant sized tissue, the provisioning of a perfusable vascular network that provides nutrients to cells in the tissue is a major challenge. This review summarizes the recent vascularization strategies for engineering 3D cardiac tissues. RECENT FINDINGS: Considerable steps towards the generation of macroscopic sizes for engineered cardiac tissue with efficient vascular networks have been made within the past few years. Achieving a compact tissue with enough cardiomyocytes to provide functionality remains a challenging task. Achieving perfusion in engineered constructs with media that contain oxygen and nutrients at a clinically relevant tissue sizes remains the next frontier in tissue engineering. The provisioning of a functional vasculature is necessary for maintaining a high cell viability and functionality in engineered cardiac tissues. Several recent studies have shown the ability to generate tissues up to a centimeter scale with a perfusable vascular network. Future challenges include improving cell density and tissue size. This requires the close collaboration of a multidisciplinary teams of investigators to overcome complex challenges in order to achieve success.

Genome Editing Redefines Precision Medicine in the Cardiovascular Field.

Genome editing is a powerful tool to study the function of specific genes and proteins important for development or disease. Recent technologies, especially CRISPR/Cas9 which is characterized by convenient handling and high precision, revolutionized the field of genome editing. Such tools have enormous potential for basic science as well as for regenerative medicine. Nevertheless, there are still several hurdles that have to be overcome, but patient-tailored therapies, termed precision medicine, seem to be within reach. In this review, we focus on the achievements and limitations of genome editing in the cardiovascular field. We explore different areas of cardiac research and highlight the most important developments: (1) the potential of genome editing in human pluripotent stem cells in basic research for disease modelling, drug screening, or reprogramming approaches and (2) the potential and remaining challenges of genome editing for regenerative therapies. Finally, we discuss social and ethical implications of these new technologies.

Reactivation of the Nkx2.5 cardiac enhancer after myocardial infarction does not presage myogenesis.

Aims: The contribution of resident stem or progenitor cells to cardiomyocyte renewal after injury in adult mammalian hearts remains a matter of considerable debate. We evaluated a cell population in the adult mouse heart induced by myocardial infarction (MI) and characterized by an activated Nkx2.5 enhancer element that is specific for multipotent cardiac progenitor cells (CPCs) during embryonic development. We hypothesized that these MI-induced cells (MICs) harbour cardiomyogenic properties similar to their embryonic counterparts. Methods and results: MICs reside in the heart and mainly localize to the infarction area and border zone. Interestingly, gene expression profiling of purified MICs 1 week after infarction revealed increased expression of stem cell markers and embryonic cardiac transcription factors (TFs) in these cells as compared to the non-mycoyte cell fraction of adult hearts. A subsequent global transcriptome comparison with embryonic CPCs and fibroblasts and in vitro culture of MICs unveiled that (myo-)fibroblastic features predominated and that cardiac TFs were only expressed at background levels. Conclusions: Adult injury-induced reactivation of a cardiac-specific Nkx2.5 enhancer element known to specifically mark myocardial progenitor cells during embryonic development does not reflect hypothesized embryonic cardiomyogenic properties. Our data suggest a decreasing plasticity of cardiac progenitor (-like) cell populations with increasing age. A re-expression of embryonic, stem or progenitor cell features in the adult heart must be interpreted very carefully with respect to the definition of cardiac resident progenitor cells. Albeit, the abundance of scar formation after cardiac injury suggests a potential to target predestinated activated profibrotic cells to push them towards cardiomyogenic differentiation to improve regeneration.

DNA methylation signatures follow preformed chromatin compartments in cardiac myocytes.

Storage of chromatin in restricted nuclear space requires dense packing while ensuring DNA accessibility. Thus, different layers of chromatin organization and epigenetic control mechanisms exist. Genome-wide chromatin interaction maps revealed large interaction domains (TADs) and higher order A and B compartments, reflecting active and inactive chromatin, respectively. The mutual dependencies between chromatin organization and patterns of epigenetic marks, including DNA methylation, remain poorly understood. Here, we demonstrate that establishment of A/B compartments precedes and defines DNA methylation signatures during differentiation and maturation of cardiac myocytes. Remarkably, dynamic CpG and non-CpG methylation in cardiac myocytes is confined to A compartments. Furthermore, genetic ablation or reduction of DNA methylation in embryonic stem cells or cardiac myocytes, respectively, does not alter genome-wide chromatin organization. Thus, DNA methylation appears to be established in preformed chromatin compartments and may be dispensable for the formation of higher order chromatin organization.

Region and cell-type resolved quantitative proteomic map of the human heart.

The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular level. Analysis of cardiac fibroblasts identifies cellular receptors as potential cell surface markers. Application of our heart map to atrial fibrillation reveals individually distinct mitochondrial dysfunctions. The heart map is available at maxqb.biochem.mpg.de as a resource for future analyses of normal heart function and disease.

Cardiac fibroblasts: more than mechanical support.

Fibroblasts are cells with a structural function, synthesizing components of the extracellular matrix. They are accordingly associated with various forms of connective tissue. During cardiac development fibroblasts originate from different sources. Most derive from the epicardium, some derive from the endocardium, and a small population derives from the neural crest. Cardiac fibroblasts have important functions during development, homeostasis, and disease. However, since fibroblasts are a very heterogeneous cell population no truly specific markers exist. Therefore, studying them in detail is difficult. Nevertheless, several lineage tracing models have been widely used. In this review, we describe the developmental origins of cardiac fibroblasts, comment on fibroblast markers and related lineage tracing approaches, and discuss the cardiac cell composition, which has recently been revised, especially in terms of non-myocyte cells.

MicroRNAs: pleiotropic players in congenital heart disease and regeneration.

Congenital heart disease (CHD) is the leading cause of infant death, affecting approximately 4-14 live births per 1,000. Although surgical techniques and interventions have improved significantly, a large number of infants still face poor clinical outcomes. MicroRNAs (miRs) are known to coordinately regulate cardiac development and stimulate pathological processes in the heart, including fibrosis or hypertrophy and impair angiogenesis. Dysregulation of these regulators could therefore contribute (I) to the initial development of CHD and (II) at least partially to the observed clinical outcomes of many CHD patients by stimulating the aforementioned pathways. Thus, miRs may exhibit great potential as therapeutic targets in regenerative medicine. In this review we provide an overview of miR function and elucidate their role in selected CHDs, including hypoplastic left heart syndrome (HLHS), tetralogy of Fallot (TOF), ventricular septal defects (VSDs) and Holt-Oram syndrome (HOS). We then bridge this knowledge to the potential usefulness of miRs and/or their targets in therapeutic strategies for regenerative purposes in CHDs.

A novel de novo TBX5 mutation in a patient with Holt-Oram syndrome leading to a dramatically reduced biological function.

BACKGROUND: The Holt-Oram syndrome (HOS) is an autosomal dominant disorder affecting 1/100.000 live births. It is defined by upper limb anomalies and congenital heart defects with variable severity. We describe a dramatic phenotype of a male, 15-month-old patient being investigated for strict diagnostic criteria of HOS. METHODS AND RESULTS: Genetic analysis revealed a so far unpublished TBX5 mutation, which occurs de novo in the patient with healthy parents. TBX5 belongs to the large family of T-box transcription factors playing major roles in morphogenesis and cell-type specification. The mutation located in the DNA-binding domain at position 920 (C→A) leads to an amino acid change at position 85 (proline → threonine). Three-dimensional analysis of the protein structure predicted a cis to trans change in the respective peptide bond, thereby probably provoking major conformational and functional alterations of the protein. The p.Pro85Thr mutation showed a dramatically reduced activation (97%) of the NPPA promoter in luciferase assays and failed to induce NPPA expression in HEK 293 cells compared to wild-type TBX5 protein. The mutation did not interfere with the nuclear localization of the protein. CONCLUSION: These results suggest that the dramatic functional alteration of the p.Pro85Thr mutation leads to the distinctive phenotype of the patient.