High cardiomyocyte diversity in human early prenatal heart development.

Cardiomyocytes play key roles during cardiogenesis, but have poorly understood features, especially in prenatal stages. Here, we characterized human prenatal cardiomyocytes, 6.5-7 weeks post-conception, by integrating single-cell RNA sequencing, spatial transcriptomics, and ligand-receptor interaction information. Using a computational workflow developed to dissect cell type heterogeneity, localize cell types, and explore their molecular interactions, we identified eight types of developing cardiomyocyte, more than double compared to the ones identified in the Human Developmental Cell Atlas. These have high variability in cell cycle activity, mitochondrial content, and connexin gene expression, and are differentially distributed in the ventricles, including outflow tract, and atria, including sinoatrial node. Moreover, cardiomyocyte ligand-receptor crosstalk is mainly with non-cardiomyocyte cell types, encompassing cardiogenesis-related pathways. Thus, early prenatal human cardiomyocytes are highly heterogeneous and develop unique location-dependent properties, with complex ligand-receptor crosstalk. Further elucidation of their developmental dynamics may give rise to new therapies.

De novo spatiotemporal modelling of cell-type signatures in the developmental human heart using graph convolutional neural networks.

With the emergence of high throughput single cell techniques, the understanding of the molecular and cellular diversity of mammalian organs have rapidly increased. In order to understand the spatial organization of this diversity, single cell data is often integrated with spatial data to create probabilistic cell maps. However, targeted cell typing approaches relying on existing single cell data achieve incomplete and biased maps that could mask the true diversity present in a tissue slide. Here we applied a de novo technique to spatially resolve and characterize cellular diversity of in situ sequencing data during human heart development. We obtained and made accessible well defined spatial cell-type maps of fetal hearts from 4.5 to 9 post conception weeks, not biased by probabilistic cell typing approaches. With our analysis, we could characterize previously unreported molecular diversity within cardiomyocytes and epicardial cells and identified their characteristic expression signatures, comparing them with specific subpopulations found in single cell RNA sequencing datasets. We further characterized the differentiation trajectories of epicardial cells, identifying a clear spatial component on it. All in all, our study provides a novel technique for conducting de novo spatial-temporal analyses in developmental tissue samples and a useful resource for online exploration of cell-type differentiation during heart development at sub-cellular image resolution.

A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart.

The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.

Wnt/ß-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells.

The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/ß-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

Human fetal cardiac progenitors: The role of stem cells and progenitors in the fetal and adult heart.

The human fetal heart is formed early during embryogenesis as a result of cell migrations, differentiation, and formative blood flow. It begins to beat around gestation day 22. Progenitor cells are derived from mesoderm (endocardium and myocardium), proepicardium (epicardium and coronary vessels), and neural crest (heart valves, outflow tract septation, and parasympathetic innervation). A variety of molecular disturbances in the factors regulating the specification and differentiation of these cells can cause congenital heart disease. This review explores the contribution of different cardiac progenitors to the embryonic heart development; the pathways and transcription factors guiding their expansion, migration, and functional differentiation; and the endogenous regenerative capacity of the adult heart including the plasticity of cardiomyocytes. Unfolding these mechanisms will become the basis for understanding the dynamics of specific congenital heart disease as well as a means to develop therapy for fetal as well as postnatal cardiac defects and heart failure.

Meta-Analysis of Cell-based CaRdiac stUdiEs (ACCRUE) in patients with acute myocardial infarction based on individual patient data.

RATIONALE: The meta-Analysis of Cell-based CaRdiac study is the first prospectively declared collaborative multinational database, including individual data of patients with ischemic heart disease treated with cell therapy. OBJECTIVE: We analyzed the safety and efficacy of intracoronary cell therapy after acute myocardial infarction (AMI), including individual patient data from 12 randomized trials (ASTAMI, Aalst, BOOST, BONAMI, CADUCEUS, FINCELL, REGENT, REPAIR-AMI, SCAMI, SWISS-AMI, TIME, LATE-TIME; n=1252). METHODS AND RESULTS: The primary end point was freedom from combined major adverse cardiac and cerebrovascular events (including all-cause death, AMI recurrance, stroke, and target vessel revascularization). The secondary end point was freedom from hard clinical end points (death, AMI recurrence, or stroke), assessed with random-effects meta-analyses and Cox regressions for interactions. Secondary efficacy end points included changes in end-diastolic volume, end-systolic volume, and ejection fraction, analyzed with random-effects meta-analyses and ANCOVA. We reported weighted mean differences between cell therapy and control groups. No effect of cell therapy on major adverse cardiac and cerebrovascular events (14.0% versus 16.3%; hazard ratio, 0.86; 95% confidence interval, 0.63-1.18) or death (1.4% versus 2.1%) or death/AMI recurrence/stroke (2.9% versus 4.7%) was identified in comparison with controls. No changes in ejection fraction (mean difference: 0.96%; 95% confidence interval, -0.2 to 2.1), end-diastolic volume, or systolic volume were observed compared with controls. These results were not influenced by anterior AMI location, reduced baseline ejection fraction, or the use of MRI for assessing left ventricular parameters. CONCLUSIONS: This meta-analysis of individual patient data from randomized trials in patients with recent AMI revealed that intracoronary cell therapy provided no benefit, in terms of clinical events or changes in left ventricular function. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01098591.

Rosuvastatin inhibits TIMP-2 and promotes myocardial angiogenesis.

BACKGROUND: Angiogenesis is usually driven by inflammation. Matrix metalloproteinases MMP-3 and MMP-9 and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 are implicated in vascular remodeling. TIMP-2 exhibits antiangiogenic properties. Statins show benefits that are additional to lipid lowering including pro- and antiangiogenic properties. Atherosclerotic lesions in the coronary arteries have been well studied, but less is known about the fine terminal branches of the myocardial vasculature. METHODS: To examine this, we studied rosuvastatin (RSV) treatment in ApoE knockout (ApoE(-/-)) mice fed a high cholesterol (HC) diet. Hearts from ApoE(-/-) mice on a normal diet, HC diet and HC diet with RSV were harvested to determine MMP-3, MMP-9, TIMP-1, TIMP-2, vascular endothelial growth factor (VEGF)-A and estrogen receptor-α (ER-α) mRNA. RESULTS: RSV inhibited TIMP-1 and TIMP-2 expression and enhanced myocardial VEGF-A and ER-α expression, independently of plasma lipid level changes, but had no effect on MMP-3 and MMP-9 expression. CONCLUSIONS: These modulations of TIMPs, VEGF and ER-α expression induced by RSV may act as local stimulating factors for arteriolar growth in the myocardium.

Relation between N-terminal pro-brain natriuretic peptide levels and response to enhanced external counterpulsation in chronic angina pectoris.

OBJECTIVE: Although enhanced external counterpulsation (EECP) provides symptom reduction in many patients with severe angina pectoris, one-quarter of patients fail to respond. Earlier reports have not clearly established whether and how EECP responders may be identified pre-hoc. We hypothesized that clinical and biochemical data may be used to predict EECP response. METHODS: We explored a database of n=53 patients who had undergone clinically indicated EECP during 35 1-h sessions in our unit (65±7 years; 49 male), and sought to clarify which factors are predictive of response. Efficiency of counterpulsation was measured as the diastolic augmentation (DA) ratio, and was recorded both at beginning and end of the EECP treatment course. An increase in 6-min walk (6MW) distance of 5% was indicative of clinical response. RESULTS: Response occurred in 28 patients (53%; nonresponse in n=25, 47%). Responders had shorter baseline 6MW distance (377±81 vs. 445±62 m; P<0.01), lower left ventricular ejection fraction (48±9 vs. 54±8%; P<0.05), frequently had an increase in DA ratio during the EECP treatment course (23/28 vs. 5/28 with unchanged or decreased DA ratio; P<0.05), and higher levels of N-terminal pro-brain natriuretic peptide [NT-proBNP; 256 (123-547) vs. 62 (26-444) ng/l, P<0.01]. In multivariate logistic regression, response was independently predicted by baseline 6MW distance and baseline NT-proBNP levels (P<0.05 for both; model sensitivity: 82%, specificity: 72%, accuracy: 79%). CONCLUSION: There is larger clinical benefit of EECP in patients with greater functional impairment and higher levels of NT-proBNP.

Electrocardiographic artefacts mimicking atrial tachycardia resulted in unnecessary diagnostic and therapeutic measures.

Electrocardiographic (ECG) artefacts may closely simulate both supraventricular and ventricular tachycardias. We describe a case initially diagnosed as rapid atrial fibrillation, based on 12-lead surface ECG (especially the limb leads) and monitor tracing. The arrhythmia was resistant to beta blockers. Because of the at times apparently regular rhythm, an esophageal ECG recording was performed, and adenosine was administered. When the presumed atrial fibrillation terminated after sodium pentothal was administered while preparing for electrical cardioversion, the oesophageal ECG recordings and the ECGs during adenosine administration were reviewed. An ECG artefact diagnosis was suspected, and then confirmed, during relapse of the "arrhythmia," with simple palpation of the radial pulse and cardiac auscultation.

Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism.

AIMS: Several drugs blocking the rapidly activating potassium (K(r)) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species. METHODS AND RESULTS: Patch-clamp and quantitative-mRNA measurements of K(r) and slowly activating K (K(s)) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The K(r) channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific K(r)-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed K(r)-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The K(s) channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit. CONCLUSION: This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific K(r)-channel blocker in human, rat, and rabbit embryos provides evidence of K(r)-channel inhibition as a common mechanism for embryonic malformations and death.

Ischemia-reperfusion injury and pregnancy initiate time-dependent and robust signs of up-regulation of cardiac progenitor cells.

To explore how cardiac regeneration and cell turnover adapts to disease, different forms of stress were studied for their effects on the cardiac progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5, and mast cells. Adult female rats were examined during pregnancy, after myocardial infarction and ischemia-reperfusion injury with/out insulin like growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac sub-domains were analyzed at one and two weeks post-intervention, both at the mRNA and protein levels. While pregnancy and myocardial infarction up-regulated Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion injury induced the strongest up-regulation which occurred globally throughout the entire heart and not just around the site of injury. This response seems to be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary to c-Kit, Isl1 was not up-regulated by pregnancy or myocardial infarction while ischemia-reperfusion injury induced not a global but a focal up-regulation in the outflow tract and also in the peri-ischemic region, correlating with the up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1. c-Kit expression was not further influenced by the exogenous growth factors. This indicates that there is a spatial mismatch between on one hand c-Kit and Nkx2.5 expression and on the other hand Isl1 expression. In conclusion, ischemia-reperfusion injury was the strongest stimulus with both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregnancy induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes throughout the heart. Utilization of these pathways could provide new strategies for the treatment of cardiac disease.

Estrogen receptors do not influence angiogenesis after myocardial infarction.

BACKGROUND: There is controversy on whether estrogen receptors are present and functioning in the myocardium. Aims. To explore if after myocardial infarction (MI) estrogen receptors α (ERα) and ß (ERß) are upregulated in myocardial tissue and to explore if the presence/ absence of ERα or ERß influences angiogenesis after MI. METHODS: MI was induced by ligation of the left anterior descending artery in knockout (KO) mice, ERαKO and ERßKO, respectively, and non-KO littermate-controls, C57Bl/6 mice. The hearts were harvested after 12 days. A part of the periinfarct tissue was collected for ERα and ERß mRNA expression determination by real-time polymerase chain reaction. Using immunohistochemistry, ERα and ERß protein expression and capillary and arteriolar densities were blindly determined in the periinfarct area. RESULTS: In myocardium disrupted mRNA was upregulated in both ERαKO and ERßKO, (p < 0.005) and did not change after MI. There was no change in mRNA expression of ERα or ERß in wild type mice after MI. Expression of ERß in ERαKO and of ERα in ERßKO did not change. Following MI ERα or ERß could not be demonstrated by immunohistochemistry in either wild type or ERαKO or ERßKO. The capillary and arteriolar densities after MI did not differ between the groups in the periinfarct area. CONCLUSIONS: Although disrupted ER mRNA is upregulated in myocardium of ER knockout mice, no change in these or native receptors occurs following MI. At least in this model ER therefore seems not to have a role in myocardial arteriogenesis and angiogenesis after MI.

A Novel Descriptive, Intelligible and Ordered (DINO) classification of coronary bifurcation lesions. Review of current classifications.

BACKGROUND: Several classification systems for coronary artery bifurcation lesions (CABL) have been described in the literature, but despite the commendable effort to simplify a difficult subject in interventional cardiology, all of them have certain limitations and shortcomings. METHODS AND RESULTS: The proposed Descriptive, INtelligible and Ordered (DINO) is a new descriptive and clinically oriented system of classifying CABLs. This classification system takes into consideration more details of the side branch angulation relative to the main branch. It uses self-explanatory terms and mnemonic characters (acronyms related to the branches of the bifurcation and the shape of side branch angulation). The DINO classification describes the extent of CABL distribution and designates its localization at the bifurcation region. Moreover, systematized simple and easy to remember terms may form a relevant classification basis for multicenter and meta-analysis investigations. CONCLUSIONS: The DINO is the first verbally anchored, all-inclusive classification system of CABLs. It describes precisely side branch angulation, using self-explanatory and instructive terms that describe both the extent of the lesion's distribution and its localization. The current coronary bifurcation lesion classifications are reviewed.

Cyclic variability of stromal cell-derived factor-1 and endothelial progenitor cells during the menstrual cycle.

The endometrium goes through a unique cycle of physiological angiogenesis during the normal menstrual cycle (MC). We studied whether there is a correlation between endothelial progenitor cells (EPCs) and plasma and endometrial levels of angiogenic growth factors during the MC. Ten healthy, regularly menstruating women provided blood samples and another 16 supplied endometrial biopsies. Blood samples were obtained over a single MC: twice in the proliferative and once in the secretory phase and at ovulation. Endometrial biopsies were provided in the proliferative or in the secretory phase. We assessed plasma levels of vascular endothelial and fibroblast growth factors, granulocyte and granulocyte-macrophage colony-stimulating factors and stromal cell-derived factor-1 (SDF-1) by ELISA; EPCs by a colony-forming unit (CFU) assay; immunostaining for endometrial SDF-1 by computer-assisted software; and endothelial cell (EC) markers by flow cytometry. In the proliferative phase, SDF-1 levels were significantly higher than during the secretory phase. EPC-CFUs correlated negatively to SDF-1 levels. Endometrial SDF-1 expression tended to be higher in the secretory than in the proliferative phase. Furthermore, vascular endothelial growth factor receptors and Tie-2 EPCs showed a cyclic pattern over the MC. Our results point to SDF-1 as a novel mediator of EPC trafficking during the MC.

Coronary artery perforation and regrowth of a side branch occluded by a polytetrafluoroethylene-covered stent implantation.

Stenting of the right coronary artery stenosis caused coronary perforation and profound dye (blood) extravasation in a 69-year-old female patient. Instantaneous balloon inflation followed by implantation of a polytetrafluoroethylene- (PTFE-)covered stent sealed the coronary perforation, restored the blood flow, and perceivably caused acute occlusion of a large side branch (SB). The immediate in situ balloon inflation prevented the development of cardiac tamponade. Surprisingly, followup coronary angiography 4 and 11 months later showed spontaneous recanalization of the SB occluded by PTFE-covered stent. The SB was filled through a channel beginning at the end of the covered stent streaming retrogradely beneath it toward the SB ostium. Up to the best of our knowledge, this is the first described case of late spontaneous recanalization of as SB occluded by a PTFE-covered stent.

Anti-endothelial cell antibodies are increased in patients with previous myocardial infarction.

OBJECTIVES: Atherosclerosis is an inflammatory disease of multifactorial origin, in which immune cells together with metabolic risk factors may initiate, propagate, and activate lesions in the arterial tree. We investigated the role of auto-antibodies against endothelial cells in patients with previous myocardial infarction. DESIGN: One hundred and four patients were studied four to five years after acute myocardial infarction (aged 36-84 years) and 83 sex-matched healthy controls (aged 32-70 years). Anti-endothelial cells IgM and IgG auto-antibodies (AECA) were quantified in plasma using flow cytometry. RESULTS: IgM and IgG AECA were significantly higher (23.08 ± 0.81 and 10.63 ± 0.31 channel shifts, respectively; p < 0.001) in patients as compared to controls (13.40 ± 0.95 and 3.53 ± 0.54 channel shifts, respectively). Further, patients who got an invasive treatment had significantly higher levels of AECA as compared to patients with only medical treatment. Plasma concentration of IgG was positively (p < 0.05) correlated to the levels of high-sensitivity C-reactive protein (hsCRP). CONCLUSIONS: We report for the first time evidence that AECA are related to signs of inflammation and are increased in patients with atherosclerosis and previous myocardial infarction and with further increase with severity of disease.

Islet-1 cells are cardiac progenitors present during the entire lifespan: from the embryonic stage to adulthood.

The aim of this study was to longitudinally characterize the distribution of cells actively expressing the progenitor transcription factor islet-1 (Isl1+) during the embryonic life, the postnatal period, and adulthood. In this study, we have used direct immunohistochemical staining toward the protein Isl1 in a longitudinal rat model. Cells actively expressing Isl1 were traced in embryos from gestational day (GD) 11 until adulthood. In early cardiac development (GD 11), the Isl1+ progenitors were located in a greater abundance in the paracardiac regions, areas suggested to be the second heart field. To a lesser extent, Isl1+ cells were present within the bulbotruncal region and the truncus arteriosus. During the following days until GD 15, the Isl1+ cells were mainly observed at the proximal outflow tract (OFT) and at the inflow area of the right atrium. No Isl1+ cells were detected in the left ventricle. Compared with GD 11, more Isl1+ cells seemed to co-express cardiomyocyte markers and a minority of the Isl1+ cells was undifferentiated. Unexpectedly, only few undifferentiated Isl1+ cells were Ki67+ while a lot of TnT+ cardiomyocytes were proliferating in the ventricles. After birth, immature Isl1+ cells were still present in the OFT where they resided until adulthood. Our data suggest that during embryogenesis, Isl1+ cells migrate from extracardiac regions into the proximal part of the heart, proliferating and giving rise to cardioblasts. Unexpectedly, only a minority of the Isl1+ cells while a majority of ventricular cardiomyocytes were proliferating. The Isl1+ cell pool persists into adulthood, which might open up new strategies to repair damaged myocardium.

Early first trimester human embryonic cardiac Islet-1 progenitor cells and cardiomyocytes: Immunohistochemical and electrophysiological characterization.

The aims of this study were to systematically characterize the distribution, proliferation, and differentiation of Islet-1(+)(Isl1(+)) progenitor cells in the early first trimester human embryonic heart during which period most of the organogenesis takes place. In hearts of gestational week 5 to 10 Isl1(+)cells were identified and mainly clustered in the outflow tract and to a lesser extent in the atria and in the right ventricle. Some of the clusters were also troponin T(+). Unexpectedly a only few Isl1(+)cells were Ki67(+)while in the ventricles a majority of Isl1(-)troponinT(+)cells were Ki67(+). Cultures derived from the digested embryonic heart developed into spontaneously beating cardiospheres. At harvest cells in these cardiospheres showed frequent expression of troponin T(+)and Nkx2.5(+), while Isl1 was expressed only in scattered cells. Only a minority of the cultured cells expressed Ki67. The cardiospheres could be frozen, thawed, and recultured to beating cardiospheres. In a multielectrode array system, the beating cardiospheres were responsive to adrenergic stimulation and exhibited rate-dependent action potential duration. In conclusion, the early first trimester human embryonic heart expresses clusters of Isl1(+)cells, some of which differentiate into cardiomyocytes. Unexpectedly, only a minority of the Isl1(+)cells, while a majority of ventricular cardiomyocytes, were proliferating. Spontaneously beating cardiospheres could be derived from the human embryonic heart and these cardiospheres showed functional frequency control.