Disease incidence and not case fatality drives the rural disadvantage in myocardial-infarction-related mortality in Germany.

OBJECTIVE: Demographic and infrastructural developments might compromise medical care provision in rural regions, particularly for acute health conditions. Studying the case of myocardial infarction (MI), we investigated how MI-related mortality at ages 65+ varies between rural and urban regions in Germany and to what extent differences are driven by varying case fatality and disease incidence. METHODS: The study relies on data containing all hospitalizations, cause-specific deaths and population counts for the total German population between years 2012-2018 and ages 65+. MI-related mortality, MI incidence and case fatality are compared between urban and rural regions in a population-wide analysis. The impacts of changing incidence and case fatality on rural-urban MI-related mortality differences are assessed using a counterfactual approach. RESULTS: Rural regions in Germany show systematically higher MI-related death rates and MI incidence at ages 65+ compared to urban regions. Higher mortality is primarily the result of higher MI incidence in rural regions, while case fatality is largely similar. The rural excess in MI-related death rates would be nullified and 1 out of 6 MI-related deaths in rural regions could be prevented if rural regions in Germany would have at least the median MI incidence of urban regions. CONCLUSIONS: MI incidence and not case fatality drives the rural disadvantage in MI-related mortality in Germany. Higher MI incidence points towards potential regional variation in the effectiveness of disease prevention. The findings highlight that improving disease prevention at the patient level carries larger opportunities for reducing regional MI-related mortality inequalities in Germany.

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.

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.

Long-term outcome of patients with invasive electrophysiology procedure-related cardiac tamponade.

AIMS: Iatrogenic cardiac tamponades are a rare but dreaded complication of invasive electrophysiology procedures (EPs). Their long-term impact on clinical outcomes is unknown. This study analysed the risk of death or serious cardiovascular events in patients suffering from EP-related cardiac tamponade requiring pericardiocentesis during long-term follow-up. METHODS AND RESULTS: Out of 19 997 invasive EPs at the Karolinska University Hospital between January 1998 and September 2018, all patients with EP-related periprocedural cardiac tamponade were identified (n = 60) and matched (1:3 ratio) to a control group (n = 180). After a follow-up of 5 years, the composite primary endpoint - death from any cause, acute myocardial infarction, transitory ischaemic attack (TIA)/stroke, and hospitalization for heart failure - occurred in significantly more patients in the tamponade than in the control group [12 patients (20.0%) vs. 19 patients (10.6%); hazard ratio (HR) 2.53 (95% confidence interval, CI 1.15-5.58); P = 0.021]. This was mainly driven by a higher incidence of TIA/stroke in the tamponade than in the control group [HR 3.75 (95% CI 1.01-13.97); P = 0.049]. Death from any cause, acute myocardial infarction, and hospitalization for heart failure did not show a significant difference between the groups. Hospitalization for pericarditis occurred in significantly more patients in the tamponade than in the control group [HR 36.0 (95% CI 4.68-276.86); P = 0.001]. CONCLUSION: Patients with EP-related cardiac tamponade are at higher risk for cerebrovascular events during the first 2 weeks and hospitalization for pericarditis during the first months after index procedure. Despite the increased risk for early complications tamponade patients have a good long-term prognosis without increased risk for mortality or other serious cardiovascular events.

Perspectives and Challenges of Pluripotent Stem Cells in Cardiac Arrhythmia Research.

PURPOSE OF REVIEW: The promises of human-induced pluripotent stem cells (hiPSCs) for modeling arrhythmogenic disease, but also for drug discovery and toxicity tests, are straightforward and exciting. However, the full potential of this new technology has not been fully realized yet. The purpose of this review is to provide an overview of the state-of-the-art research in arrhythmogenic disease modeling and drug discovery and an outlook of what can be expected from the second decade of hiPSC-based arrhythmia research. RECENT FINDINGS: Remarkable advances in genomic discoveries, stem cell biology, and genome editing via sequence-specific nucleases have been made in recent years. Together, these breakthroughs have allowed us to progress from studying monogenetic diseases with a direct genotype-phenotype relationship to genetically more complex diseases such as arrhythmogenic right ventricular dysplasia and atrial fibrillation. In addition, newly developed tools for arrhythmia research such as optical action potential recordings have facilitated the use of hiPSCs for drug and toxicity screening and their eventual clinical use. These advances in in vitro assay development, genome editing, and stem cell biology will soon enable the implementation of hiPSC-based findings into clinical practice and provide us with unprecedented insights into mechanisms of complex arrhythmogenic diseases.

Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.

Impact of immature platelets on platelet response to ticagrelor and prasugrel in patients with acute coronary syndrome.

AIMS: The influence of reticulated platelets (RPs) on platelet inhibition by ticagrelor when compared with prasugrel is unknown. We aimed to determine the influence of RPs on adenosine diphosphate- (ADP-) induced platelet aggregation in patients with acute coronary syndrome who were randomly assigned to receive either ticagrelor or prasugrel for P2Y12 receptor inhibition. METHODS AND RESULTS: One hundred and twenty-four patients were prospectively enrolled. The immature platelet fraction (IPF) was measured by an automated haematoanalyzer and platelet aggregation was assessed by multiple electrode aggregometry. In a subgroup of patients (n = 28) ADP-induced P-selectin expression was measured in dependence of the time point of study drug intake in RPs that were discriminated from mature platelets by thiazole-orange (TO) staining. The primary endpoint was the correlation of platelet aggregation and IPF in ticagrelor- vs. prasugrel-treated patients. Platelet aggregation significantly correlated with IPF in patients who received prasugrel (r = 0.41, P < 0.001). In contrast, no correlation between IPF and platelet aggregation was observed in ticagrelor-treated patients (r = 0.08, P = 0.51, P for difference of correlation coefficients P = 0.05). Within the TO-positive RP fraction, P-selectin expression was significantly higher in prasugrel when compared with ticagrelor-treated individuals (prasugrel 18.6 ± 16.0% vs. ticagrelor 11.5 ± 6.0%, P = 0.047). A time-dependent P-selectin expression was observed in prasugrel but not in ticagrelor-treated patients (prasugrel early 11.9 ± 9.4% vs. late 26.3 ± 19.0%, P = 0.031, ticagrelor early 9.6 ± 4.9% vs. late 13.5 ± 6.6%, P = 0.127). CONCLUSION: Reticulated platelets show a greater impact on platelet reactivity in response to prasugrel when compared with ticagrelor.

Induced pluripotent stem cell-derived cardiomyocytes: a versatile tool for arrhythmia research.

Induced pluripotent stem cells offer the possibility to generate patient-specific stem cell lines from individuals affected by inherited disorders. Cardiomyocytes differentiated from such patient-specific induced pluripotent stem cells lines have been used to study the pathophysiology of arrhythmogenic heart diseases, such as the long-QT syndrome or catecholaminergic polymorphic ventricular tachycardia. Testing for unwanted drug side effects or tailoring medical treatment to the specific needs of individual patients with arrhythmogenic disorders may become future applications of this emerging technology.

Induced pluripotent stem cells in cardiovascular research.

The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSC) by overexpression of a combination of transcription factors bears the potential to spawn a wealth of new applications in both preclinical and clinical cardiovascular research. Disease modeling, which is accomplished by deriving iPSC lines from patients affected by heritable diseases and then studying the pathophysiology of the diseases in somatic cells differentiated from these patient-specific iPSC lines, is the so far most advanced of these applications. Long-QT syndrome and catecholaminergic polymorphic ventricular tachycardia are two heart rhythm disorders that have been already successfully modeled by several groups using this approach, which will likely serve to model other mono- or polygenetic cardiovascular disorders in the future. Test systems based on cells derived from iPSC might prove beneficial to screen for novel cardiovascular drugs or unwanted drug side effects and to individualize medical therapy. The application of iPSC for cell therapy of cardiovascular disorders, albeit promising, will only become feasible if the problem of biological safety of these cells will be mastered.

Patient-specific induced pluripotent stem-cell models for long-QT syndrome.

BACKGROUND: Long-QT syndromes are heritable diseases associated with prolongation of the QT interval on an electrocardiogram and a high risk of sudden cardiac death due to ventricular tachyarrhythmia. In long-QT syndrome type 1, mutations occur in the KCNQ1 gene, which encodes the repolarizing potassium channel mediating the delayed rectifier I(Ks) current. METHODS: We screened a family affected by long-QT syndrome type 1 and identified an autosomal dominant missense mutation (R190Q) in the KCNQ1 gene. We obtained dermal fibroblasts from two family members and two healthy controls and infected them with retroviral vectors encoding the human transcription factors OCT3/4, SOX2, KLF4, and c-MYC to generate pluripotent stem cells. With the use of a specific protocol, these cells were then directed to differentiate into cardiac myocytes. RESULTS: Induced pluripotent stem cells maintained the disease genotype of long-QT syndrome type 1 and generated functional myocytes. Individual cells showed a "ventricular," "atrial," or "nodal" phenotype, as evidenced by the expression of cell-type­specific markers and as seen in recordings of the action potentials in single cells. The duration of the action potential was markedly prolonged in "ventricular" and "atrial" cells derived from patients with long-QT syndrome type 1, as compared with cells from control subjects. Further characterization of the role of the R190Q­KCNQ1 mutation in the pathogenesis of long-QT syndrome type 1 revealed a dominant negative trafficking defect associated with a 70 to 80% reduction in I(Ks) current and altered channel activation and deactivation properties. Moreover, we showed that myocytes derived from patients with long-QT syndrome type 1 had an increased susceptibility to catecholamine-induced tachyarrhythmia and that beta-blockade attenuated this phenotype. CONCLUSIONS: We generated patient-specific pluripotent stem cells from members of a family affected by long-QT syndrome type 1 and induced them to differentiate into functional cardiac myocytes. The patient-derived cells recapitulated the electrophysiological features of the disorder. (Funded by the European Research Council and others.)

Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease.

The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration.

Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools.

Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.

Recapitulating long-QT syndrome using induced pluripotent stem cell technology.

The generation of patient-specific stem cells by reprogramming somatic cells to induced pluripotent stem cells (iPSC) provides the basis for a promising new type of in vitro disease models. Patient-specific iPSC derived from individuals with hereditary disorders can be differentiated into somatic cells in vitro, thus allowing the pathophysiology of the diseases to be studied on a cellular level. Different types of long-QT syndrome have been successfully modeled using this approach, demonstrating that the iPSC-derived patient-specific cardiomyocytes recapitulated key features of the disease in vitro. This approach will likely serve to model other monogenetic or polygenetic cardiovascular disorders in the future. Moreover, test platforms based on patient-specific iPSC could be used to test the potential of drug candidates to induce QT-interval prolongation or other unwanted side effects, screen for novel cardiovascular drugs, or to tailor medical therapy to the specific needs of a single patient.

Unlocking the promise of mRNA therapeutics.

The extraordinary success of mRNA vaccines against coronavirus disease 2019 (COVID-19) has renewed interest in mRNA as a means of delivering therapeutic proteins. Early clinical trials of mRNA therapeutics include studies of paracrine vascular endothelial growth factor (VEGF) mRNA for heart failure and of CRISPR-Cas9 mRNA for a congenital liver-specific storage disease. However, a series of challenges remains to be addressed before mRNA can be established as a general therapeutic modality with broad relevance to both rare and common diseases. An array of new technologies is being developed to surmount these challenges, including approaches to optimize mRNA cargos, lipid carriers with inherent tissue tropism and in vivo percutaneous delivery systems. The judicious integration of these advances may unlock the promise of biologically targeted mRNA therapeutics, beyond vaccines and other immunostimulatory agents, for the treatment of diverse clinical indications.

Meta-Analysis of Short vs. Prolonged Dual Antiplatelet Therapy after Drug-Eluting Stent Implantation and Role of Continuation with either Aspirin or a P2Y12 Inhibitor Thereafter.

AIM: The optimal duration of dual antiplatelet therapy (DAPT) after drug-eluting stent (DES) implantation is an ongoing debate and novel data has emerged. The aim of this meta-analysis was to assess outcomes of short vs. control DAPT duration. In addition, the role of single antiplatelet therapy (SAPT) after DAPT with either aspirin or P2Y12 inhibitor monotherapy was analyzed. METHODS: The authors searched MEDLINE and Cochrane databases and proceedings of international meetings for randomized controlled trials (RCT) comparing ≤ 3 months with ≥ 6 months DAPT after DES implantation. The primary and co-primary outcomes of interest were definite or probable stent thrombosis (ST) and bleeding. In addition, we performed an analysis on studies who continued with either aspirin or P2Y12 monotherapy after DAPT. RESULTS: 9 RCTs comprising 41,864 patients were included and we analyzed a short DAPT duration of median 1.5 months vs. 12.1 months in the control group. The risk for ST was similar with short vs. control DAPT duration (0.5 vs. 0.5%; hazard ratio 1.17[95% CI 0.89-1.54]; p=0.26). Bleeding was significantly reduced with short vs. control DAPT duration (1.9 vs. 3.0%; 0.65[0.54-0.77]; p＜0.0001).ST was not different between short vs. control DAPT duration in the analysis of the 4 RCTs who continued with aspirin after DAPT and the 5 P2Y12 RCTs, respectively, and no heterogeneity was detected (p=0.861). Bleeding was also reduced with short vs. control DAPT in both the aspirin (1.2 vs. 1.7%; 0.71[0.51-0.99]; p=0.04) and P2Y12 inhibitor studies (2.1 vs. 3.4%; 0.62[0.47-0.80]; p=0.0003) and no heterogeneity was detected (p=0.515). CONCLUSIONS: Our meta-analysis shows that short DAPT ≤ 3 months followed by SAPT reduces bleeding and is not associated with an increase in ST. The results were consistent within the aspirin and P2Y12 SAPT studies.

Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders.

The contribution of the epicardium, the outermost layer of the heart, to cardiac regeneration has remained controversial due to a lack of suitable analytical tools. By combining genetic marker-independent lineage-tracing strategies with transcriptional profiling and loss-of-function methods, we report here that the epicardium of the highly regenerative salamander species Pleurodeles waltl has an intrinsic capacity to differentiate into cardiomyocytes. Following cryoinjury, CLDN6+ epicardium-derived cells appear at the lesion site, organize into honeycomb-like structures connected via focal tight junctions and undergo transcriptional reprogramming that results in concomitant differentiation into de novo cardiomyocytes. Ablation of CLDN6+ differentiation intermediates as well as disruption of their tight junctions impairs cardiac regeneration. Salamanders constitute the evolutionarily closest species to mammals with an extensive ability to regenerate heart muscle and our results highlight the epicardium and tight junctions as key targets in efforts to promote cardiac regeneration.

Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development.

Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.

Amnion signals are essential for mesoderm formation in primates.

Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.