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.

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.

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.

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.

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.

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.

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.

Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to TTN mutations.

Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cell­derived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9­generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.

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.

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.

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.

In search of the next super models.

The advent of pluripotent stem cell biology and facile genetic manipulation via CRISPR technology has ushered in a new era of human disease models for drug discovery and development. While these precision "super models" hold great promise for tailoring personalized therapy, their full potential and in vivo validation have remained elusive.

Monocyte-platelet aggregates affect local inflammation in patients with acute myocardial infarction.

The local inflammatory response following acute myocardial infarction (AMI) is increasingly being recognized as a central factor determining infarct healing. Myocardial inflammation can be visualized in patients using fasting 18F-FDG PET/MRI. Although this novel biosignal correlates with long-term functional outcome, the corresponding cellular substrate is not well understood. Here we present a retrospective analysis of 29 patients with AMI who underwent revascularization, suggesting a connection between post infarction myocardial fasting 18F-FDG uptake, monocyte platelet aggregates (MPA), and P2Y12 inhibition. In detail, patients with high MPA percentages of CD14highCD16+ and CD14lowCD16+ monocytes had significantly higher local 18F-FDG uptake (SUVmean) in the infarcted myocardium than patients with low MPA (p < 0.05). Furthermore, there was an association of high MPA percentage in all monocyte subpopulations with deteriorating ΔLV-EF after 6 months (p < 0.01), which was confirmed in an extended analysis with additional 29 patients without PET/MRI data available. In this analysis, administration of Ticagrelor was associated with lower MPA percentage of CD14high monocyte subpopulations than Clopidogrel (p < 0.01) or Prasugrel (p < 0.05). Taken together, the findings from this analysis suggest that platelet aggregability may affect monocyte extravasation into the infarcted myocardium and influence long-term functional outcome. P2Y12 inhibition may intervene in this pathophysiologic process. Prospective studies are needed to further examine this important relationship.

Enhanced platelet inhibition by clopidogrel and risk of bleeding in patients requiring oral anticoagulation after drug-eluting stent implantation.

AIMS: Clopidogrel is the P2Y12 inhibitor of choice in patients who undergo PCI and have an indication for oral anticoagulation (OAC). Prediction of the bleeding risk is of major interest in this population. The aim of this analysis was to investigate whether an enhanced platelet inhibition by clopidogrel measured by platelet function testing (PFT) with the Multiplate Analyzer is associated with an increased bleeding risk in patients on triple antithrombotic therapy. METHODS AND RESULTS: This investigation was performed in a cohort of 524 patients from the randomised ISAR-TRIPLE trial; 458 (87.4%) had PFT results available in the first 24 hours after PCI. Patients belonging to the lowest quintile according to PFT were considered as enhanced responders to clopidogrel. The primary endpoint was major bleeding according to TIMI criteria at nine months. The median of ADP-induced platelet aggregation in the whole population was 163 AU*min (107-241). Patients in the lowest quintile had values below 93 AU*min. These enhanced responders (92 patients) had a significantly higher risk of TIMI major bleeding (hazard ratio [HR] 3.13, 95% confidence interval [CI]: 1.38-7.09, p=0.01) and overall mortality (HR 3.42, 95% CI: 1.55-7.52, p=0.004) compared with the remaining patients (366 patients). No significant difference was observed for the secondary combined ischaemic endpoint (HR 1.27, 95% CI: 0.47-3.47, p=0.64). CONCLUSIONS: Enhanced platelet inhibition delivered by clopidogrel is associated with an increased risk for major bleeding and death in patients on OAC who undergo PCI. These results support the use of PFT to identify patients with an increased risk for bleeding.

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes.

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

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.

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.