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.

Polyscore of autonomic parameters for risk stratification of the elderly general population: the Polyscore study.

AIMS: Present society is constantly ageing and elderly frequently suffer from conditions that are difficult and/or costly to treat if detected late. Effective screening of the elderly is therefore needed so that those requiring detailed clinical work-up are identified early. We present a prospective validation of a screening strategy based on a Polyscore of seven predominantly autonomic, non-invasive risk markers. METHODS AND RESULTS: Within a population-based survey in Germany (INVADE study), participants aged ≥60 years were enrolled between August 2013 and February 2015. Seven prospectively defined Polyscore components were obtained during 30-min continuous recordings of electrocardiogram, blood pressure, and respiration. Out of 1956 subjects, 168 were excluded due to atrial fibrillation, implanted pacemaker, or unsuitable recordings. All-cause mortality over a median 4-year follow-up was prospectively defined as the primary endpoint. The Polyscore divided the investigated population (n = 1788, median age: 72 years, females: 58%) into three predefined groups with low (n = 1405, 78.6%), intermediate (n = 326, 18.2%), and high risk (n = 57, 3.2%). During the follow-up, 82 (4.6%) participants died. Mortality in the Polyscore-defined risk groups was 3.4%, 7.4%, and 17.5%, respectively (P < 0.0001). The Polyscore-based mortality prediction was independent of Framingham score, diabetes, chronic kidney disease, and major stroke and/or myocardial infarction history. It was particularly effective in those aged <75 years (n = 1145). CONCLUSION: The Polyscore-based mortality risk assessment from short-term non-invasive recordings is effective in the elderly general population, especially those aged 60-74 years. Implementation of a comprehensive Polyscore screening of this age group is proposed to advance preventive medical care.

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.

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.

Impact of sympathetic renal denervation: a randomized study in patients after renal transplantation (ISAR-denerve).

BACKGROUND: Sympathetic overactivity is frequently observed following renal transplantation (RTx), and post-transplant hypertension is a major contributing factor to graft failure and cardiovascular morbidity. This process is perpetuated by preservation of sympathetic afferent activity from the native non-functional kidneys, in the absence of efferent feedback to the renal transplant, which would otherwise modulate neurohumoral activity. We investigated the feasibility and efficacy of renal sympathetic denervation (RDN) in renal transplant recipients. METHODS: Patients (n = 18) with post-transplant hypertension were randomized 1:1 to receive RDN or medical treatment alone. The primary efficacy end point was change in office systolic blood pressure (SBP) and mean 24-h ambulatory blood pressure monitoring (ABPM) at 6 months. Safety end points were changes in renal function or renovascular complications. RESULTS: After 6 months, patients in the RDN group had a significant reduction in office SBP of 23.3 ± 14.5 mmHg (P = 0.001 for change difference between the groups). In ABPM, nocturnal blood pressure was reduced in the RDN group by -10.38 ± 12.8 mmHg (P = 0.06), whereas no change was measured during the day. In the RDN group, significantly more patients converted from non-dippers to dippers (P = 0.035). There were no adverse safety events in either group. CONCLUSION: RDN is feasible and safe in renal transplant recipients. However, larger sham-controlled studies will be necessary to clarify the potential role of RDN in this population. CLINICAL TRIAL REGISTRATION: NCT01899456.

Genetically Encoded Voltage Indicators in Circulation Research.

Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

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.

In-silico assessment of the dynamic effects of amiodarone and dronedarone on human atrial patho-electrophysiology.

AIMS: The clinical efficacy in preventing the recurrence of atrial fibrillation (AF) is higher for amiodarone than for dronedarone. Moreover, pharmacotherapy with these drugs is less successful in patients with remodelled substrate induced by chronic AF (cAF) and patients suffering from familial AF. To date, the reasons for these phenomena are only incompletely understood. We analyse the effects of the drugs in a computational model of atrial electrophysiology. METHODS AND RESULTS: The Courtemanche-Ramirez-Nattel model was adapted to represent cAF remodelled tissue and hERG mutations N588K and L532P. The pharmacodynamics of amiodarone and dronedarone were investigated with respect to their dose and heart rate dependence by evaluating 10 descriptors of action potential morphology and conduction properties. An arrhythmia score was computed based on a subset of these biomarkers and analysed regarding circadian variation of drug concentration and heart rate. Action potential alternans at high frequencies was observed over the whole dronedarone concentration range at high frequencies, while amiodarone caused alternans only in a narrow range. The total score of dronedarone reached critical values in most of the investigated dynamic scenarios, while amiodarone caused only minor score oscillations. Compared with the other substrates, cAF showed significantly different characteristics resulting in a lower amiodarone but higher dronedarone concentration yielding the lowest score. CONCLUSION: Significant differences exist in the frequency and concentration-dependent effects between amiodarone and dronedarone and between different atrial substrates. Our results provide possible explanations for the superior efficacy of amiodarone and may aid in the design of substrate-specific pharmacotherapy for AF.

Sex differences in the non-invasive risk stratification and prognosis after myocardial infarction.

BACKGROUND: Women have unfavorable prognosis after myocardial infarction (MI). This text describes sex differences in mortality and in the power of risk predictors in contemporarily-treated MI patients. METHODS: A population of 4141 MI patients (26.5% females) was followed up for 5years. Effects of sex and age on total mortality were investigated by multivariable Cox analysis. Mortality predictors were investigated by receiver-operator characteristics analysis. Stepwise multivariable Cox regression was used to create sex-specific predictive models. RESULTS: Thirty-day mortality was 1.5-fold higher in women. However, sex was not a significant mortality predictor in a model adjusted for age. Predictors for 5-year mortality performed differently in male and female patients. In women, a sex-specific model provided better risk stratification than a sex-neutral model. CONCLUSION: The unfavorable prognosis of female MI patients can be explained by advanced age. Sex-specific predictive models might improve risk stratification in female survivors of acute MI.

Assessment of mean respiratory rate from ECG recordings for risk stratification after myocardial infarction.

BACKGROUND: We recently reported that nocturnal respiratory rate (NRR) predicts non-sudden cardiac death in survivors of myocardial infarction (MI). Here, we present the details of the technique deriving NRR from ECG recordings. METHODS: Continuous ECG and respiratory chest excursions were simultaneously recorded in 941 MI survivors who were followed-up for 5-years. Mean respiratory rate was derived from the ECG based on RR intervals, QRS amplitudes, and QRS vectors and compared to chest belt measurements. NRR was calculated from Holter-ECGs accordingly using the same ECG processing. RESULTS: Directly-measured and ECG-derived respiratory rates were in good agreement. Areas under the ROC curve for 10-min-ECG- and Holter-derived respiratory rate were well in the confidence intervals of that of the chest belt measurement. The optimum dichotomy of NRR for the prediction of mortality was ≥18.6 breaths per minute. CONCLUSIONS: The mean respiratory rate can be precisely derived from continuous ECGs.

QRS fragmentation does not predict mortality in survivors of acute myocardial infarction.

BACKGROUND: Despite advances in coronary revascularization and in heart failure management, myocardial infarction survivors remain at substantially increased mortality risk. Precise risk assessment and risk-adapted follow-up care are crucial to improve their outcomes. Recently, the fragmented QRS complex, i.e. the presence of additional spikes within the QRS complexes on a 12-lead electrocardiogram, has been discussed as a potential non-invasive risk predictor in cardiac patients. HYPOTHESIS: The aim of this study was to evaluate the prognostic meaning of the fragmented QRS complex in myocardial infarction survivors. METHODS: 609 patients with narrow QRS complexes <120 ms were included in a prospective cohort study while hospitalized for myocardial infarction and followed for 5 years. RESULTS: The prevalence of the fragmented QRS complex in these patients amounted to 46.8% (285 patients). These patients had no increased hazard of all-cause death (HR 0.84, 95%-CI 0.45-1.57, p = 0.582) with a mortality rate of 6.0% compared to 7.1% in patients without QRS fragmentations. Furthermore, the risks of cardiac death (HR 1.28, 95%-CI 0.49-3.31, p = 0.613) and of non-cardiac death (HR 0.6, 95%-CI 0.26-1.43, p = 0.25) were not significantly different in patients with QRS fragmentations. However, patients with QRS fragmentations had increased serum creatine kinase concentrations (1438U/l vs. 1160U/l, p = 0.039) and reduced left ventricular ejection fractions (52% vs. 54%, p = 0.011). CONCLUSIONS: The hypothesis that QRS fragmentation might be a prognostic parameter in survivors of myocardial infarction was not confirmed. But those with QRS fragmentation had larger myocardial infarctions, as measured by creatine kinase and left ventricular ejection fraction.

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.)

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.

Generation of heterozygous (MRli003-A-5) and homozygous (MRli003-A-6) voltage-sensing knock-in human iPSC lines by CRISPR/Cas9 editing of the AAVS1 locus.

Assessment of the electrophysiological properties of cardiomyocytes is necessary for phenotyping cardiac disorders and for drug screening. Optical action potential imaging using a genetically encoded voltage-sensing fluorescent protein (VSFP) allows for high-throughput functional characterization of cardiomyocytes, which offers an advantage over the traditional patch-clamp technique. Here, we knocked VSFP into the AAVS1 safe harbor locus of human iPSCs, generating two stable voltage indicator lines - one heterozygous (MRIi003-A-5) and the other homozygous (MRI003-A-6). Both lines can be used for optical membrane potential recordings and provide a powerful platform for a wide range of applications in cardiovascular biomedicine.

High-throughput optical action potential recordings in hiPSC-derived cardiomyocytes with a genetically encoded voltage indicator in the AAVS1 locus.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.

The Polyscore of autonomic parameters predicts mortality and identifies low-risk individuals among diabetic survivors of acute myocardial infarction.

Survivors of an acute myocardial infarction with diabetes mellitus retain an increased mortality risk. Reliable assessment of individual risk is required for effective and cost-efficient medical care in these patients. The Polyscore is a previously established risk predictor consisting of seven autonomic tests derived from electrocardiogram, blood pressure, and respiration. The Polyscore allows classification of survivors of myocardial infarction in groups at low, intermediate and high mortality risk. The aim of this study was to investigate the prognostic value of the Polyscore in diabetic survivors of acute myocardial infarction, which may be impaired by the presence of diabetic autonomic neuropathy. Survivors of an acute myocardial infarction were included in a prospective cohort study during hospitalisation due to the index event at two university hospitals in Munich, Germany. The Polyscore was determined from simultaneous non-invasive 30-min recordings of electrocardiogram, continuous arterial blood pressure, and respiration which were performed in all participants. Patients were followed for 5 years. The primary and secondary outcomes were all-cause mortality and cardiac mortality. 184 of 941 enrolled patients (19.6%) suffered from diabetes mellitus. 5-year-mortality was higher in diabetic patients (15.2%) compared to non-diabetic patients (5.8%). A multivariable Cox regression model confirmed the Polyscore as a strong predictor of mortality in diabetic post-MI patients (intermediate risk: HR 6.56, 95% CI 1.61-26.78, p = 0.004, mortality 22.8%; high risk: HR 18.76, 95% CI 4.35-80.98, p < 0.001, mortality 68.8%). There was no interaction between diabetes mellitus and the Polyscore regarding mortality prediction (p = 0.775). Interestingly, in contrast to the groups at intermediate and high risk (73 patients, 39.7%), the Polyscore identified a majority of diabetic patients (111, 60.3%) with a low mortality risk, comparable to that of low-risk non-diabetic patients (3.6% and 2.1%, respectively, p = 0.339). Consistent results were observed for cardiac mortality. This analysis shows that the Polyscore predicts all-cause and cardiac mortality in diabetic survivors of acute myocardial infarction. Within these patients it identifies a large population not affected by the excess mortality associated with diabetes in this setting. Thus, the Polyscore may facilitate risk-adapted follow-up strategies in diabetic survivors of myocardial infarction.