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.

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.

Migratory and anti-fibrotic programmes define the regenerative potential of human cardiac progenitors.

Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host-graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.

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.

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.

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.

Nocturnal respiratory rate predicts ICD benefit: A prospective, controlled, multicentre cohort study.

BACKGROUND: Implantable cardioverter defibrillators (ICDs) prevent sudden cardiac death. ICD implantation decisions are currently based on reduced left ventricular ejection fraction (LVEF≤35%). However, in some patients, the non-arrhythmic death risk predominates thus diminishing ICD-therapy benefits. Based on previous observations, we tested the hypothesis that compared to the others, patients with nocturnal respiratory rate (NRR) ≥18 breaths per minute (brpm) benefit less from prophylactic ICD implantations. METHODS: This prospective cohort study was a pre-defined sub-study of EU-CERT-ICD trial conducted at 44 centers in 15 EU countries between May 12, 2014, and September 6, 2018. Patients with ischaemic or non-ischaemic cardiomyopathy were included if meeting primary prophylactic ICD implantation criteria. The primary endpoint was all-cause mortality. NRR was assessed blindly from pre-implantation 24-hour Holters. Multivariable models and propensity stratification evaluated the interaction between NRR and the ICD mortality effect. This study is registered with ClinicalTrials.gov (NCT0206419). FINDINGS: Of the 2,247 EU-CERT-ICD patients, this sub-study included 1,971 with complete records. In 1,363 patients (61.7 (12) years; 244 women) an ICD was implanted; 608 patients (63.2 (12) years; 108 women) were treated conservatively. During a median 2.5-year follow-up, 202 (14.8%) and 95 (15.6%) patients died in the ICD and control groups, respectively. NRR statistically significantly interacted with the ICD mortality effect (p = 0.0070). While the 1,316 patients with NRR<18 brpm showed a marked ICD benefit on mortality (adjusted HR 0.529 (95% CI 0.376-0.746); p = 0.0003), no treatment effect was demonstrated in 655 patients with NRR≥18 brpm (adjusted HR 0.981 (95% CI 0.669-1.438); p = 0.9202). INTERPRETATION: In the EU-CERT-ICD trial, patients with NRR≥18 brpm showed limited benefit from primary prophylactic ICD implantation. Those with NRR<18 brpm benefitted substantially. FUNDING: European Community's 7th Framework Programme FP7/2007-2013 (602299).

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.

Placebo and Nocebo Effects in Patients With Takotsubo Cardiomyopathy and Heart-Healthy Controls.

The etiology of takotsubo cardiomyopathy (TTC)-a rare, reversible, and acquired form of cardiac diseases-is not yet fully explained. An exaggerated activation of the sympathetic-nervous-system (SNS) following stressful psychosocial life events is discussed to be of key importance. In this experimental study, we tested whether TTC patients, compared to heart-healthy controls, respond more strongly to supporting placebo interventions and stressful nocebo interventions targeting cardiac function. In a single experimental session, 20 female TTC patients and 20 age matched (mean age 61.5 years, ± 12.89) catheter-confirmed heart-healthy women were examined. Saline solution was administered three times i.v. to all participants, with the verbal suggestion they receive an inert substance with no effects on the heart (neutral condition), a drug that would support cardiac functions (positive condition), and a drug that would burden the heart (negative condition). Systolic and diastolic blood pressure (DBP/SBP), heart rate (HR), endocrine markers cortisol (µg/dl), copeptin (pmol/l), and subjective stress ratings (SUD) were assessed to examine alterations of the SNS and the hypothalamic-pituitary-adrenal axis (HPA). Before and after each intervention SUD was rated. One pre and three post serum cortisol and copeptin samples were assessed, and a long-term electrocardiogram as well as non-invasive, continuous blood pressure was recorded. The study design elucidated a significant increase of SUD levels as a response to the nocebo intervention, while perceived stress remained unaffected during the preceding neutral and positive interventions. Increasing SUD levels were accompanied by higher SBP and an anticipatory increase of HR shortly prior to the nocebo intervention. SBP increased also as a response to positive verbal suggestions (Bonferroni-corrected p-values > .05). Alterations of cortisol and copeptin due to the interventions and significant placebo effects failed to appear. Interestingly no differences between TCC patients and controls could be found.These findings do not support the assumption of an exaggerated activation of the SNS as a discriminatory factor for TTC. Since especially the nocebo intervention revealed negative subjective and objective effects, our results underscore the urgent need to consider carefully the impact of verbal suggestions in the interaction with cardiac patients in daily clinical routine. This study is registered at the Deutsches Register Klinischer Studien (DRKS00009296).

Effects of smoking status, history and intensity on heart rate variability in the general population: The CHRIS study.

BACKGROUND: Heart rate variability (HRV) reflects the autonomous nervous system modulation on heart rate and is associated with several pathologies, including cardiac mortality. While mechanistic studies show that smoking is associated with lower HRV, population-based studies present conflicting results. METHODS: We assessed the mutual effects of active smoking status, cumulative smoking history, and current smoking intensity, on HRV among 4751 adults from the Cooperative Health Research In South Tyrol (CHRIS) study. The HRV metrics standard deviation of normal-to-normal (NN) inter-beat intervals (SDNN), square root of the mean squared differences of consecutive NN intervals (RMSSD), total power (TP), low (LF) and high frequency (HF) power, and their ratio (LF/HF), were derived from 20-minute electrocardiograms. Smoking status, pack-years (PY), and tobacco grams/day from standardized questionnaires were the main exposures. We fitted linear mixed models to account for relatedness, non-linearity, and moderating effects, and including fractional polynomials. RESULTS: Past smokers had higher HRV levels than never smokers, independently of PY. The association of HRV with current smoking became apparent when accounting for the interaction between smoking status and PY. In current smokers, but not in past smokers, we observed HRV reductions between 2.0% (SDNN) and 4.9% (TP) every 5 PY increase. Furthermore, current smokers were characterized by dose-response reductions of 9.8% (SDNN), 8.9% (RMSSD), 20.1% (TP), 17.7% (LF), and 19.1% (HF), respectively, every 10 grams/day of smoked tobacco, independently of common cardiometabolic conditions and HRV-modifying drugs. The LF/HF ratio was not associated with smoking status, history, or intensity. CONCLUSIONS: Smoking cessation was associated with higher HRV levels. In current smokers, heavier smoking intensity appears gradually detrimental on HRV, corroborating previous evidence. By affecting both the sympathetic and parasympathetic nervous system indexes, but not the LF/HF balance, smoking intensity seems to exert a systemic dysautonomic effect.

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.

Polyscore of Non-invasive Cardiac Risk Factors.

Non-invasive risk stratification of cardiac patients has been the subject of numerous studies. Most of these investigations either researched unique risk predictors or compared the predictive power of different predictors. Fewer studies suggested a combination of a small number of non-invasive indices to increase the accuracy of high-risk group selection. To advance non-invasive risk assessment of cardiac patients, we propose a combination score (termed the Polyscore) of seven different cardiac risk stratifiers that predominantly quantify autonomic cardiovascular control and regulation, namely the slope of heart rate turbulence, deceleration capacity of heart rate, non-invasively assessed baroreflex sensitivity, resting respiration frequency, expiration triggered sinus arrhythmia, post-ectopic potentiation of systolic blood pressure, and frequency of supraventricular and ventricular ectopic beats. These risk stratification tests have previously been researched and their dichotomies defining abnormal results have been derived from previous reports. The Polyscore combination was defined as the number of positive tests among these seven risk predictors, giving a numerical scale which ranges from 0 (all tests normal) to 7 (all tests abnormal). The Polyscore was tested in a population of 941 contemporarily treated survivors of acute myocardial infarction (median age 61 years, 182 females) of whom 72 (7.65%) died during a 5-year follow-up. In these patients, all the risk predictors combined in the Polyscore were assessed during in-hospital 30-min simultaneous non-invasive recordings of high-frequency orthogonal electrocardiogram, continuous blood pressure and respiration. Compared to Polyscore 0 stratum, the hazard ratios of mortality during follow-up increased almost exponentially in strata 1 through 7 (vs. stratus 0, the hazard ratios were 1.37, 1.96, 7.03, 15.0, 35.7, 48.2, and 114, in strata 1 to 7, respectively; p < 0.0001). This allowed selecting low-risk (Polyscore ≤ 2), intermediate risk (Polyscore 3 or 4) and high-risk (Polyscore ≥ 5) sub-groups of the population that differed greatly in the Kaplan-Meier probabilities of mortality during follow-up. Since the Polyscore was derived from recordings of only 30-min duration, it can be reasonably applied in different clinical situations including population-wide screening. We can therefore conclude that the Polyscore is a reasonable method for cardiac risk stratification that is ready for prospective validation in future independent studies.

Risk Prediction After Myocardial Infarction by Cyclic Variation of Heart Rate, a Surrogate of Sleep-Disordered Breathing Assessed From Holter ECGs.

AIMS: Sleep-disordered breathing (SDB) is common among cardiac patients, but its role as an independent risk predictor after myocardial infarction (MI) is unclear. SDB causes cyclic variation of heart rate (CVHR). The aim of this study was to score Holter ECGs of a large cohort of MI survivors for SDB-related CVHR to investigate its value for mortality prediction. METHODS: A total of 1590 survivors of acute MI in sinus rhythm were prospectively enrolled and followed for 5-year all-cause mortality. Heart rate (HR) tachograms were generated from nocturnal (00:00-06.00 am) segments of Holter ECGs, and the minutes with CVHR were quantified by a previously developed algorithm. According to a pre-specified cutpoint, SDB was assumed if CVHR was present during ≥72 min. RESULTS: Seventy-seven patients (4.8%) had flat HR tachograms which prohibited analysis for SDB. Of the remaining 1513 patients, 584 (38.6%) were classified as having SDB. Mortality rates in groups stratified according to ECG-derived SDB did not differ significantly. Taken as a continuous variable, low CVHR duration was associated with increased mortality.The mortality of patients with flat HR tachograms was significantly increased, even after adjustment for age, sex, LVEF, GRACE score and diabetes mellitus. Mortality prediction by a flat HR tachogram was also independent of heart rate variability (HRV), heart rate turbulence (HRT), and deceleration capacity (DC). CONCLUSION: In Holter ECG recordings of survivors of acute MI, signs suggestive of SDB were frequently present, but not associated with mortality. A flat nocturnal HR tachogram was a strong, independent predictor of 5-year all-cause mortality.

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.

Change in Left Ventricular Ejection Fraction Following First Myocardial Infarction and Outcome.

OBJECTIVES: This study hypothesizes that a lack of left ventricular ejection fraction (LVEF) recovery after myocardial infarction (MI) would be associated with a poor outcome. BACKGROUND: A reduced LVEF early after MI identifies patients at risk of adverse outcomes. Whether the change in LVEF in the weeks to months following MI provides additional information on prognosis is less certain. METHODS: Change in LVEF between the early (2 to 7 days) and later (2 to 12 weeks) post-MI periods in patients with a first MI was assessed in 3 independent cohorts (REFINE [Risk Estimation Following Infarction Noninvasive Evaluation]; CARISMA [Cardiac Arrhythmia and Risk Stratification after Myocardial Infarction]; ISAR [Improved Stratification of Autonomy Regulation]). Patients were categorized as having no recovery (Δ ≤0%), a modest increase (Δ 1% to 9%), or a large increase (Δ ≥10%) in LVEF. The relationship between change in LVEF and risk of sudden cardiac arrest (SCA) and all-cause mortality were assessed in Cox multivariable models. RESULTS: In REFINE, patients with no LVEF recovery had a higher risk of sudden cardiac arrest (hazard ratio: 5.8; 95% confidence interval: 2.1 to 16.6; p = 0.001) and death (hazard ratio: 3.9; 95% confidence interval: 1.5 to 10.1; p < 0.001), independent of revascularization, baseline LVEF, and medical therapy compared with patients with recovery. Similar findings were observed in the other cohorts. LVEF reassessments beyond 6 weeks post-MI were more predictive of outcome than were earlier reassessments. CONCLUSIONS: The degree of LVEF recovery after a first MI provides important prognostic information. Patients with no recovery in LVEF after MI are at high risk of sudden cardiac arrest events and death.

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.

Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers. METHODS AND RESULTS: Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca2+ dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca2+-dependent inactivation (CDI) of ICaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. CONCLUSION: The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to ß-adrenergic stimulation. Impaired CDI of ICaL underlined the electrical abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.

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.