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Aims The development of acute heart failure (AHF) is a critical decision point in the natural history of the disease and carries a dismal prognosis. The lack of appropriate risk-stratification tools at hospital discharge of AHF patients significantly limits clinical ability to precisely tailor patient-specific therapeutic regimen at this pivotal juncture. Machine learning-based strategies may improve risk stratification by incorporating analysis of high-dimensional patient data with multiple covariates and novel prediction methodologies. In the current study, we aimed at evaluating the drivers for success in prediction models and establishing an institute-tailored artificial Intelligence-based prediction model for real-time decision support. Methods and results We used a cohort of all 10 868 patients AHF patients admitted to a tertiary hospital during a 12 years period. A total of 372 covariates were collected from admission to the end of the hospitalization. We assessed model performance across two axes: (i) type of prediction method and (ii) type and number of covariates. The primary outcome was 1-year survival from hospital discharge. For the model-type axis, we experimented with seven different methods: logistic regression (LR) with either L1 or L2 regularization, random forest (RF), Cox proportional hazards model (Cox), extreme gradient boosting (XGBoost), a deep neural-net (NeuralNet) and an ensemble classifier of all the above methods. We were able to achieve an area under receiver operator curve (AUROC) prediction accuracy of more than 80% with most prediction models including L1/L2-LR (80.4%/80.3%), Cox (80.2%), XGBoost (80.5%), NeuralNet (80.4%). RF was inferior to other methods (78.8%), and the ensemble model was slightly superior (81.2%). The number of covariates was a significant modifier (P < 0.001) of prediction success, the use of multiplex-covariates preformed significantly better (AUROC 80.4% for L1-LR) compared with a set of known clinical covariates (AUROC 77.8%). Demographics followed by lab-tests and administrative data resulted in the largest gain in model performance. Conclusions The choice of the predictive modelling method is secondary to the multiplicity and type of covariates for predicting AHF prognosis. The application of a structured data pre-processing combined with the use of multiple-covariates results in an accurate, institute-tailored risk prediction in AHF

Cardio-oncology in a dish: cardiovascular tissueengineering strategies for modeling cancer therapyrelated cardiac dysfunction.Over the recent years, there was a robust proliferation of oncological therapies leading to asignificant reduction in cancer related death. Unfortunately, there was a parallel increase in thenumber of patients developing cardiotoxicity & heart failure making it the main cause of deathamong cancer survivors. Current strategies for predicting cardio-toxic side effects are limitedand robust pre-clinical, personalized platforms for assessing both harmful drugs and protectiveagents are lacking.In the current study by using human pluripotent stem cells technology, we developed a uniquehuman-engineered cardiac tissue (ECT) platform generated from cardiomyocytes, endothelialcells and authentic cardiac fibroblasts derived from the epicardial cell layer. Using this platform,we are examined the ability of the model to recapitulate the cardio-toxic effects of Doxorubicinand the cardio protective capabilities of Dexrazoxane and SGLT2i; a novel agent, that wasrecently shown to prevent heart failure in patients with diabetes and high risk forcardiovascular diseases.p Results: Paired, long-term, consecutive force evaluations of the ECTs demonstrated the cardio-toxiceffect of Doxorubicin resulting in a lower total active force for each preload compared toBaseline control. Moreover, concomitant exposure to Doxorubicin and the cardio-protectiveagents (Dexrazoxane and SGLT2 inhibitor ‘Empagliflozin’) resulted in considerable recovery ofthe active force generation compared to control. Conclusion: This report is an initial proof of concept demonstrating the ability of ECTs to serve as a modelfor functional evaluation induced by cardio toxic drugs as well as the effect of cardio-protective agents. High-throughput based on this platform may be used for identifying novel cardio-protective agents as well as for evaluating patient-specific cardio-toxic effects.

Patients with heart failure (HF) are at an increased risk of hospital admissions. The aim of this report is to describe the feasibility, safety and accuracy of a novel wireless left atrial pressure (LAP) monitoring system in patients with HF.The V-LAP Left Atrium Monitoring systEm for Patients With Chronic sysTOlicDiastolic Congestive heart Failure (VECTOR-HF) study is a prospective, multicenter, single-arm, open-label, first-in human clinical trial to assess the safety, performance and usability of the V-LAP system (Vectorious Medical Technologies) in patients with New York Heart Association class III HF. The device was implanted in the interatrial septum via a percutaneous, trans-septal approach guided by fluoroscopy and echocardiography. Primary endpoints included the successful deployment of the implant, the ability to perform initial pressure measurements and safety outcomes.To date, 24 patients have received implants of the LAP-monitoring device. No device-related complications have occurred. LAP was reported accurately, agreeing well with wedge pressure at 3 months (Lin concordance correlation coefficient = 0.850). After 6 months, New York Heart Association class improved in 40% of the patients (95% CI = 16.4%-63.5%), while the 6-minute walk test distance had not changed significantly (313.9 ± 144.9 vs 232.5 ± 129.9 meters; P = 0.076).The V-LAP left atrium monitoring system appears to be safe and accurate.

Immune responses generally decline with age. However, the dynamics of this process at the individual level have not been characterized, hindering quantification of an individual’s immune age. Here, we use multiple ‘omics’ technologies to capture population- and individual-level changes in the human immune system of 135 healthy adult individuals of different ages sampled longitudinally over a nine-year period. We observed high inter-individual variability in the rates of change of cellular frequencies that was dictated by their baseline values, allowing identification of steady-state levels toward which a cell subset converged and the ordered convergence of multiple cell subsets toward an older adult homeostasis. These data form a highdimensional trajectory of immune aging (IMM-AGE) that describes a person’s immune status better than chronological age. We show that the IMM-AGE score predicted all-cause mortality beyond well-established risk factors in the Framingham Heart Study, establishing its potential use in clinics for identification of patients at risk. REPORTING SUMMARY. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Problem The COVID-19 pandemic has challenged health care systems in an unprecedented way by imposing new demands on health care resources and scientific knowledge. There has also been an exceedingly fast accumulation of new information on this novel virus. As the traditional peer-review process takes time, there is currently a significant gap between the ability to generate new data and the ability to critically evaluate it. This problem of an excess of mixed-quality data, or infodemic, is echoing throughout the scientific community. Approach The authors aimed to help their colleagues at the Rambam Medical Center, Haifa, Israel, manage the COVID-19 infodemic with a methodologic solution: establishing an in-house mechanism for continuous literature review and knowledge distribution (March-April 2020). Their methodology included the following building blocks: a dedicated literature review team, artificial intelligence-based research algorithms, brief written updates in a graphical format, large-scale webinars and online meetings, and a feedback loop. Outcomes During the first month (April 2020), the project produced 21 graphical updates. After consideration of feedback from colleagues and final editing, 13 graphical updates were uploaded to the center's website; of these, 31% addressed the clinical presentation of the disease and 38% referred to specific treatments. This methodology as well as the graphical updates it generated were adopted by the Israeli Ministry of Health and distributed in a hospital preparation kit. Next steps The authors believe they have established a novel methodology that can assist in the battle against COVID-19 by making high-quality scientific data more accessible to clinicians. In the future, they expect this methodology to create a favorable uniform standard for evidence-guided health care during infodemics. Further evolution of the methodology may include evaluation of its long-term sustainability and impact on the day-to-day clinical practice and self-confidence of clinicians who treat COVID-19 patients.

Objective To analyze the correlation between COVID-19 vaccination percentage and socioeconomic status (SES). Methods A nationwide ecologic study based on open-sourced, anonymized, aggregated data provided by the Israel Ministry of Health. The correlations between municipal SES, vaccination percentage, and active COVID-19 cases during the vaccination campaign were analyzed by using weighted Pearson correlations. To assess the adequacy of first dose vaccination rollout relative to the municipality COVID-19 disease burden, a metric termed the vaccination need ratio was devised by dividing the total number of active cases (per 10,000 people) by the vaccination percentage of the population over 60 in each municipality, and its correlation with the SES was examined. Results 23 days after initiation of the vaccination campaign, 760,916 (56.8%) individuals over the age of 60 were vaccinated in Israel with the first dose of the BNT162b2 COVID-19 vaccine. A negative correlation was found between the COVID-19 active case burden and the vaccination percentage of the study population in each municipality (r=-0.47, 95% confidence interval [-0.59 to -0.30]). The vaccination percentage significantly correlated with the municipal SES (r=0.83, 95% confidence interval [0.79 to 0.87]). This finding persisted but was attenuated over a five-week period. A negative correlation between the vaccination need ratio and municipal SES (r=-0.80, 95% confidence interval [-0.88 to -0.66]) was found. Conclusion Lower COVID-19 vaccination percentage was associated with lower SES and high active disease burden. Vaccination efforts should focus on areas with lower SES and high disease burden to assure equality of vaccine allocation and potentially provide a more diligent disease mitigation.

COVID-19 vaccination acceptance has a key role in mitigating the pandemic. Concern has been raised that vaccination rates will be limited in demographically defined areas of lower income. Israel’s rapid vaccination campaign may allow to assess these assumptions in real-world and to devise tools for effectively focusing the vaccination efforts. We analyzed the correlation between COVID-19 vaccination rates, socioeconomic status (SES) and active COVID-19 disease burden. We carried out a nationwide study, based on data provided by Ministry of Health of COVID-19 vaccination rates in all municipalities in Israel up to January 12th, 2021. Municipal Vaccination rates of population older than 60 significantly correlated with the socioeconomic status (r=0.83, 95% confidence interval [0.79 to 0.87]). Finally, we established a novel metric for focusing the vaccination efforts based on % vaccinations and active disease burden. In Israel, a case-model country for COVD-19 vaccinations, vaccination rates were strongly correlated with SES. The study findings demonstrate the need to directly target vaccination acceptance to socio-economically disadvantaged populations and suggest potential tools for policymakers to focus their efforts.

Introduction The development of acute heart failure (AHF) is a critical decision-point in the natural history of heart failure and carries a dismal prognosis. The lack of appropriate risk-stratification tools for AHF patients limits physician ability to precisely tailor patient-specific therapy regimen at this important juncture. Machine learning (ML) based strategies may enhance risk stratification by incorporating analysis of high-dimensional patient data with multiple covariates and novel prediction methodologies. In this study, we aimed at evaluating the drivers for success in prediction models and establishing an institute-tailored ML-based prediction model for real-time decision support. Methods We used a cohort of all AHF patients admitted during a 12 years period including 10,868 patients. A total of 372 covariates were collected from admission to the end of the hospitalization (demographics, lab-tests, medical therapies, echocardiographic and administrative data). Data preprocessing included features cleaning, train-test split, imputation and normalization. We assessed model performance across two axes (1) type of prediction method and (2) type and number of covariates. The primary outcome was one-year survival from hospital discharge. For the model-type axis we experimented with seven different methods: Logistic Regression (LR), Random Forest (RF), Cox model (Cox), XGBoost, a deep neural-network (NeuralNet) and an ensembled model. Results Data pre-processing methodology combined with multiple-covariates achieved an out-ofsample AUROC prediction accuracy of more than 80% with almost all prediction models: L1/L2-LR (80.4%/80.3%); Cox (80.1%); XGBoost (80.7%); NeuralNet (80.5%). The number of covariates was a significant modifier of prediction success (p Conclusions The choice of the predictive modeling method is secondary to the multiplicity and type of covariates for predicting AHF prognosis. The application of a structured data pre-processing combined with the use of multiple-covariates results in an accurate, institute-tailored, risk prediction in AHF. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Yad Hanadiv

The coronavirus disease 2019 (COVID-19) pandemic has remarkably challenged health care organizations and societies. A key strategy for confronting the disease implications on individuals and communities was based on harnessing multidisciplinary efforts to develop technologies for mitigating the disease spread and its deleterious clinical implications. One of the main challenging characteristics of COVID-19 is the provision of medical care to patients with a highly infective disease mandating the use of isolation measures. Such care is complicated by the need for complex critical care, dynamic treatment guidelines, and a vague knowledge regarding the disease’s pathophysiology. A second key component of this challenge was the overwhelming surge in patient burden and the relative lack of trained staff and medical equipment which required rapid re-organization of large systems and augmenting health care efficiencies to unprecedented levels. In contrast to the risk management strategies employed to mitigate other serious threats and the billions of dollars that are invested in reducing these risks annually by governments around the world, no such preparation has been shown to be of effect during the current COVID-19 pandemic. Unmet needs were identified within the newly opened COVID-19 departments together with the urgent need for reliable information for effective decision-making at the state level. This review article describes the early research and development response in Israel under the scope of in-hospital patient care, such as non-contact sensing of patients’ vital signs, and how it could potentially be weaved into a practical big picture at the hospital or national level using a strategic management system. At this stage, some of the described technologies are still in developmental or clinical evidence generation phases with respect to COVID-19 settings. While waiting for future publications describing the results of the ongoing evidence generation efforts, one should be aware of this trend as these emerging tools have the potential to further benefit patients as well as caregivers and health care systems beyond the scope of the current pandemic as well as confronting future surges in the number of cases.

The use of opioids in acute pulmonary oedema is considered standard therapy by many physicians. The immediate relieving effect of morphine on the key symptomatic discomfort associated with acute heart failure, dyspnoea, facilitated the categorisation of morphine as a beneficial treatment in this setting. During the last decade, several retrospective studies raised concerns regarding the safety and efficacy of morphine in the setting of acute heart failure. In this article, the physiological effects of morphine on the cardiovascular and respiratory systems are summarised, as well as the potential clinical benefits and risks associated with morphine therapy. Finally, the reported clinical outcomes and adverse event profiles from recent observational studies are discussed, as well as future perspectives and potential alternatives to morphine in the setting of acute heart failure.

BackgroundCOVID-19 outbreak poses an unprecedented challenge for societies, healthcare organizations and economies. In the present analysis we coupled climate data with COVID-19 spread rates worldwide, and in a single country (USA).MethodsData of confirmed COVID-19 cases was derived from the COVID-19 Global Cases by the CSSE at Johns Hopkins University up to March 19, 2020. We assessed disease spread by two measures: replication rate (RR), the slope of the logarithmic curve of confirmed cases, and the rate of spread (RoS), the slope of the linear regression of the logarithmic curve.ResultsBased on predefined criteria, the mean COVID-19 RR was significantly lower in warm climate countries (0.12±0.02) compared with cold countries (0.24±0.01), (PConclusionsOur analysis suggests a plausible negative correlation between warmer climate and COVID-19 spread rate as defined by RR and RoS worldwide. This initial correlation should be interpreted cautiously and be further validated over time, the pandemic is at different stages in various countries as well as in regions within these countries. As such, some associations may be more affected by local transmission patterns rather than by climate. Importantly, we provide an online surveillance dashboard (https://covid19.net.technion.ac.il/) to further assess the association between climate parameters and outbreak dynamics worldwide as time goes by.Research in contextEvidence before this studyThe coronavirus, COVID-19 pandemic caused by the novel SARS-CoV 2, challenges healthcare organizations and economies worldwide. There have been previous reports describing the association between seasonal climactic variance and SARS-CoV 1 as well as the MERS infections, but the association with SARS-CoV 2 and climate has not been described extensively.Added value of this studyOur analysis demonstrates a plausible negative correlation between warmer climate and COVID-19 spread rate as defined by RR and RoS worldwide in all countries with local transmission as of March 9, 2020. This initial correlation should be interpreted cautiously and be further validated over time. Importantly, we provide an online surveillance dashboard available at (https://covid19.net.technion.ac.il/) for further dynamic tracking of climate effect on COVID-19 disease spread rate worldwide and on intra-country analysis between USA states.Implications of all the available evidenceOur findings of decreased replication and spread rates of COVID-19 in warm climates may suggest that the inevitable seasonal variance will alter the dynamic of the disease spread in both hemispheres in the coming months. However, we warrant a cautious interpretation of these findings given the fact that we are in the initial steps of this outbreak in many “warm” climate countries, the high variance of the data and the dynamic changes in the disease surveillance and the lack of correlation based on the limited data in the US. We hope that the online tool coupling COVID-19 data with climate data will assist in tracking the disease and tailoring the needed measures to contain it.

In this issue of Cell Stem Cell, Mills et al. (2019) use multidimensional functional screening to identify pro-proliferative compounds in cell-cycle-arrested human cardiac organoids. Using this model, the authors identify two hit compounds that restart cardiomyocyte proliferation by synergistically activating the mevalonate pathway and cell-cycle-related pathways.

Background Morphine has been a pivotal therapy in acute heart failure (AHF) for more than a century. The evidence for morphine therapy in AHF remains controversial. This study sought to assess the therapeutic effect of morphine on patients with AHF. Methods The study used a cohort of 13,788 patients admitted with a primary diagnosis of AHF. Propensity-score-matching was generated using 26 clinical variables. Primary endpoints included in-hospital mortality and invasive mechanical ventilation. Secondary endpoints included non-invasive ventilation, need for inotropes and acute kidney injury (AKI). Results 761 (5.5%) patients were treated with morphine in the first day following hospital admission. Propensity score matching yielded 672 patient pairs. The incidence of invasive ventilation was higher in the morphine-treated patients (7.4%) than in matched patients in the no-morphine cohort (3.6%), OR 2.13 (95% CI 1.32–3.57, P = 0.007). In-hospital mortality was also higher in the morphine group (17.4%) than in the matched no-morphine group (13.4%), OR 1.43 (95% CI 1.05 to 1.98, P = 0.024). For both the endpoint of invasive ventilation ( P trend = 0.005) and mortality ( P trend = 0.004), there was a significant linear dose-response relationship for the adverse effect of morphine. Morphine was associated with a significant increase in all secondary outcomes: Non-invasive ventilation (OR 2.78, 95% CI 1.95–3.96); Inotrope use (OR 3.50, 95% CI 2.10–5.82) and AKI (OR 1.81, 95% CI 1.39–2.36). A landmark analysis demonstrated no difference in post-discharge survival between cohorts. Conclusions Morphine administration is associated with significant dose-dependent risk for in-hospital mortality and need for mechanical ventilation.

Background Acute kidney injury ( AKI ) following primary percutaneous coronary intervention ( pPCI ) is frequently interpreted as contrast‐induced AKI but may result from other insults. We aimed to determine the causal association of contrast material exposure and the incidence of AKI following p PCI using a control group of propensity score–matched patients with ST‐segment–elevation myocardial infarction who were not exposed to contrast material. Methods and Results We studied 2025 patients with ST‐segment–elevation myocardial infarction who underwent pPCI and 1025 patients receiving fibrinolysis or no reperfusion who were not exposed to contrast material during the first 72 hours of hospital stay (control group). AKI was defined as creatinine of ≥0.5 mg/dL or >25% rise within 72 hours. AKI rates were similar in the pPCI and control groups (10.3% versus 12.1%, respectively; P =0.38). Propensity score matching resulted in 931 matched pairs with PCI and no PCI, with balanced baseline covariates (standardized difference AKI rates were not significantly different with and without PCI (8.6% versus 10.9%, P =0.12). In the pPCI cohort, independent predictors of AKI included age ≥70 years, insulin‐treated diabetes mellitus, diuretic therapy, anterior infarction, baseline estimated glomerular filtration rate, and variables related to the presence of pump failure (higher Killip class, intra‐aortic balloon pump use) and reduced left ventricular ejection fraction but not contrast material dose. A risk score based on the PCI cohort had similar discriminatory capacity for AKI in the control group (C statistic 0.81±0.02 and 0.78±0.02, respectively; P =0.26). Conclusions The development of AKI in patients with ST‐segment–elevation myocardial infarction undergoing pPCI is mainly related to older age, baseline estimated glomerular filtration rate, heart failure, and hemodynamic instability. Risk for AKI is similar among ST‐segment–elevation myocardial infarction patients with and without contrast material exposure.

Objectives The goal of this study was to establish a patient-specific human-induced pluripotent stem cells (hiPSCs) model of catecholaminergic polymorphic ventricular tachycardia (CPVT). Background CPVT is a familial arrhythmogenic syndrome characterized by abnormal calcium (Ca2+) handling, ventricular arrhythmias, and sudden cardiac death. Methods Dermal fibroblasts were obtained from a CPVT patient due to the M4109R heterozygous point RYR2 mutation and reprogrammed to generate the CPVT-hiPSCs. The patient-specific hiPSCs were coaxed to differentiate into the cardiac lineage and compared with healthy control hiPSCs-derived cardiomyocytes (hiPSCs-CMs). Results Intracellular electrophysiological recordings demonstrated the development of delayed afterdepolarizations in 69% of the CPVT-hiPSCs-CMs compared with 11% in healthy control cardiomyocytes. Adrenergic stimulation by isoproterenol (1 μM) or forskolin (5 μM) increased the frequency and magnitude of afterdepolarizations and also led to development of triggered activity in the CPVT-hiPSCs-CMs. In contrast, flecainide (10 μM) and thapsigargin (10 μM) eliminated all afterdepolarizations in these cells. The latter finding suggests an important role for internal Ca2+ stores in the pathogenesis of delayed afterdepolarizations. Laser-confocal Ca2+ imaging revealed significant whole-cell [Ca2+] transient irregularities (frequent local and large-storage Ca2+-release events, broad and double-humped transients, and triggered activity) in the CPVT cardiomyocytes that worsened with adrenergic stimulation and Ca2+ overload and improved with beta-blockers. Store-overload–induced Ca2+ release was also identified in the hiPSCs-CMs and the threshold for such events was significantly reduced in the CPVT cells. Conclusions This study highlights the potential of hiPSCs for studying inherited arrhythmogenic syndromes, in general, and CPVT specifically. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care, and aid in the development of new therapies.

Myocardial cell-replacement strategies are hampered by limited sources for human cardiomyocytes and by significant cell loss following transplantation. We tested the hypothesis that a combined delivery of cardiomyocytes with an in-situ polymerizable hydrogel into a post-MI rat heart will result in better functional outcomes than each intervention alone. A photopolymerizable, biodegradable, PEGylated-fibrinogen (PF) hydrogel matrix was used as the carrier for the cardiomyocytes [neonatal rat ventricular cardiomyocytes (NRVCMs) or human embryonic stem cell-derived cardiomyocytes (hESC-CMs)]. Infarcted rat hearts (LAD ligation) were randomized to injection of saline, NRVCMs, biopolymer, or combined biopolymer-cell delivery. Echocardiography revealed typical post-infarction remodeling after 30 days in the saline-injected control group [deterioration of fractional shortening (FS) by 31.0 ± 3.6%]. Injection of NRVCMs or PF alone significantly (p

Background— The ability to derive human induced pluripotent stem (hiPS) cell lines by reprogramming of adult fibroblasts with a set of transcription factors offers unique opportunities for basic and translational cardiovascular research. In the present study, we aimed to characterize the cardiomyocyte differentiation potential of hiPS cells and to study the molecular, structural, and functional properties of the generated hiPS-derived cardiomyocytes. Methods and Results— Cardiomyocyte differentiation of the hiPS cells was induced with the embryoid body differentiation system. Gene expression studies demonstrated that the cardiomyocyte differentiation process of the hiPS cells was characterized by an initial increase in mesoderm and cardiomesoderm markers, followed by expression of cardiac-specific transcription factors and finally by cardiac-specific structural genes. Cells in the contracting embryoid bodies were stained positively for cardiac troponin-I, sarcomeric α-actinin, and connexin-43. Reverse-transcription polymerase chain reaction studies demonstrated the expression of cardiac-specific sarcomeric proteins and ion channels. Multielectrode array recordings established the development of a functional syncytium with stable pacemaker activity and action potential propagation. Positive and negative chronotropic responses were induced by application of isoproterenol and carbamylcholine, respectively. Administration of quinidine, E4031 ( I Kr blocker), and chromanol 293B ( I Ks blocker) significantly affected repolarization, as manifested by prolongation of the local field potential duration. Conclusions— hiPS cells can differentiate into myocytes with cardiac-specific molecular, structural, and functional properties. These results, coupled with the potential of this technology to generate patient-specific hiPS lines, hold great promise for the development of in vitro models of cardiac genetic disorders, for drug discovery and testing, and for the emerging field of cardiovascular regenerative medicine.

Pro-arrhythmia (development of cardiac arrhythmias as a pharmacological side effect) has become the single most common cause of the withdrawal or restrictions of previously marketed drugs. The development of new medications, free from these side effects, is hampered by the lack of an in vitro assay for human cardiac tissue. We hypothesized that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) assessed with a combination of single cell electrophysiology and microelectrode array (MEA) mapping can serve as a novel model for electrophysiological drug screening. Current-clamp studies revealed that E-4031 and Sotalol (IKr blockers) significantly increased hESC-CM's action potential duration and also induced after-depolarizations (the in vitro correlates of increased arrhythmogenic potential). Multicellular aggregates of hESC-CMs were then analyzed with the MEA technique. Application of class I (Quinidine, Procaineamide) and class III (Sotalol) antiarrhythmic agents, E-4031, and Cisapride (a noncardiogenic agent known to lengthen QT) resulted in dose-dependent prolongation of the corrected field potential duration (cFPD). We next utilized the MEA technique to also assess pharmacological effects on conduction. Activation maps demonstrated significant conduction slowing following administration of Na channel blockers (Quinidine and Propafenone) and of the gap junction blocker (1-heptanol). While most attention has been focused on the prospects of using hESC-derived cardiomyocytes for regenerative medicine, this study highlights the possible utilization of these unique cells also for cardiac electrophysiological studies, drug screening, and target validation.

Myocardial cell replacement strategies are emerging as novel therapeutic paradigms for heart failure but are hampered by the paucity of sources for human cardiomyocytes. Human embryonic stem cells (hESC) are pluripotent stem cell lines derived from human blastocysts that can be propagated, in culture, in the undifferentiated state under special conditions and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. The current review describes the derivation and properties of the hESC lines and the different cardiomyocyte differentiation system established so far using these cells. Data regarding the structural, molecular, and functional properties of the hESC-derived cardiomyocytes is provided as well as description of the methods used to achieve cardiomyocyte enrichment and purification in this system. The possible applications of this unique differentiation system in several cardiovascular research and applied areas are discussed. Specific emphasis is put on the descriptions of the efforts performed to date to assess the feasibility of this emerging technology in the fields of cardiac cell replacement therapy and tissue engineering. Finally, the obstacles remaining on the road to clinical translation are described as well as the steps required to fully harness the potential of this new technology.

Objectives We evaluated the ability of human embryonic stem cells (hESCs) and their cardiomyocyte derivatives (hESC-CMs) to engraft and improve myocardial performance in the rat chronic infarction model. Background Cell therapy is emerging as a novel therapy for myocardial repair but is hampered by the lack of sources for human cardiomyocytes. Methods Immunosuppressed healthy and infarcted (7 to 10 days after coronary ligation) rat hearts were randomized to injection of undifferentiated hESCs, hESC-CMs, noncardiomyocyte hESC derivatives, or saline. Detailed histological analysis and sequential echocardiography were used to determine the structural and functional consequences of cell grafting. Results Transplantation of undifferentiated hESCs resulted in the formation of teratoma-like structures. This phenomenon was prevented by grafting of ex vivo pre-differentiated hESC-CMs. The grafted cardiomyocytes survived, proliferated, matured, aligned, and formed gap junctions with host cardiac tissue. Functionally, animals injected with saline or nonmyocyte hESC derivatives demonstrated significant left ventricular (LV) dilatation and functional deterioration, whereas grafting of hESC-CMs attenuated this remodeling process. Hence, post-injury baseline fractional shortening deteriorated by 50% (from 20 ± 2% to 10 ± 2%) and by 30% (20 ± 2% to 14 ± 2%) in the saline and nonmyocyte groups while improving by 22% (21 ± 2% to 25 ± 3%) in the hESC-CM group. Similarly, wall motion score index and LV diastolic dimensions were significantly lower in the hESC-CM animals. Conclusions Transplantation of hESC-CMs after extensive myocardial infarction in rats results in the formation of stable cardiomyocyte grafts, attenuation of the remodeling process, and functional benefit. These findings highlight the potential of hESCs for myocardial cell therapy strategies.

Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity95%, viability85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.

Transplantation of a tissue-engineered heart muscle represents a novel experimental therapeutic paradigm for myocardial diseases. However, this strategy has been hampered by the lack of sources for human cardiomyocytes and by the scarce vasculature in the ischemic area limiting the engraftment and survival of the transplanted muscle. Beyond the necessity of endothelial capillaries for the delivery of oxygen and nutrients to the grafted muscle tissue, interactions between endothelial and cardiomyocyte cells may also play a key role in promoting cell survival and proliferation. In the present study, we describe the formation of synchronously contracting engineered human cardiac tissue derived from human embryonic stem cells containing endothelial vessel networks. The 3D muscle consisted of cardiomyocytes, endothelial cells (ECs), and embryonic fibroblasts (EmFs). The formed vessels were further stabilized by the presence of mural cells originating from the EmFs. The presence of EmFs decreased EC death and increased EC proliferation. Moreover, the presence of endothelial capillaries augmented cardiomyocyte proliferation and did not hamper cardiomyocyte orientation and alignment. Immunostaining, ultrastructural analysis (using transmission electron microscopy), RT-PCR, pharmacological, and confocal laser calcium imaging studies demonstrated the presence of cardiac-specific molecular, ultrastructural, and functional properties of the generated tissue constructs with synchronous activity mediated by action potential propagation through gap junctions. In summary, this is the first report of the construction of 3D vascularized human cardiac tissue that may have unique applications for studies of cardiac development, function, and tissue replacement therapy.

The adult human heart has limited regenerative capacity and therefore any significant myocardial damage results in progressive deterioration in its function. Cell-based cardiac repair represents an exciting approach for rebuilding the injured heart. Consequentially, during the last decade, significant efforts have been made to assess the potential role of skeletal myoblasts, foetal cardiomyocytes, bone marrow stem cells, endothelial progenitors, resident cardiac progenitor cells, and embryonic stem cells (ESC) to restore the myocardial performance. Clinical translation of these basic studies has progressed rapidly with several Phase I and II clinical trials, utilizing autologous bone marrow cells and skeletal myoblasts, being well underway. In this review, we will describe these basic and clinical efforts. A specific emphasis will be placed on the potential role of human ESC for myocardial repair. These unique pluripotent cell lines can be propagated in the undifferentiated state in culture and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. The potential applications of this unique differentiating system in the emerging field of cardiovascular regenerative medicine will be discussed with special emphasis on the steps required to fully harness their unique potential.

The recent establishment of the human embryonic stem cell lines and the demonstration of their ability to differentiate in vitro to cardiomyocytes brings a unique promise to both basic and clinical research. The present report describes the characteristics of the human embryonic stem cell lines and focuses on the structural and functional properties of their cardiomyocyte derivatives. In addition, the possible signals and cues involved in the commitment and early differentiation of cardiomyocytes will be discussed. Finally, the potential applications of this unique differentiating system in several research and clinical areas are discussed, with special emphasis on the steps required to fully harness their potential for myocardial regeneration strategies.

Background— Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder resulting from desmosomal protein mutations. ARVC is characterized pathologically by fibrofatty infiltration and clinically by arrhythmias and sudden cardiac death. We aimed to establish a patient-/disease-specific human induced pluripotent stem cell (hiPSC) model of ARVC. Methods and Results— Dermal fibroblasts were obtained from 2 patients with ARVC with plakophilin-2 ( PKP2 ) mutations, reprogrammed to generate hiPSCs, coaxed to differentiate into cardiomyocytes (CMs), and then compared with healthy control hiPSC-derived CMs (hiPSC-CMs). Real-time polymerase chain reaction showed a significant decrease in the expression of PKP2 in the ARVC-hiPSC-CMs. Immunostainings revealed reduced densities of PKP2, the associated desmosomal protein plakoglobin, and the gap-junction protein connexin-43. Electrophysiological assessment demonstrated prolonged field potential rise time in the ARVC-hiPSC-CMs. Transmission electron microscopy identified widened and distorted desmosomes in the ARVC-hiPSC-CMs. Clusters of lipid droplets were identified in the ARVC-CMs that displayed the more severe desmosomal pathology. This finding was associated with upregulation of the proadipogenic transcription factor peroxisome proliferator-activated receptor-γ. Exposure of the cells to apidogenic stimuli augmented desmosomal distortion and lipid accumulation. The latter phenomenon was prevented by application of a specific inhibitor of glycogen synthase kinase 3β (6-bromoindirubin-3'-oxime). Conclusions— This study highlights the unique potential of the hiPSC technology for modeling inherited cardiac disorders in general and ARVC specifically. The hiPSC-CMs were demonstrated to recapitulate the ARVC phenotype in the dish, provide mechanistic insights into early disease pathogenesis, and provide a unique platform for drug discovery and testing in this disorder.

A 79-year-old woman has known severe tricuspid regurgitation (TR) for 10 years. TR was due to a combination of left chest radiation (treatment of breast carcinoma 20 years ago) and permanent atrial fibrillation. There was no history of rheumatic heart disease, systemic diseases (e.g. carcinoid), or the use of medications causing valve dysfunction. She has mild dyspnoea on exertion but no right-sided heart failure symptoms. Transthoracic echocardiography revealed moderate dilatation of the right ventricle (RV) and …

Cell replacement strategies are promising interventions aiming to improve myocardial performance. Yet, the electrophysiological impact of these approaches has not been elucidated. We assessed the electrophysiological consequences of grafting of two candidate cell types, that is, skeletal myoblasts and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). The fluorescently labeled (DiO) candidate cells were grafted into the rat's left ventricular myocardium. Two weeks later, optical mapping was performed using the Langendorff-perfused rat heart preparation. Images were obtained with appropriate filters to delineate the heart's anatomy, to identify the DiO-labeled cells, and to associate this information with the voltage-mapping data (using the voltage-sensitive dye PGH-I). Histological examination revealed the lack of gap junctions between grafted skeletal myotubes and host cardiomyocytes. In contrast, positive Cx43 immunostaining was observed between donor and host cardiomyocytes in the hESC-CMs-transplanted hearts. Optical mapping demonstrated either normal conduction (four of six) or minimal conduction slowing (two of six) at the hESC-CMs engraftment sites. In contrast, marked slowing of conduction or conduction block was seen (seven of eight) at the myoblast transplantation sites. Ventricular arrhythmias could not be induced in the hESC-CM hearts following programmed electrical stimulation but were inducible in 50% of the myoblast-engrafted hearts. In summary, a unique method for assessment of the electrophysiological impact of myocardial cell therapy is presented. Our results demonstrate the ability of hESC-CMs to functionally integrate with host tissue. In contrast, transplantation of cells that do not form gap junctions (skeletal myoblats) led to localized conduction disturbances and to the generation of a proarrhythmogenic substrate.

A decade has passed since the initial derivation of human embryonic stem cells (hESC). The ensuing years have witnessed a significant progress in the development of methodologies allowing cell cultivation, differentiation, genetic manipulation, and in vivo transplantation. Specifically, the potential to derive human cardiomyocytes from the hESC lines, which can be used for several basic and applied cardiovascular research areas including in the emerging field of cardiac regenerative medicine, attracted significant attention from the scientific community. This resulted in the development of protocols for the cultivation of hESC and their successful differentiation toward the cardiomyocyte lineage fate. In this chapter, we will describe in detail methods related to the cultivation, genetic manipulation, selection, and in vivo transplantation of hESC-derived cardiomyocytes.

The ability to generate patient-specific human induced pluripotent stem cells (iPSCs) offers a new paradigm for modelling human disease and for individualizing drug testing. Congenital long QT syndrome (LQTS) is a familial arrhythmogenic syndrome characterized by abnormal ion channel function and sudden cardiac death. Here we report the development of a patient/disease-specific human iPSC line from a patient with type-2 LQTS (which is due to the A614V missense mutation in the KCNH2 gene). The generated iPSCs were coaxed to differentiate into the cardiac lineage. Detailed whole-cell patch-clamp and extracellular multielectrode recordings revealed significant prolongation of the action-potential duration in LQTS human iPSC-derived cardiomyocytes (the characteristic LQTS phenotype) when compared to healthy control cells. Voltage-clamp studies confirmed that this action-potential-duration prolongation stems from a significant reduction of the cardiac potassium current I(Kr). Importantly, LQTS-derived cells also showed marked arrhythmogenicity, characterized by early-after depolarizations and triggered arrhythmias. We then used the LQTS human iPSC-derived cardiac-tissue model to evaluate the potency of existing and novel pharmacological agents that may either aggravate (potassium-channel blockers) or ameliorate (calcium-channel blockers, K(ATP)-channel openers and late sodium-channel blockers) the disease phenotype. Our study illustrates the ability of human iPSC technology to model the abnormal functional phenotype of an inherited cardiac disorder and to identify potential new therapeutic agents. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care (personalized medicine), and aid in the development of new therapies.

A decade has passed since the initial derivation of human embryonic stem cells (hESC). The ensuing years have witnessed a significant progress in the development of methodologies allowing cell cultivation, differentiation, genetic manipulation, and in vivo transplantation. Specifically, the potential to derive human cardiomyocytes from the hESC lines, which can be used for several basic and applied cardiovascular research areas including in the emerging field of cardiac regenerative medicine, attracted significant attention from the scientific community. This resulted in the development of protocols for the cultivation of hESC and their successful differentiation toward the cardiomyocyte lineage fate. In this chapter, we will describe in detail methods related to the cultivation, genetic manipulation, selection, and in vivo transplantation of hESC-derived cardiomyocytes.

Myocardial regeneration strategies have been hampered by the lack of sources for human cardiomyocytes (CMs) and by the significant donor cell loss following transplantation. We assessed the ability of a three-dimensional tissue-engineered human vascularized cardiac muscle to engraft in the in vivo rat heart and to promote functional vascularization. Human embryonic stem cell-derived CMs alone or with human endothelial cells (human umbilical vein endothelial cells) and embryonic fibroblasts (triculture constructs) were seeded onto biodegradable porous scaffolds. The resulting tissue constructs were transplanted to the in vivo rat heart and formed cardiac tissue grafts. Immunostaining studies for human-specific CD31 and alpha-smooth muscle actin demonstrated the formation of both donor (human) and host (rat)-derived vasculature within the engrafted triculture tissue constructs. Intraventricular injection of fluorescent microspheres or lectin resulted in their incorporation by human-derived vessels, confirming their functional integration with host coronary vasculature. Finally, the number of blood vessels was significantly greater in the triculture tissue constructs (60.3 +/- 8/mm(3), p < 0.05) when compared with scaffolds containing only CMs (39.0 +/- 14.4/mm(3)). In conclusion, a tissue-engineered human vascularized cardiac muscle can be established ex vivo and transplanted in vivo to form stable grafts. By utilizing a multicellular preparation we were able to increase biograft vascularization and to show that the preexisting human vessels can become functional and contribute to tissue perfusion.

Cell replacement and tissue engineering strategies are emerging as novel therapeutic paradigms for myocardial repair. Here we tested the hypothesis that a combined in situ tissue engineering and cell delivery strategy utilizing transplantable hydrogel-embedded cardiomyocytes [either neonatal rat ventricular cardiomyocytes (NRVCMs) or human embryonic stem cell-derived cardiomyocytes (hESC-CMs)] will improve functional performance in the rat infarction model. Methods and results : A novel liquid biodegradable PEGylated fibrinogen was developed, which was proved to be compatible with cardiomyocyte survival and maturation in vitro . Photopolymerization using UV beam results in a rapid liquid to hydrogel transformation. Animals were randomized to injection of saline, NRVCMs alone, the biopolymer alone, or the combined delivery of the biopolymer and the NRVCMs. Histological studies demonstrated the presence of the hydrogel-embedded cardiomyocytes within the scar tissue. The biopolymer was fully absorbed one month following delivery. Echocardiography revealed typical post-infarction remodeling in the control group (saline injection) as manifested by the deterioration of ejection fraction (EF) by 28±3% (from 43±2% at post-injury baseline to 30±2% at 4 weeks). Injection of the biopolymer or NRVCMs alone prevented this remodeling process (39±2% to 39±2% and 43±3% to 43±2% respectively; p Conclusions: We describe a novel injectable in-situ-forming hydrogel that functions as an efficient cardiomyocyte carrier for both NRVCM and hESC-CMs and acts synergistically with the grafted cells to prevent the unfavorable post-infarction cardiac remodeling and improve ventricular function in rats.

The adult human heart has limited regenerative capacity and, therefore, functional restoration of the damaged heart presents a great challenge. Despite the progress achieved in the pharmacological and surgical treatment of degenerative myocardial diseases, they are still considered a major cause of morbidity and mortality in the western world. Repopulation of the damaged heart with cardiomyocytes represents a novel conceptual therapeutic paradigm but is hampered by the lack of sources for human cardiomyocytes. The recent derivation of pluripotent human embryonic stem cell lines may provide a solution for this cell sourcing problem. This review will focus on the derivation of the hESC lines, their mechanism of self-renewal, and their differentiation to cardiomyocytes. The possible signals and cues involved in the commitment and early differentiation of cardiomyocytes in this model will be discussed as well as the molecular, structural and electrophysiologic characteristics of the generated hESC-derived cardiomyocytes. Finally, the hurdles and challenges toward fully harnessing the potential clinical applications of these unique cells will be described.

The derivation of the hESC lines and the resulting cardiomyocyte differentiation system may bring a unique value to several basic and applied research fields. Research based on the cells may help to elucidate the mechanisms involved in early human cardiac lineage commitment, differentiation, and maturation. Moreover, this research may promote the discovery of novel growth and transcriptional factors using gene trapping techniques, functional genomics, and proteomics as well as providing a novel in vitro model for drug development and testing. Finally, the ability to generate, in vitro for the first time, human cardiac tissue provides an exciting and promising cell source for the emerging discipline of regenerative medicine and myocardial repair.

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.

Human embryonic stem cells are capable of unlimited proliferation in culture in the undifferentiated state and under the proper conditions can differentiate into different cell types including spontaneously beating cardiac myocytes (Kehat et al. 2001, 2002). Spontaneous beating or automaticity is not normally exhibited by mature atrial or ventricular myocytes. In contrast, embryonic heart cells display spontaneous activity (DeHaan & Gottlieb, 1968). The main requirement for automaticity is the presence of inward current at diastolic potentials. Although two recent reports of human embryonic stem cell-derived cardiac myocytes (hES-CMs) document action potentials (He et al. 2003; Mummery et al. 2003), there are no studies of ionic currents in hES-CMs. Moreover, the more thoroughly studied mouse ES-derived CMs exhibit AP morphologies that broadly reflect either atrial-like, ventricular-like, or nodal-like parameters (Hescheler et al. 1999; Sachinidis et al. 2003) that differ markedly from their human counterparts. The cardiac Na+ channel (termed NaV1.5) is expressed in relatively high density on surface membrane of mature heart cells in atria and ventricle. Despite the high NaV1.5 expression these cell types are not normally automatic because a high density of inward rectifier K+ channels (Kir) clamps the membrane potential to a value near the K+ reversal potential. At such hyperpolarized potentials the NaV1.5 channel's open probability approaches 0. In quiescent, mature heart cells, the initiating depolarization originates from neighbouring cells via gap junctions. Depolarization of membrane potential (Vm) activates NaV1.5 rapidly, driving the rapid AP upstroke, and within a few milliseconds of sustained depolarization NaV1.5 inactivates. Thus, NaV1.5 serves the role of generating a pathway for a rapid influx of depolarizing current. The maximum diastolic potential (MDP) is a key control point for NaV1.5, if MDP is relatively depolarized then NaV1.5 will be largely inactivated and unable to contribute to the AP upstroke. For this reason, there is a correlation between Na+ channel current density (not simply channel density), and the maximum upstroke velocity of the cellular AP (dV/dtmax). Moreover, in the developing heart there is a an increase in dV/dtmax from < 20 to 100–150 V s−1 that is concomitant with the onset of TTX sensitivity (McDonald et al. 1973), an increase in Na+ current density (Fujii et al. 1988), and a negative shift in the MDP (McDonald et al. 1973; DeHaan, 1980; Sperelakis, 1984). Given the exciting potential use of hES-CMs as replacement tissue in diseased heart (Gepstein, 2002; Kehat & Gepstein, 2003), and as an in vitro model for the study of early human cardiac development it is important to characterize their functional properties. We assessed the electrical properties of these cells in spontaneously beating embryoid bodies (EBs) using a multielectrode array mapping technique and detailed patch-clamp recordings and pharmacologically dissected the critical pathways in these structures. Our results provide the first description of the ionic currents in hES-CMs and show that the basis for spontaneous electrical activity in these cells is the absence of Kir conductance, a phenomenon that provides the substrate for a relatively large voltage-gated Na+ current to drive activity.