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Dilated cardiomyopathy (DCM) is characterized by reduced cardiac output, as well as thinning and enlargement of left ventricular chambers. These characteristics eventually lead to heart failure. Current standards of care do not target the underlying molecular mechanisms associated with genetic forms of heart failure, driving a need to develop novel therapeutics for DCM. To identify candidate therapeutics, we developed an in vitro DCM model using induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) deficient in B-cell lymphoma 2 (BCL2)-associated athanogene 3 (BAG3). With these BAG3-deficient iPSC-CMs, we identified cardioprotective drugs using a phenotypic screen and deep learning. From a library of 5500 bioactive compounds and siRNA validation, we found that inhibiting histone deacetylase 6 (HDAC6) was cardioprotective at the sarcomere level. We translated this finding to a BAG3 cardiomyocyte–knockout (BAG3 cKO ) mouse model of DCM, showing that inhibiting HDAC6 with two isoform-selective inhibitors (tubastatin A and a novel inhibitor TYA-018) protected heart function. In BAG3 cKO and BAG3 E455K mice, HDAC6 inhibitors improved left ventricular ejection fraction and reduced left ventricular diameter at diastole and systole. In BAG3 cKO mice, TYA-018 protected against sarcomere damage and reduced Nppb expression. Based on integrated transcriptomics and proteomics and mitochondrial function analysis, TYA-018 also enhanced energetics in these mice by increasing expression of targets associated with fatty acid metabolism, protein metabolism, and oxidative phosphorylation. Our results demonstrate the power of combining iPSC-CMs with phenotypic screening and deep learning to accelerate drug discovery, and they support developing novel therapies that address underlying mechanisms associated with heart disease.

SUMMARY Carney complex (CNC) is a rare syndrome of multiple endocrine and non-endocrine tumors. In this paper we present a 23-year-old Iranian woman with CNC who harbored a novel mutation (c.642dupT) in PRKAR1A gene. This patient presented with pituitary macroadenoma, acromegaly, recurrent atrial myxoma, Cushing's syndrome secondary to primary pigmented nodular adrenocortical disease and pigmented schwanoma of the skin. PRKAR1A gene was PCR amplified using genomic DNA and analyzed for sequence variants which revealed the novel mutation resulting in substitution of amino acid cysteine instead of the naturally occurring valine in the peptide chain and a premature stop codon at position 18 (V215CfsX18). This change leads to development of tumors in different organs due to lack of tumor suppressive activity secondary to failure of synthesis of the related protein.

Hydatid disease is a common health problem in sheep-farming countries including Iran. The liver and lungs are the most frequent primary sites of hydatid cysts in humans. Cardiac involvement is an uncommon manifestation and the right ventricle outflow tract is rarely involved. This is a case report of a 34-year-old man who presented to the Heart Clinic, Tehran, Iran, in 2019 with a history of dyspnea and fatigue. Following imaging study, the patient was diagnosed with a right ventricular outflow tract hydatid cyst. He underwent surgical resection of the cyst. The post-operative course was uneventful.Keywords: Hydatid cyst; Right ventricular; Outflow obstruction; case report; Iran.

Given the nature of heart disease and the importance of continuing heart surgery during the pandemic and its aftermath and in order to provide adequate safety for the surgical team and achieve the desired result for patients, as well as the optimal use of ICU beds, the medical team, blood, blood products, and personal protective equipment, it is essential to change the usual approach during the pandemic. There are still a lot of evidences and experiences needed to produce the perfect protocol. Some centers may have a special program for their centers during this period of epidemics that can be respected and performed. Generally, in pandemic conditions, the use of non-surgical approaches is preferred if similar outcomes can be obtained.

Advancing maturation of stem cell-derived cardiac muscle represents a major barrier to progress in cardiac regenerative medicine. Cardiac muscle maturation involves a myriad of gene, protein, and cell-based transitions, spanning across all aspects of cardiac muscle form and function. We focused here on a key developmentally controlled transition in the cardiac sarcomere, the functional unit of the heart. Using a gene-editing platform, human induced pluripotent stem cell (hiPSCs) were engineered with a drug-inducible expression cassette driving the adult cardiac troponin I (cTnI) regulatory isoform, a transition shown to be a rate-limiting step in advancing sarcomeric maturation of hiPSC cardiac muscle (hiPSC-CM) toward the adult state. Findings show that induction of the adult cTnI isoform resulted in the physiological acquisition of adult-like cardiac contractile function in hiPSC-CMs in vitro. Specifically, cTnI induction accelerated relaxation kinetics at baseline conditions, a result independent of alterations in the kinetics of the intracellular Ca2+ transient. In comparison, isogenic unedited hiPSC-CMs had no cTnI induction and no change in relaxation function. Temporal control of adult cTnI isoform induction did not alter other developmentally regulated sarcomere transitions, including myosin heavy chain isoform expression, nor did it affect expression of SERCA2a or phospholamban. Taken together, precision genetic targeting of sarcomere maturation via inducible TnI isoform switching enables physiologically relevant adult myocardium-like contractile adaptations that are essential for beat-to-beat modulation of adult human heart performance. These findings have relevance to hiPSC-CM structure-function and drug-discovery studies in vitro, as well as for potential future clinical applications of physiologically optimized hiPSC-CM in cardiac regeneration/repair.

Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism for such protection remains elusive. One of the variants in this allele block is coding, changing cysteine to arginine at position 151 of BAG3 (rs2234962-BAG3C151R). Here, we use induced pluripotent stem cells (iPSC) to test if the BAG3C151Rvariant alters protein and cellular function in human cardiac myocytes. Quantitative protein interaction network analysis identified specific changes in BAG3C151Rprotein interaction partners in cardiomyocytes but not in iPSCs or an immortalized cell line. Knockdown of BAG3 interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3C151Rassociates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3C151Rvariant have improved response to proteotoxic stress in an allele dose-dependent manner. This study suggests that the BAG3C151Rvariant increases cardiomyocyte protection from stress by enhancing the recruitment of factors critical to the maintenance of myofibril integrity, hinting that this variant could be responsible for the cardioprotective effect of the haplotype block. By revealing specific changes in preferential binding partners of the BAG3C151Rprotein variant, we also identify potential targets for the development of novel cardioprotective therapies.

Right atrial diverticulum is a very rare anomaly. It is an outpouching arising from the right atrial free wall. Clinical presentations vary widely but some cases are associated with supraventricular tachycardia and atrial flutter/fibrillation. The incidence/prevalence of this anomaly is not available because only a few cases have been reported. We report a 38-year-old female patient who presented to the Heart Clinic, Tehran, Iran in 2019 with a history of dyspnea and chest pain. Electrocardiography revealed left bundle branch block. Following a magnetic resonance imaging study, the patient was diagnosed with a right atrial diverticulum. She underwent surgical resection of the diverticulum. The post-operative course was uneventful and no recurrence of the arrhythmia was detected during the six months of follow-up. To the best of the authors’ knowledge, this combination has not been described in the literature. Keywords: Right Atrium; Diverticulum; Left Bundle Branch Block; Case Report; Iran.

BACKGROUND The patients undergo Coronary Artery Bypass Graft Surgery fail to adhere their treatment regimen for many reasons. Among these, one of the most important reasons for non-adherence is improper training of such patients. Therefore, it is necessary for nurses to train these patients by choosing the appropriate approach. OBJECTIVE The aim of study is to compare the effect of gamification and teach-back training methods on adherence of therapeutic regimen of patients after coronary artery bypass graft(CABG) METHODS The present randomized clinical trial was conducted on 123 patients undergo CABG, Tehran, Iran, in 2019.Training was presented in teach-back group individually using teach-back training approach. In the gamification group, the developed application was installed on the patient's smartphone and the training was given in this way. The control group received routine training of the hospital. Finally, the adherence of therapeutic regimen was assessed using the adherence of therapeutic regimen questionnaire (physical activity and dietary regimen) and Morisky Medication Adherence Scale (MMAS) as pre-test and post-test (after one month later). RESULTS The sampling process continued from August 2019 to murch 2020. A total of 123 people were included in the study. One-way analysis of variance test for comparing the mean scores of teach-back and gamification training methods showed that the mean normalized scores for dietary regimen (P CONCLUSIONS Based on the results of the present study, the use of teach-back and gamification training approaches can be suggested for patients after CABG for adherence of therapeutic regimen. CLINICALTRIAL Iranian Registry of Clinical Trail (IRCT20111203008286N8).

Drug-induced cardiotoxicity and hepatotoxicity are major causes of drug attrition. To decrease late-stage drug attrition, pharmaceutical and biotechnology industries need to establish biologically relevant models that use phenotypic screening to predict drug-induced toxicity. In this study, we sought to rapidly detect patterns of cardiotoxicity using high-content image analysis with deep learning and induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). We screened a library of 1280 bioactive compounds and identified those predicted to have cardiotoxic liabilities using a single-parameter score based on deep learning. Compounds with major predicted cardiotoxicity included DNA intercalators, ion channel blockers, epidermal growth factor receptor, cyclin-dependent kinase, and multi-kinase inhibitors. We also screened a diverse library of molecules with unknown targets and identified chemical frameworks with predicted cardiotoxic liabilities. By using this screening approach during target discovery and lead optimization, we can de-risk early-stage drug discovery. We show that the broad applicability of combining deep learning with iPSC technology is an effective way to interrogate cellular phenotypes and identify drugs that protect against diseased phenotypes and deleterious mutations.GRAPHICAL ABSTRACTCONTRIBUTION TO THE FIELDIn this article, Grafton and colleagues use induced pluripotent stem cell technology and deep learning to train a neural network capable of detecting patterns of cardiotoxicity. To identify bioactive and chemical classes that lead to cardiotoxicity, they combine the neural network with high-content screening of 2560 compounds. The methods described in this study can be used to de-risk early-stage drug development, triage hits, and identify drugs that protect against disease. This screening paradigm will serve as a useful resource for drug discovery and phenotypic interrogation of stem cells and stem cell–derived cell types.

BACKGROUND Patients undergoing coronary artery bypass graft surgery (CABGS) may fail to adhere to their treatment regimen for many reasons. Among these, one of the most important reasons for nonadherence is the inadequate training of such patients or training using inappropriate methods. OBJECTIVE This study aimed to compare the effect of gamification and teach-back training methods on adherence to a therapeutic regimen in patients after CABGS. METHODS This randomized clinical trial was conducted on 123 patients undergoing CABGS in Tehran, Iran, in 2019. Training was provided to the teach-back group individually. In the gamification group, an app developed for the purpose was installed on each patient’s smartphone, with training given via this device. The control group received usual care, or routine training. Adherence to the therapeutic regimen was assessed using a questionnaire on adherence to a therapeutic regimen (physical activity and dietary regimen) and an adherence scale as a pretest and a 1-month posttest. RESULTS One-way analysis of variance (ANOVA) for comparing the mean scores of teach-back and gamification training methods showed that the mean normalized scores for the dietary regimen (PF=71.80), movement regimen (PF=124.53), and medication regimen (PF=9.66) before and after intervention were significantly different between the teach-back, gamification, and control groups. In addition, the results of the Dunnett test showed that the teach-back and gamification groups were significantly different from the control group in all three treatment regimen methods. There was no statistically significant difference in adherence to the therapeutic regimen between the teach-back and control groups. CONCLUSIONS Based on the results of this study, the use of teach-back and gamification training approaches may be suggested for patients after CABGS to facilitate adherence to the therapeutic regimen. CLINICALTRIAL Iranian Registry of Clinical Trials IRCT20111203008286N8; https://en.irct.ir/trial/41507

Mediastinal thymic cysts are uncommon lesions. Thymic cysts are usually diagnosed incidentally, and their origin could be congenital or acquired. Herein we present the case of a patient who presented with dyspnea. Chest computerized scan showed a large cystic mass. Surgical excision was performed. Pathology findings were consistent with congenital thymic cyst.

The integration of in vitro cardiac tissue models, human induced pluripotent stem cells (hiPSCs) and genome-editing tools allows for the enhanced interrogation of physiological phenotypes and recapitulation of disease pathologies. Here, using a cardiac tissue model consisting of filamentous three-dimensional matrices populated with cardiomyocytes derived from healthy wild-type (WT) hiPSCs (WT hiPSC-CMs) or isogenic hiPSCs deficient in the sarcomere protein cardiac myosin-binding protein C (MYBPC3-/- hiPSC-CMs), we show that the WT microtissues adapted to the mechanical environment with increased contraction force commensurate to matrix stiffness, whereas the MYBPC3-/- microtissues exhibited impaired force development kinetics regardless of matrix stiffness and deficient contraction force only when grown on matrices with high fibre stiffness. Under mechanical overload, the MYBPC3-/- microtissues had a higher degree of calcium transient abnormalities, and exhibited an accelerated decay of calcium dynamics as well as calcium desensitization, which accelerated when contracting against stiffer fibres. Our findings suggest that MYBPC3 deficiency and the presence of environmental stresses synergistically lead to contractile deficits in cardiac tissues.

We describe a 72-year-old woman, a known case of rheumatic heart disease with a history of mitral and aortic valve replacement 8 years previously, who underwent mitral valve-in-valve replacement supported by a transapically snared guidewire through septostomy.

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Background Patients undergoing coronary artery bypass graft surgery (CABGS) may fail to adhere to their treatment regimen for many reasons. Among these, one of the most important reasons for nonadherence is the inadequate training of such patients or training using inappropriate methods. Objective This study aimed to compare the effect of gamification and teach-back training methods on adherence to a therapeutic regimen in patients after CABGS. Methods This randomized clinical trial was conducted on 123 patients undergoing CABGS in Tehran, Iran, in 2019. Training was provided to the teach-back group individually. In the gamification group, an app developed for the purpose was installed on each patient’s smartphone, with training given via this device. The control group received usual care, or routine training. Adherence to the therapeutic regimen was assessed using a questionnaire on adherence to a therapeutic regimen (physical activity and dietary regimen) and an adherence scale as a pretest and a 1-month posttest. Results One-way analysis of variance (ANOVA) for comparing the mean scores of teach-back and gamification training methods showed that the mean normalized scores for the dietary regimen (P Conclusions Based on the results of this study, the use of teach-back and gamification training approaches may be suggested for patients after CABGS to facilitate adherence to the therapeutic regimen. Trial Registration Iranian Registry of Clinical Trials IRCT20111203008286N8; https://en.irct.ir/trial/41507

Rationale: During each beat, cardiac myocytes (CMs) generate the mechanical output necessary for heart function through contractile mechanisms that involve shortening of sarcomeres along myofibrils. Human-induced pluripotent stem cells (hiPSCs) can be differentiated into CMs (hiPSC-CMs) that model cardiac contractile mechanical output more robustly when micropatterned into physiological shapes. Quantifying the mechanical output of these cells enables us to assay cardiac activity in a dish. Objective: We sought to develop a computational platform that integrates analytic approaches to quantify the mechanical output of single micropatterned hiPSC-CMs from microscopy videos. Methods and Results: We micropatterned single hiPSC-CMs on deformable polyacrylamide substrates containing fluorescent microbeads. We acquired videos of single beating cells, of microbead displacement during contractions, and of fluorescently labeled myofibrils. These videos were independently analyzed to obtain parameters that capture the mechanical output of the imaged single cells. We also developed novel methods to quantify sarcomere length from videos of moving myofibrils and to analyze loss of synchronicity of beating in cells with contractile defects. We tested this computational platform by detecting variations in mechanical output induced by drugs and in cells expressing low levels of myosin-binding protein C. Conclusions: Our method can measure the cardiac function of single micropatterned hiPSC-CMs and determine contractile parameters that can be used to elucidate mechanisms that underlie variations in CM function. This platform will be amenable to future studies of the effects of mutations and drugs on cardiac function.

CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

OBJECTIVES: The aim of the present study was to ascertain whether the length of anterior mitral leaflet second-order chordae (SOC) could be considered as a predictor of the incidence of post-repair systolic anterior motion (SAM) and left ventricular outflow tract obstruction (LVOTO) in patients with myxomatous mitral valve disease. METHODS: With the implementation of preoperative transoesophageal echocardiography (TEE), the length of anterior mitral leaflet SOC, anterior leaflet (AL) and posterior leaflet (PL) as well as the distance from the coaptation point to the septum (C-S distance) before and after mitral valve repair (MVR) surgery were measured in 190 patients, comprising 12 who developed SAM and 178 who did not. RESULTS: The results revealed that, in patients who developed SAM, SOC were significantly higher (2.76 ± 0.15 vs 1.83 ± 0.32 mm, P< 0.001) and the C-S distance was significantly lower (2.18 ± 0.36 vs 2.91 ± 0.36 mm, P< 0.001) in comparison to the obtained results for those who did not develop SAM. SOC and the C-S distance were independent risk factors of developing SAM and had the largest area under the receiver operating characteristic (ROC) curve (P< 0.001). With application of a cut-off ROC curve analysis, the cut-offs selected for the two variables of C-S distance and SOC were 2.5 and 2.6, respectively. Sensitivity and specificity of SAM development were 100% [95% confidence interval (CI): 73.5–100] and 87.1% (95% CI: 81.0–91.4) for SOC ≥2.6 and 83.3% (95% CI: 51.6–97.9) and 73.6% (95% CI: 66.4–79.9) for the C-S distance ≤2.5. CONCLUSIONS: The two variables of the second-order chordae and the distance from the coaptation point to the septum were associated with an increased risk of the post-repair systolic anterior motion after mitral valve repair.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.

Cardiac and hepatic toxicity result from induced disruption of the functioning of cardiomyocytes and hepatocytes, respectively, which is tightly related to the organization of their subcellular structures. Cellular structure can be analyzed from microscopy imaging data. However, subtle or complex structural changes that are not easily perceived may be missed by conventional image-analysis techniques. Here we report the evaluation of PhenoTox, an image-based deep-learning method of quantifying drug-induced structural changes using human hepatocytes and cardiomyocytes derived from human induced pluripotent stem cells. We assessed the ability of the deep learning method to detect variations in the organization of cellular structures from images of fixed or live cells. We also evaluated the power and sensitivity of the method for detecting toxic effects of drugs by conducting a set of experiments using known toxicants and other methods of screening for cytotoxic effects. Moreover, we used PhenoTox to characterize the effects of tamoxifen and doxorubicin-which cause liver toxicity-on hepatocytes. PhenoTox revealed differences related to loss of cytochrome P450 3A4 activity, for which it showed greater sensitivity than a caspase 3/7 assay. Finally, PhenoTox detected structural toxicity in cardiomyocytes, which was correlated with contractility defects induced by doxorubicin, erlotinib, and sorafenib. Taken together, the results demonstrated that PhenoTox can capture the subtle morphological changes that are early signs of toxicity in both hepatocytes and cardiomyocytes.

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis., eLife digest Embryos begin as a collection of similar cells, which progress in stages to form a huge variety of cell types in particular arrangements. These patterns of cells give rise to the different tissues and organs that make up the body. Although we often use ‘model’ organisms such as mice and frogs to study how embryos develop, our species has evolved unique ways to control organ development. Investigating these processes is difficult: we cannot experiment on human embryos, and our development is hard to recreate in test tubes. As a result, we do not fully understand how developing human cells specialize and organize. Libby et al. have now created a new system to study how different genes control cell organization. The system uses human pluripotent stem cells – cells that have the ability to specialize into any type of cell. Some of the stem cells are modified using a technique called inducible CRISPR interference, which makes it possible to reduce the activity of certain genes in these cells. Libby et al. used this technique to investigate how changes to the activity of two genes – called ROCK1 and CDH1 – affect how a mixed group of stem cells organized themselves. Cells that lacked ROCK1 formed bands near the edges of the group. Cells that lacked CDH1 segregated themselves from other cells, forming ‘islands’ inside the main group. The cells retained their ability to specialize into any type of cell after forming these patterns. However, specific groups of cells were more likely to become certain cell types. The method developed by Libby et al. can be used to study a range of complex tissue development and cell organization processes. Future work could create human tissue model systems for research into human disease or drug development.

Morphogenesis results from the interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events offer little control over parallel cell type co-emergence and do not offer the capability to selectively perturb gene expression in specific subpopulations of cells. We have developed anin vitrosystem that can spatiotemporally interrogate cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined the effects of independently knocking down molecular regulators of cortical tension and cell-cell adhesion using inducible CRISPRi: Rho-associated kinase-1 (ROCK1) and E-cadherin (CDH1), respectively. Induced mosaic knockdown of ROCK1 or CDH1 in hiPSC populations resulted in differential patterning events within hiPSC colonies indicative of cell-driven population organization. Patterned colonies retained an epithelial phenotype and nuclear expression of pluripotency markers. Gene expression within each of the mixed populations displayed a transient wave of differential expression with induction of knockdown that stabilized in coordination with intrinsic pattern formation. Mosaic patterning of hiPSCs enables the genetic interrogation of emergent multicellular properties of pluripotent cells, leading to a greater mechanistic understanding of the specific molecular pathways regulating the dynamics of symmetry breaking events that transpire during developmental morphogenesis.SIGNIFICANCEHuman embryonic development entails a series of multicellular morphogenic events that lead to primitive tissue formation. Attempts to study human morphogenic processes experimentally have been limited due to divergence from model organisms and the inability of current humanin vitromodels to accurately control the coincident emergence of heterogeneous cell populations in the spatially controlled manner necessary for proper tissue structure. We developed a human induced pluripotent stem cell (iPSC)in vitromodel that enables temporal control over the emergence of heterotypic subpopulations of cells. We examined mosaic knockdown of two target molecules to create predictable and robust cell-patterning events within hiPSC colonies. This method allows for dynamic interrogation of intrinsic cell mechanisms that initiate symmetry breaking events and provides direct insight(s) into tissue developmental principles.

Tracheoesophageal disorders and diseases are prevalent in humans such that an organoid model of human esophagus could be greatly beneficial. We therefore established a three-dimensional esophageal organoid culture system through the directed differentiation of human pluripotent stem cells (hPSCs). We identified that sequential manipulation of several signaling pathways was needed to first pattern definitive endoderm into foregut, then into anterior foregut (AFG), then dorsal AFG spheroids (dAFG). Outgrowth of dAFG spheroids for 1-2 months resulted in human esophageal organoids (HEOs) with a stratified squamous epithelium comparable to a late gestation mouse embryonic esophagus. We then used HEOs and mouse embryos to identify how SOX2 mediates separation of the esophageal and respiratory lineages and found that loss of endodermal Sox2 results in complete esophageal agenesis. At the transcriptional level, SOX2 acts to promote esophageal specification in both mice and humans in part by inhibiting Wnt signaling in the dorsal AFG and promoting survival of esophageal epithelium. In addition to this use of hPSC-derived esophageal organoids to study development, HEOs can be used for future studies of esophageal pathologies, such as Barrett’s metaplasia and carcinoma.

Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility. The relative performance of these algorithms, in terms of improving signal to noise, was tested. Applying these algorithms allowed analysis of contractility in iPS-CM cultured over multiple spatial scales from single cells to three-dimensional constructs. This open source software was validated with analysis of isoproterenol response in these cells, and can be applied in future studies comparing the drug responsiveness of iPS-CM cultured in different microenvironments in the context of tissue engineering.

Summary We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes., Graphical Abstract, Highlights • Non-invasive characterization of cardiomyocytes using video motion analysis • Assessment of single-cell, monolayer, or tissue cardiomyocytes in standard plates • Drug screening by a label-free, contact-free imaging assay, In this article, Maddah and colleagues present a non-invasive method to characterize the function of stem-cell-derived cardiomyocytes based on video microscopy and image analysis. They demonstrate automated measurements of beating frequency, duration, amplitude, and variation and show that the platform can be used for cardiotoxicity drug screening and studying physiologically relevant phenotypes.

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

A very focused function for lncRNAs The human genome generates many thousands of long noncoding RNAs (lncRNAs). A very small number of lncRNAs have been shown to be functional. Liu et al. carried out a large-scale CRISPR-based screen to assess the function of ∼17,000 lncRNAs in seven different human cell lines. A considerable number (∼500) of the tested lncRNAs influenced cell growth, suggesting biological function. In almost all cases, though, the function was highly cell type—specific, often limited to just one cell type. Science , this issue p. 10.1126/science.aah7111

Background The ideal management of ischemic mitral regurgitation (MR) remains a clinical dilemma because of the suboptimal available therapeutic options. Recently, new concepts have emerged, pointing to the benefits of a patient selection approach when debating the management of moderate ischemic MR. We investigated the predictability of low-dose dobutamine stress echocardiography (DSE) in selecting candidates for CABG with moderate MR for valve repair. Methods From November 2002 to May 2010, 110 candidates for first-time CABG, who were admitted to the cardiac surgery department in Day General Hospital (Tehran, Iran), were enrolled in the present cross-sectional study. DSE was performed for each case before CABG. Those with positive findings underwent CABG alone and those with negative results underwent concomitant CABG and mitral valve repair. The patients were followed up for a minimum of 60 months. Results Of the 110 patients, 47 (42.72%) had positive test results and underwent CABG alone and 63 (57.28%) had negative DSE results and underwent concomitant CABG and mitral valve repair. The MR degree had decreased from 2.8 ± 0.3 preoperatively to 1.46 ± 0.6 early during the hospital stay and 1.9 ± 0.7 during late follow-up in the CABG group. It had decreased from 2.84 ± 0.4 preoperatively to 0.93 ± 0.65 postoperatively but then increased to 1.41 ± 0.9 during late follow-up, for a significant decrease in the combined group ( P Conclusions Despite its utility in selecting CABG patients with moderate ischemic MR for valve repair from a short-term perspective, the use of DSE cannot predict the long-term outcomes of these patients.

Amac: Bu calismada cinsiyetin izole koroner arter baypas greftlemenin (KABG) erken cerrahi sonuclari uzerindeki muhtemel etkileri arastirildi. Ca­lis­ma­ pla­ni:­ Kalp hastalarini kapsayan mevcut veri tabaninin retrospektif incelemesine, Agustos 2007 ve Mart 2011 tarihleri arasinda Iran, Tahran Shariati Hastanesi’nde izole KABG yapilan 1390 ardisik olgu (393 erkek, 997 kadin; ort. yas 58.7±10.1 yil; dagilim 23-87 yil) alindi. Erkek ve kadin cinsiyetleri ameliyat oncesi ozellikler acisindan karsilastirildi. Cinsiyetin ameliyat sonrasi mortalite ve morbidite uzerindeki muhtemel etkilerini degerlendirmek uzere basamakli cok degiskenli lojistik regresyon analizi yapildi. Bul gu lar: Kadin hastalarin yasi daha ileriydi ve diabetes mellitus, hipertansiyon, hiperkolesterolemi ve hiperlipidemi prevalansi daha yuksekti. Mortalite orani, erkeklere kiyasla, kadinlarda anlamli duzeyde daha yuksekti (%2.3’e kiyasla %6.9; p

BACKGROUND: We prospectively studied a group of patients with myocardial infarction (MI), who were candidates for surgical revascularization, to compare the efficacy of dobutamine gated myocardial SPECT with dobutamine stress echocardiography (DSE) for the detection of myocardial viability. MATERIALS AND METHODS: We investigated 224 segments from 14 patients with MI using resting echocardiography and low dose dobutamine stress echocardiography as well as resting, low and high dose dobutamine stress 99mTc-Sestamibi gated SPECT. RESULTS: In total, 13 men and 1 women with a mean age 54.57 years (range, 43 to 71 years) entered the study. Of the 125 dysfunctional segments, as assessed by ECG-gated examination, 53 (23.66% of total) were hypokinetic at rest, 64 (28.57% of total) were akinetic, and 8 (3.57% of total) were dyskinetic. The number of segments with resting wall motion abnormality (considered viable by low dose dobutamine ECG-gated examination) was significantly greater than those showing a contractile improvement in response to dobutamine in echocardiography (39.2% versus 32.8%, respectively, p < 0.05). In addition, in high dose ECG-gated examination, 42 of the 125 dysfunctional segments (33.6%) were viable. In general, the methods were well correlated. CONCLUSION: We found a good agreement between low dose dobutamine gated SPECT and stress dobutamine echocardiography for the detection of inotropic reserve in infarcted areas.

Background: The saphenous vein remains the most commonly used conduit for coronary artery bypass surgery (CABG). However, the long-term success of surgical revascularization is largely limited by development of occlusion in vein grafts. Objectives: We sought to reduce graft ischemia by maintaining the blood flow into the harvested vein throughout surgery at lowest costs and without special devices. Patients and Methods: This study was conducted on three hundred patients aged 58.5 ± 8 years undergoing elective first-time off-pump CABG with saphenous veins. Results: In addition to preserving nutritional materials and oxygen, the veins harvested via this novel technique did not go into spasm and were not subjected to high-pressure distension, eventually resulting in minimal damage to the endothelium. Conclusions: This technique confers favorable myocardial function and protection in the presence of left ventricular dysfunction, especially in elderly patients.

Dilated cardiomyopathy (DCM) is often complicated by the appearance of functional mitral regurgitation (FMR). Although mitral ring annuloplasty (MAP) is the most widely used surgical procedure for the surgical treatment of FMR, there are still reports of patients who suffer recurrent FMR at later follow-ups. We sought to investigate the efficacy of papillary muscle approximation (PMA) combined with MAP in preventing the recurrence of FMR in high-risk patients.One hundred patients with ischaemic (74%) or non-ischaemic (26%) DCM along with severe (4+/4+) or moderately severe (3+/4+) FMR were enrolled in this prospective, cross-sectional study. According to the interpapillary muscle distance (iPMD) and coaptation depth (CD), the patients were risk stratified as low (iPMD + CD 30 mm, n= 69) and high-risk (iPMD + CD30 mm, n= 31) groups. The low-risk patients underwent only MAP, whereas the high-risk patients underwent MAP plus PMA.After a mean +/- SD follow-up of 40.8 +/- 12.5 months, recurrence of 3+ to 4+ MR was observed in 8 (8.7%) and 7 (11.1%) patients in the annuloplasty group (MAP-only) and one (3.4%) patient in the combination group (MAP plus PMA) (P= 0.428). At the final follow-up, the New York Heart Association (NYHA) function class was 1.57 +/- 0.62 in the annuloplasty group and 1.45 +/- 0.57 in the combination group; there was no significant difference in NYHA function class between the first and final follow-ups (P0.05).iPMD is a valuable index in the riskstratification of the recurrence of post-MAP MR in patients with DCM complicated by FMR.The patients treated with MAP plus PMA had more favourable outcomes and lower recurrence rates than those treated via the traditional route of MAP only.

Despite huge progress in the surgical management of ischemic mitral regurgitation, there is still controversy regarding the ideal treatment option. Our study aimed to define an algorithmic approach in order to select those patients who will benefit the most from concomitant mitral valve procedure. Patients with mitral regurgitation (MR) who met the inclusion criteria were included in our study. Patients with structural MR including ruptured chordae or papillary muscle, abnormal leaflet thickening, annular calcification, or other valvular or congenital heart diseases, ventricular aneurysms and those who were candidates for other surgical procedures were excluded. A total of 350 patients (about 12 %) were classified as having ischemic mitral regurgitation (IMR) and were enrolled in our analysis. All the patients underwent coronary artery bypass grafting and by the designed algorithmic approach were also decided to have additive procedures for their concomitant IMR. Six months survival was 91.5 % among all the patients in this study. During the follow-ups 86.9 % of the patients survived . Overall mortality rate was 13.1 % (n = 46), of which 7.1 % (n = 25) occurred due to cardiac and 6 % (n = 21) as a result of a non-cardiac cause. Individualising methods to select patients suffering from IMR for concomitant mitral valve procedure and coronary artery bypass grafting (CABG) seems to serves more effectively by reducing unnecessary surgeries and at the same time not missing absolute indications for concomitant valve repairs. Our algorithm showed a promising efficacy to effectively select patients for CABG and concomitant mitral valve procedures.