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Synthetic polymers play an indispensable role in modern society, finding applications across various sectors ranging from packaging, textiles, and consumer products to construction, electronics, and industrial machinery. Commodity plastics are cheap to produce, widely available, and versatile to meet diverse application needs. As a result, millions of metric tons of plastics are manufactured annually. However, current approaches for the chemical recycling of postconsumer plastic waste, primarily based on pyrolysis, lag in efficiency compared to their production methods. In recent years, significant research has focused on developing milder, economically viable methods for the chemical recycling of commodity plastics, which involves converting plastic waste back into monomers or transforming it into other valuable chemicals. This Perspective examines both industrial and cutting-edge laboratory-scale methods contributing to recent advancements in the field of chemical recycling., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Cardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele ( Taz PM ) that phenocopies BTHS clinical traits. As Taz PM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile Taz PM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile Taz PM hearts. However, adult Taz PM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult Taz PM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy.

Background: The genetic basis of hypertrophic cardiomyopathy (HCM) is complex, and the relationship between genotype status and clinical outcome is incompletely resolved., Methods and Results: We assessed a large international HCM cohort to define in contemporary terms natural history and clinical consequences of genotype. Consecutive patients (n=1468) with established HCM diagnosis underwent genetic testing. Patients with pathogenic (or likely pathogenic) variants were considered genotype positive (G+; n=312; 21%); those without definite disease-causing mutations (n=651; 44%) or variants of uncertain significance (n=505; 35%) were considered genotype negative (G-). Patients were followed up for a median of 7.8 years (interquartile range, 3.5-13.4 years); HCM end points were examined by cumulative event incidence. Over follow-up, 135 (9%) patients died, 33 from a variety of HCM-related causes. After adjusting for age, all-cause and HCM-related mortality did not differ between G- versus G+ patients (hazard ratio [HR], 0.78 [95% CI, 0.46-1.31]; P =0.37; HR, 0.93 [95% CI, 0.38-2.30]; P =0.87, respectively). Adverse event rates, including heart failure progression to class III/IV, heart transplant, or heart failure death, did not differ (G- versus G+) when adjusted for age (HR, 1.20 [95% CI, 0.63-2.26]; P =0.58), nor was genotype independently associated with sudden death event risk (HR, 1.39 [95% CI, 0.88-2.21]; P =0.16). In multivariable analysis, age was the only independent predictor of all-cause and HCM-related mortality, heart failure progression, and sudden death events., Conclusions: In this large consecutive cohort of patients with HCM, genotype (G+ or G-) was not a predictor of clinical course, including all-cause and HCM-related mortality and risk for heart failure progression or sudden death. G+ status should not be used to dictate clinical management or predict outcome in HCM.

Background: Cardiolipin is a mitochondrial-specific phospholipid that maintains integrity of the electron transport chain (ETC) and plays a central role in myocardial ischemia/reperfusion injury. Tafazzin is an enzyme that is required for cardiolipin maturation. Venoarterial extracorporeal membrane oxygenation (VA-ECMO) use to provide hemodynamic support for acute myocardial infarction has grown exponentially, is associated with poor outcomes, and is under active clinical investigation, yet the mechanistic effect of VA-ECMO on myocardial damage in acute myocardial infarction remains poorly understood. We hypothesized that VA-ECMO acutely depletes myocardial cardiolipin and exacerbates myocardial injury in acute myocardial infarction., Methods: We examined cardiolipin and tafazzin levels in human subjects with heart failure and healthy swine exposed to VA-ECMO and used a swine model of closed-chest myocardial ischemia/reperfusion injury to evaluate the effect of VA-ECMO on cardiolipin expression, myocardial injury, and mitochondrial function., Results: Cardiolipin and tafazzin levels are significantly reduced in the left ventricles of individuals requiring VA-ECMO compared with individuals without VA-ECMO before heart transplantation. Six hours of exposure to VA-ECMO also decreased left ventricular levels of cardiolipin and tafazzin in healthy swine compared with sham controls. To explore whether cardiolipin depletion by VA-ECMO increases infarct size, we performed left anterior descending artery occlusion for a total of 120 minutes followed by 180 minutes of reperfusion in adult swine in the presence and absence of MTP-131, an amphipathic molecule that interacts with cardiolipin to stabilize the inner mitochondrial membrane. Compared with reperfusion alone, VA-ECMO activation beginning after 90 minutes of left anterior descending artery occlusion increased infarct size (36±8% versus 48±7%; P <0.001). VA-ECMO also decreased cardiolipin and tafazzin levels, disrupted mitochondrial integrity, reduced electron transport chain function, and promoted oxidative stress. Compared with reperfusion alone or VA-ECMO before reperfusion, delivery of MTP-131 before VA-ECMO activation reduced infarct size (22±8%; P =0.03 versus reperfusion alone and P <0.001 versus VA-ECMO alone). MTP-131 restored cardiolipin and tafazzin levels, stabilized mitochondrial function, and reduced oxidative stress in the left ventricle., Conclusions: We identified a novel mechanism by which VA-ECMO promotes myocardial injury and further identify cardiolipin as an important target of therapy to reduce infarct size and to preserve mitochondrial function in the setting of VA-ECMO for acute myocardial infarction., Competing Interests: Disclosures Dr Kapur receives institutional grant support and speaker/consulting honoraria from Abbott, Abiomed, Boston Scientific, Edwards, Getinge, LivaNova, Medtronic, Teleflex, and Zoll. The other authors report no conflicts.

To mitigate pollution by plastic waste, it is paramount to develop polymers with efficient recyclability while retaining desirable physical properties. A recyclable poly(methyl methacrylate) (PMMA) is synthesized by incorporating a minimal amount of an α-methylstyrene (AMS) analogue into the polymer structure. This P(MMA- co -AMS) copolymer preserves the essential mechanical strength and optical clarity of PMMA, vital for its wide-ranging applications in various commercial and high-tech industries. Doping with AMS significantly enhances the thermal, catalyst-free depolymerization efficiency of PMMA, facilitating the recovery of methyl methacrylate (MMA) with high yield and purity at temperatures ranging from 150 to 210 °C, nearly 250 K lower than current industrial standards. Furthermore, the low recovery temperature permits the isolation of pure MMA from a mixture of assorted common plastics.

Alpha-B-crystallin, a member of the small heat shock family of proteins, has been implicated in a variety of cardiomyopathies and in normal cardiac homeostasis. It is known to function as a molecular chaperone, particularly for desmin, but also interacts with a wide variety of additional proteins. The molecular chaperone function is also enhanced by signal-dependent phosphorylation at specific residues under stress conditions. Naturally occurring mutations in CRYAB , the gene that encodes alpha-B-crystallin, have been suggested to alter ionic intermolecular interactions that affect dimerization and chaperone function. These mutations have been associated with myofibrillar myopathy, restrictive cardiomyopathy, and hypertrophic cardiomyopathy and promote pathological hypertrophy through different mechanisms such as desmin aggregation, increased reductive stress, or activation of calcineurin-NFAT signaling. This review will discuss the known mechanisms by which alpha-B-crystallin functions in cardiac homeostasis and the pathogenesis of cardiomyopathies and provide insight into potential future areas of exploration.

Hypertrophic Cardiomyopathy (HCM) is a common inherited disorder that can lead to heart failure and sudden cardiac death, characterized at the histological level by focal areas of myocyte disarray, hypertrophy and fibrosis, and only a few disease-targeted therapies exist. To identify the focal and spatially restricted alterations in the transcriptional pathways and reveal novel therapeutic targets, we performed a spatial transcriptomic analysis of the areas of focal myocyte disarray compared to areas of normal tissue using a commercially available platform (GeoMx, nanoString). We analyzed surgical myectomy tissue from four patients with HCM and the control interventricular septum tissue from two unused organ donor hearts that were free of cardiovascular disease. Histological sections were reviewed by an expert pathologist, and 72 focal areas with varying degrees of myocyte disarray (normal, mild, moderate, severe) were chosen for analysis. Areas of interest were interrogated with the Human Cancer Transcriptome Atlas designed to profile 1800 transcripts. Differential expression analysis revealed significant changes in gene expression between HCM and the control tissue, and functional enrichment analysis indicated that these genes were primarily involved in interferon production and mitochondrial energetics. Within the HCM tissue, differentially expressed genes between areas of normal and severe disarray were enriched for genes related to mitochondrial energetics and the extracellular matrix in severe disarray. An analysis of the gene expression of the ligand-receptor pair revealed that the HCM tissue exhibited downregulation of platelet-derived growth factor (PDGF), NOTCH, junctional adhesion molecule, and CD46 signaling while showing upregulation of fibronectin, CD99, cadherin, and amyloid precursor protein signaling. A deconvolution analysis utilizing the matched single nuclei RNA-sequencing (snRNA-seq) data to determine cell type composition in areas of interest revealed significant differences in fibroblast and vascular cell composition in areas of severe disarray when compared to normal areas in HCM samples. Cell composition in the normal areas of the control tissue was also divergent from the normal areas in HCM samples, which was consistent with the differential expression results. Overall, our data identify novel and potential disease-modifying targets for therapy in HCM.

Barth Syndrome, a rare X-linked disorder affecting 1:300,000 live births, results from defects in Tafazzin, an acyltransferase that remodels cardiolipin and is essential for mitochondrial respiration. Barth Syndrome patients develop cardiomyopathy, muscular hypotonia and cyclic neutropenia during childhood, rarely surviving to middle age. At present, no effective therapy exists, and downstream transcriptional effects of Tafazzin dysfunction are incompletely understood. To identify novel, cell-specific, pathological pathways that mediate heart dysfunction, we performed single-nucleus RNA-sequencing (snRNA-seq) on wild-type (WT) and Tafazzin -knockout (Taz-KO) mouse hearts. We determined differentially expressed genes (DEGs) and inferred predicted cell-cell communication networks from these data. Surprisingly, DEGs were distributed heterogeneously across the cell types, with fibroblasts, cardiomyocytes, endothelial cells, macrophages, adipocytes and pericytes exhibiting the greatest number of DEGs between genotypes. One differentially expressed gene was detected for the lymphatic endothelial and mesothelial cell types, while no significant DEGs were found in the lymphocytes. A Gene Ontology (GO) analysis of these DEGs showed cell-specific effects on biological processes such as fatty acid metabolism in adipocytes and cardiomyocytes, increased translation in cardiomyocytes, endothelial cells and fibroblasts, in addition to other cell-specific processes. Analysis of ligand-receptor pair expression, to infer intercellular communication patterns, revealed the strongest dysregulated communication involved adipocytes and cardiomyocytes. For the knockout hearts, there was a strong loss of ligand-receptor pair expression involving adipocytes, and cardiomyocyte expression of ligand-receptor pairs underwent reorganization. These findings suggest that adipocyte and cardiomyocyte mitochondria may be most sensitive to mitochondrial Tafazzin deficiency and that rescuing adipocyte mitochondrial dysfunction, in addition to cardiomyocyte mitochondrial dysfunction, may provide therapeutic benefit in Barth Syndrome patients.

Selective depolymerization of lignin remains a significant challenge in biomass conversion. The biosynthesis of lignin involves the polymerization of monolignol building blocks through oxidative radical coupling reactions. A strategy for lignin degradation leverages photoredox deoxygenative radical formation to trigger reverse biosynthesis, which cleaves model compounds of the β-O-4 and β-5-β-O-4 linkages to produce monolignols, precursors to flavoring compounds. This mild method preserves important oxygen functionality and serves as a platform for achieving selective lignin depolymerization.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray and cardiac fibrosis, HCM is a highly complex disease with heterogenous clinical presentation, onset and complication. While mutations in sarcomere genes can account for a substantial proportion of familial cases of HCM, 40%-50% of HCM patients do not carry such sarcomere variants and the causal mutations for their diseases remain elusive. Recently, we identified a novel variant of the alpha-crystallin B chain ( CRYAB R123W ) in a pair of monozygotic twins who developed concordant HCM phenotypes that manifested over a nearly identical time course. Yet, how CRYAB R123W promotes the HCM phenotype remains unclear. Here, we generated mice carrying the Cryab R123W knock-in allele and demonstrated that hearts from these animals exhibit increased maximal elastance at young age but reduced diastolic function with aging. Upon transverse aortic constriction, mice carrying the Cryab R123W allele developed pathogenic left ventricular hypertrophy with substantial cardiac fibrosis and progressively decreased ejection fraction. Crossing of mice with a Mybpc3 frame-shift model of HCM did not potentiate pathological hypertrophy in compound heterozygotes, indicating that the pathological mechanisms in the Cryab R123W model are independent of the sarcomere. In contrast to another well-characterized CRYAB variant (R120G) which induced Desmin aggregation, no evidence of protein aggregation was observed in hearts expressing CRYAB R123W despite its potent effect on driving cellular hypertrophy. Mechanistically, we uncovered an unexpected protein-protein interaction between CRYAB and calcineurin. Whereas CRYAB suppresses maladaptive calcium signaling in response to pressure-overload, the R123W mutation abolished this effect and instead drove pathologic NFAT activation. Thus, our data establish the Cryab R123W allele as a novel genetic model of HCM and unveiled additional sarcomere-independent mechanisms of cardiac pathological hypertrophy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Chou, Martin, Perera, Awata, Larson, Blanton and Chin.)

Whether extracorporeal membrane oxygenation (ECMO) with Impella, known as EC-Pella, limits cardiac damage in acute myocardial infarction remains unknown. The authors now report that the combination of transvalvular unloading and ECMO (EC-Pella) initiated before reperfusion reduced infarct size compared with ECMO alone before reperfusion in a preclinical model of acute myocardial infarction. EC-Pella also reduced left ventricular pressure-volume area when transvalvular unloading was applied before, not after, activation of ECMO. The authors further observed that EC-Pella increased cardioprotective signaling but failed to rescue mitochondrial dysfunction compared with ECMO alone. These findings suggest that ECMO can increase infarct size in acute myocardial infarction and that EC-Pella can mitigate this effect but also suggest that left ventricular unloading and myocardial salvage may be uncoupled in the presence of ECMO in acute myocardial infarction. These observations implicate mechanisms beyond hemodynamic load as part of the injury cascade associated with ECMO in acute myocardial infarction., Competing Interests: This work was supported by National Institutes of Health grants R01HL159089-01 and R01HL139785-01. Dr Kapur has received consulting and speaker honoraria and institutional grant support from Abbott Laboratories, Abiomed, Boston Scientific, Edwards Lifesciences, LivaNova, Getinge, and Zoll. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

Hypertrophic cardiomyopathy is one of the most common inherited cardiomyopathies and a leading cause of sudden cardiac death in young adults. Despite profound insights into the genetics, there is imperfect correlation between mutation and clinical prognosis, suggesting complex molecular cascades driving pathogenesis. To investigate this, we performed an integrated quantitative multi-omics (proteomic, phosphoproteomic, and metabolomic) analysis to illuminate the early and direct consequences of mutations in myosin heavy chain in engineered human induced pluripotent stem-cell-derived cardiomyocytes relative to late-stage disease using patient myectomies. We captured hundreds of differential features, which map to distinct molecular mechanisms modulating mitochondrial homeostasis at the earliest stages of pathobiology, as well as stage-specific metabolic and excitation-coupling maladaptation. Collectively, this study fills in gaps from previous studies by expanding knowledge of the initial responses to mutations that protect cells against the early stress prior to contractile dysfunction and overt disease.

The intention of this Special Issue is to highlight novel approaches and new paradigms for understanding the pathogenesis of hypertrophic cardiomyopathy (HCM) [...].

Hypertrophic cardiomyopathy (HCM) is considered a primary disorder of the sarcomere resulting in unexplained left ventricular hypertrophy but the paradoxical association of nonmyocyte phenotypes such as fibrosis, mitral valve anomalies and microvascular occlusion is unexplained. To understand the interplay between cardiomyocyte and nonmyocyte cell types in human HCM, single nuclei RNA-sequencing was performed on myectomy specimens from HCM patients with left ventricular outflow tract obstruction and control samples from donor hearts free of cardiovascular disease. Clustering analysis based on gene expression patterns identified a total of 34 distinct cell populations, which were classified into 10 different cell types based on marker gene expression. Differential gene expression analysis comparing HCM to Normal datasets revealed differences in sarcomere and extracellular matrix gene expression. Analysis of expressed ligand-receptor pairs across multiple cell types indicated profound alteration in HCM intercellular communication, particularly between cardiomyocytes and fibroblasts, fibroblasts and lymphocytes and involving integrin β1 and its multiple extracellular matrix (ECM) cognate ligands. These findings provide a paradigm for how sarcomere dysfunction is associated with reduced cardiomyocyte secretion of ECM ligands, altered fibroblast ligand-receptor interactions with other cell types and increased fibroblast to lymphocyte signaling, which can further alter the ECM composition and promote nonmyocyte phenotypes., (© 2022. The Author(s).)

Hypertrophic Cardiomyopathy (HCM) is a common inherited disorder characterized by unexplained left ventricular hypertrophy with or without left ventricular outflow tract (LVOT) obstruction. Single-nuclei RNA-sequencing (snRNA-seq) of both obstructive and nonobstructive HCM patient samples has revealed alterations in communication between various cell types, but no direct and integrated comparison between the two HCM phenotypes has been reported. We performed a bioinformatic analysis of HCM snRNA-seq datasets from obstructive and nonobstructive patient samples to identify differentially expressed genes and distinctive patterns of intercellular communication. Differential gene expression analysis revealed 37 differentially expressed genes, predominantly in cardiomyocytes but also in other cell types, relevant to aging, muscle contraction, cell motility, and the extracellular matrix. Intercellular communication was generally reduced in HCM, affecting the extracellular matrix, growth factor binding, integrin binding, PDGF binding, and SMAD binding, but with increases in adenylate cyclase binding, calcium channel inhibitor activity, and serine-threonine kinase activity in nonobstructive HCM. Increases in neuron to leukocyte and dendritic cell communication, in fibroblast to leukocyte and dendritic cell communication, and in endothelial cell communication to other cell types, largely through changes in the expression of integrin-β1 and its cognate ligands, were also noted. These findings indicate both common and distinct physiological mechanisms affecting the pathogenesis of obstructive and nonobstructive HCM and provide opportunities for the personalized management of different HCM phenotypes.

End stage, nonobstructive hypertrophic cardiomyopathy (HCM) is an intractable condition with no disease-specific therapies. To gain insights into the pathogenesis of nonobstructive HCM, we performed single nucleus RNA-sequencing (snRNA-seq) on human HCM hearts explanted at the time of cardiac transplantation and organ donor hearts serving as controls. Differential gene expression analysis revealed 64 differentially expressed genes linked to specific cell types and molecular functions. Analysis of ligand-receptor pair gene expression to delineate potential intercellular communication revealed significant reductions in expressed ligand-receptor pairs likely affecting the extracellular matrix, growth factor binding, peptidase regulator activity, platelet-derived growth factor binding and protease binding in the HCM tissue. Changes in Integrin-β1 receptor expression were responsible for many observed changes related to extracellular matrix interactions, by increasing in dendritic, smooth muscle and pericyte cells while decreasing in endothelial and fibroblast cells, suggesting potential mechanisms for fibrosis and microvascular disease in HCM and a potential role for dendritic cells. In contrast, there was an increase in ligand-receptor pair expression associated with adenylate cyclase binding, calcium channel molecular functions, channel inhibitor activity, ion channel inhibitor activity, phosphatase activator activity, protein kinase activator activity and titin binding, suggesting important shifts in various signaling cascades in nonobstructive, end stage HCM.

Barth Syndrome is an X-linked disorder of mitochondrial cardiolipin metabolism caused by pathogenic variants in TAFAZZIN with pleiotropic effects including cardiomyopathy, neutropenia, growth delay, and skeletal myopathy. Management requires a multidisciplinary approach to the organ-specific manifestations including specialists from cardiology, hematology, nutrition, physical therapy, genetics, and metabolism. Currently, treatment is centered on management of specific clinical features, and is not targeted toward remediating the underlying biochemical defect. However, two clinical trials have been recently undertaken which target the mitochondrial pathology of this disease: a study to examine the effects of elamipretide, a cardiolipin targeted agent, and a study to examine the effects of bezafibrate, a peroxisome proliferator-activated receptor (PPAR) agonist. Treatments to directly target the defective TAFAZZIN pathway are under development, including enzyme and gene therapies., (© 2021 SSIEM.)

Background: Hypertrophic cardiomyopathy (HCM) is a common inherited heart disorder complicated by left ventricle outflow tract (LVOT) obstruction, which can be treated with surgical myectomy. To date, no reliable biomarkers for LVOT obstruction exist. We hypothesized that metabolomic biomarkers for LVOT obstruction may be detectable in plasma from HCM patients., Methods: We conducted metabolomic profiling on plasma samples of 18 HCM patients before and after surgical myectomy, using a commercially available metabolomics platform., Results: We found that 215 metabolites were altered in the postoperative state (p-value < 0.05). 12 of these metabolites were notably significant after adjusting for multiple comparisons (q-value < 0.05), including bilirubin, PFOS, PFOA, 3,5-dichloro-2,6-dihydroxybenzoic acid, 2-hydroxylaurate, trigonelline and 6 unidentified compounds, which support improved organ metabolic function and increased lean soft tissue mass., Conclusions: These findings suggest improved organ metabolic function after surgical relief of LVOT obstruction in HCM and further underscore the beneficial systemic effects of surgical myectomy., (© 2021. The Author(s).)

Heart disease is the leading cause of death with no method to repair damaged myocardium due to the limited proliferative capacity of adult cardiomyocytes. Curiously, mouse neonates and zebrafish can regenerate their hearts via cardiomyocyte de-differentiation and proliferation. However, a molecular mechanism of why these cardiomyocytes can re-enter cell cycle is poorly understood. Here, we identify a unique metabolic state that primes adult zebrafish and neonatal mouse ventricular cardiomyocytes to proliferate. Zebrafish and neonatal mouse hearts display elevated glutamine levels, predisposing them to amino-acid-driven activation of TOR, and that TOR activation is required for zebrafish cardiomyocyte regeneration in vivo . Through a multi-omics approach with cellular validation we identify metabolic and mitochondrial changes during the first week of regeneration. These data suggest that regeneration of zebrafish myocardium is driven by metabolic remodeling and reveals a unique metabolic regulator, TOR-primed state, in which zebrafish and mammalian cardiomyocytes are regeneration competent., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Purpose: Leptomeningeal disease (LMD) in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma is associated with a poor prognosis and limited treatment options. Osimertinib is a potent third-generation EGFR tyrosine kinase inhibitor with confirmed CNS penetration. This study reports on outcomes of patients with EGFR-mutated non-small-cell lung cancer who developed LMD and were subsequently treated with osimertinib., Methods: We identified patients treated with osimertinib 80 mg PO daily under a compassionate access scheme across nine tertiary Australian institutes between July 2017 and July 2020. Patient demographics, tumor characteristics, and treatment history were collected. Median overall survival, median progression-free survival, disease control rates (DCR), and overall response rates (ORR) were assessed. Kaplan-Meier analysis was performed and descriptive statistics were used., Results: Thirty-nine patients were analyzed of which 74% were female. Exon 19 deletions (49%) and L858R point mutations (41%) were the most common EGFR mutations. Forty-nine percentage of patients were Eastern Cooperative Oncology Group 1. The median duration of osimertinib therapy was 6 months. The extracranial DCR and ORR were 60% and 54%, and the intracranial DCR and ORR were 68% and 53%, respectively. Median overall survival was 10.5 months (95% CI, 8.17 to 15.05 months)., Conclusion: There are limited treatment options for LMD in EGFR-positive lung cancer, and osimertinib at a dose of 80 mg daily is an active therapeutic option for these patients.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, affecting 1 in 500 people in the general population. Although characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray, and cardiac fibrosis, HCM is in fact a highly complex disease with heterogenous clinical presentation, onset, and complications. While HCM is generally accepted as a disease of the sarcomere, variable penetrance in families with identical genetic mutations challenges the monogenic origin of HCM and instead implies a multifactorial cause. Furthermore, large-scale genome sequencing studies revealed that many genes previously reported as causative of HCM in fact have little or no evidence of disease association. These findings thus call for a re-evaluation of the sarcomere-centered view of HCM pathogenesis. Here, we summarize our current understanding of sarcomere-independent mechanisms of cardiomyocyte hypertrophy, highlight the role of extracellular signals in cardiac fibrosis, and propose an alternative but integrated model of HCM pathogenesis.

Background: Single cell sequencing of human heart tissue is technically challenging and methods to cryopreserve heart tissue for obtaining single cell information have not been standardized. Studies published to date have used varying methods to preserve and process human heart tissue, and have generated interesting datasets, but development of a biobanking standard has not yet been achieved. Heart transcription patterns are known to be regionally diverse, and there are few single cell datasets for normal human heart tissue., Methods: Using pig tissue, we developed a rigorous and reproducible method for tissue mincing and cryopreservation that allowed recovery of high quality single nuclei RNA. We subsequently tested this protocol on normal human heart tissue obtained from organ donors and were able to recover high quality nuclei for generation of single nuclei RNA-seq datasets, using a commercially available platform from 10× Genomics. We analyzed these datasets using standard software packages such as CellRanger and Seurat., Results: Human heart tissue preserved with our method consistently yielded nuclear RNA with RNA Integrity Numbers of greater than 8.5. We demonstrate the utility of this method for single nuclei RNA-sequencing of the normal human interventricular septum and delineating its cellular diversity. The human IVS showed unexpected diversity with detection of 23 distinct cell clusters that were subsequently categorized into different cell types. Cardiomyocytes and fibroblasts were the most commonly identified cell types and could be further subdivided into 5 different cardiomyocyte subtypes and 6 different fibroblast subtypes that differed by gene expression patterns. Ingenuity Pathway analysis of these gene expression patterns suggested functional diversity in these cell subtypes., Conclusions: Here we report a simple technical method for cryopreservation and subsequent nuclear isolation of human interventricular septum tissue that can be done with common laboratory equipment. We show how this method can be used to generate single nuclei transcriptomic datasets that rival those already published by larger groups in terms of cell diversity and complexity and suggest that this simple method can provide guidance for biobanking of human myocardial tissue for complex genomic analysis.

Left Ventricular Outflow Tract (LVOT) obstruction occurs in approximately 70% of Hypertrophic Cardiomyopathy (HCM) patients and currently requires imaging or invasive testing for diagnosis, sometimes in conjunction with provocative physiological or pharmaceutical stimuli. To identify potential biomarkers of LVOT obstruction, we performed proteomics profiling of 1305 plasma proteins in 12 HCM patients with documented LVOT obstruction, referred for surgical myectomy. Plasma was collected at the surgical preoperative visit, approximately one month prior to surgery and then at the post-surgical visit, approximately 3 months later. Proteomic profiles were generated using the aptamer-based SOMAscan assay. Principal Component Analysis using the highest statistically significant proteins separated all preoperative samples from all postoperative samples. Further analysis revealed a set of 25 proteins that distinguished the preoperative and postoperative states with a paired t -test p -value of <0.01. Ingenuity Pathway analysis facilitated the generation of protein interaction networks and the elucidation of key upstream regulators of differentially expressed proteins, such as interferon-γ, TGF-β1, and TNF. Biological pathways affected by surgery included organ inflammation, migration, and motility of leukocytes, fibrosis, vasculogenesis, angiogenesis, acute coronary events, endothelial proliferation, eicosanoid metabolism, calcium flux, apoptosis, and morphology of the cardiovascular system. Our results indicate that surgical relief of dynamic outflow tract obstruction in HCM patients is associated with unique alterations in plasma proteomic profiles that likely reflect improvement in organ inflammation and physiological function.

A unique clinical circumstance involving middle-aged male identical twins with obstructive hypertrophic cardiomyopathy (HC) is reported. The concordance of morphologic (i.e., phenotype) findings and clinical course between the 2 patients is remarkable, including timing of the onset and progression of heart failure due to left ventricular outflow tract obstruction, frequency of paroxysmal atrial fibrillation and beneficial response to surgical myectomy and Cox-Maze IV procedure (performed 14 days apart). Histopathology of resected ventricular septal muscle showed identical hallmarks of HC including myocyte disorganization, small vessel disease, and myocardial fibrosis. A missense variant of the CRYAB gene was identified as potentially relevant to the pathogenesis of HC in the twins. Taken together, these observations support a powerful genetic determinant for the morphologic and clinical expression of HC, with little or no environmental influence., (Copyright © 2020. Published by Elsevier Inc.)

Tafazzin , an enzyme associated with the rare inherited x-linked disorder Barth Syndrome, is a nuclear encoded mitochondrial transacylase that is highly conserved across multiple species and plays an important role in mitochondrial function. Numerous studies have elucidated the mechanisms by which Tafazzin affects mitochondrial function, but its effects on development and susceptibility to adult disease are incompletely understood. The purpose of this review is to highlight previous functional studies across a variety of model organisms, introduce recent studies that show an important role in development, and also to provide an update on the role of Tafazzin in human disease. The profound effects of Tafazzin on cardiac development and adult cardiac homeostasis will be emphasized. These studies underscore the importance of mitochondrial function in cardiac development and disease, and also introduce the concept of Tafazzin as a potential therapeutic modality.

Inhibition of mammalian target of rapamycin complex I (mTORC1) by rapamycin improves cardiac function in both aging and heart failure. While the protective mechanisms are not fully understood in mammals, they are presumably mediated through metabolic regulation and suppression of protein translation by reduced phosphorylation of 4EBP1, a target of mTORC1. Using transverse aortic constriction (TAC) and Gαq overexpression-induced heart failure models, we examined the effect of cardiac-specific heterozygous deletion (het) of Raptor, a component of mTORC1, and cardiac-specific transgenic overexpression of wild type or phosphorylation site mutant 4EBP1. In wild-type mice with TAC-induced heart failure, quantitative shotgun proteomics revealed decreased abundance of proteins of mitochondrial metabolism and increased abundance of proteins in oxidative stress response, ubiquitin, and other pathways. The Raptor het ameliorated both TAC- and Gαq overexpression-induced heart failure and the associated proteomic remodeling, especially those pathways involved in mitochondrial function, citric acid cycle, and ubiquitination. In contrast, transgenic overexpression of either wild type or mutant 4EBP1 aggravated TAC and Gαq, consistent with reduced adaptive hypertrophy by suppression of protein translation, in parallel with adverse remodeling of left ventricular proteomes. Partial mTORC1 inhibition by Raptor heterozygous deletion ameliorates heart failure and is associated with better preservation of the mitochondrial proteome; however, this effect does not appear to be mediated through suppression of protein translation by increased 4EBP1. Increased activity of 4EBP1 reduced adaptive hypertrophy and aggravated heart failure, suggesting that protein translation is essential for adaptive hypertrophy in pressure overload.

Background: Developmental exposure to particulate matter air pollution is harmful to cardiovascular health, but the mechanisms by which this exposure mediates susceptibility to heart disease is poorly understood. We have previously shown, in a mouse model, that gestational exposure to diesel exhaust (DE) results in increased cardiac hypertrophy, fibrosis and susceptibility to heart failure in the adult offspring following transverse aortic constriction., Results: In this study, we have analyzed gene expression in neonatal cardiomyocytes after gestational exposure by RNA-sequencing and have identified 300 genes that are dysregulated, including many involved in cardiac metabolism. We subsequently determined that these cardiomyocytes exhibit reduced metabolic activity as measured by Seahorse extracellular flux analysis. We also surveyed for modifications in DNA methylation at global regulatory regions using reduced representation bisulfite sequencing and found hypomethylation of DNA in neonatal cardiomyocytes isolated from in utero DE exposed neonates., Conclusion: We have demonstrated that in utero exposure to diesel exhaust alters the neonatal cardiomyocyte transcriptional and epigenetic landscapes, as well as the metabolic capability of these cells. Understanding how exposure alters the developing heart through dysregulation of gene expression, metabolism and DNA methylation is vital for identifying therapeutic interventions for air pollution-related heart failure.

Vascular calcification is the deposition of calcium phosphate minerals in vascular tissue. Vascular calcification occurs by both active and passive processes. Extent and tissue-specific patterns of vascular calcification are predictors of cardiovascular morbidity and mortality. The placenta is a highly vascularized organ with specialized vasculature that mediates communication between two circulatory systems. At delivery the placenta often contains calcified tissue and calcification can be considered a marker of viral infection, but the mechanisms, histoanatomical specificity, and pathophysiological significance of placental calcification are poorly understood. In this review, we outline the current understanding of vascular calcification mechanisms, biomedical consequences, and therapeutic interventions in the context of histoanatomical types. We summarize available placental calcification data and clinical grading systems for placental calcification. We report on studies that have examined the association between placental calcification and acute adverse maternal and fetal outcomes. We then review the intersection between placental dysfunction and long-term cardiovascular health, including subsequent occurrence of maternal vascular calcification. Possible maternal phenotypes and trigger mechanisms that may predispose for calcification and cardiovascular disease are discussed. We go on to highlight the potential diagnostic value of placental calcification. Finally, we suggest avenues of research to evaluate placental calcification as a research model for investigating the relationship between placental dysfunction and cardiovascular health, as well as a biomarker for placental dysfunction, adverse clinical outcomes, and increased risk of subsequent maternal and offspring cardiovascular events.

Mutations in the gene tafazzin (TAZ) result in Barth syndrome (BTHS). Patients present with hypotonia, cyclic neutropenia, 3-methyglutaconic aciduria, and cardiomyopathy, which is the major cause of mortality. The recessive, X-linked TAZ gene encodes a mitochondrial membrane-associated phospholipid modifying enzyme, which adds unsaturated fatty acid species to monolysocardiolipin to generate mature cardiolipin in the mitochondrial membrane that is essential for mitochondrial morphology and function. To identify intrinsic mitochondrial localization sequences in the human TAZ protein, we made sequential TAZ peptide-eGFP fusion protein expression constructs and analyzed the localization of eGFP fluorescence by confocal microscopy. We assessed these fusion proteins for mitochondrial localization through cotransfection of H9c2 cells with plasmids encoding organellar markers linked to TdTomato. We have identified two peptides of TAZ that are independently responsible for mitochondrial localization. Using CRISPR-generated TAZ knock out cell lines, we found that these peptides are able to direct proteins to mitochondria in the absence of endogenous TAZ. These peptides are not located within the predicted enzymatic clefts of TAZ, implying that some BTHS disease causing mutations may affect mitochondrial localization without affecting transacylase activity. These novel peptides improve our understanding of TAZ intracellular trafficking, provide insight into the molecular basis of BTHS and provide molecular reagents for developing targeted mitochondrial therapies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In utero exposure to diesel exhaust air pollution has been associated with increased adult susceptibility to heart failure in mice, but the mechanisms by which this exposure promotes susceptibility to heart failure are poorly understood. To identify the potential transcriptional effects that mediate this susceptibility, we have performed RNA sequencing analysis on adult hearts from mice that were exposed to diesel exhaust in utero and that have subsequently undergone transverse aortic constriction. We identified 3 target genes, Mir133a-2 , Ptprf , and Pamr1 , which demonstrate dysregulation after exposure and aortic constriction. Examination of expression patterns in human heart tissues indicates a correlation between expression and heart failure. We subsequently assessed DNA methylation modifications at these candidate loci in neonatal cultured cardiac myocytes after in utero exposure to diesel exhaust and found that the promoter for Mir133a-2 is differentially methylated. These target genes in the heart are the first genes to be identified that likely play an important role in mediating adult sensitivity to heart failure. We have also shown a change in DNA methylation within cardiomyocytes as a result of in utero exposure to diesel exhaust.-Goodson, J. M., Weldy, C. S., MacDonald, J. W., Liu, Y., Bammler, T. K., Chien, W.-M., Chin, M. T. In utero exposure to diesel exhaust particulates is associated with an altered cardiac transcriptional response to transverse aortic constriction and altered DNA methylation., (© FASEB.)

A number of copper reagents were compared for their effectiveness in trifluoromethylating 4-iodobiphenyl, 4-iodotoluene, and 2-iodotoluene. Yields over time were plotted in order to refine our understanding of each reagent performance, identify any bottlenecks, and provide more insight into the rates of the reactions. Interestingly, differences in reactivity were observed when a well-defined [LCuCF 3 ] complex was employed directly or generated in situ from precursors by published reports. Relative reactivities were also found to highly dependent on the nature of the iodoarenes.

The technique of Cre-mediated DNA recombination at loxP sites has been used widely in manipulation of the genome in cultured cells and in living organisms. Local delivery of Cre recombinase protein tagged with a cell-penetrating (or permeable) peptide (Cre-CPP) has the advantage of additional spatial and temporal control when compared to genetic delivery methods. In this chapter, we describe protocols for injection-based intramuscular delivery of Cre-CPP dissolved in hydrogel to skeletal muscle and by ultrasound-guided injection to cardiac muscle in mice.

The new organonickel complexes [(R-PyMA)Ni(Mes)X] [R-PyMA = N-aryl-1-(pyridin-2-yl)methanimine; aryl = phenyl, 2,6-Me 2 -, 3,5-Me 2 -, 2,4,6-Me 3 -, 2,6- i Pr 2 -, 3,5-(OMe) 2 -, 2-NO 2 -4-Me-, 4-NO 2 -, 2-CF 3 -, and 2-CF 3 -6-F-phenyl; Mes = 2,4,6-trimethylphenyl; X = F, Cl, Br, or I] were obtained as approximate 1/1 cis and trans isomeric mixtures or pure cis isomers depending on the PyMA ligand and X. The [(R-PyMA)Ni(Mes)X] complexes with X = Br or Cl were directly synthesized from the precursors trans-[(PPh 3 ) 2 Ni(Mes)X], while [(PyMA)Ni(Mes)X] derivatives with X = F or I were obtained from [(PyMA)Ni(Mes)Br] through X exchange reactions. Although density functional theory (DFT) calculations show a preference for the sterically favored cis isomers, both isomers could be observed in many cases; in three cases, even single crystals for X-ray diffraction could be obtained for the trans isomers. Possible intermediates for the isomerization were investigated by DFT calculations. All complexes were studied by multiple spectroscopic means, electrochemistry, and spectroelectrochemistry (for the reduction processes). The long-wavelength metal-to-ligand charge-transfer (MLCT) absorptions vary markedly with the R substituent of the ligand and the cathodic electrochemical potentials to a far smaller degree. Both are almost invariable upon variation of X. All of this is in line with Ni-based and π*-based lowest unoccupied molecular orbitals (LUMOs). In line with the unsymmetric character of the N Py ^N methanimine ligand, electrochemistry and MLCT transitions seem to not correspond to the same type of π* LUMO, making these PyMA ligands more interesting than the symmetric heteroaromatic polypyridine ligands such as 2,2'-bipyridine (bpy; N Py ^N Py ) and N,N-diaryl-substituted aliphatic α-diimines (N methanimine ^N methanimine ) such as the diaza-1,3-butadienes (DAB). First attempts to use these complexes in Negishi-type cross-coupling reactions were successful.

Background: We have previously reported that in utero and early life exposure to diesel exhaust particulates predisposes mice to adult heart failure, and that in utero exposure alone is sufficient to confer this predisposition. This follow up study addresses whether neonatal exposure alone can also confer this predisposition. Methods: Newborn male C57BL/6 mice were exposed to diesel exhaust (DE) particulates immediately after birth until weaning at 21 days of age, whereupon they were transferred to filtered air (FA) conditions. At the age of 12 weeks, transverse aortic constriction (TAC) was performed followed by weekly echocardiography for three weeks. After the last echocardiogram, mice were euthanized for organ harvest, gravimetry and histology. Results: Neonatal exposure to DE particulates did not increase susceptibility to cardiac hypertrophy or heart failure after TAC when compared to FA exposed controls (ventricular weight/body weight ratio 7.505 vs. 7.517 mg/g, p = Not Significant (NS)). The left ventricular ejection fraction after TAC was similar between groups at one week, two weeks, and three weeks after procedure. Histological analysis showed no difference in the degree of cardiac hypertrophy or fibrosis. Conclusions: Neonatal exposure to DE particulates does not predispose mice to TAC-induced cardiac hypertrophy and heart failure in adulthood, in contrast to previously published results showing susceptibility due to in utero exposure., Competing Interests: The authors declare no conflict of interest.

Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

Exposure to air pollution has long been known to trigger cardiovascular events, primarily through activation of local and systemic inflammatory pathways that affect the vasculature. Detrimental effects of air pollution exposure on heart failure and cardiac remodeling have also been described in human populations. Recent studies in both human subjects and animal models have provided insights into the basic physiological, cellular and molecular mechanisms that play a role in adverse cardiac remodeling. This review will give a brief overview of the relationship between air pollution and cardiovascular disease, describe the clinical effects of air pollution exposure on cardiac remodeling, describe the basic mechanisms that affect remodeling as described in human and animal systems and will discuss future areas of investigation.

Air pollution is a significant cause of cardiovascular morbidity and mortality worldwide. Although the epidemiologic association between air pollution exposures and exacerbation of cardiovascular disease (CVD) is well established, the mechanisms by which these exposures promote CVD are incompletely understood. This review provides an overview of the components of air pollution, an overview of the cardiovascular effects of air pollution exposure, and a review of the basic mechanisms that are activated by exposure to promote CVD., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Purpose: Dabrafenib is a selective, potent ATP-competitive inhibitor of the BRAFV600-mutant kinase that has demonstrated efficacy in clinical trials. We report the rationale for dose selection in the first-in-human study of dabrafenib, including pharmacokinetics, tissue pharmacodynamics, 2[18F]fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) pharmacodynamics, and dose-response relationship., Experimental Design: Dabrafenib was administered orally once, twice (BID), or three times daily (TID). Selected dose cohorts were expanded to collect adequate data on safety, pharmacokinetics, or pharmacodynamics. A recommended phase II dose (RP2D) was chosen based on safety, pharmacokinetic, pharmacodynamic, and response data., Results: One hundred and eighty-four patients were enrolled and treated with doses ranging from 12 mg once daily to 300 mg BID in 10 cohorts. Pharmacokinetic assessment of dabrafenib demonstrated a less-than-dose-proportional increase in exposure after repeat dosing above 150 mg BID. Similar to parent drug concentrations, exposure for all metabolites demonstrated less-than-dose-proportional increases. Predicted target inhibition of pERK (>80%) was achieved at 150 mg BID, with a similar magnitude of inhibition at higher doses in BRAFV600 mutation melanoma biopsy samples. Although there was large variability between patients, FDG uptake decreased with higher daily doses in patients with BRAFV600 mutation-positive melanoma. A favorable activity and tolerability profile was demonstrated at 150 mg BID. There was no improvement with TID dosing compared with BID dosing, based on FDG-PET and tumor response analyses in patients with melanoma., Conclusion: The RP2D of dabrafenib was determined to be 150 mg BID after considering multiple factors, including pharmacokinetics, tissue pharmacodynamics, FDG-PET pharmacodynamics, and the dose-response relationship. A maximum tolerated dose for dabrafenib was not determined., (©2014 American Association for Cancer Research.)

Direct reprogramming of adult, lineage-determined cells from one cell fate to another has long been an elusive goal in developmental biology. Recent studies have demonstrated that forced expression of lineage-specific transcription factors in various differentiated cell types can promote the adoption of different lineages. These seminal findings have the potential to revolutionize the field of regenerative medicine by providing replacement cells for various degenerative disorders. Current reprogramming protocols, however, are inefficient in that relatively few cells in a given population can be made to undergo reprogramming and the completeness and extent of reprogramming that occurs has been questioned. At present, the fundamental molecular mechanisms involved are still being elucidated. Although the potential clinical applications are extensive, these issues will need to be addressed before direct reprogramming may be used clinically. This review will give an overview of pioneering studies in the field, will describe what is known about direct reprogramming to specific lineage types, will summarize what is known about the molecular mechanisms involved in reprogramming and will discuss challenges for the future.

CHF1/Hey2 is a Notch-responsive basic helix-loop-helix transcription factor involved in cardiac development. Common variants in Hey2 are associated with Brugada syndrome. We hypothesized that absence of CHF1/Hey2 would result in abnormal cellular electrical activity, altered cardiac conduction system (CCS) development, and increased arrhythmogenesis. We isolated neonatal CHF/Hey2-knockout (KO) cardiac myocytes and measured action potentials and ion channel subunit gene expression. We also crossed myocardial-specific CHF1/Hey2-KO mice with cardiac conduction system LacZ reporter mice and stained for conduction system tissue. We also performed ambulatory ECG monitoring for arrhythmias and heart rate variability. Neonatal cardiomyocytes from CHF1/Hey2-KO mice demonstrate a 50% reduction in action potential dV/dT, a 50-75% reduction in SCN5A, KCNJ2, and CACNA1C ion channel subunit gene expression, and an increase in delayed afterdepolarizations from 0/min to 12/min. CHF1/Hey2 cKO CCS-lacZ mice have a ∼3-fold increase in amount of CCS tissue. Ambulatory ECG monitoring showed no difference in cardiac conduction, arrhythmias, or heart rate variability. Wild-type cells or animals were used in all experiments. CHF1/Hey2 may contribute to Brugada syndrome by influencing the expression of SCN5A and formation of the cardiac conduction system, but its absence does not cause baseline conduction defects or arrhythmias in the adult mouse.-Hartman, M. E., Liu, Y., Zhu, W.-Z., Chien, W.-M., Weldy, C. S., Fishman, G. I., Laflamme, M. A., Chin, M. T. Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias., (© FASEB.)

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation.