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Background: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy with a high prevalence of ventricular arrhythmias. How the R14 deletion causes dilated cardiomyopathy is poorly understood, and there are no disease-specific therapies. Methods: We used single-cell RNA sequencing to uncover PLN R14del disease mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We used both 2-dimensional and 3-dimensional functional contractility assays to evaluate the impact of modulating disease-relevant pathways in PLN R14del hiPSC-CMs. Results: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response (UPR) pathway in PLN R14del compared with isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the 3 main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP (binding immunoglobulin protein) inducer X. PLN R14del hiPSC-CMs treated with BiP protein inducer X showed a dose-dependent amelioration of the contractility deficit in both 2-dimensional cultures and 3-dimensional engineered heart tissues without affecting calcium homeostasis. Conclusions: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

Background: The clinical outcome of hypertrophic cardiomyopathy patients is not only determined by the disease-causing mutation but influenced by a variety of disease modifiers. Here, we defined the role of the mutation location and the mutant protein dose of the troponin T mutations I79N, R94C and R278C. Methods and results: We determined myofilament function after troponin exchange in permeabilized single human cardiomyocytes as well as in cardiac patient samples harboring the R278C mutation. Notably, we found that a small dose of mutant protein is sufficient for the maximal effect on myofilament Ca2+-sensitivity for the I79N and R94C mutation while the mutation location determines the magnitude of this effect. While incorporation of I79N and R94C increased myofilament Ca2+-sensitivity, incorporation of R278C increased Ca2+-sensitivity at low and intermediate dose, while it decreased Ca2+-sensitivity at high dose. All three cTnT mutants showed reduced thin filament binding affinity, which coincided with a relatively low maximal exchange (50.5 ± 5.2%) of mutant troponin complex in cardiomyocytes. In accordance, 32.2 ± 4.0% mutant R278C was found in two patient samples which showed 50.0 ± 3.7% mutant mRNA. In accordance with studies that showed clinical variability in patients with the exact same mutation, we observed variability on the functional single cell level in patients with the R278C mutation. These differences in myofilament properties could not be explained by differences in the amount of mutant protein. Conclusions: Using troponin exchange in single human cardiomyocytes, we show that TNNT2 mutation-induced changes in myofilament Ca2+-sensitivity depend on mutation location, while all mutants show reduced thin filament binding affinity. The specific mutation-effect observed for R278C could not be translated to myofilament function of cardiomyocytes from patients, and is most likely explained by other (post)-translational troponin modifications. Overall, our studies illustrate that mutation location underlies variability in myofilament Ca2+-sensitivity, while only the R278C mutation shows a highly dose-dependent effect on myofilament function.

BACKGROUND: Calcific aortic valve disease (CAVD) is a rapidly growing global health problem with an estimated 12.6 million cases globally in 2017 and a 112% increase of deaths since 1990 due to aging and population growth. CAVD may develop into aortic stenosis (AS) by progressive narrowing of the aortic valve. AS is underdiagnosed, and if treatment by aortic valve replacement (AVR) is delayed, this leads to poor recovery of cardiac function, absence of symptomatic improvement and marked increase of mortality. Considering the current limitations to define the stage of AS-induced cardiac remodeling, there is need for a novel method to aid in the diagnosis of AS and timing of intervention, which may be found in metabolomics profiling of patients.METHODS: Serum samples of nine healthy controls and 10 AS patients before and after AVR were analyzed by untargeted mass spectrometry. Multivariate modeling was performed to determine a metabolic profile of 30 serum metabolites which distinguishes AS patients from controls. Human cardiac microvascular endothelial cells (CMECs) were incubated with serum of the AS patients and then stained for ICAM-1 with Western Blot to analyze the effect of AS patient serum on endothelial cell activation.RESULTS: The top 30 metabolic profile strongly distinguishes AS patients from healthy controls and includes 17 metabolites related to nitric oxide metabolism and 12 metabolites related to inflammation, in line with the known pathomechanism for calcific aortic valve disease. Nine metabolites correlate strongly with left ventricular mass, of which three show reversal back to control values after AVR. Western blot analysis of CMECs incubated with AS patient sera shows a significant reduction (14%) in ICAM-1 in AS samples taken after AVR compared to AS patient sera before AVR.CONCLUSION: Our study defined a top 30 metabolic profile with biological and clinical relevance, which may be used as blood biomarker to identify AS patients in need of cardiac surgery. Future studies are warranted in patients with mild-to-moderate AS to determine if these metabolites reflect disease severity and can be used to identify AS patients in need of cardiac surgery.

Background Nowadays, multiple omics data are measured on the same samples in the belief that these different omics datasets represent various aspects of the underlying biological systems. Integrating these omics datasets will facilitate the understanding of the systems. For this purpose, various methods have been proposed, such as Partial Least Squares (PLS), decomposing two datasets into joint and residual subspaces. Since omics data are heterogeneous, the joint components in PLS will contain variation specific to each dataset. To account for this, Two-way Orthogonal Partial Least Squares (O2PLS) captures the heterogeneity by introducing orthogonal subspaces and better estimates the joint subspaces. However, the latent components spanning the joint subspaces in O2PLS are linear combinations of all variables, while it might be of interest to identify a small subset relevant to the research question. To obtain sparsity, we extend O2PLS to Group Sparse O2PLS (GO2PLS) that utilizes biological information on group structures among variables and performs group selection in the joint subspace. Results The simulation study showed that introducing sparsity improved the feature selection performance. Furthermore, incorporating group structures increased robustness of the feature selection procedure. GO2PLS performed optimally in terms of accuracy of joint score estimation, joint loading estimation, and feature selection. We applied GO2PLS to datasets from two studies: TwinsUK (a population study) and CVON-DOSIS (a small case-control study). In the first, we incorporated biological information on the group structures of the methylation CpG sites when integrating the methylation dataset with the IgG glycomics data. The targeted genes of the selected methylation groups turned out to be relevant to the immune system, in which the IgG glycans play important roles. In the second, we selected regulatory regions and transcripts that explained the covariance between regulomics and transcriptomics data. The corresponding genes of the selected features appeared to be relevant to heart muscle disease. Conclusions GO2PLS integrates two omics datasets to help understand the underlying system that involves both omics levels. It incorporates external group information and performs group selection, resulting in a small subset of features that best explain the relationship between two omics datasets for better interpretability.

Supplemental Digital Content is available in the text., Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ≈50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, HCMSMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, HCMSMN). Genotype-specific differences have been reported in cardiac function. Moreover, HCMSMN patients have later disease onset and a better prognosis than HCMSMP patients. To define if genotype-specific derailments at the protein level may explain the heterogeneity in disease development, we performed a proteomic analysis in cardiac tissue from a clinically well-phenotyped HCM patient group. Methods: A proteomics screen was performed in cardiac tissue from 39 HCMSMP patients, 11HCMSMN patients, and 8 nonfailing controls. Patients with HCM had obstructive cardiomyopathy with left ventricular outflow tract obstruction and diastolic dysfunction. A novel MYBPC32373insG mouse model was used to confirm functional relevance of our proteomic findings. Results: In all HCM patient samples, we found lower levels of metabolic pathway proteins and higher levels of extracellular matrix proteins. Levels of total and detyrosinated α-tubulin were markedly higher in HCMSMP than in HCMSMN and controls. Higher tubulin detyrosination was also found in 2 unrelated MYBPC3 mouse models and its inhibition with parthenolide normalized contraction and relaxation time of isolated cardiomyocytes. Conclusions: Our findings indicate that microtubules and especially its detyrosination contribute to the pathomechanism of patients with HCMSMP. This is of clinical importance since it represents a potential treatment target to improve cardiac function in patients with HCMSMP, whereas a beneficial effect may be limited in patients with HCMSMN.

Background H3K27ac histone acetylome changes contribute to the phenotypic response in heart diseases, particularly in end-stage heart failure. However, such epigenetic alterations have not been systematically investigated in remodeled non-failing human hearts. Therefore, valuable insight into cardiac dysfunction in early remodeling is lacking. This study aimed to reveal the acetylation changes of chromatin regions in response to myocardial remodeling and their correlations to transcriptional changes of neighboring genes. Results We detected chromatin regions with differential acetylation activity (DARs; Padj. < 0.05) between remodeled non-failing patient hearts and healthy donor hearts. The acetylation level of the chromatin region correlated with its RNA polymerase II occupancy level and the mRNA expression level of its adjacent gene per sample. Annotated genes from DARs were enriched in disease-related pathways, including fibrosis and cell metabolism regulation. DARs that change in the same direction have a tendency to cluster together, suggesting the well-reorganized chromatin architecture that facilitates the interactions of regulatory domains in response to myocardial remodeling. We further show the differences between the acetylation level and the mRNA expression level of cell-type-specific markers for cardiomyocytes and 11 non-myocyte cell types. Notably, we identified transcriptome factor (TF) binding motifs that were enriched in DARs and defined TFs that were predicted to bind to these motifs. We further showed 64 genes coding for these TFs that were differentially expressed in remodeled myocardium when compared with controls. Conclusions Our study reveals extensive novel insight on myocardial remodeling at the DNA regulatory level. Differences between the acetylation level and the transcriptional level of cell-type-specific markers suggest additional mechanism(s) between acetylome and transcriptome. By integrating these two layers of epigenetic profiles, we further provide promising TF-encoding genes that could serve as master regulators of myocardial remodeling. Combined, our findings highlight the important role of chromatin regulatory signatures in understanding disease etiology.

Microfluidic organ-on-a-chip designs are used to mimic human tissues, including the vasculature. Here we present a novel microfluidic device that allows the interaction of endothelial cells (ECs) with pericytes and the extracellular matrix (ECM) in full bio-matrix encased 3D vessel structures (neovessels) that can be subjected to continuous, unidirectional flow and perfusion with circulating immune cells. We designed a polydimethylsiloxane (PDMS) device with a reservoir for a 3D fibrinogen gel with pericytes. Open channels were created for ECs to form a monolayer. Controlled, continuous, and unidirectional flow was introduced via a pump system while the design facilitated 3D confocal imaging. In this vessel-on-a-chip system, ECs interact with pericytes to create a human cell derived blood vessel which maintains a perfusable lumen for up to 7 days. Dextran diffusion verified endothelial barrier function while demonstrating the beneficial role of supporting pericytes. Increased permeability after thrombin stimulation showed the capacity of the neovessels to show natural vascular response. Perfusion of neovessels with circulating THP-1 cells demonstrated this system as a valuable platform for assessing interaction between the endothelium and immune cells in response to TNFα. In conclusion: we created a novel vascular microfluidic device that facilitates the fabrication of an array of parallel soft-channel structures in ECM gel that develop into biologically functional neovessels without hard-scaffold support. This model provides a unique tool to conduct live in vitro imaging of the human vasculature during perfusion with circulating cells to mimic (disease) environments in a highly systematic but freely configurable manner.

Aims Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis. Methods and results Using short interference RNA-mediated loss-of-function assays, the function and mechanism of signaling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neovessel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G0/G1 cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of canonical Wnt signaling, non-canonical Wnt/Ca2+-mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1. Conclusions The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2. Electronic supplementary material The online version of this article (10.1007/s10456-018-9625-6) contains supplementary material, which is available to authorized users.

Aims The formation of cell–cell and cell–extra cellular matrix (ECM) contacts by endothelial cells (ECs) is crucial for the stability and integrity of a vascular network. We previously identified cingulin-like 1 (Cgnl1) in a transcriptomic screen for new angiogenic modulators. Here we aim to study the function of the cell–cell junction associated protein Cgnl1 during vessel formation. Methods and results Unlike family member cingulin, Cgnl1 expression is enriched in ECs during vascular growth. Cgnl1 is important for the formation of multicellular tubule structures, as shown in vitro using loss-of function assays in a 3D matrix co-culture system that uses primary human ECs and supporting mural cells. Further studies revealed that Cgnl1 regulates vascular growth by promoting Ve-cadherin association with the actin cytoskeleton, thereby stabilizing adherens junctions. Cgnl1 also regulates focal adhesion assembly in response to ECM contact, promoting vinculin and paxillin recruitment and focal adhesion kinase signalling. In vivo, we demonstrate in a postnatal retinal vascular development model in mice that Cgnl1 function is crucial for sustaining neovascular growth and stability. Conclusions Our data demonstrate a functional relevance for Cgnl1 as a defining factor in new vessel formation both in vitro and in vivo.

Indoxyl sulfate (IS) is an accumulative protein-bound uremic toxin found in patients with kidney disease. It is reported that IS impairs the vascular endothelium, but a comprehensive overview of all mechanisms active in IS-injury currently remains lacking. Here we performed RNA sequencing in human umbilical vein endothelial cells (HUVECs) after IS or control medium treatment and identified 1293 genes that were affected in a IS-induced response. Gene enrichment analysis highlighted pathways involved in altered vascular formation and cell metabolism. We confirmed these transcriptome profiles at the functional level by demonstrating decreased viability and increased cell senescence in response to IS treatment. In line with the additional pathways highlighted by the transcriptome analysis, we further could demonstrate that IS exposure of HUVECs promoted tubule formation as shown by the increase in total tubule length in a 3D HUVECs/pericytes co-culture assay. Notably, the pro-angiogenic response of IS and increased ROS production were abolished when CYP1B1, one of the main target genes that was highly upregulated by IS, was silenced. This observation indicates IS-induced ROS in endothelial cells is CYP1B1-dependent. Taken together, our findings demonstrate that IS promotes angiogenesis and CYP1B1 is an important factor in IS-activated angiogenic response.

A typical task arising from main effect analyses in a Genome Wide Association Study (GWAS) is to identify single nucleotide polymorphisms (SNPs), in linkage disequilibrium with the observed signals, that are likely causal variants and the affected genes. The affected genes may not be those closest to associating SNPs. Functional genomics data from relevant tissues are believed to be helpful in selecting likely causal SNPs and interpreting implicated biological mechanisms, ultimately facilitating prevention and treatment in the case of a disease trait. These data are typically used post GWAS analyses to fine-map the statistically significant signals identified agnostically by testing all SNPs and applying a multiple testing correction. The number of tested SNPs is typically in the millions, so the multiple testing burden is high. Motivated by this, in this study we investigated an alternative workflow, which consists in utilizing the available functional genomics data as a first step to reduce the number of SNPs tested for association. We analyzed GWAS on electrocardiographic QRS duration using these two workflows. The alternative workflow identified more SNPs, including some residing in loci not discovered with the typical workflow. Moreover, the latter are corroborated by other reports on QRS duration. This indicates the potential value of incorporating functional genomics information at the onset in GWAS analyses.

Background: Regulatory elements may be involved in the mechanisms by which 52 loci influence myocardial mass, reflected by abnormal amplitude and duration of the QRS complex on the ECG. Functional annotation thus far did not take into account how these elements are affected in disease context. Methods: We generated maps of regulatory elements on hypertrophic cardiomyopathy patients (ChIP-seq N=14 and RNA-seq N=11) and nondiseased hearts (ChIP-seq N=4 and RNA-seq N=11). We tested enrichment of QRS-associated loci on elements differentially acetylated and directly regulating differentially expressed genes between hypertrophic cardiomyopathy patients and controls. We further performed functional annotation on QRS-associated loci using these maps of differentially active regulatory elements. Results: Regions differentially affected in disease showed a stronger enrichment ( P =8.6×10 −5 ) for QRS-associated variants than those not showing differential activity ( P =0.01). Promoters of genes differentially regulated between hypertrophic cardiomyopathy patients and controls showed more enrichment ( P =0.001) than differentially acetylated enhancers ( P =0.8) and super-enhancers ( P =0.025). We also identified 74 potential causal variants overlapping these differential regulatory elements. Eighteen of the genes mapped confirmed previous findings, now also pinpointing the potentially affected regulatory elements and candidate causal variants. Fourteen new genes were also mapped. Conclusions: Our results suggest differentially active regulatory elements between hypertrophic cardiomyopathy patients and controls can offer more insights into the mechanisms of QRS-associated loci than elements not affected by disease.

BACKGROUND: Mutations in MYBPC3 are the most common cause of hypertrophic cardiomyopathy (HCM). These mutations produce dysfunctional protein that is quickly degraded and not incorporated in the myofilaments. Most patients are heterozygous and allelic expression differs between cells. We hypothesized that this would lead to cell-to-cell variation in cardiac myosin binding protein-C (cMyBP-C, encoded by MYBPC3 gene) protein levels.METHODS: Twelve HCM patients were included (six had no sarcomere mutations (HCMsmn) and served as the control group and six harbored mutations in the MYBPC3 gene (MYBPC3mut). Western blot and RNA sequencing analysis of cardiac tissue lysates were performed to detect overall cMyBP-C protein and mRNA levels. Cellular expression of cMyBP-C and α-actin was obtained by immunofluorescence staining. Quantification of cell-to-cell variation of cMyBP-C expression between cardiomyocytes was measured by determining the ratio of cMyBP-C:α-actin stained area of each cell.RESULTS: Protein and mRNA analysis revealed significantly reduced cMyBP-C levels in MYBPC3mut patients compared with HCMsmn patients (0.73 ± 0.09 vs. 1.0 ± 0.15, p CONCLUSION: This is the first study to demonstrate intercellular variation of myofilament cMyBP-C protein expression within the myocardium from HCM patients with heterozygous MYBPC3 mutations.

The lymphatic system plays a crucial role in interstitial fluid drainage, lipid absorption, and immunological defense. Lymphatic dysfunction results in lymphedema, fluid accumulation, and swelling of soft tissues, as well as a potentially impaired immune response. Lymphedema significantly reduces quality of life of patients on a physical, mental, social, and economic basis. Current therapeutic approaches in treatment of lymphatic disease are limited. Over the last decades, great progress has been made in the development of therapeutic strategies to enhance vascular regeneration. These solutions to treat vascular disease may also be applicable in the treatment of lymphatic diseases. Comparison of the organogenic process and biological organization of the vascular and lymphatic systems and studies in the regulatory mechanisms involved in lymphangiogenesis and angiogenesis show many common features. In this study, we address the similarities between both transport systems, and focus in depth on the biology of lymphatic development. Based on the current advances in vascular regeneration, we propose different strategies for lymphatic tissue engineering that may be used for treatment of primary and secondary lymphedema.

Female gender, post-menopause, chronic kidney disease (CKD) and (CKD linked) microvascular disease are important risk factors for developing heart failure with preserved ejection fraction (HFpEF). Enhancing our understanding of the interrelation between these risk factors could greatly benefit the identification of new drug targets for future therapy. This review discusses the evidence for the protective role of estradiol (E2) in CKD-associated microvascular disease and related HFpEF. Elevated circulating levels of uremic toxins (UTs) during CKD may act in synergy with hormonal changes during post-menopause and could lead to coronary microvascular endothelial dysfunction in HFpEF. To elucidate the molecular mechanism involved, published transcriptome datasets of indoxyl sulfate (IS), high inorganic phosphate (HP) or E2 treated human derived endothelial cells from the NCBI Gene Expression Omnibus database were analyzed. In total, 36 genes overlapped in both IS- and HP-activated gene sets, 188 genes were increased by UTs (HP and/or IS) and decreased by E2, and 572 genes were decreased by UTs and increased by E2. Based on a comprehensive in silico analysis and literature studies of collected gene sets, we conclude that CKD-accumulated UTs could negatively impact renal and cardiac endothelial homeostasis by triggering extensive inflammatory responses and initiating dysregulation of angiogenesis. E2 may protect (myo)endothelium by inhibiting UTs-induced inflammation and ameliorating UTs-related uremic bleeding and thrombotic diathesis via restored coagulation capacity and hemostasis in injured vessels.