tbuckley/cabot-search huggingface dataset:
Connecting the dots: molecular pathways in bicuspid aortopathy explored
Authors: B Pozo-Vilumbrales, M T Soto-Navarrete, L Martin-Chaves, M A Lopez-Unzu, J Pavon-Moron, J Rodriguez-Capitan, B Fernandez
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía, Ministerio de Ciencia e Innovación Background Bicuspid aortic valve (BAV) is the most common congenital cardiac malformation. It predisposes to thoracic aortic dilatation (TAD), aneurysm and dissection. The current consensus is that the genetic defect that leads to the aortic valve malformation also causes structural anomalies of the ascending aorta. Altered hemodynamics caused by the abnormal valve morphology would accelerate aortic media degeneration. Molecular markers and pathways involved in the aetiology and pathophysiology of bicuspid aortopathy are poorly understood. Objective To elucidate the molecular and cellular mechanisms of the disease and to identify potential predictive molecular markers using a well-established isogenic hamster model (T strain) with a high (∼40%) incidence of BAV TAD. Methods Comparative quantitative proteomics combined with Western blot and morpho-molecular analyses in the ascending aorta of tricuspid aortic valve (TAV) and BAV animals from the T strain and TAV animals from a control strain. This strategy allows exploring independently the genetic and hemodynamic effects on the aorta in genetically homogeneous populations. Results The major molecular defect in the aorta of genetically homogeneous BAV individuals is PI3K/AKT overactivation caused by alterations in the EGF, ANGII and TGF-β signalling pathways. PI3K/AKT affects the downstream eNOS, MAP2K1/2, NF-κB, mTOR and WNT pathways. Most of these alterations are seen in independent patient studies with different clinical presentations, but not in TAV hamsters from the T strain, which mainly show downregulation of the WNT pathway. Conclusions We identify a combination of defective interconnected molecular pathways, directly linked to the central PI3K/AKT pathway, common to both BAV-associated TAD patients and hamsters. The defects indicate a shift of smooth muscle cells towards the synthetic phenotype induced by endothelial-to-mesenchymal transition, oxidative stress and inflammation. WNT signalling represents a genetic factor that may cause aortic structural abnormalities and aneurysm predisposition, whereas hemodynamics is the main trigger of molecular changes, likely determining the progression of aortopathy. We identify twenty-seven novel potential biomarkers with high predictive value.
Long-range gene editing of LMNA
Authors: A A M B Rooijakkers, Y M Pinto, A J M Tijsen
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Hartstichting (Dutch Heart Foundation) Background Mutations in the LMNA gene commonly affect the cardiac muscle tissue, which leads to the development of a severe dilated cardiomyopathy with life-threatening arrhythmias. The lack of response to conventional heart failure therapy in these patients indicates the urgent need for new therapies. Gene editing has the potential to become a curative treatment for these patients by correcting the genetic cause of the disease. Prime editing is a promising new gene editing technology, which uses a prime editing guide RNA (pegRNA) to target a specific genetic locus and creates edits using a reverse-transcriptase enzyme linked to a Cas9-nickase. Purpose Conventional prime-editing technology targets one mutation per pegRNA. However, with more than 600 disease-causing mutations described in LMNA, the feasibility of applying this technology to each mutation is limited. Therefore, we aim to target not a single, but a set of mutations with one single pegRNA. Methods The pegRNAs are optimised by selecting the optimal target regions, improving their stability and 3D structure. The in vitro editing efficiency is assessed using the fluoPEER reporter plasmid, transfected into HEK293T cells. Using flow cytometry, expression of mCherry fluorescent protein is detected following successful editing of the target mutation. Results Flow-cytometry analysis of treated cells revealed an editing efficiency of 12% with a long-range pegRNA targeting a full exon, compared to 15% with a conventional short pegRNA targeting the same mutation &amp;lt;10bp downstream of the Cas9 nick site. This editing efficiency using the same long-range pegRNA was retained at 9% when editing at a distant position &amp;gt;100bp downstream of the nick site. To increase the editing efficiency, we improved the stability of the pegRNAs by the addition of different 3’ structural motifs. This resulted in an increased editing efficiencies of 15% and 17% for nearby and distant mutations, respectively. Conclusion We show that it is possible to edit different mutations within a range of up to 120 bp with a single pegRNA. By optimisations of the pegRNA 3D structure and optimal selection of target locations we aim to further improve the editing efficiency of this system. This work could eventually provide a prime editing therapy able to cure disease in a subset of patients carrying different mutations in the same genetic region. For LMNA-associated diseases this would substantially reduce the number of pegRNAs that have to be developed to cover all mutations, greatly improving the feasibility of prime editing therapy.
Preoperative examination of inflammatory biomarkers in relation to the prognosis of postoperative complications after heart transplantation
Authors: Z Tarjanyi, D Bernath, A Szappanos, T Teszak, A Kiraly, S Z Kugler, E Pallinger, G Y Nagy, B Sax, B Merkely
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): Semmelweis University Heart transplantation (HTX) is the definitive therapy for end-stage heart failure. Although complications have drastically decreased with modern immunosuppressive therapy, surgical and anesthetic techniques, significant complications such as graft failure, acute rejection or infection must still be expected. The purpose of the study is to examine cytokine pathways linked to inflammation from direct preoperative serum samples, which can serve as prognostic factors in relation to septic events and mortality in the early postoperative period. The study was conducted using the blood samples of 102 patients who underwent HTX. Blood samples were taken from the patients immediately before HTX. The amounts of a total of 32 cytokines and related proteins were determined using LEGENDplex assays. After that, we correlated the biomarker amounts determined in the serum samples with the end points we indicated. In patients undergoing HTX who developed septic events during postoperative intensive care, the preoperative MMP2 serum concentration was significantly higher compared to other patients undergoing HTX (n=23, p=0.04). Independent predictors of mortality in the intensive care unit (n=5) were preoperative MRP8/14 (AUC=0.8), NGAL (AUC=0.75), IGFBP-4 (AUC=0.73), cystatin C (AUC=0.7) and the MMP2 (AUC=0.74) serum concentrations. Patients transplanted from left ventricular mechanical circulatory support (LVAD) (n=11) were examined as a separate group, where early postoperative mortality was significantly higher (n=4, p=0.02), the independent predictor of which was the concentration of IGFBP-4 (p=0.04 , AUC=0.73). Based on our results, it can be said that the differences in the preoperative concentration of certain inflammatory biomarkers that can be measured in the serum show correlations with the postoperative septic events and mortality of patients undergoing HTX.
Hypertension-induced cardiac pro-arrhythmic disorders of connexin-43 and extracellular matrix are attenuated in hairless versus wild-type of spontaneously hypertensive rats
Authors: K Andelova, M Sykora, B Szeiffova Bacova, V Farkasova, T Stankovicova, V Knezl, M Pravenec, N Tribulova
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Development Agency under the contract no. 21-0410 VEGA grants no. 2/0002/20, 2/0006/23 Background Mechanical dysfunction and occurrence of malignant ventricular fibrillation (VF) are hallmarks of hypertensive heart disease and major health issue. Spontaneously hypertensive rats (SHR) that imitates human primary hypertension are prone to develop VF, mainly due to myocardial remodelling associated with disorders of electrical coupling protein connexin-43 (Cx43) and extracellular matrix (ECM) resulting in electrical instability. However, it is not known whether postnatal acclimation of hairless strain of SHR to surrounding temperature may induce cardio-protection. Therefore, we aimed to explore Cx43 and ECM alterations as well as susceptibility to inducible VF in hairless versus wild type SHR. Design and Methods Six-month-old wild type SHR males and females and hairless SHR housed at standard 22°C were examined. Biometric parameters, including electro- and echocardiography were registered. Left ventricular tissue was used for immunolabeling of Cx43 to reveal its topology and for western blotting to determine protein levels of Cx43 and its function modulating protein kinases, PKCε, PKCδ and PKCα as well as for ECM markers, TGF-β, SMAD2/3, MMP-2, collagen-1 and its histological deposition. Susceptibility of the heart to electrically-inducible VF was tested using isolated perfused heart technique. Key results Comparing to wild type males, hairless SHR exhibited less pronounced prolongation of QT and QRS interval as well as an increase of ejection fraction. Myocardial Cx43 and its variant phosphorylated at serine-368, which preserve electrical coupling, were increased and pro-arrhythmic cardiomyocyte Cx43 topology was attenuated in males and females hairless vs wild type SHR. Moreover, the level of β-catenin, which ensures mechano-electrical coupling, was also increased in hairless SHR. On the other hand, collagen-1 and hydroxyproline were lower and the TGF-β1 and SMAD2/3 proteins were suppressed in males hairless vs wild type SHR. Myocardial ECM remodelling was less pronounced in females regardless rat strain. Hairless SHR males were less susceptible to inducible VF compared to wild type SHR, while females were less prone to develop VF regardless the strain. In conclusion, our findings imply that postnatal acclimation of hairless SHR is accompanied by attenuation of hypertension-induced adverse down-regulation of Cx43 and up-regulation of extracellular matrix proteins. It may contribute, at least in part, to the protection from malignant cardiac arrhythmias and mechanical dysfunction.
miR-1 modulates HDAC4, calmodulin and MAPK1/Erk2 during early atrial differentiation
Authors: D Franco, C Garcia-Padilla, E Lozano-Velasco, V Garcia-Lopez, A Aranega, V Garcia-Martinez, C Lopez-Sanchez
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements None. Introduction A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. Purpose The aim of this work is to analyze the miR-1 modulation role in cardiac chamber differentiation through specific signaling pathways. Methods By means of microinjections in both primitive endocardial tubes of chick embryo culture, gain- and loss-of-function experiments were performed with premiR-1 and anti-miR-1, respectively. Subsequently, embryos were subjected to whole mount in situ hybridization with Tbx5, Gata4 and AMHC1 probes, as well as immunohistochemistry with Mef2c, HDAC4, Calmodulin/Calm1 and MAPK1/Erk2 antibodies. Also, we carried out RT-qPCR analysis of control and experimental embryos, including CRABPI, CRABPII and retinoic acid receptors (RAR and RXR). Results Our results reveal that miR-1 increases specific atrial gene expression such as Tbx5, Gata4 and AMHC1, while this microRNA diminishes Mef2c expression. Furthermore, we observed that miR-1 upregulates CRABPII and RARß, and downregulates CRABPI, which are three crucial factors in retinoic acid signaling pathway. Interestingly, we also observed that miR-1 actively interacts with HDAC4, Calmodulin and Erk2/MAPK1, key factors involved in Mef2c regulation. Conclusion All these data suggest that miR-1 functions as an epigenetic factor modulating complementary actions performed by retinoic acid and Mef2c, which are required to properly assign cells as sinoatrial precursors.
Postnatal overfeeding in mice induces early lipidic changes in heart tissue and impacts cardiomyocyte proliferation
Authors: M Josse, E Rigal, N Rosenblatt, F Rochais, J P Pais De Barros, L Rochette, C Vergely
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): French Federation of Cardiology Foundation of France Background Shortly after birth, cardiomyocytes lose their ability to proliferate to switch towards a hypertrophic growth and this ability is known to be influenced by nutritional factors. Postnatal overfeeding (PNOF) induced by litter size reduction in rodents reproduces the impact of childhood overnutrition. While PNOF was shown to induce major cardiac and metabolic alterations in adulthood, little is known about its impact during the first days of life. Purpose The objective of this project was to investigate the early changes induced by PNOF induced in heart tissue in terms of lipid profile and cardiomyocyte proliferation. Methods Two days after birth, C57BL/6 male pups were randomly distributed in litters adjusted to either 9 or 3 pups to form normally fed (NF) or postnatally overfed (PNOF) group, respectively. At 7, 10 and 24 postnatal days (D7, D10 and D24, respectively), hearts were harvested for immunostaining of the proliferation marker Ki67. Cardiac and plasma lipid composition of NF and PNOF was evaluated using liquid chromatography coupled to mass spectrometry technique. Finally, cardiomyocytes from D24 hearts were isolated and counted on Malassez counting chamber. Results At D7, D10 and D24, body weight and cardiac mass of PNOF pups were significantly higher than these of NF controls. Interestingly, at D7, lipid composition of PNOF hearts was decreased in cardiolipins, fatty acids and cholesterol derivatives. Moreover, plasma triglyceride levels were lower in D10 PNOF pups. These modifications were associated with a significant reduction of cardiomyocytes’ proliferation rate and an increased hypertrophy in PNOF hearts at D7, resulting in the reduction of cardiomyocyte number at D24 in PNOF mice. Conclusion Only five days after litter size reduction, PNOF induced major lipidic changes both in the cardiac tissue and in the plasma. In parallel, PNOF shortened the cardiomyocyte proliferative window and induced earlier cardiomyocyte maturation. Given the importance of fatty acid metabolism in cardiac development and function, these early changes could be linked to the long-term cardiometabolic alterations observed later in life.
SGK1 inhibition normalizes action potential duration in transgenic LQT2 rabbits but not in LQT1, suggesting a novel gene-specific therapeutic approach in long QT syndrome
Authors: M Barbieri, J Lourdaour, N Alerni, L Giammarino, S Nimani, L Matas, A Horvath, K E Odening
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SNSF (Swiss National Science Fundation) Background Increasing evidence suggests that secondary remodeling in Na+ and Ca2+ homeostasis may further enhance the propensity to develop arrhythmias in K-channelopathies LQT1 and LQT2 syndrome. In this context, the Serum and Glucocorticoid regulated Kinase-1 (SGK-1) was recently identified as a major regulator of sodium channels in cardiomyocytes. It is mainly activated in pathological conditions, leading to increased INaLate, prolongation of action potential duration (APD) and increased susceptibility to ventricular arrhythmias, recapitulating LQTS phenotype. SGK1 inhibition might hence be a novel therapeutic target also in genetic LQTS subtypes. Methods In cardiomyocytes (CM) isolated from wild type (WT), LQT1 and LQT2 adult rabbits patch-clamp experiments were performed to record AP with SGK1-inhibitor(SGK1-inh, 3 µM) or DMSO vehicle control. Results In the perforated patch configuration at 37°C, 3 μM of SGK1-inh shortened APD90 in LQT2 CMs at 1 Hz (DMSO-control [ms]: 477.3 vs treated cells[ms]: 362.8 p &amp;lt; 0.0001) and at 2 Hz (DMSO-cells: 325.7 vs treated cells:295.4,p &amp;lt;0.05). In contrast, in WT and LQT1 CMs, no effect on APD90 was observed neither at 1 Hz (WT, DMSO-cells: 329.2 vs treated cells: 301.6 p=ns; LQT1, DMSO-cells: 429.8 vs treated cells: 431.6, p=ns) nor at 2 Hz (WT, DMSO-cells:256.8 vs treated cells:242 p=ns; LQT1,DMSO cells:310.4 vs treated cells: 315.8,p=ns). Importantly, the SGK1-inhibitor effect in LQT2 rabbit CMs was so pronounced that it normalized APD to the wildtype level (LQT2+SGK1-Inh: 362.8 vs WT:329.2, ps=ns). Furthermore, short-term-variability in APD was markedly reduced by SGK1-Inh in LQT2, indicating an antiarrhythmic effect. Conclusions SGK1-inhibition exerts a beneficial APD-shortening effect only in LQT2, indicating a gene-specific efficacy. The underlying mechanism is still unclear. We hypothesize that the SGK1 pathway/INaLate activation in LQT1 might not be as active as in LQT2. We are currently conducting more experiments on potential genotype differences in SGK1 activity and SGK1-mediated INaLate inhibition to further elucidate the differences.Effects of SGK1-inh on AP characteristic
Deeper insights into heart failure with preserved ejection fraction through rna-sequencing of circulating endothelial cells - advanced protocol establishment enables complete transcriptome analysis
Authors: L Wurmbrand, K Kalies, K Knoepp, J Dutzmann, D Sedding
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements None. Heart failure with preserved ejection fraction (HFpEF) continues to pose a significant challenge with a bleak prognosis and a rising incidence, particularly among the aging population in Western countries. An in-depth comprehension of cellular-level pathogenesis, with a specific emphasis on the pivotal role of cellular senescence in endothelial dysfunction, is imperative for the evolution of innovative therapeutic interventions. Many previous approaches to investigate the genesis of HFpEF are based only on HFpEF-mouse models, which cannot directly be transferred to humans. The isolation of circulating endothelial cells from peripheral blood samples represents a more translatable, feasible, less invasive way to study endothelial dysfunction, a central component of HFpEF-pathogenesis, in human material for the identification of new therapeutical targets. This study focuses on establishing a new protocol for isolating circulating endothelial cells (CEC) in HFpEF patients to address endothelial dysfunction and pinpoint novel therapeutic targets through a comprehensive whole transcriptome analysis. Methodologically, CEC isolation from whole blood samples was achieved using a newly established Fluorescence-Activated Cell Sorting (FACS) panel. HFpEF patients were recruited based on their HFA-PEFF score, with non-diseased controls comprising both young and old subjects meticulously matched for sex and age. Subsequently, the isolated CECs were subjected to transcriptome analysis. Our new protocol is based on red blood cell lysis followed by magnetic cell sorting to deplete CD45+ cells. This is followed by staining with fluorescent-conjugated monoclonal antibodies. This FACS panel includes known endothelial-specific markers including CD11b- and CD45-, CD146+, CD34+ and CD31+ as well as DAPI- as a nucleus marker. The prepared CECs were sorted using FACS and a specially developed gating. We isolated the RNA in preparation for sequencing. Our protocol was validated by immunofluorescence staining of isolated CECs (markers: CD31, vWF, CD146, Phalloidin) and PCR analysis (markers: CD31, CD146 and vWF). In summary, we have successfully established a new, robust and highly targeted workflow for consistently isolating circulating endothelial cells from blood and performing RNA sequencing on the collected samples. This is a key advance towards a deeper understanding of the pathophysiology of HFpEF. The application of our methodology may enable the characterization of patients with HFpEF at the transcriptome level. The whole transcriptome analyses provide potential starting points for further in vitro and in vivo studies. As we extend our investigations to larger patient cohorts in the future, there is potential for the identification of novel therapeutic targets.
Deep proteome profiling of reticulated platelets in patients with chronic coronary syndrome using mass cytometry
Authors: J Han, K Kirmes, M Klug, J Bernett, Q Manz, L Arend, O Lazareva, M Rosenbaum, J Ruland, K L Laugwitz, M List, I Bernlochner, D Bongiovanni
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Else Körner Fresenius Stiftung (EKFS) Background Immature or reticulated platelets (RPs), a subgroup of larger and RNA-rich platelets, which are newly released by megakaryocytes from the bone marrow into blood, have gained rising interest because of the further decryption of RPs’ prothrombotic biology and the potential as biomarkers for predicting adverse events in cardiovascular disease. Purpose The biological features of RPs in patients with chronic coronary syndrome (CCS) are largely unknown. With this project, we aimed to perform the first high resolution proteomic characterisation of reticulated platelets using mass cytometry(CyTOF). Method Two samples of thrombocytes from peripheral blood (non-stimulated, TRAP-stimulated) were prepared from each patient with a custom-made CyTOF-antibody panel of 20 antibodies targeting essential transmembrane proteins(anti-CD9, anti-CD29, anti-CD31, anti-CD36-, anti-CD40, anti-CD41, anti-CD42a, anti-CD42b-, anti-CD47, anti-CD61, anti-CD62P-, anti-CD63, anti-CD69, anti-CD107a, anti-CD154, anti-GPVI, anti-GPIIbIIa complex, anti-Par1, anti-PEAR-1 and the negative control anti-CD3 coupled with different metal isotopes). RPs and mature platelets (MPs) were detected based on their RNA content. Earth mover’s distance (EMD) was assessed as a measure of differential expression. Results CyTOF detected an upregulation of important transmembrane receptors in RPs compared to MPs in quiescent platelets (Fig.1A and B). The collagen receptor GPVI (EMD 33.95), the collagen integrin receptor unit ITGB1 (EMD 32.37), the adhesion protein CD9 (EMD 31.78) and the von Willebrand receptor unit CD42b (EMD 30.08) were upregulated in RPs which all serve as crucial regulators of platelet activation and aggregation. RPs show higher median signal intensities of typical activation markers compared to MPs upon TRAP stimulation (Fig.1C). Conclusion We provide the first proteomic characterization of RPs’ pro-thrombotic profile offering the first biomolecular explanation of their hyperactivity and detrimental role in CCS patients.