German Research Foundation Research Articles

Restoring myocardial infarction-induced long-term memory impairment by targeting the cystic fibrosis transmembrane regulator.

Cognitive impairment is a serious comorbidity in heart failure patients, but effective therapies are lacking. We investigated the mechanisms that alter hippocampal neurons following myocardial infarction (MI). MI was induced in male C57Bl/6 mice by left anterior descending coronary artery ligation. We utilised standard procedures to measure cystic fibrosis transmembrane regulator (CFTR) protein levels, inflammatory mediator expression, neuronal structure, and hippocampal memory. Using invitro and invivo approaches, we assessed the role of neuroinflammation in hippocampal neuron degradation and the therapeutic potential of CFTR correction as an intervention. Hippocampal dendrite length and spine density are reduced after MI, effects that associate with decreased neuronal CFTR expression and concomitant microglia activation and inflammatory cytokine expression. Conditioned medium from lipopolysaccharide-stimulated microglia (LCM) reduces neuronal cell CFTR protein expression and the mRNA expression of the synaptic regulator post-synaptic density protein 95 (PSD-95) invitro. Blocking CFTR activity also down-regulates PSD-95 in neurons, indicating a relationship between CFTR expression and neuronal health. Pharmacologically correcting CFTR expression invitro rescues the LCM-mediated down-regulation of PSD-95. Invivo, pharmacologically increasing hippocampal neuron CFTR expression improves MI-associated alterations in neuronal arborisation, spine density, and memory function, with a wide therapeutic time window. Our results indicate that CFTR therapeutics improve inflammation-induced alterations in hippocampal neuronal structure and attenuate memory dysfunction following MI. Knut and Alice Wallenberg Foundation [F 2015/2112]; Swedish Research Council [VR; 2017-01243]; the German Research Foundation [DFG; ME 4667/2-1]; Hjärnfonden [FO2021-0112]; The Crafoord Foundation; Åke Wibergs Stiftelse [M19-0380], NMMP 2021 [V2021-2102]; the Albert Påhlsson Research Foundation; STINT [MG19-8469], Lund University; Canadian Institutes of Health Research [PJT-153269] and a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award.

Neoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile in pancreatic cancer.

BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. At diagnosis, only 20% of patients with PDAC are eligible for primary resection. Neoadjuvant chemotherapy can enable surgical resection in 30%-40% of patients with locally advanced and borderline resectable PDAC. The effects of neoadjuvant chemotherapy on the cytokine production of tumor-infiltrating T cells are unknown in PDAC.METHODSWe performed multiplex immunofluorescence to investigate T cell infiltration in 91 patients with PDAC. Using flow cytometry, we analyzed tumor and matched blood samples from 71 patients with PDAC and determined the frequencies of T cell subsets and their cytokine profiles. Both cohorts included patients who underwent primary resection and patients who received neoadjuvant chemotherapy followed by surgical resection.RESULTSIn human PDAC, T cells were particularly enriched within the tumor stroma. Neoadjuvant chemotherapy markedly enhanced T cell density within the ductal area of the tumor. Whereas infiltration of cytotoxic CD8+ T cells was unaffected by neoadjuvant chemotherapy, the frequency of conventional CD4+ T cells was increased, and the proportion of Tregs was reduced in the pancreatic tumor microenvironment after neoadjuvant treatment. Moreover, neoadjuvant chemotherapy increased the production of proinflammatory cytokines by tumor-infiltrating T cells, with enhanced TNF-α and IL-2 and reduced IL-4 and IL-10 expression.CONCLUSIONNeoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile. Combinational treatment strategies incorporating immunotherapy in neoadjuvant regimens may unleash more effective antitumor responses and improve prognosis of pancreatic cancer.FUNDINGThis work was supported by the Jung Foundation for Science and Research, the Monika Kutzner Foundation, the German Research Foundation (SE2980/5-1), the German Cancer Consortium, and the Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden.

The hepatitis delta virus and chronic hepatitis D.

The hepatitis delta virus and chronic hepatitis D.

Observation of the Y(4230) and a new structure in * *Supported in part by National Key R&D Program of China (2020YFA0406300, 2020YFA0406400); National Natural Science Foundation of China (NSFC) under Contracts Nos. (11625523, 11635010, 11735014, 11822506, 11835012, 11935015, 11935016, 11935018, 11961141012, 12022510, 12025502, 12035009, 12035013, 12061131003); the Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program; Joint Large-Scale Scientific Facility

The cross sections of at center-of-mass energies from 4.127 to 4.600 GeV are measured based on 15.6 fb data collected with the BESIII detector operating at the BEPCII storage ring. Two resonant structures are observed in the line shape of the cross sections. The mass and width of the first structure are measured to be () MeV and () MeV, respectively. They are consistent with those of the established . The second structure is observed for the first time with a statistical significance greater than 8σ, denoted as . Its mass and width are determined to be () MeV and () MeV, respectively. The first presented uncertainties are statistical and the second ones are systematic. The product of the electronic partial width with the decay branching fraction is reported.

The Preference Survey Module: A Validated Instrument for Measuring Risk, Time, and Social Preferences

Incentivized choice experiments are a key approach to measuring preferences in economics but are also costly. Survey measures are a low-cost alternative but can suffer from additional forms of measurement error due to their hypothetical nature. This paper seeks to leverage the strengths of both approaches by proposing a new survey module on risk aversion, time discounting, trust, altruism, positive and negative reciprocity, in which survey items are selected based on ability to predict choices in corresponding, incentivized experiments. The methodology and results provided in the paper can also potentially provide a model for researchers who have specific requirements and want to design their own modules. This paper was accepted by Yan Chen, behavioral economics and decision analysis. Funding: The project received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7-2007-2013) [Grant 209214]. A. Falk and T. Dohmen acknowledge funding from the Deutsche Forschungsgemeinschaft (German Research Foundation) [Grant CRC TR 224 (Project A01)] and Germany’s Excellence Strategy [Grant EXC 2126/1-390838866]. Supplemental Material: Data and the online appendices are available at https://doi.org/10.1287/mnsc.2022.4455 .

Ulf Diederichsen (1963–2021)

“For he was our own! … Fruitful both in counsel and deed; this we experienced and appreciated.” With the death of Ulf Diederichsen on November 11, 2021, after a short but severe illness, we lost a great scientist in chemical biology and good friend. He was a generous and extremely gifted man whose strength was to tackle essential biological problems at a molecular level with simple model systems and to draw insightful conclusions realizing a hallmark of chemical biology by addressing biological challenges with chemical approaches derived from simple principles. Ulf Diederichsen was born on October 7, 1963, in Munich and grew up in Göttingen, enjoying excellent scholar education at the Max-Planck Gymnasium. He moved to Freiburg im Breisgau in 1983 to study Chemistry at the Albert-Ludwigs-University. For his doctorate he moved to Zurich in 1988 to work with Prof. Eschenmoser at the ETH on the question why Nature chose pentose—but not hexose nucleic acids. He received the Dr. rer. nat. in 1993 on the Base pairing of hypoxanthine in HOMO DNA oligonucleotides and the question of the pairing behavior of glucopyranosyl oligonucleotides. He then joined the group of Prof. Dennis P. Curran at the University of Pennsylvania (Pittsburgh/USA) as postdoctoral researcher (1993–1994) investigating radical chemistry. Back to Europe he started his habilitation on “Linear nucleic acid analogues with peptidic backbones” under the mentorship of Prof. Horst Kessler at the Technical University of Munich, which was finished in 1999. He then joined the University of Würzburg as Professor of Organic Chemistry, and finally, in 2001, the University of Göttingen. He served as Dean (2005 to 2007) and Vice President for Research (2015 to 2021). In addition, he was Guest Professor at the Ludwig-Maximilians-University in Munich (1998–1999) and Goering Visiting Professor at the University of Wisconsin, USA (2000). He was elected member of the Academy of Sciences in Göttingen in 2012 and became its president in 2020. His scientific interest was mainly centered on the non-covalent interaction of peptide helices with respect to the incorporation, organization, and dynamics in lipid membranes. He successfully employed spin-labeled peptides for distance measurements. Similarly, he addressed the membrane attachment and fusion processes of SNARE proteins with model systems (conjugates of transmembrane peptides with coiled coil peptides or peptide nucleic acids). He was interested in functionalized β-peptides as artificial matrices for aggregation and modification of model membranes. The excellent results of this outstanding research were summarized in largely over 100 publications in highly renowned international journals. Besides his exceptional scientific achievements and admirable mentorship for a large number of doctoral students and postdocs, he deserves high recognition for his excellent research management particularly as Vice President of the University of Göttingen. He also served as a highly esteemed reviewer in various panels, including the German Research Foundation. The large experience collected in this important activity, particularly also for the German peptide community, he adapted in a very successful manner also in his editorial engagement as Deputy Editor of the Journal of Peptide Science since 2012, where he was supposed to take over the full responsibility as the Editor-in-Chief from 2022. Those of us who knew him are saddened by his sudden passing away and mourn the great loss of a dear friend and colleague. He will always be remembered as an inspiring teacher and mentor, as an enthusiastic scientist, but first and foremost as a warm and sincere human being, as a “feiner Kerl.”

Continuum Modelling As Tool for Optimizing the Cell Design of Magnesium Batteries

Magnesium-based next-generation batteries are of great interest since magnesium is not only very abundant, which allows economic and sustainable applications, but also less prone to dendrite formation than many other metals. Together with the bivalency of the magnesium cations the resulting possibility to safely use a metal anode enables batteries with high specific capacities.However, for a successful commercialization of magnesium batteries there are still some challenges to overcome. The high charge density of the bivalent cation causes strong coulomb interactions with anions and solvent molecules. Therefore, magnesium salts are prone to form ion pairs and bigger clusters – especially at high concentrations, which may adversely affect the transport in the electrolyte and the electrochemical reaction at the electrode.[1] Moreover, energetic barriers for desolvation and solid-state diffusion of the double-charged magnesium ion are usually very high, which can have a crucial impact on the battery performance. Former can significantly hinder the electron-transfer reaction,[2] whereas latter makes the choice of suitable cathode materials very challenging.Consequently, a good understanding of the limiting processes in rechargeable magnesium batteries is key to develop novel high-capacity / high-voltage cathode materials. For instance, it is well-known that the morphology of an intercalation material can strongly influence the battery performance and smaller particles as well as thinner electrodes are common strategies for avoiding adverse effects of transport limitations. However, high mass loadings as well as suitable separators are still essential bottlenecks for commercialization of magnesium-ion batteries.Up to date Chevrel phase (CP) Mo6S8 is considered as benchmark intercalation cathode and Mg[B(hfip)4]2 / DME is seen as most promising chloride-free magnesium electrolyte.[3,4] In our contribution we carefully study this model system of a magnesium-ion battery to get a better understanding of how to overcome undesired limitations. Therefore, we present a newly-developed continuum model, which is able to describe the complex intercalation process of magnesium cations into a CP cathode. The model considers not only the different thermodynamics and kinetics of the two intercalation sites of Mo6S8 and their interplay but also the impact of the desolvation on the electrochemical reactions and possible ion agglomeration. The parameterization and validation of the model is based on DFT calculations and experimental data. Different kind of (transport) limitations and their impact on the battery performance are studied in detail. All in all, the combination of different modelling techniques with experimental measurements provides important insights into the operation of magnesium ion batteries and enables an optimization of the cell design. Acknowledgements This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 824066 (E-MAGIC). Furthermore, this work contributes to the research performed at CELEST (Center for Electrochemical Energy Storage Ulm-Karlsruhe) and was funded by the German Research Foundation (DFG) under Project ID 390874152 (POLiS Cluster of Excellence). The simulations were carried out at JUSTUS 2 cluster supported by the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant No INST 40/575-1 FUGG. References Drews, T. Danner, P. Jankowski et al., ChemSusChem, 3 (2020), 3599-3604.Drews, P. Jankowski, J. Häcker et al., ChemSusChem, 14 (2021), 4820-4835.Aurbach, Z. Lu, A. Schlechter et al., Nature, 407 (2000), 724-727.Zhao-Karger, R. Liu, W. Dai et al., ACS Energy Lett. 3 (2018), 2005-2013.

Dysferlin facilitates the de novo biogenesis of tubular membranes in hypertrophic remodelling

Abstract Introduction Ferlins are transmembrane proteins with multiple C2 domains that may mediate Ca2+ dependent membrane fusion and repair events. In ventricles, Dysferlin expression is dominant, and patient mutations have been associated with cardiomyopathies. Hence, we hypothesized that Dysferlin is essential for the functional integrity of the tubular endomembrane system in ventricular myocytes (VM), and may facilitate the cellular hypertrophic remodelling in pressure-overload. Results In 30 weeks old Dysferlin knockout (KO) mice, echocardiography revealed dilative cardiomyopathy with reduced left-ventricular ejection fractions (difference between means KO vs. wildtype (WT) −13.45±2.17%, p<0.001, n=16/20 mice). Asking for the subcellular relevance of Dysferlin, we investigated VM nanodomains by immunolabelling in STimulated Emission Depletion (STED) nanoscopy and visualized punctual Dysferlin clusters decorating the T-tubule system of mouse and human VM. Precisely, Dysferlin clusters localized to junctions of the tubular system and the sarcoplasmic reticulum (SR) in nanometric proximity to RyR2 Ca2+ release units. Complexome profiling (LC-MS/MS) also suggested interactions of Dysferlin with RyR2 and the SR membrane-tethering protein Junctophilin-2 (JP2) in high molecular weight complexes. In JP2-overexpressing mice, live-cell STED membrane imaging captured polyadic junctional membrane structures in VM, specifically surrounded by dense Dysferlin clusters, which proposes Dysferlin as stabilizing protein of tubule-SR junctions. Further, we studied the ultrastructural remodelling of the tubular system in a mouse model of cardiac hypertrophy and heart failure induced by transaortic constriction (TAC). Interestingly, mortality four weeks post TAC was not increased in KO mice. However, echocardiography uncovered that Dysf-KO prevents left-ventricular hypertrophy in response to pressure-overload, which was confirmed by single cell measurements (VM area: WT 3559±120 μm2 vs. KO 2671±87 μm2, n=163/158 VM, p<0.001). Thus, we assumed Dysferlin to mediate tubular network proliferation during hypertrophic remodelling. Indeed, WT VM showed an augmented Dysferlin expression post TAC (158±0,125%, p=0,005, n=7/7 mice), and immunofluorescence STED nanoscopy detected an increased Dysferlin network complexity. In detail, we visualized highly abundant Dysferlin clusters decorating newly shaped axial tubules, which were quantified by component-specific tubule network analysis. Conclusion Nanoscale imaging proposes Dysferlin as stabilizer of tubule-SR junctions, thereby promoting functional excitation-contraction coupling, and demonstrates the crucial importance of Dysferlin in pressure-overload induced hypertrophic remodelling, facilitating the de novo biogenesis of axial tubule membranes and tubule-SR junctions necessary for VM growth. Thus, Dysferlin may emerge as potential novel therapeutic rationale to control subcellular membrane remodelling in cardiac diseases. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Multiscale Bioimaging Cluster of Excellence: From Molecular Machines to Networks of Excitable Cells;German Research Foundation (DFG) - Collaborative Research Centre 1002 (SFB 1002): Modulatory Units in Heart Failure

BNIP3 regulates cardiac energy metabolism by interaction with F1FO ATP-synthase

Abstract Background The oxidative phosphorylation (OXPHOS) that takes place in the mitochondria produces chemical energy in form of ATP, the main energy source of the heart. Proper mitochondrial function determines the contractility of the heart. Changes in myocardial ATP levels are often associated with cardiovascular disease. However, mitochondrial dysfunction remains an unmet therapeutic challenge. The F1FO ATP-synthase protein complex catalyses the last step of OXPHOS, synthesising ATP and determining the respiratory function of cardiac mitochondria. An increased ATP pool during cardiovascular challenges, regulated by F1FO ATP-synthase, could therefore significantly improve the clinical outcome of affected patients. In neurons, Bcl2-familiy members like BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) have been suggested to be involved in the regulation of metabolic efficiency through interaction with the mitochondrial F1FO ATP synthase. A similar metabolic regulation in the cardiac context remains unclear. Furthermore, BNIP3 has a significant impact on the loss of cardiomyocytes under various pathological conditions and is thus a potential mediator of energy metabolism in cardiomyocytes. Methods and results With native gel-electrophoresis, cardiac BNIP3 of C57BL/6J mice was first identified in higher oligomeric complexes in similar molecular weight range as mitochondrial F1FO ATP-synthase. Using 60Å-precise proximity-ligation assays to determine possible binding partners, F1FO ATP-synthase was uncovered for the first time as an interacting protein of BNIP3. This interaction was confirmed by co-immunoprecipitation of BNIP3 and F1FO ATP-synthase and by spatial localization using electron microscopy. Peptide microarray studies elucidate the f subunit of F1FO ATP-synthase as the structural interaction site. Functional analysis using extracellular flux analyser technology revealed significantly elevated mitochondrial respiratory activity in Bnip3 knock out mice and human cardiomyocytes during BNIP3 inhibition. Increased ATP levels in C57BL/6J mice after acute inhibition of BNIP3 confirmed the observed regulatory effect of BNIP3 on F1FO ATP-synthase activity. Finally, depletion of BNIP3 in mice improves stress resistance with an augmented chronotropic capacity during dobutamine-induced cardiac stress. Conclusion Depletion of BNIP3 suggests a regulatory role of BNIP3 in cardiac mitochondrial energy homeostasis via interaction with F1F0 ATP-synthase in oligomeric complexes. Since acute inhibition of BNIP3 activity allows positive modulation of cardiac performance by elevating the available ATP pool, this may serve as a beneficial treatment for patients with cardiovascular disease in the future. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German research foundation (DFG)

OAT3A1/NKD2 mediates increased calcification of aortic valvular interstitial cells under in vitro conditions of uremia

Abstract Background Chronic kidney disease is closely associated with cardiovascular disease which contributes to a high mortality and morbidity in these patients. Calcific aortic valve stenosis is the most common valve disease among adults. CKD leads to a higher incidence of AS and is associated with impaired clinical outcomes after valve replacement. CKD leads to retention of uremic toxins, such as indoxyl sulfate (IS), which is known to induce inflammatory and pro-calcific processes. The influence of IS on AS pathophysiology is currently not known. Methods and results Human aortic valvular interstitial cells (VICs) were stimulated by 4 different conditions: Control medium (CM), CM and IS in a concentration of 50 mmol/l, pro-calcifying medium (PCM) which was produced by adding NaH2PO4 and L-ascorbic acid to CM and PCM+IS. After 7 days of incubation, VICs were fixed in formalin and calcification was evaluated through staining with alizarin red solution (B). Staining was quantified by photometric measurements at 540 nm. We observed a significantly higher degree of VIC calcification under the influence PCM+IS compared to PCM alone (C). VICs were stimulated for 7 days and next-generation RNA Sequencing (RNA Seq) was performed. Analysis of RNA Seq dataset identified naked cuticle homologue 2 (NKD2) to be strongly regulated by IS. This effect was confirmed by qPCR using VICs from a total of three different human donors and immunoblot analysis (D, E). We observed a more pronounced upregulation of NKD2 gene expression under PCM+IS vs. PCM alone (p<0.0001) (D). To investigate the influence of NKD2 on VIC calcification genetic knockdown of NKD2 was performed by transfecting VICs with NKD2 siRNA compared and scrambled siRNA as negative control (nc siRNA) (F). After 21 days of stimulation and subsequent alizarin staining, we observed a significant decrease in VIC calcification under NKD2 knockdown compared to negative control under PCM+IS conditions (G, H). To identify upstream mechanisms that lead to NKD2 upregulation by IS we investigated the influence of organic anion transporters (OAT) which are responsible for cellular uptake of IS. Probenecid, known as a potent inhibitor of OATs showed a dose dependent suppression of NKD2 upregulation under PCM+IS (I). Systematic analysis of RNA Seq data regarding SLCO genes coding for OAT and OAT-peptides revealed that only SLCO3A1, coding for OAT3A1 is highly abundant in VICs (J). SLCO3A1 knockdown showed a significant decrease of NKD2 expression after stimulation with PCM+IS for 7 days compared to negative control (K). Conclusion Our findings show that in vitro conditions of uremia increase calcification of aortic VICs. NKD2 expression is regulated by PCM and IS, and PCM+IS treated cells show the strongest upregulation of NKD2. Genetic knockdown of NKD2 attenuates VIC calcification suggesting a role of NKD2 in this process. Further, our findings suggest that OAT3A1 is involved in IS mediated regulation of NKD2 expression. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Medical faculty of the University of Bonn, German Cardiac Society (DGK), German Research Foundation (DFG), TRR259

Coronary artery disease ameliorates extracellular vesicle lncRNA PUNISHER regulates angiogenic response and endothelial cells function via NFkB-dependent mechanism

Abstract Augmenting evidence indicates that long noncoding RNAs are playing a crucial role in diverse cellular/pathological processes. Intercellular transfer of extracellular vesicles transmitted lncRNA regulates vascular health and diseases. However, whether lncRNA expression in EVs is regulated in patients with coronary artery disease, is unknown. A PCR-based lncRNA array analysis revealed that EV-PUNISHER was significantly upregulated in patients with CAD (n=221) compared to healthy subjects. Our data showed that PUNISHER is the most upregulated lncRNA in CAD in accordance with other highly expressed lncRNAs. To examine the specific role of PUNISHER in EC phenotypic regulation, siRNA-mediated silencing in ECs revealed that depletion of PUNISHER suppresses the migration and proliferation of ECs. Moreover, depletion of PUNISHER decreases cell survival by reducing cell viability and proliferation through increased apoptosis and cytotoxicity. In order to investigate EC function in PUNISHER depleted cells, in vitro sprouting and tube formation assay revealed that PUNISHER is an important mediator EC functions, which acts as an anti-angiogenic factor. In vitro atherosclerotic stimuli increased PUNISHER expression in EV/EC in a dose-dependent way. Microarray analysis identified a series of pro- and anti-angiogenic genes as well genes directly involved in cell viability that are differentially regulated. We confirmed that PUNISHER is incorporated into endothelial microvesicles (EMVs) and transferred to recipient cells by using different experiments. By using lncRNA-FISH and vesicle degradation assays, we showed that PUNISHER is incorporated into EMVs, augmented recipient EC function in vitro upon transfer via EMVs. To examine whether EMV-PUNISHER promotes the EC function, different in vitro functional experiments with ECs confirmed that PUNISHER is an important regulator. Mass spectrometry analysis has revealed EMV contains numerous proteins, including RNA binding proteins such as hnRNPU, hnRNPK, hnRNPA2/B1, etc. By using RNA-pulldown and RNA immunoprecipitation (RIP), we confirmed that PUNISHER interacts with hnRNPU, which facilitates packaging into EMV prior to transfer to recipient EC. The interaction acts as an important positive regulator of cell viability and survival of recipient cells, identified using functional assay (migration, viability, proliferation, and angiogenesis). Transcription factor array has shown that PUNISHER regulates NFkB to control cellular viability and apoptosis. A murine re-endothelialization model revealed that EV-PUNISHER promotes reendothelialization. Ex vivo internalization/uptake of PKH-67-labeled EVs revealed that EVs are detectable in the cross-section of EV-perfused carotid artery. Our study unveiled for the first time that EV-incorporated PUNISHER exerts its function in endothelial cells which might be beneficial in cardiovascular pathologies where the endothelial function is dysregulated. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): German Research Foundation (DFG)German Society of Cardiology (DGK)

Body fluid status in patients with pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension

Abstract Introduction Body fluid status can be determined by plasma volume status and extracellular water to total body water (ECW/TBW) ratio and is a well-known parameter associated with survival in patients with chronic heart failure (1–3). However, its prognostic impact in patients with pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) is not fully understood. Methods 409 patients with PAH and CTEPH who entered the Giessen PH registry were included in this study (4). Plasma volume status was estimated using Duarte formula (ePVS = (1 − hematocrit) / hemoglobin × 100). ECW was calculated via Peters formula ((−2.47 × 0.842 + 8.76 × body surface area) for men and (−1.96 × 0.572 + 8.05 × body surface area) for women) and TBW via Watson formula ((2.447 − [0.09516 × age] + [0.1073 × height] + [0.3362 × body weight]) for men and (−2.097 + [0.1069 × height] + [0.2466 × body weight]) for women). Statistical analyzes were performed with R (The R Foundation). Results Median age of the included patients was 65 [53, 74] years. Median ePVS and ECW/TBW ratio were 4.5 [3.9, 5.5] and 0.39 [0.37, 0.40]. Correlation analyses revealed that ePVS correlates with pulmonary artery wedge pressure (PAWP), pulmonary vascular resistance (PVR), cardiac index, and mixed venous oxygen saturation (SvO2), whereas ECW/TBW ratio did not correlate with pulmonary hemodynamics. Accordingly, univariate Cox regression revealed that ePVS but not ECW/TBW ratio was associated with mortality (HR: 1.18 [1.02, 1.37]). ePVS remained as an independent prognostic parameter in multivariate Cox regression analysis. Patients with high ePVS showed significantly decreased survival rates (log-rank p<0.001). Conclusion ePVS but not ECW/TBW ratio is associated with prognostic parameters such as PVR, cardiac index and SvO2. Thus, ePVS may be an independent prognostic parameter in patients with PAH and CTEPH. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

Novel assay for quantifying the cholesterol crystal dissolution capacity of serum predicts outcomes in patients with severe aortic stenosis undergoing TAVR

Abstract Background Aortic stenosis (AS) is the most common valvular heart disease (VHD) in developed countries. The pathophysiology of calcific AS has several clinical and pathobiological findings in common with atherosclerosis including chronic inflammation and lipoprotein deposition. Histopathological examination has revealed atherosclerosis-like lesions, that mainly contain cholesterol crystals (CC) in resected calcific aortic valve cusps. Previous studies have demonstrated that CCs can activate the NLRP3 inflammasome, resulting in an IL-1 driven systemic inflammation, that leads to the development of atherosclerotic plaques. Purpose In this study, we sought to validate a novel assay for measuring the serum capacity to dissolve cholesterol crystals (CCDC) in patients with AS and to examine whether this biomarker may be associated with clinical outcomes. Methods Our study cohort included 348 patients with AS undergoing transcatheter aortic valve replacement. The CCDC was measured using flow cytometry to enumerate CC, that were added to a 50% serum solution, at baseline and after two hours of incubation. The dissolution capacity was indicated as percentage change in CC count at baseline and after incubation. The study cohort was stratified according to the median CCDC into high and low CC dissolvers. Results The study population was 47.7% female and had a mean age of 80.9±6.2 years. The primary end point, a composite of one-year all-cause mortality and major vascular complication occurred less frequently in the high CCDC group (7.0%) as compared with the low CCDC group (15.3%, p=0.01). This was mainly driven by lower rates of one-year mortality in patients with a high CCDC (7.0% vs 13.6%, p=0.05), as presented in Figure 1. Furthermore, unplanned endovascular interventions were significantly less frequent in high CC dissolvers in contrast to low CC dissolvers (12.2% vs 20.5%, p=0.04). Although LDL cholesterol (101.8±37.3 mg/dL vs 97.9±37.6 mg/dL, p=0.35) and total cholesterol levels (158.1±43.8 mg/dL vs 154.1±40.2, p=0.41) were comparable in the high and low CCDC group, only patients with a low CCDC showed a benefit from statin treatment (Figure 2). In multivariate analysis, only low CCDC (OR: 2.51 [95% CI: 1.02–6.15], p=0.05) and Albumin (OR: 0.88 [95% CI: 0.79–0.98], p=0.03) were independently associated with one-year all-cause mortality Conclusion The CCDC is a novel biomarker associated with clinical outcome in patients with AS undergoing TAVR. It may provide new insights into patient's preventative anti-inflammatory capacity and additional prognostic information to identify vulnerable patients beyond classic risk assessment. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).

Circulating microRNA-122-5p is associated with a lack of improvement in left-ventricular function after TAVR and regulates viability of cardiomyocytes via extracellular vesicles

Abstract Transcatheter aortic valve replacement (TAVR) is a well-established treatment option for high- and intermediate-risk patients with severe symptomatic aortic valve stenosis (AVS). However, a specific role for circulating microRNAs (miRNAs) in the improvement of cardiac function for patients after TAVR has not yet been investigated. Herein, we profiled the differential expression of miRNAs in circulating extracellular vesicles (EV-miRNAs) in patients after TAVR and, in particular, the novel role of circulating miR-122-5p in cardiomyocytes. Circulating EV-associated miRNAs were investigated by using an unbiased Taqman-based human miR array. Several EV-miRNAs (miR-122-5p, miR-26a, miR-192, miR-483-5p, miR-720, miR-885-5p, and miR-1274) were significantly deregulated in aortic stenosis patients at day seven after TAVR in comparison to the pre-procedural levels in patients without LVEF-improvement. The higher levels of miR-122-5p were negatively correlated with LVEF improvement at both day seven (r=−0.264 and p=0.015) and at six months (r=−0.328 and p=0.0018) after TAVR. At the three-year follow-up, patients with a higher level of miR-122-5p displayed significantly increased cardiovascular mortality (p=0.03). By utilization of patient-derived samples and a murine aortic-stenosis model, we observed that the expression of miR-122-5p correlates negatively with cardiac function, which is associated with LVEF. Graded wire-injury-induced aortic-valve-stenotic mice demonstrated a higher level of miR-122-5p, which was related to cardiomyocytes dysfunction. Murine ex vivo experiments revealed that miR-122-5p is highly enriched in endothelial cells in comparison to cardiomyocytes. Gain- and loss-of-function experiments suggested that EV-mediated shuttling of miR-122-5p increases the level of miR-122-5p in recipient cardiomyocytes and regulates the viability of cardiomyocytes. Mechanistically, miR pulldown, electrophoretic mobility shift assay, and RNA immunoprecipitation confirmed that miR-122-5p interacts with the RNA-binding protein, hnRNPU, in a sequence-specific manner to encapsulate miR-122-5p into large EVs. Upon shuttling into recipient cells, miR-122-5p reduces the expression of the anti-apoptotic gene BCL2, by binding to its 3' untranslated region to inhibit its translation, and thereby decreasing the viability of target cardiomyocytes. Increased levels of circulating pro-apoptotic EV-incorporated miR-122-5p are associated with reduced LVEF after TAVR. Extracellular vesicular shuttling of miR-122-5p regulates the viability and apoptosis of cardiomyocytes in a BCL2-dependent manner. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): German Research Foundation (DFG)German Society of Cardiology (DGK)

Myocardial inflammation in ischaemic and non-ischaemic heart failure relates to impaired mitochondria

Abstract Background Mitochondrial dysfunction is a driving factor in the development of heart failure (HF) and relates to poor cardiac function. Through elevated oxidative stress it is linked to myocardial inflammation. Both mechanisms promote the development of cardiac fibrosis, a key contributor to adverse outcomes in ischaemic and non-ischaemic HF. Differences in myocardial mitochondrial function and inflammation between patients with ischaemic and non-ischaemic HF have been indicated. While immunosuppression in non-ischaemic HF with myocardial inflammation has improved long-term results, little is known about possible benefits in ischaemic HF. Purpose We hypothesised that: 1. Myocardial lymphocytic inflammation and fibrosis differ in patients with HF due to ischaemic cardiomyopathy (ICM) compared to patients with dilated cardiomyopathy (DCM). 2. Inflammation and fibrosis are associated with markers of mitochondrial dysfunction. Methods Myocardial tissue specimens were obtained from the left ventricular (LV) apex of 65 patients (n[ICM] = 33, n[DCM] = 32) with HF requiring an LV assist device or heart transplantation. We assessed oxidative phosphorylation capacity (OXPHOS) via high resolution respirometry in saponine-permeabilised myocardial fibres (OROBOROS Oxygraph-2k). Reactive oxygen species (ROS) production was measured fluoroscopically via the Amplex Red method. Immunohistochemistry staining for CD3 was performed on snap-frozen tissue and digitally analysed. Azan staining was used for quantification of fibrotic area. Statistical analysis was conducted with GraphPad Prism v9.0 and IBM SPSS v27.0. Results ICM and DCM patients did not differ significantly regarding age (60.9±5.4 vs. 55.8±12.4 years, p=0.11), sex (91% vs. 90% male, p=0.93), BMI (27.6±5.1 vs. 26.5±6.0 kg/m2, p=0.82), or diabetic status (31% vs. 20% Type 2 Diabetes mellitus, p=0.31). Tissue specimens of ICM patients displayed about three times larger fibrotic areas compared to DCM (20.9±21.2 vs. 7.2±5.6%, p=0.001). Cellular inflammation (>14 CD3+ cells/mm2) was significantly more common in ICM than DCM patients. (27 vs. 6%, p=0.024). Inflammation and fibrosis did not correlate significantly (r=0.09, p=0.46). Fibrosis was not associated with OXPHOS capacity (r=−0.129, p=0.33). In patients with inflammation, OXPHOS capacity was lower compared to patients without inflammation (92.3±40.1 vs. 131.6±55.6 pmol/(s*mg), p=0.031) and inflammation was associated with lower OXPHOS capacity (r=−0.296, p=0.019). There was a trend towards lower electron transfer capacity in patients with inflammation (98.6±43.1 vs. 129.7±48.3 pmol/(s*mg), p=0.053). ROS production was not significantly different between tissue specimens with or without inflammation (0.51±0.21 vs. 0.67±0.33 pmol/(s*ml), p=0.17). Conclusions Myocardial cellular inflammation is prominent in ICM patients and relates to impaired mitochondria. Future studies may evaluate possible benefits from immunosuppression in ICM patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG)

Targeting RNA elongation in endothelial-to-mesenchymal transition

Abstract Background and purpose EndMT describes a process in which endothelial cells lose their endothelial features such as the maintenance of the vascular barrier function and trans-differentiate into mesenchymal like cells. ECs which have underwent EndMT are enriched in vulnerable plaques in humans. Several stress stimuli such as hypoxia or inflammation demand immediate induction of adaptive gene expression to improve cell survival. In contrast to the usual induction of gene expression which requires several hours, release of paused RNA Polymerase II (RNAPII) from promoter allows rapid gene induction via RNA elongation. This rapid response mechanism is mediated by the super elongation complex (SEC), which is built around the scaffold proteins AFF1 and AFF4. Here, we hypothesized that RNA elongation regulates EndMT and therefore investigated on the contribution of the SEC scaffold proteins during the process of EndMT. Methods and results After induction of EndMT in human primary endothelial cells (EC) the elongation factors AFF1 and AFF4 were induced (AFF1: 1.9-fold and AFF4: 2.7-fold, P<0.05). Overexpression of AFF4 resulted in increased expression of the mesenchymal marker genes SM22 (1.8-fold, P<0.05) and VCAN (2.0-fold, P<0.05). Accordingly, treatment of EC with two small molecules inhibiting the SEC reduced the expression of several mesenchymal markers SM22 (−87.6%, P<0.05) and Calponin (−93.8%, P<0.05) during EndMT. After induction of EndMT, a large number of newly synthesized RNA transcripts were upregulated, indicating regulation of gene expression on the level of RNA elongation during EndMT. Sequencing of newly transcribed RNA (4sU-sequencing) after treatment with the SEC inhibitors revealed a lower transcript density downstream of promoter sites, suggesting a decreased RNA elongation rate in ECs. SEC inhibitors decreased the expression of the majority of EndMT-induced transcripts (63.6%). Among these rapid induced genes we found the mRNA of RE1 (+10,8-fold). We validated the regulation of RE1 by qPCR (2.8-fold, P<0.05) and single-cell sequencing. Chromatin immunoprecipitation (ChIP) sequencing revealed a reduced occupation of RNAPII in proximity of promotor sites of RE1 upon induction of EndMT. These data suggest a SEC-mediated regulation on the level of RNA elongation of RE1 during EndMT. Conclusion SEC scaffold proteins AFF1 and AFF4 are upregulated during EndMT. Targeting the SEC during EndMT reduces the expression of common mesenchymal markers, consistent with an overall higher occupancy of paused RNAPII at promoter-proximal sites. We identify RE1 as a rapidly induced gene during EndMT that is mediated by the SEC scaffold proteins AFF1 and AFF4. Our findings suggest that RNA elongation is an inducer of EndMT. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

Optimal protamine-to-heparin dosing ratio for the prevention of bleeding complications in patients undergoing TAVR – a multicentre experience

Abstract Background Despite major advances, transcatheter aortic valve replacement (TAVR) is associated with procedure-related vascular and bleeding complications, that have a significant impact on mortality. A recently published study has shown that heparin antagonization using protamine resulted in significantly lower rates of serious bleeding events in patients undergoing TAVR as compared to those without heparin reversal. However, the optimal protamine-to-heparin dosing ratio to prevent bleeding complications without increasing ischemic complications in patients undergoing TAVR is unknown. Accordingly, daily clinical practice varies between selective to routine administration of protamine in different dosing ratios. Purpose The aim of this observational multicentre study was to compare the safety and efficacy of two different protamine-to-heparin dosing ratios for the prevention of bleeding complications after TAVR. Methods The study included 1446 patients undergoing TAVR, of whom 623 (43.1%) received partial and 823 (56.9%) full heparin antagonization (0.4–0.6 mg versus 0.9–1.0 mg protamine/100 units of heparin). The indication for partial or full heparin antagonization was left to the discretion of the operator, who made the decision according to the patient's individual thrombotic and bleeding risk. The primary endpoint was a composite of 30-day mortality, life-threatening and major bleeding. Safety endpoints included stroke and myocardial infarction at 30 days. Results The overall study population had a mean age of 81.1±6.0 years; 47.9% were of female gender. The baseline characteristics were well balanced between the two groups. Full antagonization of heparin resulted in significantly lower rates of the primary endpoint as compared to partial heparin reversal (5.6 vs. 10.4%, p<0.01), mainly driven by lower rates of life-threatening (0.5 vs 1.6%, p=0.05) and major bleeding (3.2 vs 7.5%, p<0.01). The incidence of major vascular complications was significantly lower in patients with full heparin reversal (3.5 vs 7.5%, p<0.01), as presented in Figure 1. Accordingly, the post-interventional drop in hemoglobin level and the need for red-blood-cell transfusion was lower in patients receiving full as compared to partial heparin reversal (1.5±1.2 vs 1.7±1.2 g/dl, p<0.01; 10.4 vs 15.9%, p<0.01, respectively). Regarding safety endpoints, no differences were observed in the incidence of stroke and myocardial infarction between the groups (2.2 vs 2.6%, p=0.73 and 0.2 vs 0.4%, p=0.64, respectively). Multivariate regression analyses revealed that full antagonization of heparin (OR: 0.43 [95% CI: 0.24–0.81], p<0.01) was independently associated with the primary end point Conclusion Full heparin antagonization resulted in significantly lower rates of life-threatening and major bleeding after TAVR as compared to partial heparin reversal. The occurrence of stroke and myocardial infarction was low and comparable between both groups. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).

Comparison of the antiarrhythmic potential of doxapram and its metabolite ketodoxaparam

Abstract Background A few years ago, the TASK-1 channel has been established as a potential new target for the therapy of atrial fibrillation (AF). In the heart, TASK-1 is almost exclusively expressed in the atria and is significantly upregulated in AF patients. Therefore, it plays an important role in the shortening of the atrial action potential observed during AF, making TASK-1 a promising target for AF therapy. This could be proven in a porcine model of persistent AF in which an intravenous application of the TASK-1 inhibitor doxapram led to a termination of AF and the restoration of sinus rhythm (SR). Doxapram's metabolite ketodoxapam is described in the literature as being active with limited data available on its usage. Therefore, the effect of ketodoxapram on TASK-1 and its possible use in AF therapy still needs to be investigated. Purpose The purpose of the present study was to assess the potential of ketodoxapram in the termination of AF. Furthermore, a comparison between doxapram and ketodoxapram was performed based on electrophysiological and pharmacological data. Methods UPLC-MS/MS assays were developed and validated for the measurement of doxapram and ketodoxapram in porcine plasma and brain tissue. Using these assays, the pharmacokinetics of both substances, after intravenous injections of 1 mg/kg, were determined in pigs. Furthermore, brain and plasma concentrations were measured to assess brain-to-plasma ratios. A porcine AF model was used to estimate the antiarrhythmic potential. Electrophysiological properties were evaluated, using two-electrode voltage clamp experiment on Xenopus laevis oocytes which heterologously expressed atrial potassium channels, to calculate the effect of doxapram and ketodoxapram on channel function. Furthermore, whole-cell patch clamp measurements were performed on isolated human cardiomyocytes. Results Doxapram and ketodoxapram showed strong inhibitory effects on TASK-1 (IC50 1.0 μM and 0.8 μM) and TASK-3 (5.9 μM and 1.5 μM), but no significant effect on the other measured ion channels. The maximal inhibition on TASK-1 was 96%. The brain-to-plasma ratio for doxapram (0.58) was almost ten-fold higher than for ketodoxapram (0.065), hinting at a reduced crossing of the blood-brain barrier (BBB) for ketodoxapram. The terminal half-life (t1/2) of ketodoxapram (1.71 h) was longer and the maximal concentration (Cmax; 4,604 ng/ml) was higher than for doxapram (1.38 h; 1,780 ng/ml). In a porcine model of AF, ketodoxapram led to a significantly reduced AF burdens comparable to doxapram. Conclusions Doxapram and ketodoxapram both show strong inhibitory effects on TASK-1, making them good candidates for a TASK-1 based AF therapy. Ketodoxapram with its longer t1/2, reduced crossing of the BBB and higher Cmax points towards a possible superiority in the treatment of AF compared to doxapram. Studies in a porcine AF model showed promising results for the use of doxapram and ketodoxapram in AF therapy. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation

Chronic insulin resistance deteriorates cardiac mitochondrial quality control in a mouse model of SEC-NAFLD-IR

Abstract Background Insulin resistance and nonalcoholic fatty liver disease (NAFLD) both relate to cardiovascular mortality. Using a mouse model of chronic lipid overload and secondary-NAFLD-induced insulin resistance (SEC-NAFLD-IR), we recently deciphered that SEC-NAFLD-IR already at young age provoked myocardial lipotoxicity with reduced mitochondrial efficiency and increased vulnerability to cardiac ischemia. However, long-term consequences of SEC-NAFLD-IR remain elusive. Purpose Here we aimed to elucidate the impact of long-term SEC-NAFLD-IR on multiple mitochondrial quality control (mQC) mechanisms in the heart and its consequences for cardiac function. Methods We studied 36 SEC-NAFLD-IR mice (72-week-old). For mechanistic experiments, we applied palmitate-induced insulin resistant murine HL-1 cells. Cardiac mitochondrial dynamics were measured via quantification of mitochondrial morphology and expression of mitochondrial fusion and fission factors (Opa1, Drp1, Fis1, Mfn 1 & 2). Mitophagy level was evaluated via immunofluorescence and protein expression of key mitophagy-related genes (Parkin, NIX, LC3). Mitochondrial biogenesis and mass were examined via quantitation of PGC-1α expression, mtDNA and citrate synthase activity. Results 72-week-old SEC-NAFLD-IR mice exhibited 21% (p=0.001) and 32% (p<0.001) higher body weight and heart weight compared with controls. Along with elevated oxidative stress, hepatic lipid accumulation and inflammation, 6h-fasted SEC-NAFLD-IR mice were characterized by increased plasma glucose, insulin and cholesterol. SEC-NAFLD-IR mice displayed a cardiac phenotype with 21% higher left ventricular mass (normalized to body weight, p<0.001) and 6% lower ejection fraction compared to controls (73.5% SEM 0.90 vs 69.4% SEM 1.65, p=0.04). We found several advantageous mQC mechanisms suppressed in aged SEC-NAFLD-IR mice including long form OPA1-mediated mitochondrial fusion, Parkin- and NIX-mediated mitophagy. Likewise, mitochondrial biogenesis was suppressed in the aged insulin-resistant heart, which was connected to a 65% downregulation of PGC-1α1 expression (p=0.01). Interestingly, downregulation of cardiac PGC-1α1 in aged SEC-NAFLD-IR mice coincided with upregulation of PARIS, indicating the crucial participation of the Parkin/PARIS pathway in mQC of the insulin-resistant heart. In addition, induction of insulin resistance in murine HL-1 cardiomyocytes also led to increased mitochondrial fragmentation and decreased PGC-1α1 expression. Conclusion This study demonstrated that regulation of mitochondrial network and turnover is hampered by SEC-NAFLD-IR in the hearts of aged mice, which may contribute to hypertrophy and cardiac dysfunction in insulin resistance. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

Leptin mediates protective effects on the vasculature

Abstract Introduction and purpose Lipodystrophy (LD) syndromes are characterized by the loss of adipose tissue resulting in metabolic complications and accelerated atherosclerosis. The systemic concentration of the adipokine leptin is reduced in LD as a result of adipose tissue deficiency. A therapeutical option to treat LD is the substitution of leptin, which improves metabolic complications and reduces mortality. However, the vascular effects of leptin remain largely unknown. Here we analyze the direct effects of leptin on the vascular system and the development of atherosclerosis. Methods and results Treatment of human endothelial cells (ECs) with leptin reduced endothelial inflammation and the process of endothelial-to-mesenchymal transition (EndMT) (CNN1, −41.4%, p<0.05, n=4). In addition, leptin administration prevented the EndMT-induced increase of endothelial permeability. The protective effect of leptin on EndMT was confirmed in vivo in a combined lipodystrophic and atherosclerosis-prone mouse model (LDLR−/−; aP2-nSrebp1c). Treatment of the mice with leptin (3.0 mg/kg body weight daily for 8 weeks) decreased EndMT. Leptin showed no effect on plaques size but reduced the protrusion of plaques in atherosclerotic areas of the aortic roots (−31%, p<0.05, n=4–6). Cytokine screening revealed an increase of the growth differentiation factor 15 (GDF15) in serum of LD patients (+26.2%, p<0.05, n=53–58) and in ECs after EndMT (+138%, p<0.05, n=6743–10920). This increase was reversed using leptin treatment in ECs undergoing EndMT, in the LD mice model, and in LD patients after 4 weeks of leptin administration. Indeed, treatment of endothelial cells with GDF15 induced EndMT (CNN1, +7.7-fold-control, p<0.05, n=3), and impaired EC barrier function. Neutralizing antibodies targeting GDF15 inhibited EndMT-mediated expression of mesenchymal genes (CNN1, −54%, p<0.05, n=4). The treatment of ECs with serum from LD patients induced EndMT and the increase of mesenchymal marker expression was inhibited with additional administration with neutralizing antibodies targeting GDF15 (CNN1, −28%, p<0.05, n=3). Conclusion Our findings indicate that EndMT is part of the cardiovascular disease progression in lipodystrophy syndromes. Leptin treatment has direct protective vascular effects by preventing inflammation, EndMT, and maintaining endothelial integrity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)