tbuckley/cabot-search huggingface dataset:
Zebrafish dspb -/- tert +/- mutant as model for arrhythmogenic cardiomyopathy. Impact of aging in the development of the disease
Authors: J Wagih Gomez, S Munteanu, E Nicolas Rocamora, C Gil Ortuno, A Bernabe Garcia, M L Cayuela Fuentes, J R Gimeno Blanes, M Sabater Molina
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background/Objectives Zebrafish has been reported as model to study the Arrythmogenic Cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic Cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. On the other hand, tert gene enables telomerase activity and telomere maintenance and mutations in this gene have been used for studying aging effects. After multiple crossings of our dspb model with tert mutants we have obtained dspb-/-tert+/- model line. Purpose Our aim was the characterization of dspb-/-tert+/- mutants, studying the impact of aging in the development of the disease. Methods The model was obtained by crossing dspb-/- and tert-/- mutants, crossing the heterozygous mutants and genotyping for the selection of the dspb-/-tert+/- mutants. The characterization of the phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) young adult (8 months old) and old adult (3 years old) individuals. Results Viability assays showed that dspb-/-tert+/- mutants suffer a premature death, with much lower survival rates than the dspb-/- group (survival dspb-/- 61.3% vs dspb-/-tert+/- 35.6%, p&amp;lt;0.01). Larvae heart rate analysis showed no significant differences. Echocardiography in young adults showed no significant differences between dspb-/-tert+/- and dspb-/- mutants. In old adults, echocardiography showed differences in heart rate (dspb-/- 142.5 ± 15.5 vs dspb-/-tert+/ -97.5 ± 3.5, p&amp;lt;0.01), respiratory frequency (dspb-/- 288.7 ± 36.1 vs dspb-/-tert+/- 95 ± 49.4, p&amp;lt;0.01) and atrial contraction velocity (dspb-/-155.7 ± 26.09 vs dspb-/-tert+/ -227.2 ± 17.6, p&amp;lt;0.05). Gene expression analysis in larvae showed that dspb expression drastically decreased (dspb-/- 0.3 ± 0.1 vs dspb-/-tert+/ -0.01 ± 0.008, p&amp;lt;0.01). In adults, dspb expression drastically decreased in heart tissue (dspb-/- 0.4 ± 0.01 vs dspb-/-tert+/- 0.06 ± 0.004, p&amp;lt;0.01) and skin (dspb-/ -0.34 ± 0.02 vs dspb-/-tert+/- 0.25 ± 0.01, p&amp;lt;0.01). Conclusion The dspb-/-tert+/- zebrafish presented worse survival rates, lower dspb gene expression levels and worse pathological cardiac phenotype at adult stages than dspb-/- mutants, proving that age is an important factor in the development of the disease and proving that dspb-/-tert+/- zebrafish is an appropriate model to study the impact of aging.
Impaired autophagy mechanisms associated with phospholamban-R14del disease
Authors: E Vafiadaki, E G Kranias, A G Eliopoulos, D Sanoudou
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq Foundation Phospholamban (PLN) is a key modulator of sarcoplasmic reticulum Ca2+ homeostasis and cardiac contractility. The PLN variant c.40_42delAGA (p.R14del) leads to arrhythmogenic cardiomyopathy characterized by life-threatening ventricular arrhythmias and sudden cardiac death. A large number of PLN-R14del carriers have been identified worldwide, although the expanding use of genetic testing and increased awareness is unveiling a higher frequency. PLN-containing aggregates are emerging as a hallmark of this disease, and are increasingly believed to be associated with disease pathogenesis. However, the mechanism leading to the formation of aggregates is unknown. We performed an in depth molecular and cellular analysis utilizing in vitro systems to determine if these aggregates result from aberrations in autophagy, and the underlying mechanisms. Utilizing cell culture systems we studied the expression level and subcellular distribution of fundamental autophagy-related proteins and evaluated the impact of autophagy inducers and inhibitors. Expression of PLN-R14del in different cell culture systems (HEK293 and H9c2 cells) was found to result in PLN aggregate formation, which were determined to contain the autophagy marker proteins sequestosome-1 (p62) and microtubule-associated protein light chain 3 (LC3), indicating inefficient degradation by the autophagy pathway. In support of this, PLN-R14del expressing cells were shown to exhibit increased p62 and LC3-II protein levels. This was associated with significantly reduced autophagic flux, as demonstrated by examination of LC3-II protein levels after autophagy inhibition. Moreover, by detailed immunofluorescence analysis, we determined reduced co-localization of the autophagosomal protein LC3 and the lysosomal associated membrane protein 1 (LAMP1) in PLN-R14del cells, suggesting impaired autophagosome-lysosome fusion. We next examined the distribution of key proteins mediating autophagosome-lysosome membrane fusion including the Ras-related protein Rab7, the ultraviolet radiation resistance-associated gene (UVRAG) and membrane tether proteins. In PLN-R14del transfected cells, significant alterations in their co-localization with LC3 were observed, indicating their impaired recruitment to the autophagosomes. Importantly, in PLN-WT cells changes in cytosolic Ca2+ levels by thapsigargin treatment were found to recapitulate the autophagic defects of PLN-R14del, suggesting that Ca2+ alterations are a contributing factor of PLN-R14del mediated aberrant autophagy. In conclusion, our findings reveal a causal relationship between PLN-R14del and autophagy dysregulation, through inhibition of autophagosome-lysosome fusion and autophagic flux reduction. This ultimately leads to aggregate formation, an early pathological sign in human patients.
EZH2 inhibition reduces macrophage inflammatory responses in atherosclerosis
Authors: R W M Kempkes, L C M Rief, C P A A Roomen, G R Griffith, W G Vos, L A Bosmans, M J J Gijbels, M A Hoeksema, K H M Prange, M P J De Winther, A E Neele
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation (Hartstichting) Aim Epigenetic processes are essential modulators of macrophage inflammatory responses. We postulate that interference in the epigenetic machinery of macrophages might offer novel approaches to combat atherosclerosis. Here, we investigate the repressive histone modification H3K27Me3 deposited by the polycomb repressive complex 2 (PRC2) with its catalytic component EZH2. We studied the therapeutic potential of macrophage EZH2 inhibition in the context of atherosclerosis. Methods Human monocyte-derived macrophages and murine peritoneal and bone-marrow derived macrophages were treated with the EZH2-specific inhibitor GSK126 and subsequently activated with LPS to mimic TLR4-inflammatory responses. The impact of Ezh2 inhibition on macrophage differentiation and activation compared to vehicle (DMSO) was assessed by RNA-seq, ChIP-seq, flow cytometry, western blot, and ELISA. To study its impact on atherosclerosis, female Ldlr-/- mice on a high-cholesterol diet (9weeks) were treated the last four weeks with GSK126 or control. Results Ezh2 inhibition by GSK126 in vitro lowered global H3K27Me3 levels without altering macrophage viability and differentiation, showcasing effective EZH2 inhibition. RNA-seq revealed that of more than one-third of the LPS-induced genes were significantly downregulated by EZH2 inhibition. Subsequent pathway analysis identified cytokine and interferon signaling, co-stimulation, and cell migration as the top down-regulated pathways (padj&amp;lt;0.05). Furthermore, ChIP-seq revealed considerably altered regulation of H3K27Me3 deposition. Indeed, we confirmed that gene and cytokine expression of the inflammatory mediators IL-6, IL-12, and TNF were reduced. Furthermore, membrane marker expression of co-stimulatory CD40, CD80, and CD86 were significantly decreased. In murine atherosclerosis, we observed a reduction in the Virmani plaque severity score and are currently assessing lesion size and composition. Conclusion Overall, we show that EZH2 inhibition reduces inflammatory responses in human and murine macrophages in vitro. We are currently assessing the impact of EZH2 inhibition on lesion size and composition in murine atherosclerosis. Additionally, we are performing ex vivo experiments on human endarterectomy plaques to assess the therapeutic potential of EZH2 inhibition on human atherosclerosis.
Untangling the inflammatory responses of liver x receptor (LXR) activation in human macrophages
Authors: L F Zijlstra, R W M Kempkes, M A Giera, P C N Rensen, S Kooijman, M P J De Winther, A E Neele
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Rembrandt Institute for Cardiovascular Science (grant 2022) The regulation of cholesterol and fatty acid homeostasis is essential and the Liver X Receptor (LXR) plays a crucial role in this process. When activated, LXR promotes cholesterol efflux and suppresses the transcription of pro-inflammatory genes in mouse macrophages, making it a promising therapy against atherosclerosis. Studies indeed have shown that LXR agonism is effective in pre-clinical atherosclerotic mouse models. However, the limited data on human macrophages suggests that LXR activation may have additional pro-inflammatory effects in humans, and the mechanisms contributing to this are not yet fully understood. To investigate the inflammatory properties of LXR activation in human macrophages, monocytes were isolated from buffy coats of healthy donors and differentiated into macrophages using M-CSF. The macrophages were exposed to synthetic and endogenous LXR ligands, such as GW3965, T0901317, and desmosterol, respectively, followed by stimulation with lipopolysaccharide (LPS) for six hours. We used qPCR and ELISA to analyze experiments and plan to supplement our data with RNA-Seq and multiplex assays. Preliminary results show that LXR activation reduces LPS-induced activation of interferon-mediated genes like CXCL9 and CXCL10, similar to mice. However, it promotes the transcription of NF-kB target genes like IL1B and IL12B upon LPS activation. These findings suggest that LXR activation in human macrophages not solely suppresses inflammatory responses in macrophages and that LXR activation has different effects on the regulation of genes mediated by interferon or NF-kB. Our research aims to further uncover the regulatory mechanisms behind LXR activation in human macrophages as central regulators in atherosclerosis.
Unraveling the metabolic pattern of angiopoietin-like 3 knock-out mouse models under fasting and fed conditions
Authors: O Terenghi, L Da Dalt, F Fantini, A Moregola, F Bonacina, G D Norata
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): University of Milan Background Angiopoietin-like 3 (ANGPTL3) is a hepatokine and a regulator of lipid and lipoprotein metabolism through lipoprotein lipase and endothelial lipase inhibition, preventing the hydrolyzation of lipoprotein-derived triglycerides. A lack of ANGPTL3 results into a hypolipidemic phenotype, with a lower profile of lipids and lipoproteins, and this is evident in humans carrying a loss of function mutation, or in cellular or in vivo models. ANGPTL3 action, from the liver, is finely regulated between oxidative and storage tissues in the fasting and postprandial phases; from the current pharmacological treatments a possible ANGPTL3 intrahepatic role besides the lipases’ inhibition is emerging, thus possibly affecting the systemic metabolism with different implications. Purpose An axis between ANGPTL3 and other Angiopoietin-like proteins is known to regulate the triglyceride partitioning after a meal or during fasting, however the effect on tissues and the following metabolic and pathologic implications are still not known. Therefore, we aim to describe the impact of ANGPTL3 deficiency in fasting or in a postprandial state, and in different compartments of the organism. Methods As models, we utilized Angptl3 Knock-out (KO) mice (C57BL6/J background) and their littermate controls (wild-type, WT) fed a chow diet for 16 weeks. We assessed the metabolic phenotype with indirect calorimetry and with lipids, glucose, and insulin tolerance tests; we evaluated changes in plasma lipids under fast, fed, and fast-refeed settings and we investigated the histology of key metabolic organs. Results ANGPTL3 KO mice fed ad libitum a chow diet are hypolipidemic (plasma triglycerides levels: 42,42±8,80 mg/dL in ANGPTL3 KO mice vs. 122,02±55,09 mg/dL in WT mice; plasma cholesterol levels: 44,00±9,11 mg/dL in ANGPTL3 KO mice vs. 76,51±15,87 mg/dL in WT mice). The oral charge lipid challenge suggests that KO mice have lower lipid levels at all time points. Conclusions This preliminary profiling of ANGPTL3 KO mice under fasting and fed conditions highlights the hypolipidemia of these models, and the potentially beneficial impact of ANGPTL3 deficiency on their metabolism compared to controls.
The structural remodeling of the mitochondrial Ca2+ uniporter complex underlies its functional impairment during metabolic cardiomyopathy
Authors: M Panel, L Fossier, M Lacote, L Luong-Azria, A C Meli, P Sicard, J Thireau, A Lacampagne, J Fauconnier
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ANR Introduction Calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter complex (MCUC) controls key steps of oxidative metabolism to adapt energy production to cardiac demand. To ensure this critical function, MCUC assembly and maturation is a tightly regulated process involving the AAA proteases. We recently demonstrated that mitochondrial Ca2+ uptake is impaired during the early stages of metabolic cardiomyopathy in the atria. Purpose Our aim was to investigate mitochondrial Ca2+ handling in the left ventricle during the early stages of metabolic cardiomyopathy and to characterize the mechanisms underlying the functional remodeling of the MCUC. Methods Male C57Bl6/J mice were fed a high-fat sucrose diet (HFS) for two weeks to induce metabolic cardiomyopathy. Mitochondrial Ca2+ uptake was then studied (1) in isolated ventricular cardiomyocytes using whole-cell patch-clamp coupled with confocal microscopy and (2) in isolated mitochondria using Ca2+ Green-5N. The biophysical properties of MCUC were recorded in a planar lipid bilayer. MCUC structure and AAA-proteases expression were assessed by Western blot and co-immunoprecipitation. Control mice were treated with AAV9 harboring shRNA to downregulate the expression of MAIP1. Results Metabolic cardiomyopathy was associated with reduced beat-to-beat mitochondrial Ca2+ uptake in ventricular cardiomyocytes, which was further confirmed in isolated mitochondria. Single-channel recordings of the native cardiac MCUC embedded in a planar lipid bilayer revealed a reduction in ionic conductance and open probability, demonstrating functional impairment of the MCUC. Biochemical analysis of the MCUC revealed an accumulation of the unprocessed form of the EMRE subunit and a decreased interaction of the EMRE-MICU1 subcomplex with the pore-forming MCU subunits, indicating a structural remodeling of the MCUC. The expression of the AAA proteases YME1L and AFG3L2/SPG7, which are responsible for EMRE maturation and insertion into the MCUC, was unchanged. However, the expression of m-AAA protease-interacting protein 1 (MAIP1), which orchestrates EMRE processing, was reduced. In control mice, shRNA-mediated downregulation of MAIP1 recapitulated the structural and functional changes of the MCUC. Conclusions The structural remodeling of the MCUC integrity by downregulation of its assembly affects mitochondrial Ca2+ homeostasis in the early stages of metabolic cardiomyopathy. MAIP1 is a key regulator of MCUC assembly and its downregulation underlies the functional remodeling of the MCUC. These findings provide a novel post-translational paradigm of MCUC regulation in pathophysiology and suggest novel therapeutic targets for the treatment of the metabolic cardiomyopathy.
Unbiased clustering and molecular characterisation of novel metabolic phenotypes in a heart failure cohort
Authors: E Esenkova, T Koeck, G Valentini, P Wild, E Casiraghi, E Araldi
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DIASyM cluster Heart Failure is a heterogeneous clinical syndrome characterized by the inability to pump enough blood and thus provide enough oxygen to the human body. The current classification of heart failure is based on the left ventricular ejection faction (LVEF). Although the classification clearly defines heart failure subtypes and stages, and clinical guidelines are available for their management, it is preferable to prevent the disorder or take control over it from its onset. Evidence suggests strong molecular connections between human metabolism and heart failure. Exploiting the unique and diverse molecular characteristics that drive heart failure could be an effective strategy for patient stratification, to identify new therapeutic targets and to personalize treatments. The current research attempts to identify clinically relevant metabolic subgroups of individuals with the help of a similarity network-based approach, based on targeted proteomics data of a heart failure cohort. With Olink targeted proteomics technology, six protein abundance panels (547 distinct proteins) from 3063 subjects (1103 females) were obtained. Using nonlinear combination methods, the created networks were integrated into a single similarity network and then used for clustering with unbiased network-based approaches. The results have distinguished the subgroups with worse outcome prediction, supported by significantly different clinical profiles. Surprisingly, the significant clinical parameters have shown the dominance of not only classical metabolic signatures (diabetes) but also parameters related to kidney and liver function. The differential protein abundance profiles were then compared among clusters to derive novel insights into clinical subtypes, and have highlighted a significant contribution of inflammatory proteins to the unbiased subtyping rationale. The study attempted to improve previous patient stratification efforts, by having a large sample size with a broader variety of molecular variables. These results will pave the way to precision medicine, and will allow indirectly to integrate and/or adjust the analysis by adding clinically relevant social parameters such as lifestyle, parents’ anamnesis, demographics that are often left out due to their lower contribution at the pre-processing steps in comparison to more objective laboratory parameters. Previous studies have already clustered the heart failure population, based on various clinical and biochemical parameters. However, the clustering was guided by well-established metabolic parameters and thus, the results can be highly biased. The current project allows an unbiased, data-driven approach that holds more potential to identify novel and clinically relevant metabolic subgroups and to define new molecular targets.
Aging related changes in cardiovascular system in healthy female population: photoplethysmography method and DFA analysis
Authors: B Djuric, K Zikic, N Milosevic, Z Nestorovic, D Zikic
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science Technological Development and Innovation Background The cardiovascular system undergoes dynamic changes during the biological aging in humans, leading to chronic cardiovascular and cerebrovascular diseases. Purpose Developing the novel method for non-invasive monitoring of age induced changes in arterial vessels. Methodology The pilot study was performed on 58 women, in good health, non-smokers, between the ages of 20 and 70, divided into three age groups. All subjects underwent arterial blood pressure measurement using a photoplethysmography sensor. Recordings were performed in left carotid artery and the left index finger area. Arterial blood flow waveform was then analyzed using DFA analysis, calculating the values of the scalar coefficients α1 and α2. Based on the waveform time interval shift between the standing and supine positions, the pulse wave velocity (PWV) ratio was determined. Results Systolic and diastolic arterial pressure showed a slight linear increase with the age, but without significant difference between the three age categories (p &amp;gt; 0.5). Also, the PWV ratio between the supine and standing positions did not show a statistically significant increase between the age groups. Only scalar coefficients ratio α1/α2 demonstrated a significant difference in value between all age categories. Conclusion In subjects included in this pilot study, only the scalar coefficients ratio varied significantly with the age, indicating the occurrence of structural changes in the cardiovascular system caused by biological aging.