bluuebunny/crossref_metadata_2025_split huggingface dataset:
An invertebrate model for TMEM43-induced arrhythmogenic right ventricular cardiomyopathy (ARVC) type 5
Authors: Klinke N , Reinhardt M , Ratnavadivel S , Harten H , Malmendal A , Milting H , Paululat A 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG

Objectives
TMEM43 encodes for a transmembrane protein located in the ER membrane and the nuclear rim. TMEM43 has predicted four transmembrane domains. The missense variant TMEM43 p.S358L is fully penetrant in males and associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) type 5. Even though, TMEM43 is a phyllogenetically conserved protein throughout different species, little is known on the molecular mechanism of the mutation. TMEM43 is highly homologous to the single CG8111 gene in Drosophila melanogaster. The clincal relevant amino acid serine at position p358 is situated in the third transmembrane domain, the same applies to the corresponding amino acid p.S333L in the Drosophila homologue. Here, we established the first invertebrate model to study the pathophysiological effects of the human variant in the Drosophila homologue CG8111.

Methods
To investigate the effect of this missense mutation in flies, we introduced the variant CG8111 p.S333L in Drosophila and overexpressed the mutant variant CG8111p.S333L in transgenic flies, either ubiquitously or in a tissue specific manner. Cardiac specific physiological effects of the mutant variant were examined by Semi-automatic Optical Heartbeat Analysis (SOHA) for heart rate, systolic and diastolic interval, fractional shortening and arrhythmicity index. Mutant and wildtype flies were further compared by mass-spectrometry for differentially regulated proteins. Metabolites were analysed by NMR accordingly.

Results
Our studies showed that in comparison to the wild type the ubiquitously overexpressed variant leads to lethal effects at pupal stages. Individuals died from reduced food uptake and excessive lipid droplet accumulation in adipocytes. CG8111p.S333L animals up-regulate proteins, involved in fatty acid metabolism pathways. Metabolomic data revealed increased amounts of fatty acids accumulation in mutant versus wild-type animals. Furthermore, heart specific overexpression of CG8111p.S333L was associated with arrhythmias in five week old male flies.

Conclusions
CG8111p.S333L overexpression is a lethal genotype in Drosophila. Our data suggest a metabolic dysfunction associated with the mutant form of CG8111p.S333L, which shares pathophysiological characteristics of the human condition in TMEM43 p.S358L carriers.
Beneficial effects of exercise training on expression of fibrosis-related genes in a mouse model of hypertrophic cardiomyopathy
Authors: Andreassen K , Rixon C , Hauge-Iversen IM , Sjaastad I , Christensen G , Edvardsen T , Haugaa KH , Lunde IG , Stokke MK 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority (HSØ)
KG Jebsen Cardiac Research Center

Background
The effects of exercise training prior to manifestation of phenotype in hypertrophic cardiomyopathy (HCM) are unknown. In mice carrying the HCM-pathogenic α-MHC R403Q/+ (R403Q) genetic variant, accelerated progression of the HCM phenotype is induced by cyclosporine A (CsA). We used this model to determine if exercise training initiated before phenotype development could limit disease severity.

Methods
R403Q mice were allocated to treadmill exercise training (N=23) or sedentary behavior (N=23) for 6 weeks. For the last three weeks of the 6-week period, all mice received CsA to induce the HCM phenotype. Cardiac imaging (echocardiography or MRI) and exercise testing were performed at the 0, 3 and 6-week time points. After the 6-week exercise protocol, left ventricles were harvested for molecular analyses.

Results
After 6 weeks, exercise trained mice ran 2-fold further during testing than sedentary mice (p&lt;0.0001), and 2-fold further than at baseline (p&lt;0.0001). There was no difference in VO2 max between groups. Cardiac imaging did not reveal differences in hypertrophy between exercised and sedentary HCM mice. However, we observed a reduction in cardiac expression of key fibrosis-related genes in the exercise group, i.e. Col1a2 to 49 % (p=0.03), Col1a1 to 50 % (p=0.03), Col3a1 to 46 % (p=0.02), Fn1 to 49 % (p=0.01) and Lox to 58 % (p=0.05) of levels in the sedentary group. HPLC did not reveal differences in collagen protein.

Conclusion
Exercise training initiated prior to manifestation of the HCM phenotype in mice results in lowered production of key fibrosis-related genes, including structural collagens. Further analysis is needed to determine the functional consequences of these beneficial changes in gene expression.
Myocardial tissue slices for modeling of the human PLN p.Arg14del associated cardiomyopathy
Authors: Van Der Geest JSA , Lei Z , Doevendans PAFM , Sluijter JPG 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO - The Dutch Research Council

Background
Current in vivo preclinical models lack the predictability of the clinical efficacy resulting in a high dropout rate of therapeutic candidates. Advanced human-based models are required to bridge this gap and indicate the potential of novel therapeutic approaches[1]. Myocardial tissue slices are an in vitro model that recapitulates the native multicellular architecture of the heart. This allows for modeling cellular processes in a macroscopic context entailing great promise. The most common cardiomyopathy-related mutation in the Netherlands is the loss of arginine at position 14 (p.Arg14del) in the phospholamban protein (PLN). PLN is a critical regulator of calcium cycling and contractility in the heart. The p.Arg14del mutation results in a super inhibition of SERCA2a and thus aberrant calcium handling and reduced contractile force. Mice models harbouring the PLN p.Arg14del do not completely recapitulate the human manifestation mainly due to the difference between species; heart rate, Calcium-cycling and ion properties, and different myosin heavy chain isoforms, showing the unmet need for a human-based model[2,3].

Methods & Results
300 µm thick viable myocardial tissue were sectioned from a PLN p.Arg14del patient’s left ventricle. Although the myocardial tissue slices were kept alive for eight days in static culture, these conditions initiated cell death and dedifferentiation. The tissue slices show the greatest resemblance to the intact architecture of the in vivo human heart, it is the most relevant model for viral transduction in the human heart, and proof-of-principle of this is performed. Myocardial tissue slices of a PLN p.Arg14del patient retain the structural phenotype shown by the fibrofatty deposition. Similarly, functional patient characteristics, aberrant calcium handling, and reduced contractile force are preserved.

Conclusion
Myocardial tissue slices recapitulate the (patho)physiology of the heart, as shown here with the PLN p.Arg14del case. However, the static culture conditions induce remodeling of the heart and thus only allow for acute measures in the native heart. To prolong the period that the slices recapitulate the native heart, culture conditions should mimic the environment of the heart. The tissue slices allow for a currently unmet need to modulate the complex architecture of the human heart with e.g. novel delivery tools or therapeutic interventions. All in all, myocardial tissue slices are a promising model that can give novel insights into the physiology of the human heart, and therapeutic intervention on induced or genetic cardiomyopathies.
Proteomics to assess myocardial remodelling in human heart failure and explore the effect of medications and comorbidities
Authors: Barallobre-Barreiro J , Radovits T , Fava M , Daroczi L , Hasman M , Schmidt L , Baig F , Theofilatos K , Crespo-Leiro MG , Domenech N , Merkely B , Mayr M 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Background
Myocardial remodelling is a hallmark of heart failure (HF). Our previous proteomics analyses demonstrated a profound effect of beta blockers on cardiac extracellular matrix composition in ischaemic HF patients (1). No comprehensive proteomics characterizations has been performed in non-ischaemic HF patients.

Methods
Mass spectrometry was used to analyze intracellular and extracellular protein extracts from left ventricular samples obtained from patients with ischemic (n=65) and non-ischaemic (n=114) HF after heart transplantation, as well as non-failing controls (n=19). All HF patients had ventricular dilatation and reduced ejection fraction (EF%).

Results
The proteomics comparison across 198 cardiac samples with two tissue extracts revealed hallmarks of HF in both groups of patients compared to controls, including elevated levels of atrial natriuretric peptides and the fibroblast marker vimentin, as well as decreases in creatine kinase M/B, troponin C, and myosin light chain 2 (Figure 1). In comparison to ischaemic HF patients, non-ischaemic HF patients showed elevated levels of proteins involved in proteasome activation, consistent with the notion of increased protein degradation in cardiomyocytes even at the end stage of the disease (2). Despite similar clinical characteristics, myocardial remodelling in response to HF medications and the presence of comorbidities was different between the two HF groups. In ischaemic HF patients the use of β-blockers resulted in reduced proteoglycan deposition. In non-ischaemic patients, few protein changes were associated with medication. Instead, comorbidities such as atrial fibrillation and hypertension were critical determinants of myocardial protein remodelling in these HF patients (Figure 2). Notably, hypertensive HF patients showed a marked reduction in angiotensin converting enzyme 2 (ACE2) compared to normotensive patients, and this was accompanied by a reduction in Cysteine And Glycine Rich Protein 3 (CSRP3), a key player in the organization of cytosolic structures in cardiomyocytes, and the nuclear lamina proteins lamin A/C and B. The inflammation-related galectin 3-binding protein was reduced in hypertensive patients as were the extracellular proteases matrix metalloprotease 2, cathepsin G and chymase 1.

Conclusion
Our study constitutes the largest proteomics analysis of HF to date. We observed distinct protein remodelling processes in ischaemic and non-ischaemic HF patients and discerned the myocardial effects of medications and comorbidities such as hypertension.
Investigating novel roles for nesprin-1 and the LINC complex in Dilated Cardiomyopathy
Authors: De Silva S , Catibog N , Shanahan C , Zhang Q 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): British Heart Foundation
Cardiomyopathies are an important cause of heart failure and sudden cardiac death. Recent studies have demonstrated the importance of the "passive" mechanical components of cardiomyocytes (CMs) as new causes for dilated cardiomyopathy (DCM). Nesprin-1/-2 are highly expressed in skeletal and cardiac muscle and together with SUN (Sad1p/UNC84)-domain containing proteins form the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex at the nuclear envelope (NE), which, in association with lamin A/C and emerin, mechanically couples the nucleus to the cytoskeleton. We have recently identified novel nesprin-1 mutants in DCM patients, which cause LINC complex disruption, leading to defects in nuclear organisation and myogenesis in vitro. We aim to investigate mechanisms through which these mutations lead to DCM. Therefore, we have generated a nesprin-1 mutant R8253Q knock-in (KI) mouse line (equivalent to human SYNE1 R8272Q) as the first clinically relevant animal model. 2. R8272Q is in close proximity to the binding regions to microtubule (MT) associated proteins kinesin light chain-1/2 and Akap6 at the nuclear envelope, which is potentially in involved in MT organization. Preliminary mouse echocardiography data showed significantly reduced thickness of left ventricle (LV) posterior wall in diastole, and reduced % ejection fraction in the KIs at 15 weeks after birth, suggesting LV dysfunction and a tendency of DCM. Immunofluorescence (IF) of isolated adult cardiomyocytes (ACM) from KI mice showed perinuclear MT localization was significantly reduced, KLC-1/2 and AKAP6 were mislocalized from the NE and altered nuclear positioning. Therefore, we propose to investigate novel roles of nesprin-1 and the LINC complex in cardiomyocyte mechanotransduction using this KI model to explore roles of nesprin-1 in microtubule organization, nuclear positioning, and CM homeostasis, which will yield insights into signalling leading to DCM and heart failure as well as inform strategies for translational approaches.
Unravelling sex-dependent mechanisms in calcific aortic valve stenosis
Authors: Moschetta D , Massaiu I , Valerio V , Bozzi M , Myasoedova V , Poggio P 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public Institution(s). Main funding source(s): Italian Ministry of Health

Background
Calcific aortic valve stenosis (CAVS) is the most common heart valve disease that affects 3% of the population. CAVS is a progressive, multifactorial disorder driven by several cellular and molecular pathological processes. Currently, there is no pharmacological therapy able to prevent CAVS progression. Little is known about the sex-related difference in molecular mechanisms. Recent studies reported that men showed a higher aortic valve calcium load than women, which in turn, developed more fibrosis in the stenotic aortic valve leaflets. Hence, it has been hypothesized that the mechanisms underlying CAVS progression could be different between the two sexes.

Purpose
To investigate sex-differences in CAVS molecular pathways, finding new sex-related pharmacological targets.

Methods
Valve interstitial cells (VIC) were isolated from 26 patients who underwent aortic valve replacement for aortic valve stenosis. RNA, extracted from VICs, was sequenced by third generation sequencing technology. We performed pro-fibrotic (TGFβ) treatment for 5 days and we evaluated soluble collagen expression by a colorimetric assay and αSMA expression by western blot. We evaluated extracellular calcium extent by a colorimetric assay on VICs exposed to pro-calcific (inorganic phosphate) medium for 7 days. We assessed the mitochondrial damage with mitoSOX staining by Imagestream.

Results
The gene expression analysis from sequenced VICs revealed 65 genes upregulated in men and 89 genes upregulated in women. The functional analysis showed that the TGFβ signaling resulted enriched in woman VICs, while the mitochondrial related genes were upregulated in man VICs. Only woman VICs secreted higher amount of collagen under TGFβ treatment respect to the untreated (1.22±0.03 fold change (FC) vs. untreated, p&lt;0.001). αSMA expression (marker of VICs activation) was significantly higher both in men and women exposed to different concentrations of TGFβ (TGFβ 5ng/ml: 1.88±0.34 in men and 2.66±0.32 in women, FC vs. untreated, p=0.003 and p&lt;0.0001, respectively; TGFβ 10ng/ml: 2.96±0.55 in men and 2.16±0.35 in women, FC vs. untreated, p=0.0003 and p=0.0006, respectively). The calcification potential of VICs exposed to pro-calcific medium was higher in men than in women (8.23±0.45 vs. 4.69±0.59 FC vs. untreated, respectively, p&lt;0.0001). The mitochondrial superoxide formation in man VICs was higher than in woman VICs at steady state (51.0±2.1% vs. 29.5±0.9% of positive cells, respectively, p&lt;0.0001)

Conclusion
CAVS progression seems to be driven by sex-dependent molecular pathways activation. In particular, pro-fibrotic pathways are upregulated in women, while pro-calcific pathways and mitochondrial damage in men. Our data pave the way to sex-dependent pharmacological target research, aiming to treat CAVS patients.
Single cell RNA-seq analysis reveals involvement of the interferon-I pathway and cellular senescence in calcific progression of human aortic valve cells
Authors: Ferrari SF , Agrifoglio MA , Manteiga JM , Giannese FG , Pesce MP 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ricerca corrente

Introduction
Valve interstitial cells (VICs) are heterogeneous fibroblast-like cells populating the aortic valve (AoV), involved in the extracellular matrix homeostasis. Under pathological conditions, VICs acquire the phenotype of calcific cells causing calcific aortic valve disease (CAVD) [1].

Purpose
In order to find distinctive traits of the phenotype in calcific VICs, we employed single cell RNA-sequencing (scRNASeq) to compare the gene expression signatures of VICs from stenotic AoVs (sVICs) vs. valves with insufficiency (iVICs), a pathology characterized by a lower level of calcification.

Methods
Before being used for scRNAseq profiling, VICs from the two pathological settings were amplified for three passages, after which they were loaded into the Drop-seq platform that encapsulated them in oil droplets with lysis buffer and barcoded primer beads. Beads were then subjected to reverse transcription, followed by library preparation. Libraries were sequenced with a coverage ranging from 50,000 to 100,000 reads per cell.

Results
A total of 5844 iVICs and 5610 sVICs were successfully separated and barcoded. Four independent VICs donors/pathology were processed and UMAP representation/clustering of cells coming from the eight samples identified the existence of 8 distinct cellular phenotypes, including embryonic progenitor mesenchymal (EMBVICs), ‘activated’ (ACVICs), ‘osteoblastic’ (OBVICs) and ‘quiescent’ (QVICs). The analysis of the cellular composition did not reveal substantial differences in the abundance of the different phenotypes in the two pathologies. On the other hand, differential gene expression followed by functional pathways enrichment showed a significant upregulation in sVICs vs. iVICs of genes/pathways related to Interferon-I (INF-I) in all the identified cellular phenotypes with the exception of QVICs. These included e.g for ACVICs, IFITM1 (log2FC = 1.76 P=3e-101), IFI6 (log2FC = 2.38; P = 1e-69), IFITM3 (log2FC 0.9 P = 5e-58), ISG15 (log2FC = 1.7 P= 4e-53) and IFI27 (log2FC = 1.54 P = 6e-41) genes.

Conclusions
This study reveals the involvement of the interferon-I pathway signaling in CAVD, supporting an autophagic/inflammatory/senescent phenotype switching in relevant VICs types resident in the human AoV.
CD69 expression on Treg cells prevents chronic heart damage after myocardial infarction
Authors: Blanco Dominguez R , Martin-Aguado L , De La Fuente H , Rodriguez C , Jimenez-Alejandre R , Rodriguez-Arabaolaza I , Garcia-Guimaraes MM , Vera A , Cuesta J , Cecconi A , Alfonso F , Sanchez-Madrid F , Martinez-Gonzalez J , Martin P 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e Innovación (MCIN), through the Carlos III Institute of Health (ISCIII)-Fondo de Investigación Sanitaria (PI19/00545)

Background
Increasing evidences advocate for an important function of T cells in controlling immune homeostasis and pathogenesis after myocardial infarction (MI), although the molecular mechanisms remain elusive.

Result and Methods
In this study, a broad analysis of immune markers in 283 patients show a significant CD69 overexpression on Treg cells after MI. Our results in mice demonstrate that CD69 expression on Treg cells increases survival after left-anterior-descending coronary artery (LAD)-ligation. Cd69-/- mice develop strong IL17A+ gdT cell responses after ischemia that increase myocardial inflammation and, consequently, worsen cardiac function. CD69+ Treg cells induce apoptosis and decrease IL-17A production in gdT cells by a CD39-dependent mechanism. Adoptive transfer of CD69+ Treg cells to Cd69-/- mice after LAD-ligation reduces IL17A+ gdT cell recruitment increasing survival. Consistently, clinical data from two independent cohorts of patients indicate that increased CD69 expression in peripheral blood cells after acute MI is associated with a lower risk of re-hospitalization for chronic heart failure (CHF) after 2.5 years of follow-up. This result remained significant after adjustment for age, sex and traditional cardiac damage biomarkers (OR 0.929, 95% CI, 0.838-0.980; p&lt;0.0409).

Conclusion
Our data highlight CD69 expression on T cells as a therapeutic and prognostic target to prevent CHF after MI.
The role of an alpha-selective phosphoinositide-3 kinase inhibitor in vascular inflammation
Authors: Chaudhry H , Zarban A , Kodji X , Cerutti C , Argunhan F , Ridley A , Brain SD 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public Institution(s). Main funding source(s): British Heart Foundation

Introduction
Cardiovascular inflammation is associated with endothelial cell (EC) damage, resulting in leukocyte trafficking and oedema formation. Inflammation disrupts EC junctions, increasing microvascular permeability, and resulting in a positive-feedback loop of inflammatory events. The phosphoinositide 3-kinase pathway stimulates this endothelial response and this study examined the actions of BYL-719, a PI3K-α selective inhibitor, on inflammatory responses.

Method
Confocal imaging using immunofluorescence and permeability assays were undertaken to quantify the effect of inflammatory cytokines, TNF-α and IL-1β, in the presence of the PI3K inhibitor using fluorescein isothiocyanate-dextran (40 kDa, 0.1 mg/ml) on human microvascular endothelial cells (HMVEC) (Lonza, derived from dermal tissue). Cells were treated with either cytokine for 16-18 h, followed by a 1 h treatment of drug and a final 1 h treatment with FITC-dextran. In vivo analysis was carried out per the UK Home Office Animals (Scientific Procedures) Act 1986. Male WT CD1 mice were anaesthetised initially using 5% isoflurane and maintained at 2% for procedures, to test BYL-719 in a model of dorsal skin inflammation, to determine neutrophil accumulation (measured by myeloperoxidase) and oedema formation (measured by Evans Blue accumulation). All statistical significance was determined using one-way or two-way ANOVA followed by Tukey’s post hoc test.

Results
BYL-719 significantly reduced cytokine-induced EC permeability and shape changes, including cell area and elongation (Table 1), impeding in vitro cytokine-induced inflammation. The inhibitor, abrogated effects of the inflammatory cytokines in vivo of both TNF-α and IL-1β, but interestingly had no effect on the neutrophil accumulation or oedema formation in the presence of C5a (Figure 1).
Figure 1: The effect of BYL-719 in the dorsal skin inflammation model. Mice were pre-treated with 50 mg/kg BYL-719 intraperitoneally for 30 min, injected intravenously with Evans Blue dye, intradermally injected with TNF-α (100ng/50ul), IL-1β (10ng/50ul) and C5a (300ng/50ul) for 4 h, followed by ex vivo MPO assay (A) and oedema volume measurements (B). Data are mean ±SEM two-way ANOVA by Tukey test., n=6 independent experiments in duplicates; p* &lt; 0.05 and p**** &lt; 0.0001 between control (DMSO) and BYL-719 treated groups.

Conclusions
Our findings show that the PI3K-α inhibitor, BYL-719, reduces endothelial activation and inhibits inflammatory oedema formation in the presence of TNF-α and IL-1β. We conclude that there is a potential for PI3K inhibitors to act as anti-neutrophil and oedema agents in cardiovascular-related inflammatory conditions.