bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of electrocardiographic repolarization parameters in patients with polycystic ovary syndrome
Authors: Orosz A , Szabo LA , Vagvolgyi A , Magony S , Nyiraty SZ , Toth B , Pesei F , Abraham GY , Nemes A , Lengyel CS , Varkonyi T , Baczko I
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Office

Introduction

Polycystic ovary syndrome (PCOS) is a multifactorial, endocrine disease associated with metabolic disturbances (e.g. hyperinsulinemia, insulin resistance) and increased cardiovascular risk. Recent data strongly suggest that different QT variability parameters characterizing cardiac repolarization instability represent novel markers in proarrhythmic risk assessment.

Purpose

In the present study we investigated ECG repolarization parameters, including QT variability parameters in patients with polycystic ovary syndrome.

Methods

Fifty-five PCOS patients (age: 29±6 years) and 55 age-matched healthy volunteers (age: 29±10 years) were enrolled in the study. Five-minute 12-lead resting electrocardiograms were recorded, the ECGs were digitized and evaluated off-line using the Cardiosys-A01 system (Cardiosys-A01, MDE Heidelberg GMBH, Heidelberg, Germany). The following parameters were determined: the frequency corrected QT interval (QTc) using Bazett’s, Fridericia, Framingham and the Hodges formulas; QT dispersion (QTd) and T wave peak-to-end distance (Tpeak-Tend). Among QT variability parameters we analyzed the QT variance (QTv), the QT variability index (QTVI), the short-term beat-to-beat QT and RR interval variability (STV-QT, STV-RR) based on constructed Poincaré plots and the variability ratio (VR).

Results

The RR interval did not differ significantly in PCOS patients compared to controls (821±129 ms vs. 847±99 ms), however the QT interval (373±30 ms vs. 391±27 ms, p<0.01), the QTc calculated with Bazett’s, Framingham, Fridericia and Hodges correction formulas (QTc Bazett’s: 413±18 ms vs. 426±21 ms, p<0.01) and the Tpeak-Tend intervals were significantly shorter (76±10 ms vs. 83±12 ms, p<0.01). The QTd, QTv, and STV-RR did not differ significantly. However, the VR (0.3±0.4 vs. 0.2±0.2, p<0.05), the QTVI (-0,9±0.5 vs. -1,3±0.4, p<0.001), and importantly, the STV-QT were significantly higher in PCOS patients compared to controls (4.0±0.9 ms vs. 3.2±0.9 ms, p<0.0001).

Conclusion

Some of the alterations in repolarization parameters and the significant increase in the short-term beat-to-beat QT interval variability and the QT variability index may indicate increased repolarization instability in patients with polycystic ovary syndrome compared to age-matched controls, however, further studies are needed to establish the exact relation of this finding to increased arrhythmia propensity in this population.
Modeling of an SCN5A founder mutation in iPSC-derived cardiomyocytes
Authors: Simons E , Nijak A , Vandendriessche B , Van De Sande D , Sieliwonczyk E , Labro A J , Saenen J , Snyders D , Schepers D , Loeys B , Alaerts M
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Research Foundation - Flanders (FWO)

Introduction

SCN5A encodes the α-subunit of voltage-gated cardiac sodium channel Nav1.5. Mutations in SCN5A are identified in about 20% of patients with Brugada syndrome (BrS), an inherited cardiac arrhythmia. We have identified an SCN5A founder mutation (c.4813+3_4813+6dupGGGT), leading to a loss-of-function of Nav1.5 in 25 different families. Mutation carriers show variable expression of the phenotype: from asymptomatic to syncopes and sudden cardiac death. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) to investigate the underlying pathophysiology.

Material & Methods

Dermal fibroblasts of six patients with different disease severity, and two unrelated healthy control individuals were reprogrammed using a commercially available reprogramming kit. iPSC-CMs were differentiated following a published protocol. We performed several differentiation rounds and investigated expression of cardiac markers using qPCR and immunocytochemistry and electrophysiological properties using patch-clamping.

Results

All iPSC-CMs expressed the tested markers. We observed reduction in sodium current density in patient iPSC-CMs compared to the control cells. However, our data display variability in AP characteristics between the differentiation batches, as well as between clones generated from one donor.

Conclusions

We established iPSC-CM models for a unique Belgian SCN5A founder mutation. Despite the observed variability, we could detect expected differences in electrophysiological properties of patient cells compared to controls.
Specific and common molecular alterations in knock outs of 3 desmosomal genes in HL1 cell model of Arrhythmogenic Cardiomyophathy
Authors: Vallverdu-Prats M , Brugada R , Alcalde M
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Obra Social “La Caixa Foundation”

Introduction

Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a heritable cardiac disease characterized by a progressive loss of myocardium and its replacement by fibrofatty tissue that predominantly affects the right ventricle (1,2). Worldwide prevalence of ARVC is ranging from 1:1000 to 1:5000 (3,4), and it is responsible for around 20% of all cases of sudden cardiac death (SCD) among people under 35 years of age (5). The main genetic cause are mutations in desmosomal genes (4). It is known that mutations in those genes could alter important molecular functions such as calcium homeostasis (6), and they also could cause important structural and electrical alterations such as fibrosis, adipogenesis, inflammation and arrhythmogenesis (7). We lack better understanding of those pathophysiological mechanisms and their relationship with the causal gene.

Purpose

This study aims to elucidate molecular pathways triggered by genetic defects in three of the main genes in ARVC and determine common and gene-specific molecular features.

Methods

Cardiac HL-1 cell line was edited by CRISPR/Cas9 to generate Knock Outs (KO) of three of the most frequent causal genes in ARVC: PKP2, DSG2 and DSC2. Expression levels of ARVC-related genes were tested by RT-PCR: desmosome, calcium handling and connexome related genes. Changes in the total amount of protein were checked by western blot using total protein lysate. Moreover, calcium transients were performed on all clones.

Results

HL1 KO-PKP2 showed downregulation of SCN5A, CX43, RyR2, Atp2a2, Slc8a1, Casq2, ANK2, DSC2 DSG2 and DSP. HL1 KO-DSG2 showed a downregulation of ANK2, DSC2, CX43 and PG and HL1 KO-DSC2 clones presented a downregulation of CASQ2 and ANK2. Functionally, calcium transients showed significant differences in 10mM peak of caffeine in PKP2 but still not conclusive for DSC2 and DSG2 clones.

Conclusions

Results suggest that pathophysiological mechanisms of ARVC are partially depending on the causal gene. Loss of function of PKP2 and DSC2 may be related to a dysregulation of calcium homeostasis and loss of function of DSG2 may cause alterations in connexome genes expression related to electrical dysfunction. Moreover, the absence of ANK2 expression in all HL1 cell lines might suggest a common molecular hallmark of ARVC in the presented cell model.
The ARVC causing TMEM43-p.S358L mutation has impact on the lipid metabolism
Authors: Ratnavadivel S , Klinke N , Meyer H , Paululat A , Kassner A , Gummert J , Milting H
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 450999875

Background

Transmembrane protein 43 (TMEM43) is a phylogenetically highly conserved and ubiquitously expressed protein with unknown function. The protein contains four transmembrane domains located in the inner nuclear envelope and the endoplasmic reticulum (ER). A large acidic domain located between the first and second transmembrane domains, with unknown function, is exposed to the ER lumen. TMEM43-c.1073C>T is a rare fully penetrant mutation that leads to arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). ARVC5 is associated with myocardial fibrosis, fibrofatty replacement and progressive loss of right ventricular myocardial tissue and severe arrhythmias causing sudden cardiac death (SCD) especially in males. The pathomechanism of ARVC5 is not well understood.

Methods

We isolated and cultivated primary dermal fibroblasts of three different individuals carrying TMEM43-c.1073C>T (p.S358L). Proteins were isolated from cell cultures and analysed by Orbitrap mass spectrometry. Proteome data of the mutation carriers were compared to TMEM43 wildtype control fibroblasts. Additionally, TMEM43-p.S358L and control fibroblasts were investigated by transmission electron microscopy (TEM).

Results

A total of 9 proteins were upregulated in fibroblasts expressing TMEM43-p.S358L (p<0.003). Of note, proteins involved in fatty acid metabolism, such as acyl-coenzym A (CoA) thioesterase 1 encoded by the gene ACOT1, and Malonate-CoA ligase encoded by ACSF3 were strongly upregulated (FC>3 or >2, respectively). ACOT1 catalyses hydrolysis acyl-CoAs to long chain fatty acids and CoA. ACSF3 catalyses the formation of thioesters between fatty acids and CoA. ACOT1 and ACSF3 regulate the intracellular levels of free fatty acids and are expressed in the cardiomyocytes of the human myocardium. TEM images of TMEM43-p.S358L carriers show an accumulation of multilamellar vesicles, which may support the dysregulation of the lipid metabolism.

Conclusion

The differentially regulated proteins in dermal fibroblasts and the accumulation of multilamellar vesicles suggest a dysregulation of the lipid metabolism by TMEM43-p.S358L, which may also have relevance for the development of myocardial fibrofatty replacement contributing to ARVC5.
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

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

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<0.0001), and 2-fold further than at baseline (p<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

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

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

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

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<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<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<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<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

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

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<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

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* < 0.05 and p**** < 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.
Metabolic regulation of endothelial reactive oxygen species production in obesity
Authors: Padgett C , Speese A , Rosewater C , Corley Z , Mintz J , Fulton D , Stepp D
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institutes of Health (NIH)

Introduction

The prevalence of obesity continues to rise to unprecedented levels, and with it, a corresponding increase in the incidence of cardiovascular disease (CVD). While obesity is indeed accepted as one of the most prominent risk factors for CVD, the precise molecular mechanisms by which aberrant metabolism is linked to cardiovascular function remain incompletely understood. One prevailing hypothesis is that overproduction of reactive oxygen species (ROS) from increased metabolism significantly contributes to endothelial dysfunction, which precedes many cardiovascular events such as atherosclerosis and stroke.

Purpose

In this study, we hypothesized that a receptor for advanced glycation end products, Galectin-3 (GAL3), acts as a metabolic sensor and regulates endothelial ROS production in obesity.

Methods

Obese db/db mice were crossed with mice lacking GAL3, and endothelial gene expression, microvascular reactivity, and ROS production were assessed.

Results

We demonstrate that NADPH Oxidase I (NOX1), the predominant source of endothelial superoxide production, is down-regulated by GAL3 deletion, thereby rescuing endothelial function and ameliorating endothelial ROS production in obesity. Furthermore, we demonstrate that GAL3-mediated NOX1 over-expression is amenable to improvements in metabolic status, such as lowering blood glucose with metformin, improving glucose handling by augmenting muscle mass, or improving insulin signaling through deletion of Protein Tyrosine Phosphatase 1B (PTP1B).

Conclusion

Taken together, these data demonstrate that the overproduction of superoxide by endothelial NOX1 is regulated by the metabolic sensor GAL3 in obesity, leading to endothelial dysfunction and CVD. This pathway presents an attractive target for therapeutic intervention to break the link between aberrant metabolism in obesity and its corresponding vascular pathologies.
Assessing compartmentalisation of small non-coding RNAs by fractionation of lipoproteins and extracellular vesicles of human plasma
Authors: Teng I , Gutmann C , Takov K , Mayr M
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Background/Introduction

Circulating small non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and YRNAs, have been implicated in intercellular communication and as biomarkers for cardiometabolic diseases. The putative carriers of ncRNAs are lipoproteins, extracellular vesicles (EVs) and RNA-binding proteins. However, the lack of reliable separation methods for EVs and lipoproteins from human blood hampers the assignment of small ncRNA signatures to different carriers.

Purpose

To study which classes of ncRNAs are carried by EVs or lipoproteins in the circulation.

Methods & Results

In order to minimise the co-isolation of lipoproteins and EVs, size-exclusion chromatography (SEC) was established to improve resolution of nanoparticles from plasma. Mass spectrometry and immunoblotting for EV markers (syntenin-1 and CD9) and apolipoproteins (ApoA1 and ApoB for high- and low-density lipoproteins, respectively) were used to characterise the isolated preparations and identify co-isolating species. Quantitative real-time PCR of each carrier-rich fraction after SEC showed that miRNAs were mainly detected in the late-eluting fractions, enriched in ApoA1-containing lipoproteins. Only a small proportion of miRNAs was associated with fractions rich in albumin or in EVs. On the other hand, YRNAs were predominantly found in the albumin-rich and ApoB-containing fractions. To assess whether lipoproteins are key carriers for ncRNAs, HDL and ApoB immuno-depletion methods were employed. Both lipoprotein depletion methods failed to substantially deplete small ncRNAs from plasma.

Conclusion

We studied the distribution of circulating ncRNAs amongst different SEC fractions from human plasma. We have observed a minor role for lipoproteins as carriers of ncRNAs. Further investigation will focus on using SEC fractionation to assess EVs and RNA-binding proteins and their role as ncRNA carriers in plasma.
Elderberry extract inhibits tumour necrosis factor induced monocyte adhesion to endothelial cells via modulation of the NF-κB pathway
Authors: Festa J , Singh H , Hussain A , Da Boit M
Published: June 2022

Funding Acknowledgements

Type of funding sources: Other. Main funding source(s): De Montfort PhD funded scholarship scheme

Introduction

During the early stages of atherosclerosis, monocytes attach to endothelial cells (EC) before differentiating into macrophages causing the accumulation of foam cells and plaque growth. NF-κB and vascular cell adhesion molecule 1 (VCAM-1) are some of the main mediators in EC that are involved in the monocyte adhesion process. Elderberry extract (EE) are rich in anthocyanins a subclass polyphenol with vascular-protective properties.

Purpose

The main of aim of this study is to examine if EE can prevent TNF-α induced inflammation and monocyte adherence to EC.

Methods

Primary Human Umbilical Vein Endothelial Cells (HUVEC) were pre-treated for 1-h and stimulated with or without TNF-α 10 ng/ml for western blot. Cell lysates from the treated cells were then subjected to Western blotting and probed for total and phospho-NF-κB. For monocyte adherence cells were pre-treated for 24-h, then stimulated with/or without TNF-α 10 ng/ml for 24-h. Fluorescently labelled THP-1 cells were then added to the HUVECs for an additional 30 minutes, and samples were measured in a fluorescence plate reader. Cells were put through flow cytometry for measuring reactive oxygen species (ROS) using DCFHFDA assay or VCAM-1 levels using Anti-CD106.

Results

An increase in ROS production and NF-κB phosphorylation was found after stimulation TNF-α 10 ng/ml (p = 0.01). However, cells pre-incubated with EE (50 μg/ml) for 1-hour before TNF-α stimulation caused a reduction in ROS as well as inhibition of NF-κB phosphorylation (p < 0.01). TNF-α 10 ng/ml increased the monocyte adherence to the HUVECs by a 2-fold although, EE prevented TNF-α monocyte adherence (mean value, 589.7 vs 408 p = 0.0033). This was associated with suppressed VCAM-1 expression found in the EE pre-treatment with TNF-α (p = 0.02).

Discussion

Our Preliminary data demonstrates that EE, can prevent monocytes binding onto EC potentially by inhibiting TNF-α induced NF-κB and VCAM-1 levels. Our findings postulate that NF-κB and VCAM-1 could be the direct link for targeting the prevention of monocyte adherence to EC, although this would need confirmation by blocking key cellular signalling pathways to confirm its role. This preliminary data suggests that EE and potentially other polyphenols could be a useful strategy for targeting the initial stages of atherosclerosis.
Serotype-specific microvascular changes in the liver due to dengue virus (DENV) non-structural protein-1 (NS1)
Authors: Duruanyanwu J , Campagnolo PC , Maringer KM
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): University of Surrey

Dengue virus is the most common arbovirus in humans worldwide, with a minor proportion developing severe dengue, which includes the cardiovascular complications of vascular leakage, haemorrhage, organ failure and shock, all of which could prove fatal. The mechanisms that lead to microvascular leakage remain incompletely understood. While DENV-2 NS1 has been shown by our group to disrupt endothelial-pericyte interactions leading to enhanced endothelial cell permeability in vitro, it is unclear whether this is true across all four serotypes of DENV and to what degree. It is also unclear whether DENV NS1 similarly affects all types of pericytes across the body. Our previous work was performed in saphenous venous pericytes, which we here extend to include hepatic stellate cells (HSCs), since the liver is a major site of dengue pathology. In this project, transendothelial electrical resistance readings of co-cultures of endothelial cells with either SVPs or HSCs both revealed the DENV serotypes to significantly lower endothelial membrane function, most notably at DENV-2. Additionally, preliminary data from the 3D Matrigel angiogenesis assay revealed the DENV serotypes to alter the ability of endothelial cells and SVPs to form vascular structures. Interestingly, our alamar blue proliferation assay utilising hepatic stellate cells (HSCs) showed that liver pericytes display increased proliferation in response to the various DENV serotypes, with immunostaining of HSCs also revealing an increase in myofibroblast protein alpha-smooth muscle actin. These results collectively show the ability to disrupt endothelial-pericyte interactions is shared across the DENV serotypes and suggest a potential loss of endothelial-pericyte interaction due to pericyte differentiation in the liver. This information gives further insight to the mechanisms related with DENV interactions with the vasculature, which could aid in establishing a diagnostic test or treatment for those affected with severe dengue fever.
Myostatin deletion reverses myosteatosis and improves angiogenesis in obese mice
Authors: Speese A , Padgett C A , Rosewater C L , Corley Z L , Mintz J D , Fulton D J , Stepp D W
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): T32 Training Grant: Multi-Disciplinary Training Program in the Mechanisms of Cardiometabolic Disease

Objective

To test the hypothesis that increased muscle mass achieved through deletion of myostatin improves ischemic angiogenesis in obese mice.

Methods

db/db mice, a well-described model of obesity, were crossed with mice lacking myostatin (MSTN KO), a myokine that negatively regulates muscle differentiation and growth, to generate lean and obese mice with and without elevated muscle mass (MSTN KO). We utilized confocal and electron microscopy (EM) and nuclear magnetic resonance (NMR) spectroscopy to assess morphology and histology in the skeletal muscle (SKM) of db +/-_MSTN +/- mice. Muscle lipid contents were assessed via mass spectrometry. Femoral artery ligation was used to stimulate in vivo angiogenesis in response to ischemia. Gene expression was assessed by RNA Seq with mRNA and protein expression follow-up in isolated gastrocnemius skeletal muscle via RT-qPCR and western blotting.

Results

As previously described, myostatin deletion results in significantly increased muscle mass without altering whole-body mass, fat percentage, or activity levels in obese db/db mice. Blood glucose is markedly improved but plasma lipids remain elevated.  In parallel with these improvements, we observed that obesity inhibits and MSTN deletion restores vascularization following hind limb ischemia. Despite persistent plasma lipemia, ectopic lipid deposition in skeletal muscle was largely ameliorated in obese mice lacking myostatin, a reduction largely explained by a decrease in tissue triglyceride levels.  EM and confocal microscopy revealed accumulation of lipid in obese mouse muscle cells that was lacking in obese mice with MSTN KO. The resolved deposition occurred predominantly inside muscle cells with fat cell infiltration in external spaces largely unaffected.  RNA Seq analysis of skeletal muscle revealed marked upregulation of lipogenesis pathways, notably the expression of stearoyl-CoA desaturase-1 (SCD1), the rate limiting enzyme for the oleate synthesis and subsequent triglyceride production. SCD1 was elevated in muscle from normal obese mice but not those lacking myostatin at both the protein and RNA level.

Conclusion

In summary, increased muscle mass in obese mice provides potent protection to vascular and metabolic health.  A potent correlate of this improvement is the resolution of myosteatosis in obese, hypermuscular mice.  These data suggest that resolution of myosteatosis independently of muscle mass may afford similar protection, possibly by targeted deletion or blockade of SCD1.