bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cardiomyocyte fusion as a new mechanism contributing to pathological cardiac hypertrophy
Authors: Colliva A , Vodret S , Bongiovanni W , Zacchigna S 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: None.

Introduction
Pathological cardiac hypertrophy is a maladaptive remodeling response, induced by multiple pathogenetic mechanisms, including genetic mutations and hypertension. Despite the high prevalence of this condition, the precise mechanisms driving its onset and progression remain elusive. Recent evidence indicates that homotypic cell fusion between cardiomyocytes occurs in the heart of lower vertebrates in response to injury, as well as in the rodent heart during the first week after birth, when cardiomyocyte size and ploidy increase. We hypothesized that homotypic fusion of cardiomyocytes could be triggered by pressure overload and contribute to cardiac hypertrophy.

Methods and Results
We exploited a genetic mouse model (MHC-CreER/mTmG mice) to genetically label individual cardiomyocytes by either red or green fluorescence. These mice express a tamoxifen inducible Cre recombinase (CreER) under the control of the myosin heavy chain 6 promoter (MHC). Controlled injection of tamoxifen resulted in a mosaic heart, composed by either red or green cardiomyocytes. In this genetic background, we performed transverse aortic constriction (TAC) to induce cardiac hypertrophy and analyzed cardiomyocyte size and fluorescence at one month.
The systematic analysis of more than 5000 cardiomyocytes from 6 independent experiments showed a significant increase in the number of yellow cardiomyocytes after TAC compared to sham hearts, consistent with cell-cell fusion events occurred between green and red cardiomyocytes. Moreover, these yellow hypertrophic cardiomyocytes displayed a higher rate of polynucleation, with many cells bearing three or more nuclei.

Conclusions and future perspective
Collectively, these data point to cardiomyocyte fusion as a new mechanism responsible for to the onset of cardiac hypertrophy upon pressure overload. We cannot exclude that yellow, polynucleated cardiomyocytes derived from the heterotypic fusion between a green cardiomyocyte and other red cell types. To rule out this possibility we are currently performing a new a set of experiments in which 100% of cardiomyocytes are labelled in green. In the case of homotypic fusion, we should not observe any yellow cells upon TAC. Proving that cardiomyocyte fusion contributes to cardiac hypertrophy in response to increased pressure load and dissecting the underlying molecular mechanisms will pave the way to novel strategies to both prevent and revert this highly prevalent pathological condition.
Identification of small-molecule ERBB4 agonists for the treatment of heart failure
Authors: Feyen E , Cools J , Van Fraeyenhove J , Tubeeckx M , De Winter H , Audenaert D , De Keulenaer GW , Segers VF 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Dehousse fellowship

Introduction
Although progress has been made in the treatment of heart failure, morbidity and mortality remain high, requiring new therapeutic targets. The neuregulin-1 (NRG1)/ERBB4 axis is cardioprotective and antifibrotic when activated in the myocardium, and therefore a possible target for therapy. Phase 2 and 3 clinical trials with NRG1 are ongoing, but require intravenous administration regimens, limiting applicability and efficacy.

Purpose
To develop small-molecule ERBB4 agonists with cardioprotective and antifibrotic properties.

Methods
A high-throughput screening (HTS) of 10,240 compounds was performed on a ERBB4/ERBB4 dimerization assay. Hit compounds were co-administered with NRG1 or fluorescently labeled NRG1 to determine competitive binding. Selectivity, receptor phosphorylation, cell proliferation and toxicity were determined using Luminex RTK phosphoprotein, ERBB2/ERBB3 dimerization, WST-1 colorimetric, and adenylate kinase assays. Antifibrotic effects were studied in vitro on TGF-β-induced collagen synthesis in human dermal and atrial fibroblasts, and in a mouse model of angiotensin II (AngII, 1000 ng/kg/min)-induced left ventricular (LV) myocardial fibrosis with selected compounds (83 µg/kg/h), administrated with osmotic minipumps (N=4–5/group). mRNA expression was evaluated after 7 days; LV myocardial fibrosis area, cardiomyocyte cross sectional area (CSA), echocardiographic parameters and heart- to bodyweight ratio (HW:BW) were analyzed at 28 days. Antiapoptotic effects were studied on rat atrial cardiomyocytes (AM) after hydrogen peroxide (H2O2)-induced cardiotoxicity.

Results
The HTS (Z’=0.7) resulted in 8 similar pyrimidine derivatives (EF-1–8) inducing ERBB4/ERBB4 dimerization (Emax 9–33% relative to NRG1, EC50 6E-6 to 2E-7 M). Competition assays indicate allosteric binding. The compounds also significantly potentiated NRG1-induced ERBB4 receptor dimerization up to 2.7 fold. Two compounds were excluded because of in vitro toxicity. The other 6 compounds were non-toxic and induced ERBB4, but neither ERBB1, ERBB2 or ERBB3 phosphorylation, nor tumor growth–inducing ERBB2/ERBB3 dimerization. Selected compounds showed significant dose-dependent antiapoptotic properties on H2O2-stimulated AM, and antifibrotic effects on human atrial and dermal fibroblasts. In vivo, compound EF-1 significantly decreased myocardial fibrosis (by 76±26%) and Col1a1, Col3a1 (-70±17%; -61±20%), and Nppa (-78±32%) mRNA expression, and significantly enhanced cardiomyocyte CSA (+24±8%). No differences were observed in cardiac function or HW:BW ratio.

Conclusion
We identified novel pyrimidine derivative small-molecule ERBB4 agonists with cardiomyocyte protective effects and antifibrotic properties in vitro and in AngII-induced myocardial fibrosis in vivo.
The development of systolic heart failure in case of pressure overload-induced left ventricular myocardial hypertrophy is associated with a unique microRNA expression profile in a rat model
Authors: Ruppert M , Korkmaz-Icoz S , Agg B , Sayour AA , Olah A , Nagy D , Benke K , Ferdinandy P , Merkely B , Szabo G , Radovits T 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): New National Excellence Program of the Ministry of Human Capacities

Introduction
Growing body of evidence suggests that distinct alterations in myocardial microRNA (miRNA) expression contribute to pressure overload (PO)-induced pathological cardiac remodeling. Nevertheless, it is still under intense investigation whether the changes in miRNA expression patterns are also associated with the decompensation of LV systolic function in case of PO-evoked LV hypertrophy (LVH). Hence, we aimed to characterize miRNA expression in PO-induced LVH with and without systolic heart failure (HF).

Methods
PO was evoked by abdominal aortic banding (AB) in male Sprague-Dawley rats. Age-matched, sham-operated animals served as controls. Functional and morphological alterations were assessed by echocardiography and histology. At the end of the experimental period, rats in the AB group were subcategorized based on ejection fraction [EF] into ABLVH (EF&gt;40%) and ABHF groups (EF&lt;40%). Global miRNA expression profiling was performed using next generation sequencing. Bioinformatics analysis was carried out to predict miRNA-target interactions. Expression of selected target genes was measured by qRT-PCR.

Results
Increased heart weight-to-tibial length, LV mass and fibrosis confirmed the development of pathological LVH in both the ABLVH and ABHF groups. Nevertheless, increased lung weight-to-tibial length, chamber dilatation and severely reduced EF was noted only in the ABHF and not in the ABLVH, when compared to the sham group. 50 miRNA showed different expression in the ABHF compared to the ABLVH group. Based on the altered gene expression profile, in silico bioinformatics analysis predicted several target genes. Among them, reduced mRNA expression level of Fmr1 (FMRP translational regulator 1), Zfpm2 (zinc finger protein, multitype 2), Wasl (WASP like actin nucleation promoting factor), Ets1 (ETS proto-oncogene 1) and Atg16l1 (Autophagy Related 16 Like 1) was confirmed in ABHF compared to ABLVH.

Conclusions
Decompensation of systolic function in PO-induced LVH is associated with unique miRNA profile leading to specific regulation of gene expression.
Calcium, contraction, and hemodynamic alterations induced by arrhythmogenic drugs in the zebrafish larvae
Authors: Salgado-Almario J , Vicente M , Molina Y , Martinez-Sielva A , Vincent P , Domingo B , Llopis J 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): MCIN/AEI/ 10.13039/501100011033 and “ERDF A way of making Europe” by the European Union

Background
Ca2+ ion is pivotal in excitation-contraction coupling. The dysregulation of Ca2+ fluxes is at the core of inherited and acquired arrhythmias. In recent years zebrafish have become an attractive vertebrate model to study heart development, cardiotoxicity, and heart diseases. We recently reported that the transgenic zebrafish line Tg(myl7:Twitch-4) expressing the ratiometric Ca2+ indicator Twitch-4 in the heart, allowed imaging Ca2+ transients (CaT) simultaneously with heart contraction in zebrafish larvae, thus maintaining the physiological mechano-electrical feedback.

Purpose
This study aimed to detect subtle disturbances in Ca2+ cycling, contractility, and hemodynamics induced by arrhythmogenic drugs in zebrafish larvae.

Methods
We have used the zebrafish transgenic line Tg(myl7:Twitch-4) and a custom software to acquire and analyze fluorescence emission ratio images of the heart. Larvae of 3- and 5-days post-fertilization (dpf) were incubated with arrhythmogenic drugs, and their effects on the systolic and diastolic Ca2+ and the CaT amplitude in the atrium and ventricle were determined. As the ventricular diameter was assessed in the same fluorescence images, a simultaneous, real-time, measurement of contraction correlated with Ca2+ was obtained. The systolic and diastolic areas, measured independently, were used to estimate hemodynamic parameters.

Results
Dofetilide caused time-dependent bradycardia and ultimately a 2:1 arrhythmia in 3 and 5 dpf larvae, with an increase in the CaT amplitude. the ejection fraction and the stroke volume increased, but the cardiac output decreased due to the low heart rate (2:1 arrhythmia). The T-type Ca2+ channel antagonist ML218 induced bradycardia and decreased systolic and diastolic Ca2+ levels in 3 dpf larvae, but the CaT amplitude did not change. Interestingly, some larvae (3 out of 14) displayed a progressive lengthening of the time between the start of atrial and ventricular CaT, analogous to increased PR interval of the electrocardiogram, until a ventricular systole failed. This is reminiscent of a second-degree atrioventricular block in humans. At higher ML218 concentration, a 2:1 arrhythmia was observed and increased ventricular CaT amplitude in all larvae.

Conclusions
We characterized Ca2+ changes induced by arrhythmogenic drugs and correlated them with contractility and hemodynamic parameters. The zebrafish line Tg(myl7:Twitch-4) allowed investigating drug-induced arrhythmias in this animal model with unprecedented detail. This transgenic line will serve to screen cardiotoxicity during drug development and to study conduction and rhythm defects in zebrafish, and their correlation with human disease.
Artificial light pollution and obesity as a risk factors of malignant cardiac arrhythmias - antiarrhythmogenic effect of omacor
Authors: Szeiffova Bacova B , Sykora M , Egan Benova T , Zurmanova J , Andelova K , Tribulova N 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: None.
Light pollution disrupts circadian rhythms and increases the development of obesity, but their connection to cardiovascular disease, in particular to malignant arrhythmias, is not well known.
Herein, we investigated if female rats exposed to continuous light and high-sucrose diet had altered myocardial gene transcripts and/or protein expression which affects arrhythmogenesis. We then assessed if Omacor® supplementation benefitted affected rats.
In a first experiment, adult female spontaneously hypertensive (SHR) and normotensive Wistar rats (WR) were housed under standard 12 h/12 h light/dark cycles or exposed to 6-weeks continuous 300 lux light for 24 h. In a second experiment, adult female WR rats received sucrose diet (30% sucrose solution) continuously (24 h) performed for 8 weeks. Half the rats from both experiments were throughout the experiment treated with 200 mg/100g b.w. Omacor®.
Continuous light resulted in higher rat vulnerability to malignant ventricular fibrillation (VF). This was linked with myocardial connexin-43 (Cx43) down-regulation and deteriorated intercellular electrical coupling, due in part to increased pro-inflammatory NF-κB and iNOS transcripts and decreased sarcoplasmic reticulum Ca 2+ ATPase transcripts. Omacor® treatment increased the electrical threshold to induce the VF linked with amelioration of myocardial Cx43 mRNA and Cx43 protein levels and the suppression of NF-κB and iNOS.
8 weeks lasting intake of 30% sucrose solution downregulated Cx43 and PKCε signaling along with an upregulation of myocardial PKCδ and miR-30a rendering the heart prone to ventricular arrhythmias. There was a clear benefit of Omacor® supplementation due to their antiarrhythmic effects associated with the attenuation of myocardial Cx43, PKC, and miR-30a abnormalities.
This indicates that rat exposure to continuous light and high-sucrose result in deleterious cardiac alterations jeopardizing intercellular Cx43 channel-mediated electrical communication, thereby increasing the risk of malignant arrhythmias. The adverse effects were attenuated by treatment with Omacor®, thus supporting its potential benefit and the relevance of monitoring omega-3 index in human populations at risk.
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

Abstract

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&lt;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&lt;0.01) and the Tpeak-Tend intervals were significantly shorter (76±10 ms vs. 83±12 ms, p&lt;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&lt;0.05), the QTVI (-0,9±0.5 vs. -1,3±0.4, p&lt;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&lt;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

Abstract

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

Abstract

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

Abstract

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&gt;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&gt;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&lt;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&gt;3 or &gt;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.