bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel endothelial-enriched long non-coding RNA AL590004.3 (IRENE) is dysregulated in HFpEF and regulates endothelial enhancement of cardiomyocyte function
Authors: Juni R P , Phelp P , Busscher D , Belluomo R , Kremer V , Van Bergen A , Jalink L , Boon R A 
Published: November 2023

Abstract

Background
Heart failure with preserved ejection fraction (HFpEF) is the most prevalent form of HF to date. However, there is a lack of medication which stages HFpEF as an unmet clinical need. HFpEF is characterized by cardiac microvascular dysfunction, promoted by co-morbidities-associated low-grade pro-inflammatory state. We have shown that cardiac endothelial cells regulate cardiomyocyte relaxation, which is impaired when endothelial cells are inflamed. Long non-coding RNAs (lncRNAs) have been implicated in various cardiovascular diseases. However, their role in HFpEF is unknown. Here, we discovered a novel lncRNA AL590004.3 which is significantly reduced in the heart of patients with HFpEF. We hypothesized that AL590004.3 is important for cardiac endothelial function, and dysregulation of endothelial AL590004.3 impairs cardiomyocyte relaxation.

Methods and results
We demonstrated that AL590004.3 is significantly downregulated (∼7 fold, p&lt;0.001) in HFpEF as assessed by in silico analysis of RNA sequencing of HFpEF patient cardiac biopsies. To assess the tissue distribution, we performed RNA sequencing comparing cells from various tissues and found the highest expression of AL590004.3 in the cardiac microvascular endothelial cells (CMECs), indicating its importance in cardiac endothelial cell biology. The expression of AL590004.3 was reduced when CMECs were exposed to pro-inflammatory cytokine TNFα and IL1β, indicating the detrimental effect of inflammation, as displayed by patients with HFpEF. Further, replicative senescent of CMECs led to reduced AL590004.3 expression (2 fold, p&lt;0.005), showing the deleterious contribution of ageing, similar to what occurs in HFpEF patients. We showed that AL590004.3 is mostly expressed in the chromatin fraction (∼90%) of CMECs, suggesting its function in chromatin modification. To assess the function of AL590004.3, we performed loss-of-function. Using endothelial-cardiomyocyte co-culture set-up, we showed silencing of AL590004.3 in CMECs impaired endothelial function to enhance cardiomyocyte relaxation (∼1.5 fold, p&lt;0.01). To dissect the molecular mechanism, we performed RNA sequencing of CMECs devoid of AL590004.3 and revealed four protein-coding genes (PXDC1, FAM50, ECI2, PRPF4B) that are differentially co-regulated, indicating their potential as protein-binding partners, which warrant further investigation.

Conclusion
We showed that AL590004.3 is a cardiac endothelial cell-specific lncRNA that is significantly downregulated in cardiac biopsy from HFpEF patients. AL590004.3 is important for cardiac endothelial cell function as loss-of-function in CMECs impaired endothelial enhancement of cardiomyocyte relaxation. Herewith, we name this lncRNA Important for the Regulation of ENdothelial function in HFpEF (IRENE).
The dominant-negative effect of Loss of function Nav 1.5 variants is not a critical determinant of phenotype severity
Authors: Kato K , Tano A , Jinzai H , Kubo T , Ohno S , Nakagawa Y , Horie M 
Published: November 2023

Abstract

Introduction
Loss-of-function (LOF) variants of SCN5A cause various arrhythmic diseases. Recent studies have demonstrated that some LOF-SCN5A variants can exert a dominant-negative (DN) effect through channel dimerization, resulting in higher penetrance of disease phenotypes. However, the association between the DN effect and phenotype severity remains unclear. In this study, we identified a proband with compound heterozygous SCN5A variants, p.G833R and p.T1396P, in a family with bradyarrhythmia. We analyzed the electrophysiological function of these variants and their association with channel-dimerization to elucidate their effects on phenotype severity.

Purpose
The purpose of this study is to investigate the effect of sodium channel dimerization on the phenotype severity in a family with bradyarrhythmia

Methods
We performed functional analysis of the mutant Nav1.5s induced in HEK cells utilizing a whole-cell patch-clamp method. The DN effect was also assessed by co-expressing WT and mutants in each heterozygous combinations, with or without difopein, a peptide working as a channel-coupling canceler. Cell surface expression of mutated channels were measured by flowcytometry.

Results
We identified SCN5A p.T1396P and p.G833R variants segregating in a family suffering from severe bradyarrhythmia. The proband carried both variants in a compound heterozygous manner, while her mother carried only p.T1396P. The clinical phenotype was more severe and had early onset in the proband; the compound heterozygous case.
In the patch-clamp study, HEK 293 cells solely expressing T1396P did not show any measurable sodium current, while those with G833R had comparative current with that of WT channel. Cells expressing WT/T1396P showed significant reduction and delay of inactivation decay, which was normalized by co-expression with difopein. The results implied that loss-of-function variant T1396P manifested DN-effect via channel dimerization.
Cells co-expressing T1396P and G833R did not demonstrate a DN-effect, suggesting that Nav1.5-G833R did not form a dimer. Considering more severe phenotype of compound heterozygous member in the family, the DN-cancelation by monomerized Nav1.5-G833R was not associated with a mild phenotype.

Conclusion
Our result supports the current Nav1.5-dimerization concept and consequent DN-effect by LOF-SCN5A variants. However, the phenotype severity was not simply explainable by the presence of a dominant negative effect. Further multiplanar analyses including assays for channel clustering, localization, and gating modification would be necessary.
Trapped re-entry challenges the binary view on cardiac arrhythmias
Authors: De Coster T , Teplenin A S , Feola I , Bart C I , Ramkisoensing A A , Ypey D L , Panfilov A V , Zeppenfeld K , De Vries A A F , Pijnappels D A 
Published: November 2023

Abstract

Background
There is currently a binary view on cardiac arrhythmias: (1) arrhythmias are present and thereby affect the atria or ventricles as a whole, or (2) they are not present and therefore the heart is in sinus rhythm. However, complex fractionated atrial electrograms (CFAEs) under sinus rhythm (SR) are observed in patients with atrial fibrillation (AF), raising the question whether both cardiac states (arrhythmia and SR) could coexist. Although CFAEs in AF patients have been associated with functional and structural heterogeneities (e.g. dense fibrotic regions), the underlying mechanisms of fractionation under SR remain incompletely understood.

Purpose
To test the hypothesis that an arrhythmia can exist locally with the majority of cardiac tissue in sinus rhythm, made possible through dense local fibrotic regions forming a small electrically isolated re-entrant circuit that can "trap" excitation waves, which can only be "released" under dynamic tissue changes at an isthmus connected to the bulk of the tissue.

Methods
By harnessing the unique possibilities of light-gated depolarizing ion channels (CatCh) to precisely control in vitro cardiac excitability in time and space, complemented with advanced computational modelling of whole human atria, we explored the geometry of such an electrically isolated circuit and assessed its clinical relevance.

Results
Optical mapping studies, in monolayers of CatCh-activated neonatal rat atrial cardiomyocytes (n=8), revealed that re-entry can be established and trapped by creating an electrically isolated pathway with a bulk-connecting isthmus causing source-sink mismatch. A tachyarrhythmia was shown to exist locally with SR prevailing in the bulk of the monolayer. Next, conditions were found under which re-entry could escape this pathway, thereby converting a local dormant arrhythmic source into an active driver with global impact. Escape could be established by overcoming the source-sink mismatch through widening of the isthmus or a reduction of the gap junctional coupling. In a digital twin of the human atria, it was revealed that the conditions for "trapped re-entry" and its release can be realized as well. Unipolar pseudo-electrograms derived from these complementary computational 3D studies showed CFAEs at the site of "trapped re-entry" in coexistence with normal electrograms of SR in the bulk of the atria. Upon release of the re-entry, acute arrhythmia onset occurred, affecting the complete atria as evidenced by wave front and electrogram visualization.

Conclusion
Through the concept of "trapped re-entry", we not only present a new mechanism of acute manifestation of focal tachyarrhythmias, but also provide novel insight into the origin of fractionated electrograms. This insight may provide new rationales for treatment of cardiac arrhythmias, especially for ablation by site-directed targeting.Trapped re-entry in vitro and in silico
Light transmittance in human atrial tissue and transthoracic illumination in rats support translatability of optogenetic cardioversion of AF
Authors: Portero V , Nyns E C A , Harlaar N , Deng S , Tianyi T , Bart C I , Van Brakel T J , Palmen M , Hjortnaes J , Ramkisoensing A A , Zhang G Q , Poelma R H , Ordog B , De Vries A A F , Pijnappels D A 
Published: November 2023

Abstract

Background
Optogenetics could offer a solution to the current lack of an ambulatory method for rapid automated cardioversion of AF, but key translational aspects remain to be studied.

Objective
To investigate whether optogenetic cardioversion of AF is effective in the aged heart and whether sufficient light penetrates the human atrial wall.

Methods
Atria of adult (7 weeks old) and aged (2 years old) rats were optogenetically modified to express light-gated ion channels (i.e. ReaChR), followed by AF induction and atrial illumination to determine effectivity of optogenetic cardioversion. The irradiance level was determined by light transmittance measurements on human atrial tissue.

Results
AF could be effectively terminated in remodelled atria of aged rats (97%, n=6). Next, in terms of ReaChR activation, it was shown that light pulses of 25mW/mm2 are expected to fully penetrate the human atrial wall (∼2 mm). Applying such irradiation onto the chest of adult rats resulted in full transthoracic illumination (also ∼2 mm) as evidenced by optogenetic cardioversion of AF (90%, n=4).

Conclusion
Optogenetic cardioversion of AF is effective in the aged rat heart and can also be realized with irradiation levels compatible with human atrial transmural light penetration.
Cardiomyocyte - stromal cells interplay as a novel platform for mechanistic insights in arrhythmogenic cardiomyopathy
Authors: Maione A S , Iengo L , Sala L , Lodola F , Lippi M , Zaza A , Tondo C , Pompilio G , Sommariva E 
Published: November 2023

Abstract

Background
Arrhythmogenic Cardiomyopathy (ACM) is an inherited heart disorder characterized by a high incidence of sudden death at young age. Mutations linked to ACM occur primarily in desmosomal genes (e.g. PKP2) [1]. Different cell types individually contribute to set the ACM phenotype with either functional abnormalities (cardiomyocytes; CM) [2]; or fibro-fatty substitution (cardiac mesenchymal stromal cells; CMSC) [3]; these two cell types interact and show a reciprocal influence [4]. The relative contribution of the different cell type derangements to determine disease evolution and electrical instability is still unclear.

Purpose
Based on this evidence, we hypothesize that the use of a multicellular cardiac system, modelling the functional interaction between CM and CMSC, will allow the understanding of the relative contribution of the different cell types to the ACM phenotypes.

Methods
Primary stromal cells (carrier of the PKP2 mutation c.2013delC) have been collected from a right ventricle biopsy sample; control stromal cells obtained from a biobank. iPSC reprogrammed from the same ACM patient and the isogenic line in which the PKP2 mutation c.2013delC was corrected [5] were used. Cardiac cocultures (cc) were assembled by combining 85% iPSC-CM and 15% primary CMSC, either mutated or control, in different combination (as in Table 1). Movies of contracting clusters have been collected and analysed by the MUSCLEMOTION tool [6]. Fibro-fatty accumulation have been evaluated by immunofluorescence analysis by confocal microscopy.

Results
Immunofluorescence analysis highlighted that ACM cc (cc 4) accumulate more lipids and collagen than healthy control (HC) ones (cc 3). Since intermediate fibro-fatty levels were observed in ‘mixed cc’ (cc 1 and 4), we conclude that ACM iPSC-CM are able to influence fibro-adipo-commitment of CMSC. Contraction studies showed a high propensity for cc monolayers which include ACM iPSC-CM (cc1 and cc2) to display arrhythmic events. An increase in the percentage of arrhythmic events was triggered when the ACM CMSC were replacing HC CMSC, indicating that ACM CMSC can affect iPSC-CM rhythm.

Conclusion
Overall, our data confirm the importance of the cardiomyocytes and stromal cells interplay during ACM pathogenesis. Furthermore, our novel advanced ACM cell model can recapitulate different phenotypes of ACM and offers a unique opportunity for validating pharmacological therapies to modulate ACM-related characteristics (e.g. fibro-adipose replacement, contractile defects and proarrhythmic events).
Single-nucleus RNA sequencing of TMEM43 induced arrhythmogenic cardiomyopathy reveals altered cell compositions and transcriptional states in mice
Authors: Lalaguna L , Lopez-Olaneta M , Villalba-Orero M , Garcia-Pavia P , Lara-Pezzi E 
Published: November 2023

Abstract

Introduction and purpose
TMEM43 induced Arrhythmogenic cardiomyopathy, also referred to as arrhythmogenic cardiomyopathy type 5 or ACM5, is caused by a point mutation in TMEM43 (p.S358L). The disease is characterised by chamber dilation and a progressive dystrophy of the ventricular myocardium causing life-threatening ventricular arrhythmias and sudden cardiac death. Loss of cardiomyocytes and fibrofatty replacement of the myocardium is clearly observed in our transgenic mouse model of ACM5 overexpressing TMEM43-S358L in postnatal cardiomyocytes. However, the mechanisms underlying cardiomyocyte death and fibroblast activation remain elusive, precluding the development of effective therapeutic options. Here we applied the state-of-the-art analysis on single-nucleus RNA sequencing (snRNA-seq) to characterise the cellular compositions and molecular states in ACM5 mouse hearts.

Methods
Left ventricular heart tissues of severely affected ACM5 mice and their corresponding transgenic controls overexpressing wild-type TMEM43 were used for snRNA-seq analysis. We identified 10 major cell types, which aggregated into 23 distinct clusters on the basis of transcriptional similarity.

Results
ACM5 nuclei showed significant depletion of cardiomyocytes and expanded populations of fibroblasts and myeloid cells. Fibroblasts from mutant mice acquired an activated phenotype related to remodelling of the extracellular matrix with a large number of genes like LOX, POSTN, NOX4 or COL1A1 significantly upregulated. In addition, the mutant cardiomyocytes showed altered transcriptional states characterized by cardiac stress and heart failure markers such as NPPB or MYH7. Interestingly, we were able to identify a cluster of mutant cardiomyocytes with a highly apoptotic profile. This cluster is also enriched in genes related to the unfolding protein response (UPR) stress sensors and the DNA damage response (DDR), elucidating a new potential mechanism of myocyte death in ACM5. Furthermore, heterogeneity of the myeloid populations is also observed. The number of tissue resident macrophages is increased in mutant samples and myeloid cells show additional disease-associated transcriptional states that are almost entirely absent in control samples.

Conclusion
Together these data illuminated shared and distinct cell and molecular architectures of ACM5 in mouse hearts. Further confirmation on diverse gene expression profiles like the UPR and DDR pathways related to cardiomyocyte death will establish a valuable resource to explore novel therapeutic targets and personalized therapies for ACM5.
Unraveling the pathogenesis of novel ANKRD1 missense mutation causing familial dilated cardiomyopathy using human-induced pluripotent stem cells derived cardiomyocytes
Authors: Ballan N , Shaheen N , Ghiringhelli M , Carmon T , Shiti A , Arabel G , Gepstein A , Huber I , Gepstein L 
Published: November 2023

Abstract

Introduction
Familial dilated cardiomyopathy(f-DCM) can be caused by mutations in 30 different genes encoding sarcomeric, cytoskeletal, mechanotransducers and transcription factors. One of the genes ANKRD1 encode Cardiac ankyrin repeat protein (CARP) which is involved in the mechanosensory unit located on the I-band of the sarcomere. Here we demonstrate a novel mutation in ANKRD1 gene causing f-DCM, and the usage of human-induced pluripotent(hiPSC) technology unraveling the pathogenesis of the disease.

Purpose
of the study:
1) Establishing patient specific hiPSC-derived cardiomyocytes from a family harboring a mutation in ANKRD1 gene causing f-DCM; 2) Phenotypical assessment of the morphological and ultrastructural properties of the diseased cells upon CARP activation; 3) Functional assessment of the 3 component of excitation-contraction coupling at the single-cell and tissue level.

Methods and Results
Patient-specific hiPSC were generated from a family harboring missense mutation in the ANKRD1 gene, with a substitution of the amino acid arginine to cysteine at position 182 of the Cardiac ankyrin repeat protein (CARP). ANKRD1-mut hiPSC were differentiated into cardiomyocytes and later matured using hormonal treatment. Morphological assessment using immunofluorescence(IF) techniques revealed that ANKRD1-mut hiPSC-CMs demonstrate an attenuated response to hypertrophy (upon Angiotensin-2 and Phenylephrine treatment). In addition ANKRD1-mut hiPSC-CMs displayed a higher cellular death rate upon increasing substrate stiffness and starvation. Electrophysiological assessment of the ANKRD1-mut cardiac tissues exhibit no alternations in action potential durations and conduction velocities while assessment of calcium transients demonstrated slower kinetics. In addition forces were quantified at the single-cell and tissue level , and demonstrated reduced active tension values and abnormal post-extrasystolic potentiation, while only single-cell experiments have demonstrated decreased passive tensions. These phenotypical alternations might be attributed to decreased nuclear translocation of CARP upon phenylephrine treatment as detected in IF experiments.

Conclusion
Novel mutation in ANKRD1 were found and validated to be causing f-DCM, by revealing phenotypical alternation using hiPSC-CM technology. Increased cellular death rate and impaired nuclear translocation of CARP might be the underlying causes of the morphological and contractile abnormalities detected in-vitro, leading to systolic dysfunction in the affected family.
N-terminal pro-B-type natriuretic peptide for prediction of ventricular arrhythmias: data from the SMASH Study
Authors: Sourour N , Riveland D R , Nasgaard D R , Kjekshus P H D , Larsen P H D , Omland P H D , Rosjo P H D , Myhre P H D 
Published: November 2023

Abstract

Background
Elevated N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations predict heart failure (HF) and mortality, but whether NT-proBNP predicts ventricular arrhythmias (VA) is not clear.

Purpose
We hypothesize that high NT-proBNP concentrations associate with the risk of incident VA, defined as adjudicated ventricular fibrillation or sustained ventricular tachycardia.

Methods
In a prospective, observational study of patients treated with implantable cardioverter defibrillator (ICD), we analyzed NT-proBNP concentrations at baseline and after mean 1.4 years in association to incident VA.

Results
We included 491 patients (age 66±12 years, 83% men) out of whom 51% had a primary prevention ICD indication. The median NT-proBNP concentration was 567 (25-75 percentile 203-1480) ng/L, and patients with higher concentrations were older with more HF and ICD for primary prevention. During mean 3.1±0.7 years, 136 patients (28%) had ≥1 VA. Baseline NT-proBNP concentrations were associated with risk of incident VA (HR 1.39 [95%CI 1.22-1.58], p&lt;0.001) (Figure1), HF hospitalizations (HR 3.38 [95%CI 2.69-4.25], p&lt;0.001) and all-cause mortality (HR 2.49 [2.05-3.04], p&lt;0.001) (Figure2), which persisted after adjusting for age, sex, BMI, CAD, HF, renal function, and LVEF. The association with VA was stronger in secondary vs primary prevention ICD indication: HR 1.59 (95% CI 1.34-1.88 C-statistics 0.71) vs HR 1.25 (1.02-1.52, C-statistics 0.55), p-for-interaction=0.06. Changes in NT-proBNP during the first 1.4 years did not associate with subsequent VA.

Conclusions
NT-proBNP concentrations are associated with the risk of incident VA after adjustment for established risk factors, with the strongest association in patients with a secondary prevention ICD indication.
RNA surveillance by ADAR1 is required for vascular endothelial resilience
Authors: Tual-Chalot S , Ciliberti G , Polycarpou-Schwarz M , Amponsah-Offeh M , Turchinovich A , Dimmeler S , Gatsiou A , Stellos K 
Published: November 2023

Abstract

Introduction
Long dsRNAs are recognised as danger-associated molecular patterns by the cytosolic innate immune sensors inducing thus innate immune responses. RNA editing is a post-transcriptional modification induced by the binding of Adenosine Deaminases Acting on RNA (ADARs) to double-stranded RNA structures (dsRNAs). We have recently reported that ADAR1 is the main RNA editor in endothelial cells (ECs) but the role of ADAR1 in vascular homeostatic processes remains yet elusive.

Methods
Genetic deletion strategies were employed by crossing mice carrying a conditional (floxed) Adar1 , Ifh1 or Tlr3 allele with either a Tie2-Cre or with a tamoxifen-inducible VE-Cadherin-CreERT2 mouse line. Primary human and murine vascular EC culture assays, dsRNA metabolism studies, gene-silencing and expression techniques, immunohistochemistry and confocal microscopy were used to assess the EC-specific ADAR1 effects.

Results
EC-specific ADAR1 deletion resulted in prenatal embryonic lethality evidencing an absolute requirement of EC-ADAR1 in life. Inducible EC-ADAR1 ablation in adult mice elicited premature sudden death due to a widespread lung vascular leakage and pleural effusion, reflecting a compromised EC barrier function. Mechanistically, silencing of endothelial ADAR1 disrupted long-to-short dsRNA metabolism witnessed by the striking accumulation of cytoplasmic long dsRNAs. Subsequently, the cytoplasmic accumulation of endogenous long dsRNAs induced aberrant levels of interferon-β and activation of innate immune system. Finally, activation of the cytosolic dsRNA sensors inflicted the dissociation of β-catenin from VE-cadherin in EC junctions, while, co-silencing of the dsRNA sensor, MDA5, was sufficient to restore the integrity of the ADAR1-deficient endothelium. While double EC-specific ADAR1 and TLR3 did not fully rescue the lethality of the EC-ADAR1 animals, in vivo experiments using double EC-specific ADAR1 and MDA5 KO mice fully rescued the phenotype. Validation experiments using murine vascular ECs isolated from the mouse strains of our studies confirmed our mechanistic and phenotypic findings.

Conclusion
Endogenous suppression of double-stranded RNA-induced MDA5 autoinflammatory signaling by the RNA editor ADAR1 is essential for the maintenance of vascular integrity in adult mice.