bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of CACNA1C variants in Brugada syndrome: clinical aspects and genetic testing strategies
Authors: Novelli V , Memmi M , Malovini A , Mazzanti A , Liu N , Yanfei R , Bongianino R , Monteforte N , Bloise R , Morini M , Napolitano C , Priori S.G 
Published: November 2020

Abstract

Background
Inconsistent data support the role of CACNA1C as a disease-causing gene responsible for Brugada syndrome (BrS). As of today, the only gene consistently linked with BrS is SCN5A. Several CACNA1c genetic variants have been reported in association wirh BrS; however, due to the limited evidence, CACNA1C is not suggested for routine genetic screening for BrS.

Purpose
In this study, we carried out a systematic screening of CACNA1C gene, including a functional evaluation of the identified variants, in order to determine the yield of screening in a large series of BrS probands and to address the hypothesis that an appropriate clinical selection of patients would substantially improve the yield of genetic testing.

Methods and results
Overall 564 consecutive patients, referred for BrS genetic testing, were sequenced for CACNA1C gene. Patients were divided in two groups: discovery cohort (n=200 patients) and confirmation cohort (n=363 patients). Furthermore, analysis of the clinical phenotypes of a matched SCN5A positive BrS cohort (n=146) was included for phenotype characterization.
In the discovery cohort we identified 11 different genetic variants of whom 2 (18%) were considered as potentially causative based on ACMG guidelines. However, a large proportion (81%) was classified as variants of unknown significance (VUS). Functional evaluation of the identified variants, including pathogenic and VUS, was assessed by patch-clamp and immunofluorescence studies. Re-evaluation of the variants, including functional studies results, indicated an increase of pathogenic or likely pathogenic variants (81%) getting a yield of screening of 5% in the discovery cohort. Results from the confirmation cohort confirmed a low rate of CACNA1C carriers with a yield of screening of 2.2%.
Analysing the clinical phenotype of all CACNA1C carriers showed a significantly shorter QTc [371 ms ± 16 ms vs. 399±18 ms; p=0.000004]. Furthermore, the prevalence of CACNA1C variants was highest (12.9%) among patients with a QTc in the lowest quartile (QTc &lt;390 ms). ROC curve showed an AUC of 0.91 for QTc a cut-off of 385 ms, suggesting a high predictive accuracy.

Conclusion
We confirmed that CACNA1C variants are not a common cause of BrS, with a yield of screening of 2–5%. However, pathogenic variants are more frequent (12.5%) in patients with a shorter QTc, suggesting a genetic testing strategy in this subgroup of BrS patients. Furthermore, our data highlights the impact of robust functional studies to improve variant classification and reduce uncertainties.

Funding Acknowledgement
Type of funding source: None
Rare variants in HCN4 identified in the general population are associated with complete atrioventricular (AV) block, 1. degree AV block and bundle branch block, results from 50.000 exomes
Authors: Jensen J , Vad O.B , Paludan-Muller C , Lundegaard P.R , Svendsen J.H , Haunsoe S , Bundgaard H , Olesen M.S 
Published: November 2020

Abstract

Background/Introduction
Cardiac conduction disorders cover disorders such as atrioventricular (AV) block and bundle branch block. Genome-wide association studies have identified more than 100 genetic loci for atrial fibrillation, including HCN4 loci associated with duration of the PR interval, a proxy for AV dysfunction. Recent candidate studies suggest an association between HCN4 variants and AV-block.

Purpose
We seek to determine the contribution of rare genetic variants in HCN4 to complete AV-block, 1. degree AV-block and bundle branch block (BBB) in the general population.

Methods
The UK Biobank is a population-based study of 500 000 individuals including a subset with genome-wide genotyping and exome sequencing (n=45,596). In this case-control study, we included persons of genetically determined white-European ancestry and analyzed their exome data. Analyses were performed using a logistic mixed-effects model. A gene-based burden analysis and single variant test were performed to examine the relationship between HCN4 variants and complete AV-block, 1. degree AV-block and BBB in the general population.

Results
The study included 43,371 persons. In an analysis of the HCN4 genes a significant association between rare variants (MAF&lt;0.01) in HCN4 gene was found (complete AV-block, P=2.5x10–5, 1. degree AV-block, P=1.3x10–3 and BBB, P=0.01) (Figure 1A). The association to complete AV block was mostly driven by the variants Ser835Phe (P=2.7e-3), Glu153Gly (P=3.5e-3) and Arg378Cys (P=6.3e-3) (Figure 1B).

Conclusions
Rare HCN4 variants contribute to the risk of complete AV-block, 1. degree AV-block and BBB in the general population. These HCN4 variants seem to confer a substantial penetrance. Clinical screening for some of these variants seems appropriate.
Figure 1

Funding Acknowledgement
Type of funding source: Private company. Main funding source(s): Hallas Møller - Novo nordisk
Neuropeptides as novel regulators of human atrial TASK-1 currents
Authors: Wiedmann F , Nietfeld J , Kraft M , Ratte A , Benda S , Katus H.A , Schmidt C 
Published: November 2020

Abstract

Background/Introduction
The neurokinin-III receptor (NK3R) was recently shown to regulate action potential duration (APD) in atrial cardiomyocytes by inhibition of a background potassium current. In the human heart, TASK-1 (hK2P3.1) two-pore-domain potassium channels display atrial-specific expression. Because of their phospholipase C (PLC)-dependent regulation, TASK-1 channels are a promising candidate to mediate APD prolongation via the Gq-coupled neurokinin-III receptor.

Purpose
To investigate whether TASK-1 channels mediate neurokinin-III receptor activation induced APD prolongation and to dissect the underlying molecular mechanisms.

Methods
Patch clamp measurements were performed in atrial cardiomyocytes isolated from patients with atrial fibrillation. Xenopus laevis oocytes heterologously expressing hTASK-1 and hNK3R were subjected to two-electrode voltage-clamp recordings.

Results
In Xenopus oocytes heterologously overexpressing hNK3R and hTASK-1 administration of substance P or neurokinin B resulted in TASK-1 current inhibition. This could be reproduced by application of the high affinity neurokinin-III receptor agonist senktide. Moreover, preincubation with the neurokinin-III receptor antagonist osanetant blunted the effect of senktide. Pharmacological experiments and mutagenesis studies could show a protein kinase C (PKC)-independent mechanism of TASK-1 current inhibition: upon NK3R activation TASK-1 channels are blocked via Gq-mediated PLC activation, in a DAG-dependent fashion. Finally, effects of senktide on atrial background currents could be reproduced in human atrial cardiomyocytes isolated from patients with atrial fibrillation.

Conclusion
Neurokinin-III receptor stimulation suppresses background potassium currents in isolated human atrial cardiomyocytes from patients with atrial fibrillation. Heterologously expressed human TASK-1 channels are inhibited by neurokinin-III receptor activation in a PLC and DAG dependent fashion, suggesting neuropeptides as novel regulators of human atrial TASK-1 currents.
Central Illustration

Funding Acknowledgement
Type of funding source: None
Noninvasive assessment of dynamic cardiac electrophysiology in normal human subjects
Authors: Stoks J , Van Rees B.D , Nguyen U.C , Peeters R.L.M , Dendale P , Cluitmans M.J.M , Volders P.G.A 
Published: November 2020

Abstract

Introduction
Electrocardiographic imaging (ECGI) has been used to investigate arrhythmia mechanisms in various conditions. Data on normal human subjects, especially in Europe, are scarce. Dynamic characteristics of ventricular activation and recovery during sinus rhythm have not been assessed before.

Purpose
To examine cardiac electrophysiology and its dynamic aspects in normal subjects using ECGI, in order to provide a range of normal patterns and values for activation (AT) and recovery times (RT), activation-recovery intervals (ARI, a surrogate for action potential duration) and their dynamicity.

Methods
11 Subjects (age 57±7 years, 27% male, all normal LVEF) with atypical chest pain who underwent a cardiac CT-scan as part of clinical care but who were negative for any pathology on full examination were included. A validated non-commercial potential-based formulation of ECGI was used to reconstruct unipolar electrograms (EGMs) on the epicardial surface for three sinus beats within minutes from each other, per individual. ATs and RTs were determined as the maximum negative upslope during QRS, and maximum positive upslope during T wave of the local EGMs. Additionally, we determined locations of first and last activation and recovery. Inter- and intra-individual differences were computed.

Results
Subjects had normal 12-lead characteristics without ST-deviations, and an average QTc interval of 415±18ms. Figure 1, panel A shows ECGI during sinus rhythm for 3 representative subjects, and panel B summarizes all findings on the entire epicardium. The first epicardial activation breakthrough typically occurred on the right ventricle (RV), consistent with the concept that the thinner RV wall accounts for a faster transmural activation. Last activation was mostly on the base of the left ventricle (LV), on the inferior to lateral wall. Earliest recovery occurred predominantly on the anterior surface, while latest recovery occurred on the inferior surface. Complete activation of the epicardial surface (from earliest to latest AT) took 41±8ms, while recovery (earliest AT to latest RT) took 317±24ms and average ARI (local AT to local RT) took 232±23ms. Thus, inter-individual variation of recovery duration was higher than of activation. Intra-individual differences between beats in ATs, RTs and ARIs of distinct sinus beats were small (2.3±3.1ms, 9.7±8.8ms and 9.8±9.1ms, respectively) suggesting that ECGI enables stable reconstruction quality (Figure 1, panel C).

Conclusion
In this cohort, noninvasive ECGI provides novel insights in ventricular electrophysiology. Electrical recovery is more variable than activation, both intra-individually and inter-individually. Overall, AT, RT and ARI differences between sinus beats were low. ECGI appears suitable to assess dynamic electrical patterns during cardiac pathology.
Figure 1

Funding Acknowledgement
Type of funding source: None
The effect of oestrogen withdrawal on cardiac Ca2+ regulation and the influence of GPER1
Authors: Francis A.J , Firth J.M , Islam N , Gorelik J , MacLeod K.T 
Published: November 2020

Abstract

Background
Post-menopausal women have an enhanced risk of developing heart failure, attributed to declining oestrogen levels during menopause. However, the signalling mechanisms remain undetermined.

Purpose
We aim to determine the role of G-protein coupled oestrogenic receptor 1 (GPER1) in intracellular Ca2+ regulation and the consequences of hormonal changes that may exacerbate the pathophysiology of heart failure.

Methods
Ovariectomy (OVx) (mimics menopausal hormone changes) or sham surgeries were conducted on female guinea pigs. Left ventricular cardiomyocytes were isolated 150-days post-operatively for experimental use. Cellular t-tubule network and structural integrity was measured using fluorescent di-8-ANEPPs staining and scanning ion conductance microscopy. GPER1 expression and localisation was measured by Western blot and immunostaining. The role of GPER1 activation was measured using selective agonist G-1 in electrophysiological and Ca2+-sensitive dye fluorescence experiments.

Results
Following oestrogen withdrawal, the t-tubule network density decreased by 13% and z-groove index reduced by 15%. GPER1 predominantly localised to the peri-nuclear endoplasmic reticulum and its expression increased by 32% in OVx. Action potential duration (APD) prolonged in OVx and following GPER1 activation, APD90 shortened by 11% and 25% in sham and OVx respectively. OVx cells had larger peak inward Ca2+ current (ICaL) (by 22%) and sarcoplasmic reticulum (SR) Ca2+ content (by 13%), compared with sham. While GPER1 activation had little effect on peak ICaL or SR content, it reduced Ca2+ transient amplitude (by 20%), SR fractional release (by 11%) in OVx cells. The frequency of occurrence of spontaneous Ca2+ waves evoked by periods of rapid stimulation reduced by 40% and wave-free survival time prolonged in OVx cells following GPER1 activation.

Conclusions
In the hearts of an animal species whose electrophysiology and intracellular Ca2+ regulation is akin to humans, we show that following oestrogen deficiency, the t-tubule network is down-regulated and becomes disorganised, GPER1 expression is increased and its activation induces negative inotropic responses in cardiomyocytes. This may limit the adverse changes to Ca2+ signalling reported in OVx that could be pro-arrhythmic and exacerbate the progression to heart failure.

Funding Acknowledgement
Type of funding source: Foundation. Main funding source(s): British Heart Foundation
Cardiac mitofusin-1 is declined in non-responding patients with idiopathic dilated cardiomyopathy
Authors: Hsiao Y , Shimizu I , Wakasugi T , Jiao S , Watanabe T , Kashimura T , Yoshida Y , Hanawa H , Ozaki K , Minamino T 
Published: November 2020

Abstract

Background/Introduction
Mitochondria are dynamic regulators of cellular metabolism and homeostasis. The dysfunction of mitochondria has long been considered a major contributor to aging and age-related diseases. The prognosis of severe heart failure is still unacceptably poor and it is urgent to establish new therapies for this critical condition. Some patients with heart failure do not respond to established multidisciplinary treatment and they are classified as “non-responders”. The outcome is especially poor for non-responders, and underlying mechanisms are largely unknown.

Purpose
Studies indicate mitochondrial dysfunction has causal roles for metabolic remodeling in the failing heart, but underlying mechanisms remain to be explored. This study tried to elucidate the role of Mitofusin-1 in a failing heart.

Methods
We examined twenty-two heart failure patients who underwent endomyocardial biopsy of intraventricular septum. Patients were classified as non-responders when their left-ventricular (LV) ejection fraction did not show more than 10% improvement at remote phase after biopsy. Fourteen patients were classified as responders, and eight as non-responders. Electron microscopy, quantitative PCR, and immunofluorescence studies were performed to explore the biological processes or molecules involved in failure to respond. In addition to studies with cardiac tissue specific knockout mice, we also conducted functional in-vitro studies with neonatal rat ventricular myocytes.

Results
Twenty-two patients with IDCM who underwent endomyocardial biopsy were enrolled in this study, including 14 responders and 8 non-responders. Transmission electron microscopy (EM) showed a significant reduction in mitochondrial size in cardiomyocytes of non-responders compared to responders. Quantitative PCR revealed that transcript of mitochondrial fusion protein, Mitofusin-1, was significantly reduced in non-responders. Studies with neonatal rat ventricular myocytes (NRVMs) indicated that the beta-1 adrenergic receptor-mediated signaling pathway negatively regulates Mitofusin-1 expression. Suppression of Mitofusin-1 resulted in a significant reduction in mitochondrial respiration of NRVMs. We generated left ventricular pressure overload model with thoracic aortic constriction (TAC) in cardiac specific Mitofusin-1 knockout model (c-Mfn1 KO). Systolic function was reduced in c-Mfn1 KO mice, and EM study showed an increase in dysfunctional mitochondria in the KO group subjected to TAC.

Conclusions
Mitofusin-1 becomes a biomarker for non-responders with heart failure. In addition, our results suggest that therapies targeting mitochondrial dynamics and homeostasis would become next generation therapy for severe heart failure patients.

Funding Acknowledgement
Type of funding source: None
High aerobic exercise capacity predicts increased mitochondrial response to exercise training
Authors: Schwarzer M , Zeeb S , Heyne E , Koch L.G , Britton S.L , Doenst T 
Published: November 2020

Abstract

Low exercise capacity is a strong predictor of all-cause cardiovascular mortality and morbidity. In contrast, high exercise capacity is protective and “physical fitness” is considered beneficial. These effects seem to be mediated through mitochondrial function. Importantly, exercise capacity consists of an intrinsic (genetic) and an extrinsic (exercise, environmental) part. In humans, these two parts cannot be truly separated. The rat model of high (HCR) and low (LCR) capacity runners allows to distinguish between the two parts. We assessed mitochondrial function in this model, specifically investigating the impact of exercise training on mitochondrial respiratory capacity.
HCR and LCR were divided into control and exercised groups. Exercise capacity was determined individually using a ramped test. Animals were trained five times a week for four weeks on a treadmill. Mitochondria were isolated from heart, M. gastrocnemius and liver. Citrate synthase activity and protein content were determined photometrically and respiratory capacity was measured using a Clark-type electrode.
At the same age and tibia length, LCR-C were heavier and had a lower heart to body weight ratio than HCR-C. Citrate synthase activity was lower in skeletal muscle of LCR but cardiac citrate synthase was not different between sedentary HCR and LCR. Respiratory capacity in heart and liver was not different between sedentary HCR and LCR but was lower in skeletal muscle in LCR compared to HCR with all selected substrates (glutamate: 86,0±17,6 vs. 63,7±8,0; succinate: 203±19 vs. 136±17 nAO/min/mg Protein). Exercise training led to an increase in body weight in HCR but did not change body weight in LCR. Similarly, gastrocnemius and soleus weights only increased with exercise in HCR. Exercise led to an increase in citrate synthase activity in hearts of HCR (0,78±0,07 vs. 1,58±0,45 U/mg Protein) but not of LCR. Consistently, mitochondrial respiratory capacity was found increased in HCR with exercise in heart with all substrates (glutamate: 261±43 vs. 305±35; succinate 417±32 vs. 539±65 nAO/min/mg Protein). Liver was not affected by exercise.

Conclusion
Our data suggest that genetic predisposition for aerobic capacity additionally affects the response of mitochondria to exercise. Thus, it may be possible that the “born runner” benefits more from aerobic exercise training than the “less genetically equipped counterpart”.

Funding Acknowledgement
Type of funding source: None
Cardioprotection by remote ischemic conditioning is transferable by plasma and mediated by exosomes
Authors: Lassen T.R , Just J , Hjortbak M.V , Jespersen N.R , Stenz K.T , Gu T , Yan Y , Su J , Nyengaard J.R , Kristiansen S.B , Drasbek K.R , Kjems J , Botker H.E 
Published: November 2020

Abstract

Background
Remote ischemic conditioning (RIC) by brief periods of limb ischemia and reperfusion protects against ischemia-reperfusion injury. However, the mechanism is unknown.

Purpose
We studied the role of exosomes for mediating the cardioprotective signal and whether they accumulate in injured myocardium.

Methods
Blood samples from 12 healthy male volunteers were obtained prior to and one hour after RIC. Plasma obtained before and after RIC (n=4) (P-Pre and P-Post) was used to evaluate the transferability of RIC. Pre- and Post-RIC plasma (n=8) was separated into an exosome rich fraction (Exo-Pre and Exo-Post) and an exosome depleted fraction (Prot-Pre and Prot-Post) by size exclusion chromatography. All studies were carried out in duplicate samples from each volunteer. Infarct size was compared in Sprague-Dawley rat hearts perfused with plasma, exosomes and exosome depleted fractions in a Langendorff model. We investigated changes in the miRNA content of the exosomes after RIC by a human miRNA panel. Additionally, fluorescently labeled exosomes isolated from C2C12 cells were used to assess accumulation in injured myocardium in an in vivo rat model. Rats were divided into an infarct group (n=6) (left anterior descending artery ligation) and a sham group (n=6) (without ligation). Labelled exosomes were injected in the femoral vein prior to reperfusion. Exosome-accumulation in infarcted or sham myocardium was evaluated.

Results
P-Post reduced infarct size by 15% points compared with P-Pre (55±4% vs 70±6%, p=0.03) (Fig. 1a). Exo-Post reduced infarct size by 16% points compared with Exo-Pre (53±15% vs 68±12%, p=0.03) (Fig. 1b). Prot-Post did not affect infarct size compared to Prot-Pre (64±3% and 68±10%, p&gt;0.99). We found miRNA-16, miRNA-144 and miRNA-451 to be upregulated in exosomes after RIC and the mTOR-pathway as a potential target for these miRNAs. In the in vivo model, labelled exosomes accumulated more intensively in the infarct area than in remote areas and sham hearts (Fig. 1c).

Conclusion
Cardioprotection by RIC is mediated by exosomes with a changed miRNA profile and exosomes accumulate in injured myocardium.
Figure 1

Funding Acknowledgement
Type of funding source: Private company. Main funding source(s): Novo synergy