bluuebunny/crossref_metadata_2025 huggingface dataset:
Inequalities after STEMI in National Health Service: is there really a postcode lottery?
Authors: Ferreira C , Baptista R , Ribeiro A.I , Freitas A , Ferreira J.A , Milner J , Martinho A , Almeida J.P , Goncalves V , Campos G , Rosa J , Goncalves F , Monteiro S , Monteiro P , Goncalves L
Published: November 2020

Background and purpose

Failure to address the impact of social determinants of health attenuates efficacy of proven prevention recommendations, namely because important considerations related to socioeconomic disadvantage are not captured by existing cardiovascular disease (CDV) risk stratification methods. We aimed to assess how socioeconomic determinants influence recurrent MI and all-cause death after myocardial infarction (MI) in Portugal.

Methods

We conducted a retrospective, observational cohort study, including all patients with a ST-elevation MI (STEMI) admitted to and discharged alive from an intensive cardiac care unit between 2004 and 2017 (n=1809). The median (interquartile range) follow-up was 6 (4–9) years. We used survival models to assess the relationship between their municipal (i) income by purchasing power per capita (PPC), (ii) geographical accessibility to health care, (iii) illiteracy, iv) residential socioeconomic deprivation and recurrent MI and all-cause mortality. To assess residential socioeconomic deprivation, each individual's residential postcode was matched to the recently validated Portuguese version of European Deprivation Index (EDI). The index was categorized into quintiles (Q1-least deprived to Q5-most deprived).

Results

The mean age was 64±14 years; 74% were male. Regarding individual socioeconomic variables, PPC (HR 1.19; 95% CI 0.97–1.47 for Tertile 1 vs Tertile 2; HR 1.28; 95% CI 1.04–1.56 for Tertile 1 vs Tertile 3 and HR 1.07; 95% CI 0.85–1.34 for Tertile 2 vs Tertile 3) and medical appointments in primary health centers per inhabitant (HR 0.90; 95% CI 0.75–1.09 for Tertile 1 vs Tertile 2; HR 1.23; 95% CI 0.95–1.61 for Tertile 1 vs Tertile 3 and HR 1.37; 95% CI 1.06–1.76 for Tertile 2 vs Tertile 3) were predictors of all-cause mortality, but not recurrent MI; however, in multivariate analysis adjusted for sex, age and ejection fraction, this association was no longer significant (HR 1.00; 95% CI 0.99–1.00 and, HR 1.00; 95% CI 0.89–1.17, respectively). Additionally, no evident association between illiteracy and all-cause mortality or MI was present. Concerning EDI, demographic data was similar among the quintiles (Table 1). Although EDI quintiles were not associated with all-cause mortality (HR 1.17; 95% CI 0.82–1.66 for Q5 vs Q1), the EDI was an independent predictor of recurrent MI (Figure 1). On multivariate analysis, adjusted for age, sex, hypertension, diabetes and LDL cholesterol, the HR for the most deprived (Q5) to the least deprived (Q1) quintile was 1.91 (95% CI 1.05–3.49) for MI.

Conclusions

Our study shows clear socioeconomic differentials in cardiovascular outcomes in patients with STEMI which suggests that accounting for socioeconomic deprivation might improve risk prediction and therefore disease prognosis.

Funding Acknowledgement

Type of funding source: None
Extended follow-up data: impact of selective loss to follow-up in a trial of secondary prevention of coronary artery disease
Authors: Tijssen A , Snaterse M , Jorstad H.T , Scholte Op Reimer W.J.M , Peters R.J.G
Published: November 2020

Background

We performed an extended follow-up of the RESPONSE-2 trial, which evaluated a new secondary prevention strategy for patients with coronary artery disease (CAD). Selective loss to follow-up (LTFU) however, may induce biased results.

Objectives

To assess LTFU and its implications on the internal validity of the trial at extended follow-up.

Methods

RESPONSE-2 (N=824) was a randomised trial of nurse-coordinated referral of CAD patients to ≤3 commercial lifestyle interventions, on top of usual-care, or to usual-care alone. The primary outcome was assessed at 1-year follow-up, after which the interventions were discontinued. An extended follow-up was performed two years later. We evaluated overall selective LTFU, between-group selective LTFU and within-group selective LTFU, by comparing baseline characteristics, compliance with the lifestyle interventions (intervention group only) and achievement of lifestyle-related risk factor (LRF) targets at 1-year follow-up for attendees and non-attendees of the extended follow-up. In all variables a standardized mean difference (SMD) of ≥0.2 with a p-value <0.05, based on an independent t-test for continuous data and fisher's test for discrete data, was considered evidence of selective LTFU.

Results

In total, 520 patients attended the extended follow-up: 266/411 in the intervention and 254/413 in the control group. Patients LTFU were generally younger (56.5 vs. 59.3 years; SMD=0.299), more often baseline smokers (37% vs. 22%; SMD=0.334) and had less often quit smoking directly after hospitalization (17% vs. 25%; SMD=0.203). We found no evidence of between-group selective LTFU and several indications of within-group selective LTFU (table). Intervention group attendees were more compliant with the lifestyle interventions compared with non-attendees (weight loss: SMD=0.450; p≤0.05 and physical activity: SMD=0.516; p≤0.01)

Conclusion

Baseline LRF control, compliance with the lifestyle interventions and achievement of LRF targets were less favourable for non-attendees of the extended follow-up. Effective handling of missing data is recommended to minimize loss of contrast due to within-group selective LTFU and to ensure generalizability of results.

Funding Acknowledgement

Type of funding source: Private company. Main funding source(s): The RESPONSE-2 trial was sponsored by Weight Watchers International, Inc. (New York, New York) and Philips Consumer Lifestyle (the Netherlands). The sponsors had no role in the design, data collection, data analysis, data interpretation, and writing of the manuscript.
The prevalence and patient outcomes of adult primary hypercholesterolaemia and dyslipidaemia in the UK: a longitudinal retrospective study using a primary care dataset from 2008 to 2018
Authors: Bilitou A , Rabe A , Inema L , Alamgir G , Dunton K
Published: November 2020

Background

Cardiovascular disease (CVD) remains the leading cause of death and morbidity in Europe and United Kingdom (UK). Increased low-density lipoprotein cholesterol (LDL-C), implicated in primary hypercholesterolaemia and mixed dyslipidaemia (PH/MD), is an extensively studied risk factor, with proven direct and linear causality of CVD. Lowering LDL-C remains a primary goal in the treatment and prevention of atherosclerotic CVD.

Objectives

This study aimed to quantify adult prevalence and incidence of PH/MD in the UK.

Methods

We used an anonymised dataset covering primary care practices across the UK, accessed through the Clinical Practice Research Datalink (CPRD GOLD). Ethics approval was sought and provided (Protocol 19_238R). Using a validated set of clinical codes as well as pre-treatment lipid profile levels for total cholesterol (TC) (>8 mmol/L), LDL-C (>4.9 mmol/L), we calculated prevalent and new adult cases starting from 2009 to 2018. Our denominator population was the CPRD GOLD GP-registered adult population each calendar year, adjusted for mortality.

Results

There were 1,514,916 adults in the CPRD GOLD GP register for the period from 2009 to 2018. During that period there were 354,444 patients diagnosed with PH/MD. Males comprised 46.5%, mean age on diagnosis was 58.2 years. Mean follow-up time was 104 months. The annual prevalence of PH/MD increased from 2009 to 2019 (see graph). The overall prevalence across the period was 22.7%. Mean annual incidence across the decade was 1.7%. Mean LDL-C levels were 4.7 mmol/L and mean TC level was 6.8 mmol/L. In this cohort, 15.9% experienced cardiovascular events (see table). Nearly all patients have used lipid lowering therapies. Only 2.2% achieved at least 40% reduction of LDL-C from baseline.

Conclusions

The prevalence of PH/MD has been increasing despite the availability of interventions. Considering guidelines, only a small proportion of patients have achieved LDL-C goals.

Est prevalence of PH/MD in the UK

Funding Acknowledgement

Type of funding source: Private company. Main funding source(s): Daiichi Sankyo Europe, Health iQ Ltd
Effect of PM2.5 for hospitalization of cardiovascular diseases and medical expenses by age group: a nationwide study from the Japanese Registry Of All cardiac and vascular Diseases (JROAD)
Authors: Kaihara T , Yoneyama K , Kuwata S , Mitarai T , Watanabe M , Koga M , Kamijima R , Ishibashi Y , Izumo M , Tanabe Y , Higuma T , Harada T , Akashi Y.J
Published: November 2020

Introduction

Although particulate matter with an aerodynamic diameter less than or equal to 2.5 μm (PM2.5) exposure influences cardiovascular events, there is limited knowledge how PM2.5 exposure is associated with cardiovascular hospitalization in Japan.

Purpose

We tested the hypothesis that PM2.5 exposure was related to the number of cardiovascular hospitalizations, hospitalization period, and medical expenses.

Methods

We included 835,405 subjects who were admitted to acute care hospitals in Japan. We classified PM2.5 exposure concentration into quintile groups (from low to high in the order of “PM-1” to “PM-5” group). Multilevel mixed-effects Poisson and linear regression analysis were used to estimate the association of PM2.5 exposure concentration with the cardiovascular hospitalization events, duration and medical expenses. We also analyzed those factors classified by age.

Results

PM-2, 3, 4, 5 group were positively related to the number of cardiovascular hospitalization events compared with PM-1 group (Table 1). PM-3, 4, 5 group were positively associated with a 0.018, 0.029, and 0.029 (all p<0.001) of the difference of log cardiovascular hospitalization period compared with PM-1 group (p for trend <0.001). These groups were also positively associated with a 0.016, 0.023, and 0.021 (all p<0.001) of the difference of log medical expenses compared with PM-1 group (p for trend <0.001). Analyzed by age group, hospitalization duration was longer (p<0.05) and medical expenses was lower (p<0.05) significantly in super-aging group (over 90 years old) than the group under 64 years old (Figure 1).

Conclusions

PM2.5 exposure concentration has harmful effect on not only cardiovascular events but cardiovascular hospitalization period and medical expenses due to big-data in Japan. Medical costs were kept low in comparison with their hospitalization period in subjects over 90 years old.

Figure 1

Funding Acknowledgement

Type of funding source: None
Single-cell RNA sequencing of human fetal epicardium reveals novel markers and regulators of EMT
Authors: Streef T.J , Van Herwaarden T , Smits A.M , Goumans M.J
Published: November 2020

Background

The heart is covered by the epicardium, consisting of epithelial cells and a mesenchymal layer. The epicardium has been shown to be essential during cardiac development by contributing cells through epithelial-to-mesenchymal transition (EMT) and the secretion of paracrine factors. In the adult, the epicardium conveys a cardioprotective response after myocardial infarction, albeit suboptimal compared to the epicardial contribution to heart development. Although the developing epicardium has been characterised in mice and zebrafish, knowledge on the human fetal epicardium derives mostly from cell culture models. Therefore, direct analysis of the human fetal epicardium is vital as it provides new insights into the cellular and biochemical interactions within the developing heart, which can potentially contribute to enhancing the post-injury response.

Aim

To study the human fetal epicardium using single-cell RNA sequencing (scRNA seq) in order to determine its cellular compositionThe data are further explored to e.g.identify regulators of epicardial EMT.

Methods

Epicardial layers were isolated from four fetal human hearts (14–15 weeks gestation, obtained under informed consent and according to local ethical approval). Tissue was digested, and single live cells were sorted into 384-wells plates and sequenced. Data analysis was performed using R-packages RaceID3 and StemID2. Findings were validated using qPCR and immunohistochemistry.

Results

Analysis of 2024 cells reveals a clear clustering of the epicardial epithelium and the mesenchymal population. Importantly, we found that “classical” markers, such as Wilms' Tumor 1 and T-box transcription factor 18, are not specific enough to reliably identify the epicardium, but our analysis has provided markers that do allow for robust identification of the epicardium. Additionally, we were able to identify epicardial subpopulations based on their expression profile, and we are currently investigating these using immunohistochemistry in human fetal and adult heart tissue sections. To establish the regulation of epicardial activation we are focussing on the process of EMT within our dataset using RaceID2. From our analysis, several regulators of epicardial EMT are proposed that will be followed up on in vitro.

Conclusions

We identify various novel markers of the fetal epithelial epicardium, as well as characterizing markers of the mesenchymal layer. We also identified novel factors involved in epicardial EMT, and these are currently being validated in our cell-culture model. These data can provide new insights into the post-injury response in the adult heart.

Funding Acknowledgement

Type of funding source: Public Institution(s). Main funding source(s): Dutch Heart Foundation
VENTHEART is required for cardiomyocyte specification and function
Authors: Jonsson Boezelman M , Dashi A , Tan W , Pan B , Ilmari A.M , Tiang Z , Hartman R.J.G , Anen C.G.O , Liu Z , Wu J.J , Bin G , Lim B , Walentinsson A , Foo R.S
Published: November 2020

Background

Long noncoding RNAs (lncRNAs) control early stages of cardiac differentiation, however their role in later specification and maturation is still not well explored.

Methods and results

We performed single cell RNA-seq for 2, 6 and 12 week-old hESC-CM. Weighted correlation network analysis (WGCNA) identified core genes significantly upregulated, along with a subset of lncRNAs. Importantly, these lncRNAs are highly abundant and unique to human heart. Through independent integrative analysis of genome-wide association studies (GWAS) and expression quantitative trait locus (eQTL) data using human hearts, we also identified a long intergenic noncoding RNA (we call VENTHEART, VHRT) as co-regulated with core cardiac contractile genes, and strongly associated with heart failure. VHRT was highly expressed in MYL2+ hESC-CMs in our single cell dataset, and its locus is antisense and downstream of MYL2. VHRT knockdown (KD) in 6-weeks old hESC-CMs downregulated MYL2 and other key cardiac genes. Patch clamp recordings with VHRT KD cells showed a loss of the ventricular-like action potential. Concordantly, CRISPR-mediated excision of the VHRT locus led to impaired CM sarcomere formation, and loss of CM specification gene programs. VHRT transcript replacement in VHRT-KO cells was however insufficient to rescue the phenotype. Instead, we established by 3C assay, that the VHRT locus loops and interacts with the MYL2 promoter, bearing histone marks characteristic of a super-enhancer.

Conclusion

Thus, we conclude that both the VHRT lncRNA transcript and its genomic locus are required for proper CM specification and function, and may play a role in heart failure progression.

Funding Acknowledgement

Type of funding source: Other. Main funding source(s): EMBO, Singapore National Research Council
Influence of perfusable microvasculature on excitation-contraction coupling in IPSC-derived myocardium
Authors: King O , Cruz-Moreira D , Kit-Anan W , Sayed A , Wang B.X , Fourre J , Randi A.M , Rasponi M , Terracciano C.M
Published: November 2020

The myocardium is one of the most densely vascularised tissues in the body, with dynamic metabolic demand from beating cardiomyocytes (CM) necessitating an intimate relationship with microvasculature. Endothelial cells (EC) produce a diverse array of cardio-active factors which acutely and chronically modulate myocardial phenotype. Disruption of CM-EC signalling results in pathological remodelling, and ultimately organ failure. However, as physiologically relevant recapitulation of CM-EC interaction has been difficult to achieve in vitro, many molecular mechanisms governing their interaction remain poorly understood.

To induce cardiac vasculogenesis in vitro, we have developed microfluidic chips which subject 3D hydrogel cultures to precisely controlled flow. We then co-cultured human cardiac microvascular ECs, human left ventricular fibroblasts (FB), and human induced pluripotent stem cell-derived cardiomyocytes for 5 days under a pro-vasculogenic protocol (0.5 ul/min flow rate, 50ng/ml VEGF, 100ng/ml Ang-1). Via live and fixed immunofluorescence microscopy, we observed spontaneous formation of a microvasculature network with a continuously open lumen embedded within beating myocardium. Simultaneous quantification of iPSC-CM contractility and perfused red blood cell velocity reveals biomimetic pulsatile flow profile within the microvasculature. To evaluate the influence of microvasculature on CM function, we incorporated CMs differentiated from stem cells with the genetically encoded calcium biosensor GCaMP6F. Compared to CM only control, vascularised preparations demonstrate significantly faster calcium transient time to peak (−11.5%, p=0.007) and time to 50% relaxation (−15%, p=0.01). Under static conditions and 1Hz electrical stimulation, presence of EC was associated with reduced iPSC-CM arrhythmia at baseline (p<0.0001) and during 1uM isoprenaline treatment (p=0.0003), while maintaining isoprenaline induced Ca2+ handling quickening.

To the best of our knowledge, this work represents the first fully perfusable model of the myocardial microvasculature, and highlights the importance of EC regulatory influence on CM function. Further work aims to investigate underlying molecular mechanisms to provide therapeutically relevant insight into cardiac biology.

Funding Acknowledgement

Type of funding source: Foundation. Main funding source(s): British Heart Foundation
Cholinergic nerve regulation of heart regeneration
Authors: Mahmoud A
Published: November 2020

Background

Cardiac nerves regulate many important physiological functions of the heart such as heart rate and contractility. The emerging role of cardiac nerves during tissue homeostasis and regeneration is beginning to be appreciated. We discovered that neonatal mice are capable of regenerating their hearts following injury within a brief period after birth by proliferation of the pre-existing cardiomyocytes. Furthermore, we have demonstrated that cholinergic nerves play an important role in guiding the neonatal heart regenerative response. However, the adult mammalian heart, including the human heart, is incapable of regeneration following injury. Thus, there is great excitement about understanding the evolutionarily conserved mechanisms of endogenous cardiac regeneration, so that we can explore potential avenues to reawaken this process in adult humans.

Purpose

Our overarching goal is to define the mechanisms by which cholinergic nerves regulate heart regeneration following ischemic injury by using the neonatal mouse heart regeneration model. These studies will uncover novel pathways by which cholinergic signaling promotes cardiomyocyte proliferation and heart regeneration, which holds significant therapeutic potential for treatment of adult heart disease.

Methods

In this project, we employed genetically engineered mouse models of the critical receptors for cholinergic signaling in the heart to define the mechanisms of cholinergic nerve regulation of heart regeneration. First, we generated a cardiomyocyte-specific deletion of the muscarinic receptor (M2), the most predominant muscarinic receptor subtype present in the heart. In addition, we utilized the α7 nicotinic receptor (Chrna7) knockout mice to study the role of Chrna7 in endogenous immune cells, which is the main mediator of the cholinergic anti-inflammatory pathway. These mouse models will address how cholinergic nerves regulate heart regeneration via the M2 muscarinic receptor signaling and the inflammatory response following injury.

Results

Our results demonstrate that inhibition of two different cholinergic receptors (muscarinic and nicotinic) results in a reduction in cardiomyocyte proliferation and inhibition of the neonatal cardiac regenerative response following injury. More importantly, we demonstrate that cholinergic signaling mediates the cardiac regenerative response mainly through suppression of pro-inflammatory cytokines via the cholinergic anti-inflammatory pathway.

Conclusions

Cholinergic nerve signaling plays an important role in mounting a robust cardiac regenerative response following injury. These results have significant therapeutic potential, which will forge new paradigms with respect to the role of cardiac nerves during mammalian cardiac regeneration and reveal potential mechanisms regarding the benefits of nerve stimulation following cardiac injury in humans.

Funding Acknowledgement

Type of funding source: Foundation. Main funding source(s): American Heart Association, Wisconsin Partnership Program
ADAM10 inhibition improves survival and augments cardiac function after myocardial infarction
Authors: Klapproth E , Kuenzel S , Guenscht M , Lorenz K , Weber S , Guan K , El-Armouche A
Published: November 2020

Background and purpose

Following myocardial infarction (MI), adverse fibrotic remodeling with extensive deposition of extracellular matrix (ECM) components has substantial consequences for the contractility of the ventricle finally leading to terminal heart failure (HF). Recently, inhibition of ECM-remodeling enzymes is discussed as potential treatment option for HF, especially following MI. The metalloprotease ADAM10 plays a crucial role in the development of the cardiovascular system and HF patients show elevated serum levels of the ADAM10 substrates CXCL16 and FasL. However, the causal role of ADAM10 in cardiovascular diseases has not been investigated. Here we evaluate the so far unknown role of ADAM10 in heart failure and after MI.

Methods and results

Our study capitalized from human atrial tissue biopsies, a cardiomyocyte-specific ADAM10 knockout (ADAM10 KO) mouse model as well as pharmacological ADAM10 inhibition following MI. ADAM10 expression analysis revealed elevated protein levels in HF patients compared to non-failing hearts. Upon MI, ADAM10 KO and pharmacological ADAM10 inhibition (GI254023X) significantly improved overall survival, significantly enhanced cardiac function (fractional area shortening - FAS, ejection fraction - EF) and significantly reduced infarct sizes. Compared to the high potential angiotensin receptor neprilysin inhibitor (ARNi) LCZ696, ADAM10 inhibition and combined ADAM10i/LCZ696 treatment resulted in preservation of cardiac function that was superior to sole LCZ696 treatment. Mechanistically, this functional improvement was due to reduced shedding of the ADAM10 substrate Notch1, induction of angiogenesis and an ADAM10-dependend inactivation of the NLRP3 inflammasome

Conclusion

Our data suggest that ADAM10 targeting is highly efficient for improving post-infarction cardiac function. Due to its overexpression in heart tissue of HF patients, ADAM10 could be a potential molecular target to improve therapy after MI. In terms of overall survival and pathophysiological remodeling following MI, our data suggest a greater potential of the ADAM10i/LCZ696 combinatorial therapy than sole LCZ696 treatment.

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Heart Foundation/ German Foundation of Heart Research
Protective action of the microbial metabolite butyrate against cardiomyocyte hypertrophy
Authors: Umei M , Akazawa H , Saga-Kamo A , Yagi H , Liu Q , Kadowaki H , Matsuoka R , Matsunaga H , Nomura S , Komuro I
Published: November 2020

Introduction

Short-chain fatty acids are one of the gut microbial metabolites that may influence host physiology. We previously reported that gut dysbiosis was associated with heart failure, and that the proportions of butyrate-producing bacteria diminished prominently in the gut of patients with heart failure.

Purpose

We investigated the molecular mechanism of butyrate and investigated the protective mechanism against heart failure.

Methods

We searched for G protein-coupled receptors for short-chain fatty acids using single-cell transcriptome analysis of cardiomyocytes and non-cardiomyocytes isolated from murine hearts. In addition, we examined the effects of butyrate on endothelin-1 (ET1) or isoproterenol-induced hypertrophic responses and histone deacetylase (HDAC) activities in cultured neonatal rat cardiomyocytes.

Results

Single-cell transcriptome analysis and co-expression network analysis revealed that G protein-coupled receptors for short-chain fatty acid receptors were not expressed in cardiomyocytes and that Olfr78 was expressed in vascular smooth muscle cells in the heart. Treatment with butyrate inhibited ET1-induced hypertrophic growth and up-regulation of the genes such as Nppa, Acta1, and Myh7 in cultured rat neonatal cardiomyocytes. Moreover, butyrate increased the acetylation levels of histone H3, indicating that butyrate has an inhibitory effect on HDAC in cardiomyocytes. In addition, treatment with butyrate caused up-regulation of Inpp5f, encoding inositol polyphosphate-5-phosphatase f, which was associated with a significant decrease in the phosphorylation levels of Akt. These results suggest that butyrate may act as HDAC inhibitor to increase Inpp5f gene expression, leading to the activation of Akt-glycogen synthase kinase 3beta (Gsk3beta) pathway, and thereby protect against hypertrophic responses.

Conclusion

There was no known GPCR for short-chain fatty acid expressed in cardiomyocytes. However, butyrate suppressed cardiomyocyte hypertrophy through epigenetic modification of gene expression. Our results may uncover a potential role of the dysbiosis of intestinal microbiota in the pathogenesis of cardiac hypertrophy and failure.

Funding Acknowledgement

Type of funding source: None
Redox-activated protein kinase A Type I (PKA RI) exerts a protective role during pathological pressure overload
Authors: Tarnowski D , Islam M.M.T , Trum M , Eaton P , Maier L.S , Wagner S , Sag C.M
Published: November 2020

Background

Phosphorylation of L-type calcium channels (LTCC) by cAMP-dependent protein kinase A (PKA) increases calcium current (ICa). However, it is unclear if PKA-dependent regulation of ICa is impaired in heart failure (HF) despite evidence for impaired β-adrenergic signaling. Recently, a novel PKA activation pathway by oxidation of regulatory subunit I (RI) has been identified.

Objective

We investigated the impact of redox-activated PKA for regulation of ICa, intracellular calcium (Ca) handling and contractile function in a pressure overload heart failure mouse model.

Methods

Knock-in mice (KI) that lack redox-dependent PKA activation (exchange of cysteine 17 of RI with serine) were compared to wild-type (WT) at baseline, 7 days and 6 weeks after transverse aortic constriction (TAC). Mouse echocardiography was performed to evaluate in-vivo cardiac function. ICa was measured by whole-cell patch clamp, PKA activity, cAMP levels, and protein levels of central Ca handling proteins were assessed at different time points in vitro.

Results

At baseline, no alterations of left ventricular (LV) function (echocardiography) were observed between WT and KI. TAC induced a significant RI oxidation in WT but not KI mice. Despite this difference, at 7 days after TAC, development of LV hypertrophy and impairment of systolic LV function in vivo were similar between WT and KI. Compared to baseline, 7 days after TAC a significant stimulation of peak ICa was observed in WT. In contrast to WT, the stimulation of peak ICa was absent in KI mice at 7 days after TAC. This impairment in peak ICa occurred despite a comparable increase in global PKA activity (ELISA), which was most likely due to increased cAMP levels in our TAC model (assessed by FLIM-FRET). Notably, cAMP levels were comparably increased in between groups (ELISA). Importantly, at 6 weeks after TAC, WT mice showed a mild additional deterioration of systolic LV function in vivo. In contrast, LV function was significantly more impaired in KI mice 6 weeks after TAC, which was accompanied by a significant increase in KI mice mortality. Comparing 6 weeks to 7 days after TAC, there was no stimulation of peak ICa in WT and even a significant decrease in peak ICa in KI mice. In accordance, PKA-dependent LTCC phosphorylation was absent in KI mice 6 weeks after TAC (western blotting).

Conclusion

Redox-activated PKA seems to exert a protective role by stimulation of ICa during pressure overload.

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft
Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of 5182 cases from long QT syndrome and Brugada syndrome consortia cohorts and gnomAD population controls
Authors: Walsh R , Lahrouchi N , Glinge C , Krijger C , Skoric-Milosavljevic D , Whiffin N , Mazzarotto F , Ware J , Tadros R , Bezzina C
Published: November 2020

Background/Introduction

Guidelines for variant interpretation in Mendelian disease set stringent criteria to report a variant as (likely) pathogenic, prioritising control of false positive rate over test sensitivity and diagnostic yield, and require customisation for the specific genetic characteristics of gene-disease dyads. Inherited arrhythmias like long QT syndrome (LQTS) and Brugada syndrome (BrS) are genetically heterogeneous, with missense variants constituting the preponderance of disease-causing variants. Evidence from family segregation or functional assays to confirm pathogenicity are often unavailable or impractical in clinical genetic testing, leading to high rates of variants of uncertain significance and false negative test results.

Methods

We compared rare variant frequencies from 1847 LQTS (KCNQ1, KCNH2, SCN5A) and 3335 BrS (SCN5A) cases from the International LQTS/BrS Genetics Consortia to population-specific gnomAD data. We propose disease-specific criteria for ACMG/AMP evidence classes – rarity (PM2/BS1 rules) and enrichment of individual (PS4) and domain-specific (PM1) variants in cases over controls.

Results

Rare SCN5A variant prevalence differed between BrS cases with spontaneous (28.7%) versus induced (15.8%) type 1 Brugada ECG (p=1.3x10–13) and between European (20.8%) and Japanese (8.9%) patients (p=8.8x10–18). Transmembrane regions and specific N-terminus (KCNH2) and C-terminus (KCNQ1/KCNH2) domains were characterised by high enrichment of case variants and >95% probability of pathogenicity. Applying the customised rules, 17.5% of European BrS cases and 73.7% of European LQTS cases had variants classified as (likely) pathogenic, compared to estimated diagnostic yields (case excess over gnomAD) of 19.3%/82.6%.

Conclusions

Large case/control datasets enable quantitative implementation of ACMG/AMP guidelines and increased sensitivity for inherited arrhythmia genetic testing.

Classification of Brugada/LQTS variants

Funding Acknowledgement

Type of funding source: Foundation. Main funding source(s): Dutch Heart Foundation, Netherlands Organisation for Scientific Research
Increase in inflammatory T cell subsets in atrial fibrillation: the missing link underlying inflammation in AF
Authors: Dumitriu I.E , Dimou P , Kaur S , Dinkla S , Kaski J.C , Camm A.J
Published: November 2020

Background

The precise role of inflammation in the development and perpetuation of atrial fibrillation (AF) is yet to be fully uncovered. T lymphocytes have pivotal roles in orchestrating inflammation. Specialised subsets of lymphocytes either promote or prevent inflammation. We are investigating a unique subset of lymphocytes, the CD4+CD28null T cells that expand in patients with chronic inflammation. These cells secrete high levels of pro-inflammatory cytokines and have cytolytic function. CD4+CD28null T cells are normally maintained under control by regulatory T cells (Treg), a specialised subset of T lymphocytes with suppressive function that maintain immune homeostasis and prevent pathogenic immune responses. The role of CD4+CD28null and Treg cells has not been investigated in AF.

Purpose

We hypothesised that in AF the balance between pro-inflammatory and regulatory T lymphocytes is skewed in favour of inflammatory T cells, which may sustain inflammation in AF.

Methods

Circulating CD4+CD28null T lymphocytes and Tregs were quantified by flow cytometry in paroxysmal and persistent AF patients and healthy controls (n=30). Inflammatory cytokines were quantified in serum and the function of T lymphocyte subsets was investigated using ex vivo functional assays.

Results

CD4+CD28null T lymphocytes were significantly increased in the circulation of AF patients compared to controls. Of note, a higher proportion of patients with persistent AF showed an increase in inflammatory CD4+CD28null T lymphocytes compared to patients with paroxysmal AF. A marked reduction in Treg cells was present in AF patients compared to controls. Functional assays showed that IL-7 and IL-15 cytokines were responsible for CD4+CD28null T lymphocyte expansion in AF patients.

Conclusions

We show that patients with AF have marked changes in T lymphocytes subsets: pro-inflammatory CD4+CD28null T cells increase significantly, whilst anti-inflammatory Tregs are significantly reduced. We show for the first time that the cytokines IL-7 and IL-15 are the main drivers of CD4+CD28null T cell expansion in AF patients. These novel findings may reveal novel therapeutic strategies (e.g. cytokine blockade) to re-establish the balance between pro- and anti-inflammatory mechanisms at work in AF to improve patient outcomes.

Funding Acknowledgement

Type of funding source: Foundation. Main funding source(s): British Heart Foundation
Fast delivery of flecainide via the pulmonary (bolus) or intravenous (rapid infusion) routes reduces atrial fibrillation conversion dose and minimizes negative inotropic burden
Authors: Belardinelli L , Nearing B.D , Verrier R.L
Published: November 2020

Background/Introduction

The negative inotropic effect of certain antiarrhythmic drugs limits their use for acute cardioversion of new-onset paroxysmal atrial fibrillation (AF).

Purpose

In an intact porcine model of AF, we examined the effects of pulmonary and intravenous (IV) administration of flecainide on left ventricular (LV) contractility, i.e., LV dP/dt max, at doses that are effective in converting AF to sinus rhythm. The magnitude of the decrease in LV dP/dt max and time that it remained below baseline, measured by the area under the curve (AUC), is referred to as negative inotropic burden.

Methods

Flecainide was delivered via intratracheal administration at 1.5 mg/kg bolus and compared to IV infusion at 1.0 mg/kg over 2 min (lower-dose, rapid) and 2.0 mg/kg over 10 min (ESC guideline) in 11 closed-chest, anesthetized Yorkshire pigs. These doses of flecainide have been shown effective in converting AF to sinus rhythm. Catheters were fluoroscopically positioned in the right atrium for pacing at 140 beats/min and in the LV to measure QRS complex duration and contractility (LV dP/dt). Intratracheal flecainide was delivered via a catheter positioned at the bifurcation of the main bronchi.

Results

The peak plasma levels (Cmax values) were similar but the AUC of plasma concentrations over time was greater for the higher-dose, slow IV infusion of flecainide than for either intratracheal instillation (by 32%) or lower-dose, rapid IV infusion (by 88%). Based on AUCs of LV dP/dt max (figure, left panel), the negative inotropic burden is 3.1- to 3.8-fold greater for the higher IV (1006% • min) than for the lower IV (323%·min) or the intratracheal doses (263% • min). There was a corresponding inverse increase in the AUC of QRS complex prolongation. The decrease in LV dP/dt max (Δ%) was correlated with the prolongation of the QRS complex (Δms) (y = −1.4337x + 3.6613, r2=0.69, p<0.0001) (figure, right panel).

Conclusion

Rapid delivery of pulmonary or IV flecainide reduces the dose of drug required to achieve Cmax levels associated with conversion of AF. The attendant decrease across time in exposure of the ventricles to flecainide reduces QRS complex prolongation and the accompanying negative inotropic burden.

Funding Acknowledgement

Type of funding source: Private company. Main funding source(s): InCarda Therapeutics, Inc.
Epicardial adipose tissue CD4+ T cells as novel drivers in the development of atrial fibrillation
Authors: Vyas V , Wood L , Blythe H , Hunter R.J , Lall K.S , Finlay M.C , Longhi M.P
Published: November 2020

Background

Epicardial adipose tissue (EAT) is now a well-established independent risk factor for all forms of atrial fibrillation (AF). However, the cellular profile and immune mediators within EAT remain poorly defined.

Purpose

This study sought to define the immunological signature of EAT critical to its role in the development of both persistent AF and de novo post-operative AF (POAF).

Methods

Adult patients undergoing elective cardiac surgery with no prior AF history and those with pre-existing AF were recruited to undergo EAT sampling alongside subcutaneous adipose tissue (SAT) and pre-operative blood (adipose tissue and systemic controls respectively) sampling. The blood and tissue samples were immediately taken to the laboratory for immune cell isolation, flow cytometry and quantitative polymerase chain reaction (qPCR) analysis.

Results

A total of 71 patients were recruited including 9 patients with a prior history of AF. Of the 62 a priori sinus rhythm (SR) patients, 42 remained in SR post-operatively and 20 developed de novo POAF. No differences in absolute numbers of immune cells in the blood, EAT or SAT were detected on flow cytometry analysis between the three groups of patients. However, there was a significant increase (p<0.01) in EAT-resident CD4+ memory T cell populations in pre-existing AF patients and a trend towards significance (p=0.09) in POAF patients. qPCR analysis of immune mediator expression in EAT demonstrated pre-existing AF patients uniquely showed a significant reduction (p<0.005) in the pro-fibrotic mediator transforming growth factor-β (TGF-β) compared to the pre-existing AF patients (graph 1).

Conclusions and implications

Resident CD4+ memory T cell populations are locally and exclusively elevated in the EAT of pre-existing AF and POAF patients. This would suggest primed antigen-specific CD4+ T cells are present prior to the development of AF in patients who go on to develop de novo POAF. The reduced TGF-β levels observed in the EAT of AF patients indicates the EAT immune mediator profile differs to the underlying myocardial tissue, where increased TGF-β levels have previously been described in the persistent AF cohort. Therapies targeting this EAT-resident CD4+ T cell population would provide a novel approach to the management of the inflammatory components of AF genesis.

Graph 1

Funding Acknowledgement

Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Barts Charity, Abbott
Obesity associated pro-fibrotic protein augments fibrosis in heart
Authors: Tsukano Y , Shimizu I , Yoshida Y , Hsiao Y , Ikegami R , Hayashi Y , Suda M , Katsuumi G , Nakao M , Minamino T
Published: November 2020

Chronic sterile inflammation in visceral fat has causal roles for systemic metabolic disorders in obesity. Inflamed visceral adipose tissue secretes pro-inflammatory adipokines, and this contributes to tissue remodeling under a metabolically stressed condition. Various kinds of white adipokines are broadly studied, however, roles of brown adipose tissue (BAT) derived adipokines (BATokine) remain to be explored. In this project, we tried to characterize pathogenic role of BATokine in obesity related fibrotic disorders, especially focusing on heart failure with preserved ejection fraction (HFpEF). For this purpose, we analyzed two sets of DNA microarray data, and identified an obesity associated pro-fibrotic protein (OAFP) as a possible pathogenic BATokine. Our biobank studies showed OAFP increased in patients with diastolic dysfunction, and E/e' analyzed with cardiac echo increased in direct proportion to circulating OAFP level in humans. We generated dietary obese mice model, and found OAFP increased both in BAT and circulation. We generated a murine systemic or BAT specific OAFP knockout (KO) models, and found that obesity-induced diastolic dysfunction ameliorated in these models. Cardiac fibrosis was also suppressed by genetic depletion of OAFP. We found OAFP increased in circulation in aged humans and mice, and studies in chronologically aged mice showed this molecule increased in BAT with aging. Our results indicate that OAFP is secreted predominantly from BAT, and mediates pathogenic roles by augmenting cardiac fibrosis in dietary obesity or aging. Suppression of OAFP may become a therapy for HFpEF.

Funding Acknowledgement

Type of funding source: None
Macrophage-specific IRF5 deficiency stabilizes atherosclerotic plaques in ApoE−/− mice
Authors: Hilgendorf I , Haerdtner C , Leipner J , Dufner B , Hoppe N , Wolf D , Stachon P , Zirlik A , Bode C
Published: November 2020

Background

Interferon regulatory factor (IRF) 5 is a transcription factor promoting inflammatory macrophage polarization (M1 type). Given the central role of macrophages in atherosclerotic plaque development we hypothesized that macrophage specific deletion of IRF5 will protect from atherosclerosis.

Purpose

Investigate whether intrinsic blockade of M1 macrophage polarization ameliorates atherosclerosis

Methods

Female ApoE−/−LysMCre/wtIRF5flox/floxand ApoE−/−LysMwt/wtIRF5flox/floxmice were fed a high cholesterol diet for 3 months, and atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependend bone marrow derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Aortic macrophage chimerism in irradiated ApoE−/− mice reconstituted with a mixture of CD45.1+ ApoE−/− (WT) and CD45.2+ ApoE−/− LysMCre/WtIRF5flox/flox(KO) bone marrow was evaluated to distinguish systemic from intra-plaque effects on monocyte/macrophage kinetics.

Results

Macrophage-specific IRF5 deficiency blunted LPS/IFNg-induced IL-1β and TNFα gene expression in vitro. In ApoE−/− mice, macrophage-specific IRF5 deficiency did not alter lesion size in the aortic root but significantly reduced macrophage and lipid contents by about 25% while increasing collagen deposition by over 30%. This was accompanied by relative reductions in gene expressions of pro-inflammatory (IL-1β, IL-6, IL-12) and increases in anti-inflammatory (Mertk, TGFβ, CD206) markers in atherosclerotic aortas of ApoE−/−LysMCre/wtIRF5flox/floxmice. When competing with IRF5 deficient cells in mixed irradiation bone marrow chimeras, IRF5 competent macrophages showed an advantage in accumulating in atherosclerotic aortas as disease progressed independent of monocyte recruitment.

Conclusion

Transcription factor IRF5 promotes a pro-inflammatory response in macrophages leading to vulnerable plaque formation and plaque destabilization, providing genetic evidence for targeting macrophage polarization in atherosclerosis.

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): DFG
Long non-coding RNA TERRA influences DNA damage and survival of endothelial cells and cardiomyocytes
Authors: Bink D , Pham T.P , Hofmann P , Dimmeler S , Boon R.A
Published: November 2020

Ageing is the major risk factor for cardiovascular disease. Current therapies are mainly based on proteins, while targeting long non-coding RNAs (lncRNAs) is largely unexplored. Long non-coding RNAs are emerging as novel regulators of cellular functions and contributors to cardiovascular ageing. Although telomeres are heterochromatic regions, non-coding transcripts called Telomeric repeat-containing RNA (TERRA) are transcribed from the telomeres of most chromosomes. The transcription of TERRA starts at the subtelomere and ends in the telomere, leading to molecules of 0.2–10kb. This study aims to characterize the role of TERRA in the cardiovascular system.

TERRA molecules from different chromosomes were upregulated in the hearts of old mice compared to young mice (p=0.002). An increased TERRA expression was also shown in heart tissue of patients with ischemic heart disease compared to donor heart tissue (p=0.001). In vitro an upregulation of the TERRA molecule transcribed from chromosome 20 (h20q-TERRA) was found in old passage human umbilical vein endothelial cells (HUVECs) (P15–17) compared to young HUVECs (P3) (p=0.014). IPSC-derived cardiomyocytes also increased the expression of h20q-TERRA with increasing passage (p=0.011). After knockdown of h20q-TERRA with LNA GapmeRs HUVECs show less sprout formation in a spheroid assay compared to negative control transfected HUVECs (p=0.002), without showing a change in migration (p=0.205) or proliferation (p=0.114). H20q-TERRA knockdown revealed that there was more apoptosis (p=0.015), more DNA damage as measured with the comet assay (p<0.001), increased γH2AX levels (p=0.029) and an increase in γH2AX colocalization with the telomere (p=0.011). The amount of phosphorylated P53 was also increased after knockdown (p=0.038), while it was decreased after TERRA overexpression (p=0.001). Inhibition of the ATM-yH2AX-P53 pathway did however not reduce the increased apoptosis after knockdown (KU60019 p=0.399; siP53 p=0.303). Silencing the m18-TERRA molecule in mouse endothelial H5V cells led to an increase in caspase activity (p=0.004) similar to what was shown in HUVECs. In addition, increased caspase activity (p=0.012), increased γH2AX levels (p<0.001) and increased γH2AX colocalization with the telomere (p=0.007) was also shown after silencing of h20q-TERRA in human cardiomyocytes.

In summary, our data demonstrates that TERRA is upregulated with ageing and plays a role in endothelial and cardiomyocyte function and survival. These data show that TERRA transcripts are induced in cardiovascular ageing and are essential for endothelial cell function.

Funding Acknowledgement

Type of funding source: Public grant(s) – EU funding. Main funding source(s): Horizon 2020