bluuebunny/crossref_metadata_2025 huggingface dataset:
Cis-regulatory architecture and functions of a gene desert controlling pleiotropic expression and cardiac pacemaker development
Authors: Osterwalder M , Abassah-Oppong S , Mannion B , Rouco R , Zoia M , Roland V , Barozzi I , Lopez-Rios J , Andrey G , Visel A , Pennacchio L , Cobb J
Published: October 2022

Gene deserts are extensive genomic regions spanning more than 500 kilobases and enriched near developmental genes. Despite their proposed critical roles in development, the enhancer architecture, and biological functions of most gene deserts in mammalian genomes remain unknown. The Shox2 transcription factor is a key regulator of cardiac pacemaker differentiation and at the genomic level is flanked by a centromeric gene desert harboring a multitude of predicted cis-regulatory modules (CRMs). Using CRISPR-Cas9 we demonstrate a requirement of this gene desert for pleiotropic expression and embryonic survival through full control of Shox2 in the sinoatrial node (SAN). To decode the underlying chromatin architecture and CRM logic, we performed region-specific chromatin conformation capture, epigenomic analysis and transgenic in vivo reporter assays. While the Shox2 regulatory landscape was found partitioned into largely tissue-invariant chromatin loops, we identified a surprising cardiac-specific contact domain within the gene desert. Subsequent transgenic analysis revealed clustering of non-cardiac enhancers with activities in limb, craniofacial or neuronal populations in this domain, pointing to a potential tissue-specific 3D-mechanism for enhancer attenuation. In addition, SAN-chromatin assessment and H3K27ac profiling from human fetal cardiac compartments enabled the identification of a unique cardiac enhancer located outside of the contact domain and essential for robust Shox2 levels during cardiac pacemaker development. In summary, our results serve as a blueprint to investigate gene desert function in context to cardiac defects and identify the Shox2 gene desert as a robust cis-regulatory hub indispensable for pleiotropic patterning, cardiac pacemaker differentiation and embryonic survival.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF),National Institutes of Health (NIH)
Founder effect may offer protection from ischemic heart disease in the South-Brazilian Mennonites
Authors: Da Rocha G , Vieira Junior W A , Meissner C G , Oliveira L C , Dos Santos P I , Da Silva M A , Hauser A B , McMahon F , Lopes F L , Lima Junior E , Boldt A W B
Published: October 2022

Introduction

The Mennonites are an Anabaptist population originated in Europe around 500 years ago. They are a relatively isolated population, established in Brazil in 1930, which suffered three bottleneck effects that reduced their genetic variability and may increase the frequency of diseases.

Purpose

We aimed to clarify the genetic epidemiology of heart disease in this population.

Methods

Epidemiological, biometrical and lipidogram data were collected from 2016 to 2020, based on an adapted questionnaire of the Brazilian National Health Research. In total, 485 Mennonites (MEN) and 45 non-Mennonites from three different settlements in Curitiba, Witmarsum, and Colônia Nova (CON) were enrolled in the study. DNA was extracted from blood and 104 exomes from CON, sequenced to >30x coverage.

Results

The prevalence of hypertension (20.5 vs. 31.8%, p<0.001) and dyslipidemia (41.1% vs. 65.1%, p<0.001) were lower compared to Germans (GER). When compared to Brazilians (BRA), dyslipidemia was higher in Mennonites (23% vs. 41.3%, p<0.001) and hypertension was higher in Brazilians (20.5% vs. 25.7%, p=0.02). The prevalence of overweight among the three populations doesn't have a significant statistical difference (59% MEN, 55.7% BRA, 59.7% GER, p=0.09). Myocardial infarction (MI) prevalence is drastically different between the three (0.6% MEN, 7.1% BR, 4.7% GER, p<0.001). The immigration route had an impact on the cardiovascular disease (CVD) risk when adjusted to, hypertension, sex, first degree relative with CVD, and age. The more extenuating the immigration route of the Mennonites, the higher is the CVD risk (OR 1.57, IC95 1.22–2.03, p=0.001). Forty-five variants of 35 genes were associated with a dominant effect for CVD (p<0.01) independently of sex, age, body mass index, and weekly physical activity. Between those, 38 are known to be associated with mRNA levels on heart/arteries (eQTLs in GTEX), 14 are missense mutations, one causes a frameshift and 18 destroy or create CpG sites with a possible epigenetic effect. The frequency of 14 out 16 alleles differed from non-Finish Europeans and Brazilians, indicating a possible founder effect. Eight are associated with CVD protection (p<0.001). These variants are related to less inflammation of atherosclerotic plaques.

Conclusion

The South-Brazilian Mennonite population seems to have some kind of protection for ischemic heart disease, which may rely on founder effects that raised the frequencies of protective alleles.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): CAPES for PROAP finance Code 001, and scholarships for WAVJ and LCO. ABWB receives a research productivity scholarship from CNPq (protocol 314288/2018-0).
Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells
Authors: Funakoshi S , Keller G K
Published: October 2022

Introduction

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Although human pluripotent stem cell (hPSC) represents a promising source to generate target cells in vitro, there has been no optimal protocols to generate mature compact cardiomyocytes derived from hPSCs.

Purpose

To establish the protocol to generate mature compact ventricular cardiomyocytes derived from hPSCs.

Methods

By mimicking the early ventricular development and postnatal maturation process observed in the heart in vivo, we searched for the specific factors to induce compact lineage and promote their maturation level.

Results

We established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from hPSCs. The treatment of Wnt signaling activator, CHIR, and IGF2 significantly increased the expression level of a compact marker, MYCN (p=0.006), and suppressed the trabecular markers, NPPA (p=0.0117) and BMP10 (p=0.0032). Those factors induced a proliferation of compact cardiomyocytes by two folds (p<0.0001). Additionally, we identified that the combination of PPARα agonist, dexamethasone, T3 hormone, and palmitate in the low glucose containing media following the compact specification period significantly induced the fatty acid transporter, CD36, in the compact cardiomyocytes (p<0.0001). The CD36-positive compact cardiomyocytes generated under these conditions displayed the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These mature compact cardiomyocytes showed metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts.

Conclusions

We successfully generated the mature compact cardiomyocytes derived from hPSCs. These findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Canadian Institutes of Health Research (CIHR)
High prevalence of deleterious variants in cardiomyopathy genes in patients with early onset atrial fibrillation
Authors: Vad O B , Ahlberg G , Paludan-Muller C , Refsgaard L , Sajadieh A , Haunsoe S , Bundgaard H , Svendsen J H , Olesen M S
Published: October 2022

Background

Atrial Fibrillation (AF) is a common cardiac arrhythmia associated with increased morbidity and mortality. AF has a significant heritable component and genome-wide association studies have associated numerous loci in the human genome with AF. The arrhythmia is relatively rare in younger individuals, but studies have shown that individuals with early-onset AF may harbour a considerable burden of pathogenic genetic variants.

In recent years, the concept of atrial cardiomyopathy has emerged as a mechanism involved in AF pathogenesis. Genes well-known to be related to ventricular structure, including cardiomyopathies have now also been associated with AF.

Purpose

Using targeted genetic sequencing, this study aimed to elucidate the role of deleterious genetic variants in cardiomyopathy genes in early-onset AF, and provide new insights into AF pathogenesis.

Methods

We performed targeted genetic sequencing of 445 Danish individuals with onset of AF before age 40 years and no other cardiovascular co-morbidities, and of 387 controls with no history of AF. Based on guidelines for genetic testing for clinical use, we focused on 30 genes with well-established associations with dilated cardiomyopathy, hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. We examined the prevalence of loss-of-function variants (defined as variants leading to premature stop-codon, frameshift or splice-site variants), as these are most likely to be disease-causing. We filtered for rare variants using a minor allele frequency <0.1%. The difference in prevalence in the two groups was analyzed using a logistic regression model.

Results

We found that 38 of the 445 early-onset AF patients carried loss-of-function variants in well-established cardiomyopathy genes. The prevalence of rare, loss-of-function variants was enriched in cases compared with controls (8.5%. vs. 1.0%, P=8.27x10–7). The variants were identified in eight different genes, with most rare variants found in the TTN gene (Table 1). In sensitivity analyses excluding TTN variants, we found that 12 individuals (∼2.7%) with AF harbored deleterious loss-of-function variants (P=0.0396).

Conclusions

Individuals with early onset of AF have a considerable burden of rare, deleterious variants in established cardiomyopathy genes. These new insights could help inform future recommendations for genetic testing and follow-up to detect early cardiomyopathy manifestations to prevent adverse outcomes in patients with early onset of AF. These findings support the presence of atrial cardiomyopathy.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): Hallas-Møller emerging investigator grant, The Novo Nordisk Foundation (NNF: NNF17OC0031204)The John and Birthe Meyer Foundation
Temporal trends and outcomes in ST-segment elevation myocardial infarction: a cardiac magnetic resonance imaging study over the course of 15 years
Authors: Lechner I , Reindl M , Tiller C , Holzknecht M , Fink P , Troger F , Mayr A , Klug G , Bauer A , Reinstadler S J , Metzler B
Published: October 2022

Aims

Development of evidence-based treatments in ST-elevation myocardial infarction (STEMI) patients during the last 30 years have been associated with improved outcome; however, there are data suggesting a plateauing since around 2008 (1). Moreover, contemporary data are very scarce regarding the temporal trends of infarct outcomes. This study sought to describe the temporal trends in infarct severity at myocardial tissue level over the course of 15 years by means of cardiac magnetic resonance imaging (MRI).

Methods

This study analyzed STEMI patients treated with percutaneous coronary intervention (PCI) in our Medical University who underwent a cardiac MRI between 2005 and 2021. The 15-year study period was divided into sequential 2-years blocks. Infarct characteristics were measured using MRI at 3 days [IQR 2–5] after PCI.

Results

A total of 844 STEMI patients (17% female) with a median age of 57 (interquartile range [IQR]: 51–66) years were included. The rate of evidence-based treatments was high for aspirin (99%), P2Y12i (99%), beta-blockers (91%), ACEi/ATi (92%) and statins (100%) and did not change significantly over the study period (p>0.05) with the exception for ACEi/ATi (p=0.03) and prasugrel (p<0.001), which increased and clopidogrel, which decreased during the study course (p<0.001). TIMI risk score did not change over the study period (p=0.43). Overall median infarct size was 16 [9–25]% and did not change (p=0.39) significantly. MVO, a marker of severe reperfusion injury, was also comparable (p=0.16). Accordingly, LV ejection fraction remained virtually unchanged (p=0.23)

Conclusion

Although further implementation of evidence-based treatments was seen also during the last 15 years, there has been no effect on infarct size, reperfusion injury and LV ejection fraction for patients who undergo primary PCI due to STEMI. Novel treatment strategies are needed to address this unmet therapeutic need.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund (FWF)Austrian Society of Cardiology
Homozygosity predominantly affects hypertrophic cardiomyopathy minor genes in an Egyptian clinical cohort
Authors: Allouba M , Walsh R , Afify A , Halawa S , Galal A , Hosny M , Fathy M , Theotokis P , Whiffin N , Anwar S , Elguindy A , Ware J , Barton P , Aguib Y , Yacoub M
Published: October 2022

Background

Consanguinity is prevalent in Egypt (35%) resulting in a high incidence of homozygosity. The influence of homozygosity on the genetics of Hypertrophic Cardiomyopathy (HCM) has not been adequately studied.

Purpose

The purpose of this study is to define the genetic architecture of HCM in Egypt using ethnically-matched case and control cohorts.

Methods

Prospective Egyptian patients (n=514) and controls (n=400) were recruited to Aswan Heart Centre for clinical phenotyping and genetic testing for 174 genes implicated in inherited cardiac conditions (Illumina). Rare variation (gnomAD filtering allele frequency ≤4x10–5) in 13 validated HCM genes were classified according to the American College of Medical Genetics (ACMG) guidelines and compared with a prospective HCM cohort of predominantly European ancestry (n=684).

Results

Significantly fewer rare variants detected in Egyptian patients could be classified as (likely) pathogenic compared to Europeans (40.8% vs. 61.6%, p-value=1.6x10–5). Incorporating analysis from these Egyptian case-control cohorts into the ACMG guidelines increased this yield to 53.8%. Homozygous variants were more frequently observed in Egyptian patients (4.1% vs 0.1%, p-value=2x10–7), with variants in the minor HCM genes MYL2, MYL3 and CSRP3 more likely to present in homozygosity than the major genes (MYH7, MYBPC3 and troponins), suggesting such variants are less penetrant in the heterozygous state.

Conclusions

The integration of Egyptian-specific genetic and phenotypic data significantly improves variant interpretation in HCM and consequently the precision of genetic testing. The observed prevalence of homozygosity and rare variation in minor HCM genes in Egyptian patients provides important insights into its disease-mechanisms and genetics.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Science and Technology Development FundAl Alfi Foundation
An emerging role for DPP6: reciprocal regulation of INa-Ito and implications for arrhythmogenesis
Authors: Rossetti A , Spatjens R L H , Kammerer S , Stoks J , Firneburg R , Seyen S R , Helderman-Van Den Enden A T J M , Wilde A A M , Loeys B L , Saenen J , Heijman J , Volders P G A
Published: October 2022

Background

Since the association of a chromosomal risk haplotype harboring dipeptidyl peptidase-like protein-6 (DPP6) to familial idiopathic ventricular fibrillation (iVF), a growing number of DPP6 missense variants has been reported in patients with ventricular tachyarrhythmias. The mechanisms underlying DPP6 mediated-arrhythmogenesis are not yet fully elucidated. DPP6 is a subunit of the transient outward potassium (Ito) channel complex in Purkinje cells (PC) and ventricular myocytes (VM).

Purpose

Since other Ito-channel subunits (Navβ1, KChIP2, KCNE4 and DPP10) are also known to antagonize INa, we examined whether DPP6 could play a broader role in the inter-regulation of Kv4.3 and Nav1.5 channels. We identified two novel DPP6 variants (p.Arg274His and p.His213Tyr), each segregating in families with QT/QU prolongation. DPP6 p.Arg274His carriers suffered from iVF, ectopic beats from the conduction system, and mitral valve prolapse. Other DPP6 variants (p.Ala751Val identified in this study; p.Gln526His and DPP6-T p.His332Arg published) are associated with Brugada syndrome (BrS). We hypothesized that DPP6 has opposing effects on INa and Ito displaying a reciprocal regulation of these currents.

Methods and results

First, we determined the effect of the DPP6 variants on INa and Ito in transfected CHO cells. Ito density was significantly reduced only when PC subunits were co-expressed with the DPP6 p.Arg274His or p.His213Tyr variants. Indeed, DPP6 modulates Nav1.5 channels in CHO cells by reducing INa Peak and INa Late, whereas DPP6 mutants p.Arg274His or p.His213Tyr resulted in an increase of both components compared to WT. Co-immunoprecipitation experiments in human endocardium confirmed an interaction between DPP6 and Nav1.5 channels. Computing of mutant DPP6-driven Ito-INa changes in a published human PC model led to significant prolongation of the action potential duration, mainly caused by increased INa Late.

On the other hand, the DPP6 p.Gln526His and p.Ala751Val variants, linked to BrS, led to a decreased INa Peak compared to the WT, while there was a tendency towards increased Ito density in both PC and VM molecular setups.

DPP6 (p.Arg274His and p.Ala751Val) transfection experiments in hiPSC cardiomyocytes, expressing endogenous INa and Ito, confirmed the reciprocal results obtained in CHO cells.

Conclusions

DPP6 regulates INa and Ito in a reciprocal manner. The cardiac phenotype of DPP6 variants could encompass a spectrum between two opposite poles: 1) QT/QU prolongation by DPP6 variants causing loss of Ito and gain of INa, like p.Arg274His and p.His213Tyr versus 2) BrS by DPP6 variants leading to gain of Ito and loss of INa, like p.Gln526His and p.Ala751Val.

Funding Acknowledgement

Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): ESC Personal research grant, obtained in 2019
Heterotypic Cx43/Cx45 gap junction channel conductance depends on pulse rate and could rescue from arrhythmogenic activity in cardiac tissue
Authors: Maciunas K , Snipas M , Kraujalis T , Kraujaliene L , Gudaitis L
Published: October 2022

Introduction

Gap junction (GJ) channels, formed of connexin (Cx) proteins, conduit electrical excitation in the heart. It is well established that GJ conductance (gj) depends on transjunctional voltage (Vj), but little is still known about its dynamics during the spread of cardiac excitation.

Transitional tissue of atrioventricular (AV) node expresses Cx43 and Cx45, which could form heterotypic GJs, which are highly sensitive to Vj.

Purpose

Evaluation of heterotypic gj dynamics of Cx43/Cx45 channels during the propagation of excitation in cardiac tissue.

Methods

The CE-4SM model of cardiac tissue, consisting of discrete cells connected through dynamic GJs, was developed by combining Fenton-Karma equations, which define cardiac excitability (CE), and a 4-state model (4SM) of GJ channel gating, which describes the dependence of gj on Vj and its kinetics.

Global optimization methods were applied for 4SM parameter evaluation to fit gj timecourses obtained from electrophysiological recordings.

Experiments were performed using dual-whole-cell patch clamp in Novikoff cells, endogenously expressing Cx43, and Hela cells transfected with Cx45.

Results

We developed a combined cardiac excitability model (CE-4SM), that allowed us to study an interaction between the spread of cardiac excitation and GJ channel voltage. Modeling results shows that during propagation of excitation, the phase difference between each action potential (AP) in adjacent cells, generates two subsequent Vj spikes. First spike causes small decrease of gj, while the second one, a minor increase. These impulses can accumulate into a large overall decay until the impulse rate-dependent steady-state is reached. This phenomenon is caused by variation of delays between APs and consequentially, different durations and amplitudes of Vj spikes developed across GJ channels. Interestingly, such gj decrease was capable of causing drift of spiral wave rotor or termination of fibrillation-like arrhythmia during our modeling experiments.

Also, we performed in-vitro experiments to evaluate 4SM model parameters and validate simulation results that showed gj decay at a high pulse rate, which could occur during reentry-like tachycardia.

Conclusions

Voltage gating of Cx43/Cx45 GJ channels could act as a control circuit, that lowers gj between the transitional cells of AV node at high impulse rate. Presumably, this could help to reduce the conduction or even act as a block of high-rate impulses passing through AV node. Also, such gj decrease could cause a drift of spiral waves and disruption of fibrillation-like processes.

Funding Acknowledgement

Type of funding sources: Public Institution(s). Main funding source(s): Lithuanian University of Health Sciences, Institute of Cardiology
Association of late-gadolinium enhancement in cardiac magnetic resonance with ventricular arrhythmias and mortality in patients with non-ischemic cardiomyopathy
Authors: Aslam F , Al-Sadawi M , Tao M , Aleem S , Almasry I , Singh A , Rashba E , Fan R
Published: October 2022

Background

Late-gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR) is a predictor of adverse events such as cardiovascular mortality, cardiovascular related hospitalization and defibrillation shocks in patients with non-ischemic cardiomyopathy (NICM). The correlation between LGE and ventricular arrhythmia and mortality has not been completely established.

Purpose

This meta-analysis assessed the relationship between LGE in CMR with ventricular arrhythmias: sustained, non-sustained and ICD therapy; and mortality in patients with NICM.

Methods

Databases were queried for studies reporting the association between LGE in CMR in NICM and ventricular arrhythmias and mortality, including Ovid MEDLINE, EMBASE, Web of Science, and Google Scholar. The search was not restricted to time or publication status. The minimal follow up duration was one year.

Results

A total of 46 studies and 10,548 patients (4,610 with LGE vs 5,938 without LGE) were included; mean follow up was 3 years (ranging between 13 to 71 months) and mean left ventricular ejection fraction 33%. LGE in NICM was associated with increased risk of ventricular arrhythmias and sudden cardiac death (odds ratio 4.595, 95% confidence interval 3.54–5.97; P<0.01) and mortality (odds ratio 2.949, 95% confidence interval 2.285–3.806; P<0.01). Heterogeneity is low to moderate: χ2=82.2, df =45 (P=0.001), I2=45% (Figures 1, 2).

Conclusions

Our results suggest that LGE is associated with increased risk of ventricular arrhythmias, sudden cardiac death and mortality in long-term follow up. These results further substantiate the need for larger prospective randomized trials using LGE to decide ICD indication regardless of EF. There are two ongoing trials testing this indication: CMR-ICD and CMR-GUIDE.

Funding Acknowledgement

Type of funding sources: None.
BNIP3 regulates cardiac energy metabolism by interaction with F1FO ATP-synthase
Authors: Beerlage A , Settelmeier S , Doepper H , Heinen-Weiler J , Giorgio V , Rassaf T , Hendgen-Cotta U B
Published: October 2022

Background

The oxidative phosphorylation (OXPHOS) that takes place in the mitochondria produces chemical energy in form of ATP, the main energy source of the heart. Proper mitochondrial function determines the contractility of the heart. Changes in myocardial ATP levels are often associated with cardiovascular disease. However, mitochondrial dysfunction remains an unmet therapeutic challenge. The F1FO ATP-synthase protein complex catalyses the last step of OXPHOS, synthesising ATP and determining the respiratory function of cardiac mitochondria. An increased ATP pool during cardiovascular challenges, regulated by F1FO ATP-synthase, could therefore significantly improve the clinical outcome of affected patients. In neurons, Bcl2-familiy members like BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) have been suggested to be involved in the regulation of metabolic efficiency through interaction with the mitochondrial F1FO ATP synthase. A similar metabolic regulation in the cardiac context remains unclear. Furthermore, BNIP3 has a significant impact on the loss of cardiomyocytes under various pathological conditions and is thus a potential mediator of energy metabolism in cardiomyocytes.

Methods and results

With native gel-electrophoresis, cardiac BNIP3 of C57BL/6J mice was first identified in higher oligomeric complexes in similar molecular weight range as mitochondrial F1FO ATP-synthase. Using 60Å-precise proximity-ligation assays to determine possible binding partners, F1FO ATP-synthase was uncovered for the first time as an interacting protein of BNIP3. This interaction was confirmed by co-immunoprecipitation of BNIP3 and F1FO ATP-synthase and by spatial localization using electron microscopy. Peptide microarray studies elucidate the f subunit of F1FO ATP-synthase as the structural interaction site. Functional analysis using extracellular flux analyser technology revealed significantly elevated mitochondrial respiratory activity in Bnip3 knock out mice and human cardiomyocytes during BNIP3 inhibition. Increased ATP levels in C57BL/6J mice after acute inhibition of BNIP3 confirmed the observed regulatory effect of BNIP3 on F1FO ATP-synthase activity. Finally, depletion of BNIP3 in mice improves stress resistance with an augmented chronotropic capacity during dobutamine-induced cardiac stress.

Conclusion

Depletion of BNIP3 suggests a regulatory role of BNIP3 in cardiac mitochondrial energy homeostasis via interaction with F1F0 ATP-synthase in oligomeric complexes. Since acute inhibition of BNIP3 activity allows positive modulation of cardiac performance by elevating the available ATP pool, this may serve as a beneficial treatment for patients with cardiovascular disease in the future.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German research foundation (DFG)
The regenerative response of cardiac interstitial cells
Authors: Jopling C
Published: October 2022

Understanding how certain animals are capable of regenerating their hearts will provide much needed insights into how this process can be induced in humans in order to reverse the damage caused by myocardial infarction. Currently, it is becoming increasingly evident that cardiac interstitial cells play crucial roles during cardiac regeneration. To understand how interstitial cells behave during this process, we performed single-cell RNA sequencing (scRNA-seq) of regenerating zebrafish hearts. Using a combination of immunohistochemistry, chemical inhibition and novel transgenic animals, we were able to investigate the role of cell type specific mechanisms during cardiac regeneration. This approach allowed us to identify a number of important regenerative processes within the interstitial cell populations. Here, we provide detailed insight into how interstitial cells behave during cardiac regeneration which will serve to increase our understanding of how this process could eventually be induced in humans.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence National pour la Recherche ANR-20-CE14-003 MetabOx-HeartAgence National pour la Recherche ANR-11-LABX-0015
Perivascular nerve fibres are increased in the epicardium while perivascular adipose tissue is decreased in the myocardium in patients with acute myocardial infarction
Authors: Fiet M D , Krijnen P A J , Niessen J W M
Published: October 2022

Background

Vasospasm is an important etiological factor in the induction of acute myocardial infarction (AMI). PeriVascular Nerve Fibres (PVNF) and PeriVascular Adipose Tissue (PVAT) play an important role in vasoregulation, although detailed information of their role in the heart is missing in AMI patients. In the present study we have analysed PVNF and PVAT in deceased AMI patients.

Methods

Sections of epicardial coronary arteries were obtained at autopsy from AMI patients (N=16) and controls without heart disease (N=7). Infarct age was between 3 and 6 hours old. Stenosis of epicardial coronary arteries was quantified in a HE stained slide. To analyse the intramyocardial vasculature samples were taken from the left ventricle of the heart (in AMI patients the infarcted area). Nerve fibres were stained using a specific S100 antibody. Putative activation of these nerves was stained using a specific antibody against neuropeptide Y (NPY). S100+ and NPY+ nerve fibres were quantified by light microscopy and were then calculated per square mm of the adventitia surface areas. PVAT was quantified using an Elastica Von Gieson (EvG) staining.

Results

Stenosis did not correlate with PVNF. AMI patients had a significant increase in PVNF in the epicardium but not in the myocardium. NPY was non-significantly increased in PVNF in the epicardium and myocardium in AMI patients compared with controls. PVAT was significantly decreased in AMI patients compared with controls.

Conclusion

The increase in PVNF in the epicardium and the decrease of PVAT in the myocardium suggest that they might play a role in AMI induction, independent of the atherosclerotic changed epicardial coronary arteries.

Funding Acknowledgement

Type of funding sources: None.
Cardiac magnetic resonance characterization and prediction of left ventricular thrombus after ST-segment elevation myocardial infarction
Authors: Bertolin-Boronat C , Marcos Garces V , Perez N , Gavara J , Lopez-Lereu M P , Monmeneu J V , Rios-Navarro C , De Dios E , Merenciano-Gonzalez H , Gabaldon-Perez A , Iraola Viana D , Bonanad C , Moratal D , Chorro F J , Bodi V
Published: October 2022

Background

Left ventricular thrombus (LVTh) is an uncommon yet serious complication after ST-segment elevation myocardial infarction (STEMI). Late gadolinium enhancement (LGE) sequences in cardiac magnetic resonance (CMR) imaging allows for accurate detection of LVTh. However, the implications of CMR to predict and characterize LVTh occurrence is this population is unclear.

Purpose

We aim to characterize the incidence, outcomes, and predictors of LVTh after STEMI by CMR imaging.

Methods

Our registry comprised 455 patients admitted for a first reperfused STEMI in our university hospital. Baseline characteristics were recorded. All patients underwent early (1-week) and late (6-month) CMR. Left ventricular ejection fraction (LVEF, %), infarct size (% of left ventricular mass) and microvascular obstruction (MVO, number of segments) were measured. LGE sequences were used to analyze the presence of LVTh. Patients with LVTh at 6-month CMR underwent an additional CMR 1 year after admission. Univariate and multivariate comparisons were performed to study the ocurrence of LVTh in the first 6 months after STEMI.

Results

Mean age was 58.24±11.69 years, most patients were male (82.6%) and anterior infarction occurred in more than half of the cohort (52.7%). LVTh was detected in 36 (7.9%) patients in the first 6 months after STEMI. Anticoagulation was initiated in all cases. Of these, 27 patients had LVTh at early (1-week) CMR, but 9 had LVTh at late (6-month) CMR with no prior evidence of LVTh at early CMR. A total of 6 patients had persisting LVTh at 1-year CMR (37.5% of patients with 6-month LVTh). In multivariable analysis, anterior infarction (HR 6.6 [1.91–22.83], p<0.001) and 1-week CMR-LVEF (HR 0.97 [0.93–0.99], p=0.04) and MVO (HR 1.19 [1.02–1.39], p=0.03) independently predicted the occurrence of LVTh in the first 6 months after STEMI. We computed a risk score of LVTh assigning 1 point to each of these variables (anterior infarction, CMR-LVEF <50% and MVO >3.5 segments), which allowed us to stratify the risk of LVTh in the first 6 months after STEMI (0.6% if 0 points, 3.8% if 1 point, 14.4% if 2 points, and 31.2% if 3 points).

Conclusions

CMR imaging soon after STEMI can contribute relevant prognostic value regarding LVTh occurrence after the acute event. Patients with anterior infarction, LVEF <50% and MVO in >3.5 segments at early (1-week) CMR have the highest risk of LVTh in the first 6 months after STEMI.

Funding Acknowledgement

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Instituto de Salud Carlos III and “Fondos Europeos de Desarrollo Regional FEDER” and Conselleria de Educaciόn – Generalitat Valenciana.
The cardioprotective effect of inhibiting SGLT1 in hyperglycemia ischemia reperfusion injury
Authors: Almalki A , Arjun S , Bell R , Yellon D
Published: October 2022

Background

Diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Hyperglycemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated.

Purpose

Investigate if SGLT1 is involved in a glucotoxicity injury during IRI and whether inhibiting SGLT1 with an SGLT1 inhibitor may reduce infarct size.

Method

RT-PCR and in-situ hybridization (RNAScope) combined with Immunofluorescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and isolated primary cardiomyocytes.

An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22mmol) on myocardium at reperfusion. Canagliflozin (CANA) a non-selective SGLT inhibitor (1μmoL/L to block the SGLT1 receptor and SGLT2 and 5nmol/L to block only the SGLT2 receptor) and Mizagliflozin a selective SGLT1 inhibitor (100nmol/L) was introduced following ischemia at two different glucose concentration concentrations at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining.

Results

We showed that SGLT1 is homogenously expressed throughout the myocardium and is particularly evident within the vasculature. we demonstrate that hyperglycemia at reperfusion is injurious to myocardium with an increase of myocardial infarction. Our data reveal that glucose exacerbation of injury appears to be mediated via SGLT1. We have also demonstrated that high-glucose mediated injury in the isolated, perfused heart model is abrogated through the administration of a clinically available mixed SGLT2/SGLT1 inhibitor, canagliflozin, at a dose that inhibits both SGLT2 and SGLT1, but by the SGLT2-selective concentration.

Conclusion

We have shown that SGLT1 is present in the myocardium. Hyperglycemia appears to augment myocardial infarction and inhibition of SGLT1 attenuates this incre

Funding Acknowledgement

Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): The government of saudi Arabia
Prognostic implications of unrecognized myocardial infarction before elective percutaneous coronary intervention
Authors: Nogami K , Hoshino M , Kanaji Y , Sugiyama T , Misawa T , Hada M , Yamaguchi M , Nagamine T , Teng Y , Ueno H , Matsuda K , Sayama K , Kakuta T
Published: October 2022

Background

A non-negligible proportion of myocardial infarction (MI) is not clinically recognized and unrecognized myocardial infarction (UMI) is associated with adverse outcomes.

Purpose

To determine the prevalence and prognostic significance of UMI by delayed-enhancement cardiac magnetic resonance (DE-CMR) before elective percutaneous coronary intervention (PCI).

Methods

In this prospective, single-center study, 236 patients with stable coronary artery disease undergoing elective and uncomplicated PCI were studied. All patients underwent DE-CMR before PCI. The prevalence of UMI was evaluated and the association of clinical and CMR-derived variables with primary MACE, defined as cardiovascular death, nonfatal MI, hospitalization for heart failure, unplanned late revascularization, and ischemic stroke was investigated.

Results

In the final analysis of 213 patients, 63 patients (29.6%) showed UMI. Target territory UMI was observed in 38 (17.8% of total, 60.3% of patients with UMI). UMI was significantly associated with sex, diabetes mellitus, left ventricular ejection fraction, SYNTAX score and fractional flow reserve in target vessels. During follow-up periods (median, 23 months), MACE was observed in 17 (27.0%) of patients with UMI, and 17 (11.3%) without (P=0.001). In a multivariable model, UMI (hazard ratio [HR] 2.18, 95% confidential interval, 1.10–4.33, P=0.001) remained as an independent predictor of MACE. Kaplan–Meier analysis indicated that the presence of UMI was significantly associated with higher incidence of MACE.

Conclusions

The prevalence of UMI in patients undergoing elective PCI was 29.6%. UMI was independently associated with an increased risk of MACE after successful PCI. Given the non-negligible prevalence and potential clinical significance of UMI, clinical studies comparing PCI and guideline directed medical therapy (GDMT) versus GDMT only strategy might have to take the presence of UMI into consideration.

Funding Acknowledgement

Type of funding sources: None.
Effect of sacubitril/valsartan and empagliflozin on systemic inflammation and scar remodeling in a swine model of myocardial infarction
Authors: Martinez-Falguera D , Monguio-Tortajada M , Ferrer Curriu G , Aranyo J , Teis A , Fadeuilhe E , Rodriguez-Leor O , Diaz-Guemez I , Courageux Y , Roura S , Bayes-Genis A , Bisbal F , Galvez-Monton C
Published: October 2022

Background

Angiotensin receptor neprilysin inhibitor (ARNI) and sodium glucose cotransporter 2 inhibitors (SGLT2i) lead to reduced cardiovascular death and hospitalization rates for heart failure, but mechanisms of action remain unclear.

Purpose

To evaluate the effect of ARNI and SLGT2i on cardiac function, systemic inflammation and scar remodeling in a porcine model of acute myocardial infarction (MI).

Methods

Thirty-three pigs (18 female, 15 male) with non-revascularized MI were randomized to receive treatment with beta-blocker (BB) only (BB group, n=8), empagliflozin + BB (SGLT2i group, n=8), sacubitril/valsartan + BB (ARNI group, n=9) or empagliflozin + sacubitril/valsartan + BB (SGLT2i + ARNI group, n=8) for 30 days post-MI. Cardiac function parameters were studied at 2- and 30-days post-MI by cardiac magnetic resonance imaging. The systemic immune response was analysed at baseline, 2-, 15- and 30-days post-MI to estimate 1) the absolute number of neutrophils and lymphocytes by FSC-A/SSC-A, 2) the circulating monocytes (CD172a+) and their phenotype according to CD73+, and CCR2+ expression. Animals were euthanized at 30 days, interstitial collagen and vascular density of the scar were quantified.

Results

Left ventricular ejection fraction (LVEF) and stoke volume (LVSV) improved in SGLT2i + ARNI group (P=0.02, P=0.002, respectively, 2 vs 30 days post-MI). Right ventricular ejection fraction (RVEF) and stroke volume (RVSV) improved in ARNI treatment arm (P=0.03; P=0.02, respectively, 2 vs 30 days post-MI). Significant increase in LV mass was only observed in BB group (P=0.02; 2 vs 30 days post-MI). SGLT2i and ARNI treatment prevented the acute increase in circulating leukocytes 2 days post-MI (P=0.0095; P=0.0091, respectively). Moreover, SGLT2i reduced the CCR2+ activated monocytes count at 15 days post-infarction (P=0.01). In contrast, the combined treatment increased the number of circulating monocytes (CD172a+) at 30 days (P=0.008). Reduced collagen I (P=0.04), and Col I/Col III ratio (P=0.02) was observed in ARNI group, and higher collagen III content (P=0.02) in SGLT2i group. Vascular density was significantly increased in SGLT2i + ARNI group (P=0.03).

Conclusions

SGLT2i modulates systemic inflammatory state. ARNI reduces myocardial fibrosis and improves right ventricular function. Combined treatment improves vascular density and left cardiac function in a post-MI porcine model.

Funding Acknowledgement

Type of funding sources: Other. Main funding source(s): Boehringer Ingelheim Spain, S.A, and Instituto de Salud Carlos III, CIBER Cardiovascular
Therapeutic effects of nebulization-based delivery of anti-miR-146a in experimental pulmonary arterial hypertension
Authors: Santos Peixoto Gomes J , Mendes-Ferreira P , Adao R , Maia-Rocha C , Vasconcelos I , Leite-Moreira A , Bras-Silva C
Published: October 2022

Pulmonary arterial hypertension (PAH), is a chronic disorder characterized by excessive pulmonary vascular remodelling, resulting in elevated pulmonary vascular resistance and right ventricle (RV) overload and failure. PAH remains incurable, and new therapeutic approaches are required. The microRNA-146a (miR-146a) promotes vascular smooth muscle cell proliferation and vascular neointimal hyperplasia, both important hallmarks of PAH.

This study aimed to investigate the role of miR-146a in the pathophysiology of PAH and in the progression to RV hypertrophy and failure.

Sprague Dawley rats received a subcutaneous injection of monocrotaline (MCT group) or vehicle (CTRL group) and, after fourteen days they were treated, by intratracheal nebulization, with an anti-miR-146a, forming four distinct groups: CTRL-treated; CTRL-untreated, MCT-treated, and MCT-untreated. Twenty-seven days after MCT injection, echocardiographic and invasive hemodynamic evaluations were performed in all animal groups. Also, wild-type (WT) and miR-146a knock-out (KO) (miR-146a−/−) mice were submitted to either 3 weeks of chronic hypoxia-induced PAH with weekly Sugen 5416 administration (SuHx).

Compared to MCT-untreated rats, the MCT-treated rats showed decreased RV hypertrophy, (as measured by the Fulton index),decreased RV end-diastolic diameter/left ventricle end-diastolic diameter (RVEDD/LVEDD) ratio, and decreased in RV diastolic pressures. KO-SuHx group, when compared to WT-SuHx group, showed decreased RV hypertrophy (as measured by the Fulton index), and decreased RV systolic pressures and dilation, as well as lower RV end end-diastolic diameter (RVEDDi) and right atria area (RAAi).

Our findings show that miR-146a pharmacological or genetic inhibition reduces RV remodelling and improves cardiac function. Thus, miR-146a represents a promising therapeutic target in PAH.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): This research was supported by the Portuguese Foundation for Science and Technology (FCT).
Association between plasma desmosine, a marker of elastin degradation, and total atherosclerotic burden measured by whole body magnetic resonance angiography
Authors: Ali K , Weir-Mccall J R , Khan F , Belch J , Houston J G , Mordi I R , Lang C C , Huang J T J , Choy A M
Published: October 2022

Background

Elastin degradation has been implicated in the pathophysiology of vulnerable atherosclerotic plaque. Desmosine is the cross-link component in the elastin molecule and is exclusively released from mature elastin breakdown, thus has the potential to be a physiologically relevant biomarker of atherosclerosis.

Objectives

The aim of the present study was: 1. To investigate whether patients with known cardiovascular disease (CVD) have higher elastin degradation as indicated by plasma desmosine (pDES); 2. to determine the relationship between pDES and total atherosclerotic burden.

Methods

We measured pDES by a validated stable isotope dilution LC-MS/MS method1 in a total of 146 subjects from the SUMMIT (SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools) study2. (62% male, mean age 64±8 (SD) years). This included 62 patients with prior clinical diagnosis of CVD (including coronary artery disease (CAD), cerebrovascular disease and/or lower extremity arterial disease (LEAD)), and 84 patients without a clinical diagnosis of CVD. Total atherosclerotic burden was measured by whole body magnetic resonance angiography (WB-MRA). The WB-MRA data was divided into 31 anatomical arterial segments with each segment scored according to degree of luminal narrowing: 0 = no stenosis, 1 = <50% stenosis, 2 = 51–70% stenosis, 3 = 71–99% stenosis, 4 = vessel occlusion. The segment scores were summed and from this, a standardized atheroma score (SAS) was calculated by summing each individual segment's stenosis score, and divided by the number of diagnostic segments before dividing by 4 which is the maximum potential score. Relationship between SAS and pDES levels was investigated using multiple linear regression models

Results

pDES levels were significantly greater in patients with established clinical CVD compared to patients with no CVD (CVD patients, 0.56 (0.45–0.67) ng/mL; patients with no CVD, 0.47 (0.41–0.54)ng/mL; p=0.001). Median SAS for the study population was 4 (2–10). Overall pDES levels showed a strong correlation with SAS (r=0.44, p≤0.001). Univariable analysis suggested a significant association between pDES and whole-body atheroma score as measured by SAS. (β 29.31, p<0.001). This association persisted in the multivariable regression model adjusting for traditional cardiovascular risk factors including age, gender, BMI, systolic blood pressure, diabetes, smoking status and LDL. (adjusted β 18.75, p=0.004)

Conclusion

pDES levels are elevated in patients with clinical CVD when compared to those with no clinical history of CVD. pDES is also strongly associated with global atheroma burden. Overall, these findings support the role of pDES as a potential biomarker for atherosclerosis.

Funding Acknowledgement

Type of funding sources: None.