bluuebunny/crossref_metadata_2025 huggingface dataset:
m6A modification regulates early human cardiomyocyte lineage specification
Authors: Spano G , De Majo F , Hegenbarth JC , Tiburcy M , Zimmermann WH , De Windt LJ
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 813716.

Background

RNA modifications affect gene expression through the regulation of RNA metabolism. N6-methyladenosine (m6A) is the most abundant post-transcriptional modification that occurs in RNAs. Its dynamic expression is regulated by the "writer complex" (methyltransferases) and "erasers" (demethylases) and affects numerous biological functions, including mammalian embryonic stem cell (ESC) fate specification. However, the role of m6A in human cardiomyocyte (CM) lineage specification remains unclear.

Purpose

In this study, we aim to investigate the impact of m6A modification on early human cardiomyocyte differentiation, following the dynamic expression of m6A modification of human induced pluripotent stem cells (hiPSC) into cardiomyocytes (hiPSC-CMs).

Methods

hiPSCs were differentiated into hiPSC-CMs by mesodermal induction, followed by inhibition of WNT-signaling.  We collected hiPSC derivates at different stages of the differentiation protocol: hiPSCs, hiPSC-derived cardiac mesoderm cells, hiPSC-derived cardiomyocyte progenitors (hiPSC-CPCs), and mature hiPSC-CMs. Protein levels of m6A key regulators were analyzed. To systematically profile the expression of m6A modification, we subjected hiPSC derivates to m6A immunoprecipitation combined with deep sequencing (MeRIP-seq) and RNA-seq. Enrichment analyses of gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were conducted to elucidate the biological significance of differentially expressed and methylated genes.

Results

m6A distribution analysis on hiPSC derivates revealed a conserved pattern on a transcriptome-wide level: methylation sites are mainly located nearby the stop coding regions. However, we observed upregulated levels of writer proteins during the transition of hiPSC-derived cardiac mesoderm cells into hiPSC-CPCs. Interestingly, the dynamic changes in writer protein levels toward hiPSC-CPC transition were accompanied by a higher number of significantly upregulated and hyper-methylated mRNA transcripts. GO and KEGG analyses indicated hyper-methylated upregulated transcripts are enriched in muscle cell differentiation, cardiac physiology and calcium and MAPK signaling pathways regulating heart contraction.

Conclusion

For the first time, our study provides evidence that m6A modification is a mediator of early human cardiomyocyte differentiation. The role of specific writer regulators and individual m6A transcripts will be further investigated.
Transcriptomic analysis and comparative characterization of rat H9C2, human AC16 and murine HL-1 cardiac cell lines
Authors: Gulyas-Onodi Z , Visnovitz T , Koncz A , Varadi B , Agg B , Kiss B , Makkos A , Nagy NR , Toth VE , Leszek P , Gorbe A , Giricz Z , Buzas EI , Ferdinandy P , Varga ZV
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union's Horizon 2020 research and innovation programme

National Research, Development and Innovation Fund of Hungary

Background

Cardiac cell lines and primary cell cultures are widely used to model various cardiovascular diseases in vitro. Despite the increasing number of publications using these models, limitations of these cell lines are still undetermined.

Purpose

The aim of our study was to compare the most commonly used cardiac cell lines to primary cultures and to mature cardiac tissues by transcriptomic analysis and morphological characterization.

Methods

H9C2 (rat), AC16 (human) and HL-1 (mouse) cardiac cell lines were differentiated towards a phenotype more resembling cardiomyocytes, by methods most widely used in the literature, and cells were harvested at stages of proliferation and differentiation. Whole left ventricular tissue, neonatal primary cardiac myocytes isolated from mice and rats, or human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were applied as references. Transcriptome analysis and immunocytochemical detection of cardiac structural proteins were performed on all cell models.

Results

RNA expression of cardiac markers (e.g. Tnnt2, Ryr2, Tnni3) was markedly lower in cell lines compared to primary cells or hiPSC-CM and adult tissue controls. Differentiation procedures induced a significant increase in cardiac- and decrease in embryonic markers in AC16 and H9C2 lines; however, the overall expression pattern of investigated genes in all cell lines showed significant differences in comparison to corresponding myocardium or primary cultures. Immunocytochemistry confirmed low expressions of structural protein alpha-actinin and troponin I in cell lines.

Conclusion

Expression patterns of cardiomyocyte markers and mRNA profile indicates low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless the differentiation protocol used. These limitations should be taken into account while choosing cells as in vitro platforms to model cardiomyocytes and cardiovascular diseases.
Endothelin-1 amplifies ventricular repolarization heterogeneities in chronic myocardial infarction pigs
Authors: Olivan-Viguera A , Perez-Martinez A , Pueyo E
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e Innovación (Spain) Diputación General de Aragón

Introduction

Endothelin-1 (ET-1) is a vasoconstrictor peptide secreted by endothelial cells and cardiac myocytes and fibroblasts. It is involved in oxidative stress, apoptosis regulation and ventricular remodeling processes associated with heart failure and ischemic cardiomyopathy, including myocardial hypertrophy, fibrosis and impaired conduction. ET-1 has been shown to influence cardiac electrophysiology by modulation of calcium and potassium currents and to contribute to arrhythmogenesis and sudden cardiac death.

Purpose

We aim to characterize the functional role of ET-1 in the electrophysiology of healed myocardial infarction (MI) by analysis of porcine ventricular slices as a highly representative model of ventricular tissue with preserved cellular cross-talks and architecture.

Methods

Domestic pigs (60–80 kg, n = 3) were infarcted by temporal occlusion of the left anterior descending coronary artery. 8-12 weeks after infarct induction, animals were cardioplegically arrested under deep anesthesia and sacrificed. All animal procedures conformed to the guidelines from Directive 2010/63/EU and were approved by local authorities.

350 µm-thick ventricular slices were produced from transmural tissue blocks of healed MI ventricles. Tissue blocks were taken from remote, adjacent and border zones of the infarct area. Slices were optically mapped within 8 hours after tissue collection to record transmembrane potential and intracellular calcium. Action Potential Duration (APD) and Calcium Transient Duration (CaTD) were measured at 80% repolarization for 0.5, 1 and 2 Hz pacing frequencies in the presence and absence of 100 nM ET-1.

The notation n/N is used to denote n tissue slices from N pigs.

Results

ET-1 prolonged the APD at all frequencies in remote zones, with mean prolongation percentages of 30.5%, 32%, 26.2% at 0.5, 1 and 2 Hz, respectively, n/N=7/3. However, only minor effects were observed in adjacent (mean APD prolongation of 3.3%, 4.9% and 10.7%, n/N=5/3) and border zones (7%, 4% and 3.6%, n/N=5/3).

ET-1 caused an increase in CaTD at 1 Hz in the three zones, with no significant regional differences in the amount of CaTD increase: mean prolongation of 14.1% (n/N=7/3) in the remote zone, 12.4 % (n/N=5/3) in the adjacent zone and 20.9% (n/N=4/3) in the border zone.

Conclusions

In chronic MI pigs, ET-1 induces strong APD and moderate CaTD prolongation of remote normal myocardium at low (0.5 Hz) to high (2 Hz) frequencies. The ET-1-induced effects on the AP of normal tissue, but not on CaT, are disrupted in the border zones of the infarct area and in its proximity. Our results point to ET-1 acting to enhance ventricular repolarization dispersion in chronic MI pigs, which might contribute to increased arrhythmia vulnerability.
Increased thermogenesis in hairless SHR is associated with enhanced myocardial connexin-43 signaling and suppression of pro-fibrotic signaling
Authors: Andelova K , Szeiffova Bacova B , Sykora M , Pavelka S , Pravenec M , Barancik M , Tribulova N
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 2/0158/19, 2/0002/20 and Slovak Research and Development Agency APVV 18-0548,19-0317

Background

We have previously shown that downregulation of myocardial gap junction channel protein, connexin-43 (Cx43), and up-regulation of extracellular matrix (ECM) proteins in spontaneously hypertensive rats (SHR) hearts promote occurrence of lethal arrhythmia. Cold acclimation has a potential for reducing cardiovascular risk but there is a gap of knowledge concern the heart response to adaptive thermogenesis.

Purpose

To explore myocardial Cx43 and ECM proteins in genetically hairless strain of SHR, that represents rewarding model of increased adaptive thermogenesis.

Methods

Adult hairless males and females SHRM strain that harbors mutated desmoglein-4 gene and their wild type SHR were housed at standard 22 °C (that is below thermo-neutrality for these animals) and 12:12 hour light: dark cycle. WKY control rats were used as reference normotensive strain. Heart samples of the left ventricular tissue from euthanatized rats were used for the proteomics of Cx43, protein kinases, which phosphorylates Cx43, and for ECM markers TGF-β, SMAD2/3, MMP-2, collagen-1. Immunofluoresence detection of Cx43 was performed to examine its myocardial topology.

Key Results

Cx43 protein and its phosphorylatetd status was reduced in males and females SHR vs WKY rat hearts. In contrast, adaptive thermogenesis resulted in increased both parameters in SHRM regardless the sex. Moreover, pathological topology of Cx43 attributed to its enhanced localization at lateral sides of the cardiomyocytes in SHR was attenuated in SHRM. In addition, there were gentle differences in protein levels of PKA, PKG, PKCε, MAPK, Akt between SHR and SHRM. Protein level of TGF-β and SMAD2/3 were increased in SHR vs WKY rat hearts but decreased in SHRM unlike collagen-1 that was not altered in SHRM vs SHR.

Conclusion

Findings revealed beneficial up-regulation of myocardial Cx43 along with down-regulation of extracellular matrix proteins in response of hairless SHR to increased thermogenesis.
Exercise-induced right ventricular alterations in a rodent model of athletes heart
Authors: Olah A , Bodi B , Sayour AA , Barta BA , Ruppert M , Bottlik O , Merkely B , Papp Z , Radovits T
Published: June 2022

Funding Acknowledgements

Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00837/21) to AO

National Research, Development and Innovation Office (NKFIH) of Hungary (K120277 and K135076 to BM)

Background

Intense sports activity leads to the adaptation of cardiac structure and function, the so-called athlete’s heart. Research over the last years has focused on exercise-induced adaptation of the right ventricle (RV), because the disproportionate load on the RV - when compared with left ventricle - might lead to pathological consequences, such as interstital fibrosis or chamber dilation.

Purpose

We aimed at investigating right ventricular alterations induced by regular aerobic exercise training in a rat model of athlete's heart.

Methods

Young, adult rats were divided into control (Co) and exercised (Ex) groups. Trained rats swam 200 min/day for 12 weeks. In vivo electrophysiological study and in vitro force measurements on isolated permeabilized cardiomyocytes were carried out to investigate electrical and functional alterations, respectively. Molecular biological (qRT-PCR, Western-blot) and histological investigations were applied to reveal underlying mechanisms.

Results

Exercise training was associated with increased RV cardiomyocyte width (12.5±0.1µm Co vs. 13.8±0.2µm Ex, p<0.05) and corresponding hyperphosphorylation of protein kinase B (Akt). RV myofilaments from exercised animals showed increased maximal force development and improved calcium sensitivity. Sarcomere protein investigations revealed marked overall and site-specific hypophosphorylation of troponin I.  We found prolonged QT interval and right ventricular effective refracter period (RVERP: 44.0±1.6ms Co vs. 52.8±2.1ms Ex, p<0.05) along with decreased gene expression of potassium channels. Picrosirius staining did not reveal fibrosis, that was underlied by unchanged protein expression of connective tissue growth factor (CTGF) and gene expression of profibrotic markers. Gene expression of apoptotic markers and fetal gene program did not differ between the groups.

Conclusions

According to our data, regular swim training induced RV hypertrophy, that was associated with functional improvement adn hypophosphorilation of troponin I. Prolonged repolarization without pathological alterations in RV myocardial tissue suggest physiological remodeling after balanced training.
FGF15/FGF19 is involved in the development of cardiac hypertrophy
Authors: Planavila A , Moron-Ros S , Villarroya F , Gavalda-Navarro A
Published: June 2022

Funding Acknowledgements

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

Introduction

FGF15 (and its human orthologue, FGF19) are members of the endocrine FGF family. They are produced by ileal enterocytes in response to bile acids. The liver is its main target tissue but recent studies indicate that FGF19 also regulates skeletal muscle mass and adipose tissue plasticity.

Objective

To determine the role of the enterokine FGF15/19 during the development of cardiac hypertrophy

Methods

Circulating FGF19 levels were assessed in patients with heart failure (HF) and myocardial infarction. The hypertrophic response was characterized in Fgf15-null mice subjected to a high-fat diet for 16 weeks or isoproterenol infusion with osmotic minipumps for 7 days. Effects of experimentally increased FGF15 and FGF19 levels in vivo were determined in mice using adenoviral (Ad-Fgf15) and adeno-associated vectors (AAV8-Fgf19) for 1-week or 3-weeks injection, respectively.

Results

In humans, we found that the FGF19 levels were significantly reduced in patients suffering from heart failure or myocardial infarction, reciprocally to FGF21 levels. We found that mice lacking Fgf15 do not develop cardiac hypertrophy in response to high-fat diet or isoproterenol induced cardiac hypertrophy. Specifically, we found that the ratio heart weight/tibia length (HW/TL), the quantification of cardiomyocyte area and the hypertrophy marker gene atrial natriuretic factor (Anf) -as measures of cardiac hypertrophy development- were reduced in Fgf15-null mice subjected to hypertrophy compared to their corresponding wild-type mice. By contrast, we found that experimental increases in FGF15 or FGF19 induced cardiac hypertrophy development in vivo, characterized by an increase in the HW/TL ratio, area of the cardiomyocytes and hypertrophy marker genes.

Conclusions

We stablish a cross-talk between the intestine and the myocardium through the enterokine FGF15/19 which seems a key factor required for cardiac hypertrophy development.
Dapagliflozin alleviates left ventricular hypertrophy and cardiac dysfunction in mice
Authors: Weber L , Pilz PM , Baumgartner N , Szabo PL , Arnold Z , Dostal C , Kiss A , Podesser BK
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Ludwig Boltzmann Institute for Cardiovascular Research

Introduction

Sodium glucose cotransporter 2 inhibitors (SGLT2i) are a class of oral antidiabetic drugs. Recent clinical trials demonstrated and proved the cardiovascular benefit of SGLT2i in patients suffering from ischemic heart disease. In addition, left ventricular hypertrophy (LVH) is associated with cardiovascular events and using SGLT2i alleviated LVH in diabetic patients. However, only few studies investigated the effect of SGLT2i on regression of LVH in absence of diabetes.

Aim of the study

This study aimed to investigate whether the SGLT2i Dapagliflozin (DAPA) could attenuate LVH and cardiac dysfunction in a mouse model of pressure overload-induced LVH.

Methods

Male C57BL/6J mice (body weight 20-25g) were used. LVH was induced surgically by transverse aortic constriction (TAC). DAPA (1 mg/kg bodyweight/day) was administered through drinking water. The animals were divided in four groups: Group 1 underwent TAC for eight weeks (n=8). Group 2 concomitantly received DAPA for eight weeks after TAC (n=5). Group 3 received DAPA for only two weeks (in week 7 and 8 after TAC, n=5) to clarify if DAPA treatment could alleviate LVH at a later timepoint. Group 4 served as a sham control group (no LVH, n=8). Cardiac function was assessed using transthoracic echocardiography and invasive LV hemodynamic measurements.

Results

TAC resulted in a significant reduction in LV ejection fraction (LVEF) and significant increase in heart weight to body weight ratio (HW/BW) compared to sham (p<0.001). In addition, TAC mice showed a significant increase of LV systolic pressure and end-diastolic pressure compared to sham (p<0.01). Both the LVEF and LV functional parameters were markedly improved in mice treated with DAPA for eight weeks (p<0.05). LV mass decreased compared to the untreated group. More importantly, DAPA treatment for only two weeks also improved LVEF and alleviated LVH compared to untreated TAC mice (p<0.05). Furthermore, we also found that mice with only two weeks of DAPA treatment showed a tendency to improve LV hemodynamics.

Conclusions

DAPA was cardioprotective in a mouse model of pressure overload-induced LVH in absence of diabetes. It improved LV contractile function and LVH. DAPA also alleviated LVH and induced LV regression. Our findings uncovered that the SGLT2i DAPA contributed to the regression of LVH and cardiac fibrosis. Thus, administration of SLGT2i may be a novel adjunct therapy to boost reverse remodeling e.g. in patients with elective cardiac surgery and hypertrophic cardiomyopathy.
EDEM2 regulation of cardiac lipid metabolism in heart failure with preserved ejection fraction
Authors: Raja R , Liu W
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Background

Heart failure with preserved ejection fraction (HFpEF) is the most predominant form of heart failure (HF) worldwide, one which confers substantial mortality and has no efficacious treatments. Lipid mishandling greatly contributes to the pathophysiology of HFpEF, nevertheless, the key mechanistic features regulating cardiac lipid overload remain poorly understood. EDEM2 plays a crucial role in the ER-associated degradation (ERAD) pathway. Recent studies show that ERAD-associated proteins are implicated in cellular lipid homeostasis, however, the role of EDEM2 is cardiac lipid metabolism remains poorly understood. Here, we investigate the mechanisms by which EDEM2 regulates cardiomyocyte lipid metabolism and cardiac dysfunction in HFpEF.

Methods

In vivo two-hit disease model of HFpEF, Adeno-associated virus serotype 9 (AAV9)-mediated cardiac-specific gene overexpression, tail-cuff blood pressure recordings, echocardiography, histology and qPCR.

Results

Under HFpEF inducing conditions, AAV9-Gfp injected mice (as controls) displayed an increased E/A ratio and IVRT both of which are indicative of diastolic dysfunction. Conversely, cardiac-specific EDEM2 overexpression prevented diastolic dysfunction in mice subjected to HFpEF conditions. Systolic function remained unchanged between groups. Interestingly, EDEM2 overexpression reduced myocardial neutral lipid accumulation and prevented severe oxidative stress and cardiac pathological remodeling. More importantly, although genes participating in fatty acid uptake and lipid droplets synthesis in the heart were comparable between AAV9-Gfp and AAV9-EDEM2 groups, those involved in lipolysis and fatty acid oxidation were upregulated in mice with cardiac-specific EDEM2 overexpression.

Conclusion

Cardiomyocyte-specific EDEM2 overexpression mitigates myocardial steatosis in a two-hit murine model of HFpEF, likely through enhancing fatty acid utilisation in the myocardium.
Evaluation of the interplay between NASH and HFpEF in varied murine age groups
Authors: Kucsera D , Sayour N V , Ruppert M , Radovits T , Fabian A , Kovacs A , Ferdinandy P , Varga ZV
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union's Horizon 2020 Research and Innovation Programme, grant agreement no. 739593

New National Excellence Program of the Ministry of Human Capacities (ÚNKP-21-3-II)

Introduction

Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, left ventricular hypertrophy, left atrial enlargement and increased serum levels of NT-pro-BNP. HFpEF accounts for 50% of heart failure cases, and typically develops in patients with metabolic comorbidities. Non-alcoholic fatty liver disease and subsequent steatohepatitis (NAFLD, NASH) is the most common chronic liver disease developing due to obesity. Although clinical/epidemiological data exists in humans showing that NASH may lead to cardiac dysfunction per se, experimental data in this regard is lacking.

Purpose

We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age-dependent effects of NASH on cardiac function.

Methods

Middle aged (10 months old) and aged (24 months old) C57Bl/6J mice were fed either control diet or Choline Deficient (CDAA) diet over a period of eight weeks. Young (2 months old) mice were used as a control. Before termination, echocardiography was performed. Upon termination, organs were isolated for further analysis.

Results

CDAA diet lead to the development of NASH in both age groups, without inducing weight gain, allowing us to investigate the direct effects of NASH on cardiac function. Left ventricular end-diastolic volume (EDV) was increased in aged animals, compared to young and middle aged animals, suggesting increased ventricular pressure. Aged animals were characterized by increased posterior wall thickness (PWT) during diastole and by increased LV mass, indicating left ventricular hypertrophy. Assessment of ejection fraction showed an age-dependent decline. Pulse wave and tissue Doppler measurements showed no difference in E/e’ ratio between the groups. However, strain analysis showed that diastolic dysfunction developed only in aged mice due to NASH.

Conclusion

We conclude that there were no observed changes in cardiac diastolic function due to NASH when using standard echocardiographic evaluation; however, the more sensitive method of strain analysis with 2D speckle tracking was able to show evidence of diastolic dysfunction due to NASH in aging animals.
Identification of novel N-terminal splice variants in the DCM-associated gene RBM20
Authors: Gaertner A , Brodehl A , Sielemann K , Knoll R , Gummert J , Milting H
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Erich und Hanna Klessmann Foundation

Introduction

Mutations in human RBM20, encoding the RNA binding protein 20, cause dilated cardiomyopathy (DCM). Within the nucleus RBM20 partially colocalies with other splicing factors. Sequencing of the cardiac transcriptome of a RBM20-deficient rat model revealed RBM20-dependent regulation of myocardial alternative splicing. We identified several carriers of RBM20 mutations.

In the literature, it is postulated that in myocardial tissue only one cardiac form of RBM20 with transcription start in exon 2 is expressed. However, it remains unclear why in the heart this alternative start codon is used. Furthermore, the function of putative different transcripts is unknown.

Methods

We analyzed human myocardial cDNA by 5’RACE to identify novel 5’-terminal RBM20 transcripts using gene specific primers within exon 2. Furthermore, we performed RNA-Seq analysis to detect different RBM20 transcripts directly in the human heart.

Results

By 5’RACE we could identify additional RBM20 exons localized within the first intron. The usage of two of these exons (internal exons, IE1 and IE2) instead of exon 1 results in transcripts with translation starting in the second exon. The usage of the third alternative internal exon (IE3) might result in a protein differing from full length and cardiac RBM20. Furthermore, we identified transcripts using an alternative exon, which is localized upstream of exon 1. In addition, a transcript comprising exon 1 and 2 of RBM20 was also detected by 5’ RACE. Using RNA-Seq we verified the expression of the transcripts containing exon 1, IE1 and IE2 in the human heart, respectively.

Conclusions

Here, we present first data on different N-terminal RBM20 transcripts in the human myocardium. In contrast to previous assumptions we identified different splicing forms of RBM20 with unknown functional impact in the human heart. As RBM20 is a DCM-associated gene, the function of the different RBM20 splicing variants might be interesting with respect to cardiomyopathies.
Extracellular stiffness as a determinant of cardiac dysfunction in duchenne muscular distrophy: a study on human iPSC derived cardiomyocytes
Authors: Giammarino L , Santini L , Palandri C , Musumeci M , Langione M , Pioner JM , Ferrantini C , Coppini R , Cerbai E , Poggesi C
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Fondazione Intesa San Paolo

Introduction

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive degeneration of striated muscles; in addition to skeletal muscle impairment, DMD is also characterized by progressive myocardial disfunction. The low translational value of animal models and the low availability of human samples make DMD hard to investigate; induced pluripotent stem cells (iPSCs) represent a novel tool to model this disease, preserving the genetic heritage of the patient, including the pathogenic mutation causing dystrophy.

Aim

Our aim is to characterize cardiomyocytes differentiated from iPSCs (iPSC-CMs) derived from healthy donors (CTRL) and DMD patients, to identify the pathophysiological mechanisms of DMD-related cardiomyopathy.

Materials and Methods

Cardiomyocytes are differentiated from IPSCs obtained by reprogramming isolated mononucleated blood cells from healthy donors and DMD patients. IPSC-CMs are cultured until day 60, 75 or 90 post-differentiation after plating on nanostructured substrates with two different stiffness levels: PEG-substrates, with lower rigidity, mimicking healthy extracellular tissue, and DEG-substrates, with greater rigidity, that mimic the presence of myocardial fibrosis. Through imaging techniques, we evaluated calcium handling and action potentials (AP) on DMD and CTRL iPSC-CMs by using specific fluorescent dyes for Ca2+ (CAL630) and membrane voltage (Fluovolt). Cells were stimulated at different pacing rates.

Results

The calcium transient amplitude of CTRL-iPSC-CMs became larger during maturation. This adaptation did not occur in DMD lines, showing a deficit calcium release due to poor maturation of the sarcoplasmic reticulum (SR). AP duration was shorter in the DMD line at d75 but at d90 we observed no differences when compared with the CTRL line. CTRL iPSC-CMs showed a marked ability to adapt to different substrate stiffnesses. Indeed, the calcium transient amplitude was larger and its kinetics faster when cells were grown on the rigid DEG substrates rather than on PEG plates. In the DMD line, however, no differences were observed between the substrates.

Conclusions

Our results highlight a scarce ability of DMD iPSC-CM to adapt to different substrate stiffness, resulting in mechanical and electrical impairment, especially in the presence of stiffer substrates. This might explain why cardiac impairment is usually absent in the early stages of DMD, when cardiac structural changes are still absent. However, the electrophysiological and mechanical impairment of DMD hearts may precipitate rapidly when extracellular stiffness starts to increase due to development of cardiac fibrosis.
Topographical Mapping of the cardiac autonomic innervation for selective cardiac neuromodulation in pigs and rabbits using MicroCT
Authors: Kronsteiner B , Zopf L , Heimel P , Oberoi G , Kramer A-M , Slezak P , Reissig L , Geyer S , Weninger WJ , Podesser BK , Kiss A , Moscato F
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Horizon 2020- EU H2020-EU.1.2.2. - FET Proactive

"NeuHeart" Nr. 824071

Background & Introduction

In recent years, Vagus Nerve Stimulation (VNS) has proved to be a potential therapeutic approach for the treatment of cardiovascular diseases, such as Heart Failure or atrial fibrillation [1]. However, the lack of specific anatomical knowledge of the cervical VN [2, 3] and thus, of the cardiac autonomic innervation aggravates the side effects of unselective cervical VNS.

Purpose

The goal of this study was to investigate the cardiac vagus nerve branches for selective cardiac VNS stimulation using micro-computed tomography (µCT) and 3D renderings.

Methods

Vagus nerve specimens (n= 11 pig nerves, n= 5 rabbit nerves) were harvested from the nodose ganglion down to the cardiac branches in domestic pigs and New Zealand White rabbits on both sides, and the cardiac autonomic innervation was mapped µCT and 3D renderings.

Results

Our results provide anatomical and topographical key features on the cervical and cardiac autonomic innervation including course of the cardiac branch, cardiac branching patterns, fascicle number, and size of the autonomic nerves. We also compared these aforementioned anatomical parameters between pigs and rabbits and highlighted key anatomical differences among individuals within pigs. In pigs, the cardiac branches were partly composed of both nerves even when they branched off the VN whereas in rabbits, the two nerves were completely separated and the cardiac branch was solely parasympathetic. Finally, we generated a 3D model of various parts of the VN specimen and compared them to images of the native nerves taken during VN dissection surgery.

Conclusions

Here we present an imaging approach to map the anatomy and topography of the cardiac Vagus Nerve for selective stimulation of cardiac VN branches. We also characterized the morphology of the VN, the sympathetic trunk (ST), and the cardiac branch (CB) at the level of the cardiac branching point to highlight the complex interplay between the nerves. Our data provide one possible reason for unwanted side effects of cervical VNS. However, future studies are required to broaden the knowledge in this specific research field of selective cardiac VNS.
MicroRNA biomarkers of platelet function
Authors: Gutmann C , Barwari T , Schulte C , Singh B , Theofilatos K , Joshi A , Zampetaki A , Chan M , Armstrong P , Kiechl S , Willeit J , Warner T , Mayr M
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): British Heart Foundation (BHF), VASCage (Centre for Promoting Vascular Health in the Ageing Community) of the Austrian Research Promotion Agency FFG (COMET program - Competence Centers for Excellent Technologies)

Introduction

Antiplatelet therapy (APT) leads to reduced morbidity and mortality in patients with cardiovascular disease but some still have thrombotic events. Tailoring APT to platelet function is currently limited by a lack of suitable platelet function tests. It has been previously shown that different circulating microRNAs (miRNAs) are derived from platelets and their measurement could provide new markers of platelet reactivity.

Purpose

To compare the release of different platelet miRNAs in response to different platelet agonists.

Methods

Measurements of platelet function were performed by light transmission aggregometry (LTA) in participants of the 2015 follow-up of the Bruneck study (n=338), using the following agonists: arachidonic acid (1mM), adenosine diphosphate (5µM, 20µM), collagen (0.4 µg/ml, 4 µg/ml, 10µg/ml), TRAP-6 amide (25µM) and U46619 (10µM). LTA platelet releasates were then used for RT-qPCR measurements of five platelet-enriched miRNAs (miR-21, miR-126, miR-150, miR-197, miR-223). Platelet-poor plasma (PPP) served as negative control.

Results

Platelet activation led to aggregation and extracellular release of miRNAs, with aspirin users (n=155) showing significantly lower miRNA release than non-aspirin users (n=183). Agonist responsiveness differed among miRNAs, with miR-21 being hyperresponsive to arachidonic acid and miR-150 being hyperresponsive to adenosine diphosphate, whilst release of miR-126, miR-197 and miR-223 was strongest to collagen (10µg/ml). In non-aspirin users, inflammation markers such as granulocyte counts or C-reactive protein correlated positively with platelet-derived miRNAs measured in PPP, whilst they correlated negatively with platelet-derived miRNAs measured in releasates. These effects were absent in aspirin users.

Conclusions

MiRNAs released from activated platelets can be reliably detected in PPP and platelet releasates. Preferential release of miRNAs in response to specific agonists suggests a selective release mechanism. Elevated PPP levels and decreased releasate levels of platelet-derived miRNAs in inflammatory environments suggest platelet exhaustion ex vivo due to platelet pre-activation.
Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium
Authors: Van Der Vorst E , Gencer S , Jansen Y , Bayasgalan S , Yan Y , Bianchini M , Cimen I , Mueller M , Peters L , Megens R , Duchene J , Lemnitzer P , Soehnlein O , Weber C , Doering Y
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft SFB1123

European Research Council AdG_692511

Background

Genome wide association studies revealed a strong association between the CXCL12 gene locus and cardiovascular diseases, highlighting its receptors CXCR4 and atypical chemokine receptor-3 (ACKR3) as targets for CVD research. Although vascular CXCR4 is well studied in atherosclerosis, the exact role of ACKR3 remains elusive. Therefore, the aim of this project is to decipher the role of vascular ACKR3 in atherosclerosis.

Methods

Vascular endothelium (Bmx-cre) specific Ackr3 deficient mice on Apoe-/- background were fed with a cholesterol-rich diet. Lesions in the aortic roots and arches were analyzed via H&E staining. The phenotype was further characterized employing ex-vivo perfusion and intravital microscopy for cell adhesion and Evans-blue injection for endothelial permeability. Signaling studies are performed in human coronary artery endothelial cells (HCAECs).

Results

Endothelial Ackr3 deficiency resulted in significantly smaller atherosclerotic lesions, accompanied by a more stable plaque phenotypes (more lesional collagen and smooth muscle cell content, smaller necrotic cores). Endothelial-Ackr3-/- mice revealed a reduction in plaque macrophage content and ICAM positive endothelial cells in atherosclerotic lesions. Moreover, immune cell adhesion on carotid arteries lacking endothelial ACKR3 was decreased whilst endothelial permeability was not affected. ACKR3 silencing in TNF-stimulated HCAECs decreased adhesion molecule expression and downregulated MAPK signaling and NF-kB p65 phosphorylation. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in Ackr3 deficient mice.

Conclusion

Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways resulting in increased adhesion molecule expression.
Smooth muscle cell-specific deletion of S100A4 reroutes smooth muscle cell fate and modifies the inflammatory status of murine atherosclerotic lesions
Authors: Cardoso Dos Santos LM , Azar P , Lemeille S , Bochaton-Piallat ML
Published: June 2022

Funding Acknowledgements

Type of funding sources: Other. Main funding source(s): Swiss National Science Foundation

Foundation Centre de Recherches Médicales Carlos and Elsie de Reuter

Background aims: S100A4, a small calcium binding protein, plays an important role in vascular smooth muscle cell (SMC) phenotypic switch but its implication in atherosclerotic plaque development and particularly in SMC phenotypic plasticity is not clear yet. By neutralizing S100A4 using a monoclonal antibody, we have previously shown a reduction in overall atherosclerotic burden. However, this strategy did not distinguish the different contribution between SMCs or other S100A4-expressing cells (e.g. macrophages).

Methods

Herein, we used a lineage tracing mouse model in which we induced a SMC specific deletion of S100A4 (SMC-S100A4Δ/Δ) in an ApoE-/- background. High cholesterol diet was maintained for 12 weeks, after which, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed and aortas processed for staining and single cell RNA sequencing (scRNA-seq).

Results

We showed that S100A4 deletion modulated plaque composition rather than plaque size. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt showed a reduction in macrophages and dendritic cells and an increase in total SMCs, likely participating in fibrous cap stabilization. SMC populations showed a decrease in macrophage-like inflammatory phenotype and an increase in fibroblast-like repair phenotype, as well as retention of specific SMC markers, namely Acta2 and Myh11. Gene expression analysis revealed a reduction in the expression of inflammation markers, fatty acid metabolism, and extracellular matrix-related genes, and an increase in classical SMC markers and maintenance of the contractile phenotype.

Conclusion

We report an intrinsic function of S100A4 in the establishment of SMC fate within the plaque and, surprisingly, a global impact on the atherosclerotic plaque microenvironnement and inflammation status.
SVEP1 is a new regulator of arterial tone
Authors: Morris GE , Denniff MJ , Kostogrys RB , Bountziouka V , Rainbow RD , Samani NJ , Webb TR
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Introduction

Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins can directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated Ca2+ channels (VGCCs). Integrin α9β1 has been identified as preventing exaggerated airway bronchiole contraction. Genetic variants in ITGA9, which encodes the α9 subunit of integrin α9β1, and SVEP1, a ligand for integrin α9β1, are associated with elevated blood pressure, however, neither SVEP1 nor integrin α9β1 have a reported role in vasoregulation.

Purpose

To determine whether SVEP1 and integrin α9β1 regulate blood vessel contraction.

Methods & Results

Animal experimentation was performed according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and "the Principles of laboratory animal care". Immunocytochemical staining showed both ligand and receptor co-localised within the medial layer of the aorta, and in smooth muscle cells in the mesenteric artery. siRNA inhibition of SVEP1 or integrin α9β1 significantly enhanced real-time [Ca2+]i release in isolated human VSMCs to several Gαq/11-vasoconstrictors and to UTP in VSMCs isolated from Svep1+/- mice (Fig. 1A, n=5, P<0.0001). This enhanced cellular contraction was confirmed in blood vessels by wire myography where aortic rings and mesenteric arteries (Fig. 1B, n=10 P<0.0001) from Svep1+/- mice contracted at significantly higher levels than littermate controls. Similar responses were seen in aortic rings when integrin α9β1 was inhibited using the small molecule inhibitor BOP (n=10 P<0.001). Inhibition of VGCCs using nifidepine, or PKC using bisindolylmaleimide (I) prevented this enhanced contraction, suggesting this effect is mediated via VGCCs in a PKC dependent mechanism.

Conclusions

Our studies reveal a novel role for SVEP1 and integrin α9β1 in reducing vascular hyper-contractility in response to a range of vasoconstrictor agonists through an L-type voltage gated Ca2+ channel-mediated effect. This regulatory mechanism could suggest a possible explanation for the genetic associations with blood pressure, and provide a new treatment strategy for hypertension.
Characterization of the source(s) of lipocalin-2 mediating dietary obesity-induced hypertension
Authors: Konja D , Li H , Fu B , Singh S , Wang Y
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): This work was supported by the grants from the General Research Funds (17117017 and 17121714) and Collaborative Research Funds (C7037-17W) of Research Grant Council, the Areas of Excellence Scheme (AoE/M-707/18) of University Grants Committee

Title: Characterization of the source(s) of lipocalin-2 mediating dietary obesity-induced hypertension.

Introduction

Obesity upregulates lipocalin-2 (Lcn2), a pro-inflammatory adipokine, which in turn induces vascular and metabolic abnormalities. In mice, deletion of the Lcn2 alleles has protective effects against obesity-induced vascular and metabolic dysfunctions.

Purpose

The present study investigated the sources of lipocalin-2 production as a mediator of dietary obesity-associated perivascular adipose tissue dysfunction and hypertension.

Methods

The wild type (WT) littermates or mice with whole body knockout (LKO), adipose tissue (Adn-Cre)-, kidney (Wt1-Cre)-, liver (Alb-Cre)-, and granuloid cells (Lys-Cre)-selective deletion of the Lcn2 alleles were implanted with radio-telemetry transmitters at eight-weeks of age. Blood pressure was recorded at least four 12/12 light-dark cycles every four weeks for mice fed either standard chow (STC) or high fat diet (HFD). Wire myography was performed to evaluate the functional properties of mesenteric arteries, in the presence or absence of surrounding perivascular adipose tissues. Quantitative PCR, Western blotting as well as enzyme-linked immunosorbent assay (ELISA) were also performed to measure Lcn2 expression in tissues and blood respectively.

Results

Compared to STC, HFD feeding increased systolic, diastolic, mean arterial and pulse pressure in WT mice. The amount of Lcn2 expressed in perivascular adipose tissues and present in blood were also higher in HFD-fed WT mice when compared to those fed with STC. Whole body deletion of Lcn2 alleles attenuated HFD- induced increase in blood pressure. Liver-selective deletion of Lcn2 alleles abolished the effect of HFD feeding on blood pressure, decreased Lcn2 expression in perivascular adipose tissues, and the concentration of Lcn2 in blood. Both whole body- and liver-specific deletion of Lcn2 alleles enhanced the anti-contractile activity of perivascular adipose tissues and abolished HFD-induced adipose tissue dysfunction. However, deletion of Lcn2 alleles selectively in adipose tissues, but not Wt1-Cre and Lys-Cre, partially mitigated the effects of HFD on perivascular adipose tissue function and blood pressure regulation.

Conclusion

Lcn2 derived from different tissues are distinctively implicated in dietary obesity-induced perivascular adipose tissue dysfunction and hypertension.
Adipose mesenchymal stem cells from different fat depots in obese and lean subjects have different microRNA signatures involved in their differentiation into endothelial cells
Authors: Civit-Urgell A , Badimon L , Arderiu G
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III - Fondo de Investigaciones Sanitarias (FIS) P20/01517 to GA and Spanish Ministry of Science and Innovation PID 2019-107160RB-I00 to LB. All grants were co-financed by European Union Funds, Fondo Europeo de Desarrollo Regional (FEDER) "Una manera de hacer Europa".

Background/Introduction: Cell-based regenerative medicine has emerged as the forefront therapeutic procedure to improve ischemic tissue recovery. This approach is based on the usage of multilineage stem cells that can contribute to tissue regeneration. Revascularization of ischemic tissues is one of the objectives of cell therapy1,2. In this regard, we have investigated adipose-derived stem cells (ASCs), multipotent adult mesenchymal stems cells, as a cell source for therapy because of their capability to differentiate into endothelial cells (ECs)3–5. The regulatory mechanisms that drive the differentiation to EC are under investigation.

Purpose

Our hypothesis is that microRNAs (miRNAs) are involved in ASC to EC differentiation by a defined genetic signature in each fat tissue depot. The understanding of these mechanisms may be used to promote neovascularization in ischemic tissue.

Methods

Human ASCs from white adipose tissue (WAT) were obtained by surgical procedures from lean and obese patients. Importantly, subcutaneous-WAT and visceral-WAT were obtained from the same obese patient. ASCs from fat depots of lean and obese subjects where induced to differentiate into endothelial cells. The expression of 2025 pre-miRNAs and 2578 mature miRNAs was assessed using miRNA arrays in four biological replicates.

Results

Differentially up-regulated and down-regulated miRNAs were obtained from the respective fat depots. Seven of the most differentially expressed miRNAs were validated by qRT-PCR. The miRNA targets involved in the regulation of angiogenesis were selected by bioinformatic algorithms. We have identified 4 miRNAs that promote and stimulate ASCs differentiation into cells with endothelial cell-like phenotype through the regulation of FGFR1, NRP2, MAPK1, TGFB2, ETS1 and PTPN9.

Conclusion

In summary, the regulation of ASCs to EC differentiation (endotheliogenesis) involves a distinctive miRNA signature relying on WAT depot.
Successful treatment of patent ductus arteriosus accompanying right-sided aortic arch and aberrant left subclavian artery: a case report
Authors: Pay Levent , Calik Ali Nazmi , Akyuz Sukru , Dayi Sennur Unal
Published: June 2022

Background

The coexistence of a right-sided aortic arch (RAA), an aberrant left subclavian artery (ALSA), and a patent ductus arteriosus (PDA) is a rarely seen vascular ring anomaly. There is currently no general guideline consensus on the management and follow-up of this congenital defect, posing a challenge to the clinicians. At this point, the heart team plays a critical role in the management of the disease.

Case summary

In the present case, a 25-year-old male patient was presented to the outpatient clinic with dyspnoea and fatigue. A transthoracic echocardiography revealed PDA with a left-to-right shunt. To evaluate the anatomy thoroughly, a thoracic computed tomographic angiography was performed and showed PDA accompanying ALSA and RAA. The patient was evaluated by the Heart Team, and a percutaneous closure of PDA was recommended due to signs of left ventricular volume overload. The closure was successfully performed with Amplatzer vascular plug II. At follow-up, the patient was free of symptoms.

Discussion

Clinicians should be aware of the potential concomitant lesions during the diagnostic work-up. In selected patients, percutaneous closure of PDA may be the first-line therapy in experienced centres.