bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional regulation of macrophage inflammation in the heart failure with preserved ejection fraction
Authors: Gorica E , Mohammed S A , Mengozzi A , Mongelli A , Telesca M , Wenzl F A , Ruschitzka F , Hamdani N , Costantino S , Paneni F
Published: May 2024

Funding Acknowledgements

None.

Introduction

Heart failure with preserved ejection fraction (HFpEF) is a global public health problem with no effective treatment available. Pro-inflammatory cardiac macrophages are emerging as key determinants of adverse left ventricular remodeling; however, their role in HFpEF remains poorly understood. Mechanistically, the recruitment and activation of macrophages represent a key event in maladaptive myocardial remodeling in HFpEF patients. Evidence suggests that remodeling processes in HFpEF hearts are orchestrated and amplified by cardiac macrophages which regulate cardiomyocyte function, endothelial cell activation and fibroblast differentiation. Here we sought to determine the role of macrophage inflammation in experimental and human HFpEF.

Methods

Experiments were performed in rat cardiomyocytes (H9c2), human endothelial cells (HAECS), transgenic mice, and left ventricular (LV) myocardial samples from patients with HFpEF. H9c2 and HAECS treated with pro-inflammatory macrophage-like cells (RAW 264.7) conditional media in the presence or knockdown of a top-ranking activator of M1 type macrophages such as nuclear receptor corepressor 1 (NCOR1), were used to elucidate better the involvement of this gene in transcriptional regulation in vitro. The cardiac function of myeloid cell-specific NCOR1 knockout HFpEF mice undergoing a high-fat diet and L-NAME was investigated. LV myocardial samples were used to evaluate the pro-inflammatory activity of macrophages in human HFpEF tissues.

Results

Human and murine LV specimens showed upregulation of vascular adhesion molecules (ICAM1, ICAM2, E-selectin), recruitment of M1 macrophages to the myocardium, and decreased M2 macrophages. Notably, NCOR1 deletion in macrophages protected against diastolic dysfunction, exercise intolerance, and myocardial inflammation (TNF-a, IL6, IL-1b). Mechanistically, induction of metabolic stress by palmitic acid induced macrophage polarization, upregulation of NCOR1 and inflammatory genes in RAW cells. By contrast, Finally, NCOR1 depletion prevented pro-inflammatory transcriptional programs and secretion of TNF-a, IL6 and IL-1b in the macrophage secretome.

Conclusion

Targeting macrophage NCOR1 could represent a promising approach to blunt myocardial inflammation in HFpEF.
Study of the cardiac sodium channel in a mouse model of Scn5a haploinsufficiency towards a novel approach of therapy
Authors: Ferrand M , Mougenot N , Laporte P L , Leclerc A , Algalarrondo V , Extramiana F , Charpentier F , Neyroud N
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Sorbonne Université doctoral scolarship

INSERM

Introduction

Brugada syndrome (BrS) is an inherited cardiac arrhythmia that greatly increases the risk of sudden death as a result of ventricular fibrillation. Approximately 20% of BrS cases can be attributed to a mutation in SCN5A, the gene encoding the alpha-subunit Nav1.5 of the cardiac sodium-channel, which carries the sodium current INa. These mutations typically lead to loss-of-function of Nav1.5, resulting in a decrease in INa and most of them (»70 %) are responsible for haploinsufficiency. In the Scn5a+/- mouse model, a sodium-channel dysfunction phenotype is observed, characterized by conduction abnormalities (PR and QRS interval prolongation), a 50% reduction in INa, and an increased susceptibility to arrhythmias.

Aim

The aim of this study was to assess the impact of the overexpression of a Nav1.5-peptide, which has been found to enhance INa, in Scn5a+/- mice, as a novel approach of therapy.

Methods

We used AAV9-systemic injection to overexpress the Nav1.5-peptide into the heart of Scn5a+/- mice to gauge their susceptibility to arrhythmias induced by intracardiac pacing. We also examined the Nav1.5-peptide effects on endogenous Nav1.5 expression through molecular biology and biochemistry analyses.

Results

The duration of PR and QRS intervals in the hearts of Scn5a+/- mice was restored to normal by overexpression of the Nav1.5-peptide (PR interval = 46.3 ± 0.9 ms, n=22 in Scn5a+/- mice vs 40.4 ± 0.8 ms, n= 23 in WT mice, P<0.0001 and 42.6 ± 0.9 ms, n=11 in Scn5a+/- +Nav1.5-peptide, P<0.05; QRS interval = 15.2 ± 0.4, n=22 ms in Scn5a+/- mice vs 13.2 ± 0.4 ms, n=23 in WT mice, P<0.01 and 13.6 ± 0.4 ms, n=14 in Scn5a+/- +Nav1.5-peptide, P<0.05). Additionally, overexpression of the Nav1.5-peptide seemed to protect Scn5a+/- mice from induced arrhythmic events caused by programmed-ventricular stimulation (total premature beats = 1.3 ± 0.4 in Scn5a+/- mice, n = 13 vs 0.2 ± 0.2, n = 6 in injected Scn5a+/- mice, NS and 0.2 ± 0.2, n = 12 in WT mice, P< 0.05). There were no significant differences in total Nav1.5 expression between injected and non-injected mice as shown by RT-qPCR and Western-blots, but the expression of Nav1.5 at the plasma membrane appeared to be restored by overexpression of the Nav1.5-peptide (Nav1.5/N-cadherin ratio = 0.50 ± 0.08, n = 11 in Scn5a+/- mice vs 1 ± 0.15, n= 11 in WT mice, P< 0.05 and 0.94 ± 0.13, n = 11 in injected Scn5a+/- mice, P< 0.05).

Conclusion

Gene therapy with Nav1.5-peptide can attenuate both conduction defects and increased arrhythmia inducibility observed in Scn5a+/- mice. Although additional studies will be needed to confirm and explore further our results, this could be promising to treat patients with « sodium-channel dysfunction » such as BrS.
In vivo calcium and voltage mapping of the zebrafish heart
Authors: Schepers D , Sieliwonczyck E , Vandendriessche B , Claes C , Bastianen J , Pintelon I , De Vos W , Snyders D , Alaerts M , Huisken J , Loeys B
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Research Foundation Flanders (FWO)

Purpose

Inherited cardiac arrhythmias are characterized by a disturbance in the normal electrical functioning of the heart due to a dysfunction of cardiac ion channels, accessory proteins and/or structural components. This complex process is difficult to model in vitro, while in vivo mouse models differ substantially from humans with regards to cardiac electrophysiology. Thus, there is a need for alternative disease models in cardiac arrhythmia research. The zebrafish heart shows remarkable similarity to humans in its electrocardiogram and action potential (AP) morphology. Due to the optical translucency of zebrafish larvae, it is possible visualize the cardiac calcium and voltage dynamics across their entire hearts in vivo by use of genetically encoded calcium and voltage indicators (GECI and GEVI). We combined the fast kinetics of a next generation voltage reporter (Ace2N-mNeon) with a spectrally compatible calcium sensor (R-GECO) to create a sensitive high throughput method for in vivo characterization of the cardiac AP.

Methods

Ace2N-mNeon and R-GECO were expressed under control of a myocardial specific promoter via the Tol2kit system. The effect of sensor expression on normal physiology was assessed by patch clamp experiments. Fluorescence recordings were obtained with a Leica SP8 DLS microscope and a custom-built light sheet microscope. Our zebrafish sensor line was validated by experiments with quinidine drug exposure, as well as a novel model for long QT syndrome (LQTS) with a knock-in (KI) mutation in the cacna1c gene.

Results

Sensor expression did not significantly alter cardiac electrophysiology. The tg(myl7:Ace2N-mNeon/R-GECO) line demonstrated alterations in the heart rate, depolarization and repolarization induced by quinidine exposure. We observed a significantly prolonged ventricular repolarization in the LQTS KI line. Three-dimensional optical maps of AP characteristics across the entire zebrafish heart exhibited the physiological characteristics of the zebrafish cardiac electrophysiology, with activation initiated at the atrial venous pole and propagated towards the ventricular outflow tract, prolonged repolarization in the ventricles compared to the atria and slowed depolarization in the atrio-ventricular canal.

Conclusion

By expressing Ace2N-mNeon and R-GECO in the zebrafish heart, we were able to accurately detect drug- and mutation induced alterations in the cardiac AP, as well as AP characteristics across the entire heart, without compromising normal cardiac electrophysiology. Our transgenic line will provide a promising assay for future studies of cardiac arrhythmia.
Impaired autophagy mechanisms associated with phospholamban-R14del disease
Authors: Vafiadaki E , Kranias E G , Eliopoulos A G , Sanoudou D
Published: May 2024

Funding Acknowledgements 

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

Phospholamban (PLN) is a key modulator of sarcoplasmic reticulum Ca2+ homeostasis and cardiac contractility. The PLN variant c.40_42delAGA (p.R14del) leads to arrhythmogenic cardiomyopathy characterized by life-threatening ventricular arrhythmias and sudden cardiac death. A large number of PLN-R14del carriers have been identified worldwide, although the expanding use of genetic testing and increased awareness is unveiling a higher frequency. PLN-containing aggregates are emerging as a hallmark of this disease, and are increasingly believed to be associated with disease pathogenesis. However, the mechanism leading to the formation of aggregates is unknown.

We performed an in depth molecular and cellular analysis utilizing in vitro systems to determine if these aggregates result from aberrations in autophagy, and the underlying mechanisms. Utilizing cell culture systems we studied the expression level and subcellular distribution of fundamental autophagy-related proteins and evaluated the impact of autophagy inducers and inhibitors.

Expression of PLN-R14del in different cell culture systems (HEK293 and H9c2 cells) was found to result in PLN aggregate formation, which were determined to contain the autophagy marker proteins sequestosome-1 (p62) and microtubule-associated protein light chain 3 (LC3), indicating inefficient degradation by the autophagy pathway. In support of this, PLN-R14del expressing cells were shown to exhibit increased p62 and LC3-II protein levels. This was associated with significantly reduced autophagic flux, as demonstrated by examination of LC3-II protein levels after autophagy inhibition. Moreover, by detailed immunofluorescence analysis, we determined reduced co-localization of the autophagosomal protein LC3 and the lysosomal associated membrane protein 1 (LAMP1) in PLN-R14del cells, suggesting impaired autophagosome-lysosome fusion. We next examined the distribution of key proteins mediating autophagosome-lysosome membrane fusion including the Ras-related protein Rab7, the ultraviolet radiation resistance-associated gene (UVRAG) and membrane tether proteins. In PLN-R14del transfected cells, significant alterations in their co-localization with LC3 were observed, indicating their impaired recruitment to the autophagosomes. Importantly, in PLN-WT cells changes in cytosolic Ca2+ levels by thapsigargin treatment were found to recapitulate the autophagic defects of PLN-R14del, suggesting that Ca2+ alterations are a contributing factor of PLN-R14del mediated aberrant autophagy.

In conclusion, our findings reveal a causal relationship between PLN-R14del and autophagy dysregulation, through inhibition of autophagosome-lysosome fusion and autophagic flux reduction. This ultimately leads to aggregate formation, an early pathological sign in human patients.
The structural remodeling of the mitochondrial Ca2+ uniporter complex underlies its functional impairment during metabolic cardiomyopathy
Authors: Panel M , Fossier L , Lacote M , Luong-Azria L , Meli A C , Sicard P , Thireau J , Lacampagne A , Fauconnier J
Published: May 2024

Funding Acknowledgements

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

Introduction

Calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter complex (MCUC) controls key steps of oxidative metabolism to adapt energy production to cardiac demand. To ensure this critical function, MCUC assembly and maturation is a tightly regulated process involving the AAA proteases. We recently demonstrated that mitochondrial Ca2+ uptake is impaired during the early stages of metabolic cardiomyopathy in the atria.

Purpose

Our aim was to investigate mitochondrial Ca2+ handling in the left ventricle during the early stages of metabolic cardiomyopathy and to characterize the mechanisms underlying the functional remodeling of the MCUC.

Methods

Male C57Bl6/J mice were fed a high-fat sucrose diet (HFS) for two weeks to induce metabolic cardiomyopathy. Mitochondrial Ca2+ uptake was then studied (1) in isolated ventricular cardiomyocytes using whole-cell patch-clamp coupled with confocal microscopy and (2) in isolated mitochondria using Ca2+ Green-5N. The biophysical properties of MCUC were recorded in a planar lipid bilayer. MCUC structure and AAA-proteases expression were assessed by Western blot and co-immunoprecipitation. Control mice were treated with AAV9 harboring shRNA to downregulate the expression of MAIP1.

Results

Metabolic cardiomyopathy was associated with reduced beat-to-beat mitochondrial Ca2+ uptake in ventricular cardiomyocytes, which was further confirmed in isolated mitochondria. Single-channel recordings of the native cardiac MCUC embedded in a planar lipid bilayer revealed a reduction in ionic conductance and open probability, demonstrating functional impairment of the MCUC. Biochemical analysis of the MCUC revealed an accumulation of the unprocessed form of the EMRE subunit and a decreased interaction of the EMRE-MICU1 subcomplex with the pore-forming MCU subunits, indicating a structural remodeling of the MCUC. The expression of the AAA proteases YME1L and AFG3L2/SPG7, which are responsible for EMRE maturation and insertion into the MCUC, was unchanged. However, the expression of m-AAA protease-interacting protein 1 (MAIP1), which orchestrates EMRE processing, was reduced. In control mice, shRNA-mediated downregulation of MAIP1 recapitulated the structural and functional changes of the MCUC.

Conclusions

The structural remodeling of the MCUC integrity by downregulation of its assembly affects mitochondrial Ca2+ homeostasis in the early stages of metabolic cardiomyopathy. MAIP1 is a key regulator of MCUC assembly and its downregulation underlies the functional remodeling of the MCUC. These findings provide a novel post-translational paradigm of MCUC regulation in pathophysiology and suggest novel therapeutic targets for the treatment of the metabolic cardiomyopathy.
Characterization of a new arrhythmogenic cardiomyopathy mouse model due to filamin c haploinsufficiency
Authors: Canas R , Serrano R , Rubio B , Ochoa J P , De La Pompa J L , Garcia-Pavia P , Lara-Pezzi E
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Spanish Ministry of Universities FPU fellowship (FPU19/04044), Spanish Ministry of Science and Innovation Digital Transition call (TED2021-129774B-C22), EU HORIZON EIC Pathfinder Challenges 2022 Cardiogenomics call (Project 101115416).

Background

Filamin C (FLNC) is widely expressed in heart and skeletal muscle. It participates in cell junction and sarcomere assembly and function. We have previously shown that heterozygous FLNC truncating variants (FLNCtv) cause arrhythmogenic/dilated cardiomyopathy (ACM/DCM) in patients. Diverse studies have suggested haploinsufficiency as the potential mechanism of this disease. However, animal models of FLNCtv have only been reported to develop ACM/DCM in homozygosity.

Purpose

To develop a new model of ACM/DCM caused by a FLNCtv and study the progression of the disease in heterozygosity.

Methods

Two gRNAs flanking exon 15 of the Flnc gene and SpCas9 protein were microinjected into mouse zygotes to generate a mouse line with a constitutive deletion of Flnc exon 15 (FlncWt/Ex15del). mRNA and protein stability were determined by qRT-PCR and western blot, respectively. Cardiac function was analysed by ECG and echocardiography every 2 months, starting at 8 weeks of age (Fig. 1B). The mean ECG values were calculated using 3 time intervals and standard ECG waves.

Results

FlncWt/Ex15del mice showed a reduction in Flnc mRNA and protein similar to that observed in a human ACM/DCM patient bearing a similar mutation, suggesting that this FLNCtv causes haploinsufficiency. Both male and female FlncWt/Ex15del mice developed cardiac electrophysiological defects, including reduced amplitude of the QRS complex and the P-wave, notched R and/or S waves, and increased QT interval times, and these defects worsened with time. At 40 weeks of age, FlncWt/Ex15del mice showed some initial signs of left ventricular dilatation.

Conclusions

Mice carrying FLNCtv in heterozygosity develop electrophysiological changes resembling pathological features of ACM/DCM patients due to haploinsufficiency. The development of this pathological phenotype in heterozygosity, as opposed to previous models, paves the way for the development of new gene therapies using this model.
Zebrafish dspb -/- tert +/- mutant as model for arrhythmogenic cardiomyopathy. Impact of aging in the development of the disease
Authors: Wagih Gomez J , Munteanu S , Nicolas Rocamora E , Gil Ortuno C , Bernabe Garcia A , Cayuela Fuentes M L , Gimeno Blanes J R , Sabater Molina M
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III

Background/Objectives

Zebrafish has been reported as model to study the Arrythmogenic Cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic Cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. On the other hand, tert gene enables telomerase activity and telomere maintenance and mutations in this gene have been used for studying aging effects. After multiple crossings of our dspb model with tert mutants we have obtained dspb-/-tert+/- model line.

Purpose

Our aim was the characterization of dspb-/-tert+/- mutants, studying the impact of aging in the development of the disease.

Methods

The model was obtained by crossing dspb-/- and tert-/- mutants, crossing the heterozygous mutants and genotyping for the selection of the dspb-/-tert+/- mutants. The characterization of the phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) young adult (8 months old) and old adult (3 years old) individuals.

Results

Viability assays showed that dspb-/-tert+/- mutants suffer a premature death, with much lower survival rates than the dspb-/- group (survival dspb-/- 61.3% vs dspb-/-tert+/- 35.6%, p<0.01). Larvae heart rate analysis showed no significant differences. Echocardiography in young adults showed no significant differences between dspb-/-tert+/- and dspb-/- mutants. In old adults, echocardiography showed differences in heart rate (dspb-/- 142.5 ± 15.5 vs dspb-/-tert+/ -97.5 ± 3.5, p<0.01), respiratory frequency (dspb-/- 288.7 ± 36.1 vs dspb-/-tert+/- 95 ± 49.4, p<0.01) and atrial contraction velocity (dspb-/-155.7 ± 26.09 vs dspb-/-tert+/ -227.2 ± 17.6, p<0.05). Gene expression analysis in larvae showed that dspb expression drastically decreased (dspb-/- 0.3 ± 0.1 vs dspb-/-tert+/ -0.01 ± 0.008, p<0.01). In adults, dspb expression drastically decreased in heart tissue (dspb-/- 0.4 ± 0.01 vs dspb-/-tert+/- 0.06 ± 0.004, p<0.01) and skin (dspb-/ -0.34 ± 0.02 vs dspb-/-tert+/- 0.25 ± 0.01, p<0.01).

Conclusion

The dspb-/-tert+/- zebrafish presented worse survival rates, lower dspb gene expression levels and worse pathological cardiac phenotype at adult stages than dspb-/- mutants, proving that age is an important factor in the development of the disease and proving that dspb-/-tert+/- zebrafish is an appropriate model to study the impact of aging.
From genes to phenotype: investigating the link between smad2rs29725537:c>a and high take off in mice
Authors: Soto-Navarrete M T , Pozo-Vilumbrales B , Lopez-Unzu M A , Fernandez M C , Hernandez-Martin A , Duran A C , Fernandez B
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía, Ministerio de Ciencia e Innovación, Gobierno de España.

Introduction and Purpose

High take off (HTO) is a congenital anomaly with a prevalence of 0.36% that can cause severe ischemia and sudden cardiac death. The C57Bl/6 (B6) mouse strain shows around 60% incidence of HTO, whereas it is absent in the Balb/c (BC) strain. The aim of this study was to identify the genes responsible for the development of HTO.

Methods

Following an inheritance study, a genetic linkage study was performed in B6C mice using 134 microsatellites (MIT). After selection of Smad2 as a candidate gene, a genotype-phenotype study was performed by PCR-RFLP genotyping of 293 B6C mice. The process of coronariogenesis was examined in E13.5 and E14.5 mouse embryos of B6 (n = 27) and BC (n = 23) strains with specific markers for the vascular plexuses involved in coronariogenesis (PROX1 and ERG 1/2/3) and for SMAD2. Finally, the SMAD2 and pSMAD2 protein expression levels were analyzed by western blot in ventricle, liver, and kidney of B6C mice.

Results

The incidence of HTO in B6, BC and B6C mice were concordant with an inheritance pattern mediated by a dominant causal allele and dominant modifier in the BC strain that reduces the incidence of HTO. The genetic linkage study revealed the association of a region between markers MIT-49 and 106 on chromosome 18 with the presence of HTO. This region contained a total of 10 genes, of which Smad2 was selected as a candidate gene because of its functions in aortic root formation. Comparative sequence analysis of Smad2 between the B6 and BC mouse strains uncovered an intronic SNP (Smad2rs29725537:C>A or Smad2C>A) that predicts and error in the splicing of intron 10-11. A statistically significant association (p<0.05) between the Smad2C>A allele and the presence of HTO was found. pSMAD2 was detected in embryonic coronary primordia, the subepicardial aortic plexus (SAP) and the primary coronary plexus. Around 60% of B6 examined embryos showed an exacerbated SAP compared to the BC embryos. Additionally, we detected a differential expression of total SMAD2, but not of pSMAD2, in different organs according to the Smad2C>A allele genotype (C/C > A/C > A/A).

Conclusions

We conclude that: 1) Smad2C>A allelic variant is associated with HTO in mice, suggesting it as a major causal factor. 2) Smad2 altered expression may affect the establishment of the embryonic coronary primordia leading to a mispositioned ostium. 3) the Smad2C>A allele has a systemic effect on total SMAD2 expression without affecting its phosphorylation level, which suggest a compensatory mechanism of expression that maintain the normal activity of pSMAD2 in most tissues.
Titin mechanical unloading disrupts sarcomere tensional homeostasis triggering fast, non-canonical myocardial fibrosis
Authors: Lopez-Unzu M , Pricolo M R , Silva-Rojas R , Vicente N , Gavilan-Herrera M , Velazquez-Carreras D , Morales-Lopez C , Herrero-Galan E , Alegre-Cebollada J
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): We acknowledge funding from Ministerio de Ciencia e Innovación (Madrid, Spain) through grant FJC2021 047055-I and European Research Council (ERC) through grant H2020-ERC-2020-COG (ProtMechanics-Live).

Background/Introduction

Stresses exerted during myocardial contraction and relaxation cycles are mainly braced by mechanical proteins located in the extracellular matrix (ECM) and cardiomyocytes (CMs). Titin (TTN) is a gigantic protein that scaffolds the sarcomere (i.e. the basic unit of contraction). Since most of the CM cytoskeleton is occupied by sarcomeres, titin is an essential structural hub for the maintenance of a homeostatic level of mechanical tension.

Purpose

It has been shown that mechanical disruption of the ECM affects the equilibrium forces between CMs and the ECM, leading to morphological changes in sarcomeres. However, potential alteration of tensional homeostasis resulting from defective mechanics of CM proteins remains unknown due to the lack of tools for in vivo studies. Therefore, the aim of this study is to explore the in vivo response of the myocardium to the abrupt cessation of the mechanical properties of titin.

Methods

For this purpose, we have experimentally challenged CMs to a tensional unloading by means of a titin cleavage model (TEVs-TTN) [1], both in vitro, using neonatal CM (NMCM), and in vivo. In this model, a cassette with a tobacco etch virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (myoAAV), we were able to cleave titin in living CMs, ceasing only its mechanical properties. Samples were analyzed using an array of techniques such as histology, immunofluorescence and bulk and single-nucleus RNAseq.

Results

Our results show that while in NMCM we obtain a complete titin cleavage, in vivo we observe mosaic expression of TEVp and no more than 30% cleavage of the protein. In both cases, TEVp expression in TEVs-TTN does not affect cell viability. However, mild myocardial titin cleavage leads to a fibrotic response in less than six days, characterized by an increase in interstitial collagen and an expansion of cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this fibrotic response occurs without activation of the canonical TGFβ pathway.

Conclusion

Taken together, our results suggest that the loss of titin mechanical-structural function in cardiomyocytes derived from the cleavage of a single peptide bond elicits a fast fibrotic compensatory response in the myocardium through intercellular communication with fibroblasts.
Enhancing cellular regeneration through targeted pharmacological reprogramming of senescent endothelial cells
Authors: Kalies K , Knoepp K , Wurmbrand L , Dutzmann J , Sedding D
Published: May 2024

Funding Acknowledgements

None.

Background

Senescent endothelial cells (EC) are key players in the pathophysiology of cardiovascular diseases and characterized by a reduced angiogenic and regenerative potential. Therefore, reversing EC senescence represents a promising therapeutic strategy to restore vascular integrity and increase health and lifespan.

Purpose

Here, we show a reversal of EC senescence following the application of a pharmacological, non-genetic, partial reprogramming strategy to induce a timely restricted induction of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM).

Methods

Methods to characterize the effects of pharmacological reprogramming included the quantification of gene expression as well as functional analysis of EC in vitro. In addition, the regenerative capacity of EC was evaluated in a hind-limb ischemia model in vivo in young and old C57BL/6 mice.

Results

The application of a pharmacological reprogramming cocktail led to a strong but timely restricted activation of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc in replicative senescent ECs. This activation occurred on both mRNA and protein levels (p<0.0001), concurrently significantly reducing markers of cellular aging as p16ink4a, p14arf, TNFα, IL-1b, IL-6 and CD44 (p<0.01 respectively p<0.0001), while enhancing their cellular function. Pharmacological reprogramming notably augmented the migratory (p<0.001) and proliferative capacities (p<0.01) of the replicate senescence EC and positively influenced their angiogenic functions such as sprouting (p<0.0001) and tube formation (p<0.01). Telomere length was stabilized (p<0.05) and production of reactive oxygen species decreased (p<0.05). Non-senescent EC were not influenced by the pharmacological treatment (p>0.05). Over prolonged periods, the treated ECs maintained consistently low expression of aging markers (p16ink4a and p14arf, p<0.01) while preserving their migratory capabilities (p<0.05). In vivo, a significantly improved blood flow was observed after hind limb ischemia in 22 months old C57BL/6 mice after 7 and 14 days (p<0.001), alongside a significant increase in capillary density in the gastrocnemius muscle quantified by CD31 staining (p<0.05). In 3 months old C57BL/6 mice blood flow was not influenced by the treatment (p>0.05).

Conclusion

In conclusion, we demonstrate that a short induction of OSKM via a pharmacological approach holds the potential to reverse cellular senescence in EC in vitro with improved regenerative and angiogenic capacities and thus to enhance endothelial regenerative capacity in vivo. This might present a promising strategy for ischemic diseases.
The role of FGL2 in vascular smooth muscle cell dysfunction in coronary artery bypass vein graft failure
Authors: Jackson M , Bond A , Ascione R , Johnson J , George S J
Published: May 2024

Funding Acknowledgements

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

Rationale

The sudden exposure of venous endothelial cells (vECs) to arterial fluid shear stress (FSS) is thought to be a major contributor to coronary artery bypass vein graft failure (VGF). However, the acute effects of arterial FSS on the vEC secretome and the corresponding effects on underlying vascular smooth muscle cells (VSMCs) remains poorly characterised.

Methods & Results

Human umbilical vein endothelial cells (HUVECs; n=6) pre-conditioned to venous FSS (18h; 1.5 dynes/cm2) were further exposed to venous or arterial FSS (24h; 15 dynes/cm2) using an Ibidi pump system. Analysis of the EC secretome using Tandem Mass Tagging proteomics detected significantly increased fibroleukin (FGL2) in conditioned media from ECs exposed to arterial FSS compared to venous FSS (fold-change 1.97±0.72; P=0.0078). In addition, RT-qPCR analysis of FGL2 production showed a significant increase in vECs exposed to arterial FSS (2.11±0.27 vs. 0.05±0.04 log(copy number/mg); P=0.0312). Subsequently, pooled human saphenous vein VSMCs (3 donors per pool) were treated with conditioned media from ECs exposed to arterial FSS (n=6) which showed a significant increase in apoptosis, measured by active cleaved caspase-3 (CC3) immunocytochemistry (27±1% vs 22±1%; P=0.0391), and a significant decrease in the contractile markers smoothelin and desmin quantified by Western blotting (fold-change 0.76±0.06 and 0.44±0.11; P=0.004 and P=0.0421, respectively), in comparison to VSMCs treated with conditioned media from ECs exposed to venous FSS. Treatment of VSMCs (n=6) with recombinant FGL2 (20 ng/mL) for 72h likewise resulted in increased apoptosis (27±4% vs. 16±3%; P=0.0169) and decreased desmin and smoothelin (fold-change 0.68±0.03 and 0.61±0.07; P=0.0322 and P=0.0479, respectively).

Conclusions

The exposure of vECs to arterial FSS results in increased production and release of FGL2 which induced VSMC apoptosis and de-differentiation and therefore potentially contributes to VGF. Consequently, targeting FGL2 production or release may provide a therapeutic target to improve vein graft patency.
Metabolic plasma signatures in thoracic aortic aneurysm associated to tricuspid and bicuspid aortic valves
Authors: Martin Blazquez A , Martin Lorenzo M , Donado A , Heredero A , Diaz Navarro C , Mena Garcia P , Anfaiha Sanchez M , Santiago Hernandez A , Aldamiz Echevarria G , Alvarez Llamas G
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III with co-funding from the ERDF and Comunidad de Madrid

Background/Introduction

Thoracic aortic aneurysm (TAA) develops asymptomatic with life-threatening consequences and its diagnosis is usually fortuitous. TAA is mostly idiopathic, although its prevalence is significantly higher in subjects with bicuspid aortic valve (BAV) for unknown reasons. We hypothesize that it is essential to investigate idiopathic TAA by differentiating between BAV and tricuspid aortic valve (TAV) patients for personalized diagnosis and treatment.

Purpose

We aimed to identify plasma metabolic profiles of aortic dilatation in idiopathic TAA, differentiating between subjects with BAV or TAV.

Methods

Plasma samples from 41 idiopathic TAA patients and 57 subjects without aortic dilatation (control, C) were collected and classified according to aortic valve type (BAV or TAV). Full metabolomics coverage was carried out by 1H RMN and LC-MS/MS untargeted analysis, including reverse phase and hydrophilic interaction chromatography and positive and negative electrospray ionization modes. Significant variation in metabolite levels were considered if FDR-adjusted p-value <0.05.

Results

Our analyses highlighted a metabolic signature specific for BAV and TAV subjects. BAV patients presented a marked glycerophospholipid metabolism alteration involving different lysophospholipids (LPAs, LPCs and LPEs) and phospholipids (PCs, PEs, PAs and PIs). Particularly, LPCs and LPEs were increased in BAV-TAA vs BAV-C, being an indicator of stimulation of glucose utilization through glycolysis and suppression of fatty acid biosynthesis. In contrast, carnitine-related metabolism was the most altered pathway observed in TAV subjects. In particular, we detected an increment in plasma acylcarnitines in TAV-TAA compared to TAV-C suggesting an altered β-oxidation of fatty acids and mitochondrial overload.

Conclusions

Idiopathic TAA development is differentially reflected in plasma from BAV and TAV patients, supporting an individualized diagnosis valve-dependent and suggesting also differential therapeutic targets.
Effect and safety of PCSK9 inhibitor in-hospital use on blood lipid level in the ACS patients: a systematic review and meta-analysis
Authors: Yu Y , Li D , Ye Y , Tuo X , Chen Y
Published: May 2024

Funding Acknowledgements

Type of funding sources: None. Main funding source(s): none

Aims

To evaluate the effect and safety of in-hospital use of the proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitor on blood lipid levels in the acute coronary syndrome (ACS) patients.

Methods

We searched the PubMed, MEDLINE, Web of Science, Ovid and Cochrane Library electronic databases through July 2023 to identify randomized controlled trials (RCTs) and cohort studies using PCSK9 inhibitors during ACS hospitalization. Then we compared the PCSK9 inhibitor group with a placebo or a blank control group with the same background of lipid-lowering therapy. Two reviewers independently screened the literature, extracted data, and evaluated the risk of bias for the included studies. A software was used to analyze the changes in overall lipids from baseline and the adverse event rates.

Results

A total of 15 studies enrolling 3891 patients with ACS were eligible, including 10 RCTs (n = 1406) and 5 cohort studies (n = 2485). Treatment with PCSK9 inhibitors showed a greater reduction in low-density lipoprotein cholesterol (LDLC) (SMD=1.46, 95% CI: 1.16-1.76, P<0.01). The cumulative meta-analysis of follow-up time showed that the degree of LDLC reduction stabilized after 12 weeks (SMD=1.42 at 12 weeks, 95% CI: 1.06-1.79, P<0.01), and thus, PCSK9 inhibitors dramatically decreased LDLC levels in comparison to controls at both ≤12 weeks and 12–72 weeks. The LDLC levels with PCSK9 inhibitors that reached the recommended standards of AHA/ACC (≤1.8mmol/l) and ESC (≤1.4mmol/l) were 95% and 80% respectively, which were significantly higher than those of the control group (58% and 22%). PCSK9 inhibitors can also reduce the total cholesterol, triglycerides, apolipoprotein B, and lipoprotein (a), while increasing the high-density lipoprotein cholesterol, but they have no statistical significance in reducing apolipoprotein A1. Compared with placebo or blank control, the addition of PCSK9 inhibitor did not increase the occurrence of adverse events (RR =-0.01,95% CI:-0.19-0.18, P = 0.95) and serious adverse events (RR =-0.03, 95% CI:-0.48-0.43, P=0.91).

Conclusion

In-hospital use of PCSK9 inhibitors can significantly improve the blood lipid levels of ACS patients and increase the target LDLC attainment rate, with satisfactory safety and good tolerance.
FABP4 as potential indicator for GDM-induced fetal endothelial dysfunction
Authors: Villar Ballesteros M L , Brendel H , Carstens P , Effenberger D , Mittag J , Giebe S , Taylor P D , Morawietz H
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG project no. 288034826—International Research Training Group (IRTG) 2251

Background

Gestational diabetes mellitus (GDM) is linked to an elevated risk of developing cardiovascular diseases (CVD) both for the mother and the baby in later life. Previous studies have identified fatty acid-binding protein 4 (FABP4) as a prime candidate involved in the pathophysiology of GDM. FABP4 is known to promote an inflammatory response and vascular permeability, induce oxidative stress in endothelial cells (ECs), and impair the nitric oxide signaling pathway, thereby promoting endothelial dysfunction. Indeed, women with GDM exhibit elevated blood levels of FABP4, suggesting its potential as a biomarker for endothelial dysfunction and cardiovascular risk assessment in GDM. However, the role that FABP4 plays in the fetal endothelial function remains poorly understood.

Purpose

Thus, our aim is to understand the role of FABP4 in fetal endothelial dysfunction and its regulation to mitigate the impact of GDM on the vasculature.

Methods

Fetoplacental vessels and primary human umbilical vein endothelial cells (HUVECs) were isolated from placentas and umbilical cords obtained from both normoglycemic (NG) and GDM pregnancies. We also obtained the clinical data from the mothers and collected the cord blood. Total mRNA from HUVECs was used for bulk RNA sequencing to compare gene expression, while gene expression analysis in the vessels was conducted using RT-qPCR. Additionally, NG HUVECs were pooled and cultured under high laminar flow conditions (30 dyn/cm²) or subjected to various treatments, including insulin (5nM), glucose (25mM), simvastatin (10nm), or VEGF-A stimulation. Subsequent RT-qPCRs were performed to analyze the gene expression of several genes of interest.

Results

The bulk RNA sequencing data analysis revealed FABP4 as an overexpressed gene in GDM HUVECs compared to NG. Furthermore, the analysis of data through Gene Set Enrichment Analysis provided a list of different gene sets that are enriched in GDM samples. Among these gene sets are pathways or processes implicated in endothelial dysfunction, such as angiogenesis, PI3K/AKT/mTOR signaling, reactive oxygen species pathway, or inflammatory response. In vitro experiments provided evidence for an elevated FABP4 mRNA expression after VEGF treatment of HUVECs. Conversely, both simvastatin treatment and exposure to laminar flow reduced FABP4 mRNA expression and increased eNOS expression, suggesting a potential inhibitory effect of nitric oxide on FABP4.

Conclusion

We provided evidence that FABP4 could be involved in fetal GDM-induced endothelial dysfunction. Shear stress and statins might protect the endothelium against the negative impact of FABP4.
The role of natural anti-oxidants in gut microbiota alteration subsequent to high fat diet (HFD)
Authors: Mollace V , Mollace R , Macri R , Scarano F
Published: May 2024

Funding Acknowledgements 

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PON-MIUR 000_78

Evidence exists that the gut microbiota contributes to the alterations of lipid metabolism associated with high-fat diet (HFD). Moreover, the gut microbiota has been found to modulate the metabolism and absorption of dietary lipids, thereby affecting the formation of lipoproteins occurring at the intestinal level as well as systemically, though the pathophysiological implication of altered microbiota composition in HFD and its role in the development of atherosclerotic vascular disease (ATVD) remain to be better clarified. Recently, evidence has been collected indicating that supplementation with natural polyphenols and fibres accounts for an improvement of HFD-associated intestinal dysbiosis, thereby leading to improved lipidaemic profile. This study aimed to investigate the protective effect of a bergamot polyphenolic extract (BPE) containing 48% polyphenols enriched with albedo and pulp-derived micronized fibres (BMF) in the gut microbiota of HFD-induced dyslipidaemia. In particular, rats that received an HFD over a period of four consecutive weeks showed a significant increase in plasma cholesterol, triglycerides and plasma glucose compared to a normal-fat diet (NFD) group. This effect was accompanied by body weight increase and alteration of lipoprotein size and concentration, followed by high levels of MDA, a biomarker of lipid peroxidation. Treatment with a combination of BPE plus BMF (50/50%) resulted in a significant reduction in alterations of the metabolic parameters found in HFD-fed rats, an effect associated with increased size of lipoproteins. Furthermore, the effect of BPE plus BMF treatment on metabolic balance and lipoprotein size re-arrangement was associated with reduced gut-derived lipopolysaccharide (LPS) levels, an effect subsequent to improved gut microbiota as expressed by modulation of the Gram-negative bacteria Proteobacteria, as well as Firmicutes and Bacteroidetes. This study suggests that nutraceutical supplementation of HFD-fed rats with BPE and BMP or with their combination product leads to restored gut microbiota, an effect associated with lipoprotein size re-arrangement and better lipidaemic and metabolic profiles.
Lenti-viral overexpression of RhoGEF Trio reduces CD8 T cell extravasation into atherosclerotic vein graft lesions in mice
Authors: Sluiter T , Starink L , Van Stalborch A D , Bax C , Schoon R , Huveneers S , Quax P H A , De Vries A A F , Van Buul J D , De Vries M R
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Rembrandt Institute for Cardiovascular Sciences

Background

In advanced atherosclerotic plaques, such as vein graft lesions, neovessels grow into the hypoxic plaque. This intraplaque angiogenic neovessels lack proper endothelial cell-cell junctions and are therefore susceptible to extravasation of leukocytes, which results in inflammation and atherosclerosis. T cells are the largest leukocyte subset found in atherosclerotic plaques and are key drivers of inflammation and plaque progression. RhoGEF Trio improves endothelial barrier function by strengthening endothelial cell-cell junctions in vitro, but its effect on T cell transmigration and its therapeutic potential to impede atherogenesis remains unknown.

Methods

ApoE3*Leiden mice (n=10/group) underwent bypass surgery in which a donor caval vein was interpositioned into the arterial circulation at the site of the right common carotid artery. Immediately post-operatively, a pluronic gel containing either GFP-Lentivirus (LV) (control) or Trio-GFP-LV (Trio) was locally applied around the vein graft to induce local overexpression of Trio. At post-op day 28, vein grafts were harvested and processed for morphometric and compositional analysis of the atherosclerotic lesions. Additionally, CD4 and CD8 T cells were isolated from healthy human donors and used in vitro to assess the role of Trio on trans-endothelial migration over in vitro-cultured HUVECs.

Results

At post-op day 28, both luminal and neovessel endothelial cells were GFP positive indicating successful transfection. CD8 T cell infiltration into the atherosclerotic plaque was reduced in the Trio group (45%, p=0.0426), whilst plaque size and the number of neovessels were unaffected. Mechanistically, we observed that adherence of CD8, but not CD4 T cells to the endothelial monolayer was reduced in vitro upon transfection with Trio-LV. This resulted in a decrease in the absolute number of transmigrated CD8 T cells (56%, p=0.0197), whilst the number of transmigrated CD4 T cells was unaffected. Interestingly, CD8 T cells exhibited no difference trans-/paracellular diapedesis, whilst for CD4 T cells Trio overexpression induced transcellular, rather than paracellular, transmigration.

Conclusions

LV transfection of vein grafts by local application of a pluronic gel holds promise a therapeutic strategy to improve bypass surgery outcomes. LV transfection with RhoGEF Trio did reduce CD8 T cell transmigration (in vivo and in vitro), but not CD4 T cell transmigration (in vitro) into the atherosclerotic plaque, whilst plaque size was not affected. Ongoing investigations focus on the effect of Trio on micro-vascular leakage as well as infiltration of other immune cells into the plaque.
Impact of immune system humanization on atherosclerosis in dyslipidemic immunocompromised mice
Authors: Bonacina F , Moreola A , Nour J , Greco S , Vingiani G B , Norata G D
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Cariplo Foundation. PRIN (Italian Ministry of University and Research)

Background and aim

Given the key role of immune response during atherosclerosis and the therapeutic interest on biologics targeting human immune cells, the need of experimental models to translate molecular mechanisms and test therapeutic approaches for atherosclerosis is continuously increasing.

Here we provide an immune and metabolic characterization of an innovative immunodeficient mouse humanized with human hematopoietic cells on an atheroprone background.

Methods

LDLR-KO mice were crossed with the immunodeficient C57BL/6J strain Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) to generate an immunocompromised dyslipidemic mouse (TKO-LDLR KO mice) recipient of human hematopoietic stem cells (hCD34+).

Results

TKO-LDLR KO were first characterized for their immune and metabolic profile. TKO mice are deficient in mature lymphocytes and NK cells and this profile was conserved in TKO-LDLR KO mice. Under high cholesterol diet, TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet, develop marked dyslipidemia, steatosis and atherosclerosis. This profile confirms the suitability of TKO-LDLR KO mice for atherosclerosis studies. Next we tested the impact of immune system humanization on atherosclerosis. TKO-LDLR KO pups received a low-dose irradiation (200 cGy) and thereafter 2 x 10^5 hCD34+ were injected in the liver. Engraftment of human leukocytes (hCD45+) was evaluated after two months by flow cytometry analysis from tail blood. This approach allows to reconstitute between 10-30% of hCD45+, mainly B and T cells. The humanization with hCD34+ cells worsens atherosclerosis development as compared to non-humanized TKO-LDLR KO mice.

Conclusion

We have generated and characterized the humanized dyslipidemic TKO-LDLR KO mouse. This mouse model presents human B and T cells and represents and important toll to investigate the impact of human adaptive immune cell pharmacological modulation in the context of atherosclerosis.