bluuebunny/crossref_metadata_2025 huggingface dataset:
The interaction of receptor-type protein tyrosine phosphatases sigma and zeta1 controls cardiac fibroblast migration
Authors: Hasselbach L , Weidner J , Elsaesser A , Theilmeier G
Published: October 2022

Background

Among cardiovascular diseases as a major health burden heart failure (HF) plays a central role. Our screening of patients with HF either from ischemic – or dilatative cardiomyopathy has shown that receptor-type protein tyrosine phosphatase sigma (PTPRσ) and zeta1 (PTPRz1) are upregulated compared to healthy hearts. Inhibition of PTPRσ is known to be necessary for autonomic reinnervation after myocardial infarction [1] and for various functions in the nervous system [2]. Most recently the loss of PTPRz1 in knockout-mice has been shown to lead to impaired morphogenesis with postnatal systolic and diastolic heart failure [3].

Purpose

A crucial step in myocardial remodeling and the development of HF is fibroblast migration into the wound, where they differentiate into myofibroblasts, producing extracellular matrix proteins and forming the scar. Up to now no information is available on the role of PTPRσ and PTPRz1 in fibroblast migration and myocardial remodeling.

Methods

Neonatal cardiac fibroblasts are transfected with either scrambled-, PTPRσ-, PTPRz1-siRNA or a combination thereof. A scratch wound is inflicted at confluency of the cells. The number of migrated cells, relative to the control, is determined microscopically after 48 hours. Transfected fibroblasts are seeded into transwells to examine their transmigration. After 48 hours the number of transmigrated cells, relative to the control, is determined after DAPI staining. N=4, statistical analysis is performed using Kruskal-Wallis Test with Dunn's multiple comparison.

Results

In the scratch assay a PTPRz1 knockdown resulted in a significant increase in migrating cells (1 vs. 1,355±0,1144, p=0,0265). This promigratory effect is not present in PTPRσ knockdown cells, independent of the PTPRz1 expression (1 vs. 1,032±0,1654 and 1 vs. 1,073±0,08597, both p>0,9999). In transmigration there is a fourfold increase in transmigrated cells after PTPRz1 knockdown (1 vs 4,046±0,4865, p=0,05). Like in migration this effect is reversed after downregulation of PTPRσ (1 vs 0,9834±0,3844 and 1 vs. 1,345±0,6584, both p>0,9999).

Conclusion

PTPRσ and PTPRz1 are differentially regulated during the pathogenesis of heart failure. While a knockdown of PTPRz1 leads to an increase in fibroblast migration and transmigration, downregulation of PTPRσ inhibits this effect. These results indicate an antimigratory role for PTPRz1. We hypothesize that PTPRσ and PTPRz1 interact through shared intracellular effector molecules, affecting fibroblast migration, an important step in myocardial remodeling and the development of HF. Further investigations will be conducted to identify these shared effector molecules of PTPRσ and PTPRz1 to find a way to influence heart failure through regulation of receptor-type protein tyrosine phosphatases.

Funding Acknowledgement

Type of funding sources: Public Institution(s). Main funding source(s): Carl von Ossietzky University Oldenburg
Decrease of RNA editing in the failing heart leads to induction of circRNAs
Authors: Kokot K , Kneuer J , John D , Rebs S , Mueller M , Haas J , Thiele H , Mueller O J , Hille S , Leuschner F , Dimmeler S , Streckfuss-Boemeke K , Meder B , Laufs U , Boeckel J N
Published: October 2022

Background and purpose

Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional modification process that affects the secondary structure of RNAs. Changes in RNA editing have been associated with human diseases. We therefore aimed to analyze editing in the healthy and failing human heart.

Methods and results

Transcriptome sequencing of human heart samples of heart failure (HF) patients (n=20) and controls (n=10) revealed A-to-I editing as the major type of editing (>80%). In HF patients, RNA editing was reduced, which was primarily attributable to Alu elements in introns of protein-coding genes. We identified 166 upregulated circRNAs in HF, with the majority showing reduced RNA editing in their parental host gene (88.3%). CircRNA expression did not correlate with their corresponding host gene (R=0.07, P<0.05), suggesting that an alternative splicing mechanism gives rise to the elevated circRNA levels in HF. The RNA editing enzyme ADAR2, which binds to RNA regions that are edited from adenosine to inosine, was decreased in failing human hearts (−68.2%). In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, we examined the formation of circRNAs on one exemplary candidate. AKAP13 was among the top edited mRNAs in the human heart and gave rise to a circular transcript, which was elevated in HF. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes (−31.0%).

Conclusion

Our study shows that ADAR2 mediates A-to-I RNA editing in the human heart. We describe an alternative splicing mechanism of circRNAs in the human heart. In the healthy human heart, A-to-I RNA editing represses the formation of dsRNA structures of Alu elements thereby favoring linear mRNA splicing. Our results contribute to a better mechanistic understanding into the human-specific regulation of circRNA formation and are relevant to diseases with reduced RNA editing and increased circRNA levels.

Funding Acknowledgement

Type of funding sources: None.
Golgi Apparatus fragmentation in dilated cardiomyopathy and its relationship with the alteration of vesicular transport
Authors: Gimenez-Escamilla I , Perez-Carrillo L , Portoles M , Martinez-Dolz L , Tarazon E , Rosello-Lleti E
Published: October 2022

Background

The Golgi Apparatus (GA) is an important membrane-bound organelle implied in many physiological events, as trafficking, processing and sorting of newly membrane and secretory proteins and lipids. Nevertheless, these functions can be dysregulated in a diversity of pathologies due to the alteration of the GA morphology, such as cardiovascular diseases. Previously, we described in dilated cardiomyopathy (DCM) alterations in shape and size of the secretion vesicles of GA that was related to a worse functional situation. The patients had smaller, more ellipsoidal and more numerous vesicles with a higher content of natriuretic peptides (1). This situation can be related with Golgi fragmentation, a status characterized by the presence of a dispersed GA both in physiological and pathophysiological conditions (2). However, a greater understanding of Golgi fragmentation in DCM is necessary.

Purpose

We attempted to analyse the components of the vesicular Golgi transport and the Golgi fragmentation in cardiac tissue samples from DCM patients, paying special attention to GM130, as one of the main molecules related with the Golgi fragmentation.

Methods

We analysed vesicular Golgi transport genes in human hearts by RNA-sequencing in 13 samples from DCM patients and 10 control hearts (CNT), and the miRNAs that regulate their expression by ncRNA sequencing (DCM, n=20; and CNT, n=8). Finally, we investigated changes in the protein levels of GM130 and its phosphorylated state (pGM130) by western blot with 22 DCM and 6 CNT samples in total.

Results

We found 134 differentially expressed genes related to the GA, and 15 of them were involved in the secretion of Golgi vesicles. Among these molecules, VAMP4, a v-SNARE related with the Golgi fragmentation, is found to be downregulated (−1.27-Fold; P<0.05). Moreover, we have observed a downregulation in the expression of GOLGA2, the gene that encodes GM130 (−1.24; P<0.05). Furthermore, its regulator, hsa-miR-17-5p (−1.42; P<0.01) was also downregulated. In this sense, we have found no changes in GM130 protein levels between DCM patients and CNT group. In contrast, pGM130, molecule that it has been used as a biomarker of Golgi fragmentation, was overexpressed in DCM patients (1.19; P<0.05).

Conclusion

We found a deregulation of genes related to the vesicle secretion in the GA, which together with the changes in vesicular morphology previously observed, suggests an alteration in the rate of secretion in response to the needs of the DCM patients. In addition, the increase in the phosphorylated protein GM130 suggests the presence of fragmentation in DCM patients that could be related to the alteration in vesicular transport.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)
Radiation-induced electrophysiological remodelling in neonatal rat ventricular cardiomyocytes
Authors: Mages C , Gampp H , Rahm A K , Ullrich N D , Seidensaal K , Rhein B , Debus J , Frey N , Thomas D , Lugenbiel P
Published: October 2022

Background

Ventricular tachycardia (VT) carries a relatively high risk of recurrence and significantly impacts patients' quality of life, despite state of the art treatment including antiarrhythmic drug therapy and catheter ablation. Cardiac stereotactic body radiotherapy (SBRT) has emerged as a promising non-invasive treatment option for refractory VT. A marked reduction in VT episodes is observed within days of treating high-risk patients with a single fraction SBRT. The cellular mechanisms underlying the clinically demonstrated antiarrhythmic effect are still poorly understood. Radiation-induced fibrosis is observed several months after irradiation, suggesting other mechanisms that explain the immediate benefit of radiotherapy on the VT substrate.

Purpose

We hypothesized that early antiarrhythmic effects of single-fraction radiation are produced by direct molecular and functional electrophysiological changes, independent of fibrosis. Our aim was to investigate the effects of 20 Gy ionizing photon radiation on the electrophysiology of ventricular neonatal rat cardiomyocytes at time periods from 24 hours to 96 hours after irradiation.

Methods

Ventricular neonatal rat cardiomyocytes were isolated, cultivated for 24 to 96 hours and then exposed to photon beam with a single dose of 20 Gy. Expression changes of ion channels, calcium handling proteins and structural proteins affecting cellular electrophysiology were assessed via RT-qPCR and Western blotting. Beat frequency was evaluated by video analysis and action potentials (APD) were recorded using the whole-cell voltage clamp technique in current-clamp mode. Intracellular calcium transients in Fura-2 AM loaded cardiomyocytes were recorded with an IonOptix recording system.

Results

Irradiation lead to significant changes in the mRNA and protein level of ion channels, with most distinct changes being observed 96h after radiation. Increased mRNA and protein expression of (Cav1.2) and of the potassium channels Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1) and Kcnj2 (Kir2.1) was observed. Additionally, Connexin 43 (Cx43) was upregulated. Irradiated cardiomyocytes showed a significant increase in beat rate over time. APD was significantly shortened, with a significantly increased upstroke velocity. Regarding calcium handling, significant upregulations of Ryr2 (RYR2) and Slc8a1 (NCX) transcript and protein level was detected. Additionally, calcium handling properties were altered.

Conclusion

Our results suggest that acute irradiation effects are associated with electrophysiological remodelling in cardiomyocytes. Changes of cellular ion channel expression and calcium homeostasis lead to normalization and shortening of heart-failure associated APD prolongation. These results reveal new insights into acute antiarrhythmic effects on cardiomyocytes after SBRT-therapy.

Funding Acknowledgement

Type of funding sources: None.
Plasma miR-451a predicts atrial fibrillation recurrence after pulmonary vein ablation
Authors: Cebro-Marquez M , Vilar-Sanchez M E , Gonzalez-Melchor L , Garcia-Seara J , Martinez-Sande J L , Fernandez-Lopez X A , Martinez-Monzonis M A , Gonzalez-Juanatey J R , Rodriguez-Manero M , Lage-Fernandez R , Moscoso Galan I
Published: October 2022

Background/Introduction

Atrial fibrillation (AF) is the most common sustained arrhythmia diagnosed in European countries. Several studies have shown that catheter ablation is a more effective treatment than antiarrhythmic drugs and should be prioritised in symptomatic patients. However, AF recurrence after catheter ablation remains a challenge. MiRNAs have been identified, in recent years, as epigenetic modulators in numerous biological processes, including cardiac electric conduction. MiRNAs can be easily detected in the circulation, making them attractive as potential biomarkers for risk stratification in AF patients.

Purpose

Our purpose is to find a miRNA able to predict the AF recurrence after catheter ablation.

Methods

44 patients submitted to catheter ablation were consecutively recruited and classified based on recurrence after a follow up of 12 to 48 months. Peripheral and left atrium blood samples were collected before catheter ablation. Human Cardiovascular Disease miScript miRNA PCR Arrays were performed to analyse 84 miRNAs altered in cardiovascular disease. Significant miRNAs changes in ΔCt levels were assessed using Two-way ANOVA and post hoc Tukey tests. Simple logistic regression analysis was performed to analyse association to AF recurrence. KEGG pathway enrichment analysis was performed to identify the most enriched targets and pathways involved in AF recurrence.

Results

In our cohort, overall recurrence rate was 47.7%. qPCR expression analysis showed increased levels of miR-328-3p and decreased levels of miR-486-5p in recurrent patients, both in peripheral as well as left atrial blood. Our results also showed decreased levels of miR-let-7b-5p and miR-451a in recurrent patients, but selectively regulated in peripheral and atrial blood, respectively (Figure 1). Simple logistic regression analysis showed a significant association between blood atrial levels of miR-451a and AF recurrence (Figure 2A). KEGG pathway enrichment analysis showed that miR-451a could be involved in epigenetic regulation of myocardial electromechanic integrity (Figure 2B).

Conclusion

Expression analysis of atrial blood miR-451a levels, prior to catheter ablation, could be a good a risk stratifier of recurrence.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): IN607A2019/02. Xunta of Galicia. Grants for the consolidation and structuring of competitive research units in the universities of the Galician University System, in the public research organizations of Galicia and in other entities of the Galician R&D System 2019
Empagliflozin alleviates diabetic cardiomyopathy via inhibiting excessive autophagy and adverse remodeling in type 1 diabetic mice
Authors: Madonna R , Moscato S , Cufaro M , Pieragostino D , Mattii L , Del Boccio P , Ghelardoni S , Zucchi R , De Caterina R
Published: October 2022

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with and without diabetes mellitus, but underlying mechanisms remain unclear. Autophagy is an important cardioprotective mechanism under acute stress conditions, but excessive autophagy can accelerate myocardial cell death leading to autosis. In this study we evaluated the role of empagliflozin (EMPA) against cardiac injury in streptozotocin (STZ)-induced diabetic mice, and assessed underlying mechanisms.

EMPA (10 mg/kg/die)-treated diabetic male mice had a lower number of TUNEL-positive apoptotic cells (%TdT+ 4.9±2, p<0.05), lower percentage of SA-β-gal positive senescent cardiac area (%SA-β-gal 7.9±1, p<0.05), lower fibrotic area (%picrosirius red 0.15±0.06, p<0.05), lower autophagy (%green fluorescence 2.3±0.6, p<0.01), compared to diabetic mice (%TdT+ 4.9±2; %SA-β-gal 10±2; %picrosirius red fibrotic area 0.24±0.08; %green fluorescence autophagic area 3.98±0.5). Proteomics analysis of cardiac tissue showed downregulation of apoptosis, necrosis and 5' adenosine monophosphate-activated protein kinase (AMPK) pathways, along with upstream activation of vascular endothelial growth factor and sirtuins in the heart of EMPA+STZ treated mice compared to STZ mice. Since activation of sirtuins leads to modulation of cardiomyogenic transcription factors, we analyzed total expression of serum response factor (SRF), myocardin and myocardin-related transcription factor (MRTF), and DNA binding activity of SRF by electromobility shift assay. While EMPA only increased MRTF, EMPA (2.2±0.01, p<0.01) and EMPA+STZ (2.0±0.1, p<0.01) significantly increased SRF/SRE binding activity compared with STZ alone (0.5±0.01), which was paralleled by the increase in cardiac actin expression (4.06±0.1 vs 2.2±0.01, p<0.01). EMPA significantly reversed STZ-induced systolic cardiac dysfunction at echocardiography, and downregulation of pS368-connexin 43 active isoform (Table). In parallel in vitro experiments, EMPA inhibited excessive autophagy in high glucose-treated cardiomyocytes by inhibiting the autophagy inducer GSK3β, leading to reactivation of transcriptional complex for cardiomyocyte contractile genes. Our results indicate that EMPA plays a cardioprotective effect by inhibiting excessive autophagy through the mTOR/AMPK axis in the mouse model of STZ-induced type 1 diabetes. EMPA also promotes cardiac reverse remodeling by inhibiting fibrosis, activates cardiac Cx43 and the promyogenic transcriptional program through the sirtuins-SRF axis (Figure 1), in the context of diabetes.

Funding Acknowledgement

Type of funding sources: None.
Histology-verified myocardial fibrosis and quantification in severe AS patients: correlation with non-invasive LV myocardial tissue assessment
Authors: Maltes S , Abecasis J , Pinto D G , Santos R R , Oliveira L , Mendes G S , Guerreiro S , Lima T , Freitas P , Ferreira A , Ramos S , Felix A , Cardim N , Gil V M , Mendes M
Published: October 2022

Background

Myocardial fibrosis (MF) is a common finding and a potential adverse prognostic marker in several cardiac diseases, including in severe aortic stenosis (AS). While histological analysis obtained through endomyocardial biopsy remains the gold-standard for MF assessment, non-invasive cardiac imaging may offer surrogate biomarkers for fibrosis. We tried to assess the correlation between MF quantification at histopathology and cardiac magnetic resonance (CMR)-derived tissue characterization data in patients with severe AS.

Methodology

Single-center prospective cohort enrolling 71 patients with severe symptomatic high-gradient AS undergoing surgical aortic valve replacement (SAVR) (mean age 71±9 years; 49% male, mean valvular transaortic gradient 60±20 mmHg; mean left ventricle [LV] ejection fraction 58±9%). Those with past history of myocardial infarction or cardiomyopathy were excluded. All patients underwent pre-operative CMR study with LV tissue characterization and quantification. Normal T1 mapping value was defined as >1021ms as per center protocol. Myocardial tissue was obtained during SAVR either through myocardial biopsy at basal LV septum or harvested from surgical myectomy specimens. Masson's trichrome stain was used for collagen/fibrosis assessment. Automatic quantification was obtained at QuPathTM digital pathology software after applying a dedicated artificial intelligence algorithm on ultra-high-resolution digital slide scanning images.

Results

Histology-confirmed MF was observed in all patients (median percentage of fibrotic myocardial tissue 15% [IQR 9–22%]). Median global T1 mapping and extracellular volume (ECV) percentage was 1048ms (IQR 1027–1078) and 24% (IQR 20–30%), respectively. Late gadolinium enhancement (LGE) with a non-ischemic pattern was present in 42 patients (59%) with a median LGE mass of 5.8g [IQR 1.0–10.2]; median percentage of 3.7% [IQR 0.6–10.4]. While neither T1 mapping (global or basal LV septum), ECV nor LGE had any significant correlation with histology-confirmed MF (Figure 1) the vast majority had significantly elevated global and basal LV septum T1 mapping – 81% and 92%, respectively.

Conclusion

In this single-center prospective study, microscopic MF was present in all patients with severe symptomatic high-gradient AS, was accompanied by elevated T1 mapping values but no correlation was found between myocardial fibrosis at histopathology analysis and CMR-derived LV tissue characterization parameters. This may not only stem from sampling (single point biopsy vs. whole myocardial tissue assessment) but also from distinct evaluation of different types of fibrosis by different methods.

Funding Acknowledgement

Type of funding sources: None.
First 12-month follow-up of bioresorbable synthetic heart valves implanted in the aortic position in sheep
Authors: Vis A , De Kort B J , Szymczyk W , Van Rijswijk J W , Dekker S , Driessen R , Wijkstra N , Grundeman P F , Niessen H W M , Janssen H M , Dankers P Y W , Smits A I P M , Bouten C V C , Kluin J
Published: October 2022

Background

Replacement of diseased heart valves with the currently available valve prostheses has serious drawbacks. The use of bioresorbable synthetic “in situ tissue engineered” heart valve prostheses has been proposed to overcome the limitations of traditional heart valve prostheses. Such bioresorbable synthetic heart valve prostheses have been successfully tested as pulmonary valve replacements in preclinical studies, but data on aortic valve replacement is lacking. Here, we present the first in-vivo study on the long-term functionality of bioresorbable synthetic heart valves in the high-pressure circulation.

Methods

Approval for the animal studies was obtained by the Amsterdam University Medical Centres Animal Care Ethics Committee (AVD1180020197705) and are in agreement with the current Dutch law on animal experiments (WOD). We surgically implanted bioresorbable synthetic aortic valve prostheses in 20 female Swifter sheep in orthotopic position. The scheduled follow-up times were 1, 3, 6, 9 and 12 months.

Results

Fifteen sheep (75%) recovered well from the surgical valve implantation procedure and were included in this analysis. No sheep died due to valve failure. All valves remained free from active infectious endocarditis, thrombotic complications, and pathological calcification. A total of 10 valves (67%) were intact, thin and pliable and remained free from leaflet thickening, retraction and degradation up to 12 months after implantation. In most valves (67%), the scaffold remained sparsely- or unpopulated by cells during 12 months follow up. In some valves (33%), colonization of the valve scaffold was observed, however this was most often associated with partial degradation of the leaflet and leaflet thickening. In general, the degradation of the scaffold fibres was limited throughout the follow-up period.

Conclusion

We are the first to evaluate in vivo bioresorbable synthetic heart valves in aortic position. While valves remained functional, the study also serves as a starting point to further optimize scaffold and neotissue development for heart valve replacement in the high-pressure environment.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1. Cardiovasculair Onderzoek Nederland (grant number CVON2012-01)2. Netherlands Organization for Scientific Research (024.003.013)
Specific gene domains most commonly affected by CHIP-driver mutations predicting mortality in severe aortic stenosis after transcatheter aortic valve implantation
Authors: Mas-Peiro S , Abou-El-Ardat K , Pergola G , Berkowitsch A , Meggendorfer M , Rieger M , Vasa-Nicotera M , Dimmeler S , Zeiher A
Published: October 2022

Background

Clonal haematopoiesis of indeterminate potential (CHIP), i.e. expanded somatic blood-cell clones in persons without any other haematological abnormalities, has been shown to predict survival in patients with severe aortic stenosis (AS) undergoing transcatheter aortic valve implantation (TAVI). DNMT3A and TET2 are the two most common CHIP-driver mutations. In patients with haematological disorders, specific domains of TET2 and DNMT3A most commonly accounting for detected mutations vary across different haematological malignancies.

Purpose

The aim of our study was to analyse the distribution of the mutated domains in CHIP-driver genes in patients with severe AS undergoing TAVI, in order to gain a mechanistic insight on its specific role in AS.

Methods

We used next-generation sequencing to identify mutated domains in DNMT3A and/or TET2 genes in 453 consecutive patients with severe AS undergoing TAVI.

The site of detected mutations in DNMT3A was classified as located to the conserved proline-tryptophan-proline-tryptophan (PWWP), zinc-finger (ADD), or methyl-transferase (MTase) domains, or to other non-coding regions. For TET2, mutations were classified as involving dioxygenase (DD) or Cys-rich (CD) domains. Only mutations that are classified as moderate-to-high based on their functional impact on the protein were considered.

Results

Out of 147 patients with a CHIP-driver mutation, 92 had DNMT3A mutations and 71 had TET2 mutations, with mutations in both genes being present in 16 patients.

MTase was the most commonly involved domain in DNMT3A (61% of cases). Other affected regions were PWWP/ADD/MTase (12%), no domain/non-coding region (11%), ADD (7%), ADD/MTase (5%), and PWWP (4%) (Figure 1). Overall, MTase mutations were found in a large majority of patients (78%). Such a “hot-spot” appears to be in line with a potential recently suggested mechanism involving DNA methylation.

Most common locations for mutations in TET2 were CD/DD (48%), followed by DD (33%), CD (15%) and no domain/non-coding region (4%) (Figure 2). Overall, mutations in DD were found in a large majority of patients (81%). TET2 appears to be involved in myeloid expansion and impaired immune function with increasing age; this could play a role in its impact on heart disorders.

Conclusion

In patients with severe AS undergoing TAVI, mutations in certain sites within the most common CHIP-driver genes (MTase domain in DNMT3A gene and CD domain in TET2 gene), appear to be mostly affected. These findings could be a first step to identify “hot spots” for mutated domains associated with severe AS requiring TAVI and suggest some potential mechanistic explanations.

Funding Acknowledgement

Type of funding sources: Other. Main funding source(s): The study was further supported by the German Center for Cardiovascular Research (DZHK).
Crosstalk between PKR1 and miR-124 is crucial for EMT regulation of tc21+epicardial mesothelial cells during cardiac development and repair process
Authors: Arora H , Mammadova A , Nebigil C
Published: October 2022

Background

The epicardial-mesothelial cells (EMCs) are essential regulators of cardiac growth and repairment process. The epicardial-to-mesenchymal transition (EMT) of epicardial fate determination are controlled by both cell autonomous and cardiomyocyte-originated mechanisms. Here, by using an in vitro and in vivo model of epicardial EMT, we investigated the role of miRNAs as regulators of these process and their potential targets.

Methods

EMT was induced in mice embryonic tcf21+EMCs through an angiogenic cytokine, prokineticin treatments. Cre-dependent tracing of TCF21-tm-iCRE-EMCs was utilized to abrogate prokineticin receptor-1 (PKR1) in mice epicardium. Human tcf21+ cardiac fibroblast (mainly originated from epicardial origin also utilized to study for their repairment signaling.

Results

Upon EMC-specific abrogation of PKR1 in early stages of cardiac development the TCF21-tm-iCREPKR1−/− mice exhibited 13±4% embryonic lethality due to a disconnection of epicardial cells from compact layer, failed expansion of the sub-epicardial space, and disruption of heterotypic cell interaction between epicardium and myocardium. EMT-RT profiler and enrichment analysis revealed an impaired EMT in TCF21-tm-iCREPKR1−/− hearts. Ingenuity Pathway Analysis on the EMT molecular network (e.g., Snai1, Snai2, B-catenin, N-cadherin, vimentin), specific to cardiac development and defects revealed MiR-124 as a part of this network that was inversely associated and down-stream of PKR1 in cultured Tcf21+ cells. Furthermore, protein expression of SNAIL2 was significantly downregulated in TCF21-tm-iCREPKR1−/− hearts and cultured Tcf21+ cells upon treatment with mir124 mimic. The luciferase reporter assay showed that miR124 directly targeted the 3'-untranslated region of SNAIL2 in EMCs. In a counter experiment, PKR1 gene replacement or mir124 inhibitor was able to rescue the impaired EMT-genes, and an increase in apoptosis and impaired proliferation in epicardium of embryonic TCF21-tm-iCREPKR1−/− hearts. Importantly, miRNA mimic in cultured tcf21 cells maintained epithelial features in EMCs, and increased EMT-associated transcripts and traits, such as apoptosis. In contrast, MiR124 is involved in wound healing process in tcf21+human CFs and EMCs.

Conclusion

This study shows the importance of a crosstalk between PKR1 and miR-124 with respect to EMT regulation during cardiac development and repair. Controlling fibroblast-committed EMT and pathological fibrotic remodeling is of increasing interest within the field of regenerative tissue engineering and development of interventional strategies after cardiac injury.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): Fondation de France
The role of P2Y12 in cardiovascular disease beyond atherothrombosis: P2Y12 signaling promotes emergency hematopoiesis after myocardial infarction
Authors: Seung H , Wrobel J , Wadle C , Buehler T , Chiang D , Rettkowski J , Cabezas-Wallscheid N , Hechler B , Wolf D , Duerschmied D , Idzko M , Bode C , Von Zur Muehlen C , Hilgendorf I , Heidt T
Published: October 2022

Background

Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. The purinergic ADP-receptor P2Y12 has therefore been targeted in the treatment of cardiovascular disease (CVD) to prevent atherothrombosis (1). Beyond P2Y12 expression on platelets, purinergic receptors have also been described on hematopoietic stem and progenitor cells (LSK) (2). After myocardial infarction (MI), accelerated LSK proliferation launches emergency hematopoiesis as the driving force behind the inflammatory response to MI, increasing inflammatory cell production in the bone marrow (BM) and providing leukocyte resupply for local cell recruitment to the infarct (3). The inflammatory cascade after MI covers intricate multilayered interactions between the injured myocardium and the hematopoietic BM that still remain to be fully elucidated and may unearth novel therapeutic strategies. Whereas P2X receptors have recently been found to be involved in cell trafficking (4), the role of P2Y receptors in the hematopoietic BM have not yet been characterized.

Purpose

This study aims to characterize the influence of P2Y12 signaling on emergency hematopoiesis and cardiac remodeling after MI.

Methods

Permanent coronary ligation was performed for MI to assess BM activation, inflammatory cell composition, cardiac remodeling and function in murine global and platelet-specific P2Y12 knockout models and under pharmacological P2Y12 inhibition with prasugrel using flow cytometry, qPCR, immunohistochemistry and echocardiography. In vitro studies including colony forming unit (CFU) assays and flow cytometry allowed for investigation of ADP-dependent effects on LSK cells and intracellular pathway analysis.

Results

We identified ADP as a danger signal for the hematopoietic BM, fueling emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. Detection of P2Y12 expression in LSK implicated a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 deficiency and P2Y12 inhibition with prasugrel decelerated emergency hematopoiesis and consecutively reduced the excessive inflammatory response to MI, translating to lower numbers of hematopoietic progenitors and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition ameliorated chronic adverse cardiac remodeling and preserved cardiac function after MI.

Conclusion

ADP-dependent P2Y12-mediated activation of hematopoietic stem and progenitor cells in the BM promotes emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel role of P2Y12 antagonists in CVD beyond atherothrombosis.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)
Cardiac sympathetic afferent denervation reduces matrix metalloproteinase expression and improves cardiac remodeling in rats post myocardial infarction
Authors: Zucker I , Wang H , Mishra P K , Nandi S
Published: October 2022

In a previous study from our laboratory, we demonstrated that chronic and selective cardiac sympathetic afferent (spinal) denervation at the time of myocardial infarction (MI) using epicardial application of resiniferatoxin (RTX), a neuronal toxin capable of inducing rapid degeneration of transient receptor potential vanilloid 1 (TRPV1)-expressing afferent neurons and fibers, markedly reduced the cardiac remodeling process 9–11 weeks post-MI in rats. This included reduced cardiac hypertrophy, fibrosis and apoptosis. Here, we further investigated the effect of epicardial application of RTX at the time of MI on cardiac extracellular matrix (ECM) and remodeling post-MI. Echocardiographic and morphologic data demonstrated that, compared to MI+vehicle, MI+RTX exhibited a significantly slower LV chamber dilation (6-week echocardiographic data: left ventricular end-diastolic diameter, 10.7±0.2 vs. 9.6±0.3 mm; left ventricular end-systolic diameter: 8.8±0.2 vs. 7.8±0.3 mm; MI+vehicle vs. MI+RTX, n=18, p<0.05; mean±SE). Scanning electron microscopy showed that RTX reduced collagen deposition in the peri-infarct area in post-MI rats. Western blot and zymography were used to further evaluate the effect of RTX application on matrix metalloproteinase (MMP) expression and activity, which is responsible for degrading ECM and contributing to cardiac dilation post MI. Our data suggest time-dependent increases in MMP expression in infarcted hearts post MI. RTX application largely prevented the increase in MMP9 but not MMP2, 4 weeks post-MI. RTX prevented both increased MMP2 and MMP9 activities in the peri-infarct myocardium at 8–10 weeks post-MI. We further investigated the effect of RTX application on MI-induced cardiac inflammation, which has been reported as an upstream mechanism triggering MMP activation post-MI. The data show that RTX largely abolished MI-induced plasma extravasation and reduced macrophage infiltration and cardiac cytokine content in the peri-infarct or remote myocardium of post-MI rats. These data suggest that cardiac sympathetic afferent denervation at the time of MI exerts a local anti-inflammatory effect and reduces ECM remodeling by preventing excessive MMP activation in post-MI rats.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institutes of Health Grant HL126796
Modeling cardiac fibroblast heterogeneity in fibrotic heart using human pluripotent stem cell derived epicardial cells
Authors: Funakoshi S , Fernandes I F , Keller G K
Published: October 2022

Introduction

Our understanding of human cardiac pathophysiology is limited due to the scarcity of human heart samples. Model organisms do not faithfully recapitulate human physiology and pathophysiology to the extent that is needed. Therefore, human pluripotent stem cell (hPSC)-derived cardiac cells represent promising sources to overcome these limitations to studying heart diseases.

Purpose

To generate cardiac organoids mimicking the interaction between cardiomyocytes and non-cardiomyocytes in the developing heart and the postnatal pathological conditions.

Method

It has been well known that epicardium is a main source of non-cardiac cells in the developing heart. We therefore generated cardiac organoids by mixing hPSC-derived cardiomyocytes and hPSC-derived epicardium and test if they could mimic the developing heart and be applicable for modeling heart failure.

Results

We established a cardiac organoid system generated from human pluripotent stem cells that models the developing heart's early interactions, specifically the epicardial and non-myocyte development, as well as ventricular cardiomyocyte proliferation. Inside the cardiac organoids, epicardium spontaneously differentiated into CD90-positive cardiac fibroblasts expressing fibroblast markers, FN, COL1 and COL3, and CD90-negative smooth muscle cells (SMC) expressing SMC markers, ACTA2 and MYH11. Additionally, we observed the significant increase in cardiomyocyte proliferation in the cardiac organoids compared to in cardiomyocyte only aggregation (p<0.001). We then mature our cardiac organoids using our unique maturation method, the combination of PPARα agonist, dexamethasone, T3 hormone, and palmitate, generating metabolically mature cardiac organoids. These mature organoids recapitulated the phenotypic changes observed in the failing human heart when exposed to pathological stimuli, including fibrosis, metabolic changes, and increase in heart failure markers. We used single-cell transcriptomics to dissect the cellular heterogeneity of our organoids in a model of heart failure with comparisons to primary human data and revealed our organoids recapitulated in vivo human failing heart. This analysis allowed to confirm that our organoids contain a unique subpopulation of cardiac fibroblasts possessing reparative features, suggesting the recapitulation of in vivo heterogeneity in our model.

Conclusions

We successfully generated cardiac organoids mimicking the interaction between cardiomyocytes and non-cardiac cells. Our system enables the recapitulation of in vivo heterogeneity, providing a platform for the accurate understanding of heart development and disease in a dish.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Canadian Institutes of Health Research (CIHR)
Human epididymis protein 4 indicates progression of cardiac fibrosis in Heart Failure with preserved Ejection Fraction
Authors: Nakashima N , Hanatani S H , Yamamoto M Y , Ogata F O , Araki S A , Arima Y A , Matsushita K M , Tsujita K T
Published: October 2022

Background

Cardiac fibrosis is one of the main causes of diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). Human epididymis protein 4 (HE4) is a novel pro-fibrotic protein expressed especially in activated fibroblast, so-call myofibroblast [1]. Although our previous study showed that HE4 functioned as a secretory factor promoting cardiac fibrosis in dilated cardiomyopathy [2], little is known about the role of HE4 in HFpEF.

Purpose

The aim of this study is to evaluate the pathophysiological role of HE4 in HFpEF and the association between serum HE4 levels and clinical course of HFpEF.

Methods

In basic research, we administered high-fat diet and NOS inhibitor (L-NAME) to mice for 5 weeks to induce HFpEF. We analyzed echocardiographic findings and mRNA expressions of fibrosis-related genes including HE4 by real time PCR. In clinical research, we measured serum HE4 levels of 79 patients with HFpEF and divided these patients into low and high HE4 group using median values of HE4 (102.0 pmol/L). Then, we evaluated their mortality and cardiovascular events retrospectively.

Results

In vivo, the administration of high fat diet and L-NAME induced significant increase in left ventricular mass on echocardiography compared with control group (p=0.006). The mRNA expressions of aSMA and collagen3a1 in heart tissue was upregulated in HFpEF model mice (p=0.008 and p=0.003, respectively). Furthermore, the mRNA expression of HE4 was also upregulated in HFpEF model mice (n=6) than in control mice (n=5), although these differences did not reach statistical significance (p=0.238). In vitro, recombinant HE4 upregulated mRNA expression of pro-fibrotic genes, such as periostin (p=0.001), collagen3a1 (p=0.037), aSMA (p=0.095), and PAI-1 (p=0.17), indicating HE4 facilitate the activation of fibroblast and cardiac fibrosis although some did not reach statistical significance.

In clinical research, Kaplan-Meier curve revealed that the rate of all cause death and rehospitalization for worsening heart failure were significantly higher in the high than the low HE4 group, suggesting HE4 can predicts prognosis in patients with HFpEF.

Conclusion

HE4 induces the activation of cardiac fibroblasts and is upregulated in the heart of HFpEF model mice. Furthermore, it is a useful biomarker for predicting cardiovascular events in patients with HFpEF.

Funding Acknowledgement

Type of funding sources: None.
NLRP3 mediates atheromatous plaque macrophage proliferation
Authors: Haerdtner C , Remmersmann F , Von Ehr A , Dederichs T S , Vico T , Krebs K , Wolf D , Sager H , Bode C , Westermann D , Hilgendorf I
Published: October 2022

Background

Macrophage accumulation in atherosclerotic plaques drives disease progression largely dependent on in situ proliferation. We previously reported that systemic cholesterol lowering or reduced modified lipoprotein uptake supress atheromatous plaque macrophage proliferation. In this work we investigate the intracellular mediators of macrophage proliferation.

Methods and results

Macrophages deficient in scavenger receptors CD36 or Msr1 and impaired in cholesterol-rich lipoprotein uptake, proliferated less compared to control macrophages in the same plaque exposed to the same lipid levels in a LDLR-deficient bone marrow irradiation mixed chimera model. Proliferation of plaque macrophages deficient in the intracellular cholesterol sensor LXR was not impaired, however. As modified LDL uptake can activate the NLRP3 inflammasome, we generated mixed bone marrow knockout chimeras for components of the inflammasome. NLRP3 but not Caspase-1 or interleukin-1 receptor deficient macrophages proliferated 35% less compared to NLRP3-expressing macrophages in the same plaque in vivo. These results were confirmed in NLRP3-deficient oxLDL stimulated macrophages in vitro. In line, NLRP3 inhibition of human carotid artery plaque cultures suppressed human plaque macrophage proliferation and reduced inflammasome-dependent IL-1b secretion. However, IL-1b supplementation did not restore local macrophage proliferation in accord with our findings in IL-1 receptor deficient murine plaque macrophages.

Conclusion

We identified a novel role for NLRP3, independent of the canonical Caspase-1–IL-1b inflammasome pathway, in mediating macrophage proliferation in atherosclerotic plaques in mice and men representing a druggable target.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG
Injectable carbon nanotube-functionalized hydrogel as a tool for cardiac tissue engineering
Authors: Pena B , Knight W , Cavasin M , Ferrari I , Abdel-Hafiz M , Vagnozzi R , Bosi S , Park D , Shandas R , Song K , McKinsey T , Sbaizero O , Taylor M , Prato M , Mestroni L
Published: October 2022

Background/Introduction

Heart failure (HF) is an expensive major public health problem in the United States and around the world (1). The current treatments for HF are aimed at reducing symptoms, slowing disease progression, and reducing mortality and not aimed at repairing heart muscle or restoring function. Furthermore, even with these treatments, approximately half of patients with HF will die within 5 years of diagnosis (2). Cardiac transplantation remains the only definitive treatment for those affected with end-stage HF, but availability of donor hearts remains a major limitation (3).

Purpose

The ability of the adult heart to regenerate cardiomyocytes (CMs) lost after injury is limited, generating interest in developing tissue engineering therapies to avoid progression towards HF. Rigid carbon nanotubes (CNTs) scaffolds have been used to improve CMs viability, proliferation, and maturation (4), but require undesirable invasive surgeries for implantation. To overcome this limitation, we engineered an injectable reverse thermal gel (RTG) functionalized with CNTs (RTG-CNT) that transitions from a liquid-solution to a gel-based matrix shortly after reaching body temperature allowing for a liquid-based delivery rapidly followed by a stable-gel localization (5).

Methods and results

Here we show experimental evidences the RTG-CNT hydrogel, used as a three-dimensional (3D) niche to culture human induced pluripotent stem cells (hiPSC)-CMs, promotes hiPSC-CMs alignment and elongation with increased Cx43 localization and improved contraction function when compared with traditional two-dimensional (2D) fibronectin controls and plain 3D RTG system without CNTs. Moreover, the short-term (4-week) biocompatibility of the RTG-CNT hydrogel was also assessed in a mouse model (intracardial injection). The results confirmed that the RTG-CNT hydrogel is well tolerated by the cardiac tissue.

Conclusion

Our results indicated that the injectable RTG-CNT hydrogel has the potential to be used as a minimally invasive tool for cardiac tissue engineering efforts.

Funding Acknowledgement

Type of funding sources: Other. Main funding source(s): NATIONAL HEART, LUNG, AND BLOOD (NHLBI) INSTITUTE
Correlation between coronary artery disease and coronary microvascular dysfunction symptoms and cardiac SPECT results in Latvian women patients
Authors: Vitola B , Mintale I , Trusinskis K , Kalnina M , Kedrova J , Erglis A
Published: October 2022

Introduction

The clinical evaluation of symptomatic women is challenging due to their varying clinical presentation. Women patients with intermediate or high pre-test probability for coronary artery disease – even asymptomatic or with non-informative exercise test should be referred for further focused diagnostics. Myocardial perfusion imaging with single photon emission computed tomography (SPECT) is one of the most performed non-invasive cardiac imaging tests to diagnose CAD or coronary microvascular dysfunction (CMVD).

Purpose

To evaluate the correlation between various CAD symptoms and cardiac SPECT results in women patients undergoing examination due to diagnosis of CAD or CMVD.

Methods

During the period from 2018 till 2021 we performed cardiac SPECT for women patients with intermediate or high pre-test probability or non-informative exercise test. Patients were divided into 2 groups: normal myocardial perfusion or true myocardial perfusion defect, defined as area of ischemia ≥10% of the left ventricle myocardium mass. According to the clinical symptoms patients were categorized in “asymptomatic”, “atypical symptom” and “patients with chest pain” group. The presence of CMVD and CAD in each group was determined by invasive coronary angiography or computed tomography angiography.

Results

In 4 years, SPECT was performed in 726 women patients. The most common presented symptoms were: chest pain (n=487, 67.1%), dyspnea (n=323, 44.5%), tachycardia and palpitations (n=117, 16.1%), fatigue (n=129, 17.8%), headache (n=28, 3.86%) and excessive sweating (n=15, 2.1%). 104 (14.3%) patients were asymptomatic, 487 (67.1%) with chest pain and 135 (18.6%) with atypical symptoms. True myocardial perfusion defect was detected in 125 patients (17.2%). The obstructive CAD was found in 32 cases (25.6%; 4.4% of all SPECTs). The CMVD was diagnosed in 68 women (54.4%; 9.4% of all SPECTs). In asymptomatic patient group obstructive CAD was diagnosed in 5 (4.8%) cases, in chest pain group – 11 (2.3%) cases and in atypical symptom group – 16 (11.9%) patients (p<0.001). None of asymptomatic women had CMVD. The CMVD was detected in 53 (42.4%; 7.3% of all) cases in chest pain group and in 14 (11.3%; 1.9% of all) patients in atypical symptom group (p=0.006).

Conclusion

Women patients with typical chest pain were more likely to have CMVD, but with atypical symptoms – CAD. Since women are in a very high cardiovascular risk group in Latvia, the cardiac SPECT is highly recommended for detection of CAD and CMVD, especially in patients with atypical symptoms.

Funding Acknowledgement

Type of funding sources: None.