bluuebunny/crossref_metadata_2025_split huggingface dataset:
The RGK protein rrad regulates cardiac calcium levels in zebrafish
Authors: Salgado-Almario J , Martinez-Sielva A , Mercedes P , Vicente M , Domingo B , Llopis J 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Innovation and Universities, Spain

Background/Introduction
High voltage-activated Ca2+ (CaV) channels are essential for excitation-contraction coupling. RGK proteins (Gem, Rad, Rem, and Rem2) are small Ras-like G proteins that bind CaV β-subunits and profoundly inhibit CaV1/CaV2 channels. A recent study showed that Rad inhibits CaV1.2 channels in cardiomyocytes, thus mediating the enhanced inotropy and chronotropy by β-adrenergic agonists during the fight-or-flight response. The zebrafish has emerged as a useful in vivo animal model in cardiovascular physiology and pathophysiology. The role of zebrafish rrad, the orthologe of the human RAD, in the regulation of cardiac Ca2+ homeostasis in zebrafish has not been investigated.

Purpose
The aim of this study was to investigate the role of rrad in cardiac Ca2+ regulation in zebrafish.

Methods
We evaluated the cardiac Ca2+ dynamics, heart rhythm, and hemodynamics in transgenic zebrafish larvae that overexpress rrad in the heart, as well as in heterozygous rrad knockout larvae. Measurements were done in the beating heart of 3 dpf transgenic zebrafish larvae expressing the fluorescent Ca2+ biosensor Twitch-4.

Results
In some larvae, overexpression of rrad induced a severe enlargement of the atrium with pericardial edema and a high mortality rate. In addition, the ventricle displayed low Ca2+ levels and reduced contractility. The remaining larvae had no altered morphology, but the ventricular Ca2+ levels were reduced. On the other hand, preliminary results showed that down regulation of rrad in zebrafish larvae increased atrial and ventricular Ca2+ levels, like what was found in Rad knockout mice.

Conclusion
Taken together, these findings suggest that rrad in the zebrafish heart, as in mammals, is an inhibitor of the Ca2+ entry, presumably through CaV1/CaV2 channels, and demonstrate the zebrafish's utility as a model to study the involvement of rrad in cardiac function.
Modulating sialylation as therapeutic approach for preventing age-associated cardiac dysfunction
Authors: Tarantino A , Melgari D , Ghiroldi A , Lavota I , Rota P , Calamaio S , Piccoli M , Pappone C , Anastasia L 
Published: May 2024

Abstract
Funding Acknowledgements
Type of funding sources: Public hospital(s). Main funding source(s): Ministry of Health
Voltage-gated sodium channel Nav1.5 plays a pivotal role in the proper functioning of cardiac muscle cells. Sialylation, a post-translational modification involving the addition of negatively charged sialic acid moieties, is essential for Nav1.5 functionality. Altered sialylation has been observed in various cardiovascular diseases, including Chagas disease and congenital disorders of glycosylation affecting the heart. Furthermore, sialylation status has been shown to change during human adult life, impacting brain and muscle structural properties. Cardiac arrhythmias increase in prevalence with age, contributing to higher morbidity and mortality in older people. Using an aging mouse model, we observed a decline in cardiac sialic acid levels and NaV1.5 associated with the aging process. In compelling in vitro experiments on HEK293 cells and induced pluripotent stem cell-derived cardiomyocytes, diminished external sialic acid induces Nav1.5 channel internalization, activating endocytic pathways and leading to proteasome-mediated degradation. Intriguingly, proteomic analysis revealed that introducing a specific sialic acid precursor increased membrane protein trafficking, restoring Nav1.5 protein levels and the sodium current. Electrophysiological experiments demonstrated functional recovery and highlighted the rapid response, observable within just 2 hours of sialic acid stimulation. In summary, this study provides novel insights into the complex relationship between sialic acid and NaV1.5 channel trafficking, offering new perspectives and promising therapeutic strategies to prevent cardiac dysfunction in elderly.Modulating sialylation on NaV1.5 channelOur project goal
Human induced pluripotent cardiomyocytes as a model for cardiomyopathy with a Myozap gene mutation
Authors: Zakzook F , Maria Cherian R , Aalto-Setala K 
Published: May 2024

Abstract
Funding Acknowledgements
Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): 1- Central Department of Missions, Egyptian Ministry of Higher education and Scientific Research. 2- small fund from Tampere University.
The contractility of the heart is based on cardiomyocytes (CM). These cardiomyocytes are connected through a complex network of proteins forming the intercalated disc (ID) which is crucial to ensure mechanical and electrochemical coupling. Dilated cardiomyopathy (DCM) is a disease of the myocardium that can lead to heart failure and life-threatening arrhythmias. Up to 30% of DCM cases are caused by mutations in genes encoding proteins of the sarcomere (the basic contractile unit) or desmosome (a cell-to-cell adhesion structure) and the structural cytoskeleton. Myozap (Myocardium-Enriched Zonula Occludens-1-Associated Protein) is a distinctive cardiac-enriched ID protein. It is crucial for biomechanical stress adaption in CMs due to its outstanding role in the Rho/SRF (Serum Response Factor) signalling pathway. In this study, we have identified two Finnish families with DCM and carrying a novel variant in myozap. Our aim is to characterize myozap gene mutation-both heterozygous and homozygous truncating variants-as well as its implication in DCM. We have established patient-specific human induced pluripotent stem-cell lines and derived cardiomyocyte (hiPSC-CM) to study the disease at the cellular level. Myozap expression, compared to the control cell line, was evaluated by immunohistochemistry. Electrophysiological methods, including microelectrode array (MEA) and patch clamping, were implemented to measure various parameters with emphasis on those ones related to cell-cell connection e.g., the conduction velocity. Moreover, metabolic stress indicators, including oxygen consumption rate (OCR), were estimated by Seahorse. Additionally, qPCR and ELISA were performed to measure the colocalised and closely related genes to ID and protein level of troponin, respectively. Immunohistochemistry revealed downregulation and absence of myozap expression in heterozygous and homozygous cell lines, respectively. Compared to the control one, differences in BPM, FPD were observed. Metabolic analysis of the myozap mutant elucidated mitochondrial impairment compared to the control. Some SRF and sarcomeric target genes, including MYH7, MYH6 and Myl-2 were downregulated. On the contrary, apoptotic genes (e.g., caspase 3) were upregulated. Tight junction protein (TJP) was upregulated, and this is very importance since it is closely related to the myozap in the ID proteins network. Moreover, troponin protein level, measured by ELISA, showed upregulation in mutant myozap. In conclusion, our study provides a new insight into understanding myozap’s significance in cardiac biology and in cardiomyopathy pathophysiology.
anti-interleukin-18 as therapy against atherosclerosis
Authors: Van Ingen E , Foks A C , Bernabe Kleijn M N A , Kuiper J , Bot I 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): Established Investigator of the Dutch Heart Foundation

Background
Interleukin-18 (IL-18) is a pro-inflammatory cytokine and is secreted by macrophages after NLRP3-inflammasome activation. IL-18 is important for the induction of the Th1 response and overexpression of IL-18 aggravates atherosclerosis, suggesting that inhibition of IL-18 may act atheroprotective. In this study, we investigated the therapeutic potential of an IL-18 neutralizing antibody (anti-IL18) on atherosclerosis development.

Methods and results
Female ApoE-/- mice were fed a Western-type diet for 8 weeks and simultaneously received an antibody against IL-18 (100 µg/mouse; n=15) or a rat IgG2a control (100 µg/mouse; n=15) 3x per week intraperitoneally, after which the mice were sacrificed. First, we assessed macrophage phenotype in the peritoneal cavity. Expression of the proinflammatory markers CD80 (control: 46.3±1.4 *103; p&lt;0.05 vs. anti-IL18: 40.4 ±1.7 *103) and iNOS (control: 15.5±1.8 *102 vs. anti-IL18: 8.0±1.9 *102; p&lt;0.01) were reduced. In the aorta, MFIs of anti-inflammatory macrophage markers Arginase-1 (control: 389±99 vs. anti-IL18: 654±85; p=0.06) and CD206 (control: 55.2±2.4 *103 vs. anti-IL18: 62.8±3.7 *103; p=0.1) tended to be increased expression in the ascending aorta of anti-IL18 mice compared to controls. In addition, anti-IL18 treatment promoted the number of CD4 T cells (control: 20±1% vs. anti-IL18: 28±1%; p&lt;0.0001) in the mediastinal lymph node and increased it’s regulatory phenotype (Tregs; Foxp3+) of CD4 (control: 23±1% vs. anti-IL18: 26±1%; p=0.07) and CD8 T cells (control: 0.4±0.02% vs. anti-IL18: 0.9±0.09%; p&lt;0.0001). These effects however, did not translate into an effect on plaque size in the aortic root, as measured using an Oil-Red-O staining, which did not differ between the control and anti-IL18 treatment group (control: 47.1±2.4 *104 µm2 vs. anti-IL18: 43.4±2.3 *104 µm2).

Conclusions
Anti-IL18 treatment reduced the proinflammatory phenotype of peritoneal macrophages and promoted Tregs in the mediastinal lymph node. Further analyses on plaque stability and intraplaque macrophage subsets are currently being performed.
Asporin expression is associated with human atherosclerotic plaque integrity
Authors: Fountas P , Gialeli C , Sun J , Nitulescu M , Persson A , Bengtsson E , Edsfeldt A , Goncalves I 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Other. Main funding source(s): This work was supported by the Swedish Research Council, the Swedish Heart and Lung Foundation, Skåne University Hospital funds

Background
Calcification serves as a clinical marker of atherosclerotic plaque burden. Recent studies have also shed light on its intricate association with plaque vulnerability, likely depend on calcification structures and the surrounding environment. Asporin (ASPN) is a member of the small-leucine-rich proteoglycans that contains a unique and well-conserved stretch of aspartate (D) residues, which mimic the role of glycosaminoglycans. Via its D-repeat, ASPN can interact with calcium and several other extracellular matrix molecules, thus influencing collagen mineralization and TGF-β signaling. ASPN expression has been shown to be higher in asymptomatic compared to symptomatic atherosclerotic plaques, suggesting a potential protective role. However, the role of ASPN in maintaining plaque integrity remains unknown.

Purpose
Here we aimed to explore the role of ASPN in atherosclerosis, by investigating if ASPN was expressed in human carotid plaques and by determining if ASPN is associated with risk for postoperative cardiovascular events and death.

Methods
ASPN and TGF-β protein levels were measured by ELISA and Milliplex in 176 human plaque tissue homogenates obtained from the Carotid Plaque Imaging Project (CPIP) biobank. Plaque tissue sections were stained for ASPN, destabilizing (CD68, oil red O, glycophorin A) and stabilizing plaque components (Movat, α-actin). Plaque tissue RNA expression levels were assessed in 82 plaques using bulk RNA sequencing. Plaque levels of phosphate, elastin and collagen were measured using ELISA.

Results
ASPN protein levels were higher in plaques from asymptomatic patients compared to symptomatic patients (p=0.011). ASPN RNA levels correlated positively to RNA levels of the smooth muscle cell markers ACTA2, MYH11 and TAGLN. Moreover, protein levels correlated with plaque levels of phosphate (r=0.258, p=0.001), elastin (r=0.423, p&lt;0.001), collagen (r=0.248, p=0.002) and stability factors like the TGF-β family members, TGF-β1 (r=0.176, p=0.035), TGF-β2 (r=0.581, p&lt;0.001) and TGF-β3 (r=0.396, p&lt;0.001). Furthermore, plaque ASPN protein concentration, correlated inversely with a histological plaque vulnerability index (r=-0.265, p=0.0029), which is ratio between the plaque area of destabilizing (macrophages, lipids, intra-plaque hemorrhage) and stabilizing plaque components (collagen and smooth muscle cells). Additionally, above median ASPN levels were associated with lower risk for postoperative cardiovascular events (p=0.008) and death (p=0.009).

Conclusion
Our findings suggest that elevated levels of ASPN might have a crucial role in maintaining plaque stability, affecting plaque calcification.
Drug-interacting gene expression levels differ between atherosclerotic plaque subtypes
Authors: Palm K C A , Marsman J , Engels S , Boltjes A , De Borst G J , De Kleijn D P V , Den Ruijter H M , Van Steenbeek F G , Mokry M , Pasterkamp G 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): The Dutch Heart foundation
Recent transcriptomic-based clustering of human atherosclerotic plaques revealed that plaques have a diversity and complexity that cannot be accurately described by classical concepts. Previous plaque RNA sequencing that we performed in 654 lesions identified five major plaque types that reflect different mechanisms of progression of atherosclerotic disease. These include the following plaque subtypes: 0. Fibro-collagenous, 1. Intermediate, 2. Lipo-necrotic, 3. Fibro-inflammatory, and 4. Fibro-cellular. We hypothesize that these different plaque types may respond differentially upon drug treatment.

Objective
To assess differential expression levels of drug-responsive genes between the earlier described five plaque subtypes. For this in silico experiment, the differential expression of drug-responsive genes was examined after colchicine treatment in smooth muscle cells and endothelial cells.

Methods
To begin, a web-based search identified 46 published colchicine interacting genes. Then, colchicine was applied to cultured smooth muscle and endothelial cells to verify the publicly reported drug responsive genes. Next, we modelled the effect of colchicine on the five types of human atherosclerotic plaques for those genes that were considered drug responsive. This was achieved by projecting the in vitro derived expression changes onto the gene expression data from the carotid plaques of 654 patients who participated in the ATHERO-EXPRESS study. Besides colchicine, the same experimental approach was applied for atorvastatin and rofecoxib drug treatment. Finally, changes in drug responsive gene expression were validated with mice gene expression data.

Results
34 out of the 46 publicly known colchicine interacting genes were differently expressed between plaque types (Figure 1). These genes included established determinants of cell motility and atherosclerosis development (SCD, ITGB1, Tubulins). Furthermore, in-silico colchicine treatment revealed colchicine's strong effect on the atherosclerotic plaques' transcriptome. We are currently assessing if these perturbations may lead to shifts in plaque subtypes and if these changes are reflected in mice transcriptomic data.

Conclusion
The pilot data demonstrated major differences in drug-interacting gene expression levels in advanced atherosclerotic lesions. Therefore, novel and standard drugs used to treat atherosclerosis may have varying effects depending on the atherosclerotic plaque subtype.
Timing of capillary enlargement after adenoviral VEGF gene transfer does not support post-ischemic skeletal muscle recovery in hyperlipidemic mice
Authors: Wirth G , Juusola G , Laakso H , Korpisalo P , Yla-Herttuala S 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Finish Academy (Clinical Researcher 324070 and Research Project 339560). Finnish Foundation for Cardiovascular Research

Background
Transient capillary enlargement has been shown to be essential for successful post-ischemic tissue recovery as it may facilitate the return of arterial driving pressure by supporting collateral maturation. It also leads to an intussusceptive increase in capillary density that supports efficient oxygen delivery and muscle regeneration. Hyperlipidemia has been shown to blunt post-ischemic capillary reactivity and hinder skeletal muscle recovery from ischemia. Therefore, it can be hypothesized that correction of capillary reactivity might improve post-ischemic muscle recovery under hyperlipidemia.

Purpose
The aim of this study was to test the ability of angiogenic VEGF gene therapy in improving post-ischemic muscle recovery under hyperlipidemia.

Methods
AdVEGF gene transfer was performed into the calf muscles of aged, hyperlipidemic LDLR-/-ApoB100/100 mice after acute ischemia induction. The effects of AdVEGF on capillary phenotype, tissue edema, restoration of blood flow parameters, microvascular hemoglobin oxygenation and tissue damage/regeneration were evaluated for up to 29 days post-procedure as compared to AdLacZ control using immuno- and histological analyses, magnetic resonance, contrast-enhanced ultrasound and photoacoustic imaging, respectively.

Results
AdVEGF gene therapy was able to promote capillary enlargement that led to recovery of arterial driving pressure in ischemic LDLR-/-ApoB100/100 muscles. However, capillary enlargement induced by AdVEGF appeared late, lasted up to 29 days and did not promote intussusception. Consequently, the negative effect of long-lasting enlargement, i.e., massive tissue edema, delayed blood flow recovery and aggravated ischemic tissue damage.

Conclusion
Proper time of induction and duration of capillary enlargement are crucial to yield a functional vascular response supporting post-ischemic tissue recovery.
Unresectable cardiac metastasis from melanoma responds well to combination immunotherapy—a case report
Authors: Oh Ek Leone , Dias Peter , Al-Ogaili Zeyad , Otto Jacobus , Warburton Lydia 
Published: May 2024

Abstract

Background
Melanoma can metastasize to distal organs including the heart although presentation with a symptomatic cardiac metastasis is rare. The optimal management remains uncertain particularly in the era of immunotherapy.

Case summary
We report a case presenting with a large unresectable cardiac metastasis from melanoma that responded well to treatment with immunotherapy.

Conclusion
Melanoma can metastasize to the heart and is often challenging to diagnose. Combination immunotherapy can be an effective treatment option even in the setting of a symptomatic and unresectable cardiac metastasis.
Genomic analysis of Salmonella bacteriophages revealed multiple endolysin ORFs and importance of ligand-binding site of receptor-binding protein
Authors: Guzel Mustafa , Yucefaydali Aysenur , Yetiskin Segah , Deniz Aysu , Yaşar Tel Osman , Akçelik Mustafa , Soyer Yeşim 
Published: May 2024

Abstract
Salmonella is a prevalent foodborne pathogen causing millions of global cases annually. Antimicrobial resistance is a growing public health concern, leading to search for alternatives like bacteriophages. A total of 97 bacteriophages, isolated from cattle farms (n = 48), poultry farms (n = 37), and wastewater (n = 5) samples in Türkiye, were subjected to host-range analysis using 36 Salmonella isolates with 18 different serotypes. The broadest host range belonged to an Infantis phage (MET P1-091), lysing 28 hosts. A total of 10 phages with the widest host range underwent further analysis, revealing seven unique genomes (32−243 kb), including a jumbophage (&gt;200 kb). Except for one with lysogenic properties, none of them harbored virulence or antibiotic resistance genes, making them potential Salmonella reducers in different environments. Examining open reading frames (ORFs) of endolysin enzymes revealed surprising findings: five of seven unique genomes contained multiple endolysin ORFs. Despite sharing same endolysin sequences, phages exhibited significant differences in host range. Detailed analysis unveiled diverse receptor-binding protein sequences, with similar structures but distinct ligand-binding sites. These findings emphasize the importance of ligand-binding sites of receptor-binding proteins. Additionally, bacterial reduction curve and virulence index revealed that Enteritidis phages inhibit bacterial growth even at low concentrations, unlike Infantis and Kentucky phages.
Streptococcal pyrogenic exotoxin B is a superantigen that induces murine splenocyte proliferation and secretion of IL-2 and IFN-γ ex vivo
Authors: Rasooly Reuven , Do Paula , He Xiaohua , Hernlem Bradley 
Published: May 2024

Abstract
Streptococcus pyogenes is a significant human pathogen, producing a range of virulence factors, including streptococcal pyrogenic exotoxin B (SpeB) that is associated with foodborne outbreaks. It was only known that this cysteine protease mediates cleavage of transmembrane proteins to permit bacterial penetration and is found in 25% of clinical isolates from streptococcal toxic shock syndrome patients with extreme inflammation. Its interaction with host and streptococcal proteins has been well characterized, but doubt remains about whether it constitutes a superantigen. In this study, for the first time it is shown that SpeB acts as a superantigen, similarly to other known superantigens such as staphylococcal enterotoxin A or streptococcal pyrogenic exotoxin type C, by inducing proliferation of murine splenocytes and cytokine secretion, primarily of interleukin-2 (IL-2), as shown by cytometric bead array analysis. IL-2 secretion was confirmed by enzyme-linked immunosorbent assay (ELISA) as well as secretion of interferon-γ. ELISA showed a dose-dependent relationship between SpeB concentration in splenocyte cells and IL-2 secretion levels, and it was shown that SpeB retains activity in milk pasteurized for 30 min at 63°C.
Medicare Advantage enrollees’ reports of unfair treatment during health care encounters
Authors: Mathews Megan , Beckett Megan K , Martino Steven C , Brown Julie A , Orr Nate , Gaillot Sarah , Elliott Marc N 
Published: May 2024

Abstract
We investigated unfair treatment among 1863 Medicare Advantage (MA) enrollees from 21 MA plans using 2022 survey data (40% response rate) in which respondents indicated whether they were treated unfairly in a health care setting based on any of 10 personal characteristics. We calculated reported unfair treatment rates overall and by enrollee characteristics. Nine percent of respondents reported any unfair treatment, most often based on health condition (6%), disability (3%), or age (2%). Approximately 40% of those reporting any unfair treatment endorsed multiple categories. People who qualified for Medicare via disability reported unfair treatment by disability, age, income, race and ethnicity, sex, sexual orientation, and gender/gender identity more often than those who qualified via age. Enrollees dually eligible for Medicare and Medicaid or eligible for a Low-Income Subsidy (DE/LIS) reported unfair treatment by disability, income, language/accent, race and ethnicity, culture/religion, and sex more often than non-DE/LIS enrollees. Compared with White respondents, racial and ethnic minority respondents more often reported unfair treatment by race and ethnicity, language/accent, culture/religion, and income. Female respondents were more likely than male respondents to report unfair treatment based on age and sex.