bluuebunny/crossref_metadata_2025_split huggingface dataset:
FgF23 and soluble klotho effects on the myocardium: an ex vivo study
Authors: Sen P , D Alessio C , Hamers J , Sun Z , Dendorfer A , Merkus D 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): German Center for Cardiovascular research

Background
Fibroblast Growth Factor 23 (FGF23) and soluble Klotho (sKL) play a critical role in mineral homeostasis in the body by acting on the kidney to regulate phosphate levels. Circulating FGF23 levels rise in patients with kidney disease and dysregulation of the FGF23 –sKL- axis is a common phenomenon in uremic cardiomyopathy. Studies have shown that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4), while sKL mitigates these effects and may also exert an independent protective effect on the heart(1,2).

Purpose
We aimed to study the effects of FGF23 and sKL on myocardial contractility, calcium handling and molecular signaling pathways in the human heart using an ex vivo model of cultured contracting myocardial slices.

Methods
We obtained human heart slices from explanted hearts from patients undergoing cardiac transplantation at the LMU Klinikum Munich, Germany. The 300 µm thick slices were cultured under continuous stimulation (1 Hz, 3 ms pulse width) with 1 mN preload, and contractile function were recorded continuously. Slices with vehicle, recombinant FGF23 protein (100ug/ml), recombinant sKL protein (200ug/ml) and both FGF23((100ug/ml)+sKL (200ug/ml) were continuously monitored over 10 days. We checked for mRNA expression using RT PCR.

Results
Treatment of human left ventricular tissue with recombinant FGF23 protein resulted in enhanced mRNA expression of the FGFR4. sKL treatment alone induced downregulation of the FGFR4 gene and mitigated upregulation of FGFR4 in FGF23+ sKL treated slices. Post pause potentiation (PPP), assessed by pausing the stimulation of the slices for 120s and then measuring the relative height of the first beat after resumption of pacing, is an indirect measure of sarcoplasmic reticulum Ca2+ handling capacity. PPP was reduced in FGF23 treated slices and heightened in the slices with sKL compared to vehicle treated ones, indicating that FGF23 and sKL play a role in intracellular Ca2+ handling.

Conclusions
sKL and FGF23 have opposite effects on human cardiac muscle with sKL mitigating FGF23-induced upregulation of FGFR4. Furthermore, sKL improves, while FGF23 impairs myocardial calcium handling . As altered calcium handling may also be involved in hypertrophic signaling altered calcium handling may provide a means by which FGF23 promotes and sKL inhibits myocardial hypertrophy. sKL is advocated as a promising candidate in exploring it therapeutic role in FGF23 mediated cardiomyopathy. We intend to advance our understanding of sKL-FGF23signaling mechanisms by combining transcriptomic analysis with functional data in this model of myocardial slice culture.
Novel method for induction of myocardial infarction in rats by ultrasound guided electrocoagulation of the left anterior descending artery
Authors: Nordfalk M , Groenager S S , Flethoej M , Hunter I , Ripa R S , Kjaer A , Pedersen P J 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Private company. Main funding source(s): Minerva Imaging

Background
Murine models of acute myocardial infarction (MI) are commonly established through ligation of the left anterior descending artery (LAD). Despite inherent procedural challenges, including high mortality rates, limited improvements of the induction procedure have been made. Ultrasound-guided electrocoagulation of the LAD could be advantageous by improving animal welfare and decreasing procedural mortality.

Purpose
To investigate the feasibility of MI induction by ultrasound guided electrocoagulation in rats in comparison to traditional LAD ligation.

Methods
Thirty-eight female Sprague Dawley rats were randomized to MI by either of the two induction methods leading to 19 rats in each group. Infarct size and left ventricular ejection fraction (LVEF) were evaluated using positron emission tomography (PET) with [18F]fluorodeoxyglucose ([18F]FDG) 9 days post-MI. Myocardial infarct size was categorized as non-infarcted, mild, moderate, or severe based on [18F]FDG uptake. Animal welfare was assessed twice daily for the initial 48h post-surgery using a behavioural scoring system.

Results
There was no significant difference in the mortality rate of rats with MI induced by electrocoagulation compared to LAD ligation (16% vs 37%, P=0.27). Among the surviving animals both methods produced an equal number of myocardial infarcts (n=5). Electrocoagulation resulted in 1 mild, 2 moderate, and 2 severe MIs, while LAD ligation produced 1 mild, 0 moderate, and 4 severe MIs. Infarct size was similar in the electrocoagulation group compared to the LAD ligation group (mean±SD electrocoagulation: 15±5% vs ligation: 28±13%, P=0.13). The number of non-infarcted surviving rats in the electrocoagulation group was 11 compared to 7 in the LAD ligation group (P=0.33). The LVEF of infarcted rats showed no significant difference between groups (electrocoagulation: 47±8% vs ligation: 38±10%, P=0.14). The LVEF for non-infarcted rats was 53±5%. Post-surgical animal welfare appeared to be improved in the electrocoagulation group, however, the accumulated behavioural score did not reveal a significant difference (median [interquartile range] electrocoagulation: 0 [0-1] vs ligation 2 [0-2], P=0.19).

Conclusion
Induction of myocardial infarction by ultrasound-guided electrocoagulation is feasible in rats and should be considered as an alternative to LAD ligation. Further development is needed to improve the rate of success and consistency in infarct size.
Role of Orai1 and Ca2+-stimulated adenylyl cyclase 8 in the adverse cardiac remodeling after myocardial infarction
Authors: Falcon Boyano D , Calderon Sanchez E , Martin-Bornez M , Rosado J A , Ordonez A , Smani T 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación.

Background
It is well established that abnormalities in the regulation of the intracellular concentration of Ca2+ ([Ca2+]i) occurs in diseased heart such as under acute myocardial infarction (AMI). Recent studies have demonstrated that proteins related to the Store Operated Calcium Entry (SOCE), such as Orai1/2/3 and TRPC channels, play a role in the adverse cardiac remodelling. Previously, we demonstrated that the expression of Orai1 increased in heart subjected to ischemia and reperfusion (I/R) syndrome. However, the signalling pathway implicated in Orai1 regulation has not been addressed.

Objective
The aim of this study was to examine the molecular mechanism and signalling pathways involved in the of Orai1 upregulation after I/R in cardiomyocytes.

Materials and Methods
The experiments were performed in adult (ARVM) and neonatal rat ventricular cardiomyocytes (NRVM), in a rat model of myocardial I/R and in ventricular biopsies of patients with ischemic heart failure. Ca2+ studies were realized using microfluorimetric technique in FURA-2AM loaded cardiomyocytes. Immunofluorescence was used to investigate CREB activation using a specific phospho-CREB antibody and Orai1 and AC8 protein expression were analysed by Western Blot. This study was performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the local Ethics Committee on human Research and the Animal Research Committee.

Results
We found that under I/R diastolic level of [Ca2+]i was significantly increased in cardiac myocytes isolated from I/R hearts, which was inhibited by SOCE blockers. Moreover, we found that Orai1 and Ca2+-sensitive adenylyl cyclase 8 (AC8) are overexpressed in the heart isolated from I/R rat model, as compared to sham. Similarly, we found significant increase in the expression of Orai1 and AC8 in ventricle biopsies of post-infarction patients with HF. We also observed that I/R stimulated cAMP production, involving Orai1 and AC8 activation in rats, indicating that Ca2+ entry through Orai1 stimulated AC8. Moreover, I/R phosphorylated cAMP response element-binding protein (CREB) through PKA activation, but not via EPAC2 or ERK1/2. Interestingly, we found that CREB facilitated I/R-induced Orai1 upregulation and related exacerbated SOCE by its activation of Orai1 promoter under I/R. Finally, the intramyocardial administration of AAV9 holding AC8 shRNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i.

Conclusions
Our results show that the activation of Orai1/AC8/CREB axis plays a key role in the I/R-induced Orai1 upregulation, which participated in diastolic [Ca2+]i mishandling related to the adverse cardiac remodelling.
Long-range gene editing of LMNA
Authors: Rooijakkers A A M B , Pinto Y M , Tijsen A J M 
Published: May 2024

Abstract

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

Background
Mutations in the LMNA gene commonly affect the cardiac muscle tissue, which leads to the development of a severe dilated cardiomyopathy with life-threatening arrhythmias. The lack of response to conventional heart failure therapy in these patients indicates the urgent need for new therapies. Gene editing has the potential to become a curative treatment for these patients by correcting the genetic cause of the disease. Prime editing is a promising new gene editing technology, which uses a prime editing guide RNA (pegRNA) to target a specific genetic locus and creates edits using a reverse-transcriptase enzyme linked to a Cas9-nickase.

Purpose
Conventional prime-editing technology targets one mutation per pegRNA. However, with more than 600 disease-causing mutations described in LMNA, the feasibility of applying this technology to each mutation is limited. Therefore, we aim to target not a single, but a set of mutations with one single pegRNA.

Methods
The pegRNAs are optimised by selecting the optimal target regions, improving their stability and 3D structure. The in vitro editing efficiency is assessed using the fluoPEER reporter plasmid, transfected into HEK293T cells. Using flow cytometry, expression of mCherry fluorescent protein is detected following successful editing of the target mutation.

Results
Flow-cytometry analysis of treated cells revealed an editing efficiency of 12% with a long-range pegRNA targeting a full exon, compared to 15% with a conventional short pegRNA targeting the same mutation &lt;10bp downstream of the Cas9 nick site. This editing efficiency using the same long-range pegRNA was retained at 9% when editing at a distant position &gt;100bp downstream of the nick site. To increase the editing efficiency, we improved the stability of the pegRNAs by the addition of different 3’ structural motifs. This resulted in an increased editing efficiencies of 15% and 17% for nearby and distant mutations, respectively.

Conclusion
We show that it is possible to edit different mutations within a range of up to 120 bp with a single pegRNA. By optimisations of the pegRNA 3D structure and optimal selection of target locations we aim to further improve the editing efficiency of this system. This work could eventually provide a prime editing therapy able to cure disease in a subset of patients carrying different mutations in the same genetic region. For LMNA-associated diseases this would substantially reduce the number of pegRNAs that have to be developed to cover all mutations, greatly improving the feasibility of prime editing therapy.
Preoperative examination of inflammatory biomarkers in relation to the prognosis of postoperative complications after heart transplantation
Authors: Tarjanyi Z , Bernath D , Szappanos A , Teszak T , Kiraly A , Kugler S Z , Pallinger E , Nagy G Y , Sax B , Merkely B 
Published: May 2024

Abstract
Funding Acknowledgements
Type of funding sources: Public hospital(s). Main funding source(s): Semmelweis University
Heart transplantation (HTX) is the definitive therapy for end-stage heart failure. Although complications have drastically decreased with modern immunosuppressive therapy, surgical and anesthetic techniques, significant complications such as graft failure, acute rejection or infection must still be expected. The purpose of the study is to examine cytokine pathways linked to inflammation from direct preoperative serum samples, which can serve as prognostic factors in relation to septic events and mortality in the early postoperative period. The study was conducted using the blood samples of 102 patients who underwent HTX. Blood samples were taken from the patients immediately before HTX. The amounts of a total of 32 cytokines and related proteins were determined using LEGENDplex assays. After that, we correlated the biomarker amounts determined in the serum samples with the end points we indicated. In patients undergoing HTX who developed septic events during postoperative intensive care, the preoperative MMP2 serum concentration was significantly higher compared to other patients undergoing HTX (n=23, p=0.04). Independent predictors of mortality in the intensive care unit (n=5) were preoperative MRP8/14 (AUC=0.8), NGAL (AUC=0.75), IGFBP-4 (AUC=0.73), cystatin C (AUC=0.7) and the MMP2 (AUC=0.74) serum concentrations. Patients transplanted from left ventricular mechanical circulatory support (LVAD) (n=11) were examined as a separate group, where early postoperative mortality was significantly higher (n=4, p=0.02), the independent predictor of which was the concentration of IGFBP-4 (p=0.04 , AUC=0.73). Based on our results, it can be said that the differences in the preoperative concentration of certain inflammatory biomarkers that can be measured in the serum show correlations with the postoperative septic events and mortality of patients undergoing HTX.
Cardioprotective effects of Empagliflozin in cardiac volume overload-induced by aortocaval fistula in rats
Authors: Sykora M , Andelova K , Farkasova V , Honetschlagerova Z , Goncalvesova E , Tribulova N , Szeiffova Bacova B 
Published: May 2024

Abstract
Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Slovak Research and Development Agency under the contract no. 21-0410
VEGA grants no. 2/0006/23
Heart failure is characterized by reduction in cardiac output and the occurrence of malignant arrhythmias, thereby contributing significantly to high morbidity and mortality worldwide. Myocardial structural remodelling is a key factor contributing to the development of these life-threatening events and belongs to unsolved clinical issues challenging to investigate in animal models. The aim of our study was to explore the impact of cardiac volume overload (VO) on structural remodelling and electrical coupling protein, connexin43 (Cx43) in the left and right heart ventricles as well as the efficacy of treatment with SGLT2i, Empagliflozin. Aortocaval fistula (ACF) was surgically induced in 6-month-old Wistar rats, to develop VO during 4 weeks, followed by oral administration of Empagliflozin 10 mg/kg/b.w. daily for 5 weeks. At the end of the experiment, biometric parameters were registered and left and right heart tissue samples were snap-frozen and kept at -80°C for western blot analysis and microscopic examination. Findings revealed an increase of heart, left and right ventricular mass due to ACF-VO versus sham controls, which was associated, with eccentric myocardial hypertrophy. Noteworthy, Empagliflozin reduced heart, left and right ventricular mass. Levels of myocardial protein Cx43, that determine electrical coupling among cardiomyocytes, were reduced by ACF-VO, while normalized by treatment with Empagliflozin. Moreover, treatment attenuated ACF-VO-induced increase of interstitial collagen visualized by van Gieson staining. It was in line with the protein expression of MMP-2, which is involved in structural remodeling. The protein levels of PKCε, concerned/associated with modulation of Cx43 and extracellular matrix, were decreased due to ACV-VO but normalized after treatment with Empagliflozin. On the other hand, protein levels of PKCδ, involved in proapoptotic and prohypertrophic pathways, were increased and normalized by Empagliflozin. The changes were more pronounced in the right ventricle compared to the left heart ventricle. In summary, our pilot results point out the benefit of Empagliflozin due to the suppression of myocardial structural remodelling and preservation of Cx43 in the rat ACF-VO model. Furthermore, this Empagliflozin-induced cardioprotection was associated with the normalization of Cx43, MMP-2, and PKC levels independent of glucose control. However, further analyses are needed to reveal the molecular mechanisms of the cardioprotective effects of Empagliflozin.
Unbiased clustering and molecular characterisation of novel metabolic phenotypes in a heart failure cohort
Authors: Esenkova E , Koeck T , Valentini G , Wild P , Casiraghi E , Araldi E 
Published: May 2024

Abstract
Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DIASyM cluster
Heart Failure is a heterogeneous clinical syndrome characterized by the inability to pump enough blood and thus provide enough oxygen to the human body. The current classification of heart failure is based on the left ventricular ejection faction (LVEF). Although the classification clearly defines heart failure subtypes and stages, and clinical guidelines are available for their management, it is preferable to prevent the disorder or take control over it from its onset. Evidence suggests strong molecular connections between human metabolism and heart failure. Exploiting the unique and diverse molecular characteristics that drive heart failure could be an effective strategy for patient stratification, to identify new therapeutic targets and to personalize treatments.
The current research attempts to identify clinically relevant metabolic subgroups of individuals with the help of a similarity network-based approach, based on targeted proteomics data of a heart failure cohort. With Olink targeted proteomics technology, six protein abundance panels (547 distinct proteins) from 3063 subjects (1103 females) were obtained. Using nonlinear combination methods, the created networks were integrated into a single similarity network and then used for clustering with unbiased network-based approaches.
The results have distinguished the subgroups with worse outcome prediction, supported by significantly different clinical profiles. Surprisingly, the significant clinical parameters have shown the dominance of not only classical metabolic signatures (diabetes) but also parameters related to kidney and liver function. The differential protein abundance profiles were then compared among clusters to derive novel insights into clinical subtypes, and have highlighted a significant contribution of inflammatory proteins to the unbiased subtyping rationale.
The study attempted to improve previous patient stratification efforts, by having a large sample size with a broader variety of molecular variables. These results will pave the way to precision medicine, and will allow indirectly to integrate and/or adjust the analysis by adding clinically relevant social parameters such as lifestyle, parents’ anamnesis, demographics that are often left out due to their lower contribution at the pre-processing steps in comparison to more objective laboratory parameters. Previous studies have already clustered the heart failure population, based on various clinical and biochemical parameters. However, the clustering was guided by well-established metabolic parameters and thus, the results can be highly biased. The current project allows an unbiased, data-driven approach that holds more potential to identify novel and clinically relevant metabolic subgroups and to define new molecular targets.
Pirfenidone treatment reduces cardiac allograft vasculopathy in a mouse model
Authors: Graebner C , Scheunchen M , Theil F , Kuckhahn A , Vogg N , Gessner A , Fritz N , Ramsperger-Gleixner M , Dewald O , Heim C 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): ISHLT research grant

Objectives
Chronic rejection in the form of cardiac allograft vasculopathy after heart transplantation remains without effective treatment. Main characteristics of cardiac allograft vasculopathy are immigration of leukocytes and proliferation of smooth muscle cells - collectively resulting in development of a neointima narrowing the vessel lumen and leading to graft ischemia. Pirfenidone is an anti-fibrotic drug with anti-inflammatory properties that is already approved for treatment of idiopathic pulmonary fibrosis.
The aim of this study was to evaluate whether application of pirfenidone leads to reduced luminal occlusion corresponding with decreased chronic rejection in a mouse transplant model.

Methods
Murine thoracic aortas were transplanted into the abdominal aorta of CBA mice (H2k). For allogeneic transplantation, C57BL/6 mice (H2b) were used as donors (n = 8 / group). After transplantation, mice were fed a 0.5% pirfenidone diet versus conventional diet. Preliminary tests were performed determining the blood pirfenidone level by mass spectrometry throughout the study period on days 1, 3, 7, 14, 21 and 28, respectively, as well as after obtaining the aortic grafts on day 30 for histological analysis.

Results
Preliminary tests confirmed the successful uptake of pirfenidone by achieving constant plasma levels of pirfenidone and its metabolite 5-carboxy-pirfenidone over the whole examination period.
Pirfenidone treated murine aortic grafts showed significantly reduced neointima formation compared to control allografts (luminal occlusion: 32.92 % ± 13.02 % vs. 42.44 % ± 19.81 %, p=0.046). As expected, syngeneic aortic grafts with and without pirfenidone treatment showed no luminal occlusion.

Conclusion
The results indicate that pirfenidone application after aortic transplantation reduces neointima formation and thus chronic rejection in a mouse aortic transplant model. Further analyses of underlying mechanisms are currently underway.