bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of IDH1 and IDH2 in macrophages induces pathological phenotypes in cardiomyocytes
Authors: Pennoyer P , Shumliakivska M , Fleck F , Zanders L , Zeiher A , Dimmeler S
Published: October 2024

Introduction

The clonal expansion of hematopoietic stem cells can be induced by a variety of age-related somatic mutations, a phenomenon known as "clonal hematopoiesis of indeterminate potential" (CHIP). CHIP is associated with an increased incidence and poor outcomes in patients with cardiovascular disease. The mechanisms underlying the crosstalk between CHIP cells and cardiac tissue are yet to be elucidated. Rare mutations in the metabolism-regulating isocitrate dehydrogenases encoding genes IDH1 and IDH2 have been identified as AML-like and cardiovascular high-risk CHIP-driving mutations in patient cohorts. However, the potential paracrine effects of these mutations in immune cells on cardiac tissue are unknown. Therefore, we assessed the impact of IDH1 and IDH2 mutations on the paracrine activities of macrophages.

Methods and Results

To gain first insights into the functional consequences of the IDH mutations, we used the AlphaMissense algorithm, which predicts the impact of mutations on protein function. IDH1 and IDH2 mutations identified in our patient cohorts were predicted to induce pathological alterations of protein function with a score >0.99. Therefore, we assessed the effects of silencing of the two genes in primary human CD14+ M0 macrophages on the paracrine activity on neonatal rat cardiomyocytes. Transfection with siRNA pools targeting IDH1 and IDH2 to mimic CHIP-driving conditions reduced mRNA abundance by 78% and 73%, respectively (both p<0.05). To assess the impact of IDH-silencing on cardiovascular cells, cardiomyocytes were treated with the supernatant of the macrophages. Interestingly, supernatants of IDH1 silenced macrophages significantly increased apoptosis of cardiomyocytes (8.6-fold increased expression of cleaved caspase-3; p<0.05) and reduced beating frequency. In addition, supernatants of IDH1 silenced macrophages induced a 1.5-fold increase in cardiomyocyte hypertrophy (p<0.01), whereas silencing of IDH2 showed no effect on cardiomyocyte hypertrophy and apoptosis. However, supernatants of both IDH1 and IDH2 silenced macrophages resulted in significantly increased numbers of bi- and multinucleated cardiomyocytes.

Conclusion: Loss of isocitrate dehydrogenases in human macrophages affects cardiomyocyte functions in a paracrine fashion. Particularly silencing of IDH1 in macrophages introduced cardiomyocyte hypertrophy, apoptosis, and bi- and multinucleation, and reduced contractile function. Ongoing studies will address how IDH1 and IDH2 silencing-induced alterations in macrophages control their paracrine activity on cardiomyocytes.
Evaluation of serum levels of sCD40, sCD40L and s4-1BB in advanced heart failure and after heart transplant
Authors: Monteiro V , Torres L , Carneiro R , Cunha C , Figueira F , Matta M , Silva I , Tchaick R , Paola A
Published: October 2024

Heart failure is one of the most important problems in public health. Although big advances had been reached in treatment, the prognosis of advanced heart failure is still reserved and the heart transplant is the main therapy for this stage of disease. Allograft rejection is responsible for mortality in transplantation patients and the diagnosis of this complication is made through an invasive diagnostic method – the cardiac biopsy. The inflammatory response has been described in cardiovascular disease and in allograft rejection process.

The objective of this study is to evaluate levels of sCD40, sCD40L and s4-1BB (inflammatory biomarkers) in advanced heart failure and post heart transplant with allograft rejection comparing to normal individuals and post heart transplant patients with no allograft rejection.

The study population comprised 38 patients undergoing heart transplant in our center. Fifteen patients had biomarkers dosed before heart transplant and twenty three patients had dosed just after procedure. In this group, six patients were enrolled in the study for clinical suspicion of rejection. Nineteen health persons composed the control group with age between 18 and 49 years. Plasma dosage of sCD40, sCD40L e s4-1BB was made by enzyme linked immunosorbentassay (ELISA). Mann-Whitney test, Wilcoxon and t test were used to analyse dates. Statistical analysis used GraphPad®Prism6 and the value p< 0,05 was considered statistical significant.

Allograft rejection occurred in 6,26% of studied patients. There was no statistical difference in age, sex, comorbities as hypertension, diabetes or etiology of cardiomyopathy. Serum levels of sCD40, sCD40L e s4-1BB were higher in patients with advanced heart failure comparing to controls (p< 0,0001). There was no difference in these levels of sCD40 (p = 0,74) e sCD40L (p = 0,44) between groups with and without rejection. Levels of s4-1BB were significantly lower in patients with allograft rejection (p= 0,02). The sCD40/PCR and s4-1BB/PCR indexs were significantly higher in the group of patients with allograft rejection (p =0,02 e p= 0,0001).

We concluded that sCD40, sCD40L e s4-1BB are biomarkers of severe heart failure. The serum level of s4-1BB could be a potential biomarker of allograft rejection and the association of sCD40 and s4-1BB with PCR could be a better strategy to this.
Detecting epicardial connection through intercaval bundle using intra-atrial activation sequence during right pulmonary vein pacing
Authors: Chikata A , Kato T , Usuda K , Fujita S , Usuda K , Matsuura S , Maruyama M , Otowa K , Kusayama T , Tsuda T , Hayashi K , Takamura M
Published: October 2024

Background

An epicardial connection (EC) through the intercaval bundle (EC-ICB) between the right pulmonary vein (RPV) and the right atrium (RA) is one of the reasons for the need of carina ablation for PV isolation.

Purpose

We evaluated the intra-atrial activation sequence during RPV pacing after failure of ipsilateral RPV isolation and sought to identify specific conduction patterns in the presence of the EC-ICB.

Methods

This study included 223 consecutive patients undergoing initial catheter ablation of atrial fibrillation. If the RPV was not isolated by circumferential ablation or reconnected during the waiting period, an exit map during mid RPV carina pacing was created. If the earliest site of the exit map was the RA, the patient was classified into the EC-ICB group. During the exit map, intra-atrial activation sequence and RPV-HRA time were evaluated.

Results

First-pass isolation of the RPV was not obtained in 36 patients (16.1%), and 22 patients (9.9%) showing reconnection. Among them, 12 patients were classified into the EC-ICB group and 28 patients into the Non-EC-ICB group after excluding multiple ablation lesion sets or incomplete mapping. Intra-atrial activation sequence showed different patterns between the two groups. RPV-HRA time was significantly shorter in the EC-ICB group compared to the Non-EC-ICB group (69.2 ± 15.2 ms v.s. 148.6 ± 51.2 ms. P<0.001), and RPV-HRA time<89.0ms was highly predictive for existence of the EC-ICB (sensitivity 91.7 %, specificity 89.3 %).

Conclusions

The EC-ICB can be effectively detected by intra-atrial sequencing during RPV pacing and RPV-HRA time<89.0ms was highly predictive.
Oxct1 regulates cardiomyocyte PANoptosis in acute myocardial infarction via succinylating Tfam
Authors: Li X , Ji Z J , Tao Z X , Huang R , Zhang R , Li Y J , Ma G S
Published: October 2024

Background

Myocardial ischemia leads to various forms of programmed cell death in cardiomyocytes, resulting in acute myocardial infarction (AMI) and the development of heart failure in the long term. PANoptosis is a unique cell death regulatory mechanism orchestrated by the PANoptosome complex, which regulates and intersects multiple forms of programmed cell death. Oxct1 has been demonstrated to play crucial roles in tumors, pressure-overloaded hearts, and cardiac endothelial cells. However, whether Oxct1 is involved in the regulation of PANoptosis and correlated mechanisms during AMI remains unclear.

Purpose

To elucidate the role of Oxct1 in cardiomyocytes during AMI and uncover the potential mechanisms.

Methods

The myocardial infarction model induced by anterior descending branch ligation and the primary cardiomyocytes hypoxia model were employed in our experiments. Small interfering RNA (siRNA) and adenovirus were constructed to knock down and overexpress Oxct1 for altering its expression in cardiomyocytes in vitro. Cardiomyocyte-specific knockout mice were constructed to alter Oxct1 expression in vivo. PANoptosis characteristic measurement, including Z-DNA binding protein 1(Zbp1), pyroptosis markers, apoptosis markers, necroptosis markers, ROS, and mitochondrial morphology, were performed to verify the ability of Oxct1 to regulate the PANoptosis of cardiomyocytes. Cardiac morphological, structural, and functional changes were measured to assess the effect of Oxct1 on AMI. RNA sequencing was performed to identify downstream pathways. Immunoprecipitation +Mass spectrometry and co-immunoprecipitation were performed to identify the specific binding factors of Oxct1.

Results

We found that the expression of Oxct1 was significantly elevated after AMI. Besides, gain- and loss-of-function experiments were constructed both in vitro and in vivo. Functionally, Oxct1 knockdown significantly enhanced cardiomyocyte vitality, reduced ROS production and mtDNA cytoplasmic release in hypoxic cardiomyocytes. In vivo, Oxct1cko mice manifested reduced infarct size, improved cardiac function and alleviated cardiac fibrosis by mitigating PANoptosis. However, Oxct1 overexpression resulted in the opposite effects. Afterwards, RNA-seq analysis results showed that cytosolic DNA-sensing pathway was significantly enriched in Oxct1 knockdown cardiomyocytes. Mechanistically, Oxct1 could bind to mitochondrial transcription factor A(Tfam) and increased its succinylation, promoting Tfam degradation. Downregulation of Tfam resulted in ROS production and mtDNA damage, while mtDNA released into cytoplasm could activate Zbp1 and induce cascade reaction of PANoptosis.

Conclusion

This study demonstrates that Oxct1 promotes Tfam degradation through succinylation, exacerbating ROS production, mtDNA damage and cytoplasmic release, ultimately leading to cardiomyocyte PANoptosis in AMI.
Clec4e deletion in cardiac resident cells leads to smaller infarct size and better cardiac remodeling following myocardial ischemia-reperfusion injury
Authors: Algoet M , Oosterlinck W , Pusovnik M , Himmelreich U , Gsell W , Veltman D , Gillijns H , Caluwe E , Blazejczyk K , Janssens S
Published: October 2024

Introduction

Clec4e-signalling in cardiac resident cells and circulating immune cells stimulates recruitment of proinflammatory leukocytes to infarcted myocardium following ischemia reperfusion injury (I/R) and contributes to ischemic myocardial damage. Clec4e deletion is associated with reduced cardiac injury and better left ventricular structural and functional remodeling. The mechanisms of this cardioprotective effect remain unknown.

Research question: We investigated whether Clec4e gene function in cardiac resident cells vs infiltrating immune cells mediates cardioprotection during myocardial ischemia-reperfusion injury.

Methods

We generated chimeric mice by performing bone marrow transfer in wild type (WT) C57Bl6/J and C57Bl6/J Clec4e-/- mice. Following total body irradiation using 9.5 Gy in 8w-old WT and Clec4e-/- receptor mice, 5 x 10^6 bone marrow cells originating from Clec4e-/- and WT donor mice, respectively, were injected IV. Four weeks later, mice were anesthetized and subjected to ischemia reperfusion injury (I/R) by transient occlusion of the LAD for 60 min. Troponin levels were measured at 90 min post I/R and 7T-magnetic resonance imaging with late gadolinium enhancement (LGE) for infarct size evaluation was performed at 24 hours and 28 days post I/R without LGE. Mice were euthanized at 28 days post I/R and the heart and long shaft bones were processed for postmortem immunohistochemistry and analysis of BM chimerism.

Results

Immunohistochemical analysis of CLEC4E expression on a bone marrow smear 8 w post bone marrow transfer confirmed successful BM chimerism (7% vs 99%, p<0.01). TnI levels at 90 minutes and LGE assessed infarct size on MRI at 24 hours were not significantly different between groups (n=17 in both groups) (42.1 vs 48.4 ng/ml, and 25.2 vs 31.5% of LVmass in Clec4e-/- mice with WT bone marrow versus in WT mice with Clec4e-/- BM, respectively) suggesting similar myocardial damage . After 28 days, mean LV end diastolic volumes (43µL vs 55µL), and mean end systolic volumes (13µL vs 25µL) were significantly (p<0,05) smaller in Clec4e-/- mice with WT bone marrow resulting in better preserved global LV function (LVEF 62% vs 47%, p<0,05, fig A). Infarct size, semiquantitatively assessed using planimetry on Sirius red stains showed a smaller infarct size in the Clec4e-/- mice with WT bone marrow (6.9% vs 19.8%, p<0.01). Figure B shows a representative example of LGE infarct size to scar tissue on MRI and Sirius red staining.

Conclusion

Clec4e gene deletion in cardiac resident cells, but not in infiltrating hematopoietic immune cells is associated with better functional and structural remodeling after myocardial I/R. Inhibition of Clec4e signaling via blocking antibodies or small molecule inhibitors in cardiac myocytes might be a promising cardioprotective strategy.
The development of small-molecule ERBB4 agonists as a treatment for heart failure
Authors: Cools J , Feyen E , Van Den Bogaert S , Goovaerts B , Fu Y , Wuelfers E , Roderick L , Vandersickel N , De Keulenaer G W , Segers V F M
Published: October 2024

The neuregulin-1 (NRG1)/ERBB4 signaling pathway has emerged as a cardioprotective pathway and is a promising target for the treatment of chronic heart failure. Activation of ERBB4 signaling is known to decrease cardiomyocyte cell death and hypertrophy, and fibroblast collagen synthesis. Recombinant NRG1 (rNRG1) is currently tested in phase III clinical trials for heart failure, but its need for intravenous administration is a disadvantage. In an attempt to circumvent this, we hypothesized that small-molecule-induced activation of ERBB4 is feasible and would recapitulate the effects of its natural ligand on myocytes and fibroblasts.

To this end, we screened 10,240 compounds for their ability to induce homodimerization of ERBB4. We identified a series of 8 similar compounds (named EF-1 – EF-8) that concentration-dependently induced ERBB4 dimerization, with EF-1 being the most potent and effective compound (n = 4-5 independent repeats in each group; Emax = 27.9 ± 4.8% relative to NRG1, EC50 = 10.5 ± 4.5 x 10-6). EF-1 showed neither cytotoxicity nor increased cell proliferation of tumor cell lines. In vitro, EF-1 significantly decreased in a concentration-dependent manner hydrogen peroxide–induced cardiomyocyte cell death (n = 4 independent repeats in each group; P<0.0001 compared to siERBB4, Fig. 1A), angiotensin-II (AngII)-induced cardiomyocyte hypertrophy (n = 20 individual cardiomyocytes in each group; P<0.0001 compared to AngII/vehicle, Fig. 1B), and collagen expression in cultured human fibroblasts (n = 3 independent repeats in each group; P=0.03 compared to siERBB4, Fig. 1C). The observed effects in cultured cardiomyocytes and fibroblasts could be abrogated by siRNA targeting ERBB4, indicating that they were mediated by ERBB4. Moreover, when used in vivo, EF-1 (2 mg/kg/day) significantly decreased AngII-induced left ventricular Col1a1 and Col3a1 mRNA expression (n = 4-5 mice in each group; P=0.02 and P=0.004 compared to AngII/vehicle, respectively) and inhibited AngII-induced myocardial total and interstitial fibrosis in wild-type mice (n = 4-5 mice in each group, Fig. 2), but not in Erbb4-null mice. Moreover, EF-1 decreased acute cardiotoxicity (assessed by troponin release) in wild-type mice treated with doxorubicin (DOX; n = 8-9 mice in each group; P<0.0001 compared to DOX/vehicle), but not in Erbb4-null mice.

In conclusion, we show that small-molecule-induced ERBB4 dimerization and activation is feasible, and recapitulates anti-fibrotic and cardiomyocyte protective effects in the heart in an ERBB4-dependent manner, both in vitro and in vivo. This could be the start for the further development of small-molecule ERBB4 agonists as a novel class of drugs to treat heart failure. Cardiomyocyte-protective effect in vitroAnti-fibrotic effect in vivo
Direct in vivo cardiac reprogramming with circular RNA of single transcription factor delivered by fibroblast-targeting lipid nanoparticles
Authors: Wang Q Z , Wang Z M , Huang Z Y , Ge J B
Published: October 2024

Background

Adult mammalian cardiomyocytes (CMs) hold limited capacity for self-regeneration, thus leading to permanent loss of functional myocardium once suffering from injury, especially myocardial infarction (MI). Therefore, cardiac regeneration therapy has been regarded as a potential path to help restore damaged tissue. Direct cardiac reprogramming is one of the most promising ways to boost cardiac regeneration as it could convert resident cardiac fibroblasts into functional induced cardiomyocytes (iCMs). However, difficulty of delivery has been one of the biggest problems that hinder the further clinical translation of this technique such as concerns of mostly used retrovirus vectors and lack of fibroblast-specific targeting ability. In our previous research, we have found that after knocking down of Ybx1 gene, single transcriptional factor Tbx5 could induce cardiac reprogramming of fibroblasts without adding traditional Mef2c and Gata4. But whether this cocktail could achieve in vivo direct cardiac reprogramming still remains to be explored.

Objective

We planned to develop fibroblast-targeting lipid nanoparticles (LNP) to delivery circular RNA(circRNA) of Tbx5 and small interfering RNA(siRNA) of Ybx1 to realize in vivo cardiac reprogramming in a mouse model of myocardial infarction.

Results

We first screened different formulations of tdTomato mRNA delivered-LNP both in primary neonatal cardiac fibroblasts and in the myocardial infarction model and found that formulation XF4d could realize specific selectively uptake by cardiac fibroblasts instead of cardiomyocytes. Then we succeeded in synthesizing XF4d LNP delivering Tbx5 circRNA and Ybx1 siRNA (LNPXF4d@Tbx5+siYbx1) and validated its stability at 4°C. Next, after infecting LNPXF4d@Tbx5+siYbx1, primary neonatal CFs and mouse embryonic fibroblasts could be reprogrammed into functional iCMs. We further measured the protein expression of Tbx5 and Ybx1 in CFs after LNPXF4d@Tbx5+siYbx1 treatment. What’s more, we used fibroblast-specific lineage tracing mice (Fsp1Cre/tdTomato mice) to prove the successful conversion of resident CFs into iCMs in the mouse model of myocardial infarction by intramyocardial injection of LNPXF4d@Tbx5+siYbx1. At last, we found that LNPXF4d@Tbx5+siYbx1 could help improve cardiac function after injury and reduce fibrosis, as measured by echocardiography and Masson’s trichrome staining.

Conclusion

We suggest that our fibroblast specific LNPs delivering single transcriptional factors could a promising method to realize in vivo cardiac reprogramming and help treat myocardial injury, with high possibility of clinical translation and less biosafety concerns.Graphical Abstract
Upregulation of the sodium potassium pump current supresses ectopic activity after stem cell delivery in the human-based heart after myocardial infarction
Authors: Riebel L , Wang Z , Martinez-Navarro H , Dasi A , Trovato C , Rodriguez B
Published: October 2024

Background

Cell therapy is being explored to restore the damaged and intrinsically irreparable human heart. Whilst preclinical studies showed improvements in cardiac function after injury, they also highlighted a critical pro-arrhythmic window when delivered cells produce ectopy-induced ventricular tachycardia. Recent experiments investigating the cells in vivo maturation suggested upregulation of the inward rectifier potassium current (IK1) and knock-out of the funny current (If) and sodium calcium exchanger (INaCa) to supress the arrhythmias[1].

Purpose

Whilst promising, such modifications may impact the cells’ calcium dynamics and ultimately, their efficacy. The goal of this study is to identify novel ionic targets that improve therapy safety whilst maintaining efficacy by employing the unique predictive capabilities of modelling and simulation.

Methods

For this, we used our established modelling and simulation framework of cell therapy in the chronically infarcted human heart with Purkinje, validated against experimental and clinical data from the action potential to the ECG. We incorporated experimentally observed gene expression of in vivo cell maturation[1] into our framework to reproduce human pluripotent stem cell-induced cardiomyocyte (hPSC-CM) electrophysiology at day 0 and day 14 after injection. To consider variability amongst experimentally reported hPSC-CMs, we also created a fast-beating hPSC-CM model by upregulating the SERCA pump current, If and INaCa.

Results

Our results showed that while day 0 hPSC-CMs did not cause spontaneous activity, day 14 hPSC-CMs produced ectopic beats in the ventricles every 1.5s. The rapid beating hPSC-CM phenotype at day 14 elicited tachycardia through spontaneous beating and intermittent re-entrant wavefronts (see Figure 1). Next, we demonstrated 65% upregulation of the sodium-potassium pump current (INaK) together with a 50% increase in IK1 and knock-out of If as a novel pathway to quiescence. Importantly, our simulations highlighted that compared to INaCa knock-out, an increase in INaK allowed for physiological calcium transients.

Conclusion

To conclude, in this work we have utilised modelling and simulation of the injured human heart to 1) capture pre-clinically observed arrhythmias after cell therapy and 2) identify INaK upregulation as a novel target to mitigate safety concerns, whilst preserving efficacy.
Exploration of vitamin D hydroxy metabolites C3 epimers in cardiovascular patients: comparative cross-sectional study
Authors: Abouzid M , Kruszyna L , Kagan L , Glowka F , Mikulska A A , Filipowicz D , Karazniewicz-Lada M
Published: October 2024

Background

Research on vitamin D deficiency has recently surged in popularity. This issue has been acknowledged as a widespread problem, often called a "pandemic", and approximately 90% of the Polish population suffers from vitamin D deficiency. The 3-epi-25(OH)D2 and 3-epi-25(OH)D3 metabolites are stereoisomers of 25(OH)D2 and 25(OH)D3 metabolites, and they may be accidentally measured when determining 25(OH)D levels and result in overestimating metabolite concentrations.

Purpose

Our study aims to identify vitamin D hydroxy metabolites (25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and their epimers 3-epi-25-hydroxyvitamin D2 and 3-epi-25-hydroxyvitamin D3) in patients with cardiovascular disease and healthy volunteers. To our knowledge, no similar studies have been conducted on this topic.

Method

We included 27 adult patients with venous thrombosis (fourteen males and thirteen females, aged 64.11±14.84 years) and 35 healthy volunteers (thirty-three females and two males, aged 36.37±12.29 years). We used our validated UPLC-MS/MS method to measure the concentrations of 25(OH)D2, 25(OH)D3, 3-epi-25(OH)D2, and 3-epi-25(OH)D3. Statistica 13.3 with Plus Kit 3.0 was used for the statistical assessment. Normal variables were represented as mean and standard deviation, and non-normal variables as the median and interquartile range. A P-value < 0.05 was considered statistically significant for all tests.

Results

Patients had significantly lower levels of epi-25(OH)D2 than the healthy control group (0.6 [0.4] ng/mL vs. 2.2 [1.3-4.3] ng/mL, p = 0.004). Similarly, they exhibited significantly lower levels of %epi-25(OH)D2 (8.6 [6-22.3] % vs. 39.4 [18.5] %, p = 0.035). Females had higher levels of 25(OH)D3 than males (26.5 [16.2-37.9] ng/mL vs. 18.8 [7.5] ng/mL, p = 0.04). They also exhibited higher levels of total 25(OH)D3 (30.3 [15.6] ng/mL vs. 19.4 [7.8] ng/mL, p = 0.016). Higher age (OR 1.12, 95% CI 1.07 to 1.18), being male (OR 17.77, 95% CI 3.54 to 89.3), and lower % 3-epi-25(OH)D2 (OR 0.92, 95% CI 0.84 to 0.998) were univariate predictors for patients with venous thrombosis.

Conclusion: Demographic factors within a given population may affect the 25(OH)D3 concentration. The results indicate that vitamin D epimeric metabolites, particularly epi-25(OH)D2, and their percentage were different in patients compared to healthy volunteers. Additional studies involving a larger population are necessary to fully comprehend the relationship between these vitamin D epimeric metabolites and cardiovascular health
The BET inhibitor Apabetalone protects against heart failure with preserved ejection fraction by suppressing myocardial inflammation
Authors: Costantino S , Gorica E , Mohammed S A , Telesca M , Mongelli A , Herwig M , Ambrosini S , Ruschitzka F , Hamdani N , Paneni F
Published: October 2024

Background

Posttranslational histone modifications, play a major role in cardiac hypertrophy and dysfunction. Apabetalone (APA), a selective inhibitor of bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs.

Purpose

We sought to investigate whether APA could be beneficial in cardiometabolic heart failure with preserved ejection fraction (cHFpEF).

Methods

Mice were subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce cHFpEF. Histology, mouse echocardiography (Vevo3100) and Treadmill exhaustion test were performed. Unbiased gene expression profiling by PCR array and proteomics analysis (Olink) was employed in left ventricular (LV) myocardial specimens from HFpEF mice and control animals. Cultured cardiomyocytes (CMs) treated with palmitic acid (PA) were used as an in vitro model of metabolic stress. cHFpEF mice were chronically treated with the BET inhibitor APA (150mg/kg/day) or vehicle (DMSO). In order to translate our findings to the human setting, passive stiffness of skinned CMs collected from cHFpEF patients was assessed before and after APA treatment.

Results

HFpEF mice displayed LV hypertrophy, diastolic dysfunction, myocardial fibrosis, lung congestion and impaired exercise tolerance as compared to controls. Interestingly, diastolic dysfunction - assessed by E/A ratio and isovolumic relaxation time (IVRT) - and lung congestion were significantly reduced in HFpEF mice treated with APA, while exercise tolerance was improved. Transcriptomic analysis in PA-treated CMs and LV mouse specimens from cHFpEF mice showed a profound deregulation of genes controlling inflammation, namely IL-6, TNF-alpha, and IL-1beta. ChIP assays showed BRD4 occupancy on the promoter of several inflammatory genes, including IL6. Treatment with APA suppressed most of inflammatory genes, with a pronounced effect on IL6 expression. Moreover, APA reduced circulating levels of several inflammatory chemokines. The beneficial effects of APA were explained by modulation of IL-6/CaMKII/STAT3 pathway both in cHFpEF hearts and PA-treated CMs. In skinned CMs from cHFpEF patients, both APA and IL6 blockade were able to attenuate passive stiffness.

Conclusions

Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in patients with cHFpEF.
The ghrelin receptor agonist AC01 improves cardiac function in non-human primates and in mice with heart failure by increasing cardiomyocyte contractility
Authors: Pironti G , Hage C , Carlstrom M , Champeroux P , Stahlberg M , Stromberg P , Lovati E , Andersson D C , Lund L H
Published: October 2024

Background

Current therapy for Heart Failure (HF) includes neurohormonal antagonist and modulator drugs that improve remodelling, symptoms and clinical outcomes. However, these drugs do not target the underlying problem of reduced contractility. Improving cardiac contractility without harmful consequences is an unmet need in heart failure therapy. The human peptide hormone ghrelin increases cardiac output in HF patients and increases contractility in murine cardiomyocytes.

Purpose

We aim to study the cardiovascular effects of a novel oral small molecule ghrelin receptor agonist, AC01, in in vivo and ex vivo mouse models and in non-human primates.

Methods

HF mice (N=11) underwent pressure-volume loops analysis of cardiac functional and hemodynamic to assess the effects of intravenous AC01 (N=7) or placebo (N=4). Primary cardiomyocytes isolated from HF (N=3) and SHAM (N=5) mice, were treated with combinations of vehicle, AC01, ghrelin receptor antagonist (D-Lys-3-GHRP-6) and pertussis toxin (PTX, a Gαi signalling inhibitor). Live cell imaging was performed to assess cardiomyocyte fractional shortening and Ca2+ signalling, while biochemistry assays were employed to measure PKA activity, and phosphorylation of cardiac Troponin I (cTnI). Cynomolgus monkeys (N=6) were instrumented with devices to measure pulse contour, central aortic blood pressure and ECG devices. Cardiovascular effects of AC01 following single oral dosing were assessed through pulse contour hemodynamic modelling and analysis of autonomic system activation.

Results

In HF mice, AC01 significantly improved cardiac output (p< 0.01), stroke volume (p< 0.001), and ejection fraction (p< 0.001), and increased nominally the end-systolic pressure-volume curve relationship. In cardiomyocytes, AC01 dose-dependently improved fractional shortening (p< 0.01) without increasing Ca2+ transient amplitudes. AC01 reduced PKA activity (p< 0.05) and reduced phosphorylation of cTnI at Serine 23-24 (p< 0.01). These effects were blocked by pre-treatment with either D-Lys-3-GHRP-6 or PTX. In monkeys, oral AC01 sustainably increased cardiac output (p< 0.05) and stroke volume (p< 0.05) and reduced heart rate without altering central systolic blood pressure or causing tachycardia or arrhythmia.

Conclusions

AC01 improved CO, SV, and EF in HF mice by increasing contractility in a load-independent fashion. AC01 increased cardiomyocyte contractility without affecting Ca2+ transient amplitudes, through ghrelin receptor Gαi signalling, leading to reduced phosphorylation of cTnI. AC01 improved left ventricle systolic function in monkeys. AC01 is a first in class novel inotrope, which improves contractility in different species without harmful mobilization of Ca2+. AC01 should be explored for the treatment of patients with heart failure.graphical abstract
CD14 blockade preserves left ventricular structure and systolic function in a murine model of ST-elevation myocardial infarction
Authors: Donner D G , D'elia A A , Bloom J , Kiriazis H , Wright D K , Tham Y K , Abbate A , Lavine K , Crowe D , Redlich G , Ziegelaar B W , Mcmullen J R , Wang B H , Kaye D M
Published: October 2024

Background

Cluster of differentiation-14 (CD14) facilitates the excessive pro-inflammatory monocyte-macrophage response to myocardial injury. Both acute and chronic infiltration of pro-inflammatory macrophages are implicated in the immunological progression of ischemic heart failure. However, investigation of clinically practicable strategies of CD14 blockade are yet to be reported.

Purpose

This fully blinded and randomised preclinical study evaluated the efficacy of a neutralising anti-CD14 antibody given at reperfusion in a murine model of reperfused ST-elevation myocardial infarction (STEMI) with key clinical features.

Methods

Large anterior STEMI was induced in 48 adult mice by left anterior descending coronary artery occlusion for 1 h prior to reperfusion and randomisation to receive anti-CD14 antibody, isotype control (IgG2a) or saline weekly for four weeks (independently blinded). Outcome measures included functional assessments by 9.4T cardiac magnetic resonance (CMR) imaging and ultra-high-resolution echocardiography at 21- and 28-days post-STEMI, respectively.

Results

Multi-plane short axis CMR at 21-days post-STEMI revealed greater left ventricular (LV) ejection fraction in the anti-CD14 antibody-treated group compared with saline- and isotype-treated controls (30±1 vs 19±1 and 20±1 %, respectively, p<0.001; Figure 1A). CMR also revealed reduced LV dilatation with anti-CD14-treatment compared to control groups (76±5 vs 102±8 and 107±7 µl EDV in saline- and isotype-treated controls, respectively, p<0.05). Parasternal long axis echocardiography at 28 days post-STEMI confirmed that anti-CD14 antibody treatment was associated with greater LV systolic function compared to saline and isotype controls (30±1 vs 24±1 and 25±2 % EF, respectively, p<0.05; Figure 1B). Preservation of LV end-diastolic (86±4 vs 111±6 and 101±5 µl, p<0.01) and end-systolic (61±3 vs 84±5 and 77±5 µl, p<0.01) volumes was also observed with anti-CD14 treatment. Global longitudinal strain (GLS) of the LV was also significantly greater at 28 days post-STEMI with anti-CD14 treatment relative to saline and isotype controls (-8.5±0.4 vs -6.8±0.3 and -6.6±0.4 %, respectively, p<0.05).

Conclusion(s)

The findings of this fully blinded and randomised preclinical study provide evidence of a clinically practicable strategy of CD14 blockade initiated at reperfusion to mitigate progressive LV systolic dysfunction and dilatation post-STEMI.Figure 1.Functional outcome measures
Secretomes from human epicardial adipocytes modulate atrial lymphatics and atrial fibrosis
Authors: Takahashi M , Abe I , Harada T , Yamasaki H , Takano M , Kondo H , Teshima Y , Miyamoto S , Takahashi N
Published: October 2024

Background

Cardiac lymphatics maintains myocardial fluid and immune cell homeostasis. Downregulation of cardiac lymphatics leads to cardiac dysfunction through impairment of inflammation and edema. On the other hand, it is well known that adipose tissue accumulation causes lymphatic dysfunction and impairs lymphedema.

Purpose

We evaluated the expression of atrial lymphatics in human left atrial appendage (LAA) histologically and biologically. Subsequently, we examined secretomes from epicardial adipose tissue (EAT)-derived adipocytes and validated if they change atrial lymphatics expression using ex vivo Organo-culture system.

Methods

Human LAA samples were collected from forty-two patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=16, 73.1±10.4 years), the paroxysmal AF (PAF) group (n=13, 69.8±12.6 years) and the persistent AF (PeAF) group (n=13, 71.2±6.5 years).

Human EAT were also excised from patients with and without AF (SR; n=5, AF; n=11) during cardiovascular surgery. Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation and maturation to adipocytes, cell culture supernatant (CCS) was collected, and was dropped onto the epicardial side of left atrial tissues excised from 8 weeks old male rats for 7 days using an ex vivo organo-culture system.

Results

In human LAA samples, mRNA expression of lymphangiogenesis-related genes (LYVE1, PROX1, VEGFC, and VEGFR3) was significantly lower in the PeAF group compared to the SR group (p=0.0370, <0.0001, 0.0157, and 0.0009). The number of LYVE1-positive atrial lymphatic endothelial cells (LECs) was also significantly lower in the PeAF group compared to the SR group and the PAF group (p=0.0186 and 0.038). There was a negative correlation between the number of LYVE1-positive atrial LECs and the area percentage of atrial myocardial fibrosis (r=-0.5350, p<0.001).

In the experiment using organo-culture system, lymphangiogenesis-related genes expression (Lyve1, Prox1, Vegfc, and Vegfr3) of rat atria treated with CCS of adipocytes from AF patients was significantly lower than those from SR patients (p=0.0391, 0.0100, 0.0034, and 0.0338). Furthermore, the CCS of adipocytes from AF patients contained higher protein levels of leptin, which is known to suppress lymphangiogenesis (p=0.0357). Leptin treatment (100ng/ml) on rat atria also decreased lymphangiogenesis-related genes expression such as Lyve1, Prox1, Vegfc, and Vegfr3 (p=0.0413, < 0.0001, <0.0001, and 0.0190).

Conclusions

Our study with human LAA demonstrated that atrial lymphatics interact with atrial myocardial fibrosis/progression of AF. Furthermore, our findings indicate that secretomes from EAT-derived adipocytes regulate atrial lymphatics expression, and leptin plays an important role in modulating atrial lymphangiogenesis.
Identification of genetic variants associated with exercise-induced atrial fibrillation in high-performing racehorses
Authors: Mattingsdal M , Nath L C , Sorensen E , Nissen S D , Fintl C , Tveit A , Aaronaes M , Myrstad M , Buhl R , Franklin S H , Christophersen I E
Published: October 2024

Background

Atrial fibrillation (AF) is the most common cardiac arrhythmia in athletes. Despite a lower burden of classical cardiovascular risk factors, the prevalence of AF in athletes is comparable to that in the general population of similar age (~13%, mean age 65.5), suggesting that exercise-induced AF may have different underlying mechanisms. The prevalence of AF in thoroughbred (TB) and standardbred (STB) racehorses is also relatively high (~5%), suggesting exercise as an independent risk factor. Due to selective breeding and complete control of pedigrees, racehorses are well-suited for genetic studies. Common and rare genetic variation explain a substantial part of AF variance, but no study has identified genetic variants associated with exercise-induced AF in any species.

Objective

In this project, we aimed to use the racehorse as a model to identify genetic variants associated with exercise-induced AF, and in a future study, possibly replicate the results in human athletes. Here we present preliminary results from the genome-wide association study (GWAS) of AF in racehorses.

Methods

We collected blood or hair samples from 181 STB and TB racehorses in which 71 had paroxysmal or persistent AF verified by ECG. We extracted DNA and performed whole-genome genotyping using a commercial equine genotyping platform (670k markers). We phased and imputed un-genotyped markers and increased the sample size of the control group, using publicly available equine data from similar breeds from the European Nucleotide Archive. We conducted a GWAS of 7.85 million markers in 71 cases and 296 controls using PLINK2 with covariates adjusted for population stratification, batch and sex.

Results

We identified one significant locus downstream of the protocadherin gene PCDH18 associated with AF in racehorses. The same locus has been associated with the ECG-trait P-wave terminal force in humans. Changes in P-wave terminal force is associated with increased atrial size, delayed interatrial conduction, left ventricular fibrosis and left atrial function, and considered a good predictor of AF occurrence. Several non-significant but interesting signals were also identified including the genes SLC6A15 (amino acid transport), MYBPH (myosin binding), MYF6 and MYOG (myoblast differentiation).

Conclusion

In these preliminary results, we have identified a new genetic locus associated with AF in racehorses. In addition, we have identified several candidate genes for further studies to elucidate the genetic background of exercise-induced AF. We aim to replicate our findings in a population of human athletes. Our results may aid the development of risk prediction tools for exercise-induced AF and new drugs for tailored treatment of AF in general.
Engineering hiPSC-derived tissue models for advanced amyloidosis research and therapy development
Authors: Montero Calle M , Anaut Lusar I , Larequi E , Coppiello G , Aranguren X , Salterain N , Garcia De Yebenes M , Prosper F , Gavira J J , Mazo Vega M M , Iglesias Garcia O
Published: October 2024

Introduction

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease currently lacking an optimal therapy, stemming from the absence of representative models. Our work aims to stablish for the first time an all-human 3D model of ATTR-CM where the main cellular and extracellular players of the pathology are present.

Methodology

A human induced pluripotent stem cell (hiPSC) line was derived from a patient carrying a p.Ser43Asn TTR mutation and differentiated into hepatocytes (hiPSC-HEPs), cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs). Cells were embedded in a hydrogel (tailored for each cell type) and combined with a designed 3D printed scaffold using Melt Electro Writing (MEW) technology. Tissues were maintained in culture for up to 4 weeks and characterized by staining, albumin production, electrophysiological, plus genomic analysis. Exposure to TTR fibrils (TTR-FIB) was evaluated.

Results

Human hepatic and cardiac functional tissues can be generated using MEW-designs. These constructs exhibited albumin production and an active spontaneous contraction, respectively. Moreover, tissues displayed well distributed cells throughout the 3D-structure, positive for specific hepatic and cardiac mature markers. Interestingly, electrophysiological analysis showed that cardiac tissues displayed shortened calcium transients after TTR-FIB exposure compared to unexposed tissues. However, no differences were found in terms of beat rate, metabolic activity and gene expression for the concentrations used.

Conclusion

We have developed an advanced model of ATTR-CM capable of recapitulating the multisystem complexity of the disease, which will contribute to advancing our understanding of the mechanisms of the disease, and help discovering and formulating new treatments.
Genome-wide association analysis reveals insights into the molecular etiology of dilated cardiomyopathy
Authors: Zheng S , Henry A , Consortium H E R M E S , Ware J S , Lumbers R T
Published: October 2024

Introduction

Dilated cardiomyopathy (DCM) is a primary heart muscle disease characterized by ventricular dilatation and impaired contractility. While considered a Mendelian disorder, recent evidence highlights the important role of common genetic variation. Better characterization of the genetic architecture will provide opportunities for improved diagnosis, risk stratification and biological understanding.

Methods

We performed a GWAS meta-analysis of 14,256 DCM cases and 1,185,671 controls of European ancestry from 16 studies (GWASDCM). To improve discovery power, we performed multi-trait analysis of GWAS (GWASMTAG) with three genetically correlated and clinically relevant cardiac imaging traits in 36,203 individuals. To identify effector genes at loci, we used a two-step approach: 1) the nearest gene and top 3 genes prioritized by PoPS or V2G were selected as candidates; and 2) totality of evidence including 5 additional methods (coding variant, co-localization with expression, TWAS, ABC-model, and known Mendelian cardiomyopathy genes) was summarized in a single score. We performed rare variant burden analysis (MAF<0.1%) for predicted truncating variants (PTV) in established DCM-causing genes to highlight DCM genetic architecture, and in prioritized GWAS genes, against DCM and quantitative CMR traits in UK Biobank (UKB) and 100,000 Genomes Project (GeL), implemented by REGENIE. A polygenic score (PGS) generated using a Bayesian framework (PRS-CS) was assessed for association with the same outcomes in UKB participants, including among PTV carriers in established DCM-causing genes.

Results

We identified 80 independent loci, of which 65 were novel (62 loci in GWASDCM, and 54 loci in GWASMTAG) (Figure 1A). High-confidence genes were prioritized at 62 loci, including in genes with established roles in Mendelian cardiomyopathies (TTN, BAG3, FLNC, MYBPC3, FHOD3, ALPK3), and were enriched in key biological processes (Figure 1B). We characterized DCM genetic architecture across the allelic spectrum (Figure 2A). Rare variant burden analysis highlighted novel associations with DCM and quantitative traits for PTVs in NEDD4L and MAP3K7 in UKB that were replicated in GeL (Figure 2B). PGS was associated with DCM and quantitative CMR traits (OR per PGS SD 1.8, P<1x10-16) (Figure 2C). Individuals in the top centile had 4-fold increased risk of having DCM compared with median risk. PGS stratified penetrance of rare variants in 1,546 carriers of pathogenic variants (top quintile 7.3%, bottom quintile 1.7%, P 0.005).

Conclusion

We have performed the largest DCM GWAS, resulting in 80 significant loci (65 novel), and prioritized genes using a systematic approach. We identify novel DCM-causing genes (NEDD4L and MAP3K7) and generate a PGS that associates with DCM and modulates penetrance in carriers of Mendelian disease-causing rare variants.Figure 1Figure 2
Disparities in the access to atrial fibrillation ablation in Denmark
Authors: Zoerner C R , Toennesen J , Riis-Vestergaard L D , Middelfart C , Hein R , Rasmussen P V , Ruwald M H , Gislason G H , Hansen M L
Published: October 2024

Background

Atrial fibrillation (AF) is a common arrhythmia that can lead to serious complications such as stroke, heart failure, as well as reduced quality of life. Radiofrequency and cryoablation are safe and effective treatments for AF, but they are not offered equally to all patients.

Purpose

The study aims to identify the demographic groups more or less likely to undergo AF ablation.

Methods

Using nationwide National Registries, all Danish patients with newly diagnosed AF between the 1st of January 2005, and the 31st of December 2018 were identified. The association between patients, stratified by gender, age, level of education and attachment to the job market, and the likelihood of receiving AF ablation was investigated using multivariable Cox proportional-hazard analysis. Cumulative incidence of AF ablation among these groups was calculated using the Aalen-Johansen estimator, taking death as a competing risk into account.

Results

A total of 176,248 patients were included in the study. Men were more likely to receive ablation than women (7% vs 3%). When analysing age, patients aged 25-44 and 45-64 were most likely to receive ablation, while only 0.7% of patients aged 80 or above received ablation. The rate of ablation significantly decreased with decreasing level of education. Patients with a Ph.D. or doctorate degree were more likely to receive ablation than patients with only basic school education (22% vs 2%). Finally full-time employed patients were most likely to receive ablation, followed by self-employed, unemployed, on sick leave, undergoing education, and early retired patients. Retired patients were the least likely to receive ablation (3%).

Conclusion

This study found that women, older patients, patients with lower levels of education, and patients who are not employed or who are retired are less likely to receive AF ablation. These findings suggest that there are significant social and economic disparities in AF ablation treatment in Denmark.Ablation by educationForest plot: Cox analysis