bluuebunny/crossref_metadata_2025 huggingface dataset:
Methotrexate associated with nanoparticles decreases NLRC4 inflammasome expression in infarcted rat hearts
Authors: Gatto M , Mota G A F , Borim P A , Santos A C C , Pagan L U , Souza L M , Rodrigues E A , Silva R C F , Oliveira J P G , Meirelles A L B , Brosler J Y , Maranhao R C , Zornoff L A M , Okoshi K , Okoshi M P
Published: November 2023

Abstract: Following myocardial infarction (MI), molecules released from necrotic myocytes sensitize nucleotide-binding receptors (NLRs), activate inflammasome complex, and initiate acute inflammatory process and interleukin (IL)-1β production, essential for tissue damage resolution. However, excessive IL-1β production impairs cardiac remodeling. After MI, IL-1β is released by the NLRP3 inflammasome; involvement of the NLRC4 inflammasome in post-MI remodeling has not been established. Methotrexate (MTX) carried by lipid core nanoparticles in low density emulsion (MTX-LDE) reduces post-MI inflammatory process and improves cardiac function in rodents.

Purpose

To evaluate the influence of early treatment with MTX-LDE on myocardial expression of NLRP3 and NLRC4 inflammasomes in infarcted rats.

Methods

Male Wistar rats were divided into three groups: control (Sham, n=6); MI (n=6); and MI treated with MTX-LDE (MI-MTX, n=6). MI was induced by ligation of the left anterior descending coronary artery. Twenty-four hours later, the MI-MTX group was treated with 1 mg/kg of MTX-LDE, intraperitoneally. Euthanasia was performed 72 hours after surgery under anaesthesia with ketamine and xylazine. Infarct size was assessed by histology. Myocardial expression of NLRC4, NLRP3, ASC, pro-caspase-1, caspase-1, pro-IL-1β and IL-1β was quantified by Western blotting. IL-1β concentration in serum and heart macerate supernatant was analyzed by ELISA. Statistical: ANOVA or Kruskal-Wallis; 5% significance level.

Results

Rats with MI lower than 30% of the left ventricle total area were excluded. Infarct size was larger in the MI-MTX group (43,8 ± 5,4) than MI (36,2 ± 6,22). NLRC4 and caspase-1 expression and IL-1β serum concentration were higher in MI than Sham and MI-MTX. Myocardial IL-1β concentration was higher in MI and MI-MTX than Sham and lower in MI-MTX than MI. NLRP3, ASC, pro-caspase-1, and IL-1β protein expression did not differ between groups.

Conclusion

Our study shows for the first time that NLRC4 inflammasome protein expression increases acutely after myocardial infarction in rats. Early treatment with nanoparticles-derived methotrexate reduces myocardial NLRC4 inflammasome expression and myocardial and serum IL-1β concentration.
Conditional knock out of cytosolic thioredoxin (Trx) in cardiomyocytes impairs cardiac function and exacerbates ischemia-reperfusion injury
Authors: Das K , Kumar S , Fnu K , Shurmur S
Published: November 2023

Introduction

Coronary heart disease with myocardial infarction (MI) is a leading cause of mortality in the world despite timely reperfusion of occluded coronary artery. Myocardial damage due to restoration of blood flow, known as reperfusion injury account for about 50% of final MI size. The role of Trx in protection against myocardial ischemia reperfusion has been reported; however, these studies were mostly performed using ex vivo isolated mouse/rat heart with retrograde perfusion or with injected rhTrx. However, the role of loss of Trx specifically in the myocytes using a conditional deletion has never been reported. Here, we determined whether loss of Trx in myocytes would promote ischemia-reperfusion injury, even if the endocardial endothelial cells express normal levels of Trx.

Purpose

We investigated the effect of specific deletion of Trx in cardiomyocytes on base-line cardiac function and cardiac function in ischemia-reperfusion of heart using conditional knockout of Trx (aMHC-Trx-KO) in the cardiomyocytes with normal levels of Trx in the endothelial cells to determine specifc contribution of Trx in cardiac function in I/R.

Methods

A Trx floxed mouse line was created with introduction of loxP sites flanking the mouse Trx gene. Further, the conditional deletion of Trx in cardiomyocytes was produced by breeding homozygous Trx-floxed (Trxfl/fl) mice with aMHC-Cre mice strain. The resulting aMHC-Trx-KO mice was identified by genotyping and conditional deletion of Trx in myocytes was achieved by injecting Tomoxifen (75mg/Kg in oil). The aMHC-Trx-KO mice were subjected to 30 minutes of ischemia by ligation of left descending coronary artery(LAD) and reperfusion was performed by release of slipknot and allowing blood flow of 60 minutes before sacrificing animals. The animal protocol was approved by the IACUC of the Texas Tech University Health Sciences Center, Lubbock. Echocardiography on animals was performed using Vevo 3100 (Visual Sonic). Statistical analysis was performed by Prism 8.0 (GraphPad).

Results

Deletion of Trx caused significant cardiac dysfunction in aMHC-Trx-KO mice compared to Trxfl/fl mice without treatment with tomoxifen. The ejection fraction (EF%, 52.82 vs. 73.83) and fractional shortening (FS%, 41.42 vs.25.89) were significantly reduced. Further, these mice subjected to I/R showed exacerbated cardiac dysfunction with significant decrease in heart rate and did not survive the reperfusion. We also found significant mitochondrial structural abnormalities and mitochondrial dysfunction in aMHC-Trx-KO mice compared to non-transgenic or Trxfl/fl mice without tomoxifen treatment.

Conclusion

Deletion of Trx in cardiomyocyte causes significant myocardial dysfunction. Further, Trx is required to protect against myocardial dysfunction in ischemia-reperfusion injury. Our data also suggest that cardiac mitochondria is specifically impacted by loss of Trx deletion.Figure-1
Adssl1-mediated purine synthesis modulates cardiomyocyte proliferation and heart regeneration
Authors: Li Z , Zhang R Y , Yan X X
Published: November 2023

Introduction

The enhancement of pre-exist cardiomyocyte proliferation has been proved to be an effective treatment of myocardial infarction(MI) (1-2). Purine bases from the de novo purine synthesis (DNPS) pathways is the critical source of nucleotide synthesis for proliferative cell (3). The muscle specific isoform of adenylosuccinate synthase (Adssl1) catalyses the last step of DNPS pathway (4). Patients with Adssl1 mutation develop cardiomyopathy at 13-15 years (5). Nevertheless, its role and mechanism in cardiomyocyte cell-cycle re-entry and heart regeneration remain unknown.

Purpose

To explore the effect and underlying mechanism of Adssl1 and its mediated DNPS pathway in cardiomyocyte proliferation and heart regeneration.

Methods

We used qRT-PCR, Western blot and Immunofluorescence to assess the expression of Adssl1 during heart developement and heart regeneration after apical resection (AR) operation. Using newly generated loss- and gain-of-function genetic tools to investigate the role of Adssl1 in cardiac regeneration after AR or MI operation. Transcriptomic RNA sequencing (RNA-Seq) and liquid chromatography-tandem mass spectrometry (LC/MS) allowed us to determine the metabolism of Adssl1 in modulating cardiomyocyte proliferation.

Results

Here, we found that Adssl1 was increased during heart regeneration. Depletion of Adssl1 in neonatal hearts significantly inhibited purine synthesis pathway and attenuated heart regenerative capacity after AR operation. Cardiomyocyte-specific overexpression of Adssl1 extended postnatal regenerative window and induced a robust cell-cycle re-entry after MI, leading to a decrease in fibrosis scar size and improvement of cardiac function. Adssl1 caused accumulation of Inosine, which could promote the cardiac repair in mice. Mechanistically, we demonstrated that Adssl1 significantly involved in modulating mTORC1/S6K pathway.

Conclusion

Collectively, our study elucidates the role of Adssl1 in modulating cardiac proliferation and heart regeneration through activating mTORC1/S6K pathway after MI. Notably, Inosine has the potential to be employed as an effective agent in regulating cardiac proliferation and myocardial repair.Heart regeneration
Long non-coding RNA SNHG18 epigenetic regulates ferroptosis in acute myocardial infarction
Authors: Chen J , Zhang B F , Liu G , Liu X P , Chen X Z
Published: November 2023

Background

Loss of terminally differentiated cardiomyocytes is a key pathogenic factor for the occurrence of acute myocardial infarction (AMI) and the development of lethal heart failure. LncRNAs have been confirmed to be critical in regulating AMI, besides, recently published studies disclosed that ferroptosis is a new form of programmed cell death that contributes to AMI. However, whether LncRNAs are involved in the regulation of ferroptosis and correlated underlying mechanisms in AMI remains unknown.

Purpose

Our present study is to investigate LncRNA that regulates cardiomyocyte ferroptosis in AMI and uncover the potential mechanisms.

Method

RNA-sequencing and bioinformatical analyses were conducted to identify the candidate LncRNAs for AMI. Adenovirus for LncRNA knockout and overexpression were constructed to alter the SNHG18 expression of cardiomyocytes in vitro. Cardiomyocyte-specific knockout and overexpression mice were also constructed to alter SNHG18 expression in vivo. Ferroptosis characteristic measurement, including glutathione peroxidase 4 (GPX4), Ferritin heavy chain (FTH-1), ptgs2, MDA, ROS, Fe2+, glutathione, and mitochondrial morphology, was performed to verify the ability of SNHG18 to regulate the ferroptosis of cardiomyocytes. Cardiac morphological, structural, functional, and hemodynamic changes as well as the viability of cardiomyocytes were also measured to assess the effect of SNHG18 on AMI. RNA-pulldown+Mass spectrometry, RNA immunoprecipitation were performed to identify the specific binding factors of SNHG18.

Results

RNA-sequencing results revealed the expression of SNHG18 was significantly and persistently elevated from 3 to 7 days after AMI. SNHG18 was mainly enriched in the nucleus of cardiomyocytes. Besides, the down-regulation of SNHG18 could specifically alleviate the cell viability loss caused by ferroptosis inducers. Thereafter, gain- and loss-of-function experiments were performed both in vitro and in vivo. Functionally, SNHG18 knockout dramatically enhanced cardiomyocyte vitality, reduced infarct size, improved cardiac performance and alleviated cardiac fibrosis by mitigating cardiomyocyte ferroptosis. However, enforced SNHG18 expression resulted in the opposite effects. Mechanistically, the subunit RBBP4 that forms the PRC2 (Polycomb Repressive Complex 2) complex was a direct target of SNHG18, which was also confirmed by RIP assays. In addition, Chip-PCR analysis also demonstrated that PRC2 could directly bind to the promoter region of the GPX4 and FTH1 gene, thus altering the level of H3K27me3 and inhibiting their transcription. Moreover, subsequent rescue experiments further verified that SNHG18 regulated the expression of GPX4 and FTH1 to ameliorate AMI-induced cardiomyocyte ferroptosis in a PRC2-dependent manner.

Conclusion

This study demonstrates that SNHG18-PRC2-GPX4/FTH1 pathway is a promising therapeutic target for the treatment of AMI by ameliorating cardiomyocyte ferroptosis.
Monocyte priming during acute myocardial infarction
Authors: Moggio A , Mauersberger C , Khangholi D , Dutsch A , Schunkert H , Kessler T , Sager H
Published: November 2023

Introduction Purpose

Myocardial infarction (MI) inflicts a sterile wound on the heart which recruits hundreds of thousands of monocytes from the blood. The bone marrow (BM) responds to cardiac ischemia with an increase in monocyte production, hence meeting the high demand of these cells. Apart from this quantitative response, little is known whether the BM supplies qualitatively different monocytes during the course of MI.

Methods & Results

We isolated BM Ly6Chigh monocytes from non-infarcted mice and from mice which were infarcted 72h prior (surgically induced by permanent coronary ligation) and contrasted monocytic transcriptomes using RNA sequencing. We found 672 differentially expressed genes (DEG) of which 289 were up- and 383 down-regulated (log2FoldChange³±1.0, adjusted p<0.05). Since most DEG were enriched in "cell adhesion and recruitment" pathways, we tested the functional relevance of our findings in the context of monocyte recruitment to the heart using competitive adoptive transfer experiments. Our data revealed that the BM provides monocytes that are primed for better recruitment (relative recruitment: 56.1% 12h after MI vs. 43.9% steady state, p<0.01) at the early inflammatory phase following coronary ligation (12h after MI). In contrast, monocytes displayed worse recruitment capacities (relative recruitment: 30.4% 72h after MI vs. 65.8% steady state, p<0.01) at the end of the inflammatory phase (72h after MI). We next sought to identify key players in this BM-controlled cardiac recruitment process. Our RNA sequencing data revealed that – in monocytes with poor recruitment capacities – the cell adhesion molecule CD209 was among the most significantly downregulated genes at 72h after MI over steady state. To investigate the molecular role of CD209 in the process of leukocyte recruitment to the myocardium during MI, we cloned human CD209 from a cDNA library into an overexpression plasmid system. After ectopic overexpression of CD209, THP-1 cells (a cell line derived from immortalized human blood monocytes) showed enhanced adhesion to endothelial cells (HUVECs) compared to THP-1 cells under control conditions. Finally, we infarcted wild type (CD209+/+) and CD209 knockout (CD209-/-) mice and found decreased numbers of Ly6Chigh monocytes in infarcted hearts of CD209-/- mice in comparison to CD209+/+ mice (491+52 vs. 915+180 Ly6Chigh monocytes/mg infarct tissue, mean+SEM, p<0.05) while numbers of blood Ly6Chigh monocytes were unchanged.

Conclusions

The BM responds to MI and provides an altered monocyte repertoire in terms of numbers and function and hence remotely controls monocyte/macrophage accumulation in the ischemic heart. Consequently, the heart–bone marrow axis may represent a novel therapeutic target to beneficially modify post-MI cardiac remodeling, a major contributor of MI-related morbidity and mortality.
Empagliflozin treatment prevented oxidative stress, macrophage infiltration, pro-inflammatory and pro-fibrotic responses in the left ventricle of isoprenaline-induced Takotsubo-like syndrome in rats
Authors: Tatarcheh T , Amissi S , Matsushita K , Mroueh A , Trimaille A , Gong D S , Fakih W , Muzammel H , Faucher L , Granier A , Pieper M P , Schini-Kerth V , Morel O
Published: November 2023

Introduction

Takotsubo Syndrome (TTS) is an acute heart failure (HF) syndrome induced by both emotional and physical stressors involving a surge of catecholamines and apical ballooning. Currently, there is no specific treatment for TTS. Sodium-glucose cotransporter2 inhibitors (SGLT2i) showed major cardiovascular protective effects in patients with HF independent of glycemic control. SGLT2i have also been shown to have anti-inflammatory and anti-oxidant effects, and to improve the endothelial function in experimental models of cardiac disease. However, a possible protective role of SGLT2i in TTS is unclear. Thus, this study evaluated the effect of SGLT2i on isoprenaline-induced TTS-like syndrome in rats, and determined the underlying mechanism.

Methods

32 female Sprague-Dawley rats (10 weeks old) were divided into 4 groups: control, empagliflozin (30 mg/kg/day provided in the diet for 2 weeks), isoprenaline (Iso, 100 mg/kg, i.p.) to induce a TTS-like syndrome, and Iso plus empagliflozin (IE group). After 6 h of Iso injection, echocardiography was performed to determine left ventricular (LV) ejection fraction (EF) by area and heart rate (HR). At day 3, rats were sacrificed, main mesenteric artery and microvessels were collected for vascular reactivity studies, and LV was divided into base and apex to study mRNA expression levels using RT-qPCR, macrophage infiltration by in situ CD68 immunofluorescence staining, and oxidative stress using dihydroethidium.

Results

The injection of Iso to rats caused at 6 h an increased LVEF and HR in the Iso and the IE groups as compared to their corresponding controls. The HR response was significantly smaller in the IE group compared to Iso group whereas LVEF was similar. In addition, in mesenteric microvessels but not the main mesenteric artery, Iso group showed significant decreased relaxations to acetylcholine and increased contractile responses to phenylephrine in intact rings both of which were not observed in the IE group. Hearts showed significantly increased mRNA levels of IL-1β, TNF-α, NOX2, VCAM-1, MMP-9, TGF-β1 and collagen-1, oxidative stress levels and CD68 staining in the apex compared to the base in the ISO group, all of which were significantly reduced in IE group.

Conclusions

The present findings indicate that ISO injection caused in the LV elevated levels of oxidative stress, macrophage infiltration, pro-inflammatory, pro-remodeling and pro-fibrosis responses that were more pronounced in the apex compared to the base, and also endothelial dysfunction in the systemic microcirculation, all of which were significantly prevented by the empagliflozin treatment. They further suggest that SGLT2 appears as an interesting target in TTS.
Characterisation of pomegranate juice polyphenols identifies oenothein B as an endothelium-dependent vasodilator and regulator of endothelial function that enhances actions of oligomeric procyanidins
Authors: Corder R , Khan N Q , Lilley S , Fox R , Tyler J , Angbohang A , Caton P W , Mullen W , Filomena F , Ahluwalia A , Crozier A , Malkinson J
Published: November 2023

Background

Cardiovascular disease mortality is reduced in individuals with high daily consumption of dietary polyphenols. Optimal daily intakes, choice of polyphenol, and mechanism of action have yet to be fully defined. Pomegranate juice (PJ) reduces blood pressure and improves endothelial function. These effects are attributed to the high levels of polyphenols, primarily ellagitannins.

Purpose

The aims of these investigations were (1) to identify PJ polyphenol(s) that altered endothelial function and could potentially lower blood pressure, and (2) to compare these endothelial actions to oligomeric procyanidins (OPC) purified from apple polyphenol extracts.

Methods

PJ polyphenols were purified by Sephadex LH-20 chromatography and RP-HPLC, and identified by LC-MS/MS and NMR. Fractions were screened for biological activity using cultured bovine aortic endothelial cells by measuring the degree of inhibition of endothelin-1 synthesis. Endothelial actions were further assessed by measuring changes in intracellular Ca2+ using FLIPR Ca2+-6 assay, qRT-PCR (EDN1, eNOS and KLF2 mRNAs), and NO synthesis (nitrite).

Results

Six PJ polyphenols were isolated. These were identified as punicalin, four punicalagin isomers, and oenothein B (OTB, a cyclic dimeric ellagitannin; LC-MS [M-H]-= 1567.1). At concentrations ≤5 µg/ml only OTB inhibited ET-1 synthesis (IC50 1.5 µg/ml). The identity of OTB was confirmed by NMR, and by comparison with OTB purified from Epilobium species. Further characterisation of the actions of OTB on endothelial cells showed pre-incubation with 1.25, 2.5 and 5 µg/ml OTB increased binding of biotinylated-OPC by 1.7, 2.3 and 3.8 fold. Subsequent studies of the interactions between OTB and OPC showed synergistic effects for decreases in ET-1 synthesis, EDN1 mRNA, and increases in KLF2 mRNA, eNOS mRNA and NO release. The effect of combining OTB (1 µg/ml) and OPC (1 µg/ml) lasted for over 24 h (relative to control: ET-1 -57%, EDN1 mRNA -63%, eNOS mRNA +69%, KLF2 mRNA +356%), when individually these polyphenols no longer had any significant effect. Intracellular Ca2+ responses also showed synergistic interactions, however the profile of [Ca2+] changes for OTB and OPC alone differed, suggesting separate signalling pathways initiating these endothelial responses. Endothelium-dependent vasodilator effects of OTB were confirmed on isolated mouse aorta (EC50 0.27 µg/ml).

Conclusions

These effects of OTB may underlie previously reported decreases in blood pressure and improvements in endothelial function after pomegranate juice consumption. Detailed understanding of the mechanism of action of OTB, and its interactions with OPC could lead to new therapeutic modalities for treating endothelial dysfunction in a broad spectrum of people at risk of cardiovascular disease.
Eight-year follow-up of cardiosphere-derived cell transplantation in patients with single ventricle congenital heart disease
Authors: Hirai K , Baba K , Kasahara S , Oh H
Published: November 2023

Background

Although transcoronary infusion of cardiosphere-derived cells (CDCs) in patients with single ventricles provides short-term improvement in cardiac function, the long-term efficacy is still unknown.

Purpose: We tested the hypothesis that CDC infusion is associated with improved long-term clinical outcome in patients with single ventricles and heart failure.

Methods: Patients were enrolled in the TICAP phase 1 (Transcoronary Infusion of Cardiac Progenitor Cells in Patients With Single Ventricle Physiology) and PERSEUS phase 2 (Cardiac Progenitor Cell Infusion to Treat Univentricular Heart Disease) trials between January 5, 2011, and January 30, 2015. The inclusion criteria were that patients were aged <6 years who were diagnosed with single ventricle lesions undergoing stage 2 (bidirectional Glenn: BDG) or stage 3 (total cavopulmonary connection: TCPC) palliation. Ventricular ejection fraction should be <60% during the initial screening through the final enrollment. Medical records of patients with single ventricles were obtained and analyzed as a control group who were eligible but were treated by staged palliation alone. We evaluated the effectiveness of CDCs using an integrated cohort study in 93 patients with single ventricles, including 41 patients who received CDC infusion and 52 controls.

Results: At the time of this study, 36 patients with CDC infusion and 42 controls were alive without cardiac transplantation, with mean follow-up of 9.7±1.2 years. Survival rate did not differ between the 2 groups (log-rank P = 0.351), whereas overall patients treated by CDCs had lower incidents of late failure defined as late death, protein-losing enteropathy, plastic bronchitis, BDG or TCPC take down, disqualified TCPC candidate, Ross classification 3 or 4, or rehospitalization for heart failure (P = 0.048) compared with controls. Adverse events defined as unplanned cardiac operation, arrhythmia, thromboembolic events, cerebral hemorrhage, or tumor formation (P = 0.057) and unplanned catheter intervention (P = 0.094) did not differ between 2 groups.

Conclusions: Intracoronary delivery of CDCs in patients with single ventricles did not improve all-cause mortality rate but significantly reduced the incidence of late failure at 8 years of follow-up.
AAV6-mediated gene transfer of HCN1-ddd generates slightly faster baseline beating rates as compared to Hcn2 yet with a potential risk for pro-arrhythmia
Authors: Cervera Barea A , Wang J , Jonker T , Klerk M , Boender A , Jansen M , Visser J , Van Nieuwburg-Fennema M , Kirzner O , Vos M , Tan H , Barnett P , Christoffels V , Neef K , Boink G
Published: November 2023

Electronic pacing is the current treatment of choice for complete heart block (CHB) that revolutionised last century’s standard of care. Despite its success, some critical issues remain such as providing suboptimal cardiac output and a lack of direct autonomic responsiveness. To develop a more physiological and hardware-free pacemaker, we focused on developing gene therapy-based biological pacemakers by means of ion channel over-expression. Previous studies with adenoviral gene transfer indicated possible superior outcomes for the engineered mutant HCN1-ΔΔΔ as compared to wild-type Hcn2, yet a head-to-head comparison was never made.

To assess the long-term biological pacemaker efficacy for AAV6-mediated gene transfer of HCN1-ΔΔΔ and Hcn2.

Functional biological pacemaker studies were conducted in a porcine model of radiofrequency ablation-induced CHB. Four weeks after the ablation, animals were studied in two different groups: AAV6-cTnT-HCN1-ΔΔΔ and AAV6-cTnT-Hcn2, both immunosuppressed with prednisolone and Cyclosporine A. All animals were then followed for another four weeks to evaluate in vivo biological pacemaker performance and then organs were harvested to assess transduction efficiency.

Biological pacing effectiveness was evaluated in view of maximal (max) and basal (mean) beating rates (HR), and pacemaker dependency that animals had shown throughout the study. Four weeks after the induction of CHB, on the day of the injection, all animals (n = 6) presented an average mean HR of 53,67 ± 7,64 bpm (Figure 1B) and a pacemaker dependency above 65% (Figure 1C). One week after gene transfer, these beating rates rapidly increased and pacemaker dependency declined. Hcn2 and HCN1-ΔΔΔ groups displayed a notable increase in max HR (Figure 1A), where HCN1-ΔΔΔ offered the most potent and sustained response (Hcn2 153,33 ± 47,26 bpm vs. HCN1-ΔΔΔ 220 ± 43,59 bpm; Figure 1A). In terms of mean HR, they similarly trended up in both groups (Hcn2 57,81 ± 5,49 bpm vs. HCN1-ΔΔΔ 59,22 ± 2,28 bpm; Figures 1B and E). Lastly, these faster beating rates in HCN1-ΔΔΔ resulted in modestly lower dependency in electronic back-up pacing (52 ± 7,94 % vs. 48,3 ± 22,37 % for HCN1-ΔΔΔ vs. HCN2; Figures C and F).

AAV6-mediated HCN1-ΔΔΔ gene transfer produced slightly faster mean heart rates as compared to Hcn2, yet HCN1-ΔΔΔ generated persistently increased maximal heart rates (in two out of three animals) that exceeded the physiological desirable range. These findings possibly relate to the much faster channel gating kinetics of HCN1-ΔΔΔ that were previously found to be profoundly pro-arrhythmic with the chimera channel HCN212.Figure 1Abstract ESC 2023
The role of latrophilin-2 in maintaining cardiac cell-to-cell junctions and its implications for dilated cardiomyopathy and sudden death
Authors: Cho H J , Kang M J , Son H J , Seo H J , Kim H S
Published: November 2023

Background

Identifying genes that are important for maintaining heart function and characterizing their roles and pathways are essential for developing therapeutic approaches. Latrophilin-2 (LPHN2), a relatively unknown GPCR, critically contributes to differentiation from pluripotent stem cells (PSC) to the cardiac lineage, but it is not known whether LPHN2 is active in the adult heart.

Purpose

In this study, we explored the functions of LPHN2 in adult hearts.

Methods and Results

To determine whether there is an association between variation in LPHN2 and cardiac phenotypes in humans, we used gnomAD and UK Biobank data. The data suggested that LPHN2 variants could be fatal. Accordingly, we developed a mouse model for further analysis of Lphn2. Deletion of Lphn2 causes embryonic lethality, so we generated a cardiac-specific, tamoxifen-inducible Lphn2 conditional knockout mouse (Lphn2icKO mouse) by cross-breeding Lphn2flox/flox mice with the TMX-inducible Cre recombinase (MerCreMer) under control of the α-Myh6 (α-myosin heavy chain) promoter mice. After administering tamoxifen, we found that the left ventricle became dilated and the systolic function was decreased, and gaps were created between cardiomyocytes. We also observed ventricular tachycardia and atrioventricular block associated with the dilated cardiomyopathy and subsequent sudden cardiac death. Cell-to-cell junctions (adherence, gap, and desmosomes,) were found to be dysregulated in the Lphn2icKO adult mouse heart and the heart at embryonic day 13.5 of Lphn2−/− (homozygous Lphn2 deficiency). To investigate Lphn2 function in a disease model, we generated a myocardial infarction (MI) in Lphn2icKO mice. MI with tamoxifen group showed wide QRS tachycardia and various types of block, and a survival reduction of 40% compared with that of the MI group. Regarding underlying mechanisms, we found that PGC-1α, a factor downstream of p38-MAPK that contributes to arrhythmia and maturation of PSC-derived cardiomyocytes, was downregulated (Figure).

Conclusions

LPHN2 is a key molecule that maintain adult heart integrity via its effects on cell-to-cell junctions, and the deficiency of Lphn2 exacerbates heart failure. Our findings provide evidence in support of LPHN2 being a pivotal regulator of heart function through the p38-MAPK-PGC-1α pathway.Schematic illustration
Wearable cardioverter defibrillator after ICD-system explantation: data from a multicenter registry
Authors: El-Battrawy I , Beiert T , Koepsel K , Kovacs B , Blockhaus C , Klein N , Kuntz T , Shin D I , Abumayyaleh M , Saguner A M , Erath J W , Duru F , Muegge A , Akin I , Aweimer A
Published: November 2023

Background

Data on the use of wearable cardioverter defibrillator (WCD) among patients after cardiac implantable electronic device explantation of 1- to 3-chamber Implantable Cardioverter-defibrillator system (ICD) are sparse. Subsequently several guidelines give a different recommendation regarding WCD indication in this cohort.

Objectives

We aimed to study the baseline characteristics and outcome of patients treated with WCD after ICD explantation. The primary outcome is appropriate WCD shock.

Methods

1104 patients were included in a multicenter registry from seven European centers. Among this cohort 109 patients received a WCD to bridge the time after ICD-system explantation until reimplantation due to a persistent ICD-indication. The mean follow-up time of the whole cohort was 824±773 days. Cardiovascular history and non-cardiovascular morbidities were evaluated. In addition, the presence of ventricular tachyarrhythmias and/or WCD shocks was evaluated in the explantation group. Furthermore, after ICD-reimplantation the rate of rehospitalization for ventricular tachyarrhythmias, atrial fibrillation, stroke and congestive heart failure was followed.

Results

In the explantation group patients were older (65±14 versus 59±15; p<0.001) and were hospitalized longer (21±15 days versus 14±12 days; p<0.001). The index LVEF differed significantly (35.7±14.1% versus 29.3±11.5%; p<0.001). The LVEF value did not differ significantly over short-term follow-up (35.7±14.2% versus 37.9±11.9%; p=0.204). Wear days among patients after ICD-system explantation were 61±46 days. An appropriate WCD shock was documented in 7.3% after ICD-system explantation during WCD use. Up to 80.6% of patients after ICD-system explantation received a reimplantation. The rate of rehospitalization due to ventricular tachyarrhythmias was 7.3%, due to heart failure 6.8% and due to atrial fibrillation 4.1%. After ICD-implantation the rate of appropriate shocks was 12/89 (13.4%). In the multivariable regression analysis myocarditis and ICD-system explantation were positive predictors for the risk of ventricular tachyarrhythmias, but nevertheless an improved left ventricular ejection fraction (LVEF) and use of aldosterone antagonists were negative predictors for the risk of ventricular tachyarrhythmias.

Conclusions

Cccurrence of malignant arrythmia after ICD-system explantation is high and the use of WCD among these patients could be beneficial in preventing sudden cardiac death.
Hepatokine selenoprotein P has an important role in cardiac remodeling
Authors: Usui S , Takashima S , Goten C , Inoue O , Takeda Y , Yamaguchi K , Hashimuko D , Takamura M
Published: November 2023

Background

In heart failure, the failure of the pump function results in reduced blood flow and dysfunction of energy metabolism in tissues and organs throughout the body. The liver is the largest organ in the human body and plays a central role in lipid and glucose metabolism. Hepatokine selenoprotein P (SeP) contributes to insulin resistance and hyperglycemia in patients with type 2 diabetes. Inhibition of SeP protects the heart from ischemia reperfusion injury and serum levels of SeP are elevated in patients with heart failure with reduced ejection fraction.

Objective

We investigated the role of SeP in the regulation of cardiac remodeling in response to pressure overload.

Methods and Results

To examine the role of SeP in cardiac remodeling, transverse aortic constriction (TAC) was subjected to SeP knockout (KO) and wild-type (WT) mice for 2 weeks. LV weight/tibial length (TL) was significantly smaller in SeP KO mice than in WT mice. Lung weight/TL was significantly smaller in SeP KO than in WT mice. TAC-induced cardiac upregulation of the fetal type genes, including atrial and brain natriuretic factors, was significantly attenuated in SeP KO compared to WT. Furthermore, azan staining revealed that there was significantly less interstitial fibrosis in hearts after TAC in SeP KO than in WT mice.

Expression of SeP in the liver of WT mice was significantly increased by TAC, while expression in the heart was unchanged. Hepatocyte-specific SeP KO mice were generated using the albumin-Cre-LoxP system. LV weight/TL was significantly smaller in hepatocyte-specific SeP KO than in WT mice. To determine whether hepatic overexpression of SeP affects TAC-induced cardiac hypertrophy, a hydrodynamic injection method was used to generate mice that overexpress SeP mRNA in the liver. Hepatic overexpression of SeP in SeP KO mice lead to a significant increase in LV weight/TL after TAC compared to that in other SeP KO mice.

Conclusions

These results suggest that cardiac pressure overload induced hepatic expression of SeP and the absence of endogenous SeP attenuated cardiac hypertrophy, dysfunction and fibrosis in response to pressure overload in mice. SeP possibly plays a maladaptive role against progression of heart failure through the cardiohepatic interaction.
Heart failure stimulates colorectal tumour growth through gut microbial alterations of specific populations
Authors: De Wit S , Geerlings L , Aboumsallem J P , Shi C , Schouten E M , Blancke G , Andries V , Yntema T , Koonen D , Vereecke L , Sillje H H W , De Boer R A
Published: November 2023

Background/purpose

The field of reverse cardio-oncology has received growing attention in the last years, since epidemiological studies showed that patients with HF are at increased risk of developing cancer. In addition, HF can stimulate tumour growth directly in distant organs, including colon cancer. The exact mechanisms underlying this phenomenon, however, remain largely unknown. The connection between the microbiome and colorectal cancer (CRC) has been studied extensively and microbial dysbiosis in CRC is linked to cancer progression. Recent studies have also shown that HF patients display microbial dysbiosis. We hypothesized that microbial dysbiosis might contribute to HF-induced CRC tumour growth.

Methods

C57BL/6J mice were subjected to myocardial infarction (MI), with sham surgery as control. After six weeks faeces were collected, processed for 16s rRNA sequencing, and pooled for faecal microbial transplantation. CRC tumour growth was provoked in germ-free mice by treating them with Azoxymethane (AOM)/Dextran sodium sulphate (DSS). The CRC mice were transplanted with faeces from MI or sham mice. Disease progression was monitored using the disease severity score and endoscopy. After ten weeks mice were sacrificed, and tumour number and proliferation were quantified. All experimental procedures were performed in accordance with the European Union guidelines (CCD_8141, the Netherlands, LA_1400637, Belgium).

Results

MI-induced HF resulted in a decrease in microbial diversity (Shannon diversity p=0.018), characterized by a decrease in short chain fatty acid-producing bacteria, including the genera Blautia (p<0.01) and Roseburia (p<0.01). In addition, caecal butyrate levels were decreased in MI mice (p=0.029). These changes were largely comparable to microbial changes observed in HF patients and CRC patients. Interestingly, faecal transplantation with faeces from MI mice resulted in faster growth of CRC tumours in mice treated with AOM/DSS. This was demonstrated by a higher endoscopy score (7.9 ±2.5 vs. 10 ±2.4, p=0.038), indicative of worse disease progression, increased tumour counts (22 ±5.3 vs. 29 ±6, p<0.01), and a higher percentage of Ki67 positive cells in tumour tissue (p=0.012), relative to AOM/DSS treated mice transplanted with faeces from sham mice. Sequencing data revealed several differences in the microbial composition of mice transplanted with faeces from MI mice compared to mice transplanted with faeces from sham mice.

Conclusion

Our results demonstrate that HF induces microbial dysbiosis in mice. In addition, we show, for the first time, using a faecal transplantation model, that HF-induced microbial dysbiosis is causally involved in CRC tumour growth in a mouse model of CRC. Targeting the microbiome may present a tool to mitigate HF associated co-morbidities, especially cancer.Graphical abstract
Photobiomodulation attenuates myocardial fibrosis and diastolic dysfunction in a mouse model of heart-failure with preserved ejection fraction
Authors: Nachman D , Gavish L , Zavdi S , Aliev E , Horesh A , Shtraikh S , Gertz S D
Published: November 2023

Introduction

Photobiomodulation (PBM) has been shown to positively effect pathways associated with heart failure with preserved ejection fraction (HFpEF), such as reducing inflammation and increasing NO, microvascular flow, mitochondrial membrane potential, and ATP synthesis.

Purpose

This study aimed to determine whether PBM can mitigate myocardial fibrosis and diastolic dysfunction in a mouse model of HFpEF.

Methods

HFpEF was induced in 26, 3-month-old C57/black male mice by a ‘2-hit-model’ that includes high-fat diet and inhibition of NO-synthase by L-NAME (Nω-nitro-l-arginine-methyl-ester). Transcutaneous PBM was administered 3X/week[wk] for 10wks using a 904nm super-pulsed laser (90-seconds, 4.2J/cm2 per diode, n=13). 10 non-model control mice were also included. Echocardiography was performed at baseline, 7wks, and 10wks. Mice were sacrificed at ten weeks. Histological cardiac sections were stained for collagen with picrosirius red and scanned in X40 resolution. The positive pixel count algorithm of the Aperio ImageScope software (Leica Biosystems Inc.) was used to analyzed each vessel and measured the area of adventitial collagen (AC), perimeter of the external and internal elastic lamina (EEL, IEL). %Media=100*(EEL area-IEL area)/EEL Area; %Lumen=(IEL area/EEL area); and Standardized adventitial collagen = Area of AC / EEL perimeter were calculated. Significance was determined by 1-way ANOVA by group for %media, %lumen, and EEL area, and by 1-way ANCOVA by group with EEL area as a covariate for the standardized AC to take into account the variability in vessel sizes. p<0.05 was considered significant.

Results

Mice that were subjected to the HFpEF 2-hit model had a significant increase in the medial area of the coronary vessels and a concomitant decrease in the lumen area compared to mice that were not subjected to the model (Mean ±SD, %Media: control 50%±11; sham: 64%±11; PBM: 63%±12, p<0.001 control vs HFpEF). The model did not affect the overall vessel area defined by the EEL (p=0.547). The HFpEF model induced a significant increase in the standardized adventitial collagen compared to controls, which was attenuated by PBM: control 3.0±1.6; HFpEF sham: 3.9±1.8; PBM: 3.4±1.9, p=0.008 control vs sham, p=0.055 PBM vs control; p=0.396 control vs PBM). Moreover, diastolic flow parameters demonstrated that E’ deteriorated significantly in the sham group but not in the PBM group (E’[mm/sec], baseline-vs-10w: sham; -32.9±8.6-vs- -22.5±5.7, p=0.004; PBM; -32.1±5.9-vs- -27.9±6.4, p=0.262, p=0.037), and delayed deterioration in E/E’ (Δbaseline-7w, PBM vs sham: -3.6±7.0-vs-7.6±15.4, p=0.006).

Conclusions

PBM attenuated perivascular fibrosis and diastolic parameters deterioration in a mice model of HFpEF. Further investigations into the biomolecular and histological mechanisms are performed.Trial designResults
The use of wearable cardioverter defibrillator among patients suffering from myocarditis: data from a multicenter registry
Authors: El-Battrawy I , Koepsel K , Kovacs B , Blockhaus C , Gotzmann M , Klein N , Shin D I , Abumayyaleh M , Saguner A M , Erath J W , Duru F , Muegge A , Aweimer A , Akin I , Beiert T
Published: November 2023

Background

Data on the use of the wearable cardioverter defibrillator (WCD) among patients suffering from myocarditis in a large cohort remains sparse. Subsequently several guidelines give a different recommendation regarding WCD indication in this cohort.

Methods

1596 patients were included in a multicenter registry from ten European centers. Among this cohort 132 (8%) patients received a WCD due to myocarditis. The mean follow-up time of the whole cohort was 620.2±607.8 days. The presence of ventricular tachyarrhythmias and/or WCD shocks during WCD use were evaluated.

Results

The mean age of patients was 52.5±16.3 years (75% males). 20% of patients suffered from atrial fibrillation, 17% diabetes mellitus, 40% were smoker and 38.9% suffered from arterial hypertension. The body-mass-index (BMI) of the cohort was 27.8±5.7 kg/m². The initial left ventricular ejection fraction (LVEF) was 33.4±14.6% and increased to 45.9±13.2% (p<0.05) over long-term follow-up. Patient were discharged on ACE-inhibitors (62.5%), ARNI (21.2%), aldosterone-antagonist (50%), beta-blockers (89.5%) and amiodarone (13.6%). An improvement of LVEF (LVEF>35%) was documented in 81.2% of patients. The BNP level was at baseline 7401±30584 pg/ml and decreased to 530±803 pg/ml (p<0.05) over long-term follow-up. The wear duration was 78.5±51.5 days and 34% of patients wore the WCD for longer than 90 days. The wear-hours per day were 21.1±4.8 hours. In 3.1% sustained ventricular tachycardia, 0.8% ventricular fibrillation and 9.1% non-sustained ventricular tachycardia were documented. Subsequently 3% of patients suffered from an appropriate WCD shock. The rate of inappropriate WCD shock was 0.8%. Patients suffering from WCD shock were at age 31, 31, 61, 24 and 51 years and suffered a former non-sustained ventricular tachycardia before receiving a WCD. Among these patients suffering from appropriate WCD shock only two patients showed an LVEF<35%. Among the myocarditis patients 48% suffered from a former ventricular tachycardia and/or ventricular fibrillation and therefore received a WCD.

Conclusions

Myocarditis patients can be effectively protected by WCD and may benefit from its’ use. WCD shock occurred also in patients with an LVEF >35%.
Impact of isorhamnetin for suppression of action potential aberrations associated with atrial fibrillation
Authors: Aonuma K , Xu D , Murakoshi N , Tajiri K , Yuan Z , Murakata Y , Tominaga K , Nogami A , Aonuma K , Isoda H
Published: November 2023

Background

Isorhamnetin, a natural flavonoid, has potent antioxidant, antifibrotic and L-type Cav1.2 channel modulation effects, but its antiarrhythmic effect has not been reported.

Purpose

The purpose of this study is to investigate the effect of isorhamnetin on angiotensin II (Ang II)-induced atrial fibrillation (AF) in mice.

Methods

Wild-type male mice (C57BL/6J, 8 weeks old) were randomly divided into the following three groups: 1) control group, 2) Ang II treated group, 3) Ang II+isorhamnetin treated group. Ang II (1000 ng/kg/min) was administered by an implantable osmotic pump for two consecutive weeks, and isorhamnetin (5 mg/kg) was administered intraperitoneally for three weeks, one week before the initiation of Ang II administration. HL-1 cells were also assigned three groups. Ang II (1 μM) was treated for one day, and isorhamnetin (10 μM) was pretreated one hour before the beginning of Ang II treatment. To evaluate the effect of isorhamnetin on AF, electrophysiologic study including AF induction and action potential duration (APD) measurement with atrial tissue and HL-1 cells was performed. The expression of ion channel genes and proteins, including Cav1.2, were also evaluated.

Results

AF induction rate was markedly increased by Ang II and significantly decreased by isorhamnetin. AF duration was remarkably prolonged by Ang II and significantly shortened by isorhamnetin. Atrial effective refractory period (AERP) shortened by Ang II and reversed by isorhamnetin. Ang II administration significantly prolonged APD; that was alleviated by isorhamnetin. In addition, Ang II increased the frequency of delayed afterdepolarization (DAD), whereas isorhamnetin reduced its frequency. Cav1.2 channel expression was enhanced by Ang II and normalized by isorhamnetin. On the other hand, Ang II and isorhamnetin had no effect on potassium channels.

Conclusion

The findings in this study suggest that isorhamnetin prevents APD prolongation via the modulation of Cav1.2 channel and reduced the frequency of DAD, which is one of the mechanisms to prevent AF vulnerability.Effect of isorhamnetin on APD
Roles of aldosterone on ventricular arrhythmias, and associated gap junction and ion channel remodeling in hypertrophic heart
Authors: Kobara M , Toba H , Nakata T
Published: November 2023

Background

Ventricular arrhythmias are critical causes of death in patients with chronic heart failure (CHF). Aldosterone (Ald) is an exacerbating factor of CHF and mineral corticoid receptor antagonist (MRA) is usual drug for the management of CHF. MRA also suppressed sudden cardiac death, however, mechanisms of these effects remain unknown. We investigated the effects of MRA on ventricular arrhythmias and arrhythmogenic factors using Dahl Salt sensitive (DS) rats.

Methods

(in vivo) DS rats were divided into three groups; low salt group (LS), high salt group (HS), and eplerenone-, an MRA, treated high salt group (HS+EPL). Low salt (0.3% NaCl) or high salt (8% NaCl) were administrated for 8 weeks and EPL was administrated in the last 4 weeks. Cardiac geometry and function were assessed by echocardiography. Susceptibility of ventricular arrhythmias were assessed using programmed stimulation. Myocyte hypertrophy and cardiac fibrosis were examined by Masson’s trichrome staining. Expression and distribution of connexin43 (Cx43) were examined by immunostaining and western blotting. mRNA expression of Ca transient-related factors, such as phospholamban, SERCA2a, and ryanosin 2, and potassium ion channel-related factors, such as KCNE1 and KCNQ1, was examined by RT-PCR. (in vitro) Ald (1-100 nM) was administrated to cultured rat neonatal cardiomyocytes. Expression of Cx43, Ca transient-related, and potassium ion channel-related factors are also examined as mentioned above. Effects of Src tyrosin kinase inhibitor, PP2, and ERK inhibitor, PD98059, on Ald-induced Cx43 reduction were examined by RT-PCR and immunostaining.

Results

Systolic blood pressure and Ald concentrations in cardiac tissue were higher in HS group than LS group, and EPL did not affect them. In HS group left ventricular hypertrophy, dilatation and systolic dysfunction were observed in association with myocyte hypertrophy and interstitial fibrosis. EPL significantly recovered cardiac function and histological impairments. In addition, elongation of QTc in ECG and susceptibility of ventricular tachycardia were remarkable in HS group rather than LS and EPL attenuated them. Distribution of Cx43 was scattered in HS group, and EPL attenuated these changes. Decrease in SERCA2a and increase in KCNE1/KCNQ1 mRNA expression were found in HS group, and these changes were attenuated by EPL. In cultured myocytes, Ald decreased Cx43 mRNA and protein expression in a dose dependent fashion. Ald-induced Cx43 reduction was inhibited by PP2 and PD98059. In addition, Ald (100 nM) also decreased SERCA2a and increased KCNE1/KCNQ1 mRNA expression.

Conclusion

These results indicate that Ald reduced and dispersed the Cx43 expression reduction and dispersion through Src tyrosin kinase and ERK signaling pathways, and also mediated SRECA2A and KCNE1/KCNQ1 expression, which induced QTc prolongation and susceptibility of lethal ventricular arrhythmias in hypertensive cardiac hypertrophy.