bluuebunny/crossref_metadata_2025 huggingface dataset:
VLDL induces EZH2/PRC2 complex-mediated cellular senescence in metabolic syndrome to enhance cardiotoxicity
Authors: Lin Y H , Lee H C
Published: October 2024

Introduction

In metabolic syndrome, very-low-density lipoprotein (VLDL) carries cardiotoxic lipids that cannot be completely modified by lipid-lowering medications. VLDL isolated from patients with metabolic syndrome (ms-VLDL) induces cardiac remodeling compared with that isolated from non-metabolic syndrome subjects (n-VLDL).

Purpose

This study focused on identifying dysregulated molecular pathways of ms-VLDL-associated with senescence.

Methods

RNA-sequencing, accompanied with IPA and KEGG pathway analysis were used to identify differentially expressed genes (DEGs) in cardiomyocytes after two different VLDL incubations, and the identified DEGs were subjected to functional annotation, including enriched molecular pathway and gene ontology analyses. A Seahorse XFe96 Analyzer was used to determine the mitochondrial biosynthesis oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Molecules involved in the mitochondrial pathway were validated using Western blotting, qPCR, and immunofluorescence.

Results

Compared to the n-VLDL group, the ms-VLDL group had a larger number of significant DEGs involved in several critical biological processes and molecular pathways, such as cardiomyocyte injury (ms-VLDL vs. n-VLDL for significant DEGs numbers, 2,642 vs. 1,725), senescence (259 vs. 184), and mitochondrial dysfunction (137 vs. 68). In terms of mitochondrial biosynthetic function, ms-VLDL induced 8.5 folds OCR reduction compared to n-VLDL, along with intra-mitochondrial calcium overload and excessive ROS. The essential genes involved in these pathways were shown to be differentially regulated, and our analysis identified the Ezh2/PRC2 complex, which is strongly linked to ms-VLDL-induced cardiomyocyte senescence and lipotoxicity.

Conclusion

This study identified the crucial roles of epigenetic modification enzyme Ezh2/PRC2 complex in induction the senescence pathway, which represents a potential mechanism for ms-VLDL-induced adverse cardiac remodeling in metabolic syndrome. Further functional studies are required to validate their roles in the pathogenesis of lipotoxicity and the connection of cardiomyopahty.RNA-seq of VLDL treated cardiomyocyteSenescence induction of VLDL
Investigating the effects of empagliflozin on myocardial metabolic and lipid profile in healthy and metabolic syndrome murine models following acute myocardial infarction
Authors: Mylonas N , Siokatas G , Drosatos K , Andreadou I
Published: October 2024

Background/Introduction

Empagliflozin (EMPA) is a sodium/glucose co-transporter type 2 inhibitor with clinically documented cardioprotective effect. However, the mechanism of cardioprotection remains elusive. We have previously shown that Western diet induces metabolic syndrome (MS) and deteriorates cardiac function in mice, whereas EMPA treatment for 6 weeks prevents this damage. Also, EMPA administration for 6 weeks suppresses ischemia/reperfusion (I/R) injury in mice, independent of the presence of MS.

Purpose

The aim of the present study is to investigate whether changes in myocardial metabolic and lipid profile, induced by EMPA treatment, are responsible for its cardioprotective effect in the setting of myocardial I/R injury.

Methods

C57Bl6 mice received chow diet or Western diet for 14 weeks. At week 8, mice from each dietary group were randomized into 3 subgroups: 1. Control - Sham surgery (5% DMSO-SHAM), 2. Control - I/R (5% DMSO-I/R) and 3. EMPA (10mg/kg/day in 5% DMSO) - I/R (EMPA-I/R) and received the corresponding treatment by daily per os administration for 6 weeks. At week 14, mice were subjected to 30 minutes of I, followed by 2 hours of R, or sham surgery. Myocardial tissue was harvested at the end of R, and metabolomic and lipidomic analysis was performed by nuclear magnetic resonance spectroscopy and mass spectrometry, respectively. Body weight, as well as blood glucose and cholesterol levels were assessed at baseline and at the end of the administrations.

Results

EMPA administration reduced body weight, as well as blood glucose and cholesterol levels in animals with MS. EMPA decreased anaerobic glycolysis products (UDP-glucose, Lactic acid) and ketone bodies (β-hydroxybutyrate, acetoacetic acid) in both healthy animals and mice with MS. Additionally, an increase in ATP, accompanied by a decrease in AMP and ADP were observed in the EMPA-I/R group, compared to the DMSO-I/R group. Regarding the lipidomic analysis, gangliosides GM3 emerged as the major lipid group decreased by EMPA treatment. Lastly, despite significant changes were presented in the metabolic and lipid profile between healthy animals and those with MS, the effects of EMPA on myocardial metabolic and lipid profile were the same, regardless of the presence of MS.

Conclusion(s)

EMPA administration induces metabolic changes in the heart tissue and improves myocardial energetics upon myocardial I/R injury. Also, EMPA suppresses gangliosides GM3, a lipid group that has been associated with neutrophil activation and recruitment. Importantly, the effects of EMPA on myocardial metabolic and lipid profile following I/R is independent of the presence of MS.
Defibrillation success with a second generation intercostal extravascular ICD lead and commercially available left pectoral pulse generator
Authors: Burke M C , Doshi S , Ebner A , Husby M , Marcovecchio A , Sanghera R , Scheck D , O'connor M , Knops R E
Published: October 2024

Background/Introduction

An investigational intercostal extravascular ICD lead has been developed for compatibility with commercially available pulse generators (PGs) that are placed in a left pectoral position in appropriate patients. This is substantially different from currently available extravascular ICD systems that require a custom PG placed in a left lateral position.

Purpose

The purpose of this analysis was to evaluate sensing and defibrillation of a second-generation intercostal, extravascular defibrillation lead (EV-ICD Lead) when used with currently available ICD PGs in a traditional left pectoral location.

Methods

Patients (n=20) undergoing an ICD implant or replacement underwent acute VF EV-ICD Lead connected to a left pectoral pulse generator. Induced VF was sensed and shocked using a 10 J safety margin, with subsequent higher-energy shocks delivered if required. Testing was also conducted in the left lateral pocket for a paired analysis with patients serving as their own control.

Results

Automatic sensing of acute, induced VF episodes from the left pectoral PG was successful in 19 of 20 patients (95%) whose pre-induction R-waves were greater than 1.0 mV and unsuccessful in one subject with a pre-induction R-wave of 0.3 mV. VF was successfully terminated with a 10 J safety margin in 17 of 20 (83%) patients, a 5 J safety margin in 2 of 20 (10%) patients and failed to convert one patient (BMI 32.9) from the pectoral pocket (however, conversion was successful from a lateral pocket with a 10 J shock, which provides a 30 J safety margin. With a left lateral ICD, 100% of patients demonstrated successful VF termination with ≥10 J safety margin.

Conclusion(s)

Sensing and defibrillation using a left pectoral PG location is feasible with a new intercostal, extravascular defibrillation lead.
Lipin1 prevents ischemic heart disease by regulating lipid homeostasis
Authors: Guo J , Ueda K , Toko H , Harada M , Hashimoto M , Ishizuka M , Bujo S , Issei K
Published: October 2024

Background

Lipid metabolism plays a crucial role in the pathophysiology of cardiovascular diseases. Lipin1 plays a pivotal role in lipid metabolism regulation. In the nucleus, Lipin1 acts as a transcriptional co-stimulatory molecule, interacting with peroxisome proliferator–activated receptor–gamma coactivator 1 alpha (PGC1α) and peroxisome proliferator–activated receptor alpha (PPARα). In the cytoplasm, Lipin1 functions as a phosphatidate phosphatase, converting phosphatidic acid to diacylglycerol to regulate lipid metabolism. Low expression of Lipin1 has been implicated in the severity of cardiac dysfunction in mice.

Purpose

This study aimed to investigate the role of Lipin1 in the cardiac remodeling after myocardial infarction (MI), revealing its impact on lipid metabolism and molecular pathway associated with cardiac dysfunction.

Methods

Two types of transgenic mice, cardiomyocyte-specific Lipin1 knockout (cKO) and overexpression (cOE) mice were used in this study. Eight to ten-week-old male mice were subjected to MI by coronary artery ligation. Cardiac lipid droplets were quantified, and triacylglycerol and free fatty acid levels were measured one week after MI surgery. Four weeks after MI, cardiac morphology and function were evaluated using echocardiography, alongside measurements of body weight (BW) and heart weight (HW). Additionally, gene expression analysis and histological evaluation were conducted to assess fatty acid metabolism, cardiac fibrosis, inflammation, and oxidative damage.

Results

Before MI surgery, there were no differences in HW/BW ratio, left ventricular end-diastolic diameter (LVDd) or LV fractional area change (LVFAC) among cKO/ cOE mice and their littermate controls. After MI, cKO mice exhibited significant cardiac dysfunction with larger LVDd, lower LVFAC and increased HW/BW ratio compared to littermate controls. This was accompanied by exacerbated cardiac fibrosis, increased inflammation, and augmented reactive oxygen species accumulation compared with their controls. Conversely, cOE mice displayed improved LVFAC, reduced cardiac fibrosis, inflammation, and reactive oxygen species accumulation compared to their littermate controls. Notably, the transgene of Lipin1 induced changes in the number of cardiac lipid droplets (LDs). cKO mice hearts showed a reduction in cardiac LDs with decreased levels of triacylglycerol and free fatty acids compared to the littermate controls. In contrast, cOE mice displayed increased cardiac LDs and upregulated expression of fatty acid metabolism–related genes after MI.

Conclusion

These results suggested that Lipin1 has a protective role against ischemic injury through controlling lipid metabolism in ischemic cardiomyocytes. Thus, Lipin1 emerges as a potential therapeutic target for myocardial infarction.
ND-13 preserves mitochondrial integrity via the RhoA/ROCK1/Drp1-S616 pathway providing acute cardioprotection in myocardial infarction
Authors: Prakash A , Loo S J , Yu F , Cong S , Kalkhoran S B , Crespo-Avilan G E , Ramachandra C J , Hernandez-Resendiz S , Hausenloy D J
Published: October 2024

Background

Ischemia-reperfusion injury (IRI) following acute myocardial infarction is a major contributor to heart failure and mortality worldwide. Drp1-driven mitochondrial fragmentation disrupts mitochondrial function, increases cardiomyocyte death, and aggravates cardiac dysfunction. We investigated whether ND-13, a novel DJ-1-derived peptide, can acutely protect the heart by maintaining mitochondrial homeostasis and modulating Drp1 activity during IRI.

Methods

The effects of ND-13 on Drp1 activity were assessed by studying mitochondrial dynamics, function and survival signalling pathways. Cardioprotective effects of ND-13 were assessed using in vitro, ex vivo and in vivo models of IRI.

Results

ND-13 demonstrated acute cardioprotective effects in murine adult cardiomyocytes subjected to simulated IRI by a 42% reduction in cell death. The excessive mitochondrial fission following IRI was attenuated by inhibiting Drp1 phosphorylation at the Ser616 residue by reduced activity of the RhoA/ROCK1 pathway. This in turn enhanced mitochondrial function as seen through increased mitochondrial respiration rates (basal, maximal and spare respiratory capacity), increased ATP production, maintained mitochondrial membrane potential and reduced reactive oxygen species levels. The acute cardioprotective effect of ND-13 was reproduced in both an ex vivo Langendorff based model of IRI with a 43% reduction infarct size and an in vivo murine model of IRI with a 35% reduction of infarct size. These results indicate the robust effect of the peptide and its potential for clinical translation through intravenous delivery.

Conclusion

In this study, we demonstrate ND-13 as a small peptide that modulates mitochondrial dynamics through the RhoA/ROCK1/DRP1-S616 pathway, which highlights a potential new therapeutic target for myocardial IRI.
Endothelial NRG-1 knock-out causes left ventricle dilation in the presence and absence of angiotensin II administration
Authors: Van Berlo B , Wesley C D , Kruger D N , Van Asbroeck B , Neutel C H G , Civati C , Esposito P , De Keulenaer G W , Guns P J D F , Segers V F M
Published: October 2024

Introduction

Neuregulin-1 (NRG-1) is a growth factor derived from endothelial cells and belongs to the epidermal growth factor (EGF) family. While NRG-1 is expressed by various cell types, including epithelial cells, glial cells, neurons, and myocytes, the primary source of NRG-1 is considered to be the endothelium. NRG-1 exerts its effects through the EGF receptors ERBB2, ERBB3, and ERBB4. Upon ligand binding, these receptors dimerize and become phosphorylated, leading to downstream signaling that impacts tissue proliferation, differentiation, and survival.

The biological role of NRG-1 is intricate due to its isoforms and differential expression across distinct tissue types. In our study, we investigated the impact of endothelial NRG-1 knock-out (KO) on the heart, both in the presence and absence of angiotensin II (ANGII) treatment. ANGII serves as a trigger for NRG-1 upregulation.

Methods

C57BL/6N-VE-cad-cre-ERT2 mice were crossbred with C57BL/6N-NRG F/F mice, enabling endothelial-specific NRG-1 knockout (KO) using a tamoxifen-inducible promoter. At eight weeks of age, C57BL/6N-VE-cad-cre-ERT2-NRG F/F mice received tamoxifen (1 mg) or vehicle injections for nine consecutive days intraperitoneally (i.p.) to induce the KO. Two days after injection, osmotic minipumps were subcutaneously implanted, releasing a stable dose of angiotensin II (ANGII) at a rate of 400 ng·kg·min⁻¹ for a four-week period. Sham operations served as controls (SHAM). In the final week of treatment, high-frequency ultrasound imaging was performed to assess cardiac function and dimensions. After four weeks of ANGII treatment, mice were sacrificed, and organs were weighed and harvested for further molecular analysis.

Results

High-frequency ultrasound revealed left ventricle dilation in the absence of endothelial NRG-1, both in ANGII-treated and SHAM-treated mice. This was demonstrated by a significant increase in systolic and diastolic left ventricle internal diameter (LVID) in the endothelial NRG-1 KO groups compared to their respective controls. Additionally, systolic and diastolic LV volume (LV-vol) was significantly increased in the endothelial NRG-1 KO groups, regardless of ANGII treatment. Notably, there was no difference in LVID and LV-vol between SHAM and ANGII-treated groups in the presence of endothelial NRG-1, indicating that LV dilation is primarily due to the absence of endothelial NRG-1 rather than ANGII. Furthermore, heart weights of the endothelial NRG-1 KO mice were significantly increased compared to the control groups, as shown in Figure 1.

Conclusion

Our study demonstrates that endothelial NRG-1 KO leads to LV dilation, suggesting a crucial role for endothelial-secreted NRG-1 in maintaining normal LV function in adulthood. Further research is needed to fully understand the precise role of NRG-1 in LV homeostasis.
Inhibition of B cell activation by Ibrutinib improves cardiac function in experimental myocardial infarction in mice
Authors: Li X , Marchini T , Mwinyella T , Heitlinger S , Abogunloko S , Mitre L , Westermann D , Wolf D
Published: October 2024

Background

Bruton's tyrosine kinase (BTK) is a key downstream mediator of B cell receptor (BCR) signaling. Upon BCR cross-linking, phosphorylated BTK modulates B cell activation, proliferation, and differentiation. Ibrutinib, which is clinically used in B-cell malignancies, irreversibly binds to the BTK kinase domain and blunts downstream BTK signaling.

Purpose

Here, we aimed to monitor B cell dynamics and activation in experimental myocardial infarction (MI) in mice, and evaluate the effect of B cell inhibition by Ibrutinib in cardiac function.

Methods

MI was induced in male 10-week-old C57BL/6 mice by permanent LAD ligation and Ibrutinib (25 mg/kg/day) was given in the drinking water. After 3, 7, 14, and 28 days, single cell suspensions were prepared for flow cytometry from peripheral blood, spleen, mediastinal lymph nodes (MLNs), and the bone marrow, as well as from the remote and infarcted myocardium. Total B cells and B cell subsets were quantified in the different locations by flow cytometry and B cell activation was monitored by BTK551 phosphorylation. After 28 days, cardiac function was evaluated by echocardiography.

Results

We found that total B cell numbers decrease in blood and spleen, and increase in MLNs, while plasma cells and plasmablasts increased in the infarcted heart on day 7 and 14 after MI. In the bone marrow, hematopoietic stem cells (HSC) increase after MI, while common lymphoid progenitors (CLP) and early-stage B cell progenitors (pre-pro and pro B cells) peak at day 7 after MI. The later-stage B cell progenitors (pre and immature B cells) showed a biphasic response, with an early decrease after 7 days and a later increase on day 14. Compared to the control group (vehicle), Ibrutinib treatment decreased B cell numbers in MLN and spleen by 27% and 14%, respectively, as well as decreased mature B cells by 28% in the bone marrow on day 28 after MI. Both BTK expression and phosphorylation in pro-B cells were inhibited by Ibrutinib by 8% and 15%, respectively. The inhibition of BTK phosphorylation was also evident in MLNs and spleen. Notably, Ibrutinib-treated mice showed an improved cardiac function on day 28 after MI, with stroke volume (SV) elevated by 41%, left ventricular ejection fraction (LVEF) elevated from 20% to 36% (p < 0.05), and fractional shortening (FS) elevated from 8.8% to 17.59% (p < 0.05).

Conclusion

Experimental MI leads to B cell lymphopoiesis in the bone marrow, mobilization into the infarcted myocardium, and activation in peripheral lymphoid organs. Ibrutinib treatment inhibits B cell development in the bone marrow, as well as B cell activation in peripheral lymphoid organs, and improves cardiac function post MI. Our data provides a meaningful insight for targeting B cells for the clinical treatment of patients with MI.Figure
Macrophage MST4 exacerbates cardiac microvascular ischaemia/reperfusion injury by phosphorylating ACLY
Authors: Chen A , Xia Y , Che X Y , Chen Z W , Qian J Y , Ge J B
Published: October 2024

Background

Cardiac microvascular dysfunction is a contributing factor in the development of cardiac ischaemia/reperfusion (I/R) injury. The STE20-type kinases play important role in cardiovascular diseases. However, the impact of mammalian Ste20-like kinase 4 (MST4) on cardiac microvascular I/R injury remains unknown.

Purpose

To decipher the role of MST4 in cardiac microvascular I/R injury and the underlying mechanisms.

Methods

Single-cell RNA sequencing of I/R mouse heart was performed to explore the expression changes of MST4 in different cell types. MST4 flox (MST4 f/Y) mice was crossed with myeloid-specific LysM-Cre mice to generate myeloid-specific MST4 knockout mice (MST4 CKO). Bone marrow transplantation was used to testify the role of myeloid-derived MST4 in cardiac microvascular I/R injury. Bone-marrow-derived macrophage (BMDM) and cardiac microvascular endothelial cell (CMEC) were used to dissect molecular mechanisms. Adeno-associated virus and adenovirus were constructed to overexpress ATP Citrate Lyase (ACLY) with mutation of serine 455 (S455A) in vivo and in vitro, respectively. Phosphoproteomics, coimmunoprecipitation coupled with liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis and metabolomics were performed to decipher the downstream of MST4.

Results

MST4 is dominantly upregulated in CCR2+ monocyte-derived macrophages. Myeloid-specific deficiency of MST4 improves cardiac performance in I/R mouse model. Wild type (WT) mice transplanted with BMDM from MST4 CKO mice also exhibited improved cardiac function after I/R, however, MST4 CKO mice transplanted with BMDM from WT mice showed deteriorated cardiac performance. Metabolomics showed that primary bile acid biosynthesis pathway was upregulated in BMDM overexpressing MST4. The level of 7α-hydroxyl3-oxo-4-cholestenoic acid (7-HOCA), an intermediate of bile acid, was dramatically increased in I/R mice or BMDM overexpressing MST4, which can be reversed by knocking down MST4. Phosphoproteomics and LC-MS/MS analysis discovered that ACLY was one of the most important substrates of MST4 in the process of I/R injury. ACLY was directly phosphorylated by MST4 at serine 455, which increased ACLY enzyme activity. Mechanistically, ACLY promoted cholesterol catabolism through the bile acid synthesis pathway, leading to the accumulation of 7-HOCA in the macrophage-endothelial cell microenvironment. High level of 7-HOCA was able to promote endothelial cell mtDNA release, leading to endothelial cell pyroptosis through inflammasome pathway. However, mutation of serine 455 of ACLY blocked the role of MST4.

Conclusions

Our data defined a previously unrecognized role of MST4/ACLY pathway in cardiac microvascular I/R injury. MST4 led to the accumulation of 7-HOCA by phosphorylating ACLY, furthermore, 7-HOCA promoted endothelial cell injury. Targeting MST4/ACLY pathway ameliorated microvascular I/R injury, which may provide a novel therapeutic strategy for I/R injury.
Takotsubo syndrome induced oxidative stress and endothelial dysfunction in left ventricle of rat model: association with NADPH oxidases/SGLT2 pro- oxidant pathway
Authors: Tatarcheh T , Amissi S , Matsushita K , Mroueh A , Muzammel H , Trimaille A , Gong D , Fakih W , Faucher L , Granier A , Pieper M P , Schini-Kerth V , Morel O
Published: October 2024

Background

Takotsubo syndrome (TTS) is characterized by apical ballooning predominantly affecting post-menopausal women following extreme physical and/or emotional stress. The phenotype of TTS is associated with akinetic apex and hyperkinetic base of the left ventricle (LV). Presently, there are no targeted therapies for TTS that can effectively reduce LV impairment. Empagliflozin (EMPA), an SGLT2 inhibitor, has shown considerable clinical benefits in managing heart failure. However, its specific effects and underlying mechanisms in treating TTS remain unclear.

Purpose

To explore the pathophysiology of TTS in the LV of rats, with a particular focus on the potential role of SGLT2, and to elucidate the underlying mechanisms involved.

Methods

Female Sprague Dawley rats (250-300 g) were allocated into four groups: a control group (n=8), a group treated with EMPA (30 mg/kg/day) (n=8), a group receiving ISO (Isoproterenol) at 100 mg/kg/day (n=18), and a group receiving both ISO and EMPA (IE) at 30 mg/kg/day (n=18). EMPA was administered orally through food two weeks prior to ISO injection. Echocardiography was conducted at 6 and 24 h, and on day 3. On day 3, rats were euthanized, and their organs were collected. Gene expression of markers was assessed using RT-qPCR, protein expression via immunofluorescence (IF) staining, and oxidative stress using dihydroethidium.

Results

In the ISO group, the success rate of induced Takotsubo syndrome (TTS) was 53% (10 out of 18 rats), with a mortality rate of 5%. Conversely, in the EMPA-treated group, TTS occurred in 22% of rats (4 out of 18) with no mortality observed. Significant reductions in the area of left ventricular (LV) ballooning and ejection fraction were noted in the IE group compared to the ISO group. Additionally, increased levels of reactive oxygen species (ROS) were observed in the LV of ISO rats, which were normalized by EMPA treatment in the IE rats. Furthermore, the ISO group exhibited higher protein expressions of CD68, SGLT2, and VCAM-1, which were attenuated by EMPA treatment. The mRNA expression of CYBA (p22phox) and NOX4, both representing subunits of NADPH oxidases, along with VCAM1, ICAM1, NOS2 (iNOS) and SELE (e-selectin) gene, endothelial activation marker, and TGF-β1, a fibrotic marker, showed elevated levels in the apex part of the left ventricle (LV) in the ISO group; these levels were significantly lower in the IE group.

Conclusions

These findings suggest that TTS is associated with oxidative stress-mediated endothelial dysfunction, inflammation, and pro-fibrotic responses. Moreover, SGLT2 inhibition appears to mitigate the pathology and progression of the syndrome. Therefore, SGLT2 inhibition emerges as an appealing and novel therapeutic approach for the treatment of TTS.
Epi-Allele: CRISPR-mediated allele-specific epigenetic editing alleviates hypertrophic cardiomyopathy
Authors: Wang Z , Fu Y , Han C , Wang Q , Huang Z , Ge J
Published: October 2024

Background

Hypertrophic cardiomyopathy (HCM) is a prevalent genetic heart disease caused by variants in sarcomere genes like MYH7, which accounts for 40% of cases with 75% being heterozygous. HCM shows incomplete penetrance, where mutant allele expression correlates with severity. Thus, allele-specific suppression is a potential therapy. While allele-specific RNAi or CRISPR/Cas9 showed moderate efficacy, low expression level of MYH7 is associated with the risk of heart failure and dilated cardiomyopathy. We hypothesized CRISPR-based epigenetic editing（dCas9 fused with DNA methylation and histone regulation elements） may potently achieve phenotypic modification without impacting overall gene expression.

Objective

We investigated allele-specific CRISPR epigenetic editing (Epi-Allele) to selectively suppress mutant Myh6 allele expression without affecting total expression, as a novel HCM therapy in a mouse model.

Results

We first generated C57R403Q/DBA mice to simulate heterozygous HCM patients. Allele-specific Myh6 gRNAs reduced C57 allele expression and increased DBA expression without impacting total Myh6 expression in primary cardiomyocytes, effects sustained for one year in report cells. HCM mice with Epigenetic editor and allele-specific gRNAs (Epi-Allele) showed reduced left ventricular wall thickness, normalized ECG, and less cardiac fibrosis. Similar therapeutic effects were observed in HCM mice treated by a dual AAV delivery system of Epi-Allele. We further found that Epi-Allele could reverse established disease in the inducible transgene mice. We’ve also seen similar surprising therapeutic effects from Epi-Alleles in patient-derived pluripotent stem cell-induced cardiomyocytes (iPSC-CMs).

Conclusion

We suggest that Epi-Allele may be a novel therapy for HCM and other dominant diseases where the mutant gene plays a vital role, overcoming the limitations of previous allele-specific suppression strategy.Figure abstract of Epi-AlleleThe universality of Epi-Alleles
Novel balloon catheter containing therapeutic mRNA with smart RNA switch alleviate restenosis after vascular injury
Authors: Fu Y , Wang Z , Jia Y , Tan Y , Wang Q , Li Q , Huang Z , Ge J
Published: October 2024

Background

Cardiovascular diseases are the leading cause of death worldwide, and vascular intervention is an effective method for ischemic heart diseases. However, vascular restenosis significantly affects the long-term therapeutic outcomes, which mainly caused by excessive proliferation of vascular smooth muscle cells (VSMCs). While drug-eluting stents and drug-coated balloons have successfully decreased the incidence of restenosis by inhibiting the VSMCs proliferation, they concurrently suppress the repair of endothelial cells (ECs) and re-endothelialization, thereby increasing the risk of late thrombotic events and mortality. Consequently, it is urged to develop novel high-selectively therapeutic devices to overcome vascular restenosis.

In current mRNA therapies of cardiovascular diseases, there are few effective systems for cell-specific expression. Therefore, we propose to utilize RNA editing (based on ADAR1 RNA editing) to investigate a smart RNA molecular switch capable of cell-specific recognition and selectively releasing payload in targeted cells or tissues for cell precision therapy. However, the application of gene editing in the cardiovascular system faces challenge of low delivery efficiency. We innovatively take the balloon as a specific and efficient tool to delivery therapeutic mRNA, which may be a potential therapy for vascular restenosis.

Objective

We investigated therapeutic mRNA containing a smart RNA switch to selectively suppress the VSMCs proliferation while promoting re-endothelialization and delivered by balloon as a novel therapy in vascular restenosis animal models.

Results

For RNA switch high-throughput screening, we utilized PiggyBac transposon system to establish monoclonal overexpressing cell lines and chose the cell strain with expression levels closely mimicking VSMCs and ECs. We found the most effective RNA switch from dozens of potential candidates which could specifically recognize VSMCs but not ECs. Therapeutic mRNA sequences containing the RNA switch efficiently initiated payload translation in VSMCs while maintaining payload silence in primary vascular endothelial cells, which effectively and selectively inhibited VSMCs excessive proliferation and promoted endothelial repair. Additionally, we developed an interventional balloon delivery system for mRNA drugs, achieving in situ delivery to cardiovascular lesions in vivo, demonstrating therapeutic effects consistent with cellular phenotypes in animal models.

Conclusion

We suggest that the smart RNA switch can serve as a novel, cell-specific therapeutic tool for restenosis and other cardiovascular diseases. Additionally, utilizing a balloon for mRNA delivery represents a new paradigm for mRNA therapy and gene editing in the cardiovascular field.
Epigenetic modifications in transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells
Authors: Shinjo Y , Takashima S , Usui S , Hashimuko D , Yamaguchi K , Takeda Y , Goten C , Inoue O , Sakata K , Takamura M
Published: October 2024

Background

Adipose tissue-derived stromal vascular fraction (adipose SVF) contains pluripotent mesenchymal stem cells and rarely transdifferentiate into beating cardiomyocytes. We developed a simple culture protocol under which the adult murine inguinal adipose SVF reproductively transdifferentiates into beating cardiomyocyte-like cells (beating CM) without inductions in the primary culture. We also reported that Mef2c is a crucial factor in this conversion. However, the characteristics of beating CM and the factors that regulate the differentiation of adipose SVF toward the cardiac lineage are unknown.

Purpose

To investigate sequential changes in global gene expression profiles of adipose SVF in primary culture and determine the mechanism of differentiation into beating CM.

Methods

Adipose SVF cells were isolated from adult mice's inguinal subcutaneous fat pad and cultured using the previously established beating CM induction method (Sci Rep. 2021). At 6 time points (days 0, 3, 7, 14, 21, and 28) in primary culture, total RNA was extracted, and RNA-sequencing was performed (n = 3). The data was analyzed using Subio Platform and MetaCore software. To assess the candidate gene and proteins, adipose SVF cells were transduced with the gene using a lentivirus vector and supplemented with the specific inhibitors. The cardiac differentiation was evaluated by quantitative PCR on day 28.

Results

The beating CM was confirmed on days 14, 21, and 28 in the primary culture. Among prefiltered 14,574 genes, the expression level of 749 genes were significantly changed (153 genes upregulated vs. 596 genes downregulated) between days 7 and 14 (2-fold, P < 0.05), in which beating CM appeared. The GO molecular functions analysis showed that O-GluNAC transferase and histone deacetylase (HDAC)-associated genes were expressed dominantly before day 7, and KLF4-associated genes were expressed prominently after day 14 in primary culture. The expression of cardiac troponin T (Tnnt2) and myosin light chain ventricular type (Myl2) increased on day 3 and day 14, respectively. The HDAC7, an epigenetic regulator, decreased the expression on day 14 or later. Treatment with the HDAC inhibitor (150 nmol/L) increased the expression of Tnnt2 (5-fold vs. control) on day 28 in adipose SVF. Furthermore, treatment of Mef2c-transduced adipose SVFs with HDAC inhibitors increased expression of cardiac troponin T (29-fold vs. control, P < 0.05).

Conclusion

The time course analysis demonstrated that a remarkable change occurred in the regulation of transcription on day 14 in adipose SVF primary culture. Alterations in epigenetic modifications increased beating CM in adipose SVFs. Adipose SVF could be applied to new cardiac regeneration therapies by increasing the efficiency of introduction into the beating CM.
Prevalence of elevated cardiac troponin in five non-cardiac diseases
Authors: Knudsen S , Hasselbalch R B , Strandkjaer N , Bundgaard H , Iversen K K
Published: October 2024

Background

Cardiac troponins (cTns) are regulatory proteins located in the cardiac muscle cells (cardiomyocytes). Since the 1990s, cTns have been the biochemical gold standard for detecting myocardial infarction. CTns are well studied among patients with cardiac illnesses and elevated concentrations are known to reflect myocardial injury. Still, high cTn concentrations are found in several non – cardiac conditions where signs of cardiac dysfunction are absent(1). This is especially found to be true for cardiac troponin T (cTnT) which is, more frequently than cardiac troponin I (cTnI), found to be elevated in patients with non – cardiac conditions(2,3). A study from 2016 of 1012 patients admitted to the emergency room, found troponin T elevations in 40.8% of patients without acute coronary syndrome vs. 7.4% with cTnI elevations(3). To our knowledge, no previous study has investigated cTn elevations across disease -groups where elevations of cTns are frequently detected.

Purpose

We aimed to investigate the prevalence of cTnT in five clinical non-cardiac disease-groups where frequent elevation of cTnT is previous described; Sepsis, rhabdomyolysis, end-stage renal disease (ESRD), stroke, and major non-cardiac surgery (MNCS).

Methods

Our study was a single-center prospective cross-sectional cohort study. Over a 6-month period, we included patients in the five above mentioned disease-groups. We excluded patients with any symptoms of cardiac disease or a prior history of cardiac diseases. All patients were included within 48 hours of admission and underwent clinical examination, vital signs, 12-lead electrocardiogram, echocardiography, and collections of blood samples. Cardiac troponin T was measured at patient’s bedside upon inclusion with the Cobas h232, cTnT point-of-care test.

Results

89 patients were eligible for inclusion, 15 with sepsis, 18 with rhabdomyolysis, 11 with end-stage renal disease, 22 with stroke and 23 with major non-cardiac surgery. 53% were men. 17 patients (19%) had signs of cardiac disease confirmed on echocardiographic and/or electrocardiographic examinations and were excluded. Out of the 72 patients without signs of cardiac disease, elevated cTnT above the 99th percentile was found in 21 (29%) patients; 4 (36%) with sepsis, 6 (60%) with rhabdomyolysis, 4 (44%) with ESRD, 2 (9.5%) with stroke and 5 (24%) with MNCS. The median of cTnT in patients with elevated cTnT across all patient groups was 79.5 ng/L (IQR 52.5ng/L – 187.8 ng/L).

Conclusions

Cardiac troponin T elevations are frequent in the five non-cardiac diseases investigated. Elevations are most frequent in patients with rhabdomyolysis and end-stage renal disease. Further investigation is warranted to elucidate the underlying etiology contributing to the elevation of cardiac troponin in non-cardiac diseases.
Scalable enzymatic lactate sensor for continuous monitoring in interstitial fluid
Authors: De Schrijver L , Saeidi A , Sheibani S , Locca D , Ionescu A
Published: October 2024

Elevated lactate levels have been shown to be an indicator of poor outcome in cardiovascular patients [1]. Moreover, continuous monitoring of lactate may be more clinically valuable than the absolute value of lactate for risk stratification [2]. A promising approach involves the implementation of a lactate monitoring system inspired by advanced continuous glucose monitoring [3] to provide real-time measurements of lactate in interstitial fluid (ISF) based on miniaturized lactate sensors with submillimeter dimensions.

The proposed amperometric lactate sensor employs Pt electrodes (WE = 0.785 mm²) on which the enzyme lactate oxidase is immobilized to selectively detect lactate. The sensing mechanism, shown in Fig. 1a, is based on the conversion of lactate to hydrogen peroxide, which then undergoes oxidation at the electrode surface, generating a current proportional to the concentration of lactate. Amperometric measurements are performed with an applied potential of 0.5 V, a sample volume of 2 µL and a flow rate of 1 µL/min, respecting real-life conditions. The response of the sensor towards lactate is shown in Fig. 1c, demonstrating excellent reversibility, long-term stability over 8 hours and negligible cross sensitivity to glucose (G), ascorbic acid (AA) and uric acid (UA). In addition, the sensor linear range can be extended by 10x by incorporating an outer Nafion layer, acting as a diffusion-limiter. As shown in Fig. 1d, by adjusting the thickness of Nafion layer, the linear range increases up to 20 mM lactate, as per ICU specifications.

The physiological challenges associated with ISF extraction prompted us to develop a minimally invasive, submillimeter-sized sensor for direct placement inside the body. To address the most critical step of miniaturization, we have successfully fabricated a three-electrode platform on a Si ribbon measuring 300x1500 µm² and a thickness of 100 µm (Fig. 2a). The sensor is also drastically scaled down, with smallest design having a total sensing area of 200x500 µm² (WE = 0.026 mm², Fig. 2b). Its response to lactate is displayed in Fig. 2c, confirming a reliable functionality. Fig. 2d presents the calibration curves obtained from sensors with different sizes, indicating that the reduction in size does not affect the sensitivity.

The developed lactate sensor demonstrates excellent performance: high sensitivity, excellent reversibility, and high stability. In addition, the linear range of the sensor is tunable by integrating an outer Nafion membrane. Finally, we demonstrate the successful fabrication and characterization of submillimeter-sized sensors, opening up their potential for minimally invasive in-vivo measurements.Working principle and characterizationCharacterization miniaturized sensors
Renal Semaphorin 3C mediates cardiac adaptive response
Authors: Otaki Y , Manabe I M , Watanabe T W , Aono T A , Sato J S , Tachibana S T , Kobayashi Y K , Watanabe M W
Published: October 2024

Cardiorenal syndrome, characterized by reciprocal heart-kidney interactions that progressively promote dysfunction in both organs, is associated with a high mortality rate. However, the mechanisms linking the two organs are insufficiently understood. Here we report that semaphorin 3C (SEMA3C) is a novel cardioprotective mediator secreted from the kidney. SEMA3C was highly expressed in the renal proximal tubules in mice. Renal Sema3c expression and circulating SEM3C were reduced by the left ventricular pressure overload that induced renal remodeling. Proximal tubule-specific secretory Sema3c deletion exacerbated cardiac hypertrophy, fibrosis and dysfunction after the pressure overload, suggesting renal SEMA3C is important for the proper cardiac adaptive response. Mechanistically, SEMA3C inhibits pathological cardiac remodeling at least partly by inhibiting Akt, ERK and Wnt/β-catenin signaling in cardiomyocytes. In heart failure patients, the plasma SEMA3C level was decreased and the low plasma SEMA3C was associated with poor cardiac outcomes. Collectively, our results show that SEMA3C produced by the kidney pivotally contributes to the cardiac stress response and its reduction contributes to the progression of heart failure. SEMA3C signaling appears to be an attractive therapeutic and diagnostic target for cardiorenal syndrome.
Milk-derived extracellular vehicles alleviate heart failure with preserved ejection fraction by modulating gut microbiota
Authors: Zhang S , Tong L , Hao H , Huang C , Tan D , Sikka V , Lou C , Yu X , Yang L , Lin Y , Storm G , Wang J
Published: October 2024

Background

Accounting for around 50% of human heart failure, heart failure with preserved ejection fraction (HFpEF) is driven by complex and heterogeneous pathological factors, including gut microbiome dysbiosis. We have previously reported that milk-derived extracellular vesicles (mEVs) modulate the gut microbiota in inflammatory bowel disease. However, whether mEVs can affect gut microbiota and thereby alleviate HFpEF remains unexplored.

Aims

We aim to uncover potential therapeutic effects of mEVs on HFpEF and if so, the underlying mechanisms.

Methods

The HFpEF mouse model was generated by feeding C57/B6 mice on a high-fat diet and L-NAME for 15 weeks. Mouse feces samples were analyzed to detect the presence of gut dysbiosis in HFpEF. Mice received bovine mEVs through oral gavage. Heart function was assessed with echocardiography. Mouse feces and plasma were collected to analyze gut microbiota and metabolomics, respectively.

Results

Gut microbiota was disturbed in HFpEF mice. Oral administration of mEVs effectively attenuated heart failure which was evidenced by less bodyweight gain, reduction in cardiac hypertrophy and lung weight, and improvement in cardiac function parameters such as ratios of E/A (1.8 vs. 1.5, p<0.05) and E/E’ (40 vs. 27, p<0.05). Moreover, the increase in mouse blood pressure in HFpEF was also inhibited upon treatment (SBP 130mmHg vs. 100mmHg, DBP 90mmHg vs. 75mmHg, p<0.05). Mechanistically, we ruled out the possibility of direct action of mEVs on the heart as orally administered mEVs were only distributed in the intestine. On the other hand, mEVs restored gut microbiota by increasing the abundance of beneficial bacteria such as Lachnospiraceae. As a result, oral administration of mEVs elevated blood levels of Lachnospiraceae metabolite, butyric acid, a type of short-chain fatty acid. To confirm the butyric acid-mediated heart-protective effects of mEVs, we administered butyric acid to HFpEF mice. Strikingly, butyric acid largely reproduced the therapeutic effects of mEVs on HFpEF.

Conclusions

Oral administration of mEVs restores gut microbiota dysbiosis, thereby attenuating heart failure in HFpEF. mEVs may represent a new biological therapeutic for the treatment of HFpEF.
The intracardiac echocardiographic findings during rewarming using catheter-based cryoablation
Authors: Sugitani M , Takanao M , Takahashi S , Terao S , Kogame T , Ishihara M
Published: October 2024

Introduction

Cryoablation unlike radiofrequency ablation (RFA) does not damage the endothelium and lining and causes less physical damage, and the mechanisms remain unclear. The increased myocardial echogenicity (iME) observed by intracardiac echocardiography (ICE) during RFA has been reported as the ablation indicator.

Purpose

To evaluate the ICE findings during ablation using catheter-based cryoablation.

Methods

In an extra vivo experiment, we set the bovine myocardium to 36°C in a constant temperature bath and experimented on a water tank that generated a non-pulsatile flow. Catheter-based cryoablation was applied to the bovine myocardium at -80 degrees for 240 seconds. The ICE images were evaluated using ViewFlex Xtra during catheter-based cryoablation.

Results

Macroscopic change (Figure 1): Four seconds after the start of cooling, a thin film of ice began to form between the myocardium and the catheter, and after 7 seconds, the tip of the catheter was covered with ice and the surface of the myocardium was covered with ice. After that, it grew rapidly and an ice ball was formed. There was no change after 18 seconds of cooling. ICE findings (Figure 2): 4 seconds after cooling started, the ME around the catheter tip began to increase, and the ME gradually increased with flickering. The iME stopped after cooling down for 18 seconds and did not change after that. Immediately after stopping cooling, severe flickering occurred and the area of iME began to shrink. The ME gradually returned to its original state with slight bubbles from the central ablation site. Macroscopic change and ICE findings were reproducibly confirmed. Myocardial findings after ablation: The area of cooling was discernible, but the color difference from the surrounding area was not obvious.

Conclusions

The catheter-based cryoablation ablation shows the severest change detected by ICE immediately after stopping cooling. In addition to tissue damage during the freezing stage, the rewarming may be more associated with tissue damage.
Chromatin remodeling by the histone methyltransferase SETD2 drives cardiometabolic heart failure with preserved ejection fraction
Authors: Costantino S , Ambrosini S , Mohammed S A , Gorica E , Ruschitzka F , Paneni F
Published: October 2024

Background

Cardiometabolic heart failure with preserved ejection fraction (cHFpEF) is highly prevalent and associates with a poor outcome. Pathological gene expression in heart failure is accompanied by changes in active histone marks without major alterations in DNA methylation. Histone 3 trimethylation at lysine 36 (H3k36me3) - a chromatin signature induced by the histone methyltransferase SETD2 – has shown a strong correlation with changes in gene expression in the human failing heart; however its role in cardiac disease is poorly understood. The present study investigates the role of SETD2/ H3k36me3 in cHFpEF.

Purpose

To investigate the role of chromatin remodelling in obese HFpEF (obHFpEF).

Methods

Mice with cardiomyocyte-specific deletion of SETD2 (c-SETD2-/-) were generated and subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce cHFpEF. Cardiac function and exercise tolerance were assessed by echocardiography and Treadmill exhaustion test, respectively. ChIP-Seq datasets were employed to determine the biological pathways regulated by H3k36me3, whereas chromatin immunoprecipitation assays (ChIP) were performed to investigate SETD2/H3k36me3 enrichment on gene promoters. SETD2 gain- and loss-of-function experiments were performed in cultured cardiomyocytes (CMs) exposed to metabolic stress (palmitic acid). SETD2/H3k36me3 axis was also investigated in left ventricular (LV) myocardial specimens from patients with cHFpEF and control donors. Moreover, pharmacological inhibition of SETD2 was performed in skinned CMs from cHFpEF patients.

Results

Bioinformatic analysis of ChIP-Seq data in mouse CMs showed a strong involvement of SETD2/H3k36me3 in lipid-related pathways. Specifically, SETD2 and H3k36me3 were upregulated in cHFpEF vs. control mouse hearts and were highly enriched on the promoter of sterol regulatory element-binding transcription factor 1 (SREBP1) gene. SETD2 activation in cHFpEF led to SREBP1 upregulation, myocardial triglyceride accumulation and lipotoxic damage. In cHFpEF mice, cardiomyocyte-specific deletion of SETD2 prevented hypertrophic remodeling, diastolic dysfunction and lung congestion while improving exercise tolerance. Mechanistically, SETD2 deletion blunted H3K36me3 enrichment on SREBP1 promoter thus preventing SREBP1-driven lipid accumulation and leading to a significant rewiring of the cardiac lipidome. These findings were associated with restoration of autophagic flux in the cHFpEF myocardium. In CMs exposed to palmitic acid, SETD2 depletion prevented SREBP1 upregulation, whereas SETD2 overexpression recapitulated lipotoxic damage. Finally, SETD2 was upregulated in LV myocardial samples from cHFpEF patients and its pharmacological inhibition attenuated CM stiffness.

Conclusions

SETD2 is a new molecular target implicated in the pathophysiology of cHFpEF.