bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

Abstract

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 &lt; 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 &lt; 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

Abstract

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

Abstract

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

Abstract

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.