Journal: Europace

Article Title: Impact of pulse duration on cardiac electroporation: nanosecond pulses enhance cardiomyocyte selectivity and promote a Raman-detected shift towards apoptotic cell death

doi: 10.1093/europace/euaf217

Figure Lengend Snippet: Time course of cell death in human cardiomyocytes and endothelial cells following nsPEF and µsPEF exposure. ( A and B ) Time-dependent cell death in human cardiomyocytes (AC16) and endothelial cells (HUVEC) exposed to nanosecond pulsed electric fields (nsPEF: 200 pulses, 10 Hz, 300 ns, 1.4 kV; A ) or microsecond pulsed electric fields (µsPEF: 20 pulses, 1 Hz, 100 µs, 300 V; B ). Graphs depict the percentage of dead cells within regions exposed to varying electric field strengths at different time points. Data are presented as mean ± SEM from four independent experiments per time point. Cardiomyocyte death increased significantly over the first 24 h after both nsPEF and µsPEF exposure, with an apparent decline between 24 and 60 h likely reflecting repopulation of the ablated area by viable cells and/or detachment of dead cells. Endothelial cells exhibited an earlier onset of death, with marked increases evident as early as 2 h after both treatments. Representative fluorescence microscopy images (right side of each panel) illustrate monolayer morphology following exposure, with magnified insets showing lesion progression over time and across the electric field gradient—from the ablation centre (highest field intensity) to the periphery (minimal field intensity).

Article Snippet: Experiments were conducted using the AC16 human cardiomyocytes (Cytion, Eppelheim, Germany) and human umbilical vein endothelial cells (HUVEC, C-12205; PromoCell, Heidelberg, Germany).

Techniques: Fluorescence, Microscopy

Journal: Europace

Article Title: Impact of pulse duration on cardiac electroporation: nanosecond pulses enhance cardiomyocyte selectivity and promote a Raman-detected shift towards apoptotic cell death

doi: 10.1093/europace/euaf217

Figure Lengend Snippet: Differential sensitivity of human cardiomyocytes and endothelial cells to µsPEF and nsPEF. ( A and B ) Dose–response curves showing the viability of human cardiomyocytes (AC16) and human endothelial cells (HUVEC) following exposure to nsPEF ( A ) and µsPEF ( B ). Data were fitted with sigmoidal curves using the Hill equation; shaded regions represent 95% confidence intervals. Each curve is based on at least five independent experiments. For µsPEF exposure, the ED₅₀ (electric field intensity causing 50% cell death) was significantly lower in HUVECs compared to AC16 cells, indicating greater sensitivity of endothelial cells to µsPEF-induced cytotoxicity. Bar graphs compare the mean ED₅₀ values for AC16 and HUVEC cells after exposure to nsPEF and µsPEF. Error bars indicate 95% confidence intervals. These comparisons further underscore the differential susceptibilities of the two cell types, with endothelial cells showing greater sensitivity to µsPEF. Representative fluorescence microscopy images show peripheral ablation zones in AC16 and HUVEC monolayers after µsPEF exposure. Insets highlight differences in cell viability and lesion extent, illustrating the distinct responses of each cell type to pulsed electric field exposure.

Article Snippet: Experiments were conducted using the AC16 human cardiomyocytes (Cytion, Eppelheim, Germany) and human umbilical vein endothelial cells (HUVEC, C-12205; PromoCell, Heidelberg, Germany).

Techniques: Fluorescence, Microscopy

Journal: Europace

Article Title: Impact of pulse duration on cardiac electroporation: nanosecond pulses enhance cardiomyocyte selectivity and promote a Raman-detected shift towards apoptotic cell death

doi: 10.1093/europace/euaf217

Figure Lengend Snippet: Interplay between plasma membrane permeabilization and ablation efficiency after nsPEF and µsPEF exposure in cardiomyocytes and endothelial cells. ( A and B ) The association between plasma membrane permeabilization and cell death in AC16 cardiomyocytes and HUVEC endothelial cells following exposure to nsPEF ( A ) and µsPEF ( B ). Green curves show the extent of membrane permeabilization, quantified by YO-PRO-1 uptake as a function of calculated electric field intensity across ROIs. Red curves indicate cell death, measured by PI uptake at the peak time point determined from the cell death time-course analysis (see Figure ). Data were fitted using sigmoidal curves generated with the Hill equation (solid lines), and shaded regions represent 95% confidence intervals. Data are presented as mean ± SEM, n = 3–6 independent experiments per fit. Non-overlapping confidence intervals indicate statistically significant differences between permeabilization and lethality curves ( P < 0.05). Representative images show the ablation zones in cell monolayers, highlighting cell death (red/PI signal) and permeabilization (green/YO-PRO-1 signal) after nsPEF and µsPEF exposure. White circles mark electrode footprints. These images visualize the spatial distribution of permeabilized vs. lethally ablated cells across the electric field gradient.

Article Snippet: Experiments were conducted using the AC16 human cardiomyocytes (Cytion, Eppelheim, Germany) and human umbilical vein endothelial cells (HUVEC, C-12205; PromoCell, Heidelberg, Germany).

Techniques: Clinical Proteomics, Membrane, Generated

Journal: Europace

Article Title: Impact of pulse duration on cardiac electroporation: nanosecond pulses enhance cardiomyocyte selectivity and promote a Raman-detected shift towards apoptotic cell death

doi: 10.1093/europace/euaf217

Figure Lengend Snippet: Multivariate spectral analysis reveals molecular differences between nsPEF- and µsPEF-induced ablation in human cardiomyocytes. ( A ) Overview of the experimental workflow and region selection for Raman spectroscopy. Human cardiomyocytes were cultured in removable eight-well chambers pre-coated with human fibronectin, electroporated with either nsPEF or µsPEF, and incubated for 4 h. Raman imaging was performed using a confocal microspectrometer. A representative brightfield image shows a cardiomyocyte monolayer with a clearly marked ablation region (PEF) and a non-ablated control region (sham) on the periphery. These regions were selected for Raman acquisition based on proximity to the electrode footprint (white-dashed lines) and morphological changes consistent with electroporation. ( B ) TCA of the spectral maps of healthy and ablated by nsPEF and µsPEF cells (blue: nuclei, yellow: lipids, green: cytochrome c), showing spatial patterns and relative expression levels. Scale bar = 10 µm. ( C ) Reference Raman spectra of cellular components (nuclei, lipids, cytochrome c). ( D ) PCA of cytochrome c spectra demonstrates distinct separation between nsPEF- and µsPEF-treated groups in the PC1 vs. PC2 scores plot. ( E ) Significant separation along the PC1 axis between nsPEF and µsPEF groups ( P = 0.0484), indicating distinct molecular signatures. ( F ) PC1 loadings plot highlight the spectral features that contribute most to group separation; positive peaks reflect features enriched in the nsPEF group, while negative peaks correspond to µsPEF-associated signatures. Statistical analysis of the average PC1 scores revealed a significant difference between the nsPEF and µsPEF groups ( n = 3 per group, unpaired t -test, mean ± SD). Notably, spectral differences were observed at multiple cytochrome c peaks, including increased signals at 751 cm⁻¹ and 1129 cm⁻¹ in µsPEF-treated cells (associated with the reduced Fe²⁺ form) and a prominent 580 cm⁻¹ peak in nsPEF-treated cells (associated with the oxygenated form). These findings suggest that µsPEF exposure favours accumulation of the reduced form of cytochrome c, potentially suppressing apoptosis and promoting acute cell death, whereas nsPEF induces higher levels of the oxidized form, facilitating apoptotic processes.

Article Snippet: Experiments were conducted using the AC16 human cardiomyocytes (Cytion, Eppelheim, Germany) and human umbilical vein endothelial cells (HUVEC, C-12205; PromoCell, Heidelberg, Germany).

Techniques: Selection, Raman Spectroscopy, Cell Culture, Incubation, Imaging, Control, Electroporation, Expressing

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Dexmedetomidine (DEX) has a protective effect on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. (A) Cell -Counting Kit 8 (CCK-8) was used to measure cell viability. (B, C) Apoptosis and apoptotic rate were determined using flow cytometry. (D) GSE126104 showed that DEX decreased ectodysplasin-A2 receptor (EDA2R) expression in the left ventricle of rats. ***, P <0.001 vs. con. (E) The expression of EDA2R in cardiomyocytes was detected by RT-qPCR and western blotting. ** P <0.01; *** P <0.001; **** P <0.0001 vs. con. ## P <0.01; #### P <0.0001 vs. H/R. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Cell Counting, CCK-8 Assay, Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown suppresses hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis. (A) The knockdown efficiency of EDA2R in cardiomyocytes was detected by RT-qPCR and western blotting. (B) Cell -Counting Kit 8 (CCK-8) was applied to detect cell viability. (C, D) Apoptosis was determined using flow cytometry, and apoptotic rate was calculated. *** P <0.001; **** P <0.0001 vs. con. ## P <0.01; ### P <0.001; #### P <0.0001 vs. H/R+shNC. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Cell Counting, CCK-8 Assay, Flow Cytometry

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown represses mitochondria-mediated apoptosis. (A) Mitochondrial morphology of cardiomyocytes was observed by transmission electron microscopy (the arrows represented mitochondrial morphology, scale bar=500 nm). (B) Mitochondrial membrane potential (MMP) was detected by JC-1 (Scale bar=100 µ m, Red: **** P <0.0001 vs. con. or hypoxia/reoxygenation (H/R)+shNC; Green: #### P <0.0001 vs. con. H/R+shNC). (C, D) The levels of cytochrome C in mitochondria and cytoplasm of cardiomyocytes were determined by western blotting. (E) The expressions of Bax and Bcl-2 were determined in cardiomyocytes by western blotting. (F, G) Caspase-3 and Caspase-9 activity in cardiomyocytes was detected. *** P <0.001; **** P <0.0001 vs. con. # P <0.05; ## P <0.01; ### P <0.001; #### P <0.0001vs. H/R+shNC. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Transmission Assay, Electron Microscopy, Membrane, Western Blot, Activity Assay

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown inhibits the activation of the NF-κB signaling pathway. (A) The levels of IκBα, p-IκBα (Ser32), NF-κB p65 and p-NF-κB p65 (Ser536) were detected in cardiomyocytes by western blotting. (B) The expression distribution of NF-κB p65 in cardiomyocytes was determined by immunofluorescence, the fluorescence intensity of p65 in the nucleus was quantified in three fields of three sections (the arrows represented the distribution of p65 in the nucleus, scale bar=50 µ m). (C) The transcriptional activity of NF-κB in cardiomyocytes was measured by the electrophoretic mobility shift assay (EMSA). **** P <0.0001 vs. con. #### P <0.0001. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Activation Assay, Western Blot, Expressing, Immunofluorescence, Fluorescence, Activity Assay, Electrophoretic Mobility Shift Assay

Journal: Biomedicines

Article Title: Saxagliptin Cardiotoxicity in Chronic Heart Failure: The Role of DPP4 in the Regulation of Neuropeptide Tone

doi: 10.3390/biomedicines10071573

Figure Lengend Snippet: Effect of DPP4 inhibition and/or neuropeptide substrates on the viability of AC16 cells. Western blot analysis of DPP4 ( A ) in healthy human left ventricle samples and AC16 cells. In vitro treatment protocol with various gliptins on AC16 cell line and cell viability (calcein assay) results ( B ). In vitro treatment protocol with neuropeptides and their combined administration with saxagliptin and their effect on the viability of AC16 cells ( C ). One-way ANOVA, Tukey’s post hoc test, and unpaired t -test. Data are presented as mean ± SEM. Group sizes: ( B ) n = 3 from 1 independent experiment, ( C ) n = 7 from 7 independent experiments. RFU: relative fluorescence unit.

Article Snippet: Human cardiac myocyte AC16 cell line was obtained from Merck (SCC109).

Techniques: Inhibition, Western Blot, In Vitro, Fluorescence