Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

Journal: Frontiers in Immunology

Article Title: Characterization of the m 6 A regulators’ landscape highlights the clinical significance of acute myocardial infarction

doi: 10.3389/fimmu.2024.1308978

Figure Lengend Snippet: ALKBH5 is highly expressed in myocardial infarction models and is associated with myocardial fibrosis and cardiomyocyte apoptosis. (A) Representative Western blot images and quantification of ALKBH5, Bax, and BCL-2 protein levels in the heart tissue of the control, sham operation, AMI, or AMI+rAAV-ALKBH5 groups. **** p < 0.0001. (B) Representative images of immunohistochemical ALKBH5 staining in control, sham operation, and AMI hearts, quantified at the right. **** p < 0.0001. Scale bar, 50 μm. (C) Representative immunofluorescent images of ALKBH5 in the heart tissue of the control, sham operation, or AMI groups, quantified at the right. Scale bar, 50 μm. **** p < 0.0001. (D) Representative micrographs of Masson staining in the heart tissue of the control, sham operation, AMI, or AMI+rAAV-ALKBH5 groups. Scar tissue and viable myocardium are identified in blue and red, respectively. Quantifications of the fibrotic areas are shown on the right. Scale bar, 50 μm. **** p < 0.0001. (E) Representative images of H&E staining of the hearts from the four groups of mice. Scale bar, 50 μm.

Article Snippet: Neonatal rat cardiomyocytes (NRCMs) were procured from the cardiac tissues of Sprague–Dawley rats aged between 1 and 3 days, sourced from Beijing HFK Bioscience Co., Ltd., China.

Techniques: Western Blot, Control, Immunohistochemical staining, Staining

Journal: ESC Heart Failure

Article Title: Calpain inhibition in a transgenic model of calpastatin overexpression facilitates reversal of myocardial hypertrophy

doi: 10.1002/ehf2.15250

Figure Lengend Snippet: Angiotensin‐II‐driven cardiac hypertrophy and recovery period in cardiac calpastatin overexpressing mice and DOX controls. (A) Induction of calpastatin expression at time point 0 by removal of doxycycline (DOX) in the calpastatin overexpressing group (CAST OE). Non‐induced littermates remained on doxycycline. CAST OE and non‐induced were challenged with AngII osmotic mini‐pumps for 3 weeks (+AngII), then recovered for three more weeks after pump removal (rec). MRI imaging on live animals was performed at Weeks 4, 7 and 10 as indicated. Examinations were performed in a time frame of 2 days. (B) Representative cardiac short axis MRI images and representative HE‐stained longitudinal heart sections to assess LV hypertrophy, for all groups. (C) Heart–body weight ratio (HW/BW). (D) Diameter of the interventricular septum (IVS). (E) Ejection fraction. IVS and EF were determined using 7 T high‐field MRI. (F) Cardiac cryosections, membrane‐stained with wheat germ agglutinin‐TRITC. (G) Average cardiac myocyte cross‐sectional area (>100 cells per heart). Data are shown as individual data points, means and SEM.

Article Snippet: Neonatal rat cardiomyocytes of Wistar rats (Harlan‐Winkelmann, Borchen, Germany) were isolated as described previously, then infected with adenoviruses at a multiplicity of infection of 25–50.

Techniques: Expressing, Imaging, Staining, Membrane

Journal: ESC Heart Failure

Article Title: Calpain inhibition in a transgenic model of calpastatin overexpression facilitates reversal of myocardial hypertrophy

doi: 10.1002/ehf2.15250

Figure Lengend Snippet: Calcineurin subcellular localization and NFAT activity in CAST OE cardiac myocytes and non‐induced controls. (A) Heart sections from mouse experimental groups as shown in Figure , immunofluorescence‐stained with anti‐CnA antibodies (red) and DAPI nuclear stain (blue). Nucleus outlines were highlighted (white) by edge detection filter based on the DAPI channel. (B) Calpastatin transgenic animal were crossbred with NFAT‐Luc mice to detect calcineurin NFAT activity. NFAT activity was determined by luciferase assay in heart lysates from calpastatin transgenic animals that were crossbred with NFAT‐Luc mice at the same time points as shown in Figure . Data are shown as individual data points, means and SEM.

Article Snippet: Neonatal rat cardiomyocytes of Wistar rats (Harlan‐Winkelmann, Borchen, Germany) were isolated as described previously, then infected with adenoviruses at a multiplicity of infection of 25–50.

Techniques: Activity Assay, Immunofluorescence, Staining, Transgenic Assay, Luciferase

Journal: ESC Heart Failure

Article Title: Calpain inhibition in a transgenic model of calpastatin overexpression facilitates reversal of myocardial hypertrophy

doi: 10.1002/ehf2.15250

Figure Lengend Snippet: Mapping of calcineurin A ubiquitinylation sites. (A) Schematic representation of calcineurin adenoviral vectors with progressive C‐terminal truncation. All constructs were N‐terminally fused to a GFP tag (not to scale). K456R indicates substitution of Lys with Arg at residue 456. AID, autoinhibitory domain; CaM, calmodulin binding domainCnB, calcineurin B binding domain; NES, nuclear export signal; NLS, nuclear localization sequence. (B) Lysates of neonatal rat cardiomyocytes transfected with the vectors shown in Part (A), immunopurified with anti‐ubiquitin antibody‐coupled beads (IP: Ub), followed by anti‐GFP western blot. ‘WB: GFP’ indicates expression control of calcineurin isoforms. ‘WB: GAPDH’ indicates the loading control.

Article Snippet: Neonatal rat cardiomyocytes of Wistar rats (Harlan‐Winkelmann, Borchen, Germany) were isolated as described previously, then infected with adenoviruses at a multiplicity of infection of 25–50.

Techniques: Construct, Residue, Binding Assay, Sequencing, Transfection, Ubiquitin Proteomics, Western Blot, Expressing, Control

Journal: ESC Heart Failure

Article Title: Calpain inhibition in a transgenic model of calpastatin overexpression facilitates reversal of myocardial hypertrophy

doi: 10.1002/ehf2.15250

Figure Lengend Snippet: Calpastatin‐dependent changes in calcineurin signalling during and after AngII stimulation. (A) Cardiomyocyte calcineurin‐NFAT signalling before (‘unstimulated’), during angiotensin‐II treatment (‘AngII’) and after angiotensin‐II removal (‘recovery phase’); in non‐induced double‐transgenic mice. (B) Same as in Part (A), but for induced CAST OE mice. Here, calpain enzymatic activity in cardiomyocytes is supressed by conditional overexpression of calpastatin (CAST). AID, autoinhibitory domain; CnA, calcineurin A; NFAT, nuclear factor of activated T cells; NFAT‐P: phosphorylated NFAT; tc CnA, truncated CnA; UPS, ubiquitin–proteasome system. Dashed arrows denote translocation.

Article Snippet: Neonatal rat cardiomyocytes of Wistar rats (Harlan‐Winkelmann, Borchen, Germany) were isolated as described previously, then infected with adenoviruses at a multiplicity of infection of 25–50.

Techniques: Transgenic Assay, Activity Assay, Over Expression, Ubiquitin Proteomics, Translocation Assay

Journal: bioRxiv

Article Title: TRIM24 preserves cardiomyocyte immune quiescence by repressing interferon/STAT signaling

doi: 10.1101/2025.11.13.688187

Figure Lengend Snippet: UMAP plot showing cell types identified ( A ) and TRIM24 expression (highlighted in orange) across major cardiac cell populations in human left ventricle single-nucleus RNA sequencing dataset (Broad Institute’s Single Cell Portal SCP1849), demonstrating highest expression in cardiomyocytes. Quantification of Trim24 transcripts from mouse hearts after left anterior descending coronary artery (LAD) ligation compared with sham controls, showing unchanged levels in the scar region ( D ) and upregulation in the remote region ( E ). ( F ) Subcellular localization of TRIM24 protein in cardiomyocytes isolated from sham and LAD-operated mouse hearts, showing predominant nuclear enrichment with minimal cytoplasmic signal. Statistical significance was determined using two-tailed Student’s t test. Error bars represent mean ± SEM. ns, non-significant; *, p < 0.05.

Article Snippet: Primary neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from the left ventricles of 1-2-day-old Wistar rat pups (Charles River, Lyon, France) following a protocol modified from Borlepawar et al. (2017) .

Techniques: Expressing, RNA Sequencing, Ligation, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: TRIM24 preserves cardiomyocyte immune quiescence by repressing interferon/STAT signaling

doi: 10.1101/2025.11.13.688187

Figure Lengend Snippet: ( A ) Heatmap of differentially expressed genes in neonatal rat ventricular cardiomyocytes (NRVCMs) overexpressing TRIM24 (T24OE) compared with LacZ controls, highlighting strong downregulation of interferon-stimulated genes, including, Mx1, Irf7 , and Ifit3 . ( B ) Functional enrichment analysis of downregulated transcripts showing suppression of immune-related Gene Ontology (GO) categories, including response to type I and type II interferon signaling, cytokine-mediated signaling, STAT-family binding. ( C ) Bar graphs of the relative FPKM values of representative pro-inflammatory genes significantly downregulated upon TRIM24 overexpression, which was further validated by quantitative real-time PCR ( D ). ( E ) Chromatin immunoprecipitation sequencing (ChIP-Seq) of TRIM24-bound regions in NRVCMs with motif enrichment analysis (DREME/Tomtom), revealing enrichment of nuclear receptor motifs (RARα) and immune-related motifs recognized by STAT transcription factors. ( F ) Enrichment analysis of STAT-bound gene sets demonstrating preferential TRIM24 occupancy at STAT target loci. Statistical significance was determined using two-tailed Student’s t test. Error bars show means ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Primary neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from the left ventricles of 1-2-day-old Wistar rat pups (Charles River, Lyon, France) following a protocol modified from Borlepawar et al. (2017) .

Techniques: Functional Assay, Binding Assay, Over Expression, Real-time Polymerase Chain Reaction, ChIP-sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: TRIM24 preserves cardiomyocyte immune quiescence by repressing interferon/STAT signaling

doi: 10.1101/2025.11.13.688187

Figure Lengend Snippet: Quantitative PCR analysis shows that TRIM24 overexpression reduces Stat1a ( A ), Stat1b ( B ), and Stat3 ( C ) transcript levels in neonatal rat ventricular cardiomyocytes (NRVCMs) under both basal conditions and following interferon γ (IFNγ) stimulation. Conversely, TRIM24 knockdown upregulates Stat1a ( D ), Stat1b ( E ), and Stat3 ( F ) expression at baseline, with further induction upon IFNγ treatment. Immunoblotting ( G ) and densitometric quantification ( H-K ) demonstrate that TRIM24 overexpression decreases total and phosphorylated STAT1 and STAT3 protein levels under resting and IFN-stimulated conditions. In contrast, immunoblotting ( L ) and corresponding densitometry ( M-P ) reveal that TRIM24 knockdown increases both total and phosphorylated STAT1 and STAT3, particularly following IFNγ stimulation. Statistical significance was determined using two-tailed Student’s t test. Error bars show means ± SEM. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Primary neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from the left ventricles of 1-2-day-old Wistar rat pups (Charles River, Lyon, France) following a protocol modified from Borlepawar et al. (2017) .

Techniques: Real-time Polymerase Chain Reaction, Over Expression, Knockdown, Expressing, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: TRIM24 preserves cardiomyocyte immune quiescence by repressing interferon/STAT signaling

doi: 10.1101/2025.11.13.688187

Figure Lengend Snippet: ( A ) Representative images from transwell migration assays in which macrophages migrated toward conditioned media derived from neonatal rat ventricular cardiomyocytes (NRVCMs) with TRIM24 overexpression, knockdown, or controls. ( B ) Quantification of migrated cells, presented as bar graphs, showing that conditioned media from TRIM24-overexpressing NRVCMs reduced macrophage migration, whereas supernatants from TRIM24 knockdown cells significantly enhanced migration. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed Student’s t -test. *, p < 0.05.

Article Snippet: Primary neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from the left ventricles of 1-2-day-old Wistar rat pups (Charles River, Lyon, France) following a protocol modified from Borlepawar et al. (2017) .

Techniques: Migration, Derivative Assay, Over Expression, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: TRIM24 preserves cardiomyocyte immune quiescence by repressing interferon/STAT signaling

doi: 10.1101/2025.11.13.688187

Figure Lengend Snippet: Super-resolution single-molecule localization microscopy was used to quantify nuclear organization of phosphorylated STAT1 (p-STAT1) and phosphorylated STAT3 (p-STAT3) in neonatal rat ventricular cardiomyocytes (NRVCMs) following interferon (IFNγ) or LPS stimulation. Spectral graphs depict ( A, B, G, H ) the absolute number of pair-wise distances of all blinking-event counts vs. distances between two points (absolute Ripley curves), showing reduced nuclear abundance of signals and clustering density (peak height and shape) of p-STAT1 and p-STAT3 in TRIM24-overexpressing cells under both stimuli. Relative Ripley analyses ( C, D, I, J ) normalized for total signal numbers reveal no or minimal reorganization of p-STAT3 and only modest alterations in p-STAT1 under IFNγ. Persistent homology analyses (relative frequency of components / holes vs. component / hole size represented by the distances of the respective bar end points) for p-STAT1 show minor changes in components ( E ) but increased maximum and width of hole distributions ( F ) under IFNγ, consistent with larger inter-cluster voids. Under LPS, notably, nuclear p-STAT3 levels were significantly reduced in TRIM24-overexpressing cells after LPS stimulation ( G ).

Article Snippet: Primary neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from the left ventricles of 1-2-day-old Wistar rat pups (Charles River, Lyon, France) following a protocol modified from Borlepawar et al. (2017) .

Techniques: Microscopy

Journal: bioRxiv

Article Title: TRIM24 preserves cardiomyocyte immune quiescence by repressing interferon/STAT signaling

doi: 10.1101/2025.11.13.688187

Figure Lengend Snippet: Immunoblotting ( A ) and densitometric analysis show that proteasomal inhibition by MG132 does not prevent TRIM24-mediated downregulation of STAT1 ( B ), whereas treatment with IACS attenuates the TRIM24-induced decrease in STAT1 protein levels ( C ). Super-resolution single-molecule localization microscopy was used to quantify nuclear and cytosolic organization of TRIM24 in neonatal rat ventricular cardiomyocytes (NRVCMs) following IACS-5971 treatment. Spectral graphs depict the absolute number of pair-wise distances of all blinking-event counts vs. distances between two points (absolute Ripley curves), showing reduced nuclear ( D ) while increased cytosolic ( E ) abundance and clustering density of TRIM24 in IACS-5971 treated cells. Persistent homology analyses show minor changes in components ( F ) and width of hole distributions ( G ) in the nucleus compared to the cytosol.

Article Snippet: Primary neonatal rat ventricular cardiomyocytes (NRVCMs) were isolated from the left ventricles of 1-2-day-old Wistar rat pups (Charles River, Lyon, France) following a protocol modified from Borlepawar et al. (2017) .

Techniques: Western Blot, Inhibition, Microscopy