Journal: bioRxiv

Article Title: PITX2C Deficiency Promotes Arrhythmogenic Remodeling via Oxidative Stress in Atrial Myocytes

doi: 10.64898/2026.03.27.714813

Figure Lengend Snippet: Sarcomere structure was assessed in neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA. [A] Confocal microscopy of immunofluorescently labelled Myomesin (M-band), F-actin (Z-disk), and DAPI (nuclei). Scale bars depict 50 microns. Quantification of [B] sarcomere length [C] sarcomere organization and [D] M-band density (as a % of total cell area) [A,B] Ctl: n=105 cells, KD: n=106 cells. [D] Ctl: n=124 cells, KD: n=126 cells [E] Confocal microscopy of immunofluorescently labelled Titin, F-actin, and DAPI. Scale bars depict 50 microns and [E’] representation of co-localization of Titin and F-actin signal along one myofibril. [F] Quantification of Titin and F-actin co-localization. Ctl: n=154 cells, KD: 140 cells. Statistical analyses were performed using Student’s t-test [B-D, F] to study differences between groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibodies used were alpha-Actinin (1:100, Sigma A7732), Myomesin (1:20, DSHB mMac) and Titin (1:100, DHSB 9 D10).

Techniques: Transfection, Confocal Microscopy

Journal: bioRxiv

Article Title: PITX2C Deficiency Promotes Arrhythmogenic Remodeling via Oxidative Stress in Atrial Myocytes

doi: 10.64898/2026.03.27.714813

Figure Lengend Snippet: Neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA were treated with 1mM N-acetylcysteine (NAC) for 24 hours. [A] Quantification of mitochondrial specific reactive oxygen species using MitoSOX relative to Ctl cells in Ctl or KD NRAMs. Ctl: 76 cells, KD: 79 cells, KD + NAC: 96 cells. Intracellular calcium rate kinetics were quantified including [B] beat rate, [C] time to peak, [D] time to 90% decay, and [E] time to 50% decay. Early [F] and delayed [G] afterdepolarizations via calcium traces and quantified. Ctl: n=68 cells, KD: n=47, KD + NAC: 72 cells. [H] Quantification of Titin and F-actin co-localization. Ctl: 60 cells, KD: 91 cells, KD + NAC: 76 cells. Statistical analyses were performed using Student’s t-test [A-E, H] or Fisher’s exact test [F,G] to study differences between groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibodies used were alpha-Actinin (1:100, Sigma A7732), Myomesin (1:20, DSHB mMac) and Titin (1:100, DHSB 9 D10).

Techniques: Transfection

Journal: bioRxiv

Article Title: Mechanobiological Specialization of Choroid Plexus Macrophages Defined by Titin Expression

doi: 10.64898/2026.01.20.700716

Figure Lengend Snippet: Titin-expressing macrophages are present in the choroid plexus. (A) Heatmap of the top eight differentially expressed genes in each of the three ChP macrophage sub-clusters. Gene expression levels are represented on a normalized gradient scale. (B) DAB immunohistochemical stain of a 5 µm FFPE section of control ChP tissue using a CD163 antibody. CD163-positive macrophages are marked by brown staining, while cell nuclei are counterstained blue with hematoxylin, indicating their distribution within the vascularized ChP tissue. Scale bar = 40 µm. (C) Violin plots illustrating TTN expression levels in ChP samples from Alzheimer’s disease and control conditions. Each plot shows the distribution, density, and variability of TTN transcript counts in ChP macrophages within each condition. (D) Schematic representation of the experimental workflow for evaluation of TTN RNA enrichment in macrophages, involving tissue dissociation, CD163 positive cell enrichment, RNA isolation, cDNA synthesis, PCR amplification using isoform-specific TTN primers, and ΔΔCq calculation with GAPDH used for normalization. (E) Fold changes, computed from ΔΔCq, in TTN isoform expression relative to GAPDH in CD163-bead enriched macrophages relative to whole tissue. The x-axis represents different primer sets (1, 3, 7, and 12), and the y-axis shows fold changes (log scale). Each point represents a single donor sample/primer set pair. (F) Schematic representation of the TTN gene with highlighted primer binding sites, with genomic coordinates on chromosome 2, indicating selected splicing sites and isoforms of TTN. (G) Immunofluorescence images of ChP with anti-CD68 (red) and anti-titin (green) primary antibodies, co-stained with DAPI (blue), showing cytoplasmic titin protein in ChP macrophage cytoplasm. Merged images are in right column. Scale bars = 20 µm.

Article Snippet: For titin immunofluorescence, primary antibody incubation with rabbit anti-titin (Proteintech Catalog #27867-1-AP) at 1:250 dilution and mouse anti-CD68 antibody (Millipore Cat.#168M-95) at 1:250 was conducted overnight at 4°C, followed by detection with secondary donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Cat. #A21206) (1:500) and goat anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, Cat. #A32728), with nuclei counterstaining with DAPI.

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Staining, Control, Isolation, cDNA Synthesis, Amplification, Binding Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Mechanobiological Specialization of Choroid Plexus Macrophages Defined by Titin Expression

doi: 10.64898/2026.01.20.700716

Figure Lengend Snippet: Titin-expressing macrophages are present in the choroid plexus. (A) Heatmap of the top eight differentially expressed genes in each of the three ChP macrophage sub-clusters. Gene expression levels are represented on a normalized gradient scale. (B) DAB immunohistochemical stain of a 5 µm FFPE section of control ChP tissue using a CD163 antibody. CD163-positive macrophages are marked by brown staining, while cell nuclei are counterstained blue with hematoxylin, indicating their distribution within the vascularized ChP tissue. Scale bar = 40 µm. (C) Violin plots illustrating TTN expression levels in ChP samples from Alzheimer’s disease and control conditions. Each plot shows the distribution, density, and variability of TTN transcript counts in ChP macrophages within each condition. (D) Schematic representation of the experimental workflow for evaluation of TTN RNA enrichment in macrophages, involving tissue dissociation, CD163 positive cell enrichment, RNA isolation, cDNA synthesis, PCR amplification using isoform-specific TTN primers, and ΔΔCq calculation with GAPDH used for normalization. (E) Fold changes, computed from ΔΔCq, in TTN isoform expression relative to GAPDH in CD163-bead enriched macrophages relative to whole tissue. The x-axis represents different primer sets (1, 3, 7, and 12), and the y-axis shows fold changes (log scale). Each point represents a single donor sample/primer set pair. (F) Schematic representation of the TTN gene with highlighted primer binding sites, with genomic coordinates on chromosome 2, indicating selected splicing sites and isoforms of TTN. (G) Immunofluorescence images of ChP with anti-CD68 (red) and anti-titin (green) primary antibodies, co-stained with DAPI (blue), showing cytoplasmic titin protein in ChP macrophage cytoplasm. Merged images are in right column. Scale bars = 20 µm.

Article Snippet: For titin immunofluorescence, primary antibody incubation with rabbit anti-titin (Proteintech Catalog #27867-1-AP) at 1:250 dilution and mouse anti-CD68 antibody (Millipore Cat.#168M-95) at 1:250 was conducted overnight at 4°C, followed by detection with secondary donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Cat. #A21206) (1:500) and goat anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, Cat. #A32728), with nuclei counterstaining with DAPI.

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Staining, Control, Isolation, cDNA Synthesis, Amplification, Binding Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Proteo-transcriptomics and morphometrics of teleost cardiac cells define regulatory networks and exercise-induced cardiomyocyte hypertrophy and hyperplasia

doi: 10.64898/2026.01.11.698907

Figure Lengend Snippet: (A) Outline of cell isolation and FACS of SSC-A Hi /GFP Hi ventricular cardiomyocytes from adult male zebrafish. (B) Representative laser confocal z-stack images of a sorted cardiomyocyte showing the colocalization of two sarcomeric proteins. α-Actinin staining is shown in cyan hot, Titin in orange hot, and nuclear staining in grey. The image on the right shows a magnified (3x zoom) area. (C) Representative laser confocal z-stack images of a sorted cardiomyocyte showing the sarcomeric localization of α-Actinin and myomesin. α-Actinin staining is shown in cyan hot, Myomesin in orange hot, and nuclear staining in grey. The images on the right (c’ and c’’) show 3x magnified regions highlighting the M-bands and Z-discs. (D) Normalized fluorescence intensity profile showing sarcomeric α-Actinin and Myomesin staining along the depicted line shown in c’’. The M-bands are labeled by Myomesin and the Z-discs by α-Actinin.

Article Snippet: Conjugated and primary antibodies used in this study include anti-Cardiac Troponin T (cTnT) Mouse Monoclonal Antibody (13-11, 1:500; ThermoFisher Scientific); Alexa Fluor® 647 Mouse anti-Cardiac Troponin T (cTnT), (1:250; BD Biosciences), PE Mouse anti-Cardiac Troponin T (cTnT), (1:250; BD Biosciences), anti-α-Actinin (Sarcomeric) Vio® R667, REAfinityTM (1:200; Miltenyi Biotec), anti-TTN (Titin) monoclonal antibody (M07), clone 2F12 (mouse, 1:250, Abnova), anti-Myomesin (mouse, mMaC myomesin B4, 1:200, DSHB).

Techniques: Cell Isolation, Staining, Fluorescence, Labeling

Journal: bioRxiv

Article Title: Proteo-transcriptomics and morphometrics of teleost cardiac cells define regulatory networks and exercise-induced cardiomyocyte hypertrophy and hyperplasia

doi: 10.64898/2026.01.11.698907

Figure Lengend Snippet: (A) Outline of adult male zebrafish ventricle isolation, dissociation, cytospin, and subsequent laser confocal imaging. (B) Representative laser confocal images of GFP+ cardiomyocytes showing intact and high-quality cells with visible sarco-meres. GFP fluorescence is shown in cyan, while nuclei are shown in yellow. (C) Representative laser confocal images of GFP+ cardiomyocytes showing intact cells with variable morphological shapes and sizes. Four main morphological features are highlighted, including rounded, rod-shaped, multipolar, and elongated cardiomyocytes. (D) Outline of cell isolation and FACS of GFP+ ventricular cardiomyocytes from adult male zebrafish. (E) Representative laser confocal images of FACS-isolated cardiomyocytes showing α-Actinin and myomesin staining. α-Actinin staining is shown in cyan hot, myomesin in orange hot, and nuclear staining in grey. The image on the right shows a resulting mask from the staining image on the left using Fiji. (F) Masks of randomly selected cardiomyocytes from (E) showing marked differences in the morphology and shape of cardiomyocytes. (G) Quantitative comparisons of perimeter, MinFeret, Circularity, and Aspect Ratio descriptors are shown (each group representing N = 6), revealing heterogeneity in male cardiomyocyte size and shape. (H) Outline of adult male zebrafish ventricle isolation, dissociation, and live-cell acoustic flow cytometry and bright field imaging of the GFP Hi SSC-A Hi cardiomyocyte cluster. Cells were run without fixation. (I) Representative bright field images of GFP Hi SSC-A Hi cardiomyocytes obtained using Attune CytPix acoustic focus.

Article Snippet: Conjugated and primary antibodies used in this study include anti-Cardiac Troponin T (cTnT) Mouse Monoclonal Antibody (13-11, 1:500; ThermoFisher Scientific); Alexa Fluor® 647 Mouse anti-Cardiac Troponin T (cTnT), (1:250; BD Biosciences), PE Mouse anti-Cardiac Troponin T (cTnT), (1:250; BD Biosciences), anti-α-Actinin (Sarcomeric) Vio® R667, REAfinityTM (1:200; Miltenyi Biotec), anti-TTN (Titin) monoclonal antibody (M07), clone 2F12 (mouse, 1:250, Abnova), anti-Myomesin (mouse, mMaC myomesin B4, 1:200, DSHB).

Techniques: Isolation, Imaging, Fluorescence, Cell Isolation, Staining, Flow Cytometry