rat cardiomyoblast cell line h9c2  (Procell Inc)

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Quantification of morphological, functional, and biochemical features of H9c2 rat cardiomyoblast retinoic acid differentiation

doi: 10.1016/j.jmccpl.2025.100486

Figure Lengend Snippet: live imaging time series of DIV5 H9c2 cells labelled with fluo-4 AM. Scale bar = 250 μm.

Article Snippet: The H9c2 rat ventricular-derived cardiomyoblast cell line was obtained from the American Type Culture Collection (ATCC, CRL-1446).

Techniques:

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Quantification of morphological, functional, and biochemical features of H9c2 rat cardiomyoblast retinoic acid differentiation

doi: 10.1016/j.jmccpl.2025.100486

Figure Lengend Snippet: live imaging time series of DIV14 H9c2 cells labelled with fluo-4 AM. Scale bar = 250 μm.

Article Snippet: The H9c2 rat ventricular-derived cardiomyoblast cell line was obtained from the American Type Culture Collection (ATCC, CRL-1446).

Techniques:

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Quantification of morphological, functional, and biochemical features of H9c2 rat cardiomyoblast retinoic acid differentiation

doi: 10.1016/j.jmccpl.2025.100486

Figure Lengend Snippet: Overview of morphological, functional, and molecular changes observed during H9c2 cell differentiation. Several morphological features, including increased cell area, length, eccentricity, multinucleation, and the emergence of actin clusters, increased with differentiation, particularly at DIV14 as illustrated by the model cell shape changes. Functional changes were marked by the onset of spontaneous Ca 2+ transients at DIV5, represented by the lightning bolt symbol, with a rightward shift observed at DIV14. Molecular changes reflected altered gene and protein expression across differentiation, including established markers (GATA4, cTnT, Myom2, Cacna1c ) and novel markers (Nkx2.5, ankyrin-B, VIM, Cx43). Shading denotes transcript/protein identity, and numbers indicate relative abundance.

Article Snippet: The H9c2 rat ventricular-derived cardiomyoblast cell line was obtained from the American Type Culture Collection (ATCC, CRL-1446).

Techniques: Functional Assay, Cell Differentiation, Expressing

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Quantification of morphological, functional, and biochemical features of H9c2 rat cardiomyoblast retinoic acid differentiation

doi: 10.1016/j.jmccpl.2025.100486

Figure Lengend Snippet: Differentiated H9c2 cells become larger, multi-nucleated, and develop localized actin intensities at DIV14. (A) Representative images of H9c2 cells at DIV0 (Ai), DIV5 (Aii), and DIV14 (Aiii) stained with phalloidin (F-actin, white) and Hoechst (nuclei, blue). (B) Length, perimeter, area, eccentricity, and the number of nuclei per cell were measured using a custom MATLAB script, which resulted in significant increases in length (Bi), area (Bii), eccentricity (Biii), and percentage of multinucleated cells (Biv). (C) Density distribution plots of cell length (Ci), area (Cii), and eccentricity (Ciii) demonstrate right-shifted distributions at DIV5 and DIV14 compared to DIV0. Circular (Di-iii) and elliptical (Div) actin-positive clusters were present in many of the cells. Insets show STED resolved clusters at 2× magnification. (E) Quantification of actin clusters, with a higher percentage of cells displaying clusters at DIV14 than DIV5 (Ei) and a greater number of clusters per cell at DIV14 (Eii). Data are presented as mean ± SEM (B, E). Density plots show probability distributions (C). Results are colour-coded: yellow = DIV0, magenta = DIV5, purple = DIV14. Scale bars: 40 μm (A); 5 μm (D) (insets zoomed 2×). N = 3 independent biological replicates (separate passages), with n = 27 total cells per group (3 segmentable cells from each of 3 fields of view [FOVs], 9 total FOVs per replicate). Experiments were performed on separate days. Statistical significance: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant; tested by one-way ANOVA with Tukey's post-hoc test, normalized to DIV5 (B), and independent t-test (E). See for detailed statistical analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The H9c2 rat ventricular-derived cardiomyoblast cell line was obtained from the American Type Culture Collection (ATCC, CRL-1446).

Techniques: Staining

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Quantification of morphological, functional, and biochemical features of H9c2 rat cardiomyoblast retinoic acid differentiation

doi: 10.1016/j.jmccpl.2025.100486

Figure Lengend Snippet: Differentiated H9c2 cells exhibit spontaneous Ca 2+ transients, subpopulations of which exhibit characteristics nearing those of primary cardiomyocytes. (A) Imaging of spontaneous Ca 2+ transients in differentiated H9c2 cells was performed using fluo-4 AM. Stills 10 s apart from 2 min H9c2 DIV5 (Ai), H9c2 DIV14 (Aii), or 1 min primary cardiomyocytes (Aiii) time series show spontaneous changes in [Ca 2+ ] i . See , , for corresponding live imaging recordings. Inlays highlight active cells in the field of view (FOV) undergoing a spike in [Ca 2+ ] i . (B) Quantification of Ca 2+ transient parameters. Mean spike amplitude (Bi) in H9c2 cells was similar to primary cardiomyocytes, whereas frequency (Bii) and synchronous firing rate (Biii) differed significantly. (C) Density distribution plots of spike amplitude (Ci), frequency (Cii), and synchronous firing rate (Ciii). Right-shifted distributions were observed for frequency (Cii) and synchronous firing rate (Ciii) at DIV14·N = 7 independent biological replicates (passages) for DIV5 (total of 56 cells), N = 6 independent biological replicates (passages) for DIV14 (total of 60 cells), and N = 5 independent biological replicates (primary cardiomyocyte cultures; total of 43 cells). For each biological replicate, 4 imaging dishes were treated as technical replicates and cells were selected across these dishes. Each experiment was performed on separate days. Data is presented as mean ± SEM. Results are graphed as Magenta = DIV5, Purple = DIV14, Violet = C (primary cardiomyocytes). Scale bar = 40 μm. ****, p < 0.0001; unlabeled, p > 0.05 by one-way ANOVA and post-hoc Tukey's multiple comparison. Data are normalized to mean primary cardiomyocyte measures, see for detailed statistical analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Differentiated H9c2 cells exhibit spontaneous Ca 2+ transients, subpopulations of which exhibit characteristics nearing those of primary cardiomyocytes. (A) Imaging of spontaneous Ca 2+ transients in differentiated H9c2 cells was performed using fluo-4 AM. Stills 10 s apart from 2 min H9c2 DIV5 (Ai), H9c2 DIV14 (Aii), or 1 min primary cardiomyocytes (Aiii) time series show spontaneous changes in [Ca 2+ ] i . See supplemental Videos 1–3 for corresponding live imaging recordings. Inlays highlight active cells in the field of view (FOV) undergoing a spike in [Ca 2+ ] i . (B) Quantification of Ca 2+ transient parameters. Mean spike amplitude (Bi) in H9c2 cells was similar to primary cardiomyocytes, whereas frequency (Bii) and synchronous firing rate (Biii) differed significantly. (C) Density distribution plots of spike amplitude (Ci), frequency (Cii), and synchronous firing rate (Ciii). Right-shifted distributions were observed for frequency (Cii) and synchronous firing rate (Ciii) at DIV14·N = 7 independent biological replicates (passages) for DIV5 (total of 56 cells), N = 6 independent biological replicates (passages) for DIV14 (total of 60 cells), and N = 5 independent biological replicates (primary cardiomyocyte cultures; total of 43 cells). For each biological replicate, 4 imaging dishes were treated as technical replicates and cells were selected across these dishes. Each experiment was performed on separate days. Data is presented as mean ± SEM. Results are graphed as Magenta = DIV5, Purple = DIV14, Violet = C (primary cardiomyocytes). Scale bar = 40 μm. ****, p < 0.0001; unlabeled, p > 0.05 by one-way ANOVA and post-hoc Tukey's multiple comparison. Data are normalized to mean primary cardiomyocyte measures, see for detailed statistical analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The H9c2 rat ventricular-derived cardiomyoblast cell line was obtained from the American Type Culture Collection (ATCC, CRL-1446).

Techniques: Imaging, Comparison

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Quantification of morphological, functional, and biochemical features of H9c2 rat cardiomyoblast retinoic acid differentiation

doi: 10.1016/j.jmccpl.2025.100486

Figure Lengend Snippet: Differentiated H9c2 cells exhibit gene expression changes consistent with cardiomyocyte differentiation. (A) Set of genes chosen for quantitative analyses. Genes are grouped based on their involvement in retinoic acid signaling, transcription, cytoskeleton, regulation of ion flux, or signal transduction. Bolded genes indicate those that exhibited significant changes in expression level between groups. (B) Transcript expression of Tgm2 (Bi), Myom2 (Bii) and Cacna1c (Biii) increased during differentiation, while Gja5 (Biv) expression decreased. (C) Representative Western blot of select cytoskeleton-associated proteins from DIV0, DIV5 h, DIV3 and DIV5 differentiated H9c2 cell lysates (Ci). Protein expression of transcription factors (Cii). Nkx2.5 decreased while GATA4 levels increased across differentiation. Cytoskeletal protein (Ciii) levels increased and then returned to base line for vinculin (VCL), decreased for ⍺-actinin (ACTN1) and vimentin (VIM), and increased for ankyrin-B (ANKB) and cardiac troponin T (cTnT). Lastly, the ion channel (Civ) connexin-43 (Cx43) levels decreased during differentiation. For RT-qPCR data: DIV0, DIV5 h (5 hours), DIV3, DIV5 N = 4 independent biological replicates (passages), each with 3 technical replicates. For Western blot data: DIV0, DIV5 h, DIV3, DIV5 (N = 6 independent biological replicates (passages), except cTnT N = 4 independent biological replicates (passages). Lysates were run independently on separate gels between replicates and quantified per replicate. Data is presented as mean ± SEM. Results are graphed as Yellow = DIV0, Orange = DIV5 h, Red = DIV3, Magenta = DIV5. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; unlabeled, p > 0.05 by one-way ANOVA and post-hoc Tukey's multiple comparison. Data are normalized to mean DIV0 measures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The H9c2 rat ventricular-derived cardiomyoblast cell line was obtained from the American Type Culture Collection (ATCC, CRL-1446).

Techniques: Gene Expression, Transduction, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Quantification of morphological, functional, and biochemical features of H9c2 rat cardiomyoblast retinoic acid differentiation

doi: 10.1016/j.jmccpl.2025.100486

Figure Lengend Snippet: Differentiated H9c2 cells at DIV14 more closely resemble primary cardiomyocytes than DIV5 . (A) Set of genes chosen for quantitative analyses. Genes are grouped based on their involvement in retinoic acid signaling, transcription, cytoskeleton, regulation of ion flux, and signal transduction. Bolded genes indicate those that exhibited significant differences in expression level between groups. (Bi) Representative Western blots of lysates from DIV5 and DIV14 H9c2 cells, primary cardiomyocytes, embryonic day 15.5 (E15.5) heart tissue, and postnatal day 2 (P2) heart tissue. Densitometry analysis revealed significant differences in the levels of several cytoskeleton-associated proteins (vinculin (VCL), ⍺-actinin (ACTN1), vimentin (VIM) and cTnT) in DIV5 H9c2 cells compared to primary cardiomyocytes. Levels of these proteins in DIV14 H9c2 cells were more similar to those in primary cardiomyocytes, with the exception of cTnT. (Biii) Levels of the transcription factor Nkx2.5 were significantly decreased at both DIV5 and DIV14 compared to primary cardiomyocytes. For Western blot data DIV5, DIV14 (N = 3 independent biological replicates (passages); primary cardiomyocytes (C) (N = 3 independent cultures); P2, E15.5 (N = 3 individual hearts for each). Lysates were run independently on separate gels between replicates and quantified per replicate. Data are presented as mean ± SEM. Results are graphed as Magenta = DIV5, Purple = DIV14, Violet = C (primary cardiomyocytes). ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; unlabeled, p > 0.05 by one-way ANOVA and post-hoc Tukey's multiple comparison. Data are normalized to primary cardiomyocytes (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The H9c2 rat ventricular-derived cardiomyoblast cell line was obtained from the American Type Culture Collection (ATCC, CRL-1446).

Techniques: Transduction, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: The phosphoinositide PI5P impairs mitochondrial function through endosome-mitochondria proximity

doi: 10.1101/2025.09.22.677701

Figure Lengend Snippet: (A) H9C2 cells were fixed and stained for mitochondria (in green) or for PI5P (in green). Zoomed region (i) and (ii) are shown below. (B) Line scan of fluorescence intensity along each arrow from inserts (i) and (ii) from (A). (C) Proportion of mitochondrial vs. non-mitochondrial PI5P. Results are from 19 cells across 3 independent experiments. (D) Cells were treated with STA (100nM for the indicated time) or with DMSO (vehicle only), fixed and stained for mitochondria (in green) or PI5P (in red). Right panel show the quantification of PI5P fluorescence intensity in the mitochondrial (mito.) or non-mitochondrial mask (non-mito.). Results are from 13-27 cells across 3 independent experiments. Statistical significance was determined by unpaired t-test. *p<0.05, ***p < 0.001.

Article Snippet: The rat embryonic cardiomyoblastic cell line H9C2 (2-1, Merck) was cultured in DMEM medium (31966, ThermoFisher Scientific) supplemented with 10% FBS and 1% penicillin– streptomycin in a 37°C, 5% CO2 incubator.

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: The phosphoinositide PI5P impairs mitochondrial function through endosome-mitochondria proximity

doi: 10.1101/2025.09.22.677701

Figure Lengend Snippet: (A) Schematic depicting the subcellular fractionation performed on H9C2 cells to obtained a post-nuclear supernatant (PNS), supernatant (SPNT) and pellet fractions. (B) The PNS, SPNT and pellet fractions were immunoblotted with the indicated antibodies. Quantification was performed from 4-8 independent experiments. (C) The pellet fractions obtained from control or 2DG-treated cells were immunoblotted with the indicated antibodies. Quantifications are shown on the right. Statistical significance was determined by unpaired t-test. *p<0.05, **p < 0.01, ns non-significant.

Article Snippet: The rat embryonic cardiomyoblastic cell line H9C2 (2-1, Merck) was cultured in DMEM medium (31966, ThermoFisher Scientific) supplemented with 10% FBS and 1% penicillin– streptomycin in a 37°C, 5% CO2 incubator.

Techniques: Fractionation, Control

Journal: BMC Pharmacology & Toxicology

Article Title: Differential effects of β-blockers nebivolol and metoprolol on mitochondrial biogenesis and antioxidant defense in angiotensin II-induced pathology in H9c2 cardiomyoblasts

doi: 10.1186/s40360-025-00970-8

Figure Lengend Snippet: Effect of nebivolol and metoprolol on Ang II-induced ROS production in H9c2 cells. ROS generation was measured by quantifying fluorometric product DCF using a fluorescence reader with excitation and emission wavelengths of 488 and 522 nm, respectively. Treatment with Ang II (1 µM) increased ROS production, which was significantly reduced by pretreatment with Neb (1 µM) and Met (10 µM) ( A - B ; one-way ANOVA, * P < 0.05). ( C and D ) Neb and Met pretreatment attenuated the Ang II-induced upregulation of NADPH oxidase (NOX4) and NOX2 mRNA expression levels (quantitative RT-PCR; one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05 vs. untreated (Con), # P < 0.05 vs. Ang II). Data are presented as means ± SEM ( n ≥ 4–8 per treatment group)

Article Snippet: The H9c2 rat cardiomyoblasts cell line (CRL-1446) was obtained from ATCC.

Techniques: Fluorescence, Expressing, Quantitative RT-PCR

Journal: BMC Pharmacology & Toxicology

Article Title: Differential effects of β-blockers nebivolol and metoprolol on mitochondrial biogenesis and antioxidant defense in angiotensin II-induced pathology in H9c2 cardiomyoblasts

doi: 10.1186/s40360-025-00970-8

Figure Lengend Snippet: Effect of nebivolol and metoprolol on Ang II-induced apoptosis in H9c2 cells. A, B and C) Apoptosis markers were assessed by qRT-PCR. Neb and Met induce mRNA expression level of pro-apoptosis marker BNIP3 ( A ), BAX ( B ), and suppress anti- apoptosis marker BCL2 ( C ) as vs. Ang II treated cells (* P < 0.05). ( D ) The ratio of BAX/BCL2 was significantly elevated in Neb- and Met-pretreated cells compared to Ang II alone, with Neb showing a more pronounced effect than Met ( $ P < 0.05 vs. Met + Ang II). * P < 0.05 vs. untreated (Con), # P < 0.05 vs. Ang II, NS: not significant vs. Ang II. Data are presented as means ± SEM ( n ≥ 3–6 per treatment group)

Article Snippet: The H9c2 rat cardiomyoblasts cell line (CRL-1446) was obtained from ATCC.

Techniques: Quantitative RT-PCR, Expressing, Marker

Journal: BMC Pharmacology & Toxicology

Article Title: Differential effects of β-blockers nebivolol and metoprolol on mitochondrial biogenesis and antioxidant defense in angiotensin II-induced pathology in H9c2 cardiomyoblasts

doi: 10.1186/s40360-025-00970-8

Figure Lengend Snippet: Effect of nebivolol and metoprolol on mitochondrial dynamics in H9c2 cells. RT-qPCR was used to assess the mRNA expression levels of genes related to mitochondrial fusion and fission. Pretreatment with Neb and Met increase mRNA levels of MFN2 ( A ), OPA1 ( B ) compared to Ang II-treated cells. However, no significant changes were observed in the expression of FIS ( C ) and DRP1 ( D ). * P < 0.05, vs. untreated (Con), # P < 0.05 vs. Ang II, $ P < 0.05 vs. Met + Ang II. Data are presented as means ± SEM ( n ≥ 3–6 per treatment group)

Article Snippet: The H9c2 rat cardiomyoblasts cell line (CRL-1446) was obtained from ATCC.

Techniques: Quantitative RT-PCR, Expressing