Genetic and Molecular Characterization of H9c2 Rat Myoblast Cell Line

Abstract

This study presents a comprehensive genetic characterization of the H9c2 cell line, a widely used model for cardiac myoblast research. We established a short tandem repeat (STR) profile for H9c2 that is useful to confirm the identity and stability of the cell line. Additionally, we prepared H9c2 metaphase chromosomes and performed karyotyping and molecular cytogenetics to further investigate chromosomal characteristics. The genetic analysis showed that H9c2 cells exhibit chromosomal instability, which may impact experimental reproducibility and data interpretation. Next-generation sequencing (NGS) was performed to analyze the transcriptome, revealing gene expression patterns relevant to cardiac biology. Western blot analysis further validated the expression levels of selected cardiac genes identified through NGS. Additionally, Phalloidin staining was used to visualize cytoskeletal organization, highlighting the morphological features of these cardiac myoblasts. Our findings collectively support that H9c2 cells are a reliable model for studying cardiac myoblast biology, despite some genetic alterations identified resembling sarcoma cells. The list of genes identified through NGS analysis, coupled with our comprehensive genetic analysis, will serve as a valuable resource for future studies utilizing this cell line in cardiovascular medicine.

Keywords: ICLAC; SKY analysis; STR profiling; cardiomyoblast; cell authentication; in vitro model; karyogram; myocardium; next-generation sequencing.

Figure 1

Light microscopic appearance of H9c2 cells. (A,B) In culture, H9c2 cells typically have a fibroblast-like morphology, with elongated and spindle-shaped cells that can form a confluent monolayer. They also show a high degree of adherence to the culture substrate, allowing them to grow in a dense configuration. The magnifications are as follows: 100× (A) and 200× (B). The scale bars represent 100 µm.

Figure 2

Electron microscopic appearance of H9c2 cells. (A–F) H9c2 cells display an elongated spindle-shaped morphology with a centrally located nucleus (N). Mitochondria (M) are visible as elongated or spherical organelles with double membranes and cristae. The cytoplasm contains lipid droplets (LD), lysosomes (Ly), ribosomes (R), and vesicles (V). The endoplasmic reticulum (rER) of H9c2 cells forms a network of membranous tubules and flattened sacs, with rough endoplasmic reticulum identifiable by ribosomes on its surface. The images were captured at (A) 2156×, (B) 10,000×, (C,D) 21,560×, and (E,F) 27,800×, respectively.

Figure 3

Protein expression in H9c2 cells. Cell protein extracts were prepared from H9c2 cells and rat heart tissue. The proteins (50 µg protein per lane) were then analyzed by Western blot to determine the expression of specific proteins. Ponceau S staining and probing with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific antibody were used as controls to ensure equal protein loading. Size markers are indicated on the left margin of each Western blot.

Figure 4

F-actin cytoskeleton staining in H9c2 cells. The cytoskeleton of cultured H9c2 cells was labeled with Alexa Fluor 488^TM-labeled Phalloidin conjugate (green) and nuclei were counterstained with DAPI (blue). Images were captured using a Nikon Eclipse E80i fluorescence microscope at 200×, 400×, or 600× magnification. Scale bars at the different magnifications are indicated in the images.

Figure 5

Karyogram analysis and mFISH results of the H9c2 cell line. The left panel displays a representative image of inverted DAPI-banding. The right panel shows a representative mFISH result of the same metaphase, generated using the commercially available 22xRat probe. In this analysis, interchromosomal rearrangements in H9c2 chromosomes are visible as color changes within single chromosomes. The color code for each chromosome is provided at the bottom of this panel. Evaluation of this analysis, according to the International System for Human Cytogenomic Nomenclature (ISCN) nomenclature, revealed the following karyotype [52,53]: 74-81<3n>,X,del(X)(q2?q3?),+1,+del(1)(q?12),del(2)(q?13),−3,−3,−3,+6,+9,+der(12)t(3;12)(q11;q1?2)×3,+der(12)(X;12)(q?3;q1?2),+14,del(20)(q10),+del(20)(p10)[cp12].

Figure 6

Virtual Comparative Genomic Hybridization results for the H9c2 cell line, translated into the human genome. Copy number alterations are depicted using a color code, with shades of red representing losses and green indicating gains.