Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype

Abstract

H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays.

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a ‘function follows form’ model of differentiation

“…1D-F). We finally observed that the duplication time of H9C2 cells was two days, in line with previous results 14 (Fig. 1G); meanwhile, after the differentiation protocol, the rate of proliferation decreased (Fig.…”

“…They confirmed the presence of a cardiomyocyte-like phenotype by identifying mRNAs related to cardiac muscle and mitochondrial metabolism as being upregulated on a transcriptional level relative to those in H9C2. 14 However, their study was restricted to investigating changes in the transcriptome, which does not always correlate with the functional proteome; 15 therefore, their findings require support from a quantitative and systematic proteomics analysis. This requirement is further reinforced by the pervasive role of micro RNAs in the post-transcriptional regulation of gene expression in cardiomyocytes during differentiation and disease.…”

Cardiac tissue regeneration: A preliminary study on carbon‐based nanotubes gelatin scaffold

“…Thus, the reduction of both proliferation rate and expression of the specific markers agree with previous reports investigating cardiomyocyte‐like differentiation and were mainly accounted for a shift in cardiomyocyte maturational status that has not been yet occurred as H9c2 required additional time to adapt to the scaffold.…”

“…The H9c2 cell line is currently used as a gold standard in vitro model as, due to its biochemical, morphological and electrical/hormonal signaling properties, it is able to mimic the behavior of skeletal and cardiac muscle . A preliminary goal of the study was to evaluate any phenotypical changes of H9c2 cell line, due to two different culture conditions: FBS reduction (from 10% to 1%) and all‐trans retinoic acid culture enrichment . In fact, one important feature of this cell line is its ability to differentiate from mononucleated myoblasts to myotubes when cultured in a low serum concentration media, getting an elongated shape and positioning in a parallel fashion .…”

Surface-modified particles loaded with CaMKII inhibitor protect cardiac cells against mitochondrial injury

“…These cells have the ability to differentiate into cardiomyocyte-like cells when they are exposed to all-trans retinoic acid in reduced-serum media. After differentiation, H9c2 cells express a more cardiac-like phenotype such as having a greater mitochondrial mass 31 , as well as having an increased expression of proteins involved in Ca 2+ handling and mitochondrial metabolism 41 . Such characteristics are of importance in these studies since the particles being tested were designed to target the mitochondria of cardiomyocytes.…”