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.
Highlights
Primary cardiomyocytes are fragile and difficult to maintain in culture for long periods
With the objective of characterizing H9c2 cell cardiac-driven differentiation, a simple visual assessment by microscopy was performed in H9c2 myoblasts growing on medium supplemented with 10% fetal bovine serum (FBS) or in differentiated H9c2 cells maintained in medium supplemented with 1% of FBS plus 1 μM retinoic acid (RA) (Fig 1)
In order to verify whether H9c2 differentiation promotes alterations in cellular viability and mitochondrial morphology, cells were incubated with the mitochondrial-selective probe tetramethylrhodamine methyl ester (TMRM) and with fluorescent probes Hoechst 33342 and Calcein-AM
Summary
Primary cardiomyocytes are fragile and difficult to maintain in culture for long periods. The different adhesion kinetics of the heterogeneous isolated fraction led to the separation of the different components in the culture dish In this stage, cells are still not fully differentiated into adult cardiomyocytes but are already predestinated, leading to the appearance of several cardiomyocyte-specific markers. The great majority of studies are performed using undifferentiated H9c2 myoblasts, raising questions on the relevance of the results obtained when compared to primary cardiomyocytes. This is especially pertinent in cardiotoxicity studies, since dose-responses are altered by the cell differentiation state [6, 7]. The results are very relevant to understand metabolic and signaling alterations occurring during H9c2 cardiomyoblast differentiation, paving the way for a more suitable use of this cell model for different experimental aims
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