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.

Fig 1. Morphologic and molecular characterization of H9c2 cell line before and after the differentiation process.

A) Representative images of the effect of serum withdrawal and RA supplentation on H9c2 cell morphology. Morphologic alterations were observed using the fluorescent probes Hoechst 33342 (nucleus, blue), Calcein-AM (cell viability, green) and TMRM (mitochondria, red). An overlay of phase contrast and Hoechst labeling is also shown. H9c2 cells cultured in 10% FBS-containing medium (top panels) showed a stellar shape, in the most cases, with one nucleus per cell. Upon culture in a medium containing 1% of FBS supplemented with 1 αM RA (bottom panels), differentiated H9c2 cells showed an elongated shape with several nuclei. Epifluorescence microscopy images are representative of 4 different cell preparations. White bar represents 10 μm. B) Decreased proliferative rate by modulating the percentage of serum in the medium. H9c2 cells were seeded at a density of 5,000 cells/ml and allowed to proliferate during 6 days in presence of RA. Results were analyzed by using the SRB assay. Undifferentiated H9c2 cells incubated in a high-serum medium proliferate fast, while incubation in a low-serum medium with daily RA additions during 5 days promoted cell cycle arrest. Statistical analysis: * p < 0.05 vs 10% FBS; the graph is representative of 5 independent experimental studies. C) Cellular content of specific cardiac marker troponin T, troponin I and cardiac calsequestrin in different cell populations. By using Western blotting, it was possible to confirm that RA-differentiated cells have increased content of cardiac markers. The images are representative of 4 different experimental studies which produced the same result. Ponceau labeling represents the loading control and was used to normalize data.

Fig 2. Semi-quantification by Western blotting of selected proteins involved on mitochondrial function and metabolism.

Protein content of PGC1α, TOM20, complex I NDUFS4 subunit, uncoupling protein 3, creatine kinase and lactate dehydrogenase was measured as described in the materials and methods section. H9c2 myoblasts were differentiated by being cultured in 1% FBS medium supplemented with 1 μM RA and maintained for 5 days. Total extracts from both cellular groups were collected. Ponceau labeling represents the loading control and was used to normalize data. Data represent the mean ± SEM of 4–5 independent experiments (*) p<0.05 versus respective undifferentiated group.

Fig 3. Semi-quantification of selected proteins involved on calcium handling and metabolism by Western blotting.

Protein content of pyruvate dehydrogenase, hexokinase II, Pink, SERCA2 and glutathione peroxidase 1 was measured by Western blotting as described in the materials and methods section. H9c2 myoblasts were differentiated by being cultured in 1% FBS medium supplemented with 1 μM RA and maintained for 5 days. Total extracts from both cellular groups were collected. Ponceau labeling represents the loading control and was used to normalize data. Data represent the mean ± SEM of 4 independent experiments (*) p<0.05 versus undifferentiated group.

Fig 4. Evaluation by Western blotting of selected proteins with probable involvement on the differentiation of H9c2 cells towards a cardiac-like cells.

Cellular content of Creb (total and phosphorylated form), PI3K (Class III and p85), PTEN and PDK1were measured as described in the materials and methods section. Ponceau labeling represents the loading control and was used to normalize data. Data represent the mean ± SEM of 4 independent experiments (*) p<0.05 vs undifferentiated group.

Fig 5. Evaluation by Western blotting of selected proteins with probable involvement on the differentiation of H9c2 cells towards a cardiac-like cells.

Cellular content of Akt, Gata 4, ERK1 (total and phosphorylated), and ERK2 (total and phosphorylated) were measured as described in the materials and methods section. Ponceau labeling represents the loading control and was used to normalize data. Data represent the mean ± SEM of 4 independent experiments (*) p<0.05 vs respective undifferentiated group.