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Under myocardial microenvironment, bone marrow-derived mesenchymal stem cells (MSCs) can transdifferentiate into cardiomyocytes (CMs). However, the role of histone deacetylase 1 (HDAC1) in this directed differentiation process remains unclear. The current study is to determine the acetylation regulatory mechanisms that may be involved in the directed CM differentiation from MSCs. MSCs isolated from male Sprague-Dawley (SD) rats were marked with Ad-EGFP and co-cultured with CMs. Flow cytometry was used to sort EGFP-positive (EGFP+) MSCs from the co-culture system. Then, the expression of cardiac troponin T (cTnT) in these MSCs was detected by immunofluorescence assay. In addition, HDAC1 levels at different co-culture times were measured by quantitative real-time polymerase chain reaction (QT-PCR) and Western blotting. At 4 days after co-culture with CMs, the MSCs began to expression detectable levels of cTnT. The expression of HDAC1 in CMs was much lower than that in MSCs. After co-culture with CMs, the expression of HDAC1 in MSCs was significantly decreased in a time dependent manner. In addition, our recent study has also identified that knockdown of the HDAC1 could promote the directed differentiation of MSCs into CMs. The results suggest that HDAC1 has a negative correlation with cardiac cell differentiation from MSCs under a myocardial microenvironment. HDAC1 might play an important role in the directed differentiation of MSCs into CMs in heart.

Failure of the directed differentiation of the transplanted stem cells into cardiomyocytes is still a major challenge of cardiac regeneration therapy. Our recent study has demonstrated that the expression of histone deacetylase 1 (HDAC1) is decreased in bone mesenchymal stem cells (BMSCs) during their differentiation into cardiomyocytes. However, the potential roles of HDAC1 in cardiac cell differentiation of BMSCs, as well as the mechanisms involved are still unclear. In current study, the expression of HDAC1 in cultured rat BMSCs is knocked down by lentiviral vectors expressing HDAC1-RNAi. The directed differentiation of BMSCs into cardiomyocytes is evaluated by the expression levels of cardiomyocyte-related genes such as GATA-binding protein 4 (GATA-4), Nirenberg, Kim gene 2 homeobox 5 (Nkx2.5), cardiac troponin T (CTnT), myosin heavy chain (MHC), and connexin-43. Compared with that in control BMSCs, the expression of these cardiomyocyte-related genes is significantly increased in these HDAC1 deficient stem cells. The results suggest that HDAC1 is involved in the cardiomyocyte differentiation of BMSCs. Knockdown of the HDAC1 may promote the directed differentiation of BMSCs into cardiomyocytes.

Objective: To isolate and culture cardiac stem cells (CSCs) in vitro and evaluate their potential of differentiation into functional cardiac myocytes., Methods: Myocardial tissues obtained from neonatal SD rats were cut into pieces of 0.5-1.0 mm(3), and digested twice for 5 min at 37 degrees C; with 0.2% trypsin and 0.1% collagenase II. The remaining tissues were cultured in complete explant culture medium (CEM) at 37 degrees C; in the presence of 5% CO(2). About a week later, a layer of fibroblast-like cells was generated from the adherent explants. These cells were passaged and seeded at about 1x10(6) cells/ml in poly-D-lysine-coated multi-well plates in cardiosphere-growing medium. When beating of the cultured cells was observed (at week 2), flow cytometry and immunohistochemistry were performed for identification of the primary and passaged cells., Results: The primary cells were successfully cultured from the digested myocardial tissue, and flow cytometry demonstrated the phenotype of c-kit(+)CD31(+)CD34(-)CD45(-)CTnT(-). After cell passage for about two weeks, single beating cells and cell clusters with synchronized contraction were seen microscopically, and their phenotype was converted to c-kit(+)CD31(-)CD34(-)CD45(-) CTnT(+). Immunohistochemistry staining identified CTnT expression in the passaged cells but not in the primary cells., Conclusions: A cell population with the phenotype c-kit(+)CD31(+)CD34(-)CD45(-)CTnT(-) has been obtained from neonatal SD rat heart, which possesses the potential to differentiate in vitro into beating cardiac myocytes and express CTnT protein.