We have established a cardiomyogenic cell line (CMG) from mouse bone marrow stromal cells that can be induced to differentiate into cardiomyocytes in vitro by 5-azacytidine treatment. A number of lines of evidence confirmed the cardiomyocyte characteristics of CMG cells. These cells expressed a number of cardiomyocyte-specific genes including ANP, BNP, GATA4, and Nkx2.5/Csx. In ventricular muscle of small mammals, there is a developmental switch from expression of β-MHC, which is the predominant fetal form, to that of α-MHC around the time of birth. There is also a developmental switch from expression of α-skeletal actin, which is the predominant fetal and neonatal form, to that of α-cardiac actin, the predominant adult form. Differentiated CMG cells mainly expressed β-MHC and α-skeletal actin. Expression of α-MHC and α-cardiac actin was detected, but at low levels. MLC-2 genes are specifically expressed in the chamber. MLC-2v is specifically expressed in ventricular cells, whereas MLC-2a was specifically expressed in atrial cells. Differentiated CMG cells expressed MLC-2v, but not MLC-2a. Moreover, skeletal muscle cells do not express α-MHC or MLC-2v. These results indicated that differentiated CMG cells had a phenotype specific to fetal ventricular cardiomyocytes.
Differentiated CMG cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C before final 5-azacytidine treatment. The MEF-2A and MEF-2D genes were expressed after final 5-azacytidine treatment. This pattern of gene expression in CMG cells was similar to that of in vivo
developing cardiomyocytes (33
). These results indicate that the stage of differentiation of the CMG cell is between cardiomyocyte-progenitor and differentiated cardiomyocytes.
Differentiated CMG cells connected to adjoining cells via intercalated discs, formed myotubes, and beat spontaneously. These differentiated CMG myotubes have a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, abundant glycogen granules, a number of mitochondria, and many atrial granules. Tagoe et al
) reported that the most common size of atrial granules observed in the adult mice atrium was 150–200 nm in diameter, but they also found that ~35% of the atrial granules in adult mice atria ranged between 50 and 150 nm in diameter. The atrial granules observed in the differentiated CMG myotubes were 70–130 nm in diameter. A previous report (35
) found that almost all atrial myocytes expressed ANP in fetal heart, whereas in the ventricular wall, cells containing immunoreactive granules were scattered. Analysis of the pattern of expression of cardiomyocyte-specific genes indicated that the phenotype of the differentiated CMG cardiomyocytes corresponded to fetal ventricular cardiomyocytes. The high-density granules observed in the differentiated CMG cells might correspond to those in fetal ventricular cardiomyocytes.
CMG myotubes have either sinus node–like or ventricular myocyte–like action potentials with a relatively long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization.
Although action potentials can be seen in noncardiomyocyte cells such as skeletal muscle cells or nerve cells, the action potential in CMG cells is characterized by duration (36
). The duration of action potentials in skeletal muscle cells or nerve cells are <5 ms (40
). The most diastolic potential, action potential amplitude, and the overshoot potential of the sinus node–like CMG cells were close to the equivalent values reported in vivo
rabbit sinus node cells (42
). In rabbit ventricular cells, the most diastolic potential and action potential amplitude were reported to be approximately between –90 and –95mV, and 120 mV, respectively. Although the most diastolic potential and action potential amplitude of the ventricular cardiomyocyte-like CMG cells were slightly shorter than these values, the shape of the action potential was very close to in vivo
ventricular cardiomyocyte. The observation of several distinctive patterns of action potential in CMG cells may reflect different developmental stages. The electrophysiological patterns of action potential and expression patterns of the ion channels in differentiated CMG cells should be clarified in the future.
Both embryonic stem (ES) cells (43
) and embryonal carcinoma (EC) cells (44
) may differentiate into cardiomyocytes in vitro
. These cells were derived from totipotent embryonal blastocyst and either required endoderm for mesodermal differentiation or could differentiate into endoderm and ectoderm by themselves. CMG cells differ from these cells in several ways. First, CMG cells were obtained from adult bone marrow. Second, they did not require endoderm for differentiation, and they only differentiated into mesoderm, as demonstrated in other marrow stromal cell lines. Third, CMG cells were easy to culture because they are adherent like fibroblasts, have a high growth rate, and do not require expensive cytokine (leukemia inhibitory factor) supplement. Finally, differentiation is easily induced by 5-azacytidine treatment. ES cells and EC cells differentiate into cardiomyocytes at rates of ~50% and 5%, respectively. CMG cells differentiate into cardiomyocyte-like cells after final 5-azacytidine treatment, and the efficiency of the differentiation into cardiomyocytes is ~30%. Thus, CMG cells provide a powerful tool for the further investigation of cardiomyocyte differentiation. Although transcription factors such as d-HAND, e-HAND (46
), MEF-2C (33
), Nkx2.5/Csx, GATA4, and TEF-1 are known to play important roles in cardiac development (48
), the lack of a model for cardiomyocyte differentiation has meant that little is known about the interactions of these genes. This simple new model for cardiomyogenesis may help clarify the cascade of transcriptional activation that regulates differentiation into cardiomyocytes.