Loss of cardiomyocytes leads to regional contractile dysfunction, and necrotized cardiomyocytes in infarcted ventricular tissues are progressively replaced by fibroblasts to form scar tissues. Recent studies revealed that transplanted fetal cardiomyocytes could survive in this heart scar tissue, and that these transplanted cells limited scar expansion and prevented postinfarction heart failure.1 The transplantation of cultured cardiomyocytes into the damaged myocardium has been proposed as a future method for the treatment of heart failure. Although this is a revolutionary idea, it remains unfeasible in the clinical setting, since it is difficult to obtain donor fetal heart. A cardiomyogenic cell line could potentially substitute for fetal cardiomyocytes in this therapy. Therefore, both developmental biologists and the cardiologists eagerly await the development of a cardiomyogenic cell line.
Recent reports have revealed that bone marrow stromal cells have many characteristics of mesenchymal stem cells. Pluripotential progenitor marrow stromal cells may differentiate into various types of cell types including bone, muscle, fat, tendon or cartilage.2 Based on these findings, we hypothesized that marrow stromal cells might also differentiate into cardiomyocytes, and repeatedly screened marrow stromal cells that began spontaneous beating after exposing them to 5-azacytidine, a cytosine analog capable of altering expression of certain genes that may regulate differentiation. We finally isolated a cell line that differentiates into cardiomyocytes in vitro, named CMG (cardiomyogenic), from adult marrow stromal cells.3 The use of adult tissues as a source of cardiomyocytes makes this system particularly appropriate for the development of cardiomyocyte transplantation.
CMG cells form myotubes and show spontaneous contraction
We repeated limiting dilutions several times, and isolated hundreds of clones, and observed several clones that could differentiate into the cardiomyocytes and show spontaneous beating. These experiments were repeated and reproducible, but the percentage of cardiomyocyte differentiation was distinct among these clones. Phase contrast photography revealed that CMG cells showed a fibroblast-like morphology before 5-azacytidine treatment (0 week), and this phenotype was retained through repeated subcultures under nonstimulating conditions. After 5-azacytidine treatment, the morphology of the cells gradually changed. Approximately 30% of the CMG cells gradually increased in size, formed a ball-like appearance, or lengthened in one direction, and formed a stick-like morphology at 1 week. They connected with adjoining cells after 2 weeks, and formed myotube-like structures at 3 weeks. The differentiated CMG myotubes maintained cardiomyocyte phenotype and beat vigorously for at least 8 weeks after final 5-azacytidine treatment, and did not de-differentiate. Most of the other nonmyocytes showed adipocyte appearance.
CMG cells have a cardiomyocyte-like ultrastructure
Figure 1.
CMG cells electron microscopy after 5-azacytidine treatment of cultured marrow cells.
Full figure and legend (35K)Transmission electron microscopy photographs revealed that the differentiated CMG myotubes had the typical striation and pale-staining pattern of the sarcomeres. Nuclei were positioned in the center of the cell, not beneath the sarcolemma. The most conspicuous feature of the differentiated CMG myotubes was the presence of membrane-bound dense secretary granules measuring 70–130 nm in diameter. These granules were thought to be atrial granules, and were especially concentrated in the juxtanuclear cytoplasm, but some were also located near the sarcolemma. These findings indicated that CMG cells had a cardiomyocyte-like ultrastructure.
CMG cells have several types of action potential
An electrophysiological study was performed on differentiated CMG cells at 2–5 weeks after 5-azacytidine treatment. There were at least two types of distinguishable morphological action potentials; sinus node-like potentials (Figure 2a), and ventricular myocyte-like potentials (Figure 2b). The sinus node-like action potential showed a relative shallow resting membrane potential with late diastolic slow depolarization, like a pacemaker potential. Peak and dome-like morphology were observed in ventricular myocyte-like cells. A cardiomyocyte-like action potential recorded from these spontaneous beating cells had the following properties: (1) a relatively long action potential duration or plateau, (2) a relatively shallow resting membrane potential, and (3) a pacemaker-like late diastolic slow depolarization.
Figure 2.
Representative tracing of the action potential of CMG cells. Action potential recordings were obtained from the spontaneous-beating cells at day 28 after 5-azacytidine treatment using a conventional microelectrode. We categorized these action potentials into two groups; a sinus-node-like action potential (a) or a ventricular cardiomyocyte-like action potential (b).
Full figure and legend (11K)All the action potentials recorded from the CMG cells until 3 weeks revealed sinus node-like action potential. The ventricular myocyte-like action potentials could be recorded after 4 weeks, and the percentage of these action potentials gradually increased thereafter. It is possible that the percentage of the ventricular myocyte-like action potentials at 5 weeks was underestimated. Most of the action potentials recorded from differentiated CMG cells revealed ventricular myocyte-like appearance, but the action potential of the differentiated CMG cells was difficult to record. The glass microelectrode was frequently damaged, because the spontaneous contraction of the differentiated cells at 5 weeks was too big.
Cardiomyocyte-specific gene expression
Differentiated CMG cells expressed both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes. Table 1 shows the summary of the expression of cardiac contractile proteins such as the 
- and 
-MHC (myosin heavy chain), -cardiac and -skeletal actin genes. Both - and -MHC expression could be detected by RT-PCR in differentiated CMG cells, but -MHC expression was overwhelmingly stronger than that of -MHC. CMG cells expressed both -cardiac and -skeletal actin. Northern blot analysis revealed that the -skeletal actin gene was expressed at markedly higher levels than the -cardiac actin gene in CMG cells. Interestingly, CMG cells expressed (myosin light chain-2v) (MLC-2v), but not -2a.
Cardiomyocytes expressed GATA4 (a GATA-motif-binding Zinc-finger-type transcription factor expressed in the early stage of the developing heart), TEF-1 (transcription enhancement factor), and Nkx2.5 (a homeobox-type transcription factor specifically expressed beginning in the early developing heart), while skeletal muscle cells only expressed TEF-1. Differentiated CMG cells expressed GATA4, TEF-1 and Nkx2.5 (Figure 3a). Interestingly, CMG cells already expressed these genes before 5-azacytidine treatment. Figure 3b shows the time course of muscle enhancement factor 2-A (MEF2-A), MEF2-C and MEF2-D gene expression. MEF-2C was already expressed before 5-azacytidine treatment, but MEF-2A and MEF-2D were induced after 5-azacytidine treatment.
Figure 3.
RT-PCR analysis of cardiomyocyte-specific transcription factors. (a) Time course of the expression of Nkx2.5 (a homeobox-type transcription factor specifically expressed beginning in the early developing heart) and GATA4 ( a GATA-motif-binding Zinc-finger-type transcription factor expressed in the early stage of the developing heart). (b) Time course of the expression of muscle enhancement factor 2-C (MEF2-C) and muscle enhancement factor 2-D (MEF2-D) genes. Since the primers were designed to demonstrate the alternative splicing forms MEF-2 genes, several bands can be observed.
Full figure and legend (20K)Discussion
These cells expressed a number of cardiomyocyte-specific genes including ANP, BNP, GATA4 and Nkx2.5. In ventricular muscle of small mammals, there is a developmental switch from expression of -MHC, which is the predominant fetal form, to that of -MHC around the time of birth. There is also a developmental switch from expression of -skeletal actin, which is the predominant fetal and neonatal form, to that of -cardiac actin, the predominant adult form. Differentiated CMG cells mainly expressed -MHC and -skeletal actin. Expression of -MHC and -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, while MLC-2a was specifically expressed in atrial cells. Differentiated CMG cells expressed MLC-2v, but not -2a. These results indicated that differentiated CMG cells had a phenotype specific to fetal ventricular cardiomyocytes.
Differentiated CMG cells expressed Nkx2.5, 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. These results indicated that the stage of differentiation of the CMG cell is between cardiomyocyte-progenitor and differentiated cardiomyocytes.
CMG cells have either sinus-node-like or ventricular myocyte-like action potential. Although action potentials can be seen in nonmyocyte cells such as skeletal muscle cells or nerve cells, the action potential in CMG cells is characterized by duration. The duration of action potentials in skeletal muscle cells or nerve cells are less than 5 ms. The most diastolic potential, action potential amplitude, and the overshoot potential of the sinus-nodal-like CMG cells were close to the equivalent values reported in vivo rabbit sinus nodal cells. In rabbit ventricular cells, the most diastolic potential and action potential amplitudes were reported to be -90 to -95 mV and 120 mV, respectively. Although the most diastolic potential and action potential amplitudes 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 from marrow cells to cardiac cells.
References
- Leor J, Patterson M, Quinones MJ et al. Transplantation of fetal myocardial tissue into the infarcted myocardium of rat. A potential method for repair of infarcted myocardium? Circulation 1996; 94 (Suppl II): 332–336. | ISI |
- Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: 71–74. | Article | PubMed | ISI | ChemPort |
- Makino S, Fukuda K, Miyoshi S et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999; 103: 697–705. | PubMed | ISI | ChemPort |
