| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Toshiyuki Keio University, Department of Internal Medicine, Instructor, 医学部, 助手 (20265804)
SANO Motoaki Keio University, Department of Internal Medicine, Instructor, 医学部, 助手 (30265798)
ANZAI Toshihisa Keio University, Department of Internal Medicine, Instructor, 医学部, 助手 (60232089)
KATO Takahiro Keio University, Department of Internal Medicine, Instructor, 医学部, 助手 (60276219)
村田 光繁 慶應義塾大学, 医学部, 助手 (30317135)
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| Budget Amount *help |
¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneous beating cells were repeatedly screened for. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells ; they connected with adjoining cells after 1 week, began spontaneous beating after 2 weeks, and beat synchronously after 3 weeks. They expressed ANP and BNP, and were stained with anti-myosin, -desmin and -actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials ; sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain and α-actin, indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1 and MEF-2C mRNA before 5-azacytidine treatment, and expressed MEF-2A and MEF-2D after treatment. They expressed adrenergic α1A, α1B and α1D receptor before 5-azacytidine treatment, and expressed adrenergic β1, β2 and muscarinic M1 and M2 receptors after the treatment. Stimulation with phenylephrine, isproterenol and carbachol increased ERK phosphorylation and second messengers. Isoproterenol increased the beating rate, which was blocked with propranolol and CGP20712A (β_1-selective blocker) by 79% and 71%, respectively. This new cell line provides a powerful model for the study of cardiomyocyte differentiation and cardiovascular tissue engineering.
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