Biomedical Engineering Reference
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the changes (Fig. 4c). Administration of verapamil decreased the beating rate of the
bone marrow-derived cardiomyocytes, and shortened the duration and plateau of the
action potential. These findings demonstrated that these cells expressed functionally
active L-type Ca
2+
hannels. Expression of the various ion channels during the differ-
entiation process is summarized in Fig. 5.
3.4 Expression and Function of
ȕ
1
-and
ȕ
2
-Adrenergic Receptors in CMG Cells
Mammalian heart expresses both
ȕ
1
-and
ȕ
2
-adrenergic receptors, the
ȕ
1
receptor being
the predominant sub-type (approximately 75-80% of total b receptors). CMG cells
did not express
ȕ
1
and
ȕ
2
receptor transcripts before 5-azacytidine exposure, but the
mRNAs were expressed from 1 week (Fig. 6a), indicating that they expressed
ȕ
1
and
ȕ
2
mRNA after acquiring the cardiomyocyte phenotype.
Isoproterenol, a
ȕ
stimulant, increased the cAMP content of the CMG cells, and
propranolol completely inhibited the isoproterenol-induced cAMP accumulation (Fig.
6b). Isoproterenol was applied to the cells to determine whether it would increase the
spontaneous beating rate, and the results showed that it increased it significantly to
48% over the rate in the control cells. Preincubation with propranolol (a nonselective
b blocker) or CGP20712A (a
ȕ
1
-selective blocker) significantly suppressed the isopro-
terenol-induced increase in beating rate (by 78 and 71%, respectively; Fig. 6c). Taken
together, these results indicated that the
ȕ
1
and
ȕ
2
-adrenergic receptors expressed in
CMG cells were functional, and that the isoproterenol-induced increase in spontane-
ous beating rate was mainly mediated by
ȕ
1
receptors.
3.5 Purification and Transplantation of Bone Marrow-Derived Cells into the
Heart
Because our method produced not only cardiomyocytes but other cells such as adipo-
cytes or osteoblasts, we needed to purify the cardiomyocytes prior to transplantation
into the recipient heart. As shown in Fig. 7a-d, The MLC-2v-EGFP plasmid-
transfected CMG cells showed EGFP signals after cardiomyocyte differentiation.
After cell sorting using the EGFP signals, more than 99% of the sorted cells showed
EGFP-positive signals (Fig. 7e-h).
Purified cells were then transplanted into the free walls of recipient left ventricles,
and the success of transplantation was analyzed histologically. Mice transplanted with
EGFP-positive cells were sacrificed at 2, 4, 8, and 12 weeks. EGFP-positive cardio-
myocytes survived in the recipient hearts for more that 12 weeks (Fig. 8a-c). The
orientation of the transplanted cells was parallel to the surrounding recipient cardio-
myocytes. The control experiment revealed no EGFP-positive cells in the heart.
Next, we marked the purified cells with adenovirusmediated LacZ gene, and trans-
planted them into recipient hearts as above. Figure 8d shows the entire murine heart
stained with LacZ at 4 weeks after transplantation, and Fig. 8e shows an enlargement
of the site of injection. LacZ-positive regenerated cardiomyocytes were observed on
the epicardial surface. Double staining with LacZ and hematoxylineosin
