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(Circulation. 2002;105:380.)
© 2002 American Heart Association, Inc.

Basic Science Reports

Bone Marrow–Derived Regenerated Cardiomyocytes (CMG Cells) Express Functional Adrenergic and Muscarinic Receptors

Daihiko Hakuno, MD, PhD; Keiichi Fukuda, MD; Shinji Makino, MD; Fusako Konishi, PhD; Yuichi Tomita, MD; Tomohiro Manabe, MD; Yusuke Suzuki, MD; Akihiro Umezawa, MD, PhD; Satoshi Ogawa, MD, PhD

From the Cardiopulmonary Division (D.H., K.F., S.M., F.K., Y.T., T.M., Y.S., S.O.), Department of Internal Medicine, Institute for Advanced Cardiac Therapeutics (K.F.), and the Department of Pathology (A.U.), Keio University School of Medicine, Tokyo, Japan.

Correspondence to Keiichi Fukuda, MD, PhD, Institute for Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail:kfukuda{at}sc.itc.keio.ac.jp

	Abstract

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Background— We recently reported that cardiomyocytes could be differentiated from bone marrow mesenchymal stem cells in vitro by 5-azacytidine treatment. In native cardiomyocytes, adrenergic and muscarinic receptors play crucial roles in mediating heart rate, conduction velocity, contractility, and cardiac hypertrophy. We investigated whether these receptors are expressed in differentiated CMG cells, and if so, whether they have downstream signaling systems.

Methods and Results— Reverse transcription–polymerase chain reaction revealed that CMG cells had already expressed _1A-, _1B-, and _1D-adrenergic receptor mRNA before 5-azacytidine treatment, whereas expression of ß₁-, ß₂-adrenergic and M₁-, M₂-muscarinic receptors was first detected at 1 day. Phenylephrine dose-dependently induced phosphorylation of ERK1/2, which was completely inhibited by prazosin, and significantly increased cell size. Isoproterenol augmented cAMP by 38-fold, which was fully inhibited by propranolol. Isoproterenol (10^-7 mol/L) increased the spontaneous beating rate by 47.6% (basal, 127±16 bpm), and propranolol and CGP20712A (ß₁-selective blocker) reduced it by 79.0% and 71.0%, respectively, whereas ICI118551 (ß₂-selective blocker) induced slight reduction. Cell motion, percent shortening, and contractile velocity were increased by 37.5%, 26.9%, and 50.6%, respectively, in response to isoproterenol. Phenylephrine and isoproterenol augmented ANP and BNP gene expressions. Carbachol increased IP₃ by 32-fold, which was markedly inhibited by atropine as well as AFDX116 (M₂-selective blocker) measured by radioimmunoassay.

Conclusions— These findings indicate that CMG cells expressed _1A, _1B, and _1D receptors before differentiation and expressed ß₁, ß₂, M₁, and M₂ receptors after they obtained the cardiomyocyte phenotype. These receptors had functional signal transduction pathways and could modulate cell function.

Key Words: cells • heart rate • receptors, adrenergic, beta • signal transduction • receptors, adrenergic, alpha

	Introduction

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A growing body of evidence shows that neurohumoral factors strongly modify heart rate, conduction velocity, myocardial contractility, and hypertrophy in cardiomyocytes.^1–3 The sympathetic and parasympathetic nerves play a critical role in modulating these cardiac functions through ₁-, ß₁-, and ß₂-adrenergic and muscarinic receptors. ₁-Adrenergic receptor agonists strongly induce cardiac hypertrophy both in vivo and in vitro.^4,5 There are 3 ₁-receptor subtypes, _1A, _1B, and _1D, all of which are expressed in the heart.⁶ The _1A and _1B subtypes are abundant in adult rat cardiomyocytes, but specific functional differences of each receptor remain unknown. There are 3 known subtypes of ß-adrenergic receptors: ß₁, ß₂, and ß₃.⁷ Cardiomyocytes express ß₁ and ß₂ receptors, and ß₁ receptors play a pivotal role in mediating heart rate, conduction velocity, and contractility.^7,8

Muscarinic receptors mediate postganglionic parasympathetic cholinergic signal transduction and have 5 subtypes; M₁ through M_5.⁹ Cardiomyocytes mainly express the M₂-receptor subtype, and this receptor is crucial to the negative regulation of heart rate, conduction velocity, and contractility.⁹ Sharma et al¹⁰ showed that M₁ receptors are also expressed in adult ventricular cardiomyocytes and increase the magnitude of calcium transients. Although the precise function of the M₁ receptor remains unknown, it may be able to modulate the function of cardiomyocytes.

A number of studies have demonstrated that cardiomyocytes can be differentiated from various multipotent stem cells such as embryonic stem (ES) cells¹¹ and embryonic carcinoma cells.¹² We recently reported that cardiomyocytes could be generated from bone marrow mesenchymal stem cells in vitro with the use of 5-azacytidine (5-AZ).¹³ These CMG cells showed spontaneous beating and expressed atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Analysis of the isoforms of contractile proteins revealed that CMG cells had a fetal ventricular phenotype. Furthermore, CMG cells expressed Nkx2.5, GATA-4, TEF-1, and MEF-2C before 5-AZ treatment and expressed other cardiac-specific transcription factors such as MEF-2A and MEF-2D and developed a cardiomyocyte phenotype after the treatment, suggesting that the developmental process of CMG cells was close to that of cardiomyoblasts.

These regenerated cardiomyocytes could be a useful and powerful tool for cardiomyocyte transplantation, but first further characterization of their cardiomyocyte phenotype is needed. Since adrenergic receptors and muscarinic receptors are critically implicated in modulating cardiac function, we determined whether these receptors are expressed in CMG cells, and if so, whether they are functional. We report that differentiated CMG cells expressed _1A-, _1B-, _1D-, ß₁-, and ß₂-adrenergic receptors and muscarinic M₁ and M₂ receptors and that stimulation of CMG cells with phenylephrine, isoproterenol, or carbachol could activate downstream signaling pathways through specific receptors.

	Methods

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Cell Culture and 5-AZ Treatment
Cells were cultured on 60-mm dishes in Iscove’s Modified Dulbecco’s Medium (GIBCO BRL) as described.¹³ To induce differentiation, cells were treated with 3 µmol/L of 5-AZ for 24 hours and were maintained for several weeks. The percentage of the cardiomyocytes after 5-AZ was approximately 20% to 30%, the same as described previously.¹³ The CMG cardiomyocytes began to spontaneously contract 2 to 3 weeks after 5-AZ treatment.

Reverse Transcription–Polymerase Chain Reaction Analysis
Total RNA was extracted, and reverse transcription–polymerase chain reaction (RT-PCR) was performed as described.¹³ The expressions of _1A, _1B, _1D, ß₁, ß₂, M₁, and M₂ receptors, ANP, and BNP mRNAs were analyzed. PCR was performed for 30 to 40 cycles, with each cycle consisting of denaturation at 94°C, annealing at 54°C to 60°C, and amplification at 72°C for 1 minute each. The PCR primers used are listed in Table 1. Before quantitative analysis, the linear range of the PCR cycles was measured for each receptor, and the appropriate number of PCR cycles was determined. Densitometric analysis was applied to quantify mRNA levels. GAPDH was used as an internal control for each sample.

View this table: [in this window] [in a new window]   Table 1. PCR Primers Used in Study

Western Blot Analysis
Polyclonal antibodies to extracellular responsive kinase (ERK)1 and phosphorylated ERK1/2 were purchased from Santa Cruz Biotechnology and New England Biolabs, respectively. Western blot analysis was performed as described previously.¹⁴

Immunostaining and Cell Sizing Protocol
Monoclonal antibody (MF20) to sarcomeric myosin was obtained from ATCC. Immunostaining and measurement of cell size (cell area, perimeter) were performed as described previously.^13,14

cAMP Accumulation Assays
Cells were incubated in serum-free medium with 10^-4 mol/L of 3-isobutyl-1-methylxanthine (Sigma) for 30 minutes and stimulated with isoproterenol (Sigma) for 10 minutes. The medium was aspirated rapidly, and incubation was terminated by addition of 1 mL of ice-cold 0.1N HCl. The lysates were centrifuged at 3000 rpm for 10 minutes, and the supernatants were used as samples. The cAMP levels were counted by radioimmunoassay with the assay kit (YAMASA).

Videotape Recording
The cultured cells were observed through an inverted-type phase-contrast video microscope (IX70, OLYMPUS) equipped with a x4 quartz objective lens and a x1 relay lens as described.¹³ Contractile parameters (cell motion distance, percent shortening, and contractile velocity) were analyzed by NIH image. Measurements of changes in cell length are not possible in CMG cells because they lack a single axis of myofibrillar alignment. Therefore, cell shortening diameter was calculated in the individual cells.

Inositol Triphosphate Production Assays
Cells were incubated in serum-free medium for 1 hour and were incubated in serum-free medium containing 10 mmol/L LiCl for 30 minutes.¹⁵ Stimulation was terminated by aspiration of medium and addition of 400 µL of ice-cold 20% perchloric acid for 20 minutes. The lysates were centrifuged at 5000 rpm, and the supernatants were titrated to pH 7.5 with 1.5N KOH. Then, they were centrifuged at 5000 rpm for 15 minutes at 4°C, and the supernatants were used as samples. Inositol triphosphate (IP₃) production was measured by radioimmunoassay with an assay kit (Amersham).

Statistical Analysis
Values are presented as mean±SEM. The significance of differences among mean values was determined by ANOVA. Statistical comparison of the control group with the treated group was carried out by means of the nonparametric Fisher’s multiple comparison tests. The accepted level of significance was P<0.05.

	Results

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CMG Cells Express ₁-Adrenergic Receptor mRNA Before Final 5-AZ Treatment
To begin to address whether ₁-adrenergic receptors were involved in modulating the function of CMG cells, we first detected ₁ receptor expression (Figure 1A). Adult murine ventricles were used as a positive control. CMG cells expressed all the ₁ receptor subtypes (_1A, _1B, and _1D) before 5-AZ treatment. A low level of expression of _1A was observed before 5-AZ treatment, but their expression was markedly augmented after the treatment. Expression of _1B was unaffected by the treatment. A high level of expression of _1D was first detected before 5-AZ treatment, but it noticeably decreased after the treatment. The quantitative analysis of their expression is shown in Figure 1B.

View larger version (24K): [in this window] [in a new window]   Figure 1. Temporal expression of 1-adrenergic receptor subtype mRNA in CMG cells. A, Each panel shows RT-PCR products for 1A, 1B, and 1D receptors and GAPDH. XHaeIII is a DNA size marker. Heart indicates adult murine left ventricles; RT (+) or RT(-), with or without reverse transcription and indicates positive or negative control; and Wk, weeks. B, Densitometric analysis was performed; ratio of RT-PCR product of 1 subtype to that of GAPDH is shown. Data were obtained from 5 separate experiments and were presented as arbitrary units over controls. Values are mean±SEM. *P<0.01 vs controls (before 5-AZ treatment).

Phenylephrine Induces Activation of ERK1/2 and Hypertrophy Through ₁-Receptors in CMG Cells
To investigate whether ₁ receptors are expressed at the protein level and can transduce signals, we stimulated CMG cells with phenylephrine (Sigma) and detected phosphorylation of ERK1/2. Phenylephrine induced phosphorylation of ERK1/2 in a time- and dose-dependent manner in the cells at 2 weeks after 5-AZ treatment (Figure 2, A and B). ERK1/2 was activated as early as 5 minutes and peaked at 10 minutes. Similar results were also obtained before 5-AZ treatment (data not shown). We preincubated the cells with prazosin (Sigma) before the stimulation and measured the phosphorylation in cells before and at 2 weeks after 5-AZ treatment. Phenylephrine-induced phosphorylation was completely inhibited by prazosin (Figure 2C). Next, we investigated whether phenylephrine is capable of inducing CMG cell hypertrophy. Phenylephrine increased the cell area and perimeter by 42.2% and 24.5%, respectively, over controls. These findings indicated that CMG cells expressed functionally active ₁-adrenergic receptors.

View larger version (32K): [in this window] [in a new window]   Figure 2. Effect of phenylephrine (PHE) on phosphorylation of ERK1/2 in CMG cells. A, Cells at 2 weeks after 5-AZ treatment were stimulated with phenylephrine (10-4 mol/L), and Western blot analysis was performed to detect phosphorylation of ERK1/2. Similar results were obtained in 3 separate experiments. B, Cells were stimulated with phenylephrine (10-8 - 10-5 mol/L) for 10 minutes, and phosphorylation of ERK was detected. Results were reproducible in 3 separate experiments. C, Prazosin (PZ) (10-6 mol/L) was added to cells 20 minutes before stimulation with phenylephrine (10-6 mol/L). Left panel shows results in CMG cells before 5-AZ treatment. Right panels show that of CMG cells at 2 weeks after 5-AZ treatment. We obtained similar results in 3 separate experiments.

CMG Cells Express ß₁- and ß₂-Adrenergic Receptor mRNA After 5-AZ Treatment
ß₁- and ß₂-adrenergic receptors play a crucial role in catecholamine-induced increases in heart rate, conduction velocity, and contractility.^7,16 To explore whether ß₁ and ß₂ receptors are functional, we examined their expression. CMG cells did not express their transcripts before 5-AZ treatment. They expressed ß₁ and ß₂ receptor mRNA from 1 day onward after treatment by PCR level and were stable after 1 week (Figure 3A). Quantitative analysis is shown in Figure 3B. The temporal expression pattern of these receptors was different from that of ₁. These findings suggest that CMG cells express ß₁ and ß₂ mRNA when they attain the cardiomyocyte phenotype.

View larger version (21K): [in this window] [in a new window]   Figure 3. Temporal expression of ß1- and ß2-adrenergic receptor mRNA in CMG cells. A, Experiments were run as described in Figure 1A. Each panel shows RT-PCR products of ß1, ß2 receptors and GAPDH. B, Densitometric analysis was performed; ratio of the RT-PCR product of ß subtype to that of GAPDH is shown. Data were obtained from 5 separate experiments and were presented as arbitrary units over controls. *P<0.01 vs controls.

Isoproterenol Increases cAMP Content, Spontaneous Beating Rate, and Contractility in CMG Cells
To examine whether ß₁ and ß₂ receptors can transduce their signals in CMG cells, we stimulated the cells with various concentrations of isoproterenol and measured intracellular cAMP content. Isoproterenol increased cAMP in a dose-dependent manner (Figure 4A). The absolute cAMP values of controls and cells stimulated with 10^-7 mol/L of isoproterenol were 8.1±1.5 and 147.4±16.2 pmol/10⁵ cells, respectively. cAMP increased by 38-fold with isoproterenol over the control. We next preincubated the cells with propranolol (Sigma) and stimulated with isoproterenol. Propranolol completely inhibited isoproterenol-induced cAMP accumulation (Figure 4B). These findings demonstrated that CMG cells not only expressed ß₁- and ß₂-adrenergic receptor protein but also expressed the signal transduction molecules for these receptors.

View larger version (12K): [in this window] [in a new window]   Figure 4. ß-Receptor–mediated cAMP accumulation in CMG cells. A, Effect of isoproterenol on cAMP accumulation in CMG cells at 2 weeks after 5-AZ treatment. B, Cells were preincubated with propranolol (10-6 or 10-5 mol/L) for 20 minutes and stimulated with isoproterenol (10-7 mol/L) for 10 minutes. Data were obtained from 5 separate experiments and were presented as arbitrary units over the controls. *P<0.01, **P<0.05 vs controls.

To ascertain whether isoproterenol could increase the spontaneous beating rate, we stimulated the cells with isoproterenol and observed them for changes in beating rate (Table 2). The beating rate of the control cells ranged from 58 to 292 (average 127±16, n=100) beats/min. The spontaneous beating rate increased significantly by 47.6% with isoproterenol. In some cells, however, isoproterenol did not increase the beating rate. Next, the cells were preincubated with either vehicle (PBS), propranolol, CGP20712A (ß₁-selective blocker, RBI-Sigma), or ICI118551 (ß₂-selective blocker, Sigma)¹⁷ and were stimulated with isoproterenol. Propranolol, CGP20712A, and ICI118551 reduced isoproterenol-induced increase in beating rate by 79.0%, 71.0%, and 21.0%, respectively.

View this table: [in this window] [in a new window]   Table 2. Isoproterenol Increases Spontaneous Beating Rate and Contractility of CMG Cells, Mainly Through ß1 Receptors

We further investigated the effect of isoproterenol on the contractile function. Isoproterenol increased the cell motion distance, percent shortening, and contractile velocity by 37.5%, 26.9%, and 50.6%, respectively. Isoproterenol-induced increase in contractility was almost completely inhibited by both propranolol and CGP20712A. Collectively, these results indicated that ß₁- and ß₂-adenergic receptors expressed in CMG cells were functional and that the isoproterenol-induced increase in spontaneous beating rate and contractility might be mediated mainly by ß₁ receptors.

Phenylephrine and Isoproterenol Induce ANP and BNP mRNA Expression
Hypertrophic stimuli are well known to induce the reprogramming of gene expression in cardiomyocytes. To investigate whether these stimuli can induce cardiac hypertrophy marker gene expression, we performed RT-PCR to quantify mRNA expression of ANP and BNP. Each value was normalized with GAPDH. Both phenylephrine (5x10^-5 mol/L) and isoproterenol (10^-4 mol/L) significantly induced the ANP (24 hours) gene by 5.1- and 6.9-fold, respectively. They also induced the BNP (1 hour) gene by 5.1- and 3.8-fold, respectively. These findings demonstrated CMG cells to have functional - and ß-adrenergic signal transduction systems.

CMG Cells Express Muscarinic Receptor mRNA After 5-AZ Treatment
To date, 5 subtypes (M₁ through M₅) of muscarinic receptors have been cloned.⁹ M₁ and M₂ receptor subtypes are expressed in murine neonatal and adult cardiomyocytes.¹⁰ Figure 5A shows the temporal expression pattern of M₁ and M₂ receptor mRNA. Neither receptor was detected before 5-AZ treatment. CMG cells began to express both receptors 1 day after the treatment by PCR level, and their expression became stable after 1 week. Quantitative analysis of their expression is shown in Figure 5B. These findings showed that the M₁ and M₂ receptors are expressed when they obtain the cardiomyocyte phenotype.

View larger version (24K): [in this window] [in a new window]   Figure 5. Temporal expression of M1 and M2 muscarinic receptor mRNA in CMG cells. A, Each panel shows RT-PCR products of M1, M2 receptors and GAPDH. B, Densitometric analysis was performed; ratio of the RT-PCR product of muscarinic subtype to that of GAPDH is shown. Data were obtained from 5 separate experiments and were presented as arbitrary units over controls. *P<0.01 vs controls.

Carbachol Stimulates IP₃ Production Through Muscarinic Receptors in CMG Cells
To explore whether these muscarinic receptors can transduce signals, we stimulated CMG cells with the muscarinic receptor agonist carbachol (Calbiochem) for 5 minutes and measured intracellular IP₃ content. Carbachol increased IP₃ content in a dose-dependent manner (Figure 6A). The absolute IP₃ values of the controls and the cells stimulated with 10^-7 mol/L of carbachol were 0.4±0.1 and 13.0±1.2 pmol/10⁵ cells, respectively. IP₃ content peaked by 32-fold over the control cells. We then preincubated the cells with atropine and the M₂-selective blocker AFDX116 (Tocris Cookson) for 5 minutes, stimulated with carbachol. Atropine and AFDX116 inhibited carbachol-induced IP₃ production by 84.5% and 89.2%, respectively (Figure 6B). These findings indicate that muscarinic receptors can transduce signals and that M₂ receptors play a critical role in this carbachol-induced IP₃ production.

View larger version (11K): [in this window] [in a new window]   Figure 6. Carbachol induced IP3 production through M2-muscarinic receptors in CMG cells. A, Effect of carbachol on IP3 production in CMG cells at 2 weeks after 5-AZ treatment. B. Effect of atropine (ATRO) (10-6 mol/L) and AFDX116 (10-7 or 10-6 mol/L) on carbachol-induced IP3 production. Data were obtained from 5 separate experiments and were presented as arbitrary units over controls. *P<0.01 vs controls.

	Discussion

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₁-Adrenergic Receptor Expression in CMG Cells
The present study demonstrates that bone marrow–derived CMG cardiomyocytes express all the ₁-receptor subtypes (_1A, _1B, and _1D) before the final 5-AZ treatment and consistently express thereafter. Moreover, phenylephrine induced ERK activation and hypertrophy in CMG cells, which was inhibited by prazosin. We supposed that expression of these ₁-receptor subtypes in undifferentiated CMG cells might be explained by their ubiquitous or wide expression in vivo.⁶ Interestingly, the present results show that expression of _1A receptors gradually increase, whereas that of _1D receptors prominently decrease after 5-AZ treatment. This transcriptional switch might result from the fact that CMG cells obtain the cardiomyocyte phenotype. Stewart et al¹⁸ showed _1A and _1B receptor mRNA to be expressed abundantly in both neonatal and adult rat hearts, whereas _1D receptors were expressed only at a low level. They also reported that hypertrophic stimuli induced an increase in _1A and a decrease in _1D receptors.¹⁹ Thus, temporal changes in the expression of the ₁-adrenergic receptor subtype in CMG cells are very similar to the postnatal changes previously observed in the neonatal rat heart.

ß₁- and ß₂-Adrenergic Receptor Expression in CMG Cells
Cardiomyocytes express both ß₁- and ß₂-adrenergic receptors in mammalian hearts, the ß₁ receptor being the predominant subtype (approximately 75% to 80% of total ß receptors) ⁷. We demonstrated that CMG cells first express both ß₁ and ß₂ receptors on 1 day when directed to differentiate into the cardiomyocyte and that stimulation of ß receptors with isoproterenol dose-dependently increases their cAMP content, which is completely blocked by propranolol. In CMG cells, the dose-response curve of cAMP accumulation by isoproterenol is similar to that in neonatal rat¹⁷ cardiomyocytes. These findings indicate that differentiated CMG cells have functionally active ß receptors. Heart-specific expression of ß₁ receptors is in accordance with the temporal expression of their mRNA in CMG cells.

We observed that isoproterenol had a positive chronotropic effect on CMG cells. The increment in beating rate (47.6%) is similar to that of adult murine cardiomyocytes (65%)²⁰ and ES cell–derived cardiomyocytes (40%).¹¹ CGP20712A significantly inhibited isoproterenol-induced increase in beating rate to the same extent as the nonselective ß-blocker propranolol. However, ICI118551 slightly inhibited this increase. These results suggest that the ß₁ receptor was the predominant subtype to mediate changes in beating rate in CMG cells. Moreover, contractility represented as cell motion, percent shortening, and contractile velocity was significantly augmented in response to isoproterenol. This positive inotropic effect was completely inhibited by propranolol as well as CGP20712A. To our knowledge, this is the first report to show the changes in contractility in regenerated cardiomyocytes. The increase in cell motion, percent shortening, and contractile velocity in CMG cells in response to isoproterenol were similar to those of neonatal rat cardiomyocyte.¹⁷ These findings confirmed that the ß₁ receptors played a critical role to mediate isoproterenol-induced signaling in differentiated CMG cells.

M₁- and M₂-Muscarinic Receptor Expression in CMG Cells
Muscarinic receptors show tissue-specific expression, and cardiomyocytes mainly express M₂ receptors in mouse and human.²¹ We found that both M₁ and M₂ muscarinic receptor mRNA was first expressed in CMG cells after 5-AZ treatment and that carbachol increased IP₃ production in a dose-dependent manner, which was markedly inhibited by atropine and AFDX116. Since the localization of muscarinic receptors is tissue-specific, it does not seem surprising that these receptors were expressed when they were directed to differentiate into cardiomyocytes, in the same pattern as ß₁- and ß₂-adrenergic receptors. Previous studies showed that M₁ receptors coupled to Gq/G11 and activated phospholipase Cß through Gq, leading to IP₃ production, and that M₂ receptors coupled to Gi/G0/Gz and activated phospholipase Cß through Giß, leading to IP₃ production.^22–24 The marked inhibition of IP₃ production by AFDX116 strongly suggested that the M₂ receptor is the predominant subtype in CMG cells.

Significance of Expression of Adrenergic and Muscarinic Receptors in CMG Cells
Cardiomyocytes in vivo respond to both sympathetic and parasympathetic nerves and change heart rate, conduction velocity, and contractility, enabling them to adapt to rapid changes in systemic oxygen demand. To date and to our knowledge, the only possible candidates for regeneration of cardiomyocytes are ES cells or mesenchymal stem cell–derived CMG cells. For regenerated cardiomyocytes to be useful for transplantation, they must express functional adrenergic and muscarinic receptors. A previous pharmacological study revealed that ₁- and ß₁-adrenergic and muscarinic receptors but not ß₂ receptors had already contributed to chronotropic responses in early-stage (7 days) murine ES cell–derived cardiomyocytes, whereas ß₂ receptors first functioned at the intermediate stage (7+4 days).¹¹ The present study demonstrated mesenchymal stem cell–derived cardiomyocytes to express _1A-, _1B-, _1D-, ß₁-, and ß₂-adrenergic and M₁- and M₂-muscarinic receptors, to maintain signal transduction pathways, and to also show cardiac hypertrophy in response to an agonist, as well as having positive inotropic and chronotropic responses to a ß agonist. In this regard, although we did not investigate all signaling pathways and their functions, CMG cells are a potential candidate for cardiomyocyte cell transplantation. Future studies are needed with the transplanted heart to analyze the adrenergic and muscarinic responses of CMG cells in vivo.

	Acknowledgments

 
This study was supported in part by research grants (10B-1) of Nervous and Mental Disorders from the Ministry of Health and Welfare and research grants from the Ministry of Education, Science, and Culture, Japan, and Health Science Research Grants for Advanced Medical Technology from the Ministry of Welfare, Japan.

Received August 15, 2001; revision received November 7, 2001; accepted November 8, 2001.

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