Bone Marrow–Derived Regenerated Cardiomyocytes (CMG Cells) Express Functional Adrenergic and Muscarinic Receptors
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 α1A-, α1B-, and α1D-adrenergic receptor mRNA before 5-azacytidine treatment, whereas expression of β1-, β2-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 (β1-selective blocker) reduced it by 79.0% and 71.0%, respectively, whereas ICI118551 (β2-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 α1A, α1B, and α1D receptors before differentiation and expressed β1, β2, M1, and M2 receptors after they obtained the cardiomyocyte phenotype. These receptors had functional signal transduction pathways and could modulate cell function.
Received August 15, 2001; revision received November 7, 2001; accepted November 8, 2001.
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 α1-, β1-, and β2-adrenergic and muscarinic receptors. α1-Adrenergic receptor agonists strongly induce cardiac hypertrophy both in vivo and in vitro.4,5⇓ There are 3 α1-receptor subtypes, α1A, α1B, and α1D, all of which are expressed in the heart.6 The α1A and α1B subtypes are abundant in adult rat cardiomyocytes, but specific functional differences of each receptor remain unknown. There are 3 known subtypes of β-adrenergic receptors: β1, β2, and β3.7 Cardiomyocytes express β1 and β2 receptors, and β1 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 M5.9 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 α1A-, α1B-, α1D-, β1-, and β2-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.
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 α1A, α1B, α1D, β1, β2, 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.
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
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).
The cultured cells were observed through an inverted-type phase-contrast video microscope (IX70, OLYMPUS) equipped with a ×4 quartz objective lens and a ×1 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).
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.
CMG Cells Express α1-Adrenergic Receptor mRNA Before Final 5-AZ Treatment
To begin to address whether α1-adrenergic receptors were involved in modulating the function of CMG cells, we first detected α1 receptor expression (Figure 1A). Adult murine ventricles were used as a positive control. CMG cells expressed all the α1 receptor subtypes (α1A, α1B, and α1D) before 5-AZ treatment. A low level of expression of α1A was observed before 5-AZ treatment, but their expression was markedly augmented after the treatment. Expression of α1B was unaffected by the treatment. A high level of expression of α1D 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.
Phenylephrine Induces Activation of ERK1/2 and Hypertrophy Through α1-Receptors in CMG Cells
To investigate whether α1 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 α1-adrenergic receptors.
CMG Cells Express β1- and β2-Adrenergic Receptor mRNA After 5-AZ Treatment
β1- and β2-adrenergic receptors play a crucial role in catecholamine-induced increases in heart rate, conduction velocity, and contractility.7,16⇓ To explore whether β1 and β2 receptors are functional, we examined their expression. CMG cells did not express their transcripts before 5-AZ treatment. They expressed β1 and β2 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 α1. These findings suggest that CMG cells express β1 and β2 mRNA when they attain the cardiomyocyte phenotype.
Isoproterenol Increases cAMP Content, Spontaneous Beating Rate, and Contractility in CMG Cells
To examine whether β1 and β2 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 β1- and β2-adrenergic receptor protein but also expressed the signal transduction molecules for these receptors.
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 (β1-selective blocker, RBI-Sigma), or ICI118551 (β2-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.
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 β1- and β2-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 β1 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 (5×10−5 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 α- and β-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.
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.
α1-Adrenergic Receptor Expression in CMG Cells
The present study demonstrates that bone marrow–derived CMG cardiomyocytes express all the α1-receptor subtypes (α1A, α1B, and α1D) 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 α1-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 α1A receptors gradually increase, whereas that of α1D 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 α1A and α1B receptor mRNA to be expressed abundantly in both neonatal and adult rat hearts, whereas α1D receptors were expressed only at a low level. They also reported that hypertrophic stimuli induced an increase in α1A and a decrease in α1D receptors.19 Thus, temporal changes in the expression of the α1-adrenergic receptor subtype in CMG cells are very similar to the postnatal changes previously observed in the neonatal rat heart.
β1- and β2-Adrenergic Receptor Expression in CMG Cells
Cardiomyocytes express both β1- and β2-adrenergic receptors in mammalian hearts, the β1 receptor being the predominant subtype (approximately 75% to 80% of total β receptors) 7. We demonstrated that CMG cells first express both β1 and β2 receptors on 1 day when directed to differentiate into the cardiomyocyte and that stimulation of β 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 β receptors. Heart-specific expression of β1 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 β-blocker propranolol. However, ICI118551 slightly inhibited this increase. These results suggest that the β1 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 β1 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 β1- and β2-adrenergic receptors. Previous studies showed that M1 receptors coupled to Gq/G11 and activated phospholipase Cβ through Gqα, leading to IP3 production, and that M2 receptors coupled to Gi/G0/Gz and activated phospholipase Cβ through Giβγ, 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 α1- and β1-adrenergic and muscarinic receptors but not β2 receptors had already contributed to chronotropic responses in early-stage (7 days) murine ES cell–derived cardiomyocytes, whereas β2 receptors first functioned at the intermediate stage (7+4 days).11 The present study demonstrated mesenchymal stem cell–derived cardiomyocytes to express α1A-, α1B-, α1D-, β1-, and β2-adrenergic and M1- and M2-muscarinic receptors, to maintain signal transduction pathways, and to also show cardiac hypertrophy in response to an α agonist, as well as having positive inotropic and chronotropic responses to a β 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.
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.
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- ↵Simpson P. Norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an alpha 1 adrenergic response. J Clin Invest. 1983; 72: 732–738.
- ↵Chien KR, Knowlton KU, Zhu H, et al. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J. 1991; 5: 3037–3046.
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- ↵Steinberg SF. The molecular basis for distinct beta-adrenergic receptor subtype actions in cardiomyocytes. Circ Res. 1999; 85: 1101–1111.
- ↵Hosey MM. Diversity of structure, signaling and regulation within the family of muscarinic cholinergic receptors. FASEB J. 1992; 6: 845–852.
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- ↵Stewart AF, Rokosh DG, Simpson PC, et al. Cloning of the rat alpha 1C-adrenergic receptor from cardiac myocytes. alpha 1C, alpha 1B, and alpha 1D mRNAs are present in cardiac myocytes but not in cardiac fibroblasts. Circ Res. 1994; 75: 796–802.
- ↵Rokosh DG, Stewart AF, Simpson PC, et al. Alpha1-adrenergic receptor subtype mRNAs are differentially regulated by alpha1-adrenergic and other hypertrophic stimuli in cardiac myocytes in culture and in vivo. Repression of alpha1B and alpha1D but induction of alpha1C. J Biol Chem. 1996; 271: 5839–5843.
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