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First published online October 4, 2007
Stem Cells Vol. 26 No. 1 January 2008, pp. 202 -211
doi:10.1634/stemcells.2007-0490; www.StemCells.com
© 2008 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Myocardin A Enhances Telomerase Activities in Adipose Tissue Mesenchymal Cells and Embryonic Stem Cells Undergoing Cardiovascular Myogenic Differentiation

Rosalinda Madonna, James T. Willerson, Yong-Jian Geng

The University of Texas Health Science Center at Houston and the Texas Heart Institute, Houston, Texas, USA

Key Words. Stem cell • Telomerase • Myocardin • Heart • Development • Myogenesis

Correspondence: Correspondence: Yong-Jian Geng, M.D., Ph.D., The Center for Cardiovascular Biology and Atherosclerosis Research, Cardiology Division, Department of Internal Medicine, University of Texas Houston Medical School, 6431 Fannin Street, MSB 6.037, Houston, Texas 77030, USA. Telephone: 713-500-6077; Fax: 713-500-6556; e-mail: yong-jian.geng{at}uth.tmc.edu

Received on June 20, 2007; accepted for publication on September 26, 2007.

First published online in STEM CELLS EXPRESS  October 4, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Acting as a reverse transcriptase that maintains nuclear telomere length and replication potential, telomerase usually decreases in expression and activities when mammalian stem cells undergo terminal differentiation. This study identified, in adult adipose tissue, a subpopulation of mesenchymal stem cells (MSCs) that coexpresses telomerase and myocardin A, a key regulator of cardiovascular myogenic development. The telomerase/myocardin A-positive MSCs differentiated into cardiovascular myogenic cells while retaining expression and activation of the telomerase catalytic unit, telomerase reverse transcriptase (TERT), at a level comparable to that of ESCs. Both myocardin A and TERT could be coimmunoprecipitated from the developing MSCs and ESC-derived EBs with either anti-TERT or anti-myocardin A antibodies, suggesting the formation of TERT-myocardin A complexes in the MSCs and EBs. The proteins pulled down with anti-myocardin antibodies showed almost the same levels of telomerase activities as those precipitated with anti-TERT antibodies. Overexpression of myocardin A by cDNA transfection significantly increased telomerase activities and promoted telomere synthesis by MSCs. The data from this study indicate a potentially novel function of myocardin A in maintaining the myogenic stemness in developing MSCs and EBs by enhancing telomerase activation and promoting myogenic gene expression.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Adult adipose tissue contains multipotent stem cells that can differentiate into mature cells of other tissues, including cardiovascular cells [1–4]. Among the most abundant adipose tissue stem cells are stromal or mesenchymal stem cells. Recent studies on MSCs from animal [5–8] and human [1, 3, 9] adipose tissues have provided several lines of evidence that adipose tissue-derived MSCs can differentiate or transdifferentiate into beating myogenic cells in culture. However, little is known about the cellular and molecular mechanisms underlying the myogenic development of these adipose tissue-derived MSCs. In culture, MSCs possess a certain degree of "stemness" or pluripotency similar to that seen in ESCs. Thus, an adult stem cell that can give rise to different types of somatic cells is expected to possess certain biological characteristics similar to those of ESCs, which express cellular proteins important for maintaining the pluripotency of growth and differentiation.

Telomerase is a ribonucleoprotein complex that catalyzes the addition of oligonucleotide (TTAGGG) repeats to telomeres, the repetitive DNA structures residing at the ends of linear chromosomes [10, 11]. This telomerase-catalyzed addition prevents telomere shortening and stabilizes chromosomes [12]. Telomerase contains RNA-dependent DNA polymerase (reverse transcriptase); the RNA component (complementary to the telomeric single-stranded overhang) serves as a template for synthesis of the TTAGGG repeats directly onto telomeric ends. This extension of the 3' DNA template permits additional replication of the 5' end of the lagging strand, thus compensating for the telomere shortening that occurs in its absence. Telomerase is expressed abundantly in embryonic or undifferentiated cells, but its expression and activity weaken, leading to gradual shortening of telomeres, in highly differentiated, mature somatic cells with aging [13]. In the heart, telomerase activity is associated with myogenic cell survival, growth, and differentiation [14–17]. Telomerase activities and telomere synthesis are altered as heart failure develops [18]. However, the role of telomerase in regulating the potency of cardiac myogenic stem cells is unclear.

Myocardin A serves as a transcriptional coactivator of serum response factor, which plays a regulatory role in the development of the heart [19, 20] and blood vessels [20–24]. As a cardiac and vascular muscle-specific transcriptional regulator, myocardin A contributes to the development of cardiovascular myocytes. The biological effects of myocardin A may be mediated through interaction with or regulation by other transcriptional factors, such as the myocardin-related transcription factors [22] and Nkx2.5 (or Csx) [19], an evolutionarily conserved cardiac transcription factor of the homeobox gene family. Derived from alternative splicing of the myocardin gene, myocardin A is the most abundant isoform of myocardin in the heart at all stages of embryonic development [19].

The present study was designed to test the hypothesis that telomerase is highly expressed and biologically activated in adult adipose tissue-derived, myogenic stem cells residing in the stromal or mesenchymal compartment characterized by coexpression of myocardin A.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Isolation and Culture
Visceral adipose tissue was harvested from male BALB/C mice at 6 months old, pigs at 2 years, and dogs at 1.5 years. Mesenchymal cells were isolated using a modified version of published methods [5, 25]. In brief, adipose tissue was mechanically minced and digested with collagenase. After adipocyte removal, the vascular stromal fraction was plated (at a density of 1,000 cells per cm2) in Iscove's modified Dulbecco's medium (Gibco, Grand Island, NY, http://www.invitrogen.com; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with L-glutamine (2 mmol/l), penicillin (100 U/ml), streptomycin sulfate (100 µg/ml), 0.1 mM nonessential amino acids, 10–4 mol/l 2-mercaptoethanol, and 20% fetal bovine serum. After 24 hours, nonadherent cells were removed, and MSCs were selected on the basis of their adherent properties. The MSCs were cultured in 100-mm dishes in high-density culture for up to four passages. Within 5–7 days after plating, spindle-shaped adherent cells were apparent and had formed colonies. Most adherent cells were negative for CD31 and CD34; a small fraction of adherent cells expressed {alpha}-smooth muscle actin. Multipotent characteristics of MSCs were confirmed by verifying in vitro their ability to differentiate into adipocytes, vascular endothelial cells, smooth muscle cells (SMCs), and spontaneous contractile myocytes. For comparison, the following cell lineages were cultured: murine endothelial cells and SMCs, human coronary SMCs, human HeLa cells, rat H9c2 myoblasts (American Type Culture Collection, Manassas, VA, http://www.atcc.org), and the murine ESC line CCE (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com).

RNA Analysis by Real-Time Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated (using RNA isolation kits; Qiagen, Valencia, CA, http://www1.qiagen.com) and reverse transcribed into single-strand cDNA by reverse transcriptase. Total RNA was processed directly to cDNA synthesis using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen) according to the manufacturer's protocol. All real-time polymerase chain reaction (PCR) primers were designed using software OligoPerfect Designer (Invitrogen). The primers for murine myocardin A were 5'-CTTCTCTCCCCCAGCTTCCA-3' (forward) and 5'-CTTGGGCTTTTGGGACAAGG-3' (reverse). All reactions were performed in triplicate in Myi Single-Color Real-Time PCR detection system (Bio-Rad, Hercules, CA, http://www.bio-rad.com). A melting curve was generated at the end of every run to ensure product uniformity. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an active and endogenous reference to correct for differences in the amount of total RNA added to the reaction and to compensate for different levels of inhibition during reverse transcription of RNA and during PCR. The primers for GAPDH were 5'-AGAACATCATCCCTGCATCC-3' (forward) and 5'-CACATTGGGGGTAGGAACAC-3' (reverse). Data are from at least three different experiments and are presented as relative fluorescence units, using copy numbers as the measurement unit, normalized to GAPDH.

cDNA Cloning and Transfection
cDNAs of murine telomerase reverse transcriptase (mTERT) (3.6 kilobase [kb]) and murine myocardin A (3.1 kb) were amplified by PCR and subcloned into TOPO cloning vector pcDNA3.1 (Invitrogen). The primers for mTERT were 5'-CACCATGACCCGCGCTCCT-3' (forward) and 5'-CGCCCAGTCCAAAATGGTCTG-3' (reverse); the primers for murine myocardin A were 5'-CACCATGACACTCCTGGGGTCTGAAC-3' (forward) and 5'-GTCCCACTGCTGTAAGTGGAGATCCAT-3' (reverse). The PCR products were confirmed by DNA sequencing. For cDNA transfection, murine MSCs were plated in 100-mm dishes at subconfluence and then incubated with 16 µg of plasmid DNA in the presence of a Lipofectamine reagent (Invitrogen). The transfected cells were selected for 15 days using 400 µg/ml G418 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Neomycin-resistant clones were used for further experiments.

Immunofluorescence Microscopy
Cells grown in eight-well glass chamber slides (Lab-Tek; OligoPerfect Designer) were fixed with 4% paraformaldehyde, permeabilized, and then blocked in phosphate-buffered saline (PBS) containing 1% bovine serum albumin for 30 minutes. Cells were incubated in this solution with primary antibodies for 1 hour at 4°C. After being incubated and washed in PBS, the slides were incubated with Texas Red- or fluorescein isothiocyanate (FITC)-conjugated anti-goat or anti-rabbit secondary antibodies. Nonimmune IgG was used as the isotype control (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). The slides were washed and mounted with a solution containing 4,6-diamidino-2-phenylindole (VectaShield; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and viewed through a fluorescence microscope.

Immunoprecipitation and Immunoblotting
Total proteins were isolated from MSCs in ice-cold RIPA buffer, separated under reducing conditions, and electroblotted onto polyvinylidene fluoride membranes (Immobilon-P; Millipore, Bedford, MA, http://www.millipore.com). After blocking, the membranes were incubated overnight at 4°C with primary antibodies against cardiac sarcomeric {alpha}-actinin (Sigma-Aldrich), TERT (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), myocardin A (extracted from rabbits inoculated with a synthetic myocardin A peptide), smooth muscle {alpha}-actin (Sigma-Aldrich), and β-actin (Sigma-Aldrich). The blots were developed using a SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL, http://www.piercenet.com). The intensity of each immunoreactive protein band was measured by densitometry. For immunoprecipitation, cell lysates were mixed with primary antibodies against myocardin A or TERT, and the immunocomplexes were pulled down with protein-G beads (Sigma-Aldrich). The immunoprecipitants were then eluted, concentrated, and subjected to immunoblotting.

Telomeric Repeat Amplification Protocol Assays
Telomerase activity was quantified using a TRAPeze telomerase detection kit (Intergen; Chemicon, Temecula, CA, http://www.chemicon.com), according to the manufacturer's protocol. Briefly, 1 x 106 cells per sample were lysed in 200 µl of ice-cold Chaps lysis buffer (0.5% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 10 mmol/l Tris-HCl [pH 7.4], 1 mmol/l MgCl2, 1 mmol/l EGTA, 10% glycerol, 5 mmol/l β-mercaptoethanol). After 30 minutes of incubation on ice, the lysates were centrifuged at 12,000g for 20 minutes at 4°C, and the supernatant was assayed for protein concentration using the Bradford method (Bio-Rad). The telomeric repeat amplification protocol (TRAP) reaction was performed using 1 µg of protein. Telomere extension was performed at 30°C for 30 minutes, followed by 36 cycles of a three-step PCR (94°C for 30 seconds, 59°C for 30 seconds, and 72°C for 1 minute). The final extension step was performed at 72°C for 3 minutes. A standard curve of telomerase activity was generated with TSR8 control templates at different concentrations. In all the PCRs, a 36-base pair template was included as the internal control. In addition, the nuclear proteins from HeLa cells were tested as positive controls for telomerase activity, and Chaps buffer was tested as negative controls for the presence of primerdimer PCR artifacts and PCR contamination carried over from other samples. The TRAP products were analyzed by electrophoresis in a nondenaturing 12% polyacrylamide gel at 5 V/cm. After electrophoresis, the gels were stained with SYBR Green and photographed. TERT activity was measured by examination of 96-well plates in a fluorescence plate reader at excitation/emission settings of 495/516 nm for fluorescein or FITC and 600/620 nm for sulforhodamine.

Southern Blot Analysis of Telomere Repeats
Telomere synthesis was evaluated by Southern blot analysis of telomeric restriction fragments [26]. Each sample, containing a total of 106 cells, was incubated at 55°C in lysis buffer containing proteinase K. After centrifugation, supernatants were exposed to isopropanol and ethanol to precipitate genomic DNA, which was digested with RsaI and HinfI, separated in 0.8% agarose gel, and transferred to nylon membranes (Roche Diagnostics, Mannheim, Germany, http://www.roche-applied-science.com). Hybridization was performed for 4 hours at 42°C in a solution containing digoxigenin-labeled probe (TTAGGG), according to the manufacturer's instructions (Roche Diagnostics), and the specific signal for telomeres was detected by chemiluminescence.

Fluorescent Flow Cytometry
Cells were labeled with primary antibodies against myocardin A or TERT, then incubated with FITC- or PE-conjugated secondary antibodies to rabbit or goat IgG for 30 minutes. Nonimmune IgG was used as the isotype control (Becton Dickinson). Cells positive for myocardin A were defined when their fluorescence was significantly greater than that of control IgG-stained cells. Both myocardin A-positive and -negative cells were sorted and collected using a Beckman Coulter flow cytometer (Beckman Coulter, Miami, http://www.beckmancoulter.com).

Statistical Analysis
Differences were analyzed by the Student's t-test for two-group comparisons, and by analysis of variance (ANOVA) for multiple group comparisons. The existence of individual differences, in case of significant F values at ANOVA, was tested by Scheffé's multiple contrasts. For all tests, statistical significance was established at p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Telomerase Expression in MSCs from Adult Adipose Tissue
Telomerase expression in MSCs derived from adult adipose tissues was examined by immunofluorescent microscopy, reverse transcription (RT)-PCR, Western blot, and TRAP assays. In primary cultures, immunofluorescent microscopy visualized the presence of telomerase reverse transcriptase (TERT) immunoreactive cells in MSCs isolated from adult murine adipose tissue (Fig. 1A–1C). The immunofluorescence was specific for the anti-TERT antibody because staining with nonimmune control IgG did not generate any microscopically detectable fluorescent signal in the same type of cells (Fig. 1D–1F). Similar TERT immunofluorescence was observed in primarily cultured MSCs from adult porcine (supplemental online Fig. 1) and canine (supplemental online Fig. 2) adipose tissues. MSCs from all three species tested had the same pattern of TERT expression. The intensity and intracellular distribution of TERT immunofluorescence varied among MSCs. Many MSCs expressed intense nuclear TERT immunofluorescence, whereas other cells showed modest signals, mainly in the cytoplasm (Fig. 1A–1C).


Figure 1
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  		Figure 1. Expression of TERT in MSCs isolated from adipose tissue stroma. (A–C): Murine MSCs were stained with rabbit polyclonal antibodies to the telomerase catalytic subunit TERT. FITC-conjugated anti-rabbit IgG was used as the secondary antibody. Nuclear counterstaining was conducted with the fluorochrome DAPI. (A): TERT immunofluorescence. (B): DAPI nuclear staining in the same field as (A). (C): Combined (A) and (B). Arrows indicate MSCs with positive TERT immunofluorescence (green) and the blue fluorescent nuclei stained with DAPI. (D–F): Murine MSCs were stained with FITC-conjugated anti-rabbit IgG, without primary antibody. Scale bar = 50 µm. Representative images from three different experiments are shown. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

 
To confirm TERT expression in MSCs as well as ESCs, total RNA was extracted from murine MSCs and ESCs and analyzed by RT-PCR for TERT. Positive TERT cDNA bands at the same molecular size occurred in the RNA samples from both MSCs and ESCs (Fig. 2A). The intensity of TERT cDNA bands in MSCs was similar to that seen in ESCs, suggesting that the steady-state expression of TERT mRNA was comparable between MSCs and ESCs. To determine the TERT protein expression in MSCs from adipose tissue, Western blot analysis was conducted using anti-TERT antibodies in total proteins extracted from MSCs of murine, porcine, and canine adipose tissues, with ESCs as positive controls. For loading controls, anti-β-actin antibodies were used to probe the same membrane following striping previous antibodies. Both TERT and β-actin protein bands were detected in MSCs from all the species tested (Fig. 2B). The TERT and β-actin protein bands appeared at almost equal intensities in MSCs from the murine, porcine, and canine adipose tissues. Although the MSC TERT protein bands were slightly lower than those seen in ESCs, the β-actin bands showed no significant difference between MSCs and ESCs.


Figure 2
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  		Figure 2. Expression and activity of TERT in MSCs isolated from adipose tissue stroma. (A): Reverse transcription-polymerase chain reaction analysis for TERT mRNA in total RNA isolated from mMSCs and mESCs. A representative run from three different experiments is shown. (B): Western blot. Total cellular proteins from mESCs, mMSCs, dMSCs, and pMSCs were analyzed by Western blot with anti-TERT antibody (upper panel) as well as anti-β-actin antibody (lower panel). A representative blot from three different experiments is shown. (C): Telomeric repeat amplification protocol assays for TERT activity in mECs, ESCs, MSCs, mSMC, and hSMC, as well as HeLa cells. A 36-bp internal control band indicating equal loading of samples is visible in all lanes. Assay shown here is representative of three independent experiments. (D): Fluorometry of the TERT activity in three separate experiments. Abbreviations: bp, base pairs; dMSC, canine mesenchymal stem cell; hSMC, human smooth muscle cell; mEC, murine endothelial cell; mESC, murine embryonic stem cell; mMSC, murine mesenchymal stem cell; mSMC, murine smooth muscle cell; pMSC, porcine mesenchymal stem cell; TERT, telomerase reverse transcriptase.

 
For determination of telomerase bioactivities, TRAP assays were performed in murine MSCs and ESCs. As demonstrated by agarose gel electrophoresis, positive signals for telomerase-mediated synthesis of telomeric fragments were detected in the reaction using the lysates of murine MSCs and ESCs, as well as HeLa cancer cells (Fig. 2C, 2D). There was no significant difference in the telomerase activity between MSCs and ESCs. In contrast, the samples from mature vascular endothelial and smooth muscle cells showed no or little telomerase activity (Fig. 2C, 2D).

Coexpression of Myocardin A and Telomerase in Adipose Tissue-Derived MSCs
Myocardin A is involved in the regulation of cardiac-specific [27–29] and vascular muscle-specific [24, 30–32] myogenic differentiation [20, 21, 24]. We hypothesized that MSCs committed to cardiac myogenesis express both myocardin A and telomerase. To test this hypothesis, double immunostaining was performed in MSCs with anti-TERT and anti-myocardin A antibodies. In the primary culture of murine MSCs, many TERT-positive cells stained positively with anti-myocardin A antibodies as illustrated by immunofluorescent scanning confocal microscopy (Fig. 3A–3D). In the double-positive cells, both myocardin A and TERT immunofluorescent signals were found in the nuclei (Fig. 3D). The percentage of myocardin A/TERT dual-positive cells was estimated to be 8%–10% in MSCs expressing the TERT protein. To confirm expression of myocardin A in TERT-positive MSCs, myocardin A mRNA was analyzed quantitatively by real-time PCR in total RNA isolated from murine adipose tissue MSCs. Positive signals for myocardin A mRNA were detected in primary MSC cultures and in the positive control fetal canine cardiomyoblasts (Fig. 4A). The expression of myocardin A mRNA increased when the cells were passed for several generations. In the MSCs, as shown by real-time PCR, TERT mRNA expression was moderately increased (Fig. 4B).


Figure 3
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  		Figure 3. Confocal immunofluorescent microscopy of coexpression of TERT and Myoc A in MSCs isolated from adipose tissue stroma. Expression of TERT and Myoc A in MSCs cultured from murine adipose tissue was detected by confocal immunofluorescent microscopy with antibodies against TERT and Myoc A. (A): DAPI nuclear staining. (B): TERT immunofluorescence (tetramethylrhodamine B isothiocyanate, red). (C): Myoc A immunofluorescence (fluorescein isothiocyanate, green). (D): Merged (A–C). Arrowheads, TERT-positive cells; arrows, Myoc A-positive cells. Scale bar = 75 µm. Representative images from three different experiments are shown. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; Myoc A, myocardin A.

 


Figure 4
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  		Figure 4. Coexpression of TERT and Myoc A in MSCs isolated from adipose tissue stroma. (A, B): Quantitative real-time polymerase chain reaction for assessment of Myoc A (A) and TERT (B) mRNA expression in dMyoblast and differentiating pMSCs in pri cultures and p3 cultures. Data are means ± SD from three experiments. (C, D): Flow cytometry of MSCs for the expression of Myoc A and TERT. (C): MSCs were stained with fluorescein isothiocyanate (FITC)-anti-rabbit IgG isotype control in the FL1-channel. (D): MSCs were stained with pri antibodies against Myoc A and then with FITC-anti-rabbit IgG in the FL1-channel. Cells were analyzed using a Beckman Coulter flow cytometer. Cells in region P1 were defined as positive for Myoc A, whereas cells in region P2 were negative. Both groups of cells were sorted and collected for analysis of protein expression. The data shown here are representative of n = 3 independent experiments. (E): Western blot with anti-Myoc A antibodies in murine MSCs and Neo hearts. Representative blot from three different experiments is shown. (F): Quantitation of TERT proteins by densitometry of TERT and β-actin protein bands of Western blot in sorted Myoc A-positive and -negative cells shown in (G). The sorted Myoc A-positive or -negative cells were analyzed by Western blot with anti-TERT antibody and then stripped and restained with anti-β-actin antibody. Intensity of immunoreactive protein bands was assessed by densitometry. Data are means ± SD from three experiments. Abbreviations: dMyoblast, dog myoblasts; Myoc A, myocardin A; Neo, neonatal; p3, three passage; pMSC, porcine MSC; pri, primary; RFU, relative fluorescence units.

 
To determine myocardin A protein expression, MSCs were subjected to flow cytometry and Western blot analysis using anti-myocardin A antibodies. In flow cytometry compared with the cells stained with control IgG (Fig. 4C), a small but distinct subpopulation of MSCs showed a much greater myocardin A immunofluorescence in the cultures (Fig. 4D, region P1). However, the majority (more than 90%) of MSCs showed little or no signal of myocardin A immunofluorescence, as their fluorescent intensity was almost equal to that seen in the same type of cells stained with normal, nonimmune IgG (Fig. 4C). Using the fluorescent intensity of the control cells stained negatively for myocardin A as the threshold to sort out myocardin A-positive cells from negative cells, myocardin A-positive cells were sorted and collected (Fig. 4D, region P1). As controls, cells negative for myocardin A with a fluorescent intensity 50% lower than that of IgG control stained cells (Fig. 4C, region P2) were also collected (Fig. 4D, region P2). The myocardin expression was confirmed by Western blot analysis, which clearly revealed two protein bands in both MSCs and neonatal heart (Fig. 4E). The higher molecular mass band migrated at approximately 100 kDa, corresponding to the molecular weight of myocardin A (myocardin A-I), and the lower molecular mass band appeared much weaker, which might represent a truncated isoform of myocardin A (myocardin A-II) at a much lower concentration. Hence, the 100-kDa myocardin A-I isoform was predominately expressed in MSCs. The myocardin A-positive and -negative MSCs were then analyzed for TERT protein expression. Interestingly, the myocardin A-positive MSCs expressed levels of TERT significantly higher than those of the myocardin A-negative cells (p < .01), whereas there was no difference in β-actin expression between the two groups of cells (Fig. 4F). Thus, myocardin A-positive MSCs possessed increased levels of TERT.

Telomerase Activity in MSC Nuclear Proteins Immunoprecipitated with Anti-Myocardin A Antibodies
The increased expression of TERT in MSCs positive in expression of myocardin A suggests that the two nuclear proteins may be closely associated and interact with each other during the myogenic development of MSCs. To explore this possibility, coimmunoprecipitation assays were conducted using nuclear proteins extracted from murine MSCs, neonatal hearts, and ESC-derived EBs that had already developed contractile myocytes. The precipitants were then subjected to TRAP analysis of telomerase activities. Positive controls were set up using Hela cell nuclear extracts, whereas negative controls were the precipitants pulled down with normal IgG or omission of nuclear proteins. TRAP assays showed that the nuclear proteins of murine ESC-derived EBs, neonatal hearts, and adipose tissue-derived MSC proteins pulled down with anti-myocardin A antibodies contained significant telomerase activities (Fig. 5A). In the same protein samples, the levels of telomerase activity in the anti-myocardin A precipitants from murine MSCs were almost equal to those in the MSC proteins pulled down with anti-TERT antibodies (Fig. 5B). Similar to the finding in the antibody immunoprecipitation study on MSCs, in the murine EBs, the level of telomerase activity in anti-myocardin A immunoprecipitants was not different from that in the anti-TERT immunoprecipitants proteins (Fig. 5B). The specificity of the TRAP assays performed in the anti-TERT and anti-myocardin A immunoprecipitants was confirmed by the negative control study with the protein fractions pulled down with normal, nonimmune IgG, which expressed little telomerase activity, and by the positive control experiments with the Hela cell proteins that exhibited a strong telomerase activity (Fig. 5A, 5B). Thus, the nuclear proteins pulled down with anti-myocardin A antibodies from MSCs, ESCs, and neonatal hearts contain specific telomerase activity, suggesting the presence of a telomerase-myocardin A complex in the nuclei of cells undergoing myogenic differentiation.


Figure 5
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  		Figure 5. Analysis of telomerase activities in proteins immunoprecipitated with antibodies against Myoc A and TERT in mMSCs and developing EBs. (A): Telomeric repeat amplification protocol assay for telomerase activity in anti-Myoc A or anti-TERT immunoprecipitants from the nuclear proteins of mMSCs (lanes 1 and 2), murine neonatal heart (lanes 4 and 5), and beating EBs at day 14 (lanes 7 and 8). Normal IgG immunoprecipitants (lanes 3, 6, and 9) were used as negative controls. Hela cells were used as the positive control (lanes 10 and 14). Standard curve of telomerase activity was generated using serial dilution of TSR8 control template (lanes 11–13). A 36-base pair band indicating the loading amounts of samples is visible in all lanes. The assay shown here is representative of three independent experiments. (B): Measurement of telomerase activities by fluorometry in the same samples shown in (A) (lanes 1–3 and 7–9). Data represent means ± SD from three independent experiments. Abbreviations: IP, immunoprecipitation; mMSC, mMSC, murine mesenchymal stem cell; Myoc A, myocardin A.

 
Myocardin A Overexpression Enhances Telomere Synthesis by Telomerase in MSCs
The association of myocardin A with TERT raises the possibility that increased myocardin A expression may modulate activities of telomerase in MSCs. To test this possibility, overexpression of TERT and myocardin A in MSCs was achieved by cDNA transfection. In comparison with mock control MSCs, MSCs transfected with TERT cDNA showed an increased level of TERT expression as demonstrated by Western blot analysis of immunoprecipitants pulled down with anti-TERT antibodies (Fig. 6A, upper panel). In MSCs transfected with myocardin A cDNA, markedly increased myocardin A expression was detected with anti-myocardin A Western blot on the immunoprecipitants pulled down with anti-TERT antibodies (Fig. 6A, lower panel). Further analysis of the telomerase activities by TRAP assays showed significantly increased telomerase activities in MSCs with overexpressed myocardin A at levels similar to those observed in MSCs transfected with TERT cDNA and in the positive control HeLa cells (Fig. 6B). By contrast, mature aortic SMCs and mock control MSCs expressed much lower telomerase activities.


Figure 6
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  		Figure 6. Analysis of telomerase and telomere synthesis in MSCs with Myoc A or TERT cDNA transfection. (A): IP with anti-TERT antibodies followed by WB with anti-Myoc A (lower panel) or TERT (upper panel) antibodies in MSCs Trans with Myoc A cDNA and in mock control cells. Representative immunoblots from three different experiments are shown. (B): Telomeric repeat amplification protocol assay for telomerase activities in MSCs transduced with Myoc A cDNA, TERT cDNA, and mock vector. HeLa cells were used as a positive control and mSMCs as a negative control. Data are means ± SD from three separate experiments. (C): Southern blots with DNA extracted from MSCs stably Trans with Myoc A (lane 2), from mock control MSCs (lane 3), and from murine smooth muscle cells (lane 4). Agarose gel electrophoresis of the predigested genomic DNA was performed for the Southern analysis of telomeres in murine adipose tissue-derived MSCs Trans with Myoc A cDNA (lane 2), insert-free vector control (lane 3), and mSMCs (lane 4). A representative blot from three different experiments is shown. Abbreviations: IP, immunoprecipitation; kBp, kilobase pairs; mSMC, murine smooth muscle cell; Myoc A, myocardin A; Tert, telomerase reverse transcriptase; Trans, transfected; WB, Western blot.

 
To determine whether increased expression of myocardin A promotes synthesis of telomeres, the level of telomere repeats was assessed by Southern blot in the genomic DNA extracted from MSCs with overexpression of myocardin A and mock control cells. Consistent with the results of TRAP assays shown above, MSCs with overexpressed myocardin A contained much greater amounts of longer telomere repeats than did control MSCs transfected with insert-free plasmid (mock) and adult aortic SMCs (Fig. 6C). All the cells used for the analysis of telomere length were in the same serial passages up to 4. Thus, MSCs with myocardin A overexpression possessed enhanced telomerase activities with augmented synthesis of telomeres.

Myocardin A/Telomerase Double-Positive MSCs Differentiate into Contractile Myocytes
Myocardin A is a promyogenic nuclear protein that regulates the development of cardiovascular myocytes [19], so the potential of myocardin A/TERT double-positive MSCs for myogenic differentiation was examined. In culture, these MSCs grew in colonies and overlapped each other without contact inhibition (Fig. 7A). Microscopically visible contractile cells developed spontaneously in some of the MSC colonies during days 7–21 (Fig. 7B; supplemental online Video A). These beating myogenic cells clustered and formed junctions with one another as they contracted rhythmically in a synchronized fashion clearly visible by microscopy. As recorded by a digital video camera (supplemental online Video B), the rhythm of the myogenic cell contraction was fairly stable, maintaining a rate of approximately 70–100 beats/minute. The visible cell contraction lasted for almost 2 weeks and then gradually declined. For comparison, the myogenesis of ESC-derived EBs, which also expressed both TERT and myocardin A, was assessed by using the three-dimensional hanging-drop culture system. The development of myogenic cells was clearly detected in the EBs that contained TERT and myocardin A (supplemental online Video B).


Figure 7
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  		Figure 7. Myogenic differentiation and expression of cardiovascular myogenic biomarkers in MSCs and ESCs during development. (A, B): High-power bright-field photographs of MSCs in primary culture (A) and in development (B). Differentiating interference contrast images were taken from MSCs in primary culture and those highly developing with contractile myocytes. Representative images from three different experiments are shown. (C–E): Western blot analysis for cardiac sarcomeric {alpha}-actinin (C), SM {alpha}-actin (D), and general β-actin (E) in the colonies of adipose tissue-derived MSCs with or without contractile myogenic cells. Total cellular proteins were extracted from MSC cultures with or without contractile cells and fractionated by SDS-polyacrylamide gel electrophoresis. Protein bands were electrotransferred onto polyvinylidene difluoride membranes and stained with specific antibodies. Representative immunoblots from three different experiments are shown. Abbreviation: SM, smooth muscle.

 
Further analysis of specific biomarkers for cardiovascular myogenesis revealed the presence of significant signals for cardiac {alpha}-sarcomeric actinin, a myogenic marker in the heart, in the MSC colonies with beating myocytes; however, the signals were low or almost undetectable in the region without beating cells (Fig. 7C). Furthermore, the colonies with beating cells expressed smooth muscle {alpha}-actin, a smooth muscle differentiation marker (Fig. 7D), but noncontractile cells did not produce the smooth muscle actin. As expected, both contractile and noncontractile cells expressed the ubiquitous actin isoform β-actin at almost equal levels (Fig. 7E). Thus, the promyogenic cells derived from MSCs were characterized by coexpression of markers for cardiac and smooth muscle.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
A variety of stem cell or stem cell-like lineages isolated from embryonic and adult tissues can differentiate into cardiovascular cells and may have potential for cardiac stem cell therapy [33, 34]. Whereas ESC studies remain laboratory-based, adult stem cells—mostly from bone marrow [35] and skeletal muscle [36]—have recently been used to treat patients with chronic heart failure. However, the potency of adult stem cells for growth and differentiation is debated. Our study provides new evidence that adult MSCs from the stroma of adipose tissue, by expressing high levels of telomerase activities, share biological features with ESCs. Furthermore, our data indicate that telomerase, which normally declines or disappears in a mature or highly differentiated cell, exists actively in certain subpopulations of MSCs, such as those expressing myocardin A, a gene product critical for cardiovascular myogenesis [19]. Hence, characterized by coexpression of telomerase and myocardin A—two nuclear proteins with unrelated biofunctions—these MSCs comprise a subset of cells with myogenic potential residing in the stroma of adipose tissue. In line with recent reports by other groups [30, 37], our finding of increased expression of TERT and myocardin A in these myogenic MSCs suggests that they likely represent an intermediate step or "biological window" by which an undifferentiated, uncommitted stem cell transitions into the commitment to myogenic development while maintaining the potency for proliferation or quiescence.

The ribonucleoprotein telomerase plays a key role in maintaining telomere length in stem cells and in immortal and actively dividing cells [11]. Adult cells usually express lower activities of telomerase and have shorter telomeres than embryonic cells [10]. Our finding that the myogenic MSCs from adipose tissue express biologically active telomerase at a level comparable to that seen in ESCs strongly suggests that MSCs from adult adipose tissue are premature and undifferentiated. In this study, we found higher levels of TERT expression in primary cultured or low passage animal MSCs. A recent report by Jun et al. has shown a low-level TERT activity in human MSCs infected with a retroviral vector [38]. The species difference or viral vector selection may account for the discrepancy in TERT expression between the animal and human cells. Synthesized in the cytoplasm, TERT protein is translocated and executes its function in the nuclei [39, 40]. The small portion of TERT in the cytoplasm may represent the TERT precursor or premature form, which may remain functionally silent or possess other unknown biological properties [41].

Expression of telomerase is developmentally regulated in the heart. Previous work by Borges and Liew [14] has shown that telomerase activity declines rapidly after birth, becoming almost undetectable within 3 weeks. The telomerase activity decline at the time that cardiomyocyte progenitors become terminally differentiated suggests that telomerase downregulation is associated with the permanent withdrawal of cardiomyocytes from the cell cycle. The coexpression of biologically active telomerase and myocardin A in adipose tissue-derived MSCs may indicate that these cells are still premature myogenic progenitor cells. Indeed, when cultured in vitro, these dually positive MSCs from the stromal compartment of adipose tissue show an increased potency for self-renewal and myogenic development. The telomerase/myocardin A dual-positive MSCs appear to represent a unique subpopulation of cells in the adipose tissue stroma, with the potency of the telomerase-associated self-renewal and myocardin A-driven cardiomyogenesis.

Myocardin A is a nuclear protein that plays a role in the regulation of cardiomyogenic cell maturation from MSCs. Its bioactivities may not, however, be limited to cardiomyogenesis. Recent studies have shown that myocardin A acts as a potent transcriptional coactivator of serum response factor [20]. Myocardin A belongs to the SAP (SAF-A/B, Acinus, PIAS) domain family of nuclear proteins that regulate diverse aspects of chromatin remodeling and transcription [32]. In embryonic tissue, myocardin is initially synthesized in the cardiac crescent at the time of cardiogenic specification and is maintained throughout the atrial and ventricular chambers of the heart during later development [32]. Myocardin A is also expressed in embryonic vascular smooth muscle cells within the cardiac outflow tract and aortic arch arteries and in the developing visceral smooth muscle of the respiratory, gastrointestinal, and genitourinary tracts. Conversely, very little myocardin exists in the skeletal muscle cells of adults. Furthermore, myocardin A is the most abundant isoform of myocardin in the heart at all stages of development [19].

Our observation that myocardin A expression promotes telomerase activity by enhancing the synthesis of telomeres points out to a potentially novel function for myocardin A. The bioactivities of these two nuclear proteins are very different; telomerase has an antisenescent or antiapoptotic function, whereas myocardin A regulates cardiovascular myogenesis. The enhancement of telomerase activity in myocardin A-expressing myogenic stem cells may play a key role in maintaining the status of myogenic stemness (i.e., the potency of myogenic cell growth and differentiation). Furthermore, this association may help explain why myogenic differentiation can occur in stem cells without compromising the potency of proliferation. Certainly, our data strongly suggest that a cell with active myogenic gene expression can simultaneously express an antisenescent gene, such as telomerase. This notion is consistent with the findings of other groups showing that ectopic expression of TERT does not affect differentiation in either ESCs [42] or adult stem cells [43]. Our results from the studies of coimmunoprecipitation and functional assays suggest that both myocardin A and telomerase may have additional bioactivities that have not been reported previously. In addition to the maintenance of telomere length [39, 44], telomerase may have other functions, including regulation of cell cycle, apoptosis, and cell-to-cell communication [40]. Our current work focuses on MSCs from younger animals and at lower passages. Whether telomerase and myocardin A are coexpressed and interplay with each other in human cells and in cells of aged animals is an interesting topic for future study.

Stem cell transplantation is emerging as a possible new treatment for cardiac patients, and recent clinical trials have documented potential benefits of stem cell therapy for patients with acute myocardial infarction and end-stage heart failure [35, 45]. Planat-Benard et al. [7] have recently shown that MSCs can differentiate into ventricular and atrial myocyte-like cells that also respond to stimulation with adrenergic agonists. As a potential alternative source of therapeutic stem cells, adipose tissue has several advantages over other sources, including abundance, accessibility, and ease of replenishment. For example, adult stem cells could easily be isolated from liposuction waste tissue. Although adipose tissue-derived adult stem cells [25, 46–50] have been shown to differentiate into adipocytes, chondrocytes, osteocytes, myocytes, and neuronal lineages, we have shown in our culture system that MSCs develop primarily into the types of cells usually seen in wound healing, including myogenic, vasogenic, and connective cells. However, the findings of these in vitro studies are limited because they do not precisely reproduce the environment within a living heart. We are currently conducting in vivo experiments in which MSCs derived from adipose tissue are transplanted into animal hearts in models of myocardial infarction or ischemia. After angioplasty, these stem cells may be delivered along with a vasodilating agent, such as prostacyclin, through the coronary arteries [51]. In addition, cholesterol lowering with statins may have an impact on cardiac stem cell properties, including the response to inflammatory stimulation [52]. Such in vivo preclinical investigations could generate important information that may, in turn, warrant clinical trials of adipose tissue-derived MSCs in selected patients with heart disease.

In summary, in this study, we have analyzed and identified a subpopulation of myogenic cells from the mesenchymal cell population of adipose tissue stroma with high levels of telomerase and myocardin expression. Similar to those from embryonic stem cells, the adipose tissue-derived myogenic cells coexpress telomerase and myocardin A with an increased potency of proliferation and differentiation. Demonstrated by confocal microscopy, telomerase exists in myogenic cells with high levels of myocardin A in the nuclei or in the cytoplasm as a premature form with unknown function. Furthermore, telomerase and myocardin A may form complexes, as they can be coimmunoprecipitated with anti-TERT or myocardin A antibodies. Finally, enhanced expression of myocardin A is associated with increased telomerase activities and telomere synthesis. These data point out a novel function of myocardin A and the potentially concerted role of telomerase and myocardin in promotion of myogenesis.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Drs. Michael Wassler, Daming Tang, and Danli Wu for kind help with manuscript preparation. We also thank Rebecca Bartow, Ph.D., for editorial assistance. This study was supported by NIH Grants R01HL59249 and R01HL69509 (to Y.-J.G.), grants from the Texas State Higher Education Coordinating Board ATP/TDP program (to Y.-J.G.), and the T5 Grant from the United States Department of Defense (to Y.-J.G. and J.T.W.).


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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