Electrophysiological deterioration and resurrection in the scarred heart. Pijnappels, D.A.

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Citation

Pijnappels, D. A. (2009, June 18). Electrophysiological deterioration and resurrection in the scarred heart. Retrieved from

https://hdl.handle.net/1887/13851

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License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

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F ibroblasts from human post-myocardial infarc- tion scars acquire properties of cardiomyocytes after transduction with a recombinant myocardin gene

John van Tuyn,^1,2,3,* Daniël A. Pijnappels,^1,* Antoine A.F. de Vries,² Ingrid de Vries,² Ietje van der Velde-van Dijke,² Shoshan Knaän-Shanzer,² Arnoud van der Laarse,¹ Martin J. Schalij,¹ Douwe E. Atsma¹

*Authors contributed equally

Department of Cardiology,¹ Virus and Stem Cell Biology Laboratory,² Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands Interuniversity Cardiology Institute of the Netherlands,³ Utrecht, the Netherlands FASEB J. 2007;21:3369-79

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Chapter

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Abstract

Myocardial scar formation impairs heart function by inducing cardiac remodeling, decreasing myocardial compliance, and compromising normal electrical conduction.

Conversion of ventricular scar fibroblasts (VSFs) into (functional) cardiomyocytes (CMCs) may be an effective alternative treatment to limit loss of cardiac performance after myocardial injury. In this study, we investigated whether the phenotype of VSFs can be modified by gene transfer into cells with properties of CMCs. To this end, fibroblasts from postmyocardial infarction scars of human (h) left ventricles were isolated and characterized by cell biological, immunological, and molecular biological assays. Cultured hVSFs express GATA4 and connexin (Cx) 43 and display adipogenic differentiation potential. Infection of hVSFs with a lentivirus vector encoding the potent cardiogenic transcription factor myocardin renders them positive for a wide variety of cardiomyocyte-specific proteins, including sarcomeric components, transcription factors, and ion channels, and induces the expression of several smooth muscle marker genes. Forced myocardin expression also endowed hVSFs with the ability to transmit an action potential and to repair an artificially created conduction block in cardiomyocyte cultures. These finding indicate that in vivo myocardin gene transfer may potentially limit cardiomyocyte loss, myocardial fibrosis, and disturban- ces in electrical conduction caused by myocardial infarction.

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Introduction

A

fter myocardial infarction (MI), the CMCs in the infarcted myocardium are replaced by fibrotic scar tissue composed of (myo)fibroblasts and a collagen- rich extracellular matrix (ECM).¹ These cells persist and remain metabolically active even in areas of long-standing fibrosis.² Myocardial scars contribute to deleterious remodeling and increased mechanical stiffness of the heart resulting in impaired sy- stolic and diastolic function which may lead to symptoms of heart failure. In addition, excessive deposition of ECM components reduces electrical coupling between CMCs thereby impairing impulse propagation.³ Currently, stem cell transplantation is explored as a new therapeutic modality to regenerate damaged myocardium.⁴ So far, most animal and clinical studies have demonstrated only a modest and some- times even transient improvement of cardiac function after infusion of somatic stem cells in infarcted myocardium.^5,6 In addition, true myocardial regeneration via the in vivo (trans)differentiation of somatic stem cells into CMCs seems to be at best a rare event.⁷ Therefore, alternative treatment strategies targeting cardiac fibroblasts have been proposed, including (i) inhibition of ECM synthesis by these cells, (ii) their conversion into (cardio)myocytes, and (iii) the use of these cells as a local source of cardioregenerative and/or angiogenic factors.⁸ Introduction of a gene encoding a developmental regulator of (cardio)myogenesis in VSFs may induce their transdif- ferentiation into (cardiac) muscle cells. Illustrating the feasibility of this concept, in vitro and in vivo experiments have shown that transfer of the gene coding for the skeletal muscle-specific transcription factor (TF) MYOD1 leads to skeletal muscle differentiation in a variety of non-muscle cell types including cardiac fibroblasts.^9-11 Given the differences in ion channel composition between skeletal muscle cells and CMCs¹² it is, however, unlikely that MYOD1-transduced fibroblasts can produce a cardiac action potential. It would therefore be preferable to use a gene encoding a TF involved in cardiomyogenesis to induce heart muscle differentiation in VSFs and to endow them with the desired electrical properties.

Several TFs, including Nkx2.5, GATA binding protein family members (e.g., GATA4), T box-containing protein family members (e.g., Tbx5), the heart- and neural crest derivative-expressed (Hand) 1 and 2 proteins, polypeptide C of the MADS box transcription enhancer factor 2 (Mef2) group of proteins (e.g., Mef2C), and serum response factor (SRF), have been found to be essential for proper heart development during embryogenesis.^13,14 Each of these cardiac TFs is directly involved in the transcriptional regulation of genes encoding the specialized structural components and channel proteins of CMCs by binding to well-established DNA sequences in the promoter/

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enhancer regions of these genes. Recently, muscle development in the roundworm Caenorhabditis elegans was found to be regulated by three functionally redundant genes: the MyoD family homologue hlh-1, the SRF/Mef2 homologue unc-120, and the HAND homologue hnd-1.¹⁵ In vertebrates, the corresponding genes have adopted more specific roles in muscle development, with the MyoD family members regulating skeletal muscle formation while the Hand genes are involved in smooth and cardiac muscle development; SRF and Mef2 family members play an important role in specifying all three muscle lineages.^13-15 However, forced expression of the genes encoding the aforementioned TFs in somatic mammalian cells of nonmuscle origin has not yet been reported to result in the formation of CMCs. Recently, a new car- diac and smooth muscle-restricted TF, designated myocardin, was identified.¹⁶ This protein, which exerts most of its effects by forming a complex with SRF and Mef2c, appears to be a key regulator of cardiac and smooth muscle differentiation.^16-18 Infec- tion with a myocardin-encoding adenovirus vector caused ±10% of human dermal fibroblasts (hDFs) and human mesenchymal stem cells (hMSCs), as well as ±100% of human epicardial cells, to adopt a cardiomyocyte-like phenotype through activation of genes specifying a wide variety of cardiomyocyte-specific polypeptides, including sarcomeric components and heart muscle-specific TFs.^19,20

In this study we used reporter gene assays to compare the ability of the cardiogenic TFs (cTFs) GATA binding protein 4 (GATA4), heart- and neural crest derivative-expressed 1 (Hand1, also known as eHand), MADS box transcription enhancer factor 2C (Mef2C), myocardin (both cardiomyocyte-enriched isoforms), Nkx2.5, and Tbx5 to transactivate the promoters of the human natriuretic peptide precursor A (ANF), myosin heavy chain 6 (MHC), and gap junction protein 1 (Cx43) genes. As myocardin outperformed all other cTFs as a transcriptional activator, we generated a self-inacti- vating (SIN) lentivirus vector (LV.CMV.myocL-HA) directing synthesis of the largest isoform of human myocardin (myocL). This lentivirus vector was subsequently used to impose a cardiomyocyte-like gene expression program on hVSFs, as evidenced by reverse transcription-polymerase chain reaction (RT-PCR) analyses and immunofluorescence microscopy (IFM). Furthermore, electrophysiological studies using multie- lectrode array (MEA) culture dishes revealed that hVSFs transduced with LV.CMV.

myocL-HA were highly effective at repairing an artificially created conduction block in monolayers of neonatal rat (nr) CMCs.

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Materials and Methods

Isolation and Culture of Human Myocardial Scar Fibroblasts

hVSFs were isolated from multiple samples of human ventricular scar tissue obtained during surgical reconstruction of the left cardiac ventricle. Each sample was cut into small pieces, which were transferred to porcine gelatin (Sigma-Aldrich)-coated 10- cm² culture dishes, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS; all from Invitrogen). A glass coverslip was placed on top of the tissue pieces to prevent them from floating. The culture medium was refreshed every 3 days. Out- growth of cells was visible 2 days after culture initiation. Three days later the coverslip and tissue pieces were removed, the hVSFs were detached with trypsin-EDTA solution (Invitrogen), and reseeded in new culture dishes. When the cultures reached 3-6×10⁶ cells, aliquots of 2×10⁵ hVSFs were frozen to -80°C at -1°C per min in DMEM containing 40% FBS and 10% dimethylsulfoxide (Sigma-Aldrich) and subsequently stored in nitrogen vapor.

Cell Culture and In Vitro Differentiation Assays

HeLa cells were propagated in DMEM supplemented with 10% FBS. hDFs were cultured as described before¹⁹; the isolation and culture of nrCMCs has been specified elsewhere.²¹ Induction and analysis of in vitro adipogenesis and osteogenesis has been carried out as previously described.¹⁹

Immunophenotyping

The surface antigen expression profile of hVSFs was determined by flow cytometry as described previously for bone marrow-derived hMSCs.¹⁹

Promoter Assays

For comparing the transactivating activity of different cTFs, we generated the luciferase-encoding reporter constructs pANF.LUC, pMHC.LUC, and pCX43.LUC containing 2241, 5505, and 3009 bp fragments of the human ANF, MHC, and Cx43 promoters, respectively. The nucleotide sequences of these pGL3-basic (Promega, Madison, WI, USA) derivatives have been deposited in the GenBank database under accession nos. EF186080, EF186081, and EF186082.

In the expression plasmids pU.CAG.hrGFP, pU.CAG.myocL, pU.CAG.myocS, pU.CAG.Hand1, pU.CAG.Nkx2.5, pU.CAG.GATA4, pU.CAG.Mef2C, and pU.CAG.

Tbx5, coding sequences of the Renilla reniformis green fluorescent protein (Strata-

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gene, San Diego, CA, USA), myocL, the small cardiomyocyte-enriched isoform of hu- man myocardin (myocS), and the human versions of Hand1, Nkx2.5, GATA4, Mef2C, and Tbx5 are placed under the transcriptional control of the CAG promoter²² and rabbit β-globin gene polyadenylation signal. For the nucleotide sequences of these constructs, see GenBank accession nos. EF186083, EF186084, EF208955, EF208956, EF186085, EF186086, EF186087, and EF186088, respectively.

One day before transfection, 3x10⁴ HeLa cells were seeded per well of a 24-well plate (Greiner, Frickenhausen, Germany). The next day the cells were transfected using Ex- Gen 500 (Fermentas, Hanover, MD, USA) with 0.2 µg pMHC.LUC DNA or the molar equivalent of pANF.LUC or pCX43.LUC supplemented with pUCBM21 (Boehrin- ger Mannheim, Mannheim, Germany) to a total of 0.2 µg DNA and 0.2 µg pU.CAG.

myocL DNA or the molar equivalent of pU.CAG.myocS, pU.CAG.Hand1, pU.CAG.

Nkx2.5, pU.CAG.GATA4, pU.CAG.Mef2C, pU.CAG.Tbx5, or pU.CAG.hrGFP, again supplemented with pUCBM21 to a total of 0.2 µg DNA. Sixteen hours later, the transfection medium was replaced by fresh culture medium. At 48 h post-transfection, the luciferase activity in each cell sample was measured using Steady-Glo (Promega) and a Lumat LB 9507 luminometer (EG & G, Berthold, Bad Wildbad, Germany). The induction factors of the various TFs were calculated for all three heart muscle-specific promoters by dividing the luciferase activity observed after cotransfection of one of the three reporter constructs and a TF-encoding plasmid by that found after cotransfection of pU.CAG.hrGFP and the same reporter construct. Each pU.CAG plasmid was tested in quadruplicate in three independent experiments to compensate for pos- sible differences in transfection efficiency.

Lentivirus Vector Production

To generate SIN human immunodeficiency virus type I vectors encoding a recombinant myocL protein carrying an epitope of the human influenza virus A hemag- glutinin (HA) at its C terminus (myocL-HA) or specifying a nuclear-targeted version of Escherichia coli β-galactosidase (nls-bGal), the shuttle plasmids pLV.CMV.myo- cL-HA and pLV.C-PGK.nls-bGal were made on the basis of construct pLV-CMV- IRES-eGFP.²³ Expression of myocL-HA from pLV.CMV.myocL-HA is controlled by the human cytomegalovirus immediate-early gene (CMV-IE) promoter and pLV.C- PGK.nls-bGal contains a hybrid promoter consisting of the enhancer of the CMV-IE fused to the human 3’-phosphoglycerate kinase gene promoter to drive transgene expression. Furthermore, to enhance transgene expression in both lentivirus vector shuttle plasmids, the 3’ long terminal repeat is preceded by the woodchuck hepatitis virus post-transcriptional regulatory element.²⁴ The nucleotide sequences of pLV.

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CMV.myocL-HA and pLV.C-PGK.nls-bGal have been deposited in the GenBank database under accession nos. EF186078 and EF186079, respectively. The lentivirus vector shuttle plasmid pLV.CMV.myocL.IRES.eGFP and pLV.CMV.bGal.IRES.eGFP were also derived from pLV-CMV-IRES-eGFP and specify bicistronic mRNAs encoding the enhanced green fluorescent protein and myocL or bGal, respectively. For the nucleotide sequences of these constructs, see GenBank accession nos. EF205035 and EF205034. Vesicular stomatitis virus G-protein-pseudotyped lentivirus vector stocks were produced by seeding six 175 cm² culture flasks with 6.6x10⁴ 293T per cm² and transfecting these producer cells the next day with 83 ng/cm² of either pLV.

CMV.myocL-HA or pLV.C-PGK.nls-bGal together with 76 ng/cm² psPAX2 (Addge- ne, Cambridge, MA, USA) and 41 ng/cm² pLP/VSVG (Invitrogen) using the calcium phosphate-DNA coprecipitation method.²⁵ Sixteen hours later, the transfection medium was replaced by new culture medium. At 48 h post-transfection, the culture fluid was collected and freed of cellular debris by centrifugation at room temperature for 10 min at 800 g and filtration through a 0.45 µm pore-sized cellulose acetate filter (Pall Corporation, East Hills, NY, USA). To concentrate the lentivirus vector particles, 31 ml of the cleared culture medium was loaded onto a 4 ml cushion of 20% sucrose in phosphate-buffered saline (PBS), then centrifuged for 90 min at 25,000 rpm and 10°C in an SW 28 rotor (Beckman Coulter, Fullerton, CA, USA). Next, the supernatant was discarded and the pellets with the vector particles were suspended in 200 µl PBS-10%

FBS by gentle rocking overnight at 4°C.

An aliquot of both vector stocks was serially diluted in culture medium containing 8 µg/ml polybrene and used to infect HeLa cells. Four hours later, the inoculum was exchanged with fresh culture medium. At 2 days postinfection, the functional titer (in terms of HeLa cell-transducing units [HTUs]) of the LV.C-PGK.nls-bGal preparation was determined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of formaldehyde-fixed cells and that of the LV.CMV.myocL-HA stock was determined by immunostaining of ethanol-fixed cells with a 1:1000 dilution of monoclonal anti- body (mAb) HA.11 (Covance, Princeton, NJ, USA) directed against the epitope tag.

Lentivirus Vector Transduction

hVSFs were seeded at a density of 2x10⁴ cells/cm²in 10 cm² culture dishes with (for IFM) or without (for RT-PCR analysis) glass coverslips. The next day, the culture fluid was replaced by 1 ml fresh growth medium containing 20 µg/ml diethylaminoethyl- dextran sulfate (GE Healthcare) and 1 HTU of LV.CMV.myocL-HA or LV.C-PGK.

nls-bGal per cell. Four hours later, the cells were washed three times with PBS and the inoculum was replaced by fresh culture medium. One week after transduction, cells were analyzed by IFM or RT-PCR analysis.

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Reverse Transcription-Polymerase Chain Reaction Analysis

Semiquantitative RT-PCR analyses were performed as previously reported²⁰ with the addition of primer pairs targeting transcripts encoding the 1C and 1D subunits of the voltage-dependent L-type calcium channels (CACNA1C or Cav1.2; 5’-AGGAGCAG- TTTTTGGGGTTT-3’ and 5’-TGGAGCTGACTGTGGAGATG-3’ and CACNA1D or Cav1.3; 5’-GCAAGATGACGAGCCTGAG-3’ and 5’-ATGGTTATGATGGTTAT- GACAC-3’, respectively), the cardiac pacemaker channel (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; HCN4; 5’-CGCCTCATTCGATA- TATTCAC-3’ and 5’-CGCGTAGGAGTACTGCTTC-3’), and the inwardly rectifying potassium channel J3 (KCNJ3 also known as Kir3.1; 5’-TCCCCTTGACCAACTT- GAACT-3’ and 5’-ACGACATGAGAAGCATTTCCTC-3’). For these new primer pairs, 35 PCR cycles and an annealing temperature of 60°C were applied. As internal controls for the quantity and quality of the RNA specimens, RT-PCR amplifications targeting transcripts of the housekeeping gene glyceraldehyde-3-phosphate dehydro- genase (GAPDH) were performed in parallel. PCRs carried out on cDNA derived from total RNA samples of human atrium, ventricle, vascular smooth muscle, and skeletal muscle served as positive controls, and PCRs in which the cDNA was replaced by water were included as negative controls. For comparing relative mRNA levels, only PCR samples from within the linear range of amplification were used.

Quantitative RT-PCR (qPCR) was performed in triplicate using the qPCR-&GO kit (Qbiogene, Irvine, CA, USA) and the LightCycler 480 real-time PCR system (Roche, Nutley, NJ, USA), according to the manufacturers’ specifications. To determine PCR efficiencies, standard curves were generated by 2-fold serial dilution of the cDNA template derived from VSFs treated with myocardin (series of 8). Both GAPDH and the β-actin (ACTB) gene were used to normalize transcription levels. Melting cur- ve analysis was carried out to verify that only a single product was formed during the qPCR reaction. Normalization and statistical analysis were performed using the Relative Expression Software Tool (REST).²⁶ Additional primers used in the qPCR reactions were 5’-AGAAGGATTCCTATGTGGGCG-3’ and 5’-CATGTCGTCC- CAGTTGGTGAC-3’ for ACTB, 5’-TGTACAATGCCGTGTCCAAC-3’ and 5’-TCT- TCATTCGGCTCACTGAG-3’ for ANF, 5’-TCTGAGTGCCTGAACTTGC-3’ and 5’-ACTGACAGCCACACCTTCC-3’ for Cx43, 5’-CCTGTTCGACAGTCAGCCG-3’

and 5’-CGACCAAATCCGTTGACTCC-3’ for GAPDH, and 5’-AACCTGAGGGAG- CGGTACTTC-3’ for smMHC.

Immunofluorescence Microscopy

Immunofluorescent labeling of cells was performed as described previously.²⁰ Nu- clei were stained with Hoechst 33342 (Molecular Probes, Carlsbad, CA, USA). HA-

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tagged myocardin was stained with a 1:10,000 dilution of mAb HA.11 in PBS-5% FBS.

The mAb directed against sarcomeric tropomyosin (clone CH1; IgG1) and the rabbit polyclonal Ab recognizing Cx43 were obtained from Sigma-Aldrich. Goat polyclonal Ab’s specific for gap junction proteins 5 (Cx40) and 7 (Cx45), CACNA1C and the subunit of the voltage-gated sodium channel type V (SCN5A, also known as Nav1.5) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and used at a 100-fold dilution (for properties of the other cardiac and smooth muscle protein- specific Ab’s, see refs. 19, 20). Ten randomly distributed recordings were made of each channel protein staining at 200-fold magnification and signal intensities were quantified using Image-Pro Plus (Version 4.1.0.0, Media Cybernetics, Silver Spring, MD, USA). All samples were stained at the same time and processed equally. The mean intensities of samples were considered significantly different when P<0.05 using a Student’s t test.

Induction and Restoration of Artificial Conduction Block

Experimental conduction blocks were created in monolayers of nrCMCs as described before.²¹ In brief, 2x10⁶ freshly isolated nrCMCs were seeded on pretreated MEA culture dishes (MultiChannel Systems, Reutlingen, Germany) with 60 titanium nitride electrodes (electrode diameter: 30 µm, interelectrode distance: 200 µm) to generate confluent monolayers of synchronously contracting cells. After 2 days of culture, a 330 µm gap spanning the entire diameter of the culture dish was created by a laser dis- section microscope (P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany) that was either left free of cells (n=10) or filled with 5x10⁴ nrCMCs (positive control;

n=9), LV.CMV.bGal.IRES.eGFP-infected hVSFs (negative control; n=10), or LV.CMV.

myocL.IRES.eGFP-transduced hVSFs (n=9). The infection of hVSFs with LV.CMV.

bGal.IRES.eGFP or LV.CMV.myocL.IRES.eGFP took place 2 days before application to the channel. After 7 days of culture, electrograms were recorded and color-coded activation maps were generated.

Results

Transactivation of Heart Muscle-Specific Promoters by Cardiogenic Transcrip- tion Factors

To identify the most suitable cTF for endowing hVSFs with properties of CMCs, expression plasmids were generated encoding human GATA4, Hand1, Mef2C, the 2 myocardin splice variants myocL and myocS, Nkx2.5, and Tbx5 and transient trans-

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fection experiments in HeLa cells were performed to test their ability to transactivate the promoters of the human genes specifying (i) the atrial peptide hormone ANF, (ii) the cardiac sarcomeric protein αMHC, and (iii) the gap junction protein Cx43, which is abundantly expressed in CMCs.

Although many promoter activation studies focus on proximal promoter elements, we also included distal promoter elements in our reporter gene constructs to more closely mimic endogenous gene regulation. Functional GATA4, Hand1-E12 complex, Mef2, and Tbx5 binding sites are present in all three promoters, whereas only the ANF and Cx43 promoter regions contain known Nkx2.5 recognition sequences.

CARG boxes, which are required for DNA binding of the myocardin/SRF complex, are present only in the ANF and MHC promoters.

As shown in Figure 1, both myocardin isoforms strongly stimulated the activity of all three heart muscle-specific promoters. GATA4 failed to induce ANF promoter acti- vity but did activate the MHC and Cx43 promoters, albeit to a lesser extent than myo- cardin. All other cTFs significantly activated only the Cx43 promoter, with Hand1 and Tbx5 showing the lowest transactivating activity toward this promoter. Based on these screenings, myocardin was used in the rest of the study.

Isolation and Characterization of Human Ventricular Scar Fibroblasts

In vitro culture of human myocardial scar tissue resulted in the outgrowth of cells with a spindle-shaped morphology (Figure 2). After one round of subculturing, the cells stained negative for sarcomeric α-actinin, sarcomeric MHC, and smooth muscle MHC (smMHC), indicating the cultures were free from contaminating cardiac and smooth muscle cells. The surface antigen profile of the hVSFs as determined by fluorescence-activated flow cytometry was similar to that of hDFs (Table 1). Both cell types expressed at their surface large amounts of the hyaluronate receptor (CD44), the major T cell antigen (Thy-1; CD90), endoglin (CD105), and human leukocyte

Figure 1. ANF, αMHC, and Cx43 promoter activation by the cTFs GATA4, Hand1, Mef2C, myocL, myocS, Nkx2.5, and Tbx5 in HeLa cells. The average induction factors and standard errors are shown (n=12)

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class I antigens (HLA-ABC), as well as low numbers of membrane cofactor protein (MCP also known as CD46), a component of the complement system. In contrast, typical MSC markers such as neural cell adhesion molecule (NCAM;CD56), β3 integrin (CD61), P-selectin (CD62P), transferrin receptor (CD71), and vascular cell adhesion molecule 1 (VCAM-1; CD106) were not present at their plasma membranes.

The surface of hVSFs and hDFs also stained negative for the hematopoietic markers CD11A, CD14, CD15, CD19, CD34, and CD45 as well as for the endothelial markers platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31), vascular endothelial growth factor receptor 2 (VEGFR-2; Flk-1), and VE-cadherin. Neither cell type expressed the CXC motif chemokine receptor 4 (CXCR-4; fusin), human leukocyte class II subtype DR antigens (HLA-DR), the Coxsackie and adenovirus receptor (CAR), or the human and murine stem cell markers CD133 and Sca-1 at their surface or con-

Table 1

Surface marker profile of hDFs and hVSFs.

CAR

CD 10 (NEP; MME) CD11A (LFA-1; integrin αL) CD14 (LPS-R)

CD15 CD19

CD31 (PECAM-1) CD34 (sialomucin) CD44 (HCAM) CD45 (LCA) CD46 (MCP) CD56 (NCAM) CD61 (integrin β3) CD62P (P-selectin) CD71 (transferrin receptor) CD90 (Thy-1)

CD105 (endoglin) CD106 (VCAM-1) CD133

CXCR4 (fusin) HLA-ABC HLA DR Sca-1 (Ly-6A/E) VE-cadherin VEGFR2 (Flk-1)

hDFs hVSFs

- -

+ -

- -

++ ++

- -

+ +

- -

++ ++

- -

++ ++

- -

tained the cardiac progenitor cell marker Isl-1. Finally, in contrast to hVSFs, hDFs displayed neutral endopeptidase (NEP; MME; CD10) at their plasma membrane. Semi-quantitative RT-PCR analyses and IFM were used to investigate the expression of cardiac, smooth, and skeletal muscle genes in hVSFs.

These experiments revealed that hVSFs naturally express the GATA4 and Mef2C genes at low levels (Figure 3 and Figure 5, lane U). Interestingly, in the majority of hVSFs the GATA4 protein is localized in the cytoplasm, while in nrCMCs this cTF is exclusively found in the nucleus (Figure 3). In addition, hVSFs abundantly express Cx43 (Figure 3 and Figure 5, lane U). In these cells, the Cx43 protein is mainly localized around the nucleus with small amounts being present at intercellular borders, while in nrCMCs Cx43 is largely con- fined to areas of cell-cell contacts (Fi- gure 3). Interestingly, hVSFs also con- tain low amounts of CACNA1C- and SCN5A-specific mRNAs (Figure 5, lane U).

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Human Ventricular Scar Fibroblasts can Undergo Adipogenesis but not Osteo- genesis

To investigate whether hVSFs have characteristics of hMSCs, their capacity to differentiate into osteoblasts and adipocytes was tested. Under conditions that induced osteogenesis in hMSCs, hVSFs as well as hDFs failed to differentiate into osteoblasts (Figure 4). When exposed to adipogenic differentiation medium hMSCs and hVSFs underwent adipogenesis in contrast to hDFs (Figure 4).

Forced Myocardin Expression Results in the Activation of Cardiomyocyte-Speci- fic Genes in Human Ventricular Scar Fibroblasts

Using a combination of RT-PCR analyses and IFM, we investigated the presence of cardiac, smooth, and skeletal muscle-specific transcripts in hVSFs infected with the SIN lentivirus vectors LV.C-PGK.nls-bGal or LV.CMV.myocL-HA. After transduc-

Figure 2. Phase contrast image of hVSFs after 30 days in culture. Scale bar represents 50 µm.

Figure 3. GATA4 (green) and Cx43 (red) staining of hVSFs and nrCMCs. Nuclei were stained with the blue-emitting fluorochrome Hoechst 33342.

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tion of the cells with the control vector LV.C-PGK.nls-bGal, no changes in the expression of the analyzed genes were observed as compared with uninfected hVSFs (compare columns U and L in Figure 5 A).

Figure 4. Adipogenesis and osteogenesis of hDFs, hMSCs, and hVSFs. Red stain labels calcium deposits in the osteogenesis assay and fatty inclusions in the adipogenesis assay.

Figure 5. Panel A, semi-quantitative RT-PCR analysis of smooth, cardiac, and skeletal muscle marker gene expression in untreated hVSFs (U) and in hVSFs infected with either LV.C-PGK.nls-bGal (L) or LV.CMV.myocL-HA (M). PCRs in which the cDNA was replaced by water were included to de- monstrate absence of cross-contamination (-). All PCR samples shown are from within the linear range of amplification.

Panel B, quantification of the relative induction levels of transcripts encoding CACNA1C, CTNT, Cx43, ANF and smMHC, in LV.CMV.myocL-HA vs LV.C-PGK.nls-bGal treated VSFs, using quantitative RT-PCR analysis. No significant differences were observed between LV.C-PGK.nls-bGal and mock treated VSFs (data not shown). Values are normalized to both GAP- DH and beta-actin (ACTB) house keeping genes, p-values are indicated for significantly upregulated genes (p<0.05).

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In contrast, infection of hVSFs with the myocardin-encoding SIN lentivirus vector LV.CMV.

myocL-HA led to activation of the genes encoding the cTFs Hand1 and Hand2, and caused an increase in GATA4 (Figure 5A and Figure 6 C) and Mef2C (Figure 5A) expression.

Figure 6. Analysis by IFM of cardiac and smooth muscle marker gene expression in hVSFs transduced with either LV.C-PGK.nls-bGal (control) or LV.CMV.myocL-HA (myocardin). (A) Co-labeling of LV.CMV.myocL- HA (left panel)- and LV.C-PGK.nls-bGal (right panel)-treated hVSFs for HA-tagged myocardin (green) and smMHC, ssTnI, CACNA1C, Cx40, Cx45, or SCN5A (red). (B) Quantification of channel protein levels following immunostaining of LV.CMV.myocL-HA- or LV.C-PGK.nls-bGal-transduced hVSFs. Values are expressed as mean±SEM relative to control samples (n=10). * indicates significant difference (p<0.001), # indicates no significant difference (p=0.23). (C) Immunostaining of LV.CMV.myocL-HA (left column)- or LV.C-PGK.nls- bGal (right column)-transduced hVSFs with antibodies specific for sarcomeric α-actinin, ANF, GATA4, SER- CA2a, or tropomyosin. (D) Co-labeling of LV.CMV.myocL-HA (3 leftmost columns)- and LV.C-PGK.nls-bGal (right columns)-transduced cells for sarcomeric α-actinin and smMHC (upper row) or ANF (lower row).

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Nkx2.5-specific transcripts were not detected in LV.CMV.myocL-HA-transduced hVSFs (Figure 5A, lane M). Furthermore, myocardin induced the expression in hVSFs of the genes encoding the cardiac sarcomeric components, cardiac troponin T (cTnT), atrial and ventricular myosin light chain 2 (Mlc2a and Mlc2v), the and β isoforms of cardiac myosin heavy chain (MHC and βMHC; Figure 5), α-actinin, and tropomyosin (Figure 6C) as well as of the ANF gene (Figure 5 and Figure 6C). LV.CMV.myocL-HA infection of hVSFs did not result in an accumulation of detectable amounts of cardiac troponin I (cTnI)-specific mRNA, normally present only in mature CMCs, but did induce expression of slow-twitch skeletal troponin I (ssTnI), which encodes the major isoform of troponin I in immature heart muscle cells (Figure 5A and Figure 6A).²⁷ Forced myocardin expression also led to the expression in hVSFs of genes spe- cifying the sarcoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a) and the cardiac ion channel proteins CACNA1D, HCN4, and KCNJ3 and caused a large increase in the intracellular levels of the gap junction proteins Cx40 and Cx45 (Figure 6A-B). In addi- tion, myocardin up-regulated the expression of CACNA1C and of the gene encoding SCN5A at both the mRNA and protein level (Figure 5 and Figure 6A-B). These effects were seen in essentially all LV.CMV.myocL-HA-transduced hVSFs as judged by IFM (Figure 6). Besides activating cardiac genes, myocardin also induced the expression of the smooth muscle genes encoding aortic smooth muscle actin (ASMA), calponin h1 (CNN1), transgelin (SM22), and smMHC (Figure 5 and Figure 6A). However, no synthesis of the skeletal muscle-specific transcripts for fast-twitch skeletal troponin I (fsTnI) and skeletal myosin heavy chain (skMHC) was observed after infection of hVSFs with LV.CMV.myocL-HA. The presence of cardiac and smooth muscle mar- kers in hVSFs was always associated with myocardin gene expression, as detected by immunostaining for the HA tag attached to myocardin (Figure 6A). Double labe- ling for smMHC and either ANF or sarcomeric α-actinin confirmed that cardiac and smooth muscle marker genes were expressed in the same cells (Figure 6D). Spontane- ous beating of LV.CMV.myocL-HA-transduced VSFs was never observed, consistent with the absence of properly organized sarcomeres as evidenced by the lack of stria- tion after immunostaining with Ab’s directed against sarcomeric components (Figure 6A, C, D).

Forced Myocardin Expression Endows Human Ventricular Scar Fibroblasts with the Capacity to Repair an Artificially Created Conduction Block In Vitro

Using MEA culture dishes (Figure 7A), we recorded extracellular electrograms throu- ghout a spontaneously and synchronously beating monolayer of nrCMCs.¹⁹ Electrical coupling between the lower and upper part of the culture dishes was lost after esta-

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blishment of a 330±20 µm-wide cell-free gap, resulting in 2 fields of asynchronously beating nrCMCs (n=10; Figure 7B-C). Filling the channel with LV.CMV.bGal.IRES.eGFP-transdu- ced hVSFs (n=10) did not restore electrical conduction between both rCMC fields.

Figure 7. Electrical conduction across monolayers of nrCMCs after establishment of a cell-free gap and its filling with different cell populations. (A) Arrangement of the MEA in the cell culture dishes. The electrodes in rows 2 and 3 cover the area in which a cell-free gap is created, while those in rows 1 and 4 are positioned underneath bordering cardiomyocytes. (B) Typical electrograms from electrodes bordering and spanning the gap region in nrCMC cultures in which the channel was left open or filled with either LV.CMV.bGal.IRES.eGFP-transduced hVSFs (hVSFs + control), LV.CMV.myocL.IRES.eGFP-transduced hVSFs, (hVSFs + myocardin) or nrCMCs. Electrograms were recorded 7 days after the cell-free gap was made and filled with cells. (C) Isochronous color-coded activation maps showing 2 electrically separated nrCMC fields after generation of the cell-free gap or after the establishment of a cellular conduction block using LV.CMV.bGal.IRES.eGFP-transduced hVSFs. Restoration of electrical conduction across the gap and consequently electrical synchronization of the culture occurred after filling the channel with LV.CMV.myocL.IRES.eGFP-transduced hVSFs or control nrCMCs.

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In contrast, seeding of nrCMCs in the gap (n=9) restored action potential transmis- sion across the channel to normal levels (conduction velocity 19±0.8 cm/s), there- by resynchronizing the 2 nrCMC fields (Figure 7B-C). Importantly, application of LV.CMV.myocL.IRES.eGFP-transduced hVSFs into the gap (n=9) also resulted in restoration of conduction across the channel and consequently in resynchronization of both nrCMC fields (Figure 7B-C). The conduction velocity across the hVSFs was only

~30% lower than that across the nrCMCs (13±1.1 cm/s and 19±0.8 cm/s, respectively.

p<0.01).

Discussion

Key findings of this study are 1) myocardin gene transfer leads to the induction of a (partial) cardiac phenotype in hVSFs from adult patients by activating genes encoding, among others, cardiomyocyte-specific TFs, sarcomeric components, and channel proteins involved in cell-to-cell communication and action potential propagation between heart muscle cells; and 2) myocardin-transduced hVSFs are able to conduct an electrical impulse over considerable distances at relatively high velocities, thereby restoring an artificially created conduction block in cardiomyocyte cultures.

In Vitro Characterization of hVSFs

The in vitro characterization of hVSFs performed in this study revealed that these cells can transdifferentiate into adipocytes in vitro. This finding may have clinical significance since substitution of compact myocardial scar tissue by fat tissue is regularly observed in human patients at late stages of heart failure.^28,29 The ability of hVSFs to undergo adi- pogenesis in culture could be used to develop an in vitro model system to investigate the molecular mechanisms underlying the lipomatous changes occurring in diseased hearts. We also found that hVSFs are positive for GATA4, but that in most cells this cTF is found outside of the nucleus, precluding a direct role in transcription activation.

Ex vivo cultured hVSFs contained small quantities of the cardiac ion channel proteins SCN5A and CACNA1C (Figure 5, Figure 6A-B) and large amounts of the gap junction component Cx43 (Figures 3 and 5, 6A-B). The expression of SCN5A and CACNA1C by cardiac scar fibroblasts has not been described before. The presence of Cx43 in hVSFs is consistent with earlier studies showing that in vitro culture and myocardial damage both lead to activation of the Cx43 gene in cardiac fibroblasts whereas the interstitial cells in healthy ventricular myocardium do not possess significant amounts of Cx43.³⁰

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Transactivating Capacity of Cardiogenic Transcription Factors

A comparative analysis of the ability of several human cTFs to transactivate three different human heart muscle-specific promoters corroborated previous experiments demonstrating that myocardin is an exceptionally strong transcription activator acting directly or indirectly on a wide variety of different cardiac genes.¹⁶ In fact, none of the other cTFs were able to induce the human ANF, MHC, or Cx43 promoters to a similar degree as either myocS or myocL (Figure 1). Furthermore, in this first-time compa- rison of the two main cardiomyocyte-enriched isoforms of myocardin, we failed to detect significant differences in their transactivating capacity, leaving the biological role of the amino acid sequence contributed by myocardin exon 11 undefined. The ac- tivation of pANF.LUC and pMHC.LUC by myocardin is compatible with the presence in the ANF and MHC promoters of SRF binding sites. Although the Cx43 promoter lacks SRF binding sites, it does contain recognition sequences for activating protein-1 (AP-1) family members such as c-Fos and c-Jun. Transcription of the c-Fos and c-Jun genes is activated by SRF, thereby indirectly regulating Cx43 gene expression.³¹ It is li- kely that regulation of the Cx43 gene by SRF-myocardin complexes is indirect as well.

Similarities between Myocardin-Transduced Human Ventricular Scar Fibro- blasts and Embryonic Cardiomyocytes

Despite the induction of numerous cardiac genes on transduction with the myo- cardin-encoding lentivirus vector, hVSFs did not acquire the phenotype of mature CMCs, as evidenced by the absence of Nkx2.5 and cTnI, coexpression of smooth muscle marker genes, and the absence of properly structured sarcomeres. However, the observed phenotype does resemble that of early CMCs. Like neonatal CMCs, myocardin-transduced hVSFs display ssTnI (Figure 5A and Figure 6A) instead of cTnI expression (Figure 5A).^32,33 Furthermore, the expression in reprogrammed hVSFs of HCN4 and CACNA1D (Figure 5A), both required for cardiac pacemaker activity, is consistent with the phenotype of immature CMCs,^34,35 which are spontaneously ac- tive. It should be noted, however, that automaticity in myocardin-transduced hVSFs was never observed. Other features shared by myocL-transduced hVSFs and cardiomyocyte precursor cells are the presence of heart muscle-specific sarcomeric components, which are not organized into sarcomeres,³⁶ and the coexpression of cardiac and smooth muscle genes^37-39 (Figure 6D). In accordance with these observations, Wu et al. recently identified a common precursor for cardiac and smooth muscle cells in the mammalian heart, which initially possesses both cardiac and smooth muscle markers.⁴⁰ However, expression of genes encoding definitive cardiac and smooth muscle marker genes like cTnT and smMHC was not observed until the bipotential precursor cells started to differentiate into heart or smooth muscle cells.

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Electrical Properties of (Myocardin-Transduced) Human Ventricular Scar Fibro- blasts

In our in vitro electrophysiological studies, hVSFs transduced with a control lentivi- rus vector were unable to conduct an electrical impulse over a distance of 330±20 µm (Figure 7) even though they contained high levels of Cx43 (Figures 3, 5, 6A-B). The rapid decline of the electrical signal (Figure 7B) is consistent with previous in vitro experiments showing that the maximum distance over which cardiac fibroblasts can conduct an electrical impulse from neighboring CMCs is 300 µm.⁴¹ Conduction under these circumstances is characterized by its low speed (4.6±1.8 mm/s) and electrotonic nature. Because fibroblasts do not generate an action potential, they function as an electrical load that reduces the speed of action potential propagation in bordering CMCs.^42,43

After transduction with a lentivirus vector encoding myocardin, hVSFs abundantly expressed the genes for the gap junction components Cx40, Cx43, and Cx45 and the ion channel proteins CACNA1C, CACNA1D, HCN4, KCNJ3, and SCN5A (Figure 5, Figure 6A-B). These data suggest that myocardin-transduced hVSFs obtained com- ponents essential for impulse propagation.¹² Although the functionality of the afore- mentioned channel proteins has not been tested, myocardin-transduced hVSFs were able to resynchronize in vitro two fields of nrCMCs >300 µm apart (Figure 7). The conduction velocity across the cell-filled gap (13±1.1 cm/s) approached that of nr- CMCs (19±0.8 cm/s), which makes purely electrotonic conduction unlikely. However, it cannot be ruled out completely that increased Cx40 and Cx45 levels are important contributors to the improved conduction velocity of these cells.

Study Limitations

Neonatal rat CMCs were used in our in vitro electrophysiological studies. Although the use of human heart muscle cells is preferable, the limited availability of this cell type and their tendency to dedifferentiate during culture prevented the use of hu- man CMCs. Furthermore, although forced myocardin expression endows hVSFs with 1) the three major connexins characteristic for nrCMCs (Figure 6A), 2) cardiac ion channel proteins necessary for the propagation of Na⁺, K⁺, and Ca²⁺ currents (Figure 5 and Figure 6A), and 3) the ability to conduct electricity at high velocities and to repair an artificially created conduction block in vitro (Figure 7), an in-depth electrophys- iological characterization of these cells is required to prove that they truly become excitable.

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Perspectives

Genetic modification of VSFs may provide a means of repairing conduction defects in damaged hearts. However, the transduction of VSFs with proper doses of all genes needed to produce a cardiac action potential represents a formidable technical challenge. Overexpression of myocardin activates an extensive cardiac gene expres- sion program in hVSFs, leading to the production of key sarcomeric and electrical components of CMCs. This coincides with the appearance of electrical conduction in these cells and the restoration of an artificially created cellular conduction block in vi- tro. With further refinements, this technology holds the promise to transdifferentiate VSFs into cardiomyocyte-like cells in situ, thereby potentially improving myocardial mechanical performance and repairing electrical conduction defects.

Acknowledgements

The authors are indebted to Dirk Ypey for critical reading of the manuscript and Binie Klein for supplying the hDFs. We also thank Robert Adelstein, Rob Hoeben, Jun-ichi Miyazaki, Didier Trono, and Tetsuya Yamagata for supplying the smMHC-specific rabbit antiserum, pLV-CMV-IRES-eGFP, pCAGGS, psPAX2, and pSSRα-GATA-4, respectively. Part of this work was financed by the Netherlands Organization for He- alth Research and Development (ZonMw; Grant MKG5942).

References

1. Frangogiannis, N. G. (2006) The mechanistic basis of infarct healing. Antioxid Redox Signal. 8, 1907-1939

2. Sun, Y., Kiani, M. F., Postlethwaite, A. E., and Weber, K. T. (2002) Infarct scar as living tissue.

Basic Res Cardiol. 97, 343-347

3. Swynghedauw, B. (1999) Molecular mechanisms of myocardial remodeling.

Physiol Rev. 79, 215-262

4. Dimmeler, S., Zeiher, A. M., and Schneider, M. D. (2005) Unchain my heart: the scientific founda tions of cardiac repair. J Clin Invest. 115, 572-583

5. de Muinck, E. D., Thompson, C., and Simons, M. (2005) Progress and prospects: cell based regene- rative therapy for cardiovascular disease. Gene Ther. 13, 659-671

6. Chien, K. R. (2006) Lost and found: cardiac stem cell therapy revisited. J Clin Invest 116, 1838-1840 7. Murry, C. E., Reinecke, H., and Pabon, L. M. (2006) Regeneration gaps. Observations on stem cells

and cardiac repair. J Am Coll Cardiol. 47, 1777-1785

8. Brown, R. D., Ambler, S. K., Mitchell, M. D., and Long, C. S. (2005) The cardiac fibroblast: thera- peutic target in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol. 45, 657-687 9. Etzion, S., Barbash, I. M., Feinberg, M. S., Zarin, P., Miller, L., Guetta, E., Holbova, R., Kloner, R. A.,

Kedes, L. H., and Leor, J. (2002) Cellular cardiomyoplasty of cardiac fibroblasts by adenoviral deli-

(23)

very of MyoD ex vivo: an unlimited source of cells for myocardial repair. Circulation. 106, I125-130 10. Kizana, E., Ginn, S. L., Allen, D. G., Ross, D. L., and Alexander, I. E. (2005) Fibroblasts can be gene

tically modified to produce excitable cells capable of electrical coupling. Circulation. 111, 394-398 11. Murry, C. E., Kay, M. A., Bartosek, T., Hauschka, S. D., and Schwartz, S. M. (1996) Muscle differen-

tiation during repair of myocardial necrosis in rats via gene transfer with MyoD. J Clin Invest. 98, 2209-2217

12. Hille, B. (2001) Ion channels of excitable membranes, Sinauer Associates, Inc, Massachusetts, USA 13. Dunwoodie, S. L. (2006) Combinatorial signaling in the heart orchestrates cardiac induction,

lineage specification and chamber formation. Seminars in Cell & Developmental Biology

14. Srivastava, D. (2006) Making or breaking the heart: from lineage determination to morphogenesis.

Cell. 126, 1037-1048

15. Fukushige, T., Brodigan, T. M., Schriefer, L. A., Waterston, R. H., Krause, M. (2006) Defining the transcriptional redundancy of early bodywall muscle development in C. elegans: evidence for an unified theory of animal muscle development. Genes Dev. 20,3395-3406

16. Wang, D.-Z., Chang, P. S., Wang, Z., Sutherland, L., Richardson, J. A., Small, E., Krieg, P. A., and Olson, E. N. (2001) Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell. 105, 851-862

17. Chen, J., Kitchen, C. M., Streb, J. W., and Miano, J. M. (2002) Myocardin: a component of a molecular switch for smooth muscle differentiation. J. Mol. Cell Cardiol. 34, 1345-1356

18. Creemers, E. E., Sutherland, L. B., Oh, J., Barbosa, A. C., Olson, E. N. (2006) Coactivation of MEF2 by the SAP domain proteins myocardin and MASTR. Mol. Cell. 23,83-96

19. van Tuyn, J., Knaan-Shanzer, S., van de Watering, M. J., de Graaf, M., van der Laarse, A., Schalij, M. J., van der Wall, E. E., de Vries, A. A., and Atsma, D. E. (2005) Activation of cardiac and smooth muscle-specific genes in primary human cells after forced expression of human myocardin. Cardio vasc Res. 67, 245-255

20. van Tuyn, J., Atsma, D. E., Winter, E. M., van der Velde-van Dijke, I., Pijnappels, D. A., Bax, N. A., Knaan-Shanzer, S., Gittenberger-de Groot, A. C., Poelmann, R. E., van der Laarse, A., van der Wall, E. E., Schalij, M. J., and de Vries, A. A. (2006) Epicardial cells of human adults can undergo an epi- thelial-to-mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells. 25, 271-278

21. Pijnappels, D. A., Schalij, M. J., van Tuyn, J., Ypey, D. L., de Vries, A. A., van der Wall, E. E., van der Laarse, A., and Atsma, D. E. (2006) Progressive increase in conduction velocity across human me- senchymal stem cells is mediated by enhanced electrical coupling. Cardiovasc Res. 72, 282-291 22. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Efficient selection for high-expression transfec-

tants with a novel eukaryotic vector. Gene. 108, 193-199

23. Vellinga, J., Uil, T. G., de Vrij, J., Rabelink, M. J., Lindholm, L., and Hoeben, R. C. (2006) A system for efficient generation of adenovirus protein IX-producing helper cell lines. J Gene Med. 8, 147-154 24. Donello, J. E., Loeb, J. E., and Hope, T. J. (1998) Woodchuck hepatitis virus contains a tripartite

posttranscriptional regulatory element. J Virol. 72, 5085-5092

25. Graham, F. L., and van der Eb, A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 52, 456-467

26. Pfaffl, M. W., Horgan, G. W., Dempfle, L. (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.

Nucleic Acids Res. 30, e36

(24)

27. Parmacek, M. S., and Solaro, R. J. (2004) Biology of the troponin complex in cardiac myocytes.

Prog Cardiovasc Dis. 47, 159-176

28. Baroldi, G., Silver, M. D., De Maria, R., Parodi, O., and Pellegrini, A. (1997) Lipomatous metaplasia in left ventricular scar. Can J Cardiol. 13, 65-71

29. Winer-Muram, H. T., Tann, M., Aisen, A. M., Ford, L., Jennings, S. G., and Bretz, R. (2004) Com puted tomography demonstration of lipomatous metaplasia of the left ventricle following myocar- dial infarction. J Comput Assist Tomogr. 28, 455-458

30. Camelliti, P., Green, C. R., and Kohl, P. (2006) Structural and functional coupling of cardiac myocytes and fibroblasts. Adv Cardiol. 42, 132-149

31. Mitchell, J. A., Shynlova, O., Langille, B. L., Lye, S. J. (2004) Mechanical stretch and progesterone differentially regulate activator protein-1 transcription factors in primary rat myometrial smooth muscle cells. Am. J. Physiol. 287, E439-E445

32. Reiser, P. J., Westfall, M. V., Schiaffino, S., and Solaro, R. J. (1994) Tension production and thin- filament protein isoforms in developing rat myocardium. Am J Physiol, 267, H1589-1596

33. Huang, X. P., Pi, Y. Q., Lee, K. J., Henkel, A. S., Gregg, R. G., Powers, P. A., and Walker, J. W. (1999) Cardiac troponin I gene knockout: a mouse model of myocardial troponin I deficiency.

Circ. Res. 84, 1-8

34. Takemura, H., Yasui, K., Opthof, T., Niwa, N., Horiba, M., Shimizu, A., Lee, J. K., Honjo, H., Kamiya, K., Ueda, Y., and Kodama, I. (2005) Subtype switching of L-Type Ca 2+ channel from Cav1.3 to Cav1.2 in embryonic murine ventricle. Circ J. 69, 1405-1411

35. Yasui, K., Liu, W., Opthof, T., Kada, K., Lee, J. K., Kamiya, K., and Kodama, I. (2001) I(f) current and spontaneous activity in mouse embryonic ventricular myocytes. Circ Res. 88, 536-542 36. Gonzalez-Sanchez, A., and Bader, D. (1990) In vitro analysis of cardiac progenitor cell differentia-

tion. Dev Biol. 139, 197-209

37. Jain, M. K., Kashiki, S., Hsieh, C. M., Layne, M. D., Yet, S. F., Sibinga, N. E., Chin, M. T., Feinberg, M.W., Woo, I., Maas, R. L., Haber, E., and Lee, M. E. (1998) Embryonic expression suggests an im- portant role for CRP2/SmLIM in the developing cardiovascular system. Circ Res. 83, 980-985 38. Li, L., Miano, J. M., Cserjesi, P., and Olson, E. N. (1996) SM22 alpha, a marker of adult smooth

muscle, is expressed in multiple myogenic lineages during embryogenesis. Circ Res. 78, 188-195 39. Samaha, F. F., Ip, H. S., Morrisey, E. E., Seltzer, J., Tang, Z., Solway, J., and Parmacek, M. S. (1996)

Developmental pattern of expression and genomic organization of the calponin-h1 gene. A con- tractile smooth muscle cell marker. J Biol Chem. 271, 395-403

40. Wu, S. M., Fujiwara, Y., Cibulsky, S. M., Clapham, D. E., Lien, C. L., Schultheiss, T. M., and Orkin, S. H. (2006) Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell. 127, 1137-1150

41. Gaudesius, G., Miragoli, M., Thomas, S. P., and Rohr, S. (2003) Coupling of cardiac electrical acti- vity over extended distances by fibroblasts of cardiac origin. Circ Res. 93, 421-428

42. Miragoli, M., Gaudesius, G., and Rohr, S. (2006) Electrotonic modulation of cardiac impulse conduction by myofibroblasts. Circ Res. 98, 801-810

43. Kizana, E., Ginn, S. L., Smyth, C. M., Boyd, A., Thomas, S. P., Allen, D. G., Ross, D. L., and Alex- ander, I. E. (2006) Fibroblasts modulate cardiomyocyte excitability: implications for cardiac gene therapy. Gene Ther. 13, 1611-1615

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