Arterial delivery of genetically labelled skeletal myoblasts to the murine heart: Long-term survival and phenotypic modification of implanted myoblasts

Cell Transplantation, Vol. 5, No. I, pp. 77-91, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rightr reserved 0890.263.6897/96 $15.00 + .OO

ELSEVIER

0963-6987(95)02016-O

Original Contribution

ARTERIAL DELIVERY OF GENETICALLY LABELLED SKELETAL MYOBLASTS TO THE MURINE HEART: LONG-TERM SURVIVAL AND PHENOTYPIC MODIFICATION OF IMPLANTED MYOBLASTS SHAWN W. ROBINSON,* PETER W. CHO,~ HYAM I. LEVITSKY,~ JEAN L. OLSON,~ RALPH H. HRUBAN,~ MICHAEL A. ACKER,?# AND PAUL D. KESSLER*’ The Peter Belfer Cardiac Laboratory, and #Division

and Departments of *Medicine, tSurgica1 Sciences, $Oncology, and SPathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA, of Cardiac Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

0 Abstract - The ability to replace damaged myocardial tissue with new striated muscle would constitute a major advance in the treatment of diseases that irreversibly injure cardiac muscle cells. The creation of focal grafts of skeletal muscle has been reported following the intramural injection of skeletal myoblasts into both normal and injured myocardium. The goals of this study were to determine whether skeletal myoblast-derived cells can be engrafted into the murine heart following arterial delivery. The murine heart was seeded with genetically labeled C2C12 myoblasts introduced into the arterial circulation of the heart via a transventricular injection. A transventricular injection provided access to the coronary and systemic circulations. Implanted cells were characterized using histochemical staining for P-galactosidase, immunofluorescent staining for muscle-specific antigens, and electron microscopy. Initially the injected cells were observed entrapped in myocardial capillaries. One week after injection myoblasts were present in the myocardial interstitium and were largely absent from the myocardial capillary bed. Implanted cells underwent myogenic development, characterized by the expression of a fast-twitch skeletal muscle sarcoendoplasmic reticulum calcium ATPase (SERCAl) and formation of myofilaments. Four months following injection myoblast-derived cells began to express a slow-twitch/ cardiac protein, phospholamban, that is normally not expressed by C2C12 cells in vitro. Most surprisingly, regions of close apposition between LacZ labeled cells and native cardiomyocytes contained structures that resembled desmosomes, fascia adherens junctions, and gap junctions. The cardiac gap junction protein, connexin43, was localized to

ACCEPTED

817195.

This work supported by The Muscular Dystrophy Association (P.D.K.), Clinical Research Grant 94-043 1 from the March of Dimes Birth Defects Foundation (P.D.K.), Harriet and David Finkelstein Collaborative Heart Disease Grant (M.A.A. and P.D.K.). and National Institute of Health Grants T32 HL-07227 (S.W.R.), and Kll HL-02379 (P.D.K.). S.W.R. is a recipient of a Robert Wood Johnson Foundation Minority Medical Faculty

some of the interfaces between implanted cells and cardiomyocytes. Collectively, these findings suggest that arterially delivered myoblasts can be engrafted into the heart, and that prolonged residence in the myocardium may alter the phenotype of these skeletal muscle-derived cells. Further studies are necessary to determine whether arterial delivery of skeletal myoblasts can be developed as treatment for myocardial dysfunction. 0 Keywords Phospholamban;

Myoblast SERCAl.

transfer;

Cardiomyoplasty;

INTRODUCTION

Cardiac and skeletal muscle differ in a number of fundamental properties, including morphology, mechanical characteristics, mechanism of excitation-contraction coupling, embryological origin, and response to injury. It is generally accepted that cardiac muscle cells are incapable of regeneration (reviewed in Ref. 16), whereas repair of skeletal muscle can occur after injury. The adult complement of cardiomyocytes is attained soon after birth, when cardiomyocytes withdraw from the cell cycle. Stem cells, or committed cardiac precursor cells, have not been identified in the adult mammalian heart. When cardiac tissue of an adult mammal is irreversibly injured, the cardiomyocytes do not re-enter the cell cycle (16). If a significant number of contractile cells are damDevelopment Fellowship. Presented in part at the 66th Annual Scientific Sessions, American Heart Association, November 1Oth, 1993. ‘Address correspondence to Paul D. Kessler, Johns Hopkins University School of Medicine, Ross Research Room 812, 720 Rutland Avenue, Baltimore. [email protected].

MD 21205. E-mail:

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aged, myocardial dysfunction ensues. Like the nuclei of cardiomyocytes, the multiple nuclei of mature skeletal muscle fibers are incapable of reentering the cell cycle. However, 1 to 5% of the nuclei in adult skeletal muscle belong to adult myoblasts, called satellite cells (35). These mitotically quiescent cells are located beneath the basal lamina of muscle fibers, and when triggered contribute to muscle growth, repair, and regeneration via fusion with established muscle fibers and de novo fusion. Methods for the isolation and transduction of myoblasts have been well described. Myoblast implantation, via direct injection into skeletal muscle, has been utilized as a potential therapy for genetic diseases of muscle (reviewed in Refs. 6 and 8) and as a vehicle for the delivery of recombinant proteins (4,13,17). In these examples, implanted myoblasts are incorporated into host skeletal muscle fibers. Similarly, the direct intramural implantation of skeletal myoblasts into normal and cryoinjured myocardium has also been reported (31,34,52). These myoblast grafts underwent differentiation and survived for periods of at least 3 mo in normal myocardium (31), and up to 8 wk in cryoinjured heart tissue (34,52). Recently, Field and coworkers (32) have demonstrated that intramyocardial grafts can be utilized to deliver a secreted protein directly to the myocardium. However, the likely need for multiple injections, coupled with the relative inaccessibility of the heart, are important limitations for the possible clinical applications of this strategy (6). Neumeyer et al. (39) recently reported the successful arterial delivery of L6 skeletal myoblasts to rat skeletal muscle. Arterial delivery of skeletal myoblasts to the myocardium possesses several theoretical advantages over their direct infiltration in the myocardium, most notably a less invasive approach to their introduction in the heart. The feasibility of this approach was suggested by the potential migratory properties of adult skeletal myoblasts, which retain their ability to traverse the basal lamina of their associated muscle fiber and migrate short distances to fuse with adjacent fibers (23). We report that stable engraftment of skeletal myoblasts into the myocardium can be achieved by delivery of myoblasts via the coronary circulation. The clonal myogenic cell line, C2C12, was genetically marked to express bacterial P-galactosidase and served as the donor cells (7). These cells have been widely used to analyze vertebrate myogenesis and more recently their use in a number of cell transplantation studies have identified myoblasts as an effective cellular vehicle for the delivery of recombinant proteins (4,6,17). Following mitogen withdrawal proliferating C2C12 cells exit from the cell cycle, fuse, and express muscle-specific proteins, recapitulating myogenic development in vivo. The fasttwitch skeletal muscle sarco-endoplasmic reticulum cal-

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cium ATPase (SERCAl) was used to identify C2C12 cells that had undergone myogenic development. This protein is not expressed in cardiac tissue or C2C12 myoblasts, but its expression is induced within 72 h after withdrawal of serum from the medium (19,29). Our experiments were designed to determine whether arterially delivered myoblasts could incorporate into the myocardium and form striated muscle. Both myoblast-intrinsic factors and environmental cues define the biochemical characteristics of skeletal muscle fibers formed in repair processes that involve adult myoblasts (reviewed in Ref. 8). We therefore also sought to discover whether or not cues originating in the cardiac milieu could modify the developmental program of the implanted cells. Finally, we evaluated these implanted cells at the ultrastructural level to confirm their residence outside of the capillary bed and to begin to characterize the structural interactions between cardiomyocytes and the myoblast-derived cells.

MATERIALS

AND METHODS

Genetically Lubelled Cell Lines C2-MFGlacZ. C2C12 myoblasts (7) were obtained from the American Type Culture Collection (CRL 1772). C2C12 myoblasts and C2-MFGlacZ transductants, 5 x lo5 cells, were grown in 10 mL of growth medium [Dulbecco’s Modified Essential Medium (DMEM) with 20% fetal bovine serum (FBS), 1% chick embryo extract (Gibco BRL), 5 kg/mL gentamicin] or modified differentiation medium (DMEM, 5% FBS, 5 bg/mL gentamitin). The replication-defective retroviral vector system MFG (14,25), a gift of R. Mulligan, was used to genetically mark C2C12 myoblasts with E. coli P-galactosidase, encoded by the LacZ gene. This vector lacks a selectable marker and mediates high-level integration and expression of transduced genes (17,25). Recombinant LacZ encoding virus was harvested from $-CRIP packaging cell lines (14) and C2C12 myoblasts were transduced as described by Jaffee and colleagues (25). Forty-eight hours prior to transduction, +-CRIP producer cells (+CRIP MFGlacZ) were plated at a density of 2 x lo6 cells per 100 mm culture dish in DMEM plus 10% FBS and grown in 10% CO, at 37°C. On the day prior to transduction the medium was replaced with fresh medium. The overnight culture supernatant was collected and filtered through a 0.45 km filter (Acrodisc, Gelman Sciences). Twenty four hours prior to retroviral infection, 5 x lo5 C2C12 myoblasts were plated in growth medium. The myoblast monolayer was exposed to the retroviral mixture supplemented with 50 pg/mL DOTAP (Boehringer Mannheim) for 3 h, and replaced with growth medium. Three days after transduction, approxi-

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mately 40% of the myoblasts expressed B-galactosidase. The C2-MFGlacZ cell line was isolated by limiting dilution cloning. The C2-MFGlacZ isolate screened negative for infectious B-galactosidase encoding helper virus using an adherence assay (4). C2-Zn Cell Line. C2-Zn cell line was a gift of E. Ralston (41). This cell line was produced by transfection and stable selection of plasmid DNA encoding LacZ fused with nucleotides that encode the hormoneindependent nuclear localization domain (amino acids 497-524) of the rat glucocorticoid receptor. Cells were grown at low density (5 x lo5 cells/l0 mL) in growth medium supplemented with 200 pg/mL G418 (Genetitin, Gibco BRL) or differentiation medium (DMEM, 2% horse serum). Myoblast Injections Male C3H mice, aged 8 to 36 wk, were used for this study. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH) publication No. 85-23, revised 1985. Freshly trypsinized C2-MFGlacZ, or C2-Zn myoblasts, were washed twice and resuspended at a concentration of 1 x IO’ cells/ml in serum-free DMEM. Mice were anesthetized with inhaled methoxyflurane, and the heart was exposed via a 1 cm left thoracotomy incision. Under direct vision, 0.1 mL of cell suspension was bolus injected into the left ventricular cavity using a 30 gauge needle attached to a Hamilton syringe. Blood was aspirated into the syringe, prior to injection, to confirm placement within the ventricular cavity. The thoracotomy was closed with silk sutures and animals were returned to their cages and observed for evidence of infection or distress. The procedure was performed without endotracheal intubation. The operative mortality was lo%, primarily related to respiratory insufficiency or bleeding. Lethal generalized seizures were observed in two animals upon emergence from anesthesia, and this was likely due to cerebral embolization. No mortality was observed beyond the immediate postoperative period. At intervals of 1 to 27 wk after the initial procedure, inhaled methoxyflurane was administered and when the mice became areflexic the hearts were removed, washed, weighed, and divided for processing. Sixty-five animals were studied after injection with genetically labelled myoblasts, and seven animals were studied after sham injections of 1 x lo6 UV-irradiated C2-MFGlacZ cells (N = 3), or conditioned media from C2-MFGlacZ cultures (N = 4). In Situ Histochemical Staining for (3-Galactosidase Activity Three-millimeter sections of cardiac tissues were fixed in 10% formaldehyde in phosphate-buffered saline, and

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B-galactosidase activity was determined as described (45), with development of the X-gal precipitate overnight at 30°C to minimize diffusion of the LacZ reaction product (9). Three randomly selected 3-mm thick samples of tissue from each animal were dehydrated with graded ethanol, embedded in paraffin, and 10 p,rn sections were stained with hematoxylin-eosin. Hearts were assigned to LacZ-positive groups if blue cells were observed after light microscopic examination of 20 lo-p,rn sections.

Estimates of the Number of LucZ Positive Cells in the Heart We estimated the number of labelled myoblasts present in the heart immediately, and 1 wk after injection using a method that had been validated for the murine transventricular injection model (50). Fifteen consecutive 20 pm X-gal stained test sections were prepared from a randomly chosen section of tissue and photographed. The number of blue-stained nuclei in these 15 sections was determined and the volume of these sections was calculated as the product of thickness and planimetered area, with appropriate correction for magnification. The total number of LacZ-positive cells contained in the whole heart was estimated as: [Number of LacZpositive nuclei in test sections/Calculated volume of test sections] x [Total volume of heart (extrapolated from the wet weight of the heart)]. Estimates of cell count reported represent pooled data from five and three animals sacrificed immediately and 1 wk after intraventricular injections, respectively.

Immunofluorescence Microscopy Hearts were frozen in melting isopentane/dry ice in Tissue-Tek (Miles, Inc.) and 5 Frn cryosections were placed on subbed slides and stored at -70°C until use. Immunofluorescence procedures were performed according to Kaprielian and Fambrough (29) except that unless noted, all incubations were performed in sodiumfree buffers (11). The frozen sections were fixed in 4% formaldehyde for 10 min, quenched in 5 mg/mL lysine, and incubated with fluorochrome-conjugated antibodies (Ab) in 0.25% triton X-100, 0.25% gelatin. Fluorescein or rhodamine fluorochromes were directly coupled to mouse monoclonal antibodies (mAbs) Al (antiphospholamban; Ref. 47), lOD1 (anti- SERCAl; Ref. 29), using NHS-rhodamine [(5- and 6)-carboxytetramethylrhodamine, succinimidyl ester] or NHSfluorescein [5-(and 6)carboxyfluorescein, succinimidyl ester] according to manufacturers directions (Pierce Chemical). For immunodetection of LacZ, fixed sections were sequentially probed with rabbit anti-bacterial B-galactosidase IgG (5 Prime 3’) and fluorescein-conjugated goat anti-rabbit IgG. For the detection of connexin43, an

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antipeptide antiserum (5) raised in a rabbit against amino acids 252-271 of rat connexin43 was used at a 1:250 dilution (a gift of E. Beyer). Conjugated and unconjugated primary Abs were used at a concentration of 10 pg/mL or 5 pg/mL, respectively. For incubations using mAbs A4.1025 (anti-myosin heavy chain; Ref. 49) we used a method described by Fung et al. designed to detect antigens in murine tissue using unconjugated mouse mAbs (20). Antibody complexes, consisting of primary antibody-fluorescinated secondary Ab complexes were generated as described (20). Tissue sections were preincubated in mouse serum and incubated with the complexed antibodies overnight at 4°C. Nonspecific background staining was negligible with this technique. Tissue sections were examined with a Zeiss Axioskop microscope fitted for epifluorescence microscopy and photographed with Kodak Tungsten 64 or Kodachrome 400 films. Confocal microscopy was performed using a Bio-Rad MRC-600 confocal microscope and images were printed with a Mitsubishi video printer. Electron Microscopy Hearts from animals euthanized at various time points after injection were cut into 3 mm sections, fixed in 2.5% glutaraldehyde, and processed for B-galactosidase activity as described above. X-gal stained samples were cut to less than 1 mm thickness, washed in 0.1 mol/L phosphate buffer, postfixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer, dehydrated in graded ethanol, and infiltrated and embedded with Araldite (Polysciences). One pm sections were cut with glass knives on a Sorval MT-2 microtome and counterstained with Fuchsin red. LacZ-positive cells remained blue following processing and were identified with light microscopy. Corresponding thin (60-90 nm) sections were cut with a diamond knife, mounted on 200 mesh copper grids, poststained in saturated uranyl acetate and modified lead citrate, and examined with a Philips CM12 electron microscope at 60 kV. RESULTS

Localization of Arterially Delivered Myoblasts in the Myocardial Interstitium To identify injected skeletal myoblasts unambiguously, C2C12 cells were genetically marked to express B-galactosidase either in the nucleus or the cytoplasm. We reasoned that cytoplasmically expressed LacZ would identify the borders of implanted cells and thus define their shape and cell-cell interfaces. Alternatively, nuclear-localized LacZ would help demonstrate multinucleate fiber formation of myoblast-derived cells. The expression of cytoplasmic or nuclear-targeted B-galactosidase did not alter myogenic development in vitro (shown in Fig. 1). Following mitogen withdrawal, both

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genetically labelled cell lines could be induced to undergo myodifferentiation and form multinucleated myotubes (Fig. 1C and lD), that express SERCAl (19). This protein is not expressed in cardiac muscle or C2C12 myoblasts, yet is expressed in C2C 12 myotubes and fasttwitch skeletal muscle (19,29). One million C2-MFGlacZ or C2-Zn myoblasts were injected into their syngeneic host, the C3H mouse (7). Within minutes of the injection, myoblasts were present throughout the left and right coronary artery distributions where they appeared to be entrapped to the lumina of small capillaries (Fig. 2A and 2B). Within 1 wk after injection, cells were no longer in capillaries and instead they appeared to be integrated into the myocardial interstitium (Fig. 2C), often as multinucleated fibers that aligned with the cardiac fiber axis (Fig. 2D and 2E). C2-MFGlacZ cells stained in situ showed a uniformly blue cytoplasm, which generally respected architectural borders (Fig. 2C). Histological staining for LacZ activity revealed occasional LacZ-positive blue-stained cells in mediastinal lymph nodes (Fig. 2F), liver, spleen, and rarely in the kidneys and lungs. As kidney has a high lysosomal B-galactosidase activity, this tissue was screened for genetically labelled cells with immunofluorescent microscopy, using an antibody to E. Coli B-galactosidase. Initial evaluations of the brain and skeletal muscle failed to identify LacZ-positive cells and thus systematic surveys of these tissues were not performed. The presence of myoblasts in visceral organs, and the characteristically dispersed appearance of arterially delivered myoblasts in tissue stained for B-galactosidase activity 16 wk after injection suggests that an inadvertent intramural injection did not occur (Fig. 2G). A comparable section of uninjected mouse heart is shown for comparison in Fig. 2H. Histological evidence of myocardial thrombosis or infarction was not identified. Lymphocytes and macrophages were occasionally seen near LacZ-positive cells. A 0.1 mm LacZ-positive tumor, raised above the epicardial surface without extension into the myocardium, was observed in one animal that received C2-MFGlacZ cells. Tumors were not observed upon gross inspection of thoracic or abdominal viscera. The specificity of the histochemical staining in cardiac tissue was reconfirmed by immunostaining with an antibody to B-galactosidase. LacZ positive cells were present in 47 of 65 experimental animals (72%) at periods up to 6 mo following injection (Table 1). The hearts of seven sham-injected animals were devoid of B-galactosidase activity. All animals injected with genetically labelled myoblasts that we have studied are included in this table. Note that animals injected with C2-Zn cells and studied at 4 and 5 wk were assigned to the LacZnegative group. As these were the first animals studied this likely reflects our inexperience with the protocol.

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A

Fig. 1. Development of C2-MFGlacZ and C2-Zn cells in vitro. (A and C) C2-Zn cells grown on cover slips in (A) growth medium or (C) differentiation medium and processed for LacZ histochemistry. The P-galactosidase fusion protein remains localized to the nucleus. (B and D) C2-MFGlacZ cells were plated on cover slips and incubated with (B) growth or (D) modified differentiation medium for 48 h and processed as above. There is formation of myotubes in modified differentiation medium. Scale bar 20 km.

We used a previously validated method to estimate the number of myoblasts remaining in the murine heart (50). Approximately 50,000 myoblasts, about 5% of the cells injected into the left ventricular cavity, were present in the heart immediately after injection. This is in agreement with the number of cells expected to partition to the coronary circulation (50). One week after the injection, we estimate that approximately 700 LacZ-positive cells were present in the heart. The cell counting method that was used to estimate the number of cells retained is only an approximation. We therefore did not estimate the number of implanted cells at each time point, and instead concentrated our efforts in describing the properties of implanted myoblasts.

Expression of a Fast-Twitch Skeletal Muscle Protein, SERCAI To determine whether the injected cells undergo myogenie development in situ, cardiac sections were stained with mAb lOD1 (29) that recognizes the isoform of the sarcoplasmic reticulum calcium ATPase (SERCA 1) ex-

pressed in C2C12 myotubes (multinucleate, differentiated muscle cells), but is not expressed in cardiac tissue or C2C12 myoblasts. Frozen tissue sections were processed for LacZ activity, and when P-galactosidase expressing cells were identified, the adjacent section was processed for immunofluorescence microscopy. Immediately after injection, cells expressing SERCAl were not identified. Three days following injection, mononuclear and multinucleate cells positive for P-galactosidase expression also stained with the anti-SERCAl mAb lOD1. One week following injection, all LacZ-positive cells expressed SERCAl. As illustrated in Fig. 3G, the expression of SERCAl in implanted cells was observed for at least 6 mo following implantation. Although immunofluorescence microscopy is not quantitative, micrographs obtained at these later time points suggest that the level of SERCAl protein in myoblast-derived cells may be reduced following prolonged residence in the heart. A consistent finding was that LacZ-positive cells located in other tissues did not express SERCAl. Visceral abdominal organs of eight animals sacrificed at 3 days to 6 mo were processed for immunostaining using mAbs

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Table 1. Numbers and percents of mice expressing

LacZ in the myocardium at different times after myoblast injection Cell Line Injected

Weeks in vivo Immediately 3 days 5 days 1 weeks 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 8 weeks 10 weeks 12 weeks 16 weeks 20 weeks 21 weeks Total

C2-Zn 516 212 212 5/l O/l 314 O/6 o/3 l/l l/l l/l l/l 212 212 2.5/39 (64%)

C2-MFGlacZ 414 214 l/l 414 213 213 313 l/l 212 l/l 22/26 (85%)

C2-Zn or C2-MFGlacZ were injected intraventricularly into the heart of syngeneic male C3H mice. After the period (weeks in vivo) shown, hearts were removed and processed for LacZ histochemistry. Twenty consecutive 10 pm sections were analyzed and mice were assigned to the LacZ positive group if blue cells were observed. All data entries are represented as number of LacZ-positive animals/number of animals processed for histochemistry.

against myosin heavy chain (mAb A4.1025; Ref. 49) and SERCAl using immunofluorescent techniques. The adjacent section of tissue was processed for B-galactosidase activity and revealed only mononuclear LacZpositive cells. There was no coexpression of musclespecific markers in these LacZ positive cells. A mediastinal lymph node is shown 6 mo after injection that contains many mononuclear cells that express B-galactosidase (Fig. 2F).

Co-Expression of the Slow-Twitch/Cardiac Protein Phospholamban in Implanted Striated Muscle Nerve and hormonal influences can alter the phenotype of muscle (27,40). Chronic electrical stimulation of fast-twitch skeletal muscle induces the expression of slow-twitch isoforms of contractile and sarcoplasmic reticulum (SR) proteins, including myosin heavy chain,

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calcium ATPase (SERCA2a) and the slow-twitch/ cardiac protein phospholamban (1,10,22,33). These proteins may determine the slower contraction and relaxation rates of slow-twitch skeletal and cardiac muscle fibers. To determine whether the cardiac environment altered the implanted cells, we probed tissue with mAb Al that is specific for mammalian phospholamban (47). Cardiac sections were processed for immunofluorescence microscopy with fluorophore-conjugated anti-phospholamban mAb and anti-SERCAl mAb. Phospholamban expression was initially restricted to host cardiac cells (Fig. 3B and 3D). However, starting at 4 mo after the injection, some myoblast-derived cells co-expressed the integral SR membrane protein phospholamban (Fig. 3F and 3H) and the skeletal muscle SR marker, SERCAl (Fig. 3E and 3G). We have never observed phospholamban expression in genetically labelled C2C12 myoblasts or myotubes in vitro. However, C2C12 myotubes could only be maintained in cell culture in our laboratory for periods up to 2 mo, because spontaneous contractile activity would eventually cause the cells to lift off the culture stratum. Nevertheless, the induction of phospholamban expression in situ suggests that some aspect of the cardiac milieu may have altered the developmental program of the implanted cells.

Expression of a Cardiac Gap Junction Protein, Connexin43 Normally, cardiac cells are electrically coupled to adjacent cells via specialized gap junctions, composed of the hexamers of the protein connexin43 (5), that allow the exchange of ions and small molecules between adjacent cells. Gap junctions are found within the intercalated disk and at sites of side-to-side contact between cardiac cells (30,37). Gap junctions are found in virtually every cell type in mammals. A notable exception is adult skeletal muscle, where myofibers do not form gap junctions with neighboring myofibers. However, gap junctions have been identified in developing avian and rodent muscle fibers and primary cultures of these cells (28,42,43), and connexin43 has been identified in cultured L6 myoblasts prior to fusion (3). We used an an-

Fig. 2. In situ histochemical staining for B-galactosidase activity in murine myocardium. (A-E) Cardiac sections were processed for B-galactosidase activity, embedded in paraffin, and 10 pm sections were counterstained with hematoxylin-eosin, except (B) which is without counterstain. Tissue was processed immediately (A,B), 1 wk (C), 3 wk (D), or 27 wk (E) after injection of genetically labeled cells. C2-MFGlacZ cells are shown in Panels A and C; C2-Zn cells are shown in Panels B, D, and E. The arrows in E denote LacZ-positive nuclei. Note the myoblast-derived myotube is aligned in parallel to the myocardial fiber. (F) Paraffin stained section of a mediastinal lymph node, 27 wk after the injection of C2-Zn cells. Lung tissue is shown in the right hand portion of the micrograph. Many B-galactosidase expressing cells are shown within the lymph node, best appreciated at the lower left hand aspect of the pane1 (G and H). Low magnification micrograph of thick (3 mm) sections of X-gal stained myocardial tissue. (G) 16 wk after injection of C2- MFGlacZ cells. Note the presence of blue-stained structures throughout the field. (H) Sample of control heart negative for X-gal staining. Scale bar 20 pm A-F, 500 pm G and H.

Fig. 3. Expression of SERCAI and phospholamban in situ. Tissue sections were prepared from animals injected with C2Cl2 myoblasts that express bacterial B-galactosidase. Sections were incubated with fluorescein-conjugated mAb lODl(anti-SERCAl) or rhodamine-conjugated mAb A 1(anti-phospholamban). Paired images of the identical field viewed through rhodamine or fluorescein filter sets are shown. (A and B) and (C and D) Paired images obtained 3 wk after implantation. The myoblast-derived cells (white arrowheads) do not co-express phospholamban. (E and F) and (G and H) Micrographs show paired fields obtained five (E and F) and 6 mo (G and H) after myoblast injection. The myoblast-derived cells (white arrowheads) co-express phospholamban. Other cells in E and G exhibit only background fluorescence. Scale bar 20 pm.

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tiserum raised against connexin43 to characterize the region of cell contact. A punctate linear pattern of staining at apparent cardiomyocyte-cardiomyocyte interfaces was observed throughout the field (arrows, Fig. 4A) and is characteristic of connexin43 immunofluorescence in the murine heart (21). Additionally, we identified connexin43 at some of the interfacial surfaces between skeletal muscle derived cells and cardiac cells, shown in Fig. 4A and 4B. Connexin43 was not identified between some cell pairs, but when present could be identified as early as 3 days and up to 6 mo postimplantation.

Fine Structure of Myoblast-Derived Fibers To determine the ultrastructure of implanted cells, genetically labelled myoblasts were identified in situ with transmission electron microscopy (TEM). The X-gal reaction product, which appears blue when visualized with light microscopy, forms a crystalloid deposit that can be identified under the electron microscope (9,38,46). Tissue sections were prepared for histochemical visualization of LacZ and processed for TEM. The 5-bromo-4chloro-3-indole reaction product forms an electron-dense crystalloid precipitate that is predominantly associated with the nuclear membrane in cells expressing nuclear localized LacZ (9,38,46). In cells that express the cytoplasmically localized LacZ, the crystalloid forms inclusions that are free in the cytoplasm (38). The micrographs in Fig. 5A and 5B show a C2-Zn cell in situ 3 wk following injection. Typical crystalloid inclusions, identical to those described (9,38,46) are found in patches associated with the outer layer of the nuclear envelope (large black arrows) and the cytoplasm (small black arrows), which identify it as a C2-Zn cell. Myofilaments have formed with ordered sarcomeric repeats signifying the differentiation of implanted cells. A myoblast-derived cell adjacent to a cardiomyocyte is shown in Fig. 5B. Typical crystalloid inclusions, associated with the nucleus (large black arrows) and cytoplasm (small black arrows) are observed, which are distinct from glycogen inclusions or endogenous organelles, such as transverse tubules. At 3 wk after implantation, mitochondria (m) were consistently larger in the cardiomyocytes, while the myoblast-derived cells contained small mitochondria (curved white arrow labeled m) and somewhat less-ordered myofilaments. In contrast to C2-Zn cells, crystalloid material was found in the cytoplasm of C2-MFGlacZ derived cells. Implanted cells are detected 5 mo after injection in Fig. 6. It is interesting to note that at these later intervals, the mitochondria in myoblast-derived cells appear similar in size to the mitochondria contained in cardiomyocytes, as illustrated in Fig. 6F. For implanted cells to contribute to mechanical func-

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tion they must couple to surrounding cardiac tissue. In cardiac muscle the intercalated disk serves not only to mechanically tether a cardiac cell to its neighbors but it also contains specialized gap junctions that serve to electrically couple adjoining cardiac cells. Along an intercalated disk, fascia adherens junctions and desmosomes are regions of durable cell-cell connections (30,37). In Fig. 5C, a myoblast-derived cell adherent to a cardiomyocyte is shown 3 wk after injection. The myoblast-derived cell is identified by the 5-bromo-4-chloro-3-indole reaction product (small black arrows). Electron-dense thickenings of the cytoplasmic face of the sarcolemmal membranes (white straight arrows), consistent with spot desmosomal junctions, are shown at the interface between the two cell types in Fig. 5C. Importantly, the density and size of mitochondria, labeled m, appear to differ between the myoblast-derived cell and cardiomyocyte. In Fig. 5D, a portion of the interface adjacent to that in Fig. 5C is shown. Note an attachment of the LacZ-positive cell to the cardiomyocyte appears to be either forming or receding. Structures that appear similar to an intercalated disk (30,37) were occasionally observed between the two cell types. Examples are shown in the electron micrographs of Fig. 6B and 6C. As noted above, connexin43 was immunolocalized to some of the interfaces between the two cell types. To search for gap junctions, multiple thin sections adjacent to that shown in Fig. 6C were prepared and analyzed with TEM. Figure 6E and 6F shows a structure, rarely observed at the interface of the two different cell types, that has the appearance of a gap junction. We were not able to detect junctions formed by the other labeled cell (#4) in Fig. 6A.

DISCUSSION

The use of genetically labelled cell lines for intravascular delivery has provided a means to address issues of particular interest to the field of myoblast transfer. The genetic labeling of donor cells offers a distinct advantage in definitive detection and was used by Soonpaa et al. (46) to demonstrate coupling of fetal cardiomyocyte grafts with host myocardium. The ability to visualize C2C 12 cells following arterial delivery provides a unique approach to define the migratory capacity of skeletal myoblasts. Second, the characterization of single implanted skeletal myoblasts, surrounded by cardiomyocytes, offers potential insights into the relative contributions of myoblast-intrinsic factors vs. cardiac environmental cues toward the eventual direction of myocyte development. Finally, the development of a method of delivering skeletal myoblasts to the heart may generate novel alternatives for myoblast transfer.

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Although this approach of searching for individual cells was tedious and labor intensive, it allowed us to identify single cells within relatively intact tissue, and may have enhanced our ability to demonstrate their integration into the myocardium. Myoblasts are capable of limited migration (23). However, there is some disagreement regarding their ability to cross intact cell barriers (18). Although satellite cells do not normally migrate into the bloodstream (36) we have demonstrated C2C12 myoblasts are able to survive in the microcirculation, and may migrate across intact basement membrane into the myocardial interstitium. Our results, and those of Neumeyer et al. (39), suggest that rodent myogenie cells are capable of traversing the capillary bed. We predicted that myoblasts introduced into the arterial circulation of the heart would behave similarly to the nearest physiological model, the intraventricular injection of tumor cells (50,51). Following a transventricular injection, B16 murine melanoma cells have been shown to arrest in the coronary capillaries, where they undergo lysis within 5 min (50). It has been emphasized that systolic obliteration of entrapped cells leads to their poor survival (50,51). In our studies LacZ-labelled cells were initially present in the capillary bed. However, after 1 wk the surviving LacZ-labelled myoblasts were predominantly localized to the myocardial interstitium. We estimate that about 700 myoblasts remain in the heart at 1 wk following transventricular injection. The mechanism of the translocation of myoblasts from the vasculature into the myocardial interstitium is unknown. Although we did not detect histological evidence of a sustained ischemic insult, transient ischemia related to capillary plugging may have rendered the capillary bed more suitable for myoblast egress. Clearly, insights into

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the mechanism of translocation may enhance arterial cell transfer. The trigger for myogenic development remains undefined. In cultured myogenic cell lines the withdrawal of mitogens is sufficient to induce fusion of myoblasts to form myotubes. Although it is a formal possibility that myogenic development is initiated while the cells are in the circulatory bed, it seems likely, based on the above arguments, that the myoblast represents the migratory species, and differentiation occurs after cells exit the vasculature. Koh et al. (31) demonstrated that C2C12 myoblasts are capable of undergoing myogenic development within the heart. In our study, differentiated cells, identified by the expression of the fast-twitch skeletal muscle-specific protein, SERCAl , were observed as early as 3 days after implantation. One week after injection all LacZ-positive cells expressed SERCAl. Interestingly, myoblasts located in nonmyogenic tissue did not undergo biochemical differentiation. In comparison, Jiao et al. (26) demonstrated that developed skeletal muscle could be sustained in a nonmyogenic environment. These workers injected myoblasts and myotubes simultaneously into the brain; therefore it is unclear whether de novo development occurred. Our results suggest that development of myoblasts in extracardiac sites is an uncommon occurrence following transventricular injection. Myotubes formed from the fusion of C2C12 myoblasts display spontaneous contractile activity. If this potential to perform work is to be exploited, engrafted skeletal muscle fibers must be mechanically and electrically coupled to the surrounding myocardium. The first criterion of mechanical coupling may be met by our identification of structures between cardiomyocytes and the

Fig. 4. Expression of connexin43 in skeletal muscle-derived cells. Tissue was processed for immunofluorescence microscopy 4 wk after injection. Frozen 5 pm sections were serially incubated in mAb anti-SERCAl, rhodamine-labeled goat-anti-mouse IgG, connexin43 antisera, and fluorescein conjugated goat-anti-rabbit IgG. (A) Image obtained on a Zeiss Axioskop epifluorescence microscope. Use of long pass filter produces superimposed fluorescein and rhodamine images. One skeletal myocyte in field is identified by anti-SERCAl staining. White arrows indicate connexin43 staining between cardiomyocytes. Note connexin43 staining at interfaces of skeletal myocyte and neighboring cardiomyocytes. Scale bar 20 pm. (B) Same field as A, but magnified and imaged using a Bio-Rad MRC-600 confocal microscope. Image is pseudocolored with green for fluorescein channel, and red for rhodamine channel. Scale bar 10 pm.

Fig. 6. Region of close membrane apposition between implanted cardiomyocytes and myoblast-derived cells, 5 mo after implantation. (A) One micron section stained with Fuchsin red. LacZ-positive cells remain blue after processing (cells labeled 2 and 4). (B) Low-magnification micrograph of thin section corresponding to that shown in (A). Cells labeled 2 and 4 are myoblast derived. Boxed area shows the region where Cell 2 abuts with a cardiomyocyte. Red blood cells within the capillary lumen (R and r) are marked to orient panels C and D. (C) Section is adjacent to that shown in Panel B. Cell number 2 is identified by crystalloid precipitate in cytoplasm (small black arrows). Large black arrows show a structure that resembles an intercalated disk. A red blood cell within the capillary (R) is marked to provide orientation. (D) High magnification view of Cell 4 shows crystalloid precipitate in cytoplasm (small black arrows), and identifies the cell as myoblast derived. (E) Section adjacent to that shown in Panel F. A structure that resembles a gap junction is shown (white arrow). Small black and white arrows show crystalloid precipitate. (F) High magnification of a portion of the boxed region in Panel A. Cell 2 is labeled with cytoplasmic precipitate (small black arrows). Note there is a small amount of precipitate that is localized at the extreme edge of the cardiomyocyte, likely due to diffusion of the precipitate during processing. Structures resembling an intercalated disk and gap junction are denoted by the large black arrows. Scale bar 1 pm.

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Fig. 5. Electron micrograph of blue cells in murine ventricular tissue. Cells were injected microscopic visualization of the 5-bromo-4-chloro-3-indole crystalloid precipitate. (A and injection. (A) The X-gal precipitate is present in the perinuclear region (large black arrows) Note the formation of myofibrils. (B) A myoblast-derived cell (M) and cardiomyocyte (C) (large black arrow) and the cytoplasmic precipitate (small black arrows) identifies the

into C3H mice and processed for electron B) C2-Zn myoblasts are shown 3 wk after and in the cytoplasm (small black arrows). are juxtaposed. The perinuclear precipitate

cell as myoblast derived. Nucleus of the myoblast-derived cell (N). Note the difference in mitochondrial (m) sizes between cell types. (C and D). Sections prepared from an animal injected with C2-Zn cells and sacrificed 3 wk after injection. (C) A myoblast-derived cell (M) and cardiomyocyte (C) is shown. A second myoblast-derived cell is in the upper left-hand region of the micrograph. The process of an interstitial cell is shown in the gap between the two myoblast-derived cells. Black arrows denote crystalloid inclusions. The formation of spot desmosomes is shown at points of contact between the cell types (white arrows). Mitochondria (m) are shown. (D) A spot desmosome is shown where the process of a myoblast-derived cell (M) shown in Panel 5C contacts a cardiomyocyte (C). Black arrows show crystalloid inclusions, and small black triangles show typical cytoplasmic glycogen inclusions. Scale bar 1 pm.

engrafted skeletal muscle cells that resemble classical mechanical junctions. In fact, mechanical coupling between dissimilar muscle types is not a novel observation. Terasaki et al. (48) have identified desmosomes and fascia adherens junctions between cardiac cells and subendocardial smooth muscle cells in intact ovine myocardium. The second criterion of electrical coupling is difficult to prove. Putative gap junctions have been identified between host myocardium and fetal cardiomyocyte implants (46), but were not detected when unlabeled C2C 12 myoblasts were directly implanted into the ventricular muscle (31). In Fig. 6E and 6F we show struc-

tures that resemble a gap junction found, albeit rarely, between P-galactosidase labeled cells and cardiomyoctes. Equally intriguing is the immunofluorescence localization of the gap junction protein, connexin43, at some sites of donor and host cell contact. This protein presumably forms a channel that allows small molecules to pass between cells. Cells that are coupled via gap junctions form a functional syncytium. Whether the connexin43 component was contributed by the myoblastderived cell remains obscure. However, myoblasts express connexin43 prior to fusion (3), and connexin40 has been identified in developing murine muscle fibers (15).

Arterial delivery of myoblasts

Taken together these data suggest that components for electrical coupling may be present in some engrafted cells that survive 5 to 6 mo. Parallels with an in vivo model of skeletal muscle conditioning is also consistent with possible electrical coupling of implanted cells. Low frequency electrical stimulation causes fast skeletal muscle to undergo a transformation to a more slow-twitch phenotype (reviewed in Refs. 27 and 40). Specifically, prolonged stimulation resulted in dramatic increases in both the protein and mRNA encoding the slow-twitch isoform (SERCA2) of SERCA, nascent expression of phospholamban, and down regulation of the fast-twitch isoform, SERCAl (22,33). Maximal elevations of phospholamban protein were observed after 70 days of chronic electrical stimulation (22). The levels of SERCAl protein were reduced to about 25% of baseline after 70 days of stimulation (22). Other workers have reported up to a 7-fold increase in mitochondrial volume density in chronically stimulated rabbit hindlimb muscle (44). By comparison, phospholamban expression in the donor myoblasts was first noted at 4 mo after injection. Whereas C2C12 myoblasts can express both fast-twitch and slow- twitch isoforms of myosin heavy-chain and SERCA in tissue culture (19,24), we have not observed phospholamban expression in genetically labelled C2C12 myotubes cultured for 2 mo. Longer periods of culture are not possible as the mechanical activity causes the cells to lift from the culture stratum. Although inhibiting spontaneous contractions with tetrodotoxin might prevent cell loss, this manipulation is associated with alterations in sarcoplasmic reticulum protein expression ( 12). A useful experiment would be to determine whether fibers formed exclusively of C2C 12 nuclei express phospholamban when formed in a skeletal muscle environment. However, this was not feasible, due to the inefficient delivery of myoblasts to skeletal muscle via the vascular system. Therefore, while it is remotely possible that this apparent induction is a property of the parental cell line, we assume that the expression of phospholamban is an effect of the cardiac environment. Although it is a possibility that the cells have been reprogrammed to a cardiac phenotype, a switch to a slow-twitch phenotype seems more likely. Antibody probes for cardiac-specific markers, such as troponin I, are currently available to answer this important question. The crystalloid inclusions are identical to that reported for cells expressing bacterial LacZ (9,38,46) and are dissimilar in appearance to endogenous organelles or inclusions present in cardiac cells, such as transverse tubules, sarcoplasmic reticulum, or glycogen inclusions. To minimize diffusion of the substrate we developed the X-gal precipitate at 30°C (9). Note that there was minimal dif-

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fusion of the substrate across cell borders, illustrated in Fig. 6F. Additionally, the blue signal in tissue sections processed for TEM shown in Fig. 6A is fainter than that observed for cells processed for hematoxylin-eosin staining (Fig. 2C). Although this may be accounted for in part by the marked difference in the thickness of sections (1 micron vs. 10 microns) or reduced expression of 1acZ at later time points (compare Fig. 2E and 2D), there may be some leeching of the signal during processing for TEM. We consider it unlikely that the putative myoblastderived cells (#2 and #4 in Fig. 6) are cardiomyocytes that have been exposed to blue precipitate derived from a myoblast that is out of the field of observation. This would require the precise placement of the myoblasts so that cell # 3 is spared. Second, putative myoblast cell #2 has an unusual shape for a cardiomyocyte. Within the accepted reliability of this technique, the cells in Fig. 6 have been identified correctly. Perhaps the limitations of this labeling technique may be circumvented in future studies using immunoelectron microscopy with a muscle-specific antibody. The selective injection of radiographic contrast material, recombinant proteins, and bacterial enzymes into the coronary arteries is a widely practiced nonsurgical procedure. The inactivation of the interferonyreceptor in murine myoblasts by homologous recombination was recently reported (2), and suggests the possibility of engineering human myoblasts with a favorable immunologic and biological profile for myocardial repair or gene transfer. Importantly, vascular delivery of myoblasts may be used as a platform for the delivery of recombinant proteins and growth factors to the myocardium. However, future studies will need to evaluate the potential of primary myoblast cultures for intravascular delivery, and improve the efficiency of the transfer pro cess. Although the constellation of observations is consistent with possible coupling of engrafted skeletal muscle-derived cells, definitive proof of electrical coupling will require demonstration of the transfer of small tracer molecules between myoblast-derived cells and cardiomyocytes or identification of high conductance communications between cells. Collectively, our findings suggest that arterially delivered myoblasts can form stable grafts in the heart and that signals originating in this environment may alter the phenotype of these skeletal muscle derived cells. Physiologic studies are necessary to determine whether implanted myoblasts form functional skeletal muscle grafts, a prerequisite for further development as a potential treatment for myocardial dysfunction. Ackno~~/rd~lner,f.~ ~ We thank R. Mulligan and E. Ralston for generous gifts of cells.We thank J. Wang, D. Fambrough. and E. Beyer for generous gifts of hybridomas and purified antibodies, and B. Byrne for

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assistance with confocal microscopy. The monoclonal antibody A4.1025, developed by H. Blau, was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biological Sciences, University of Iowa, Iowa City, IA, under Contract NOl-HD-2-3144 from the NICHD.

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