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【摘要】
In children, interruption of cardiac atrioventricular (AV) electrical conduction can result from congenital defects, surgical interventions, and maternal autoimmune diseases during pregnancy. Complete AV conduction block is typically treated by implanting an electronic pacemaker device, although long-term pacing therapy in pediatric patients has significant complications. As a first step toward developing a substitute treatment, we implanted engineered tissue constructs in rat hearts to create an alternative AV conduction pathway. We found that skeletal muscle-derived cells in the constructs exhibited sustained electrical coupling through persistent expression and function of gap junction proteins. Using fluorescence in situ hybridization and polymerase chain reaction analyses, myogenic cells in the constructs were shown to survive in the AV groove of implanted hearts for the duration of the animal??s natural life. Perfusion of hearts with fluorescently labeled lec-tin demonstrated that implanted tissues became vascularized and immunostaining verified the presence of proteins important in electromechanical integration of myogenic cells with surrounding re-cipient rat cardiomyocytes. Finally, using optical mapping and electrophysiological analyses, we provide evidence of permanent AV conduction through the implant in one-third of recipient animals. Our experiments provide a proof-of-principle that engineered tissue constructs can function as an electrical conduit and, ultimately, may offer a substitute treatment to conventional pacing therapy.
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Disruption of atrioventricular (AV) impulse propagation in the heart is a serious clinical problem in infants and children as well as in adults.1-3 Congenital complete heart block or AV block because of ischemia, endocarditis, maternal systemic lupus erythematosus, or surgery is currently treated by implanting an artificial pacemaker device.2,4 Although the efficacy of pacemakers as a palliative therapy cannot be disputed, and the range of indications requiring intervention with these devices continues to expand, their long-term performance remains primarily unsatisfactory, especially in pediatric patients.3 Children have a substantially higher incidence of reoperation compared with adults because of limited battery life, lead fractures and failure, cardiac perforation, valve dysfunction, diminished ventricular function, and thrombus formation.1,2 Additionally, the size of newborn and small children frequently requires pacemaker leads to be positioned epicardially, rather than transvenously, which results in even greater failure rates and rising capture thresholds.2 Consequently, there is a pressing need for the advancement of innovative, lasting pacing therapies designed specifically for pediatric patients. In view of that, we sought to develop an implantable tissue that would function as an electrical conduit between the atria and ventricles for eventual use in children that lack normal AV conduction. To be suitable for clinical application, the tissue should be autologously derived, easy to fabricate and implant, and pose no risk of tumor growth nor have arrhythmogenic potential. Ideally, it would account for patient growth, function for the lifespan of the individual, respond to autonomic stimuli, and allow for the orderly and sequential spread of electrical impulses from the upper to lower chambers of the heart through the insulating barrier formed by the fibrous annulus of the AV valves.
In this study, we used a tissue engineering approach to fabricate biocompatible, three-dimensional, collagen-based constructs that contained fetal rat myogenic precursor cells called myoblasts. Compared with commonly used injection-based methods, we reasoned that three-dimensional tissue would allow for more precisely targeted and abundant delivery of cells to the heart. We chose to use myoblasts because they are a therapeutically relevant cell type given that they can be autologously derived and harvested in sufficient quantities from a skeletal muscle biopsy for construct fabrication.5,6 Unlike standard cardiac muscle cell preparations, primary myoblasts are also capable of cell division, which permits expansion and enrichment before transplantation.7 To mitigate transplant rejection, we chose to use syngeneic primary cells, rather than cell lines, as they are less likely to promote tumor growth or cause inflammation.7 Lastly, myoblasts were deemed suitable for cardiac implantation because they are resistant to ischemia, electrically excitable, and have been shown to differentiate and survive when grafted into the heart.8,9
Here, we show that fetal rat myoblasts in engineered tissue constructs (ETCs) were capable of limited fusion and differentiation, unlike cultures on plates; nevertheless, they continued to express proteins important in electromechanically coupling adjacent cells. The myoblasts within the constructs maintained cell-to-cell communication through persistent expression and function of the gap junction protein connexin43 . Tissue constructs were surgically implanted in the cardiac AV groove of adult Lewis rats, and the cells contained therein were shown to survive and integrate in the heart for the duration of the recipient animal??s natural life (2 to 3 years). Furthermore, implanted ETCs possessed a blood supply and were found capable of permanently establishing an alternative conduction pathway between the right atrium and right ventricle.
【关键词】 conduction engineered
Materials and Methods
Myoblast Isolation and Fabrication of ETCs
Myoblasts were isolated from E18 to E20 rat paraspinal skeletal muscles essentially as described previously.10 Cells were plated at low density on 150-mm plates (Falcon; BD Biosciences, Bedford, MA) coated with laminin (L-2020; Sigma, St. Louis, MO). A fraction of the cells were plated at higher density on laminin-coated 12-mm no. 1 glass coverslips for immunohistochemical staining. Myoblasts were induced to differentiate into myotubes using Dulbecco??s modified Eagle??s medium (Invitrogen, Carlsbad, CA) with 2% horse serum and 1% antibiotics in the presence or absence of 10 µmol/L cytosine 1-ß-D arabinofuranoside.10 A day after cell isolation and plating, ETCs were fabricated by mixing the myoblasts with ice-cold 0.175% Cellagen (ICN, Irvine, CA), 14% Matrigel (BD Biosciences), 1% penicillin-streptomycin-glutamine (Invitrogen), 1% Fungizone (Invitrogen), 1x Ham??s F-10 media with 14 mmol/L NaHCO3 (Sigma).5 While still liquid, the mixture was cast into molds comprised of silicone tubing cut in half lengthwise with monofilament polyester mesh (0.331 opening) (McMaster-Carr, Elmhurst, IL) attached to each end with silicone adhesive (Rhodia, Cranbury, NJ). Constructs were warmed at 37??C to induce gelling and covered with myoblast culture media.10 After 3 days, constructs were used for implantation or differentiated.
Implantation of Engineered Tissue into the Cardiac AV Groove
Adult virgin Lewis rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and then intratracheally intubated with a 16-gauge intravenous catheter. Rats were ventilated with an INSPIRA small animal respirator (Harvard Apparatus, Holliston, MA), and anesthesia was maintained with 0.5 to 1.0% isoflurane and 100% oxygen. The heart was accessed through an anterior right-sided thoracotomy at the fifth intercostal space. After incision of the pericardium above the right atrium, the epicardium of both atrium and ventricle near the aorto-atrio-ventricular triangle was carefully removed. A tissue construct (2 x 2 x 2 mm) was gently inserted into the groove and held in position by a single 7-0 polypropylene monofilament stitch. The chest wall was closed in layers, the pneumothorax was evacuated with a 22-gauge intravenous catheter, and the animals were extubated and treated with buprenorphine (0.01 mg/kg, subcutaneously) every 8 to 12 hours for 3 days.
Immunohistochemical Staining
Cells on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 8.0, for 1 hour at 4??C. Cultures were permeabilized for 3 minutes with 0.1% Triton X-100 in PBS and stained with anti-desmin (D1033, Sigma), anti-N-cadherin (CADHNabmX; RDI; Concord, MA), anti-Cx40 (AB1726; Chemicon, Temecula, CA), anti-Cx43 (mAb 3067 or mAb 3068, Chemicon), anti-Cx45 (mAb 3100 or mAb 3101, Chemicon), anti--sarcomeric actin (A2172, Sigma), anti-skeletal myosin MY-32 (M4276, Sigma), anti-slow skeletal myosin 5C5 (M8421, Sigma), anti-dystrophin CAP 6C10,11 anti--actinin 2,12 anti--actinin 3,12 anti-myogenin 5FD (M3559; DAKO, Carpinteria, CA), anti-M-cadherin (611101; BD Transduction), anti-cardiac troponin I 3350 2F6.6,13 anti-MyoD (MYODabm-58, RDI), and anti-neural cell adhesion molecule (NCAM or CD56) (mAb 2120Z, Chemicon) antibodies using the manufacturer??s or author??s suggested dilutions. Primary antibodies were detected with species-appropriate Alexa 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR) mixed with Alexa 568-phalloidin (Molecular Probes) and 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes) before mounting and visualization on a multipoint spinning disk confocal system (Atto; BD Biosciences) attached to a Zeiss Axiovert 200M microscope.14 The confocal system and microscope were each illuminated with an X-Cite 120 mercury-halide light source and images were acquired using either a CoolSNAP HQ (Photometrics, Tuscon, AZ) or MicroMAX 1300YHS CCD camera (Princeton Instruments, Trenton, NJ) controlled with MetaMorph 6.2 software (Universal Imaging, Molecular Devices; Downington, PA). Image processing was accomplished with MetaMorph 6.2 and Photoshop CS (Adobe, San Jose, CA).
Some implanted hearts were retrograde-perfused at 60 mmHg constant pressure in the Langendorff mode at 37??C for 10 minutes with 10 µg/ml of fluorescein isothiocyanate-labeled Lycopersicon esculentum lectin (Vector Laboratories, Burlingame, CA) suspended in 0.2-µm filtered Krebs-Henseleit (KH) buffer (117 mmol/L NaCl, 24 mmol/L NaHCO3, 11.5 mmol/L D- or immunohistochemically stained with the above antibodies after a 30-minute incubation with Image-iT (Molecular Probes). Primary antibodies were detected with highly cross-absorbed goat anti-mouse or anti-rabbit Alexa-conjugated secondary antibodies (Molecular Probes) and visualized as described previously or using a LSM 410 confocal microscope (Carl Zeiss, Thornwood, NY).14
Immunoblot Analyses
Immunoblotting was performed as described previously using the following antibodies diluted as suggested by the supplier: anti-Cx40 (AB1726, Chemicon), anti-Cx43 (mAb 3067, Chemicon; or 13-8300; Zymed, South San Francisco, CA), anti-Cx45 (mAb 3101, Chemicon), anti-N-cadherin (CADHNabmX, RDI), anti-desmin (D-1033, Sigma), and anti-MyoD (MYODabm-58, RDI).14,16 Primary antibodies were detected with horseradish peroxidase-labeled secondary antibodies and the ECL kit (Amersham, Arlington Heights, IL).
Transmission Electron Microscopy
ETCs containing myoblasts or myotubes were fixed in 1.25% formaldehyde, 2.5% grade I glutaraldehyde, and 0.03% picric acid in 100 mmol/L cacodylate buffer overnight. Tissue was rinsed with buffer, stained with a mixture of 1% osmium tetroxide and 1.5% potassium ferrocyanide followed by 1% aqueous uranyl acetate, and dehydrated through a graded ethanol series and propylene oxide. After infiltration and embedding with Epon-Araldite (EMS, Hatfield, PA), sections (60 nm thick) were cut on an Ultracut-S ultramicrotome (Reichert, Depew, NY) and mounted on copper grids (200 mesh).17 For immunostaining studies, the constructs were fixed in 4% paraformaldehyde in PBS overnight, infiltrated with 2.3 mol/L sucrose in PBS containing 150 mmol/L glycine, and frozen in liquid nitrogen. Ultrathin sections were cut at C120??C using the Tokayasu method and incubated with anti-Cx43 (mAb 3067; Chemicon) or anti-Cx45 (mAb 3101; Chemicon) antibodies, which were detected with rabbit anti-mouse secondary antibodies (Jackson) and a protein A-gold conjugate (EMS). Transmission electron microscopy was performed on a Jeol 1200EX (80 kV).
Dye Transfer Studies
ETCs containing myotubes were incubated for 1 hour in media containing 5 µg/ml Hoechst 33342, and cells at one end of the construct were labeled for 2 minutes with 5 µmol/L calcein AM (Molecular Probes) and 20 µmol/L CM-DiI (Molecular Probes) by suspension in Dulbecco??s modified Eagle??s medium (Invitrogen) containing 2% horse serum, 1% antibiotics, and the aforementioned dyes. The ability of calcein (but not CM-DiI) to transfer from cell-to-cell through gap junctions was monitored microscopically along the length of the ETC, and some constructs were treated with 2 mmol/L 1-heptanol for 10 minutes before dye loading. Images were acquired as described above.
Conduction Velocity Measurements
ETCs differentiated for 14 days were impaled at one end with two 0.254-mm diameter 99.9% platinum wires (VWR, West Chester, PA) spaced 1 mm apart and connected to the terminals of a Grass S48 stimulator through a SIU5 stimulus isolation unit (Grass-Telefactor, West Warwick, RI). The stimulation pulse width was 0.5 ms, and voltage was set at 1.5 times that required to initiate construct twitch.18,19 Single platinum wires positioned 50 mm and 100 mm away from the paired wires served as recording electrodes referenced to the negative electrode. ETCs attached to polyester mesh at each end were submerged in aerated 37??C KH buffer for the duration of each experiment.15 Signals (mV) were amplified and band-pass filtered (10 to 2000 Hz) using an EVR recorder (E for M Corp., Torrance, CA), and data were acquired every 0.1 ms using PowerLab Chart 3.4.6 software (AD Instruments, Colorado Springs, CO). The conduction speed was calculated as the distance between the electrodes divided by the initial peak amplitudes between the recording sites. Stimulation train rates ranging from 1 to 200 Hz resulted in essentially identical rates of conduction in the six ETCs examined and constructs containing killed cells (n = 6) failed to exhibit depolarization.
Y Chromosome Detection
The rat SRY gene was detected in DNA samples as described previously.20,21 Identity of the amplified product as rSRY was confirmed by EcoRI digestion and sequencing. Fluorescence in situ hybridization of implanted hearts was performed using the fluorescein isothiocyanate-labeled Star*FISH rat Y chromosome paint probe (Cambio, Cambridge, UK). Slides were prepared as described for immunohistochemical staining and incubated with anti-desmin (D1033; Sigma), anti-Cx43 (mAb 3067; Chemicon), anti-Cx45 (mAb 3101; Chemicon), anti--sarcomeric actin (A2172; Sigma), or anti-skeletal myosin MY-32 (M4276; Sigma) antibodies detected with Alexa 568 goat anti-mouse secondary antibodies (Molecular Probes). For Y chromosome enumeration, slides were first postfixed for 2 minutes and then treated with 0.2 N HCl for 15 minutes, 0.1% Triton X-100 in PBS for 2 minutes, 10 µg/ml proteinase K in PBS for 2 minutes, and 27 mmol/L glycine in PBS for 1 minute. Slides were postfixed again and rinsed in glycine/PBS then 0.2x standard saline citrate before treatment with freshly prepared 0.1 mol/L triethanolamine, 0.25% acetic anhydride for 10 minutes. After washing in 2x standard saline citrate twice, slides were dehydrated through an ethanol series and hybridized to the probe overnight at 37??C after denaturation at 80??C for 10 minutes. Finally, slides were washed according to the Star*FISH protocols (Cambio) and visualized.
Optical Mapping of Langendorff-Perfused Hearts
Rats were anesthetized with an intraperitoneal injection of ketamine (150 mg/kg), xylazine (10 mg/kg), and heparin (500 U/kg). Hearts were rapidly excised and placed in ice-cold KH buffer. After cannulation of the aorta, the hearts were perfused as described above with filtered (0.2 µmol/L) KH buffer.15 Temperature was maintained at 37??C and monitored with a thermistor probe placed in the left ventricle via the left atrium. A continuous cavitary electrographic recording was taken between the aortic root and the left ventricular apex. The electrode signal from the latter was digitized at 1000 Hz with a DAQCard 6036E 16-bit PCMCIA data acquisition device (National Instruments, Austin, TX) and collected using LabVIEW 6 software (National Instruments). Perfused hearts were loaded with 5 µmol/L di-8-ANEPPS for 5 minutes (Molecular Probes), and optical mapping was performed using a laser-scanning system.22,23 To eliminate cardiac motion, 11 mmol/L 2,3-butanedione monoxime was added to the perfusate.24 The pattern of electrical activation on the cardiac surface was recorded during sinus rhythm or bipolar pacing using an insulated minicoaxial stimulation electrode (model BS4-73-0181; Harvard Apparatus). A coaxial bipolar stimulation electrode was used to minimize the spatial spread of electrical stimulation-induced tissue depolarization. The right atrium was stimulated either epicardially or endocardially, whereas the right ventricle only was epicardially paced. Acousto-optical deflectors (InRad, Northvale, NJ), controlled with custom-built PC-based software, focused an argon ion laser (514 nm; Coherent Innova 90C-A5, Santa Clara, CA) on a 200-µm diameter spot on the surface of the heart for 10 µs. Serial excitation of a user-defined grid of spots allowed data from 100 sites to be acquired in 1 ms.22,23 The resulting fluorescence was filtered using a 645-nm long-pass filter, detected with a cooled 16-mm avalanche photodiode (model 630-70-72-571; Advanced Photonix, Irvine, CA), and digitized with 12-bit resolution at 1000 Hz (DT2821-G-16SE; Data Translation, Marlboro, MA). Signals were analyzed off-line using customized software written in MATLab (Mathworks, Natick, MA).22
Results
Isolation and Characterization of Fetal Myogenic Precursor Cells
Initially, we examined dissociated fetal rat myoblast preparations attached to conventional laminin-coated culture plates for the expression of muscle-specific proteins and those important in electrically and mechanically coupling adjacent cells (Figure 1) . Using immunohistochemical analyses, we found that morphologically distinct myoblasts accounted for 78.1 ?? 4.16% (mean ?? SD, n = 8) of the total cell population. In contrast to contaminating cell types (eg, fibroblasts), undifferentiated myoblasts stained for the intermediate filament protein desmin, the fascia adherens junction protein N-cadherin (Figure 1A) , and the contractile apparatus protein -sarcomeric actin (Figure 1B) . We also found staining for the adhesion protein N-CAM, the muscle-specific transcription factor MyoD, and another adherens junction protein, M-cadherin (not shown). Notably, myoblasts stained for the connexin proteins Cx40(5), Cx43(1), and Cx45(6). The latter are constituents of gap junction channels, which directly connect the cytoplasmic compartments of neighboring cells and provide for regulated low-resistance intercellular electrical coupling in excitable tissues. As myoblasts begin to align, fuse, and differentiate into multinucleated myotubes, the expected pattern of expression would include a decline in MyoD as well as adherens and gap junction proteins with a concurrent, albeit transient, rise in the myogenic regulatory factor myogenin and the sequential appearance of other contractile proteins such as members of the -actinin and myosin families.25,26
Figure 1. Characterization of myogenic precursor cells under standard culture conditions. The expression of gap junction and muscle-specific proteins in myoblasts, myotubes, and fibroblasts cultured on laminin-coated plastic and glass was examined. A: Cells attached to coverslips were immunostained for nuclei (blue); filamentous actin (red); and either desmin, N-cadherin, Cx40(5), Cx43(1), or Cx45(6) (green). B: Immunostaining of myoblasts and fibroblasts (arrows) for nuclei (blue), filamentous actin (red), and -sarcomeric actin (green) 24 hours after isolation. C: Immunohistochemical staining of myoblasts differentiated into myotubes for 7 days in the absence of cytosine 1-ß-D arabinofuranoside. Nuclei (blue), -sarcomeric actin, myogenin, Cx43(1), or Cx45(6) (red), and -actinin 2 or dystrophin (green) staining is shown. D: Immunoblot analysis of Cx40(5), unphosphorylated Cx43(1), Cx45(6), N-cadherin, desmin, and MyoD in myoblasts (MB), myotubes (MT), and fibroblasts (FB) cultured on plastic. Myotubes were differentiated in the presence of arabinofuranoside to limit fibroblast proliferation. Days in culture are indicated as is the molecular weight (kd) of the major band in each blot. Results are representative of six independent experiments. Scale bars represent µm.
In our hands, plated myoblasts induced to differentiate into myotubes for 7 days (Figure 1C) showed continued expression of -sarcomeric actin, Cx45(6), and desmin. Differentiating myotubes also displayed a decrease in Cx43(1), N-cadherin, and M-cadherin levels and an increase in expression of the actin-binding proteins -actinin 2 and -actinin 3, the sarcolemmal protein dystrophin, and myogenin. Skeletal myosin was expressed in later stages of myotube differentiation (>5 days), and myogenin levels slowly declined in cultures between days 7 and 14. To more globally quantify the expression profile of proteins in differentiating myogenic cells, plated cultures were maintained in the presence of cytosine arabinofuranoside (which prevents mitosis through selective inhibition of DNA synthesis), thus allowing the initial proportion of muscle-derived cells to contaminating cells to remain constant. Immunoblot analyses revealed persistently low levels of Cx40(5), diminishing Cx43(1) levels, and increasing Cx45(6) levels (Figure 1D) . Cx40(5) protein expression in myoblast and myotube cultures, which remained constant, was likely attributable to a small percentage of contaminating fibroblasts whereas the decline in Cx43(1) was consistent with the previously reported loss of intercellular communication during differentiation.8,27,28 The unanticipated rise in Cx45(6) expression in differentiating myoblasts provided evidence that continued production of some proteins may be brought about by culture conditions (eg, attachment factors). As cadherin levels were reduced in differentiating myotubes (as expected for a myoblast-specific marker), desmin protein levels remained constant throughout differentiation. Predictably, MyoD was expressed only in proliferating and fusing cells.25,26 Cultures lacking cytosine arabinofuranoside had >95% fibroblasts within 7 days (FB d7) and a considerably different protein expression profile compared with either myoblasts or myotubes (Figure 1D) . Progression of myotubes to fully mature myofibers was not observed (nor expected) because this process requires innervation and other in vivo triggers.10,29
Electrical Coupling of Differentiated Cells in ETCs
Next, we studied myogenic precursor cells integrated within ETCs to further explore the possibility that the extracellular environment may differentially regulate gap junction expression and function. Myoblasts were cast in a mixture of collagen type I, Matrigel, culture media, and antibiotics.5 Cells in the constructs were maintained in vitro as undifferentiated precursor cells or induced to differentiate into myotubes. Histological examination of tissue constructs revealed a uniform distribution of cells throughout the hydrogel and a longitudinal cellular orientation parallel to the direction of tensile strain (Figure 2A) . Because the hydrogel and myoblast mixture is liquid at the outset, it could be cast in any shape and size before solidification and dehydration.5,30 If the hydrogel had two or more points of attachment, it showed signs of internal passive tension attributable to contraction of collagen resulting from cellular interactions with the surrounding matrix.30 Aside from these properties, the tissue constructs were mechanically rigid enough to be sutured and permitted us to control both the orientation and number of cells implanted in the heart. In short, the existing ETC design possessed a number of traits that would prove useful in subsequent implantation experiments.5,30
Figure 2. Characterization of myogenic precursor cells maintained in three-dimensional tissue constructs. The expression and function of gap junction proteins and the expression of muscle-specific proteins in myoblasts and myotubes from ETCs was examined. A: Constructs containing myotubes differentiated for 2 weeks were sectioned (5 µm) and subjected to H&E or Masson??s trichrome staining. ETC sections were immunohistochemically stained for nuclei (blue), -actinin 2, dystrophin (red), -sarcomeric actin, skeletal myosin, myogenin, Cx43(1), and Cx45(6) (green). Fluorescent images depict 10 merged optical sections (0.5 µm) and scale bars represent µm. B: Electron microscopy of ETCs revealed early sarcomere assembly in myotubes (white arrows), the presence of extracellular collagen fibrils (black arrowheads), and gap junctions in myotubes (between black arrows) that stained for Cx43(1) or Cx45(6) (black dots). C: Immunoblot analysis of total Cx43(1) (phosphorylated and unphosphorylated), Cx45(6), N-cadherin, and desmin in myoblasts (MB) and myotubes (MT) from ETCs. Fibroblast (FB) proteins were collected from cells grown on plastic. Days in culture are indicated along with the molecular weight (kd) of the major band in each blot. Results are representative of six independent experiments. D: Gap junction function as determined by the ability of calcein (green), but not CM-DiI (red), to transfer from cell-to-cell in myotubes stained for nuclei with Hoechst 33342 (blue). Interference of gap junction assembly by 1-heptanol prevented calcein transfer and intracellular dye transfer was observed between elongated, multinucleated cells consistent with myotubes.
ETCs containing myotubes (differentiated up to 2 weeks) demonstrated staining for -sarcomeric actin, -actinin 2, myogenin, dystrophin, and Cx43(1). Cx45(6) was expressed at uniformly low levels in myoblasts, myotubes, and fibroblasts when compared to the results from cells maintained in culture dishes. It was also evident that the myoblasts did not fuse to the extent seen in plated cultures because the majority of cells in the constructs contained only 2.01 ?? 1.65 nuclei (mean ?? SD, n = 15; Table 1 ). The myogenic differentiation program appeared delayed or inhibited as fully-formed sarcomeric structures were only rarely observed by -actinin 2 staining. In addition, myogenin expression was prolonged (up to 14 days), and skeletal myosin was not apparent in many of the myotubes. To better localize proteins involved in intracellular electrical conduction, electron microscopy of ETCs was performed. Early contractile apparatus formation was apparent in the majority of cells and assembly of gap junction channels was observed between differentiating myotubes (Figure 2B) . Moreover, gap junctions stained for Cx43(1) and Cx45(6), but only rarely with Cx40(5). Immunoblot analysis of ETC-derived proteins demonstrated Cx43(1), Cx45(6), and N-cadherin levels increased throughout differentiation (Figure 2C) . Despite absence of mitotic inhibition in ETCs, desmin protein levels remained constant, indicating that the relative proportion of muscle cells did not change. Analysis of -actinin 2 and desmin staining in sections of ETCs differentiated for 2 weeks confirmed the majority of cells were muscle-derived (Table 1) .
Table 1. Summary of in Vitro ETC Staining and Conduction Velocity Measurements
To assess intercellular communication between adjoining myotubes in ETCs, we first studied the ability of a dye to travel from cell-to-cell along the length of the construct (Figure 2D) . Myotubes at one end were concurrently loaded with the acetoxymethyl (AM)-ester of calcein and chloromethylated (CM)-DiI. Calcein AM was hydrolyzed to a small fluorescent molecule (calcein; molecular weight, 622.54) by intracellular esterases, while the lipophilic CM-DiI became incorporated in plasma membranes. When functional gap junctions were established, only the cytosolic calcein tracer was transferred from labeled cells to unlabeled cells. By examining the opposite end of the construct, we determined that myotubes were capable of intercellular communication (n = 12), although nuclear staining revealed that not all cells were coupled by gap junction channels (Table 1) . ETCs that contained no cells (n = 4) or killed myotubes (ie, constructs immersed in water for 48 hours to lyse cells but not affect matrix structure) (n = 6) were not able to transfer calcein. We also found dye transfer could be abolished in constructs containing viable myotubes by treatment with 1-heptanol (n = 6), an inhibitor of gap junction assembly. The dye transfer experiments also highlight the cell density in the constructs as the images presented in Figure 2D show the entire tissue construct rather than a thin section.
Constructs containing differentiated cells contracted in a coordinated manner on stimulation with paired platinum electrodes (Supplemental Movie 1, see http://ajp.amjpathol.org). Bipolar electrographic recordings of myotube-containing constructs demonstrated an apparent conduction velocity of 2.075 ?? 0.333 m?
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作者单位:From the Departments of Cardiac Surgery,* Anesthesiology, Surgery, and Cardiology,¶ Children??s Hospital Boston, and the Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts