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• W2038962536 abstract "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. 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. Disruption of atrioventricular (AV) impulse propagation in the heart is a serious clinical problem in infants and children as well as in adults.1Bevilacqua L Hordof A Cardiac pacing in children.Curr Opin Cardiol. 1998; 13: 48-55Crossref PubMed Scopus (14) Google Scholar, 2Berul CI Cecchin F Indications and techniques of pediatric cardiac pacing.Expert Rev Cardiovasc Ther. 2003; 1: 165-176Crossref PubMed Scopus (25) Google Scholar, 3Mond HG Irwin M Morillo C Ector H The world survey of cardiac pacing and cardioverter defibrillators: calendar year 2001.Pacing Clin Electrophysiol. 2004; 27: 955-964Crossref PubMed Scopus (99) Google Scholar 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.2Berul CI Cecchin F Indications and techniques of pediatric cardiac pacing.Expert Rev Cardiovasc Ther. 2003; 1: 165-176Crossref PubMed Scopus (25) Google Scholar, 4Benson DW Genetics of atrioventricular conduction disease in humans.Anat Rec A Discov Mol Cell Evol Biol. 2004; 280: 934-939Crossref PubMed Scopus (35) Google Scholar 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.3Mond HG Irwin M Morillo C Ector H The world survey of cardiac pacing and cardioverter defibrillators: calendar year 2001.Pacing Clin Electrophysiol. 2004; 27: 955-964Crossref PubMed Scopus (99) Google Scholar 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.1Bevilacqua L Hordof A Cardiac pacing in children.Curr Opin Cardiol. 1998; 13: 48-55Crossref PubMed Scopus (14) Google Scholar, 2Berul CI Cecchin F Indications and techniques of pediatric cardiac pacing.Expert Rev Cardiovasc Ther. 2003; 1: 165-176Crossref PubMed Scopus (25) Google Scholar 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.2Berul CI Cecchin F Indications and techniques of pediatric cardiac pacing.Expert Rev Cardiovasc Ther. 2003; 1: 165-176Crossref PubMed Scopus (25) Google Scholar 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.5Vandenburgh H Del Tatto M Shansky J Lemaire J Chang A Payumo F Lee P Goodyear A Raven L Tissue-engineered skeletal muscle organoids for reversible gene therapy.Hum Gene Ther. 1996; 7: 2195-2200Crossref PubMed Scopus (115) Google Scholar, 6Ozawa CR Springer ML Blau HM A novel means of drug delivery: myoblast-mediated gene therapy and regulatable retroviral vectors.Annu Rev Pharmacol Toxicol. 2000; 40: 295-317Crossref PubMed Scopus (33) Google Scholar Unlike standard cardiac muscle cell preparations, primary myoblasts are also capable of cell division, which permits expansion and enrichment before transplantation.7Kessler PD Byrne BJ Myoblast cell grafting into heart muscle: cellular biology and potential applications.Annu Rev Physiol. 1999; 61: 219-242Crossref PubMed Scopus (109) Google Scholar 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.7Kessler PD Byrne BJ Myoblast cell grafting into heart muscle: cellular biology and potential applications.Annu Rev Physiol. 1999; 61: 219-242Crossref PubMed Scopus (109) Google Scholar 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.8Reinecke H MacDonald GH Hauschka SD Murry CE Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair.J Cell Biol. 2000; 149: 731-740Crossref PubMed Scopus (297) Google Scholar, 9Murry CE Wiseman RW Schwartz SM Hauschka SD Skeletal myoblast transplantation for repair of myocardial necrosis.J Clin Invest. 1996; 98: 2512-2523Crossref PubMed Scopus (549) Google ScholarHere, 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 [Cx43(α1)] and, to a lesser extent, connexin45 [Cx45(α6)]. 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 (∼2½ 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.Materials and MethodsMyoblast Isolation and Fabrication of ETCsMyoblasts were isolated from E18 to E20 rat paraspinal skeletal muscles essentially as described previously.10Rando TA Blau HM Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.J Cell Biol. 1994; 125: 1275-1287Crossref PubMed Scopus (789) Google Scholar 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-β-d arabinofuranoside.10Rando TA Blau HM Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.J Cell Biol. 1994; 125: 1275-1287Crossref PubMed Scopus (789) Google Scholar 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), 1× Ham's F-10 media with 14 mmol/L NaHCO3 (Sigma).5Vandenburgh H Del Tatto M Shansky J Lemaire J Chang A Payumo F Lee P Goodyear A Raven L Tissue-engineered skeletal muscle organoids for reversible gene therapy.Hum Gene Ther. 1996; 7: 2195-2200Crossref PubMed Scopus (115) Google Scholar 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.10Rando TA Blau HM Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.J Cell Biol. 1994; 125: 1275-1287Crossref PubMed Scopus (789) Google Scholar After 3 days, constructs were used for implantation or differentiated.Implantation of Engineered Tissue into the Cardiac AV GrooveAdult 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 × 2 × 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 StainingCells 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-α-sarcomeric actin (A2172, Sigma), anti-skeletal myosin MY-32 (M4276, Sigma), anti-slow skeletal myosin 5C5 (M8421, Sigma), anti-dystrophin CAP 6–10,11Byers TJ Kunkel LM Watkins SC The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle.J Cell Biol. 1991; 115: 411-421Crossref PubMed Scopus (201) Google Scholar anti-α-actinin 2,12Chan Y Tong HQ Beggs AH Kunkel LM Human skeletal muscle-specific alpha-actinin-2 and -3 isoforms form homodimers and heterodimers in vitro and in vivo.Biochem Biophys Res Commun. 1998; 248: 134-139Crossref PubMed Scopus (61) Google Scholar anti-α-actinin 3,12Chan Y Tong HQ Beggs AH Kunkel LM Human skeletal muscle-specific alpha-actinin-2 and -3 isoforms form homodimers and heterodimers in vitro and in vivo.Biochem Biophys Res Commun. 1998; 248: 134-139Crossref PubMed Scopus (61) Google Scholar anti-myogenin 5FD (M3559; DAKO, Carpinteria, CA), anti-M-cadherin (611101; BD Transduction), anti-cardiac troponin I 3350 2F6.6,13Bodor GS Porter S Landt Y Ladenson JH Development of monoclonal antibodies for an assay of cardiac troponin-I and preliminary results in suspected cases of myocardial infarction.Clin Chem. 1992; 38: 2203-2214PubMed Google Scholar 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.14Cowan DB Lye SJ Langille BL Regulation of vascular connexin43 gene expression by mechanical loads.Circ Res. 1998; 82: 786-793Crossref PubMed Scopus (131) Google Scholar 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-[+]-glucose, 3.3 mmol/L KCl, 1.25 mmol/L CaCl2, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, and 10 U/L insulin) equilibrated with 95% O2 and 5% CO2 followed by a 2-minute washout in KH buffer alone.15Stamm C Cowan DB Friehs I Noria S del Nido PJ McGowan Jr, FX Rapid endotoxin-induced alterations in myocardial calcium handling: obligatory role of cardiac TNF-alpha.Anesthesiology. 2001; 95: 1396-1405Crossref PubMed Scopus (37) Google Scholar All implanted hearts were perfusion-fixed under pressure in 4% paraformaldehyde in PBS for 10 minutes before passive fixation at 4°C overnight in the same solution. ETCs containing either myoblasts or myotubes were fixed overnight at 4°C in 4% paraformaldehyde in PBS. After embedding and sectioning (5 μm thickness), slides were baked overnight at 65°C, deparaffinized in xylenes, rehydrated through a graded ethanol series, and subjected to antigen retrieval by heating three times for 5 minutes in 1 mmol/L ethylenediaminetetraacetic acid (pH 8.0) using a 700 W microwave oven set to high. Slides were used for either routine histological staining [hematoxylin and eosin (H&E) or Masson's trichrome] 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).14Cowan DB Lye SJ Langille BL Regulation of vascular connexin43 gene expression by mechanical loads.Circ Res. 1998; 82: 786-793Crossref PubMed Scopus (131) Google ScholarImmunoblot AnalysesImmunoblotting 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).14Cowan DB Lye SJ Langille BL Regulation of vascular connexin43 gene expression by mechanical loads.Circ Res. 1998; 82: 786-793Crossref PubMed Scopus (131) Google Scholar, 16Cowan DB Poutias DN Del Nido PJ McGowan Jr, FX CD14-independent activation of cardiomyocyte signal transduction by bacterial endotoxin.Am J Physiol. 2000; 279: H619-H629Google Scholar Primary antibodies were detected with horseradish peroxidase-labeled secondary antibodies and the ECL kit (Amersham, Arlington Heights, IL).Transmission Electron MicroscopyETCs 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).17Cowan DB Noria S Stamm C Garcia LM Poutias DN del Nido PJ McGowan Jr, FX Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes.Circ Res. 2001; 88: 491-498Crossref PubMed Scopus (44) Google Scholar 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 −120°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 StudiesETCs 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 MeasurementsETCs 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.18Kupa EJ Roy SH Kandarian SC De Luca CJ Effects of muscle fiber type and size on EMG median frequency and conduction velocity.J Appl Physiol. 1995; 79: 23-32PubMed Google Scholar, 19Wakeling JM Syme DA Wave properties of action potentials from fast and slow motor units of rats.Muscle Nerve. 2002; 26: 659-668Crossref PubMed Scopus (44) Google Scholar 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.15Stamm C Cowan DB Friehs I Noria S del Nido PJ McGowan Jr, FX Rapid endotoxin-induced alterations in myocardial calcium handling: obligatory role of cardiac TNF-alpha.Anesthesiology. 2001; 95: 1396-1405Crossref PubMed Scopus (37) Google Scholar 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 DetectionThe rat SRY gene was detected in DNA samples as described previously.20Muller-Ehmsen J Whittaker P Kloner RA Dow JS Sakoda T Long TI Laird PW Kedes L Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium.J Mol Cell Cardiol. 2002; 34: 107-116Abstract Full Text PDF PubMed Scopus (423) Google Scholar, 21Yao M Dieterle T Hale SL Dow JS Kedes LH Peterson KL Kloner RA Long-term outcome of fetal cell transplantation on postinfarction ventricular remodeling and function.J Mol Cell Cardiol. 2003; 35: 661-670Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar 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-α-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 0.2× standard saline citrate before treatment with freshly prepared 0.1 mol/L triethanolamine, 0.25% acetic anhydride for 10 minutes. After washing in 2× 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 HeartsRats 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.15Stamm C Cowan DB Friehs I Noria S del Nido PJ McGowan Jr, FX Rapid endotoxin-induced alterations in myocardial calcium handling: obligatory role of cardiac TNF-alpha.Anesthesiology. 2001; 95: 1396-1405Crossref PubMed Scopus (37) Google Scholar 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.22Kwaku KF Dillon SM Shock-induced depolarization of refractory myocardium prevents wave-front propagation in defibrillation.Circ Res. 1996; 79: 957-973Crossref PubMed Scopus (93) Google Scholar, 23Dillon S Morad M A new laser scanning system for measuring action potential propagation in the heart.Science. 1981; 214: 453-456Crossref PubMed Scopus (77) Google Scholar To eliminate cardiac motion, 11 mmol/L 2,3-butanedione monoxime was added to the perfusate.24Cheng Y Mowrey K Efimov IR Van Wagoner DR Tchou PJ Mazgalev TN Effects of 2,3-butanedione monoxime on atrial-atrioventricular nodal conduction in isolated rabbit heart.J Cardiovasc Electrophysiol. 1997; 8: 790-802Crossref PubMed Scopus (42) Google Scholar 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.22Kwaku KF Dillon SM Shock-induced depolarization of refractory myocardium prevents wave-front propagation in defibrillation.Circ Res. 1996; 79: 957-973Crossref PubMed Scopus (93) Google Scholar, 23Dillon S Morad M A new laser scanning system for measuring action potential propagation in the heart.Science. 1981; 214: 453-456Crossref PubMed Scopus (77) Google Scholar 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).22Kwaku KF Dillon SM Shock-induced depolarization of refractory myocardium prevents wave-front propagation in defibrillation.Circ Res. 1996; 79: 957-973Crossref PubMed Scopus (93) Google ScholarResultsIsolation and Characterization of Fetal Myogenic Precursor CellsInitially, 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 α-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(α5), Cx43(α1), and Cx45(α6). 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 an" @default.
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• W2038962536 title "Cardiac Conduction through Engineered Tissue" @default.
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