title: Morphological, electrophysiological and coupling characteristics of bone marrow-derived mononuclear cells-an in vitro-model.
abstract: OBJECTIVE
  Transplantation of bone marrow-derived mononuclear cells (BMC) may be suitable to prevent myocardial remodeling and improve left ventricular function after myocardial infarction. However, it is unknown whether or not cardiomyocytes and BMCs can form functioning cell-to-cell coupling and develop adequate electrophysiological properties.
  
  
  METHODS
  BMCs were isolated from minipig leg bones, treated with 5-azacytidine (10 microM) for 24 h, cultured for 7 days and labeled with a fluoroscopic dye (DIL). BMCs were cocultured with spontaneously beating cardiomyocytes of neonatal rats. On days 4, 7 and 14 cocultured cells were analyzed. Immunhistochemistry (Connexin 43, alpha-actinin) was used to assess cardiomyogenic differentiation. Action potential characteristics were recorded in whole cell patch clamp mode and to investigate intercellular communication a second gap junction permeable fluoroscopic dye was brought into BMCs by microinjection (Lucifer yellow, LY).
  
  
  RESULTS
  From day 7 in coculture BMCs beated synchronously with neonatal rat cardiomyocytes. On day 14, 55.9% of BMCs expressed alpha-actinin and 98.3% were positive for gap junction protein connexin 43. BMC action potential duration (APD90) was mean 11.1 ms with dV/dt(max) of 26.8 V/s and similar to atrial cardiac type. However, microinjection of LY revealed only little dye transfer into adjacent rat cardiomyocytes.
  
  
  CONCLUSIONS
  Cocultured BMCs have the potential for early expression of muscle specific proteins in about 60% after 14 days and for cardiac gap junction proteins. Synchronous beating indicates an effective electromechanical coupling. In this heterologous setting we could prove only weak metabolic coupling.
authors.affiliation: Department of Cardiac Surgery, Heart Center Leipzig, University of Leipzig, Leipzig, Germany. rastan@rz.uni-leipzig.de
authors.forename: Ardawan Julian
authors.initials: AJ
authors.lastname: Rastan
authors.forename: Thomas
authors.initials: T
authors.lastname: Walther
authors.forename: Martin
authors.initials: M
authors.lastname: Kostelka
authors.forename: Jens
authors.initials: J
authors.lastname: Garbade
authors.forename: Andreas
authors.initials: A
authors.lastname: Schubert
authors.forename: Anita
authors.initials: A
authors.lastname: Stein
authors.forename: Stefan
authors.initials: S
authors.lastname: Dhein
authors.forename: Friedrich Wilhelm
authors.initials: FW
authors.lastname: Mohr
mesh_terms: D000185:Actinin / Q000032:analysis; D000200:Action Potentials / Q000502:physiology; D000818:Animals; D016026:Bone Marrow Transplantation / Q000379:methods; D002450:Cell Communication; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D018031:Connexin 43 / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D007150:Immunohistochemistry / Q000379:methods; D007546:Isoquinolines / Q000032:analysis; D009000:Monocytes / Q000378:metabolism / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D013552:Swine; D013556:Swine, Miniature
chemical_list: D018031:Connexin 43; D005456:Fluorescent Dyes; D007546:Isoquinolines; D000185:Actinin; C017475:lucifer yellow