Morphological, electrophysiological and coupling characteristics of bone marrow-derived mononuclear cells-an in vitro-model.
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.
Rastan
Ardawan Julian
AJ
Department of Cardiac Surgery, Heart Center Leipzig, University of Leipzig, Leipzig, Germany. rastan@rz.uni-leipzig.de
Walther
Thomas
T
Kostelka
Martin
M
Garbade
Jens
J
Schubert
Andreas
A
Stein
Anita
A
Dhein
Stefan
S
Mohr
Friedrich Wilhelm
FW
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
D018031:Connexin 43; D005456:Fluorescent Dyes; D007546:Isoquinolines; D000185:Actinin; C017475:lucifer yellow