title: Pro-arrhythmogenic potential of immature cardiomyocytes is triggered by low coupling and cluster size.
abstract: OBJECTIVE
  Cell transplantation strategies to regenerate compromised myocardium take advance of in vitro generated cardiomyocytes. Common in those immature myocytes is spontaneous impulse formation and a restricted ability to establish proper electrical interaction. Spontaneous impulse formation and impaired cell-to-cell coupling have been shown to be arrhythmogenic. To investigate whether these features harbour a pro-arrhyhmogenic potential for cell transplantation, a co-culture of spontaneously active neonatal rat cardiomyocytes (NRC) and quiescent adult dog cardiomyocytes (ADC) was used.
  
  
  METHODS
  ADCs and NRCs were isolated and cultured on laminin-coated substrates. Connexin43, N-cadherin and alpha-actinin expression was evaluated with immunohistochemistry. Intercellular coupling was measured in cell pairs using the dual voltage clamp technique and fluorescent dye injection.
  
  
  RESULTS
  One day after isolation, NRCs were beating spontaneously, while ADCs remained quiescent in monoculture. ADC resting membrane potential was -80.3+/-0.2 mV (mean+/-SEM, N=24) and did not change significantly over time. NRCs had a maximal diastolic potential of -65.0+/-2.8 mV (N=4). After one day of co-culture, pseudopodia-like extensions developed at the former intercalated discs of ADCs, contacting the NRCs. Only ADCs that contacted three or more NRCs started to beat in synchrony. Expression of connexin43 and N-cadherin indicated presence of electrical and mechanical junctions at the interface between the two cell-types. Transfer of Lucifer Yellow demonstrated junctional permeability between ADCs and NRCs. Junctional conductance between ADC-ADC (31.9+/-5.1 nS, N=10) and NRC-NRC (35.0+/-9.6 nS, N=6) pairs was significantly higher compared to ADC-NRC pairs (9.7+/-2.9 nS, N=8). Gap-junctional blockade with halothane reversibly abolished NRC-triggered beating of ADCs. Computer simulations demonstrated that within a delicate 'window' of gap junctional conductance small clusters of spontaneously active cells are able to induce triggered activity in quiescent mature myocytes but also in a two-dimensional sheet of ventricular cells.
  
  
  CONCLUSIONS
  Spontaneously active immature cardiomyocytes are able to trigger mature cardiomyocytes depending on their level of electrical coupling and the amount of coupled immature myocytes.
authors.affiliation: Heart Lung Center Utrecht, Department of Medical Physiology, Yalelaan 50, 3584 CM Utrecht, The Netherlands.
authors.forename: Teun P
authors.initials: TP
authors.lastname: de Boer
authors.forename: Marcel A G
authors.initials: MA
authors.lastname: van der Heyden
authors.forename: Martin B
authors.initials: MB
authors.lastname: Rook
authors.forename: Ronald
authors.initials: R
authors.lastname: Wilders
authors.forename: Renske
authors.initials: R
authors.lastname: Broekstra
authors.forename: Bart
authors.initials: B
authors.lastname: Kok
authors.forename: Marc A
authors.initials: MA
authors.lastname: Vos
authors.forename: Jacques M T
authors.initials: JM
authors.lastname: de Bakker
authors.forename: Toon A B
authors.initials: TA
authors.lastname: van Veen
mesh_terms: D000185:Actinin / Q000378:metabolism; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000503:physiopathology*; D015820:Cadherins / Q000378:metabolism; D002450:Cell Communication; D017690:Cell Transplantation; D003198:Computer Simulation; D004285:Dogs; D004594:Electrophysiology; D006352:Heart Ventricles; D007150:Immunohistochemistry; D008955:Models, Cardiovascular; D032383:Myocytes, Cardiac / Q000502:physiology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015820:Cadherins; D000185:Actinin