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. CONCLUSION: Spontaneously active immature cardiomyocytes are able to trigger mature cardiomyocytes depending on their level of electrical coupling and the amount of coupled immature myocytes.