Programme Contents

Multiple spontaneously active Ca²⁺ waveforms in Nkx2.5-GFP cardiac lineage cells show selective modulation by I_f channel blockade, endothelin I and elevation of intracellular cAMP

Introduction: Isolated cells of the human and or mouse cardiac conduction system show location-dependent spontaneous activity, often described by changes in either electrical activity or in intracellular calcium ([Ca²⁺]_i) cycling.

Aims: To investigate the functional properties of spontaneously active cardiac lineage (Nkx2.5-eGFP⁺) cells derived from mouse embryonic stem cells.

Methods: Cardiomyocyte differentiation was initiated using the hanging drop method to generate embryoid bodies. Following 18-20 days of differentiation cell aggregates were dissociated and Nkx2.5-eGFP⁺ cells isolated by FACS. Cells were loaded with the Ca²⁺ sensitive fluorophore, Fluo4-AM (10μM), prior to high speed confocal imaging. Ca²⁺ fluorescence data were analysed for rate and changes in waveform kinetics (maximum changes in up and down slope and width at half height). Post-imaging, cells were fixed and immunolabelled with anti-PGP9.5 (a Purkinje cell marker).

Results: On the basis of frequency and width at least five distinct spontaneously active populations were present in the Nkx2.5-eGFP⁺ cardiac lineage cells. The high frequency population were less sensitive to the funny channel blocker, ZD7288 (10μM), but more sensitive to ryanodine (10μM). Endothelin I (10nM) increased oscillation frequency, except in the slowest frequency waveforms. Angiotensin II (100nM) was without effect.

Discussion: Pacemaker automaticity mainly involves I_f current and/or ryanodine receptor mediated Ca²⁺ release from the SR. The slowest frequency cells were positive for PGP9.5, indicating that they are Purkinje-like cells. This study has shown that spontaneously active cells can be readily isolated from NKx2.5-eGFP cardiac lineage cells. These cells are suitable for the investigation of the cardiac conduction system.