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Received March 28, 2002
Accepted after revision September 1, 2002
1 Department of Preclinical and Clinical Pharmacology, University of Firenze, 50139 Firenze, Italy
2 Laboratoire de Cardiologie Cellulaire et Moléculaire, INSERM U-446, Université Paris-Sud, Faculté de Pharmacie, F-92296 Chatenay-Malabry Cedex, France
3 INSERM U-446, Université Paris-Sud, Faculté de Pharmacie, F-92296 Chatenay-Malabry Cedex, France
* To whom correspondence should be addressed. E-mail: fisch{at}vjf.inserm.fr.
HL-1 cells are adult mouse atrial myocytes induced to proliferate indefinitely by SV40 large T antigen. These cells beat spontaneously when confluent and express several adult cardiac cell markers including the outward delayed rectifier K+ channel. Here, we examined the presence of a hyperpolarization-activated If current in HL-1 cells using the whole-cell patch-clamp technique on isolated cells enzymatically dissociated from the culture at confluence. Cell membrane capacitance (Cm) ranged from 5 to 53 pF. If was detected in about 30 % of the cells and its occurrence was independent of the stage of the culture. If maximal slope conductance was 89.7 ± 0.4 pS pF-1 (n = 10). If current in HL-1 cells showed typical characteristics of native cardiac If current: activation threshold between -50 and -60 mV, half-maximal activation potential of -83.1 ± 0.7 mV (n = 50), reversal potential at -20.8 ± 1.5 mV (n = 10), time-dependent activation by hyperpolarization and blockade by 4 mM Cs+. In half of the cells tested, activation of adenylyl cyclase by the forskolin analogue L858051 (20 µM) induced both a ~6 mV positive shift of the half-activation potential and a ~37 % increase in the fully activated If current. RT-PCR analysis of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) expressed in HL-1 cells demonstrated major contributions of HCN1 and HCN2 channel isoforms to If current. Cytosolic Ca2+ oscillations in spontaneously beating HL-1 cells were measured in Fluo-3 AM-loaded cells using a fast-scanning confocal microscope. The oscillation frequency ranged from 1.3 to 5 Hz and the spontaneous activity was stopped in the presence of 4 mM Cs+. Action potentials from HL-1 cells had a triangular shape, with an overshoot at +15 mV and a maximal diastolic potential of -69 mV, i.e. more negative than the threshold potential for If activation. In conclusion, HL-1 cells display a hyperpolarization-activated If current which might contribute to the spontaneous contractile activity of these cells.
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