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.
We report a novel method of engineering a highly effective collagen-cell scaffold based on RGD+ peptides cross-linked to a clinically approved collagen matrix. The main advantages were cell contractile performance, cardiomyocyte viability and differentiation.
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