Cardiac specific transcription factor Csx/Nkx2.5 regulates transient-outward K+ channel expression in pluripotent P19 cell-derived cardiomyocytes

Figures

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  Fig. 1. The expression of Csx/Nkx2.5 mRNA analyzed by semi-quantitative RT-PCR. a Representative ethidium bromide staining of PCR products of P19CL6 cells (lanes 1, 3, 5) and P19CL6-Csx cells (lanes 2, 4, 6) during differentiation period. b Relative expression of Csx/Nkx2.5 mRNA was shown after normalizing to the expression of β-actin in each lane

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  Fig. 2. Change in action potentials and cell size during differentiation. Typical action potential recordings of P19CL6-derived cardiomyocytes (a, c) and P19CL6-Csx-derived cardiomyocytes (b, d). Developmental change in cell capacitance of P19CL6-derived cardiomyocytes and P19CL6-Csx-derived cardiomyocytes (e). Action potentials were recorded in current clamp mode in the early stage (a, b) and in the late stage (c, d) of differentiation. Each bar indicates the mean of total membrane capacitance values in the early stage (filled bars) and the late stage (open bars) in panel (e). *p < 0.05 compared with P19CL6 in the early stage

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  Fig. 3. Transient outward currents (Ito) in P19CL6-derived cardiomyocytes and P19CL6-Csx-derived cardiomyocytes. a Representative current traces from P19CL6-derived cardiomyocytes in the early stage without 4-AP (i), with 5 mmol L−1 4-AP (ii), and (iii) the difference between the two (i)–(ii). Currents were elicited by depolarizing pulses from a holding potential of ‒ 80 mV to the test potentials between ‒ 30 and + 50 mV (20 mV increments). b Current densities were plotted against test potentials in P19CL6-derived cardiomyocytes (left panel) and P19CL6-Csx-derived cardiomyocytes (right panel). *p < 0.05 compared with P19CL6 cells in the early stage

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  Fig. 4. Delayed rectifier K+ currents expressed in P19CL6-derived cardiomyocytes and P19CL6-Csx-derived cardiomyocytes. Current–voltage relationships of E4031-sensitive currents (IKr) (a, b), and chromanol 293B-sensitive currents (IKs) (c, d). Each inset shows representative current traces (at 0 mV in IKr, at + 30 mV in IKs,) before (white up-pointing triangle) and after (black up-pointing triangle) application of each inhibitor. Scales of insets are 200 ms and 100 pA for IKr, 500 ms and 200 pA for IKs. IKr was elicited by 1 s-depolarizing pulses from a holding potential of ‒ 40 mV to the test potentials between ‒ 30 and + 40 mV (10 mV increments). IKs was elicited by the same protocol as IKr, except for the duration of depolarizing pulse (3 s). Current amplitude was measured at the end of the depolarizing test pulse and normalized by cell capacitance. *p < 0.05 compared with P19CL6 cells in the early stage

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  Fig. 5. Inward rectifier K+ currents (IK1) and ACh-activating K+ currents (IK,ACh) in P19CL6-derived cardiomyocytes and P19CL6-Csx-derived cardiomyocytes. Current–voltage relationships of IK1 (a, b) and IK,ACh (c, d). Scales of insets: 50 ms and 200 pA for IK1, 50 ms and 500 pA for IK,ACh. IK1 currents were elicited by hyperpolarizing and depolarizing pulses from a holding potential of ‒ 40 mV to the test potentials between ‒ 110 and + 20 mV (10 mV increments). Each inset shows representative current traces at ‒ 110 mV before (white up-pointing triangle) and after (black up-pointing triangle) application of 200 µmol L−1 Ba2+. IK,ACh was elicited by hyperpolarizing and depolarizing pulses from a holding potential of ‒ 50 mV to the test potentials between ‒ 120 and + 40 mV (20 mV increments). Each inset shows representative current traces at ‒ 120 mV before (white up-pointing triangle) and after (black up-pointing triangle) application of 20 µmol L−1 carbachol. *p < 0.05 compared with P19CL6 cells in the early stage, and †p < 0.05 compared with P19CL6-Csx cells in the early stage

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  Fig. 6. ATP-sensitive K+ currents (IK,ATP) in P19CL6-derived cardiomyocytes and P19CL6-Csx-derived cardiomyocytes. Sample current recordings at a holding potential of ‒ 40 mV (a, b). Instantaneous current–voltage relationships (c, d) were obtained by ramp pulses from + 60 mV to ‒ 120 mV at the rate of ‒ 0.18 V/s, before (i) and after (ii) application of 100 µmol L−1 cromakalim. Group data (n = 4) for the cromakalim-sensitive current, IK,ATP (ii)–(i) are plotted in the early stage (dotted line) and in the late stage (solid line)