Functional expression of potassium channels in cardiomyocytes derived from embryonic stem cells

Abstract

Royan B(1) stem cell can be differentiated to specialized cell types including cardiomyocytes. This developmental change is accompanied with expression of various K(+) channel types. The aim of this study was to detect functional expression of K(+) currents from stem cell stage and one week and two weeks after differentiation into cardiomyocyte. Mouse stem cell derived cardiomyocytes (ES-cardiomyocytes) were isolated to single cell suspension for K(+) current recording using whole cell patch-clamp technique. The predominant depolarizing current in ES-cardiomyocytes was a tetraethylammonium (TEA) (10 mM) sensitive current which was partially blocked by nifedipine (1 μM) and attenuated by increasing concentration of EGTA (10 mM) in the pipette solution. Pharmacology and electrophysiological properties of this oscillatory sustained current very well matched with characteristics of Ca(2+) activated K(+) current. In addition there was another kind of sustained outward K(+) current which was resistance to TEA but was inhibited by 3,4-diaminopyridine. The characteristic features of this current indicate that this current was due to activation of delayed rectifier K(+) channels. RT-PCR study also confirmed expression of these two types of K(+) channels in ES-cardiomyocytes. Therefore, present study shows functional expression of two types of K(+) ionic current in ES-cardiomyocytes.

Keywords: Cardiomyocytes; Embryonic stem cells; K+current; Patch-clamp; RT-PCR; Royan B1 line.

Fig. 1

(A) Current recorded from single undifferentiated stem cell when depolarized to +50 mV form holding potential of -60 mV (the recorded currrnt is mailly leak current). (B) Current-voltage (I/V) curve for outward sustanied current recorded from one old week ES-cardiomyocytes (n=6) compared with undifferentiated stem cells (n=7). The sustained current was elicited by depolarizing from holding potential of -60 mV to various test potentials for 300 ms. All the values are mean ± SEM. Mean current amplituedes were measured relative to holding current at -60 mV. There are statistically significant differences between current amplitudes in these two groups of cells (Stuedent's t-test, *P<0.05, **P<0.01).

Fig. 2

Block of sustained outward current in 7 ± 1 days old week ES-cardiomyocytes by TEA with 0.2 mM EGTA in KCl recording pipette. The sustained current was elicited by depolarising from the holding potential (-60 mV) to test potential up to +50 mV for 300 ms using 10 mV step increments. (A) Original control current records. (B) TEA (10 mM) blocked this current. (C) Current recorded after wash.

Fig. 3

Block of sustained outward current by TEA with 0.2 mM EGTA in recording pipette in 7 ± 1 days in ES-cardiomyocytes. Current-voltage (I/V) curve of the sustained current in control (circle) and presence of 10 mM TEA (triangle). The sustained current was elicited by depolarising from the holding potential (-60 mV) up to test potential of +50 mV for 300 ms using 10 mV step increment. All the values are mean ± SEM (n=6). Mean current amplitudes were measured relative to the holding current.

Fig. 4

Sustained outward current was reduced when there was 10 mM EGTA in recording pipette. Current/voltage (I/V) curve of the sustained current in control (with 0.2 mM EGTA), and test (10 mM EGTA) group cells. This comparison was made in one old week stem cell derived cardiomyocytes. All the values are mean ± SEM (n=6). Mean current amplitudes were measured relative to the holding current at -60 mV. Star shows statistically significant differences at some test potential between two groups of cells. Key: *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).

Fig. 5

Inhibition of sustained outward current by nifedipine (0.2 mM EGTA in recording pipette) in one old week ES-cardiomyocytes. (A) Original control current records. The sustained current was elicited by depolarising from the holding potential (−60 mV) to test potential up to +50 mV for 300 ms. (B) Nifedipine (1 μM) inhibited the current, but no recovery occurred on wash-out.

Fig. 6

Inhibition of sustained outward current by nifedipine (0.2 mM EGTA in recording pipette) in one old week EScardiomyocytes. Current-voltage (I/V) curve for the sustained current in; control (circle), and in the presence of nifedipine 1 μM (triangle). The sustained current was elicited by depolarising from the holding potential (-60 mV) to various test potentials up to +50 mV for 300 ms. Nifedipine (1 μM) partially inhibited the current. All values are mean ± SEM (n=5). Mean current amplitudes were measured relative to the holding current at -50 mV. Star shows where the inhibition of current is statistically significant. (* P>0.05, Student's t-test).

Fig. 7

Effect of 3,4-diaminopyridine on the TEA resistance current. 3,4-diaminopyridine (1 mM) reversibly blocked the current. Original current records before (A) and after addition of 3,4-diaminopyridine (B) when depolarised up to +40 mV for 300 ms from the holding potential (-70 mV) with 10 mM EGTA in the pipette solution. This study was made in two old weeks ES-cardiomyocytes.

Fig. 8

Current voltage (I/V) curve for TEA resistance current in; control (circle), and after perfusion of 3,4- diaminopyridine, (1 mM). This study was made in two old weeks ES-cardiomyocytes. Current was recorded by depolarised up to +40 mV for 300 ms with 10 mM EGTA in the pipette solution. Mean current amplitudes were measured relative to the holding current at -70 mV. All the values are mean ± SEM (n=7 for each group). Star shows where statistically significant differences exist between control current and current recoded after perfusion of 3,4-diaminopyrinde (*P<0.05, Student's t-test).

Fig. 9

Expression pattern of genes by reverse transcription – polymerase chain reaction (RT-PCR). Evaluation of genes expression large conductance Ca²⁺ activated K⁺ channel (kcnma1) and delayed rectifier K⁺ channel (kcna1) performed in three stages including mouse embryonic stem cells (day 0), one old week EScardiomyocytes (day 7) and two old weeks EScardiomyocytes (day 14).