Fig. 1. Expression of cardiac-specific genes. RT-PCR experiments show the expression of cardiac specific genes (MLC 2V: 499 bp, MLC 2a: 286 bp, α-MHC: 302 bp, β-MHC: 205 bp, α-cardiac actin: 99 bp and Nkx 2.5: 216 bp) and β-actin (internal standard, 510 bp) in mES-derived cardiomyocytes after 20 days in vitro induction (D-20) and adult mouse heart (heart). The identities of the amplicons were confirmed by sequencing. The minus sign (−) indicates reaction without cDNA as control.

Fig. 2. Measurement of resting [Na⁺]_i using SBFI. Cells were loaded with SBFI-AM for 2 h. The intensity of SBFI was recorded every 3 s with the cooled CCD camera. (A) In undifferentiated mES cells, the time course of fluorescence was plotted before and after changing the bath solution containing 5 mM Na⁺ and 10 μM digitonin. The intensity of SBFI was gradually increased and then abruptly decreased. (B) When the bath solution was changed to 10 mM [Na⁺]_i and 10 μM digitonin, the intensities of SBFI decreased with a time course. (C) We plotted the points of sudden change in fluorescence indicated by an arrow in A and B. The null point was determined by drawing a linear regression curve indicated by a dot line. The X-intercept indicated by an arrow means the resting [Na⁺]_i. These data were obtained from 249 mES cells.

Fig. 3. [Na⁺]_i measurement by fluorescence. (A) Cells were loaded with SBFI-AM for 2 h. SBFI fluorescence was recorded every 10 s with the cooled CCD camera. Before experiments, cells were incubated in the K⁺ free solution for 10 min to block Na⁺/K⁺ ATPase. Images were taken before (a) and 10 min after bathing the cells with 10 mM K⁺ solution to activate the Na⁺/K⁺ (pump) ATPase (b). High intensity indicates low [Na⁺]_i and low intensity does high [Na⁺]_i. White bar indicates 10 μm. (B) The time courses of [Na⁺]_i were plotted in undifferentiated mES cell (upper panel) and 15-day derived cardiomyocyte (lower panel). We used pseudo ratio −δF/F₀: −δF/F₀ = −(F − F_base)/F_base, F: measured fluorescence intensity of SBFI, F_base: the lowest level of fluorescence intensity in the cell. The ordinate (−δF/F₀) corresponds to [Na⁺]_i. (C) [Na⁺]_i changes (|δF/F₀|) before and after 10 mM K⁺ were measured in the derived cardiomyocytes at various stages. Values were 0.037 ± 0.009 (n = 24) in undifferentiated cells, 0.089 ± 0.009 (n = 63) in derived cardiomyocytes for 4–6 days in vitro induction (early stage), 0.147 ± 0.019 (n = 145) for 7–11 days induction (middle stage) and 0.157 ± 0.014 (n = 68) for 12–18 days induction (late stage). Significant changes in [Na⁺]_i were indicated as * compared with mES cells and as § compared with early stage of differentiation. Both P values were <0.001.

Fig. 4. Ouabain effects on [Na⁺]_i. (A and B) The representative fluorescent [Na⁺]_i images were taken in control (left), 10 min after application of 30 μM ouabain (middle) and 3 mM ouabain (right) in undifferentiated mES cells (mES) (A) and 15-day derived cardiomyctyes (B). Cells were bathed in the 10 mM K⁺ solution for more than 10 min before application of ouabain to activate the Na⁺/K⁺ (pump) ATPase. White bar indicates 10 μm.

Fig. 5. Functional development of Na⁺/K⁺ (pump) ATPase. (A and B) The time course of ouabain effects on [Na⁺]_i was plotted in undifferentiated mES cell (a, left row) and 15-day derived cardiomyocyte (b, right row). The ordinate (−δF/F₀) corresponds to [Na⁺]_i. (C) [Na⁺]_i changes (−δF/F₀) before and after application of 30 μM (a) or 3 mM ouabain (b) were measured in the derived cardiomyocytes at various stages of differentiation (n = 17–53 cells). Significant changes in [Na⁺]_i were indicated by * compared with mES cells and § compared with early stage of differentiation. Both P values (* and §) were <0.01.

Fig. 6. Direct recording of I_p. (A) The I_p was activated by 10 mM K⁺ solution after blocking Na⁺/K⁺ ATPase by K⁺ free solution. Ramp clamp pulses (200 ms) were applied from −70 to +70 mV at every 20 s in the whole cell patch clamp mode. (a and b) The representative ramp clamp currents in K⁺ free solution (1) and 10 min after changing the bath solution to 10 mM K⁺ one (2) were superimposed in undifferentiated mES cells (a) and 15-day derived cardiomyoctes (b). I_p was defined as a subtracting current [(2) − (1)] in each trace (right row). (B) The I_p was obtained as an ouabain-sensitive current. (a and b) An ouabain-sensitive current was obtained by subtracting currents before (2) and after application of 30 μM ouabain (1), [(2) − (1)] in undifferentiated mES cell (a) and 15-day derived cardiomyoyctes (b).

Fig. 7. Functional expression of Na⁺/Ca²⁺ exchangers. Cells were loaded with 5 μM fura red-AM for 30 min at 37 °C in the standard bath solution to monitor [Ca²⁺]_i. Fura read fluorescence was recorded every 10 s with the confocal microscopy. (A) When the function of NCXs were blocked by the application of 100 μM KB-R7943, a [Ca²⁺]_i transient was observed in undifferentiated mES cells (a). In 15-day derived cardiomyocytes, KB-R7943 induced the sustained elevation of resting level of [Ca²⁺]_i (b). (B) The numbers of the cells were summarized in: (a) showing a [Ca²⁺]_i transient pattern and (b) showing a sustained [Ca²⁺]_i elevation pattern responded to KB-R7943 at various stages of differentiation (n = 11 − 32 cells). (C) The intensities of fluorescence 30 min after application of drug were measured at various differentiated stages (n = 11 − 32 cells). Significant differences were indicated by * and § compared with mES cell and early stage of differentiation, respectively. Both P values (* and §) were <0.001. Mean values between middle stage and late stage were also significant different (P < 0.01).

Fig. 8. Ouabain effects on [Ca²⁺]_i. Cells were loaded with fluo3-AM for 30 min to monitor [Ca²⁺]_i. Fluo-3 fluorescence was recorded every 10 s with the confocal microscopy. (A) The application of 30 μM ouabain did not change [Ca²⁺]_i in undifferentiated mES cells (a) but induced [Ca²⁺]_i oscillations in 15-day derived cardiomyocytes. (B) The numbers of cells showing [Ca²⁺]_i oscillations by the application of ouabain were summarized. [Ca²⁺]_i oscillations were not observed in undifferentiated mES cells (0/63 cells), but observed in 6.81% cells (3/44), 77.8% cells (28/36) and 75% cells (12/16) in the derived cardiomyocytes at early stage, middle stage and late stage, respectively.

Fig. 9. RT-PCR analysis of Na⁺/K⁺ ATPase and NCX. A, The expression of mRNA for α1, α2, and α3 subunit Na⁺/K⁺ ATPase in mES cells and derived cardiomyocytes at various stages of differentiation (early, middle and late stage). (B) The expression of NCX1 in mES cells during the differentiation. RT-PCR analysis was performed after equalization of cDNAs for mouse β-actin. Heart: adult mouse heart cDNA. (−): negative control (without cDNA). The identity of these amplicons was confirmed by sequencing. Band size is α1, α2, and α3 isoform Na⁺/K⁺ ATPase is 255, 348 and 408 bp, respectively, and 631 bp in NCX1.

Primer used for PCR

Summary of resting [Na⁺]_i

The resting [Na⁺]_i in mES cells and derived cardiomyocytes is estimated by null-point methods same as Fig. 2. These values of resting [Na⁺]_i are not significantly different among them.