Functional expression and pharmaceutical efficacy of cardiac-specific ion channels in human embryonic stem cell-derived cardiomyocytes

Figures

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  Figure 1. Characterization of cardiomyocytes differentiated from H9 hESCs. (a) Bright field images of H9 cells at day 0 (left panel) and differentiating cells at day 30 (right panel). Scale bar = 100 μm (b) Differentiated cardiomyocytes (hESC-CMs) at day 30 were subjected to immunofluorescent staining with antibodies against α-SA (red), cTnT (green), or MLC2a (yellow). Nuclei were stained with DAPI, and the merged images are shown. Scale bar = 20 μm. (c) Protein expression in undifferentiated H9 cells, hESC-CMs, and adult human left ventricular tissue (adult hLV) was assessed by Western blot analysis with antibodies against pluripotency markers (OCT4 and NANOG) and cardiomyocyte markers (α-SA, cTnT, MLC2a, MLC2v, SERCA2a and Cx43). (d) The mRNA levels of α-MHC and β-MHC in hESC-CMs and adult hLV were accessed by quantitative RT-PCR and the ratio of β-MHC/α-MHC mRNA levels was determined (n = 3). *P < 0.05. (e) Gene expression in hESC-CMs at day 30 and hESCs was accessed by quantitative RT-PCR with cardiomyocyte markers (GATA4, GATA6, and cTnT) and pluripotency markers (OCT4 and NANOG) n = 3. *P < 0.05. (f) Flow cytometry analysis of hESC-CMs at day 30 with antibodies against cardiomyocyte…

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  Figure 2. Electrophysiological recordings of Na+ channels in hESC-CMs. (a) Superimposed current traces were evoked by 300-ms depolarizing step pulses from −100 to +70 mV from holding potential of −120 mV. (b) Representative current traces were elicited by the first step depolarizing pulses as described in (A), then returning to −40 mV for 1 s. The second identical step depolarizing pulses were applied. (c) The current-voltage (I-V) relationship for the panel (a, open circle) and (b, closed circle). n = 7. (d) Expression of Nav1.5 subtype in H9 cells and hESC-CMs was accessed by quantitative RT-PCR. n = 5. *P < 0.05.

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  Figure 3. Electrophysiological recordings of Ca2+ channels in hESC-CMs. Superimposed current traces were recorded by applying 500-ms step depolarizing pulses in the voltage range of −50 to +50 mV from a holding potential of −50 mV under control condition (a) and in the presence of 10 µM Nifedipine (b). (c) I-V relationship of peak current in the absence (open circle) and presence of Nifedipine (closed circle). n = 6. (d) Expression of Cav1.1, Cav1.2, and Cav3.1 subtypes in H9 cells and hESC-CMs was accessed by RT-PCR. n = 5. *P < 0.05.

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  Figure 4. Electrophysiological recordings of K+ channels in hESC-CMs. (a) Whole-cell K+ currents of hESC-CMs. (b) Summary of I-V relationship of panel (a). n = 5. (c) Expression of Kv11.1 subtype in H9 cells and hESC-CMs was accessed by quantitative RT-PCR. n = 5. *P < 0.05.

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  Figure 5. Action potential in hESC-CMs. Action potential was induced by minimal current injection to overcome threshold in current clamp mode. (a) Representative trace of action potential recorded in hESC-CMs. (b) Analysis of action potential. APD50 and APD90 means action potential durations at 50% and 90% of repolarization; RMP, resting membrane potential; TP, threshold potential. n = 7.

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  Figure 6. The effect of BIM (I) on L-type Ca2+ current in hESC-CMs. Superimposed current traces under control conditions (a) and in the presence of BIM (I) (1 μM) (b) were obtained by applying the same pulse protocol for Fig. 3. (c) I-V relationship at the peak Ca2+ current in the absence (open circle) and presence of BIM (I) (1 µM) (closed circle). n = 6. *P < 0.05. (d) Superimposed current traces were recorded by applying one-step depolarization pulse of 0 mV from a holding potential of −50 mV in the presence of BIM (I) (0, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μM). (e) BIM (I)-induced inhibition of the L-type Ca2+ current was measured at peak current, and normalized to the current amplitude observed in the absence of BIM (I). Normalized currents were fitted with the Hill equation. n = 6.

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  Figure 7. The effects of another PKC inhibitor, chelerythrine, on the BIM (I)-induced inhibition of L-type Ca2+ currents. (a) Superimposed current traces in control conditions, in the presence of Chelerythrine, and in the presence of Chelerythrine + BIM (I). (b) Summarized data from traces in (a). n = 6. *P < 0.05. NS = not significant.