Glucose Counteracts Isoprenaline Effects on Ion Channel Functions in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Figures

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  Figure 1. High level of glucose counteracted the effects of high-concentration isoprenaline on action potentials. “NC” represents data from hiPSC-CMs treated with normal level of glucose (5.5 mM). “Ni” represents data from hiPSC-CMs treated with normal level of glucose and high concentration (1 mM for 1 h) of Iso. “HC” represents data from hiPSC-CMs treated with high level (22 mM for 14 days) of glucose. “Hi” represents data from hiPSC-CMs treated with a high level of glucose and a high concentration of Iso. Action potentials (AP) were recorded at 1 Hz stimulation. (A) Representative traces of APs in different groups. (B) Mean values of resting potentials (RP). (C) Mean values of maximal depolarization velocity of AP (Vmax). (D) Mean values of action potential amplitude (APA). (E) Mean values of action potential duration at 10% repolarization (APD10). (F) Mean values of action potential duration at 50% repolarization (APD50). (G) Mean values of action potential duration at 90% repolarization (APD90). Data are presented as mean ± SEM and analyzed by one-way ANOVA with Holm-Sidak post-test. The cell number of every experimental group is given as “n”. ** p < 0.01, *** p < 0.001. “n.s.” means…

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  Figure 2. Effects of high-concentration isoprenaline and a high level of glucose on sodium channel currents. “NC” represents data from hiPSC-CMs treated with normal level of glucose. “Ni” represents data from hiPSC-CMs treated with normal level of glucose and a high concentration of Iso. “HC” represents data from hiPSC-CMs treated with a high level of glucose. “Hi” represents data from hiPSC-CMs treated with a high level of glucose and a high concentration of Iso. Sodium channel currents (INa) were recorded using protocols shown in (D,F,G) (inset). The late INa was measured as the area under the current curve to zero line from 300 ms to 350 ms. (A) Representative traces of peak INa at −40 mV in cells from each group. (B) Representative traces showing late INa at −40 mV in cells from each group. (C) Mean values of late INa at −40 mV. (D) I–V curves of peak INa in cells of each group. (E) Activation curves of peak INa in cells of each group. (F) Inactivation curves of peak INa in cells of each group. (G) Peak INa curves of recovery from inactivation in cells of each group. (H) Mean values of peak INa at −40 mV. (I) Mean values of voltage at 50% activation. (J) Mean values of voltage at 50%…

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  Figure 3. Effects of high-concentration isoprenaline and a high level of glucose on transient outward potassium current (Ito). “NC” represents data from hiPSC-CMs treated with normal level of glucose. “Ni” represents data from hiPSC-CMs treated with normal level of glucose and a high concentration of Iso. “HC” represents data from hiPSC-CMs treated with a high level of glucose. “Hi” represents data from hiPSC-CMs treated with a high level of glucose and a high concentration of Iso. Transient outward potassium current (Ito) was recorded using protocols shown in (A,B) (inset). (A) Representative traces of peak Ito at 40 mV in cells from each group. (B) I–V curves of peak Ito in cells of each group. (C) Mean values of peak Ito at 40 mV. Data are presented as mean ± SEM and analyzed by one-way ANOVA with Holm-Sidak post-test. The cell number of every experimental group is given as “n”. * p < 0.05.

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  Figure 4. High-concentration isoprenaline and a high level of glucose induced ROS generation in hiPSC-CMs. Cells were incubated with 5 μM DCFH-DA solution in serum-free medium at 37 °C for 30 min in the dark in each cell group. A fluorescence microscope was used to evaluate the fluorescence intensity of cells. “NC” represents data from hiPSC-CMs treated with normal level of glucose. “Ni” represents data from hiPSC-CMs treated with normal level of glucose and a high concentration of Iso. “HC” represents data from hiPSC-CMs treated with a high level of glucose. “Hi” represents data from hiPSC-CMs treated with a high level of glucose and a high concentration of Iso. (A) Representative pictures showing the fluorescence intensity of cells in different groups. (B) Statistical analyses of fluorescence intensity representing levels of ROS. Magnification, ×400. Data are presented as mean ± SEM and analyzed by one-way ANOVA with Holm–Sidak post-test. The cell number of every experimental group is given as “n”. *** p < 0.001. “n.s.” means no significance.

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  Figure 5. Immunofluorescence analysis of β1 (ADRB1) in hiPSC-CMs cultured with high concentration of glucose. “NC” represents data from hiPSC-CMs treated with normal level of glucose. “HC” represents data from hiPSC-CMs treated with a high level of glucose. (A) Immunofluorescence detecting the expressions of α-actinin (red) and ADRB1 (green). The cell nuclei were stained by DAPI (blue). (B) Statistical analyses of the fluorescence intensity representing the expression level of ADRB1. Data are presented as mean ± SEM and analyzed by t-test. The cell number of every experimental group is given as “n”. *** p < 0.001.

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  Figure 6. qPCR assay of PI3K, G protein, PKA, and CaMKII expression in hiPSC-CMs cultured with a high level of glucose. (A) The expression level of mRNA for PI3-kinase (PI3K) in hiPSC-CMs with normal (NC) and high glucose (HC). (B)The expression level of Gs (GNAS), Gi (GNAI2), Gq (GNAQ and GNA11) in hiPSC-CMs with normal (NC) and high glucose (HC). (C) The expression level of PKA (PRKACA) and CaMKII (CAMK2A) in hiPSC-CMs treated with normal (NC) and high concentration (HC) of glucose. Data are presented as mean ± SEM and analyzed by t-test. (n = 3, biological replicates). ** p < 0.01.

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  Figure 7. A PKA blocker attenuated the toxic effects of isoprenaline on action potentials in hiPSC-CMs. “Ctr” represents data from hiPSC-CMs treated with vehicle. “H-89” represents data from hiPSC-CMs treated with H-89 (10 µM). “Iso” represents data from hiPSC-CMs treated with a high concentration of Iso (1 mM). “H-89+Iso” represents data from hiPSC-CMs treated with H-89 plus a high concentration of Iso. Action potentials (AP) were recorded at 1 Hz stimulation. (A) Mean values of resting potentials (RP). (B) Mean values of maximal depolarization velocity of AP (Vmax). (C) Mean values of action potential amplitude (APA). (D) Mean values of action potential duration at 90% repolarization (APD90). (E) Mean values of action potential duration at 50% repolarization (APD50). (F) Mean values of action potential duration at 10% repolarization (APD10). Data are presented as mean ± SEM and analyzed by one-way ANOVA with Holm–Sidak post-test. The cell number of every experimental group is given as “n”. ** p < 0.01, *** p < 0.001. “n.s.” means no significance.

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  Figure 8. A PKA blocker attenuated the toxic effects of isoprenaline on transient outward currents (Ito) and late sodium currents in hiPSC-CMs. “Ctr” represents data from hiPSC-CMs treated with vehicle. “H-89” represents data from hiPSC-CMs treated with H-89. “Iso” represents data from hiPSC-CMs treated with a high concentration of Iso. “H-89+Iso” represents data from hiPSC-CMs treated with H-89 and a high concentration of Iso. (A) I–V curves of Ito in cells of each group. (B) Mean values of peak Ito at 40 mV in cells of each group. (C) Representative traces showing late INa at −40 mV in cells of each group. (D) Mean values of late INa at −40 mV in cells of each group. The late INa was measured as the area under the current curve to zero line from 300 ms to 350 ms. Data are presented as mean ± SEM and analyzed by one-way ANOVA with Holm–Sidak post-test. The cell number of every experimental group is given as “n”. ** p < 0.01, *** p < 0.001.