Progesterone Changes the Pregnancy-Induced Adaptation of Cardiomyocyte Kv2.1 Channels via MicroRNA-29b

Figures

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  Figure 1. Pregnancy-induced changes in the electrophysiological properties and Kv channel expression of cardiomyocytes. HR (a and c) and QTc interval (b and d) of human subjects (nonpregnant subjects = 135 and pregnant subjects = 336) and model mice (n = 9 − 11 animals per group). (e) Representative recordings of the APs in cardiomyocytes stimulated by the rectangular pulse from nonpregnant and pregnant mice. Comparisons of the APD30 (f), APD50 (g), and APD90 (h) between the two groups (n = 12 cells from 4 animals per group). (i) qRT–PCR analysis of Kv channels in ventricles from nonpregnant and pregnant mice (n = 8 − 9 individual experiments). ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. NP. Comparisons of electrophysiological data were conducted by Student's t test. Comparisons of qPCR data were conducted by a one-way ANOVA followed by the Bonferroni post hoc test. NP: nonpregnant group; P: pregnant group; HR: heart rate; QTc: corrected QT; APD: action potential duration.

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  Figure 2. APD and Kv channel expression in response to P4 treatment. Changes in the HR (a) and QTc interval (b) caused by P4 treatment during pregnancy (pregnant subjects = 336 and pregnant subjects who received P4 medications = 117). The pregnant mice were injected daily with P4 (50 mg/kg/d) or sesame oil during gestation for 18 days. Age-matched nonpregnant mice injected with sesame oil served as controls (c). HR (d) and QTc interval (e) of pregnant mice with or without P4 injections. (f) Representative recordings of the APs in cardiomyocytes stimulated by the rectangular pulse from pregnant mice and pregnant mice that received P4 injections. (g–i) Comparisons of APD30, APD50, and APD90 between pregnant mice with and without P4 injections. (j) qRT–PCR analysis of Kv channels in ventricles from pregnant mice and pregnant mice treated with P4. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (n = 7 − 10 from 4-6 mice per group). Comparisons of data were conducted by Student's t test or one-way ANOVA followed by the Bonferroni post hoc test. P: pregnant group; P4: progesterone; HR: heart rate; QTc: corrected QT; APD: action potential duration.

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  Figure 3. Whole-cell Kv currents and Kv channel expression in response to P4 treatment in the H9C2 myocyte line. The H9C2 cells were exposed to 1 μM or 5 μM P4 and cultured for 48 h. The H9C2 cells without treatment and cultured under normal conditions were used as controls (Ctl). (a) Representative recordings of whole-cell voltage clamp experiments in control, 1 μM P4-, and 5 μM P4-treated H9C2 cells for 48 h. (b) The current–voltage (IV) curve from each group was analyzed at a steady state of the voltage-dependent currents (150 to 180 ms). (c) The current density in each group was compared at +60 mV. ∗p < 0.05 and ∗∗∗p < 0.001 vs. Ctl (n = 12 − 15 cells per group). Comparisons of data were conducted by a one-way ANOVA followed by the Bonferroni post hoc test. (d) qRT–PCR analysis of Kv channels in the H9C2 cells cultured under 1 μM and 5 μM P4 conditions (n = 8 − 9 individual experiments per group). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. Ctl. Comparisons of data were conducted by a one-way ANOVA followed by the Bonferroni post hoc test. P4: progesterone.

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  Figure 4. Effects of RU486 on the P4-regulated expression of Kv2.1. The H9C2 cells were exposed to 1 μM P4 and 5 μM P4 and cultured with/without 5 μM RU486 for 48 h. The H9C2 cells without any treatment and cultured under standard conditions were used as controls (Ctl). (a and c) qRT–PCR analysis of Kv2.1 in the H9C2 cells cultured under 1 μM and 5 μM P4 conditions with/without 5 μM RU486 application. (b and d) WB analysis of Kv2.1 in the H9C2 cells cultured under 1 μM and 5 μM P4 conditions with/without 5 μM RU486 application. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. Ctl (n = 4 − 6 individual experiments). Comparisons of data were conducted by a one-way ANOVA followed by the Bonferroni post hoc. P4: progesterone; RU486: an antagonist of P4; qRT–PCR: quantitative real-time polymerase chain reaction; WB: western blot.

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  Figure 5. Validation of Kv2.1 miR-29b targeting. (a) Schematic illustration of the hypothesis that progesterone facilitated Kv2.1 channel expression through miR-29b. (b) Structure diagram of the luciferase reporter of kcnb1 and its mutant. Seed match regions of kcnb1 and miR-29b are indicated as vertical lines. The mutation site of kcnb1 mRNA is indicated in blue. (c) qRT–PCR analysis of miR-29b in the ventricles of pregnant mice with/without P4 injections. (d) qRT–PCR analysis of miR-29b in the H9C2 cells treated with 5 μM P4 for 48 h. The H9C2 cells cultured without treatment served as negative controls (Ctl). (e) Luciferase assay for kcnb1. miR-29b mimics were cotransfected with the wild-type 3′-UTR of kcnb1 (kcnb1-WT) or its mutant (kcnb1-M) into 293 cells. Luciferase activities were measured after 24 h of culture (n = 3 individual experiments). qPCR (f), WB (g), and immunocytochemistry analysis (h) of Kv2.1 expression were conducted in the H9C2 cells from the different transfection groups that were cultured under 5 μM P4 conditions. The H9C2 cells transfected with scrambled RNA and cultured without P4 exposure were used as controls (NC). ∗∗p < 0.01 and ##p < 0.01 (n = 4–6 individual…

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  Figure 6. Effects of P4 on human Kv2.1 channel activities. (a) Representative Kv2.1 current traces derived from control, 5 μM P4 treatment, and 5 μM P4 with miR-29b mimics. (b) Representative Kv2.1 single-channel traces derived from control, 5 μM P4, and 5 μM P4 with miR-29b mimics. (c) Current–voltage (I–V) relationships of the steady-state Kv2.1 currents. (d) The current density at +50 mV with control, 5 μM P4, and 5 μM P4/miR-29b. (e) Changes in the probability of Kv2.1 channel opening. Values shown are the mean ± S.E.M. ∗p < 0.05 and ∗∗p < 0.01 vs. Ctl; #p < 0.05 and ##p < 0.01 vs. 5 μM P4 (n = 9 − 10 cells per group). Data were compared by a one-way ANOVA followed by the Bonferroni post hoc test. P4: progesterone.

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  Figure 7. Schematic illustration summarizing the conclusion that progesterone regulated cardiomyocyte Kv2.1 channels via microRNA-29b and progesterone receptors.