Gestational diabetes triggers postpartum cardiac hypertrophy via activation of calcineurin/NFAT signaling

Figures

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  Figure 1. HIP females as a model of gestational diabetes. (A) HIP females (5–6 months of age) have reduced glucose tolerance compared to WT littermates, but fasting blood glucose is similar (n = 25 for WT, 30 for HIP). (B,C) Area under the curve in glucose tolerance tests performed in HIP (B) and WT (C) females in the pregnancy (N = 17 HIP and 14 WT in total) and control (N = 13 HIP and 11 WT in total) groups at baseline (BL), late (19–20 days) pregnancy, at the time of weaning the pups and 2 months postpartum. (D) Fasting blood glucose in HIP females from the pregnancy group at baseline and 2 months postpartum (N = 8). Statistical analysis was performed using Two-way ANOVA with Bonferroni’s multiple comparisons post-test for data in panels A-C and paired Student’s t-test for data in panel D.

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  Figure 2. Cardiac hypertrophy in two months postpartum HIP females. (A–F) Left ventricular mass (A,B), diastolic thickness of the interventricular septum (C,D) and diastolic thickness of the left-ventricular posterior wall (E,F) in WT (A,C,E) and HIP (B,D,F) females from control and pregnancy groups at baseline (BL), late pregnancy and 2 months postpartum. (G,H) Heart weight-to-body weight ratio of WT (G) and HIP (H) females at one day and 2 months postpartum. (I,J) Expression of ANP (I) and BNP (J) in hearts from control (Ctl), one day postpartum and 2 months postpartum WT and HIP females. Top panels show representative examples while bottom panels show relative band intensities; the experiment was repeated 4 times. For BNP, the intensity analysis included the bands with molecular weights between 15 and 20 kDa. In panels (A–H), statistical significance was determined using Two-way ANOVA with the Bonferroni post-test to compare the pregnancy versus control groups at each timepoint. In panels (I,J), data for animals in control (not pregnant) groups sacrificed at the time when pregnancy groups were at 1 day and 2 months postpartum were pooled. For these panels, statistical analysis was…

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  Figure 3. Nuclear versus cytosolic localization of NFATc4 in myocytes from HIP and WT females. (A) Examples of immunofluorescence images in myocytes from HIP females in the control (Ctl), 1 day postpartum (1d post) and 2 months postpartum (2mo post) groups. Cells were labeled with an anti-NFATc4 antibody. (B,C) Mean ratio of nuclear-to-cytosolic NFATc4 signal in cells from WT (B) and HIP (C) females in the control (WT: 4 females, 10–15 cells/rat; HIP: 6 females, 9–14 cells/rat), 1 day postpartum (WT: 3 females, 11–16 cells/rat; HIP: 5 females, 7–13 cells/rat) and 2 months postpartum (WT: 4 females, 10–11 cells/rat; HIP: 4 females, 9–12 cells/rat) groups. The number of cells per each rat is shown in Supplementary Table 1. (D–F) Expression of calcipressin-1 in hearts from control (Ctl), one day postpartum and two months postpartum WT and HIP females. Representative examples (D) and relative band intensities (E,F). Data for the sample loaded into Lane 7 of the WT gel were excluded from analysis. Data for animals in control groups sacrificed at the time when pregnancy groups were at 1 day and 2 months postpartum were pooled. Statistical analysis was performed with a mixed model using the…

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  Figure 4. Ca2+ transients in myocytes from control (Ctl), 1 day and 2 months postpartum females. (A,B) Representative examples of Ca2+ transients stimulated at 0.2, 0.5, 1 and 2 Hz in myocytes from control, non-pregnant HIP females (A) and two months postpartum HIP females (B). (C,D) Ca2+ transient amplitude in myocytes from HIP (C) and WT (D) females. (E,F) Decay time of Ca2+ transients triggered by pacing at 0.5 Hz in myocytes from HIP (E) and WT (F) females. (G,H) Diastolic Ca2+ as a function of stimulation frequency in myocytes from HIP (G) and WT (H) females. Measurements were done in females from control (WT: 6 females, 3-4cells/rat; HIP: 9 females, 3–7 cells/rat), 1 day postpartum (WT: 6 rats, 3–6 cells/rat; HIP: 8 rats, 2–6 cells/rat) and 2 months postpartum (WT: 3 rats, 6 cells/rat; HIP: 5 rats, 3–7 cells/rat) groups. The number of cells per each rat is shown in Supplementary Table 2. Data for animals in control groups sacrificed at the time when pregnancy groups were at 1 day and 2 months postpartum were pooled. Statistical analysis was performed with Two-way ANOVA for panels C, D, G and H, and with a mixed model using the Nested One-Way ANOVA analysis in Prism 8.1.2 for panels E…

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  Figure 5. Nuclear versus cytosolic localization of HDAC4 in myocytes from HIP and WT females. (A) Examples of immunofluorescence images in myocytes from HIP females in the control (Ctl), 1 day postpartum (1d post) and 2 months postpartum (2mo post) groups. Cells were labeled with an anti-HDAC antibody. (B,C) Mean ratio of nuclear to cytosolic signal in cells from WT (B) and HIP (C) females in the control (WT: 3 females, 10–12 cells/rat; HIP: 5 females, 9–12 cells/rat), 1 day postpartum (WT: 3 females, 10–12 cells/rat; HIP: 4 females, 9–12 cells/rat) and 2 months postpartum (WT: 3 females, 10–11 cells/rat; HIP: 4 females, 6–10 cells/rat) groups. The number of cells per each rat is shown in Supplementary Table 3. Data for animals in control groups sacrificed at the time when pregnancy groups were at 1 day and 2 months postpartum were pooled. Statistical analysis was performed with a mixed model using the Nested One-Way ANOVA analysis in Prism 8.1.2 and Dunnett’s post-test for multiple comparisons.