Analysis of maternal and fetal cardiovascular systems during hyperglycemic pregnancy in the nonobese diabetic mouse

Abstract

Preconception or gestationally induced diabetes increases morbidities and elevates long-term cardiovascular disease risks in women and their children. Spontaneously hyperglycemic (d)-NOD/ShiLtJ female mice, a type 1 diabetes model, develop bradycardia and hypotension after midpregnancy compared with normoglycemic, age- and gestational day (GD)-matched control (c-NOD) females. We hypothesized that onset of the placental circulation at GD 9-10 and rapid fetal growth from GD 14 correlate with aberrant hemodynamic outcomes in d-NOD females. To develop further gestational time-course correlations between maternal cardiac and renal parameters, high-frequency ultrasonography was applied to d- and c-NOD mice (virgin and at GD 8-16). Cardiac output and left ventricular (LV) mass increased in c-NOD but not in d-NOD mice. Ultrasound and postmortem histopathology showed overall greater LV dilation in d-NOD than in c-NOD mice at mid to late gestation. These changes suggest blunted remodeling and altered functional adaptation of d-NOD hearts. Umbilical cord ultrasounds revealed lower fetal heart rates from GD 12 and lower umbilical flow velocities at GD 14 and GD 16 in d-NOD versus c-NOD pregnancies. From GD 14 to GD 16, d-NOD fetal losses exceeded c-NOD fetal losses. Similar aberrant responses in pregnancies of women with diabetes may elevate postpartum maternal and child cardiovascular risk, particularly if mothers lack adequate prenatal care or have poor glycemic control during gestation.

Keywords: NOD mouse; cardiac adaptations; cardiovascular; cardiovascular system; developmental origins of health and disease; diabetes; pregnancy.

FIG. 1

Maternal Cardiac Analyses. (A) An example of a B-Mode image of parasternal short axis view of the heart from a gd16 d-NOD. M-Mode sample volume was placed over the middle of the chamber, slightly off the papillary muscle. (B) M-Mode cine loop of parasternal short axis of the heart, also from a gd16 d-NOD. Measurements were performed excluding papillary muscle, seen as caps above the posterior wall in systole. (C) Cardiac output normalized by viable implant site number. Increasing CO is seen over gestation in c-NOD (p=0.025), but not d-NOD mice. Effect of gestation (analyzed via regression slopes) differed between groups (p=0.024). (D) Stroke volume normalized for viable implant site number. Increasing SV is seen over gestation in c-NODs (p=0.035). Again, no increase in SV is observed over gestation in d-NODs. Effect of gestation was different between groups (p=0.030). (E) Fractional shortening. FS was increased in d-NOD compared to c-NODs across the study (p=0.0265), however no significance was reached at any specific gd. No effect of gestation is seen on FS in either d-NOD or c-NOD dams. PM = Papillary muscle, LV = Left ventricle, AW = Anterior left ventricular wall, PW= Posterior left ventricular wall. Solid line and closed circles = c-NODs, dashed lines and open circles = d-NODs. Significantly different slopes upon regression analysis indicated differing effects of gestation between groups, slopes by linear regression considered significantly non-zero at p<0.05.

FIG. 2

Left ventricular size and extent of dilation, calculated using ultrasonography and histological analyses. (A) Left ventricle diastolic diameter (mm) measured by ultrasound increased throughout gestation in c-NOD (p=0.014) but not d-NOD mice (p=0.42). Regression analysis showed a difference in slopes between the groups (p=0.017). (B) Ultrasound measurements in c-NODs showed increasing LV mass over gestation (p=0.050). No increase or trend was seen in d-NODs. Regression analysis showed a trend towards a difference in slopes (p=0.15). Values normalized to number of viable implantation sites. (C) Histological cross-sections of ventricles at the level of the papillary muscles stained with H&E, scale bar reports 500μm and refers to all panels. (D) Ratio of LV lumen area: total ventricular area was used as a measure of ventricular dilation. Overall the ratio was increased in d-NOD compared to c-NODs (p=0.028). Differences at specific gestation days reached significance at gd14. Ratios were measured using histological morphometry. Solid line, closed circles and solid bars = c-NODs, dashed lines, open circles and white bars = d-NODs. * = Difference between groups. Significance between groups were determined by two-way ANOVA with p<0.05. Increases over gestation within groups were determined by linear regression with a non-zero slope (p<0.05), non-statistically significant trends over gestation defined at p<0.15.

FIG. 3

Renal physiology assessed via ultrasonography and histolopathology. (A) Example of a B-Mode image of the left kidney of a gd12 d-NOD. (B) PW Doppler of a gd10 d-NOD renal artery, measurements taken on peak systolic velocity and end diastolic velocity. (C) Renal artery RI did not differ between c-NOD and d-NODs prior to mating (data not shown) or at any gestational time-point. (D) Renal glomeruli in histological sections stained with PAS, scale bar reports 20 μm. No qualitative differences were observed between d-NOD and c-NODs from gd8 to gd16. (E) Semi-quantitative renal histopathological analysis showing no differences in renal pathology score between d-NOD and c-NODs at any gestational time-points studied. Solid bars = c-NOD, white bars = d-NOD. Difference between groups was determined by two-way ANOVA. Effect of gestation within groups was determined by linear regression.

FIG. 4

Fetal Scan. (A) Representative B-Mode image of a gd10 c-NOD fetus. (B) Representative PW Doppler image of umbilical artery in a gd10 c-NOD fetus. (C) Representative PW Doppler image of the umbilical artery in a gd16 c-NOD fetus. (D) Umbilical artery peak flow velocities, C-NODs showed an increase with increasing gestation (p=0.0012). Within time-points, c-NOD flow velocities are increased compared to d-NODs (p<0.0001), peak differences at gd14 and gd16 (p<0.001, p<0.05, respectively). A significant interaction was present between variables (p=0.047). (E) Umbilical artery mean flow velocities. C-NOD but not d-NOD peak flow velocities increased over gestational time-points measures (p=<0.0001) and differences between groups peaked at gd14 and gd16 (p<0.001, p<0.01, respectively). (F) Fetal heart rates. Similar to umbilical flow velocity, c-NOD fetal heart rates increased throughout gestation (p<0.0001) and are, overall, higher than fetuses of d-NOD dams (p<0.001). (G) Fetal loss as measured by percentage of resorptions to viable pups. Overall, fetal loss was elevated in d-NOD compared to c-NODs (p=0.036). Early gestation days for this study were defined as gd10 and 12, later gestation days were defined as gd14 and 16. Differences between groups did not differ at early gestation days but reached significance at later gestation days (p<0.05). Minimum n=9 per group for all ultrasound data (minimum of three fetuses per dam, 3 dams per group at each gd). Significance between groups at each gestation day determined by two-way ANOVA with Bonferroni’s post-test, with p<0.05. Effect of gestation within each group determined by linear regression, increases and decreases over time defined by non-zero slope with p<0.05. Solid bars = c-NOD, white bars = d-NOD. Umb = Umbilical cord, Pl = Placenta. Single asterisks = significance between glycemic groups; hash tag = significance from gd10; dollar sign = significance from gd12.