Targeting Myadm to Intervene Pulmonary Hypertension on Rats Before Pregnancy Alleviates the Effect on Their Offspring’s Cardiac-Cerebral Systems

Pregnancy with pulmonary hypertension (PH) seriously threatens the life and safety of mothers and infants. Here, the long-term effect of maternal PH on the postpartum growth of rat offspring was focused for the first time, as well as explored the role of Myadm in PH rats before pregnancy based upon the previous findings. Patients with PH are prone to hypoxemia, leading to insufficient placental structure and function, which affects the organ function of fetuses, followed by evidence that differently expressed genes (DEGs) existed in the heart of maternal PH newborn rats and enriched in pathways related to cardiac and nerve development on human infants with similar birth outcome: low birth weight (LBW). LBW was one of the possible birth outcomes of pregnancy with PH, especially severe PH, accompanied by evidence that offspring derived from mothers with PH presented lower birth weights and slower growth rates than those derived from normal control mothers in a rat model. Besides, maternal PH rat offspring showed cardiac remodeling and a significant elevation of the expression levels of hypoxia- and inflammation-related markers in the cerebral cortex at both 10 and 14 weeks of age, respectively. What is more, the previous studies found that the overexpression of Myadm could result in the remodeling of the pulmonary artery. And targeting Myadm to intervene PH before pregnancy could alleviate sustained low weight growth in maternal PH rat offspring, and the pathological changes of the cardiac–cerebral system caused by maternal PH, including enlarged right heart cavity, loss of cardiomyocytes, abnormal heart index, as well as cerebral cortex hypoxia and the inflammatory state as they grew up to a certain extent. The findings show the pathological significance of maternal PH on offspring growth and the cardiac–cerebral development in a rat model, as well as point out the potential treatment target, which may provide a further reference for pregnancy outcomes in women with PH and healthy development of offspring to some extent.

. The establishment and evaluation of pregnancy with MCT-induced PH rat model. A. Representative example of the pulsed-wave Doppler of pulmonary flow recorded in each group. Yellow arrow indicates the second wave peak, which is one of the pulmonary flow characteristics of PH. B. Schematic of the experimental setup for the establishment of pregnancy with MCT-induced PH rat model. C. Representative H&E staining of pulmonary artery and heart of maternal rat in each group. The left images show the entire pulmonary artery (1.2× magnification) and heart (0.6× magnification). The bar indicates 1000 μm (pulmonary artery) and 2000 μm (heart), respectively. The right images show the section of pulmonary artery (20.0× magnification) and right ventricular wall (10.0× magnification). The bar indicates 50 μm (pulmonary artery) and 100 μm (heart), respectively. Yellow arrows show thickened pulmonary artery and increased right ventricular cavity. PH, pulmonary hypertension.
Supplementary Figure 2B showed the experimental setup for the establishment of shRNA-Myadm intervene maternal PH rat model. And the interference efficiency of shRNA-Myadm evaluated by western blot analysis and RT-qPCR (Supplementary Figure 2C). After they got pregnant, their right heart function were evaluated by ultrasound. Supplementary Figure 2A showed the pulsed-wave Doppler of pulmonary flow in each group. Compared with normal control, the spectrum showed decreased peak in PH group. In addition, yellow arrow indicated the second wave peak, which is one of the pulmonary flow characteristics of PH. However, silencing Myadm of PH rats could alleviate their pulmonary flow. Moreover, the maternal rats in each group were sacrificed at the end point of the study. Then, their pulmonary arteries and hearts were collected and fixed with 4% paraformaldehyde, embedded in paraffin, sliced into 5μm sections and then stained for haematoxylin and eosin (H&E) staining. Compared with normal control, pulmonary artery of PH rats were thickened, and their right ventricular cavity increased, which could be alleviated by Myadm intervention (Supplementary Figure 2D), which consisted with our previous findings.

Supplementary Figure 2.
The establishment and evaluation of shRNA-Myadm intervene maternal PH rat model. A. Representative example of the pulsed-wave Doppler of pulmonary flow recorded in each group. Yellow arrow indicates the second wave peak, which is one of the pulmonary flow characteristics of PH. B. Schematic of the experimental setup for the establishment of shRNA-Myadm intervene maternal PH rat model. C. The interference efficiency of shRNA-Myadm evaluated by western blot analysis and RT-qPCR (n=3 rats per group). D. Representative H&E staining of pulmonary artery and heart of maternal rat in each group. The left images show the entire pulmonary artery (1.4× magnification) and heart (0.4× magnification). The bars indicate 1000 μm (pulmonary artery) and 2000 μm (heart), respectively. The right images show the section of pulmonary artery (20.0× magnification) and right ventricular wall (10.0× magnification). The bars both indicate 50 μm. Yellow arrows show thickened pulmonary artery and increased right ventricular cavity. NC, normal control. PH, pulmonary hypertension.
In the main text, we explored the long-term effects of maternal PH on the whole rat offspring for the first time. Half male and half female offspring were contained in every examination. In supplementary materials, the data of male and female offspring subgroups are placed with the results of whole group offspring in the following four figures. . Each result of the whole group of offspring (n=6 rats per group with half male and half female). F. Western blot analysis of MMP2, MMP9, COX-2, iNOS and HMGB1 in the cerebral cortex of offspring in each group at 10 weeks of age (a) and 14 weeks of age (b). NC, normal pregnancy group; PH, pregnancy with PH group; M, male; F, female. * represents the statistical significance between NC and PH. # represents the statistical significance between male and female rats in the same group. *(#)p<0.05, **(##)p<0.01, ***(###)p<0.001, ****(####) p<0.0001

Supplementary
Supplementary Figure 6. Abnormal cardiac changes in offspring in the PH pregnancy group. A. The heart index (weight of heart/body weight) of offspring at ten weeks of age and fourteen weeks of age in each group. B. The expression change of ET-1 in the plasma in each group at ten weeks of age and fourteen weeks of age determined by ELISA. C. The expression change of BNP in the heart tissue in each group at ten weeks of age and fourteen weeks of age determined by ELISA. D. The mRNA expression of TGF-β1, TGF-β2, MMP2 and TIMP2 in the heart tissue of offspring in each group at ten weeks of age. E. The mRNA expression of TGF-β1, TGF-β2, MMP2 and TIMP2 in the heart tissue of offspring in each group at fourteen weeks of age. (a). Each result with sex subgroup (n=3 rats per group); (b). Each result of the whole group of offspring (n=6 rats per group with half male and half female). NC, normal pregnancy group; PH, pregnancy with PH group; M, male; F, female. * represents the statistical significance between NC and PH. # represents the statistical significance between male and female rats in the same group. *(#)p<0.05, **(##)p<0.01, ***p<0.001, **** p<0.0001 Figure 7. Boxplot of maternal PH severity and foetal birth weight.