Transcriptome and morphological analysis on the heart in gestational protein-restricted aging male rat offspring

Abstract

Background: Adverse factors that influence embryo/fetal development are correlated with increased risk of cardiovascular disease (CVD), type-2 diabetes, arterial hypertension, obesity, insulin resistance, impaired kidney development, psychiatric disorders, and enhanced susceptibility to oxidative stress and inflammatory processes in adulthood. Human and experimental studies have demonstrated a reciprocal relationship between birthweight and cardiovascular diseases, implying intrauterine adverse events in the onset of these abnormalities. In this way, it is plausible that confirmed functional and morphological heart changes caused by gestational protein restriction could be related to epigenetic effects anticipating cardiovascular disorders and reducing the survival time of these animals. Methods: Wistar rats were divided into two groups according to the protein diet content offered during the pregnancy: a normal protein diet (NP, 17%) or a Low-protein diet (LP, 6%). The arterial pressure was measured, and the cardiac mass, cardiomyocytes area, gene expression, collagen content, and immunostaining of proteins were performed in the cardiac tissue of male 62-weeks old NP compared to LP offspring. Results: In the current study, we showed a low birthweight followed by catch-up growth phenomena associated with high blood pressure development, increased heart collagen content, and cardiomyocyte area in 62-week-old LP offspring. mRNA sequencing analysis identified changes in the expression level of 137 genes, considering genes with a p-value < 0.05. No gene was. Significantly changed according to the adj-p-value. After gene-to-gene biological evaluation and relevance, the study demonstrated significant differences in genes linked to inflammatory activity, oxidative stress, apoptosis process, autophagy, hypertrophy, and fibrosis pathways resulting in heart function disorders. Conclusion: The present study suggests that gestational protein restriction leads to early cardiac diseases in the LP progeny. It is hypothesized that heart dysfunction is associated with fibrosis, myocyte hypertrophy, and multiple abnormal gene expression. Considering the above findings, it may suppose a close link between maternal protein restriction, specific gene expression, and progressive heart failure.

Keywords: arterial hypertension; cardiovascular disease; fetal programming; gestational protein-restriction; heart miRNA transcriptome; myocyte hypertrophy.

FIGURE 1

Tail systolic arterial pressure from NP (n = 15) compared to age-matched LP (n = 25) male offspring from 32nd to 62nd weeks of age was measured by an indirect tail-cuff method using an electrosphygmomanometer. The results were expressed as mean ± SD, and the two-way ANOVA statistical analysis was performed. *p < 0.05 **p < 0.01, ***p < 0.001.

FIGURE 2

In A, a graphic of LV cardiomyocytes (arrow) area (NP: 174 fields; LP: 199 fields from n = 5) and representative micrographs of NP offspring (B) and age-matched LP progeny (C) LV tissue stained with hematoxylin-eosin (HE). In D, depicted LV collagen content (100 fields for each group from n = 5) and picrosirius fast green stained LV tissue from NP (E) and LP (F) offspring. The results were expressed as mean ± SD, and Student’s t-test performed the statistical analysis. *p < 0.05, ****p < 0.0001.

FIGURE 3

Expression of genes significantly altered, grouped by their hole in hypertrophy (A), interstitial fibrosis (B), cardiac function (C), adrenergic receptor signaling (D), oxidative stress (E) and inflammatory activity (F). This analysis was performed based on relevant literature on aging and cardiac disease (the gene’s abbreviations and references are found in Supplementary Table S1). The results were expressed as mean ± SEM.

FIGURE 4

Expression of genes significantly, grouped by their role altered in carbohydrate (A), lipids metabolism (B), DNA-associated protein (C), protein synthesis (D), and Ion (E) and Calcium Channels (F). These analyzes were performed based on pertinent literature on aging and cardiac disease (the gene's abbreviations and references are found in Supplementary Table S1). The results were expressed as mean ± SEM.

FIGURE 5

Expression of genes significantly altered, grouped by their participation in cell membrane compounds (A), cytoskeleton (B), apoptosis (C), autophagy (D), oncogenes (E), and cell proliferation (F). The analysis was performed based on pertinent literature on aging and cardiac disease (the gene's abbreviations and references are found in Supplementary Table S1). The results were expressed as mean ± SEM.

FIGURE 6

Fold-change of mRNA expression in LP offspring (n = 5) compared to age-matched NP progeny (n = 5).

FIGURE 7

Effect of maternal protein restriction on AT1, AT2, NOS1, SOD2, JAK2, pJAK2, STAT3, and pSTAT3 proteins, measured using immunoblotting analysis in the isolated LV. The protein variation results from LP progeny were expressed as mean ± SD and relative to control (NP offspring), assigning a value in %). The statistical analysis was performed by Student’s t-test when comparing only two samples of independent observations; only one offspring of each litter was used for immunoblotting experiments. *p < 0.05.

FIGURE 8

Effect of maternal protein restriction on ERK 1/2, Na-K ATPase, AKT, pAKT, CREB, pCREB, AMPk, and pAMPk proteins, measured using immunoblotting analysis in the isolated LV. The statistical analysis was performed by Student’s t-test when comparing only two samples of independent observations; only one offspring of each litter was used for immunoblotting experiments. The protein variation results from LP progeny were expressed as mean ± SD and relative to control (NP offspring), assigning a value in %. *p < 0.05; **p < 0.01.

FIGURE 9

The picture depicted a schematic representation of the cyclic AMP and associated pathways investigated and biological response disorders in LV from 62nd weeks of age male rats from maternal restricted-protein intake.