Pregestational diabetes alters cardiac structure and function of neonatal rats through developmental plasticity

Abstract

Pregestational diabetes (PGDM) leads to developmental impairment, especially cardiac dysfunction, in their offspring. The hyperglycemic microenvironment inside the uterus alters the cardiac plasticity characterized by electrical and structural remodeling of the heart. The altered expression of several transcription factors due to hyperglycemia during fetal development might be responsible for molecular defects and phenotypic changes in the heart. The molecular mechanism of the developmental defects in the heart due to PGDM remains unclear. To understand the molecular defects in the 2-days old neonatal rats, streptozotocin-induced diabetic female rats were bred with healthy male rats. We collected 2-day-old hearts from the neonates and identified the molecular basis for phenotypic changes. Neonates from diabetic mothers showed altered electrocardiography and echocardiography parameters. Transcriptomic profiling of the RNA-seq data revealed that several altered genes were associated with heart development, myocardial fibrosis, cardiac conduction, and cell proliferation. Histopathology data showed the presence of focal cardiac fibrosis and increased cell proliferation in neonates from diabetic mothers. Thus, our results provide a comprehensive map of the cellular events and molecular pathways perturbed in the neonatal heart during PGDM. All of the molecular and structural changes lead to developmental plasticity in neonatal rat hearts and develop cardiac anomalies in their early life.

Keywords: RNA sequencing; cardiac dysfunction; developmental plasticity; heart development; neonates; pregestational diabetes.

FIGURE 1

Electrophysiological changes in the neonatal hearts. Changes in the electrocardiography parameters of the 2-day neonatal hearts. QTc, corrected QT interval. Data are shown as Mean ± SEM (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significant.

FIGURE 2

Echocardiography of the two-day neonatal hearts. Transthoracic echocardiographic analyses were performed using the Vevo LAZR-X ultrasound system: Representative images of M-mode (A), B-mode (B) and pulsed-wave doppler (C) traces of the left ventricles are shown, and different functional hemodynamic parameters were evaluated (D). Data are shown as Mean ± SD (n = 6), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

Networks of differentially regulated genes in the neonatal rats. (A) Gene overlap analysis by Circos plot shows the overlap between genes and functional categories based on two input gene lists: Up-regulated genes (blue arc) and the genes that were down-regulated in neonatal rats (red arc). (B) Enrichment of heart development processes in neonatal rats. Enrichment of genes matching membership terms: heart, heart rate, development, differentiation and proliferation in the GO Biological Processes, KEGG Pathway. The outer pie shows the number and percentage of genes in the background associated with the membership (in black); the inner pie shows the number and percentage of genes in the individual input gene list associated with the membership. Hierarchical cluster of statistically enriched and significant terms (C,D). Network layout from a subset of representative terms from the whole cluster. Each pie sector is proportional to the number of hits originating from a gene list. Similar clusters of upregulated (C) and downregulated (D). The term with the most significant enrichment was selected for each cluster to describe its ID (Lower panel).

FIGURE 4

Gene ontology and network mapping. Bubble diagram of enriched terms of DEGs by DAVID database relating to change in the (A) biological process, (B) molecular function and (C) KEGG pathways. Significant pathways with P-values < 0.05 were plotted by the ggplot2 (R package). (D) GOCircle plot; the inner ring is a bar plot where the bar height indicates the significance of the term (–log10 p-value) and the color indicates the z-score. The outer ring displayed scatterplots of the expression levels (logFC) for the genes in each term. (E) GOChord plot of the relationship between the list of selected genes and their corresponding GO terms, together with the logFC of the genes. The left half of GOChord displayed whether the gene was up-or down-regulated. The right half represented different GO terms with different colors. A gene was linked to a certain GO term by the colored bands.

FIGURE 5

Histopathology of the neonatal hearts. (A) Photomicrograph of hearts after Masson’s Trichrome stain (n = 3): (a) Neonatal rat heart sections from non-diabetic females showing no fibrosis. (b) Neonatal rat heart sections from diabetic females showing fibrosis (collagen deposition; see arrows). (B) Ki-67 immunostaining of neonatal rat hearts from diabetic mothers showing increased cell proliferation (see arrows). Quantification of the ki-67 positive cells is shown in the right panel (n = 3).

FIGURE 6

Gene regulatory network analysis of the DEGs and expression profile of key regulators in the human heart. (A) Circos plot showing the association among differentially expressed genes and their key regulators, including transcription factors. The upper group of sectors represents overlapped genes, while the lower one represents the key regulators. The width of individual sectors of corresponding key regulators reflects the number of links connecting to the DEGs, while their color indicates the significance of interaction. (B) Gene expression (in TPM) of human key regulators in the cardiovascular tissues as analyzed by RNA-seq data available in the GTex database.

FIGURE 7

Correlation of transcriptomic perturbation during PGDM with the phenotypic changes. Transcriptomics analyses, including differential gene expression and pathway analysis of the neonatal hearts from STZ-induced diabetic rats, demonstrated that PGDM leads to the downregulation of genes related to heart development, structure and function. The alteration in gene expression also affects cardiomyocyte proliferation, cardiac remodeling, electrophysiology and echocardiographic parameters, leading to cardiac abnormalities. Upregulated genes are colored black, the genes having a positive effect on the indicated pathway are colored green, and the genes having a negative impact on the indicated pathway are colored red.