Pregestational type 2 diabetes mellitus induces cardiac hypertrophy in the murine embryo through cardiac remodeling and fibrosis

Abstract

Background: Cardiac hypertrophy is highly prevalent in patients with type 2 diabetes mellitus. Experimental evidence has implied that pregnant women with type 2 diabetes mellitus and their children are at an increased risk of cardiovascular diseases. Our previous mouse model study revealed that maternal type 2 diabetes mellitus induces structural heart defects in their offspring.

Objective: This study aims to determine whether maternal type 2 diabetes mellitus induces embryonic heart hypertrophy in a murine model of diabetic embryopathy.

Study design: The type 2 diabetes mellitus embryopathy model was established by feeding 4-week-old female C57BL/6J mice with a high-fat diet for 15 weeks. Cardiac hypertrophy in embryos at embryonic day 17.5 was characterized by measuring heart size and thickness of the right and left ventricle walls and the interventricular septum, as well as the expression of β-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, desmin, and adrenomedullin. Cardiac remodeling was determined by collagen synthesis and fibronectin synthesis. Fibrosis was evaluated by Masson staining and determining the expression of connective tissue growth factor, osteopontin, and galectin-3 genes. Cell apoptosis also was measured in the developing heart.

Results: The thicknesses of the left ventricle walls and the interventricular septum of embryonic hearts exposed to maternal diabetes were significantly thicker than those in the nondiabetic group. Maternal diabetes significantly increased β-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, and desmin expression, but decreased expression of adrenomedullin. Moreover, collagen synthesis was significantly elevated, whereas fibronectin synthesis was suppressed, in embryonic hearts from diabetic dams, suggesting that cardiac remodeling is a contributing factor to cardiac hypertrophy. The cardiac fibrosis marker, galectin-3, was induced by maternal diabetes. Furthermore, maternal type 2 diabetes mellitus activated the proapoptotic c-Jun-N-terminal kinase 1/2 stress signaling and triggered cell apoptosis by increasing the number of terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells (10.4 ± 2.2% of the type 2 diabetes mellitus group vs 3.8 ± 0.7% of the nondiabetic group, P < .05).

Conclusion: Maternal type 2 diabetes mellitus induces cardiac hypertrophy in embryonic hearts. Adverse cardiac remodeling, including elevated collagen synthesis, suppressed fibronectin synthesis, profibrosis, and apoptosis, is implicated as the etiology of cardiac hypertrophy.

Keywords: cardiac remodeling; diabetic embryopathy; fibrosis; hypertrophy; pregestational type 2 diabetes.

Figure 1. Cardiac hypertrophy in embryos from a T2DM dam

A, Blood glucose level of T2DM mouse model. ND, Nondiabetes, DM, type 2 diabetes mellitus. B, Enlarged heart in embryos from ND and DM dam. C, Thickness of left ventricle (LV) wall, interventricular septum (IVS), and right ventricle (RV) wall of embryo from ND and DM dam. D, Cardiomyocyte size in heart of embryo from ND and DM dam. 76 and 40 hearts of embryos from 16 ND dams and 40 DM dams, respectively, were removed at E17.5. * indicates significant difference (P < 0.05) when compared to ND group.

Figure 2. Alteration of hypertrophy-associated genes in embryonic heart in T2DM pregnancy

Hyperglycemia increased the expression of fetal genes, β-MHC (A), and ANP (B). Hypertrophy-associated genes, IGF1 (C), DES (D), and ADM (E), were influenced by T2DM in the embryonic heart. Each experiment was repeated three times. * indicates significant difference (P < 0.05) when compared to ND group.

Figure 3. Collagen synthesis was increased by hyperglycemia

Hyperglycemia suppressed the expression of matrix metalloproteinases, MMP1 (A) and MMP13 (B). T2DM influenced the expression of TIMP1 (C), TIMP2 (D), TIMP3 (E), and TIMP4 (F). Hyperglycemia activated collagen genes, COL1A2 (G) and COL4A (H). Each experiment was repeated three times. * indicates significant difference (P < 0.05) when compared with the ND group.

Figure 4. Fibronectin synthesis was suppressed by hyperglycemia

mRNA expression of MMP24 (A) and FN1 (B) in the embryonic heart. Western blot of MMP24 (C) and FN1 (D) in embryonic heart. Immunofluorescence of MMP24 (E) and FN1 (F) in embryonic heart. Each group contained three samples, * indicates significant difference (P < 0.05) when compared with the ND group.

Figure 5. T2DM induced pro-fibrosis in the embryo heart

A. Masson staining of the embryo heart section. Arrow represents collagen staining (blue line). mRNA expression of pro-fibrosis markers, CTGF (B), OPN (C), and GAL3 (D). Each group contained three samples. * indicates significant difference (P < 0.05) when compared with the ND group.

Figure 6. T2DM induced apoptosis of cardiomyocytes in embryo heart

A, TUNEL assay of embryo heart from ND and DM dams. B, Quantification of TUNEL positive cells based on panel A. C, Western blot of pro-apoptotic factor, BAX, and anti-apoptotic factor, BCL-2, in embryo hearts from ND and DM dams. D, Activation of JNK and c-JUN in heart of embryos from ND and DM dams. * indicates significant difference (P < 0.05) when compared with the ND group.

Figure 7. Cardiac hypertrophy was induced by high glucose in vitro

A, The cell size was increased by high glucose (200 mg/dl) in H9C2 cell line. B, Quantification of cell hypertrophy based on panel A. High glucose influenced hypertrophy associated markers (C–G). * indicates significant difference (P < 0.05) when compared with the low glucose group.