The lifelong impact of fetal growth restriction on cardiac development

Abstract

Background: Maternal nutrient restriction (MNR) is a widespread cause of fetal growth restriction (FGR), an independent predictor of heart disease and cardiovascular mortality. Our objective was to examine the developmental and long-term impact of MNR-induced FGR on cardiac structure in a model that closely mimics human development.

Methods: A reduction in total caloric intake spanning pregestation through to lactation in guinea pig sows was used to induce FGR. Proliferation, differentiation, and apoptosis of cardiomyocytes were assessed in late-gestation fetal, neonatal, and adult guinea pig hearts. Proteomic analysis and pathway enrichment were performed on fetal hearts.

Results: Cardiomyocyte proliferation and the number of mononucleated cells were enhanced in the MNR-FGR fetal and neonatal heart, suggesting a delay in cardiomyocyte differentiation. In fetal hearts of MNR-FGR animals, apoptosis was markedly elevated and the total number of cardiomyocytes reduced, the latter remaining so throughout neonatal and into adult life. A reduction in total cardiomyocyte number in adult MNR-FGR hearts was accompanied by exaggerated hypertrophy and a disorganized architecture. Pathway analysis identified genes related to cell proliferation, differentiation, and survival.

Conclusions: FGR influences cardiomyocyte development during critical windows of development, leading to a permanent deficiency in cardiomyocyte number and compensatory hypertrophy in a rodent model that recapitulates human development.

Figure 1. Descriptive characteristics of control and MNR birth cohorts

A and B. Number of completed days of gestation at birth (A) and birth weight (B) for the control and MNR neonatal cohorts. C and D. Absolute weight gain (grams) (C) and relative weight gain (%) as a percent of birth weight (D) at four weeks of life for the control and MNR neonatal cohorts. Bars represent mean ± S.D. Analysis by Student’s t-test.

Figure 2. Maternal nutrient restriction preserves brain mass at the expense of heart mass

A–D. Comparison of brain weight to total body weight (TBW) ratio (A and C) and heart weight to total body weight ratio (B and D) for control and MNR-FGR fetuses (A and B) and neonates (C and D). Bars represent mean ± S.D. Analysis by Student’s t-test.

Figure 3. Maternal nutrient restriction impairs cardiomyocyte development in fetal and neonatal hearts

A. Photomicrographs of left ventricular myocardium from control and MNR-FGR fetuses and neonates. Black boxes indicate area magnified below. *Denotes lumen of left ventricle. Scale bar equals 200μm. B. Number of cardiomyocytes per 40X hpf in fetal and neonatal control (white bars) and MNR-FGR (black bars) hearts. (*p<0.001). C. Relative increase in cardiomyocyte number from fetal to neonatal stage in 40X hpf cross sections of control and MNR-FGR hearts. D. Percent mononucleated cardiomyocytes of total cardiomyocytes per hpf in fetal and neonatal control (white bars) and MNR-FGR (black bars) hearts. (*p<0.05). For B–D, data represent mean number ± S.D. (n=16–18 per group). Analysis by 2-way ANOVA (B and D) and Student’s t-test.

Figure 4. Maternal nutrient restriction increases the cardiomyocyte proliferative index in fetal and neonatal hearts

A. Photomicrographs of ventricular myocardium from control and MNR-FGR fetuses and neonates stained with anti-Ki-67 antibody (brown) and counterstained with hematoxylin (blue). Scale bar equals 100μm. B. Quantification of Ki-67 positive cardiomyocytes (%) per 40X hpf in fetal and neonatal control (white bars) and MNR-FGR (black bars) hearts. Data represent mean number ± S.D. (n=6–8 per group, *p<0.001). C. Photomicrographs of ventricular myocardium from control and MNR-FGR fetuses and neonates stained with anti-TUNEL antibody (brown) and counterstained with hematoxylin (blue). Scale bar equals 100μm. B. Quantification of TUNEL positive cardiomyocytes (%) per 40X hpf in fetal and neonatal control (white bars) and MNR-FGR (black bars) hearts. Data represent mean number ± S.D. (n=4 per group, 1 animal per litter, *p<0.001). Analysis by 2-way ANOVA.

Figure 5. Strongest predicted causal network by IPA analysis of neonatal hearts

A. Network contains differentially upregulated (green) and downregulated (red) proteins, as well as literature-inferred activation (blue) and inhibition (orange) proteins. Top functions of the genes were related to cellular proliferation, differentiation and survival. The node shapes denote triangle: kinase, fat diamond: peptidase, tall diamond: enzyme, rhomboid: transporter, oval: transcription regulator, circle: other. n=5 animals per group (1 animal per litter). B. Quantification of PARP-1 expression (RT-PCR) in whole hearts from fetal control (white bars) and MNR-FGR (black bars) animals. Data represent mean ± S.E.M. Analysis by Student’s t-test. C. Photomicrographs of papillary muscle and ventricular myocardium from control and MNR-FGR fetal hearts stained with anti-PARP1 antibody (brown) and counterstained with hematoxylin (blue). Scale bar equals 100μm.

Figure 6. Fetal growth restriction impairs heart growth and cardiomyocyte growth in adult hearts

A. Low (20X) and high power (40X) photomicrographs of left ventricular endocardium and epicardium from MNR-FGR adults. Scale bar equals 100μm. B. Photomicrographs of ventricular mid-myocardium from control and MNR-FGR adults. Black boxes indicate area of myocardium magnified below. *Denotes lumen of left ventricle. Scale bar equals 200μm. C. Comparison of heart weight to total body weight ratio for control and MNR-FGR adult guinea pigs. D. Number of cardiomyocytes per hpf (40X) in control and MNR-FGR adult hearts. E. Cross sectional cardiomyocyte area in control and MNR-FGR adult hearts. F. Percent mononuclear cardiomyocytes of total cardiomyocytes per hpf in control and MNR-FGR adult hearts. G. Quantification of Ki-67 positive cardiomyocytes (%) per 40X hpf in control and MNR-FGR hearts. Data represent mean number ± S.D. (n=16–18 per group). Analysis by Student’s t-test.