Neonatal Growth Restriction Slows Cardiomyocyte Development and Reduces Adult Heart Size

Abstract

Prematurity is associated with reduced cardiac dimensions and an increased risk of cardiovascular disease. While prematurity is typically associated with ex utero neonatal growth restriction (GR), the independent effect of neonatal GR on cardiac development has not been established. We tested the hypothesis that isolated neonatal GR decreases cardiomyocyte growth and proliferation, leading to long-term alterations in cardiac morphology. C57BL/6 mice were fostered in litters ranging in size from 6 to 12 pups to accentuate normal variation in neonatal growth. Regardless of litter size, GR was defined by a weight below the 10th percentile. On postnatal day 8, Ki67 immunoreactivity, cardiomyocyte nucleation status and cardiomyocyte profile area were assessed. For adult mice, cardiomyocyte area was determined, along with cardiac dimensions by echocardiography and cardiac fibrosis by Masson's trichrome stain. On day 8, cardiomyocytes from GR versus control mice were significantly smaller and less likely to be binucleated with evidence of persistent cell cycle activity. As adults, GR mice continued to have smaller cardiomyocytes, as well as decreased left ventricular volumes without signs of fibrosis. Neonatal GR reduces cardiomyocyte size, delays the completion of binucleation, and leads to long-term alterations in cardiac morphology. Clinical studies are needed to ascertain whether these results translate to preterm infants that must continue to grow and mature in the midst of the increased circulatory demands that accompany their premature transition to an ex utero existence. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Keywords: cardiac development; cardiomyocyte; growth; left ventricle; prematurity.

Figure 1. Neonatal GR reduces binucleation and cell size in newborn mice

On postnatal day 8, (A) cardiac sections were obtained from control mice (gray bars, N = 24 mice from 10 litters) and GR mice (white bars, N = 27 mice from 8 litters) to determine the Ki67 labeling index. Cardiomyocytes were isolated from additional control mice (B is representative of 1933 cells from 33 mice from 11 litters) and GR mice (C is representative of 2078 cells from 29 mice from 10 litters) to determine nuclear count, (D) area of mononucleated and binucleated cells, and (E) the percentage of binucleated cardiomyocytes with an area within discrete intervals. *p<0.05 and **p<0.01

Figure 2. Neonatal GR decreases cardiomyocyte size into adulthood

On postnatal day 70, cardiomyocytes were isolated from control mice (A is representative of 867 cells from 9 mice from 4 litters) and GR mice (B and C are representative of 1034 cells from 8 mice from 3 litters), and (D) the percentage of binucleated cardiomyocytes with an area within discrete intervals was determined for control versus GR mice (gray versus white bars, respectively). *p<0.05 and **p<0.01

Figure 3. Determination of collagen distribution

Coronal sections of the heart from (A) control and (B) GR adult mice were stained with Masson’s trichrome and four images were stitched together to maintain resolution and contextual relationships. The horizontal bar represents 2 mm. *indicates the left ventricular cavity.