Maternal nutrient restriction alters gene expression in the ovine fetal heart

Abstract

Adequate maternal nutrient supply is critical for normal fetal organogenesis. We previously demonstrated that a global 50% nutrient restriction during the first half of gestation causes compensatory growth of both the left and right ventricles of the fetal heart by day 78 of gestation. Thus, it was hypothesized that maternal nutrient restriction significantly altered gene expression in the fetal cardiac left ventricle (LV). Pregnant ewes were randomly grouped into control (100% national research council (NRC) requirements) or nutrient-restricted groups (50% NRC requirements) from day 28 to day 78 of gestation, at which time fetal LV were collected. Fetal LV mRNA was used to construct a suppression subtraction cDNA library from which 11 cDNA clones were found by differential dot blot hybridization and virtual Northern analysis to be up-regulated by maternal nutrient restriction: caveolin, stathmin, G-1 cyclin, alpha-actin, titin, cardiac ankyrin repeat protein (CARP), cardiac-specific RNA-helicase activated by MEF2C (CHAMP), endothelial and smooth muscle derived neuropilin (ESDN), prostatic binding protein, NADH dehydrogenase subunit 2, and an unknown protein. Six of these clones (cardiac alpha-actin, cyclin G1, stathmin, NADH dehydrogenase subunit 2, titin and prostatic binding protein) have been linked to cardiac hypertrophy in other species including humans. Of the remaining clones, caveolin, CARP and CHAMP have been shown to inhibit remodelling of hypertrophic tissue. Compensatory growth of fetal LV in response to maternal undernutrition is concluded to be associated with increased transcription of genes related to cardiac hypertrophy, compensatory growth or remodelling. Counter-regulatory gene transcription may be increased, in part, as a response to moderating the degree of cardiac remodelling. The short- and long-term consequences of these changes in fetal heart gene expression and induction of specific homeostatic mechanisms in response to maternal undernutrition remain to be determined.

Figure 1

Differential screening of fetal LV nutrient-restricted subtraction cDNA library This nutrient-restricted (tester) cDNA library was generated by ‘subtracting’ common cDNAs found in fetal LV from control-fed ewes. The same volume of PCR-amplified cDNA library from fetal LV derived from nutrient-restricted ewes was dot blotted onto nylon membranes. ³²P-labelled nutrient-restricted (tester) or control-fed (driver) cDNAs were then hybridized with the nutrient-restricted (tester) cDNA library. Differentially expressed cDNAs are identified with the arrows.

Figure 2. A model describing up-regulation of fetal LV mRNAs in response to hypertrophy induced by maternal undernutrition

Binding of MT1-MMP activates proMMP-2, which is then cleaved to form activated MMP-2. Caveolin is also required for the AT2-induced activation of AT1 to initiate signal transduction. Stathmin inhibits CDKI p27, which allows the cell cycle to proceed. These molecules are involved in inducing hypertrophy and mediate cell proliferation and cardiac remodelling. Cardiac α-actin and titin are up-regulated in response to these processes. Caveolin is also known to block G1 cyclin and binds to eNOS to inhibit its activity. CARP is activated by SAPKs in response to stress, which prevents protein synthesis. RNA helicase, also known as CHAMP, inhibits the cell cycle by activation of CDKI p21. These molecules are up-regulated and have inhibitory effects during hypertrophy. It is proposed that hypertrophy of fetal LV in response to maternal undernutrition is a homeostatic response between stimulatory and inhibitory signal transduction pathways. The abbreviations used are: MT1-MMP, membrane type 1 matrix metalloproteinase; AT1, angiotensin II type 1 receptor; ESDN, endothelial and smooth muscle derived neuropilin; eNOS, endothelial NO synthase; SAPKs, stress activated protein kinases; CHAMP, cardiac-specific helicase activated by MEF2. Continuous arrows denote activation and dotted arrows denote inhibition of signalling pathways.