Maternal nutrient restriction and the fetal left ventricle: decreased angiotensin receptor expression

Abstract

Background: Adequate maternal nutrition during gestation is requisite for fetal nutrition and development. While a large group of epidemiological studies indicate poor fetal nutrition increases heart disease risk and mortality in later life, little work has focused on the effects of impaired maternal nutrition on fetal heart development. We have previously shown that 50% global nutrient restriction from 28-78 days of gestation (early to mid-pregnancy; term = 147 days) in sheep at mid-gestation retards fetal growth while protecting growth of heart and results in hypertensive male offspring at nine months of age. In the present study, we evaluate LV gene transcription using RNA protection assay and real-time reverse transcriptase polymerase chain reaction, and protein expression using western blot, of VEGF and AT1 and AT2 receptors for AngII at mid-gestation in fetuses from pregnant ewes fed either 100% (C) or 50% (NR) diet during early to mid-gestation.

Results: No difference between the NR (n = 6) and C (n = 6) groups was found in gene transcription of the AngII receptors. Immunoreactive AT1 (1918.4 +/- 154.2 vs. 3881.2 +/- 494.9; P < 0.01) and AT2 (1729.9 +/- 293.6 vs. 3043.3 +/- 373.2; P < 0.02) was decreased in the LV of NR fetuses compared to C fetuses. The LV of fetuses exposed to NR had greater transcription of mRNA for VEGF (5.42 +/- 0.85 vs. 3.05 +/- 0.19; P < 0.03) than respective C LV, while no change was observed in immunoreactive VEGF.

Conclusion: The present study demonstrates that VEGF, AT1 and AT2 message and protein are not tightly coupled, pointing to post-transcriptional control points in the mid gestation NR fetus. The present data also suggest that the role of VEGF and the renin-angiotensin system receptors during conditions inducing protected cardiac growth is distinct from the role these proteins may play in normal fetal cardiac growth. The present findings may help explain epidemiological studies that indicate fetuses with low birth weight carry an increased risk of mortality from coronary and cardiovascular disease, particularly if these individuals have reduced cardiovascular reserve due to an epigenetic decrease in vascularization.

Figure 1

Amount of VEGF mRNA relative to β-actin mRNA in the LV of 78 dGA fetuses as determined by ribonuclease protection assay. Inset picture depicts representative results of RT-PCR reactions for VEGF₁₆₅ and VEGF₁₈₉ run on a 1% agarose gel. NR (■, n = 6) fetuses exhibited increased VEGF transcript in the LV compared to C (□, n = 6) fetuses. *P < 0.05.

Figure 2

Fetuses from NR (■, n = 6) ewes demonstrated less AT1 expression than C (□, n = 6) LV. Inset depicts representative immunoblot for AT1 and β-actin. Equal protein loading between lanes was demonstrated by re-probing membranes for β-actin. *P < 0.05.

Figure 3

Fetuses derived from NR (■, n = 6) ewes exhibited decreased AT2 expression in the LV compared to fetuses from the C (□, n = 6) ewes. Inset depicts representative immunoblot for AT2 and β-actin. Equal protein loading between lanes was demonstrated by re-probing membranes for β-actin. *P < 0.05.

Figure 4

Fetuses derived from NR (■, n = 6) ewes exhibited no difference in total immunoreactive VEGF, or immunoreactive VEGF₁₆₅ (46 kDa) or VEGF₁₈₉ (54 kDa) isoforms in the LV compared to fetuses from the C (□, n = 6) ewes. Inset depicts representative immunoblot for VEGF and β-actin. Equal protein loading between lanes was demonstrated by re-probing membranes for β-actin.