Implication of RAS in Postnatal Cardiac Remodeling, Fibrosis and Dysfunction Induced by Fetal Undernutrition

Abstract

Fetal undernutrition is a risk factor for cardiovascular diseases. Male offspring from rats exposed to undernutrition during gestation (MUN) exhibit oxidative stress during perinatal life and develop cardiac dysfunction in ageing. Angiotensin-II is implicated in oxidative stress-mediated cardiovascular fibrosis and remodeling, and lactation is a key developmental window. We aimed to assess if alterations in RAS during lactation participate in cardiac dysfunction associated with fetal undernutrition. Control dams received food ad libitum, and MUN had 50% nutrient restriction during the second half of gestation. Both dams were fed ad libitum during lactation, and male offspring were studied at weaning. We assessed: ventricular structure and function (echocardiography); blood pressure (intra-arterially, anesthetized rats); collagen content and intramyocardial artery structure (Sirius red, Masson Trichromic); myocardial and intramyocardial artery RAS receptors (immunohistochemistry); plasma angiotensin-II (ELISA) and TGF-β1 protein expression (Western Blot). Compared to Control, MUN offspring exhibited significantly higher plasma Angiotensin-II and a larger left ventricular mass, as well as larger intramyocardial artery media/lumen, interstitial collagen and perivascular collagen. In MUN hearts, TGF-β1 tended to be higher, and the end-diastolic diameter and E/A ratio were significantly lower with no differences in ejection fraction or blood pressure. In the myocardium, no differences between groups were detected in AT1, AT2 or Mas receptors, with MrgD being significantly lower in the MUN group. In intramyocardial arteries from MUN rats, AT1 and Mas receptors were significantly elevated, while AT2 and MrgD were lower compared to Control. Conclusions. In rats exposed to fetal undernutrition, RAS disbalance and associated cardiac remodeling during lactation may set the basis for later heart dysfunction.

Keywords: RAS receptors; angiotensin II; cardiovascular remodeling; fetal programming; fibrosis; lactation; left ventricular hypertrophy.

Figure 1

Echocardiographic parameters in 21-day old male offspring from rats exposed to maternal undernutrition during pregnancy (MUN) and control rats fed ad libitum (C). (a) IVSd, interventricular septum thickness at diastole; (b) PWd, posterior wall thickness at diastole; (c) LVMI, left ventricular mass index; (d) LVIDd, left ventricular internal diameter at diastole; (e) E/A, E, mitral peak early-filling velocity, and A, mitral peak flow velocity at atrial contraction; (f) LVEF, left ventricular ejection fraction. Sample size per group: 6 rats from 5 litters. Student’s t test, * p < 0.05 compared to C rats.

Figure 2

Myocardial and intramyocardial artery area in 21-day old male offspring from rats exposed to maternal undernutrition during pregnancy (MUN) and control rats fed ad libitum (C). (a) Myocardial area with representative images from sections stained with hematoxylin-eosin and captured with ×10 objective; (b) intramyocardial artery media/lumen and representative images stained with Masson Trichrome Stain and captured with a ×40 objective. Sample size per group: 7 rats from 5 litters. Student’s t test, * p < 0.05 compared to C rats.

Figure 3

Heart collagen content in 21-day old male offspring from rats exposed to maternal undernutrition during pregnancy (MUN) and control rats fed ad libitum (C). (a) Interstitial collagen and representative binary images; (b) perivascular intramyocardial artery collagen and representative binary images. Heart sections were stained with Sirius red; images were obtained at ×10 (myocardium) or ×40 objective (intramyocardial arteries) and transformed in binary images for quantification. Left panels show quantitative analysis of the relative area occupied by collagen. Sample size per group: 7 rats from 5 litters. Student’s t test, * p < 0.05 compared to C rats.

Figure 4

Immunohistochemistry for RAS receptors in the myocardium from 21-day old male offspring from rats exposed to maternal undernutrition during gestation (MUN), and control rats fed ad libitum (C). (a) AT1 receptors; (b) AT2 receptors; (c) Mas receptors; (d) MrgD receptors. Heart sections were stained with individual primary antibodies; the resulting immuno-complexes were detected with a biotinylated secondary antibody and amplified by ABC complex, using 3,3′-diaminobenzidine (DAB) as chromogen. Images were obtained with a ×20 objective. Left panels show quantitative analysis of MUN and C myocardium-stained fractional areas (percentage of tissue total area) using SACAIA method. Representative DAB images are shown in right panels (scale bar = 20 μm). Sample size per group: 7 rats from 5 litters. Student’s t test, * p < 0.05 compared to C.

Figure 5

Immunohistochemistry for RAS receptors in intramyocardial arteries from 21-day old male offspring from rats exposed to maternal undernutrition during pregnancy (MUN) and control rats fed ad libitum (C). (a) AT1 receptors; (b) AT2 receptors; (c) Mas receptors; (d) MrgD receptors. Heart sections were stained with individual primary antibodies; the resulting immunocomplexes were detected with a biotinylated secondary antibody, and amplified by ABC complex, using 3,3′-diaminobenzidine (DAB) as chromogen. Images were obtained with a ×20 objective. Left panels show quantitative analysis of MUN and C intramyocardial artery-stained fractional areas (percentage of tissue total area) using SACAIA method. Representative DAB images are shown in right panels (scale bar = 20 μm). Sample size per group: 7 rats from 5 litters. Student’s t test, * p < 0.05 compared to C rats.

Figure 6

Protein expression level of TGF-β1 by Western blot in cardiac tissue from 21-day old male rats. Results show the densitometric analysis, relativized to GADPH expression and representative examples. Sample size per group: 5–7 rats from 4 litters. Student’s t test.

Figure 7

Schematic diagram depicting the relationship between fetal undernutrition, alterations in Ang II and RAS cardiac receptors during lactation and development of cardiac structural and functional alterations, which may contribute to long-term heart disease.