Maternal nutrient restriction predisposes ventricular remodeling in adult sheep offspring
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Journal of Nutritional Biochemistry 24 (2013) 12581265Maternal nutrient restriction predisposes ventricular remodeling in adultsheep offspring
Wei Gea,b,1, Nan Hub,1, Lindsey A. Georgec, Stephen P. Fordc, Peter W. Nathanielszc,d,Xiao-Ming Wanga,, Jun Rena,b, c,
aDepartment of Geriatrics, Xijing Hospital, Fourth Military Medical University, Xi'an, China 710032bCenter for Cardiovascular Research and Alternative Medicine
cCenter for the Study of Fetal Programming, University of Wyoming, Laramie, WY, 82071, USAdCenter for Pregnancy and Newborn Research, University of Texas Health Sciences Center at San Antonio, San Antonio, TX, 78299, USA
Received 31 October 2011; received in revised form 31 August 2012; accepted 2 October 2012Abstract
Maternal nutrient restriction during pregnancy is associated with the development of a thrifty phenotype in offspring, conferring increased prevalence ofmetabolic diseases in adulthood. To explore the possible mechanisms behind heart diseases in adulthood following maternal nutrient restriction, dams were feda nutrient-restricted (NR: 50%) or control (100%) diet from 28 to 78 days of gestation. Both groups were then fed 100% of requirements to lambing. At 6 years ofage, female offspring of NR and control ewes of similar weight and body condition were subjected to ad libitum feeding of a highly palatable diet for 12 weeks.Cardiac geometry, post-insulin receptor signaling, autophagy and proinflammatory cytokines were evaluated in hearts from adult offspring. Our results indicatedthat maternal nutrient restriction overtly increased body weight gain and triggered cardiac remodeling in offspring following the 12-week ad libitum feeding.Phosphorylation of insulin receptor substrate-1 (IRS1) was increased in left but not right ventricles from NR offspring. Levels of signal transducer and activator oftranscription-3 were up-regulated in left ventricles, whereas expression of tumor necrosis factor- and toll-like receptor-4 was enhanced in right ventricles, inadult offspring of maternal nutrition-restricted ewes. No significant differences were found in pan-IRS1, pan-AMP-dependent protein kinase (AMPK), pan-Akt,phosphorylated AMPK, phosphorylated Akt, glucose transporter 4, phosphorylated mammalian target of rapamycin, Beclin-1 and microtubule-associated protein1 light-chain 3 II proteins in left and right ventricles between the control and NR offspring. These data revealed that maternal nutrient restriction during early tomid gestation may predispose adult offspring to cardiac remodeling possibly associated with phosphorylation of IRS1 as well as proinflammatory cytokines butnot autophagy. 2013 Elsevier Inc. All rights reserved.Keywords: Nutrition restriction; Gestation; Offspring; Insulin signaling1. Introduction
Alterations in maternal nutritional status during pregnancy arecapable of predisposing adult offspring to unfavorable permanentstructural and functional deficits in multiple organ systems [1,2].Epidemiological evidence from human studies has closely linkedmaternal undernutrition and fetal growth restriction during gesta-tion with the development of a thrifty phenotype in offspring inlater life . In particular, gestational undernutrition during the firstand second trimesters of pregnancy has been shown to predispose Corresponding authors. J. Ren is to be contacted at: University ofWyoming College of Health Sciences, Laramie, WY 82071, USA. Fax: +1 307766 2953. X.-M. Wang, Department of Geriatrics, Xijing Hospital, FourthMilitary Medical University, Xi'an 710032, China. Tel.: +86 29 84775543;fax: +86 29 84775543.
E-mail addresses: firstname.lastname@example.org (X.-M. Wang), email@example.com(J. Ren).1 Equal contribution.
0955-2863/$ - see front matter 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.jnutbio.2012.10.001the fetus to cardiovascular, metabolic and endocrine diseases lateron in postnatal life [4,5]. Undernourished ewes on rangeland usuallylose a significant portion of weight during early to mid gestation,leading to compromised health condition of their offspring .This gestational undernutrition-related postnatal health defect isconsistent with the significance of the critical period (i.e., first half ofgestation for fetal development) during gestation [6,7,9,10]. Despitethe ample clinical and agricultural observations, the precisemechanism of action behind abnormal physiological function inpostnatal life as a consequence of maternal nutrient deficiency stillremains elusive.
Recent evidence has demonstrated a unique role of fetal insulinresponsiveness and signaling in maternal-undernutrition-triggereddefects during postnatal life [11,12]. In particular, fetal pancreatic -cells may inherit a persistent secretory defect as a developmentalresponse to fetal malnutrition , a primary cause of intrauterinegrowth restriction . Earlier work from our laboratory revealedthat maternal undernutrition from early to mid gestation may changethe levels of the growth-promoting insulin-like growth factor
1259W. Ge et al. / Journal of Nutritional Biochemistry 24 (2013) 12581265receptor levels in fetal myocardium, which may contribute to cardiacgrowth and remodeling in fetal sheep heart . Nonetheless, theimpact of maternal undernutrition on insulin signaling cascadeduring postnatal life has not been examined. To this end, this studywas designed to evaluate the effect of an early gestational nutrientrestriction on postnatal cardiac geometry and insulin signalingcascade. Insulin signaling was examined at the levels of insulinreceptor substrate-1 (IRS1), and post-receptor signaling including Aktand AMP-dependent protein kinase (AMPK) [14,15]. Given thatinflammation and autophagy are known to be closely associated withinsulin sensitivity and cardiac remodeling , crucial proteinmarkers of inflammation and autophagy such as tumor necrosisfactor- (TNF), signal transducer and activator of transcription-3(STAT3), toll-like receptor-4 (TLR4), Becline-1 and microtubule-associated protein 1 light-chain 3 (LC3) were also monitored inmyocardium from offspring of control and nutrition-restricted ewes.
2. Materials and methods
2.1. Experimental animals
All animal procedures were approved by the University of Wyoming AnimalCare and Use Committee (Laramie, WY, USA). On day 20 of pregnancy, multiparousewes of mixed breeding were weighed so that individual diets could be calculatedon a metabolic body weight basis (weight0.75). The diet consisted of a pelleted beetpulp [79.7% total digestible nutrients (TDN), 93.5% dry matter (DM) and 10.0% crudeprotein]. Rations were delivered on a DM basis to meet the total TDN required formaintenance for an early pregnant ewe (NRC requirements). A mineralvitaminmixture [51.43% sodium triphosphate, 47.62% potassium chloride, 0.39% zinc oxide,0.06% cobalt acetate and 0.50% ADE vitamin premix (8,000,000 IU vitamin A, 800,000IU vitamin D3 and 400,000 IU vitamin E per pound; amount of vitamin premix wasformulated to meet the vitamin A requirements)] was included with the beet pulppellets to meet nutritional requirements. On day 21 of gestation, all ewes wereplaced in individual pens and fed control rations. From day 28, ewes were randomlyA
Left Ventricle Right Ventricle
Fig. 1. Cardiac geometric property of the whole heart, left ventricle or right ventricle from adulbody weight ratio, (C) left and right ventricular weight and (D) left and right ventricular waassigned to a control-fed group (100% NRC requirements which included 100%mineralvitamin mixture) or a nutrient-restricted (NR) group (fed 50% NRCrequirements which included 50% mineralvitamin mixture). Dams were fed anNR (50% National Research Council recommendations) or control (C: 100%) dietfrom 28 to 78 days of gestation (term ~150 days). Both groups were then fed 100%of requirements to lambing . At 6 years of age, female offspring of NR and Cewes of similar weight and body condition were subjected to an ad libitum feedingof a highly palatable diet for 12 weeks with automated monitoring of feed intake(Grow Safe System) . At necropsy, ewes were sedated with ketamine (22.2 mg/kg body weight) and maintained under isoflurane inhalation anesthesia (4%induction, 1%2% maintenance). Ewes were then exsanguinated while under generalanesthesia, and hearts were collected and weighed. The left and right ventricleswere dissected from the septum and remainder of the heart and weighed, and theirthickness was determined.2.2. Hematoxylin and eosin (H&E) staining
Following the removal of hearts, myocardial tissues were immediately placed in10% neutral-buffered formalin at room temperature for 24 h after a brief rinse withphosphate-buffered saline. The specimens were embedded in paraffin, cut in 5-msections and then stained with H&E. Cardiomyocyte cross-sectional areas werecalculated on a digital microscope (400) using the NIH Image J (version 1.34S)software .2.3. Masson trichrome staining
Hearts were harvested and sliced at the midventricular level followed by fixationwith normal buffered formalin. Paraffin-embedded transverse sections were cut in5-m thickness and stained with Masson trichrome. The sections were photographedwith a 40 objective of an Olympus BX-51 microscope equipped with an OlympusMaguaFire SP digital camera. Eight random fields from each section (three sections persheep) were assessed for interstitial fibrosis. To determine fibrotic area, pixel counts ofblue-stained fibers were quantified using the Color range and Histogram commands inPhotoshop. Fibrotic area was calculated by dividing the pixels of blue-stained area tototal pixels of nonwhite area .B
20 Control NR
Left Ventricle Right Ventricle
t offspring of control and NR ewes. (A) Whole heart weight, (B) whole heart weight-to-ll thickness. MeanS.E.M., n=4; *Pb.05 vs. respective control group.
1260 W. Ge et al. / Journal of Nutritional Biochemistry 24 (2013) 125812652.4. Western blot analysis
Tissue from sheep ventricles were homogenized and lysed in a radioimmunopreci-pitation (RIPA) lysis buffer containing20mMTris, 1mMEDTA, 1mMEGTA, 150mMNaCl,1% Triton, 0.1% sodium dodecyl sulfate (SDS) and a protease inhibitor cocktail. Proteinconcentrations were determined using a Bio-Rad protein assay reagent (Bio-RadLaboratories, Inc., Richmond, CA, USA). Samples containing equal amount of proteinswere separated on10% SDS-polyacrylamide gels in aminigel apparatus (Mini-PROTEAN II,Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to nitrocellulosemembranes. The membranes were blocked with 5% milk in TBS-T, and were incubatedovernight at 4C with anti-insulin receptor substrate 1 (IRS1), anti-phosphorylated IRS1(pIRS1, Ser307), anti-AMPK, anti-phosphorylated activated protein kinase (pAMPK,Thr172), anti-Akt, anti-phosphorylated Akt (pAkt, Ser473), anti-glucose transporter 4(GLUT4), anti-mammalian target of rapamycin (mTOR), anti-phosphorylated mTOR(pmTOR, Ser2448), anti-LC3B II, anti-Beclin-1, anti-STAT3, anti-TNF, anti-TLR4, anti--tubulin and anti-GAPDH (both as loading control) antibodies. All antibodies reacted wellwith sheepmyocardium.After immunoblotting, the filmwas scanned, and the intensity ofimmunoblot bands was detected with a Bio-Rad calibrated densitometer .
2.5. Statistical analysis
Data are presented as meanS.E.M. Differences between groups were assessed aStudent's t test. P values less than .05 were considered significant.B
500 Control NR
Left Ventricle Right Ventricle
Fig. 2. Histological examination of left ventricle (LV) or right ventricle (RV) from adultoffspring of control and NR ewes using H&E staining. (A) Representative H&E stainingimages from LV and RV in control and NR groups. (B) Quantitative analysis ofcardiomyocyte cross-sectional area. Averaged areas of at least 200 nucleated myocytesper section were used from each sheep. MeanS.E.M., n=4; *Pb.05 vs. respectivecontrol group.3. Results
3.1. General features of adult offspring from ewes subjected tonutrition restriction
Following the 12-week ad libitum feeding of a highly palatablediet, the 6-year-old female offspring of NR displayed a higher bodyweight gain (44%2%) compared with that of the control ewes(29%4%, Pb.05 between the two groups). The average daily feedintake was comparable between control (0.640.03 kg feed/day) andNR (0.670.05 kg feed/day, PN.05 vs. control) ewes. The heart weightwas slightly although significantly greater in adult offspring from NRewes than those from the control ewes (Fig. 1A). However,normalized heart weight was not significantly different between thetwo groups (Fig. 1B), possibly due to the higher body weight gain inthe NR group. Both left and right ventricular weights were elevated inNR offspring compared with those from control group (Fig. 1C).However, neither left nor right ventricular wall thickness was overtlydifferent between the NR and control groups (Fig. 1D).A
8 Control NR
Left Ventricle Right Ventricle
Fig. 3. Histological examination of myocardial fibrosis in LV or RV from adult offspringof control and NR ewes using Masson trichrome staining. (A) Representativephotomicrographs (400) of myocardial sections stained with Masson trichrome.(B) Quantitative analysis of myocardial fibrotic area (Masson trichrome stained area inlight blue color normalized to the total myocardial area; magnification=400). MeanS.E.M., n=8 random fields from four sheep per group (6 mm2); *Pb.05 vs. respectivecontrol group.
1261W. Ge et al. / Journal of Nutritional Biochemistry 24 (2013) 125812653.2. Myocardial histological findings in adult offspring followingmaternal nutrition restriction
To evaluate the impact of maternal nutrient restriction onmyocardial histology in adult offspring, myocardial sections fromcontrol and NR groups were stained with H&E. In line with thegeometric data, myocardial sections from left and right ventricles inadult offspring from nutrient-restricted ewes displayed significantlygreater cardiomyocyte cross-sectional areas compared with thosefrom control ewes (Fig. 2). Further analysis of myocardial fibrosisusing Masson trichrome staining revealed overt cardiac fibrosis inboth left and right ventricles in adult offspring from nutrient-0
3 Control NR
Left Ventricle R...