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Am J Physiol Regul Integr Comp Physiol. Author manuscript; available in PMC 2009 May 1.
Published in final edited form as:
PMCID: PMC2674931
EMSID: UKMS4649

Effect of maternal nutrient restriction from early-to-mid gestation on cardiac function and metabolism after adolescent-onset obesity

Abstract

Maternal nutrient restriction (NR) from early-mid gestation has marked effects on endocrine sensitivity and organ function of the resulting offspring. We hypothesized that early NR may re-set the expression profile of genes central to myocardial energy metabolism, influencing ectopic lipid deposition and cardiac function in the subsequent adult offspring when obese. NR offspring were exposed to an ‘obesogenic’ environment and their cardiac function and molecular indices of myocardial energy metabolism assessed to explore the hypothesis that an obese individual’s risk of heart disease may be modified after maternal NR. Pregnant sheep were fed either 100% (control) or 50% (NR) energy requirement from 30-80 days gestation and 100% thereafter. At weaning, offspring were either exposed to an obesogenic environment or remained lean. At ~one year of age their haemodynamic response to hypotension was determined, together with left ventricular expression profiles of fatty acid binding protein 3 (FABP3), peroxisome proliferator-activated receptor γ (PPARγ) and its co-activator (PGC)1α, acetyl CoA carboxylase (ACC), AMP-activated protein kinase (AMPK)α2 and voltage-dependent anion channel 1 (VDAC1). Obesity produced left ventricular hypertrophy in all animals, with increased ectopic (myocardial) lipid in NR offspring. Obesity per se significantly reduced myocardial transcript expression of PGC-1α, AMPKα2, VDAC1 and ACC and increased expression of PPARγ and FABP3. However, despite NR animals being similarly obese their transcript expression of ACC, PPARγ and FABP3 was similar to lean animals, indicating altered cardiac energy metabolism. Indeed, blunted tachycardia and an amplified inotrophic response to hypotension characterized cardiac function in obese NR offspring. The results suggest that maternal NR during early organogenesis can precipitate an altered myocardial response to hypotension and increased myocardial lipid deposition in the adult offspring after adolescent-onset obesity, potentially rendering these individuals more at risk of early heart failure as they age.

Keywords: obesity, cardiac function, maternal nutrition, lipid infiltration

Introduction

Obesity is a significant risk factor for diabetes, hypertension, cardiovascular disease and dyslipidemia (54). Sedentary lifestyle together with increased caloric intake are the main causes of obesity and related onset of the metabolic syndrome (27; 41). Obesity is well known to increase the risk of a broad spectrum of non-communicable diseases including renal (11) and cardiovascular disease through changes in sympathetic activation (2) an up-regulated renin-angiotensin system (39), cardiac hypertrophy, atherosclerosis and local inflammatory responses (1; 28). Specifically, obesity has been shown to have a substantial inhibitory effect on cardiac function partly as a consequence of excess fat accumulation in and around the heart (29). Increased peri- and epicardial fat is associated with a fetal pattern of myocardial energy utilization, i.e. a predominantly glucose, rather than fatty acid, dependence, and changes in the expression of the key intracellular molecular energy ‘switches’ e.g. peroxisome proliferator-activated receptor γ coactivator (PGC)1α (31), acetyl CoA carboxylase (ACC) and AMP-activated protein kinase (AMPK)α2 (30). At the same time obesity results in significant changes to left ventricular and diastolic function (13) that is exacerbated by the concomitant sympathetic overdrive that occurs (24; 25); leading to a myocardium that is refractory to β-adrenergic signaling (8).

Global changes in maternal macronutrient intake targeted at defined stages of pregnancy can have pronounced effects on cardiovascular control in the resulting offspring (21). Such adaptations are usually accompanied by significant changes in the density of expression of organ receptor populations and relevant enzyme activities and hence endocrine responsiveness, substrate transport and utilization, for review see (33). For example, maternal nutrient restriction in early pregnancy is associated with increased tissue-specific glucocorticoid receptor expression in the newborn (50) which subsequently go on to develop increased blood pressure later in life, but only in a pre-feeding basal state (23). Maternal nutrient restriction coincident with early heart development, however, has no obvious long term effects on cardiac function when the offspring are maintained under a natural “free-living” environment (22; 23). Importantly, the long-term programming outcomes appear to be amplified when the offspring are exposed to an obesogenic environment (43; 44).

The extent to which the prenatal nutritional environment may influence the expression of genes associated with cardiac hypertrophy has been examined in one study (26), but none have investigated potential effects on energy metabolism in the heart, especially with an early onset of obesity. This is important since cardiac hypertrophy is associated with suppressed insulin dependent glucose transporter (GLUT) 4 expression and heart type fatty acid binding protein (FABP)3 (42), but enhanced insulin independent GLUT1 transport (1) and ectopic lipid accumulation (45). These changes in myocardial energy metabolism with obesity may also impact on other ‘nutrient sensors’ within cardiac tissue such as the peroxisome proliferator-activated receptor (PPAR)γ expression and its coactivator (PGC-1α) (14). Adaptations of this type could also determine, in part, progression toward heart disease with obesity - as a consequence of changes in myocardial substrate supply and utilization, particularly fatty acids, since these account for more than half of ATP production in the heart.

Whilst the prenatal nutritional environment has been shown in many studies to alter specific aspects of growth, development and physiology per se, it is evident that the physical expression of a ‘programmed’ end-point is dependent, in part, on the postnatal environment. Specifically, the degree of physical activity, food intake and energy density of the food - in short the degree of exposure and active engagement in an ‘obesogenic’ lifestyle - may influence the levels of hypertension and/or obesity observed (38; 49; 51). In the present study we hypothesized that programming by prenatal nutrient restriction may re-set the expression profile of genes that are central to myocardial energy metabolism, influencing lipid deposition in this tissue and ultimately affecting cardiac function under duress. In allowing these ‘programmed’ animals to become overweight we sought to model a sedentary, westernized culture characterized by reduced physical activity and increased access to energy dense food. The effects of obesity on myocardial function were then assessed in vivo during a hypotensive challenge followed by a molecular characterization of myocardial energy metabolism. This included a direct comparison of the effects of obesity per se by further comparing cardiovascular function and cardiac outcomes between lean and obese individuals born to normally fed mothers.

Materials and methods

Animals and experimental design

All procedures were performed in accordance with the UK animals (Scientific Procedures) Act, 1986 and approved by the local ethics committee of the University of Nottingham at Sutton Bonington. At day 30 of gestation, 16 twin bearing ewes were randomly allocated to receive either a control (~7-8 MJ/day of metabolisable energy (ME), n=6) or nutrient restricted diet (50% of control, n=10) until day 80 of gestation. Thereafter all sheep were fed to 100% calculated metabolisable energy requirements to term (12-13 MJ/day near to term) (2). Offspring were delivered spontaneously and were reared by their mothers as singletons (one twin being euthanized) from day 7 to weaning (10 weeks). There were 3 and 2 males in the control and nutrient restricted group, respectively. After birth, all mothers were fed a diet of hay ad libitum together with a fixed amount of concentrate pellets sufficient to fully meet their own metabolisable energy requirements, plus that needed to maintain lactation. All diets contained adequate minerals and vitamins. From weaning to 12 months of age all offspring were group-housed in a barn (50m2; i.e. restricted activity) with ad lib access to hay and concentrate pellets (crude protein 140 g/kg, oil 3%, 12.7 MJ/Kg dry matter; Manor Farm Feeds, UK) to promote increased fat deposition; these sheep were designated as either ‘Obese’ (O) or ‘Nutrient Restricted Obese’ (NRO), as previously described (48). A further group of sheep (n=8) were also incorporated into the experimental design to control for the postnatal treatment structure i.e. adolescent-onset obesity. These sheep were contemporaneous to control animals i.e. the ewes were fed 100% ME requirements throughout gestation and gave birth to twin female offspring, with only one of each twin pair used for study. At weaning, however, these sheep were allowed an environment that encouraged unlimited low-moderate physical activity (a field as oppose to a barn) with supplemental feed provided as required (51). These sheep were designated as ‘lean’ (L). At one year of age i.e. as young adults, all sheep had arterial (carotid) and venous catheters (jugular) inserted. Prior to surgery, all food, but not water, was withdrawn from the animals for 24 h. Anaesthesia was induced with propofol (Rapinovet; 6 mg.kg-1) and maintained with 3-4% isoflurane in 3-4 l.min-1 O2. All sheep received a course of antibiotic (10 mg.kg-1 I.M. procaine penicillin, ‘Duphapen’; Fort Dodge Animal Health Ltd, Southampton, UK) and analgesia (2 mg.kg-1 I.M. flunixin meglumine; ‘Finadyne’; Schering-Plough, Kenilworth, UK) for 3 days post-operatively. Catheter patency was maintained by daily flushing with heparinized saline (50 I.U. heparin.ml-1). All sheep had established normal feeding patterns within 1 h after surgery and showed no visible signs of discomfort for the duration of the experimental period.

Experimental protocols

Cardiovascular experiments

No experiment was performed until after 2-4 days post-operative recovery. The investigator was blinded to the dietary origin of the sheep prior to any experiment being performed. All sheep were pre-habituated to the experimental conditions prior to the same experiment being conducted with 3 different treatments on three separate days; 1) with a background infusion of saline, 2) with pre-treatment and infusion of the muscarinic antagonist, atropine sulphate and 3) with pre-treatment and infusion of the mixed ß-antagonist, propranolol.

Cardiovascular responses to sodium nitroprusside (SNP) infusion

Sheep were habituated to a metabolic crate and after at least an hour the arterial catheter was connected to pre-calibrated pressure transducers (SensorNor 840; S 4925) attached at heart level linked to a data acquisition system (Po-Ne-Mah; Version 3, Gould Instrument Systems Inc) and a baseline recording taken over a further hour. Analogue signals for real-time systolic, diastolic, mean arterial pressure and heart rate were recorded at one-second intervals, digitized and then stored on an Excel spreadsheet for further analysis (18). Resting cardiovascular data (baseline systolic, diastolic, mean arterial pressure and heart rate) for these animals has been described elsewhere (51). The first derivative of the positive (+dp/dt) and negative (-dp/dt) change in pressure associated with each heart beat was calculated automatically and was taken to reflect the strength of myocardial contractility and relaxation, respectively. The rate pressure product [(mmHg.min-1)/103] was used as an assessment of myocardial work.

Saline infusion

Upon a background of saline infusion (1 ml-1.min-1), the sheep were infused I.V. (2.5 μg.kg-1.min-1) with the endothelium-dependent vasodilator sodium nitroprusside (SNP; Abbott Laboratories, Maidenhead, UK) for 5 min with a further 5 min recording of the recovery period.

Atropine infusion

On a separate day, the protocol for saline infusion was followed exactly except the sheep received a bolus dose of atropine I.V. (2.4 mg) followed by a constant infusion of 1 mg.ml-1. Further bolus doses (1.2 mg) were given and each failed to elicit any increase in heart rate confirming complete muscarinic blockade, as previously described in detail (53).

Propranolol infusion

On a separate day, the protocol for saline infusion was again followed except the sheep received a bolus dose of propranolol I.V. (20 mg) followed by a constant infusion of 1 mg.ml-1.min-1 as previously described in detail (53).

Blood and molecular analyses

Biochemical analyses

Whole blood was withdrawn from the jugular catheter into pre-treated (50 μl glutathione and EGTA solution (4.75 g EGTA and 3.00 g glutathione dissolved in 50 mL deionized water)) heparinised blood tubes at -5min before and +4min into the SNP infusion with saline. Plasma concentrations of catecholamines were measured by HPLC with electrochemical detection, as previously described in detail (19). At the end of all experimental protocols all sheep were humanely euthanased with electrocortical stunning and exsanguination. A representative sample of left ventricular (LV) tissue was flash frozen in liquid nitrogen and stored at -80°C until analysis.

Lipid extraction and tissue triglyceride assay

Lipids in frozen left ventricle (~ 500mg) were first extracted with a mixture (1:1) of chloroform and methanol and were dissolved in tert-butyl alcohol/Triton X-100 mixture (1/1 by volume) before measuring enzymatically (Infinity ™ Triglycerides Liquid Stable Reagent, Thermo Electron) (12; 18).

Total RNA isolation and reverse transcription

RNA was extracted from a fixed quantity of the LV (~100-200 mg) using Tri® reagent (T9424, Sigma). RNA concentration was measured spectrophotometrically and its purity confirmed by measuring the A260/A280 ratio. RNA concentration was adjusted to 3μg/μl using nuclease-free water (Ambion) and samples were stored at -80°C. First-strand cDNAs were reverse transcribed in a reaction containing 3 μg of total RNA, 200 U of SuperScript™ II reverse transcriptase (18064-014, Invitrogen), first strand buffer (250 nM tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 125 ng pd(N)6 random hexamer 5′-phosphate (27-2166-01, GE Healthcare), 10 mM dNTP mix (28-4065-64, GE Healthcare), and 40 units RNaseOUT Recombinant Ribonuclease Inhibitor (10777-019, Invitrogen). The conditions of synthesizing cDNA were therefore in accordance with the manufacturer’s protocol.

Polymerase chain reaction for standard curve generation

PCR was performed in the presence of Thermo-Start® Master Mix (AB-0938-DC-MM, ABgene). The forward and reverse primers (Sigma Genosys) are given in (Table 1). The amplification parameters were set at: 95°C (15min); 35-40 cycles of 94°C (45secs), annealing temperature (30secs), 72°C (45secs); 72°C (15min). The annealing temperature for the α/β-adrenergic receptors, PGC-1α and AMPKα2 was 55°C and for ACC was 60°C. The amplified PCR products were then extracted from agarose gels by QIAquick® gel extraction method (28706, Qiagen). The extracted amplicons were then sequenced to confirm the target region of amplification and serially diluted to generate standard curves for real-time polymerase chain reaction assay.

Table 1
Summary of ovine specific oligonucleotide primers for myocardial genes determined using real-time PCR

Quantitative Real-Time Polymerase Chain Reaction Assay

The relative abundance for each gene was measured by quantitative real-time PCR, using the Techne Quantica Real-Time Nucleic Acid Detection System (Techne, Barloworld Scientific Ltd) with QuantiTect SYBR Green PCR Master Mix (204145, Qiagen), diluted RT reactions, and 10-15 pmol of the forward and reverse primers (Table 1). A reverse transcribed (RT)-negative control was used to ensure absence of genomic DNA contamination. The amplification parameters were set at: 95°C (15 min); 45 cycles of 94°C (30 secs), using the same annealing temperature as detailed above. Melt curve analysis was performed to ensure reaction specificity. Primers were designed to amplify small fragments (150-250 bp) and ensure ~98% amplification efficiency. 18s was chosen as the housekeeping gene as the alternatives (e.g. GAPDH and ß-actin) are nutritionally sensitive (49).

Statistical analyses

Data were first analyzed for an effect of treatment group (L, O and NRO) by a univariate general linear model (GLM) procedure with treatment and, where appropriate, sex as a fixed effect using SPSS v14 (SPSS Inc, Chicago, IL). Specific contrasts selected a priori to test for effects of postnatal obesity (L vs. O) or prenatal diet (O vs. NRO) were also examined. Estimated marginal means are presented together with their respective S.E.M. unless otherwise stated. For cardiovascular responses, data were analyzed as area-under-the-response-curve (GraphPad Prism v5; San Diego, USA). For repeated measures (e.g. catecholamines before and after SNP) all data were first compared by paired-t-test followed by a repeated measures general linear model to test for any treatment effects (SPSS v14). For all comparisons, statistical significance was accepted when p<0.05.

Results

Body composition and metabolic status

The overall effect of exposure to an ‘obesogenic’ environment (~65% reduced physical activity, ~30% increased food intake (51)) from after weaning (3 months of age) to young adulthood (1 year of age) has been reported previously (43; 51), but to summarise; obese vs. lean sheep were, by definition, significantly heavier (obese, ~90 vs. lean, 58kg; ± 1.8 [s.e.d.]) and fatter (obese, ~7.0 vs. lean, 1.5kg ± 0.3 [s.e.d.] visceral fat mass) at one year of age, with marginally increased fasting plasma glucose concentration (obese, 5.47 vs. lean, 4.47 mmol.L-1 ± 0.57 [s.e.d.]) and significantly higher plasma non-esterified fatty acid (obese, 0.60 vs. lean, 0.31 mmol.L-1 ± 0.08 [s.e.d.]), leptin (obese, 20 vs. lean, 3 ng.mL-1 ± 1 [s.e.d.]) and insulin (obese, 1.34 vs. lean, 0.56 ng.mL-1 ± 0.20 [s.e.d.]), concentrations.

Left ventricular triglyceride and transcript expression for cardiac energy metabolism

The triglyceride content of the left ventricle was unaffected by postnatal obesity per se but was significantly increased (~3 fold) in those obese offspring born to nutrient restricted mothers (Figure 1a). Myocardial expression of FABP3 and PPARγ2 was raised by obesity, a response that was not seen in those obese offspring born to nutrient restricted mothers (Figure 1b&d). However, mRNA abundance for ACC was reduced with postnatal obesity, but this was not observed in nutrient restricted obese offspring (Figure 1c). Gene expression of PGC-1α, AMPKα2 and VDAC1 were all reduced by postnatal obesity per se, again an adaptation not observed in nutrient restricted obese offspring (Table 2). Gene expression of AMPKα2 was positively correlated with triglyceride content in the NRO offspring only (r2 = 0.57, p<0.05, Figure 2).

Figure 1
Left ventricular triglyceride and transcript expression for cardiac energy metabolism (a) Triglyceride content in left ventricle; mRNA abundance of (b) fatty acid binding protein 3 (FABP3), (c) acetyl CoA carboxylase (ACC), and (d) peroxisome proliferators ...
Figure 2
Positive correlation between AMPKα2 and triglyceride content in the heart of obese offspring born to mothers nutrient restricted between early-to-mid gestation.
Table 2
Mean expression of genes involved in cardiac energy metabolism in the left ventricle of lean (L), obese (O) or prenatally nutrient restricted obese (NRO) offspring

There was no effect of obesity on gene expression for GLUT 4, the insulin receptor (IR) or glucocorticoid receptor (GR), although GR and GLUT 1 mRNA abundance were both reduced in the NRO offspring (Table 2). In the O group, however, GLUT 1 gene expression was raised compared to the lean animals (Table 2).

Plasma catecholamines and myocardial adrenergic receptor gene expression

Resting plasma catecholamines were almost two-fold greater in O vs. L but individual variation prevented this achieving statistical significance (Figure 3a). With hypotension, obese animals exhibited a significantly greater increment in total plasma catecholamines (L, -0.153±0.716; O, 0.538±1.523; NRO, 1.516±0.946 nmol/L: L vs. O P<0.05). Gene expression for both the β1 and 2 adrenergic receptors was reduced with obesity, but not when the obese offspring had been born to nutrient restricted mothers (Figure 3b&c). There was no difference between dietary groups in the expression of mRNA for either the α1 or α2 adrenergic receptors (data not shown).

Figure 3
Effects of obesity and maternal nutrient restriction on (a) total plasma catecholamine concentration and cardiac gene expression of (b) β1 and (c) β2-adrenergic receptors. Values are expressed as means ± SEM and lean (n=8), obese ...

Resting cardiovascular status and response to SNP

Saline infusion

Prior to saline infusion, mean arterial pressure was higher in obese sheep (O and ONR) vs. lean (L) (O, 97±2 and ONR, 99±2 vs. L, 89±1 mmHg, P=0.03, 1-way ANOVA) with no interaction with prenatal diet. These results are similar to those published previously for systolic and diastolic pressures in these animals (51). Additionally, obese sheep had a significantly higher resting rate pressure product (L, 8.62±0.87; O, 10.07 ± 0.66; NRO, 11.96 ± 0.82 ((mmHg.min-1)3) but similar +dp/dt (L, 800±117; O, 739 ± 113; NRO, 803±103 mmHg) and -dp/dt (L, 545±74; O, 328±72; NRO, 458±66). SNP infusion elicited a rapid and significant decline in blood pressure (Figure 4a), provoking a significant increment in heart rate (Figure 4b) during the challenge that was significantly greater (0 to +5min; p<0.05, F=4.25) in lean vs. obese sheep (L, 33±8; O, 12±8; NRO, 12±7 beats.min-1). Whilst there was no effect of obesity per se on +dp/dt (Figure 4c), there was a trend (p=0.09, F=3.16) for the increment to be greater in the prenatally NRO group (O, 2689±2008 vs. NRO, 7208±1555 AUC units). In addition, there was a trend (p=0.09, F=3.15) for the rate of cardiac relaxation (-dp/dt) to be slower in obese sheep (L, 6935±1819 vs. O, 3232±1017 AUC units) (Figure 4d). During the 5 min recovery period, the return of diastolic pressure (L, -137±47 vs. O, -362±26 AUC units; p=0.001, F=17.2) and +dp/dt (L, -487±1228 vs. O, 3113±686 AUC units; p=0.01, F=6.54) toward baseline was significantly blunted in obese vs. lean sheep. There were no effects of prenatal diet on the recovery of cardiac function in obese sheep.

Figure 4
Cardiovascular response to sodium nitroprusside during saline infusion in lean (open circles, n=8), obese (closed circles, n=6) and obese offspring born to mothers nutrient restricted between early-to-mid gestation (shaded squares, n=10) at one year of ...

Atropine infusion

There were no significant effects of obesity or prenatal diet on the cardiac response to SNP during atropine infusion (Figure 5), however, the differences in response observed during saline were abolished when conducted on a background of muscarinic blockade (Figure 4avs.Figure 5a). Overall the cardiac response to SNP as reflected in heart rate (Figure 5b), +dp/dt (Figure 5c) and -dp/dt (Figure 5d) following atropine was similar to that observed during saline (i.e. a comparison of equivalent results presented in Figure 5 to Figure 4) in all groups.

Figure 5
Basal blood pressure response to sodium nitroprusside during atropine infusion in lean (open circles, n=8), obese (closed circles, n=6) and obese offspring born to mothers nutrient restricted between early-to-mid gestation (shaded squares, n=10) at one ...

Propranolol infusion

There were no significant effects of obesity or prenatal diet on the cardiac response to SNP during propranolol infusion (Figure 6a). As expected, propranolol significantly blunted the increment in heart rate (Figure 6b), cardiac contractility (+dp/dt, Figure 6c), and relaxation (-dp/dt, Figure 6d) in all groups relative to results observed during saline infusion (i.e. a comparison of equivalent results presented in Figure 6 to Figure 4). In accord with the response observed during saline, the recovery of diastolic pressure following withdrawal of SNP tended to be blunted in obese compared with lean sheep (Figure 6a).

Figure 6
Basal blood pressure response to sodium nitroprusside during propanolol infusion in lean (open circles, n=8), obese (closed circles, n=6) and obese offspring born to mothers nutrient restricted between early-to-mid gestation (shaded squares, n=10) at ...

Discussion

The major finding of our study is that fetal exposure to maternal nutrient restriction during early gestation increased ectopic lipid deposition in the left ventricle together with a resetting of cardiac energy metabolism when the offspring became obese. This was particularly striking since contemporaneous nutritional controls, that were equally obese, had as low levels of myocardial ectopic lipid as lean animals. Myocardial lipid infiltration is normally related to current diet, aging and diastolic dysfunction, and clinically is often seen in heart failure patients with diabetes or obesity (5; 29; 45). Our findings in prenatally programmed animals thus suggest they are potentially more at risk from earlier heart failure. However, despite having higher blood pressure (51) they did not show any obvious clinically significant metabolic or other health problems at ~ one year of age (43). Therefore, the observed molecular changes that accompanied raised lipid within the hearts of these in utero nutrient restricted, obese animals may provide important early evidence of the developmental mechanisms by which cardiac function can become impaired with obesity.

Obesity is the most significant and potentially preventable risk factor for suffering coronary events; increasing the incidence of other significant risk factors such as hypertension (34; 35; 48). Defined obesity is increasingly being observed in childhood and subsequently tracking into adulthood giving these individuals an exacerbated cardiovascular risk (4). In the present study juvenile-onset obesity per se led to left ventricular hypertrophy (LVH) most likely through increased afterload, myocardial workload and increased peripheral vascular resistance (47). Indeed, obese animals in the present study had significantly greater rate pressure product, indicative of increased myocardial functional stress and together with the increase in left ventricular wall thickness, but without clinical symptoms, suggests these offspring at increased risk of heart failure (1; 5; 40; 52). Functionally, the heart in all obese sheep was compromised as indicated by delayed myocardial relaxation after a hypotensive stimulus. Whilst it is accepted that the greater increment in plasma catecholamines during hypotension in obese animals may, in part, contribute to this response the fact that propranolol infusion failed to eliminate the effect suggests deficits in cardiac contractility per se as the primary mechanism.

Chronic sympathetic activation by systemic β-AR agonist administration results in cardiac hypertrophy, blunted parasympathetically-mediated cardiovascular reflexes, suppressed β-AR mediated inotropic responses and sensitivity (10; 36; 37). We also report reduced gene expression of β12-AR (but not α12-AR) in hypertrophied myocardium with obesity. Desensitization of adrenergic signalling occurs in chronic heart failure that is characterized by a rapid increase in sympathetic activation and a decrease in β1-AR abundance (7; 20). In the present study, there was a non-significant trend for the obese animals to have higher circulating catecholamine concentrations than lean animals and, in the O group only, down-regulated β12-AR. Maternal nutrient restriction combined with postnatal obesity, however, resulted in protected β12-AR gene expression and consequently a strong and sustained increase in systolic contractility with hypotension that was abolished by pre-treatment with propranolol.

In combination with reduced β12-AR signaling in the obese heart we also observed significant changes to the molecular machinery influencing myocardial energy metabolism as reflected by lower transcript levels of β12-AR, AMPKα2, ACC and PGC-1α. Myocardial energetic deficiency is the likely mechanism responsible for an apparent inability to increase cardiac work under physiological stimuli such as hypoxia, sympathetic activation and ischemia (3). Myocardial PGC-1α, has been shown to be down-regulated by obesity induced through consumption of a high fat diet and to underpin the observed cardiac dysfunction both in this model (15), and in another of LVH unrelated to obesity (31). Thus, in the current study, we propose that low PGC-1α in both groups of obese animals is a major factor contributing to LVH (51). In addition, further myocardial energetic controls are mediated by AMPK, of which α2 is the primary isoform mediating its catalytic activity in the heart (9). AMPKα2 is activated by a high AMP:ATP ratio and the subsequent phosphorylation of ACC that it enables, favours myocardial fatty acid utilization. Again, we have confirmed that obesity down-regulates AMPKα2 and ACC mRNA abundance. However, importantly, in prenatally nutrient restricted, obese animals (NRO group) there was dissociation between the two enzymes, i.e. low AMPKα2 but normalized ACC. This suggests early life exposure to a reduced maternal intake of a balanced diet, entrains a myocardial energetic imbalance when coupled with excess weight gain postnatally. Therefore, based on these data, we speculated that the myocardium of NRO animals was more susceptible to fatty acid infiltration. Indeed, this proved to be the case. Triglyceride content in the hearts of prenatally nutrient restricted, but postnatally overnourished animals was ~3-fold greater than in contemporaneous overweight controls that had similar myocardial triglyceride content to lean animals.

The primary change in oxidative metabolism in the failing heart is from a reliance on the ß-oxidation of non-esterified fatty acids to glycolysis for metabolic energy i.e. a return to a fetal pattern of oxidative metabolism (6; 17). Indirect evidence that an adaptation of this type was beginning to occur in the previously nutrient restricted obese offspring is provided by the relative changes in transcript expression for the enzymes governing myocardial energy metabolism (ACC, AMPKα2 and FABP3). Maintenance of elevated ACC in conjunction with reduced AMPKα2 in the hearts of offspring born to nutrient restricted mothers could be indicative of a shift away from fatty acid toward carbohydrate oxidation (17). Indeed, removal of the normal inhibitory effect of ACC on AMPKα2, which is the catalytic subunit in the heart, could explain the strong positive correlation between this gene and triglyceride content that was only seen in the obese animals born to nutrient restricted mothers. In addition, the apparent failure of nutrient restricted offspring to increase gene expression of FABP3 with obesity, would further suppress fatty acid oxidation in the heart. Alternatively it may be that in these animals the primary source of substrate utilization is from ectopic stores i.e. stored triglycerides rather than circulating metabolites, as GLUT 1 gene expression was also substantially suppressed in these individuals. Given the resetting of substrate metabolism in the obese animals born to nutrient restricted mothers, we indirectly determined the potential for ATP and ADP transport by measuring gene expression of the inner mitochondrial membrane protein VDAC1 (32). Its expression was suppressed in all obese animals and given that VDAC1 may also influence glucose metabolism (32), our findings suggest potential metabolic inefficiency in the hearts of previously nutrient restricted but obese offspring (16; 46). Clearly, future studies should be focused on examining the regulation of cardiac energy metabolism in nutritionally programmed animals that subsequently become obese.

Perspectives and Significance

We have shown for the first time in a relevant large animal model that maternally nutrient restricted offspring, as obese adults, develop dysfunction in their myocardial energy metabolism resulting in a tendency for elevated ectopic lipid deposition in their myocardial tissue. It is hypothesized that these individuals would therefore have an increased likelihood of heart failure as they become aged; an adverse outcome that is likely exacerbated by their accompanying insulin resistance (43) and changes to adipose tissue biology (44).

Acknowledgements

This work was supported by a British Heart Foundation Lectureship to DSG (BS/03/001) and the European Union Sixth Framework Programme for Research and Technical Development of the European Community - The Early Nutrition Programming Project (FOOD-CT-2005-007036).

Footnotes

Disclosures

No author declares any conflict of interest with regard to this manuscript.

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