The Programming of Cardiac Hypertrophy in the Offspring by Maternal Obesity Is Associated with Hyperinsulinemia, AKT, ERK, and mTOR Activation

Abstract

Human and animal studies suggest that suboptimal early nutrition during critical developmental periods impacts long-term health. For example, maternal overnutrition during pregnancy and lactation in mice programs insulin resistance, obesity, and endothelial dysfunction in the offspring. Here we investigated the effects of diet-induced maternal obesity on the offspring cardiac phenotype and explored potential underlying molecular mechanisms. Dams fed the obesogenic diet were heavier (P < 0.01) and fatter (P < 0.0001) than controls throughout pregnancy and lactation. There was no effect of maternal obesity on offspring body weight or body composition up to 8 wk of age. However, maternal obesity resulted in increased offspring cardiac mass (P < 0.05), increased heart-body weight (P < 0.01), heart weight-tibia length (P < 0.05), increased left ventricular free wall thickness and area (P < 0.01 and P < 0.05, respectively), and increased myocyte width (P < 0.001). Consistent with these structural changes, the expression of molecular markers of cardiac hypertrophy were also increased [Nppb(BNP), Myh7-Myh6(βMHC-αMHC) (both P < 0.05) and mir-133a (P < 0.01)]. Offspring were hyperinsulinemic and displayed increased insulin action through AKT (P < 0.01), ERK (P < 0.05), and mammalian target of rapamycin (P < 0.05). p38MAPK phosphorylation was also increased (P < 0.05), suggesting pathological remodeling. Increased Ncf2(p67^phox) expression (P < 0.05) and impaired manganese superoxide dismutase levels (P < 0.01) suggested oxidative stress, which was consistent with an increase in levels of 4-hydroxy-2-trans-nonenal (a measure of lipid peroxidation). We propose that maternal diet-induced obesity leads to offspring cardiac hypertrophy, which is independent of offspring obesity but is associated with hyperinsulinemia-induced activation of AKT, mammalian target of rapamycin, ERK, and oxidative stress.

Numerous studies have identified in utero and early postnatal life as periods of development when an organism is particularly vulnerable to environmental insults such as suboptimal nutrition (1–3). This can lead to increased risk of metabolic disorders such as insulin resistance, obesity, and cardiovascular dysfunction (4). Initial focus on such programming was prompted by epidemiological studies linking low birth weight to increased risk of metabolic disease. Many animal models have subsequently provided direct evidence that the early environment can mediate these relationships and have shown direct effects of early diet on cardiac structure and function. Consistent with more recent human studies, animal studies have shown that early overnutrition is as detrimental to long-term health as early undernutrition (4).

The obesity epidemic, including women of child-bearing age, has focused attention toward the increasing prevalence of obese pregnancies and the associated gestational diabetes. These have detrimental effects on the mother and baby in both the short and long term (5). Children exposed to maternal obesity and gestational diabetes during fetal life have a higher risk of insulin resistance (6), myocardial hypertrophy (7), and cardiovascular disease (8, 9). In addition, maternal obesity in humans is associated with increased risk of congenital heart defects (10). These relationships have been supported by studies in animal models. Using a mouse model of maternal diet-induced obesity, it was demonstrated that overnutrition in early life programs metabolic outcomes including obesity, insulin resistance, and hypertension at 3 months of age (11). In light of the known association between current adiposity and insulin resistance and hypertension, it is unclear whether the development of these latter parameters is purely the result of increased adiposity in the offspring at this age. Other reports have shown adverse cardiac remodeling and fetal myocardial hypertrophy directly resulting from maternal obesity.

There is therefore good evidence that maternal obesity influences the structure of key organs and metabolism in the offspring. However, molecular mechanisms underlying these phenotypic outcomes remain poorly defined and their independence from offspring obesity unclear. Therefore, the aim of this study was to investigate the effects of maternal obesity on offspring cardiac phenotype before the development of offspring adiposity and to establish the potential molecular mechanisms underlying the development of offspring cardiac hypertrophy. We hypothesized that maternal obesity would result in offspring cardiac hypertrophy and that peripheral insulin resistance leading to hyperinsulinemia could provide a potential mechanism.

Offspring were studied at 8 wk of age, when there were no differences in body mass or body composition. This eliminated potential confounding effects due to adult-onset obesity. To support the observed increase in net cardiac mass, ventricular widths and area were assessed by stereology, myocyte widths were quantified, and molecular markers of cardiac hypertrophy were measured, including the cardiac fetal genes, atrial natriuretic (Nppa) and brain natriuretic peptide (Nppb), which are reactivated in the hypertrophied heart (12). The ratio of β- to α-myosin heavy chain was also calculated because an increased ratio is another widely recognized feature of hypertrophy (13). MicroRNAs (miR)-1 and miR-133, key regulators of cardiac hypertrophy and development (14, 15) and remodeling (16–18) were also assessed. To elucidate further potential underlying mechanisms attributable to hyperinsulinemia, insulin action through AKT, ERK, and p38MAPK activation was examined. Finally, markers of metabolic and oxidative stress were measured.

Materials and Methods

Animals

All studies were approved by the local ethics committee and were conducted according to Home Office Animals (Scientific Procedures, United Kingdom) Act 1986. Female C57BL/6J mice, approximately 4 wk of age were fed ad libitum either a standard control chow [7% simple sugars, 3% fat (wt/wt)] RM1 diet or a highly palatable energy-rich obesogenic diet [10% simple sugars, 20% animal fat (wt/wt) and sweetened condensed milk [55% simple sugar, 8% fat, 8% protein (wt/wt); Nestle, Croydon, UK], fortified with mineral and vitamin mix AIN93G, for 6 wk before mating for first pregnancy. Both diets were purchased from Special Dietary Services (Witham UK), the compositions of which have been previously described (11). The dams were allowed to litter and the first litter culled after weaning. This first pregnancy ensured the mice were proven breeders. After a week, mice were remated for a second pregnancy, and d 1 of pregnancy was signified by the appearance of a plug. Dams were maintained on their respective experimental diets throughout both pregnancies and lactation. Dams were weighed at the beginning and end of pregnancy. Body composition in conscious dams was measured by time-domain nuclear magnetic resonance (Bruker minispec LF series, Bruker Optik GmbH, Ettlingen, Germany) at the end of lactation.

Forty-eight hours after delivery, the litters were reduced at random to six pups with an equal sex ratio where possible. At 21 d of age, all offspring were weaned onto standard chow (RM1 diet) fed ad libitum. The animals were maintained on this diet throughout the study. At 8 wk of age, a single male offspring was randomly selected from each litter by the animal technician who was blind to the study and killed by ketamine overdose in the fed state for body composition analysis by dual-energy x-ray absorptiometry (Lunar PIXImus; Lunar, Fitchburg, WI) and tibial length measured from the bone scans. For tissue and plasma analysis, another littermate from each litter was killed by rising CO₂ asphyxiation, blood glucose measured (OneTouch Ultra; LifeScan Inc., Milpitas, CA) and blood taken for serum insulin measurements (Ultrasensitive mouse insulin enzyme immunoassay kit; Mercodia, Uppsala, Sweden). Heart and other tissues were weighed, collected, and snap frozen in liquid nitrogen and stored at −80 C until analysis (n = 7 control litters and n = 9 litters of offspring of obese dams (Mat-Ob).

Stereology of whole heart

Eight-week-old hearts were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. All sections of the block were collected at 10 μm using a Leica microtome (Leica Microsystems, Heidelberg, Germany). Slides were processed and stained for hematoxylin and eosin. All quantitative analyses of fixed tissue were performed using an Olympus BX-50 microscope (Olympus, Ballerup, Denmark), fitted with a motorized specimen stage and microcator. All analyses were performed using the Computer Assisted Stereology Toolbox version 2.0 program (Olympus), with the observer blind to the treatment groups. Wall width (micrometers) was measured on mid cardiac sections for the left ventricle (combining both the free wall and intraventricular septum), left ventricular free wall, intraventricular septum, left lumen, right ventricle, and right lumen. Diagonal lines were superimposed onto the section at ×1.25 magnification. Width was measured where the lines crossed the edge of the wall or lumen under analysis.

Multiple sections taken throughout the heart were used to quantify the area of all heart dimensions. Points were superimposed on the sections at a ×1.25 magnification, counting the number of points falling on the left ventricle and septum, right ventricle, left lumen, and right lumen using the Cavalieri Principle (19). Area was calculated by:

Where A(p) = area for each point and ΣP = sum of each point for all wall and lumen measurements separately, averaged over the number of sections analyzed.

Quantitative real-time PCR

Snap-frozen left ventricular tissue (20 mg) was placed into RNA-later solution (QIAGEN Ltd., Crawley, UK) on ice for 30 min. Tissue was then homogenized in lysis buffer and RNA extracted using the mirVana total RNA isolation kit (Ambion, Applied Biosystems, Life Technologies, Warrington, UK) as per the manufacturer's instructions. The cDNA synthesis was carried out using oligo-deoxythymidine primers (Promega, Southampton, UK). MiRs-1 and -133 were quantified using TaqMan microRNA assays (Ambion, Applied Biosystems, Life Technologies). Primers for mRNA quantification were designed using the Universal ProbeLibrary Assay Design Centre (Roche, Welwyn Garden City, UK) and target specificity confirmed with Primer-blast (National Center for Biotechnology Information, Bethesda, MD). Primer sequences used for quantitative PCR quantification with SYBR Green (Invitrogen, Life Technologies, Paisley, UK) are listed in Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org. All data are normalized to the expression of a housekeeping gene, GAPDH, the expression of which was similar between groups.

Immunohistochemistry

Frozen tissue was sectioned for hematoxylin and eosin staining and myocyte width analysis. To examine myocyte width, wheat germ agglutinin (Texas Red-X conjugate; Molecular Probes, Invitrogen) was used to stain cell borders. Briefly, sections were fixed in 4% formaldehyde for 20 min at room temperature, washed with PBS, and incubated for 1 h at room temperature in the dark with 5 μg/ml of conjugated agglutinin with gentle rocking. Slides were washed three times with PBS, air dried before the addition of SlowFade Gold mountant (Invitrogen). Images were then analyzed double blinded using IMAGE J software (http://rsbweb.nih.gov/ij/). Only myocytes in cross-section were selected and then widths from a total of 80–100 cells from two separate images per heart sample were measured and collated.

Chromatin immunoprecipitation

Frozen tissue was cross-linked for 10 min at room temperature using 1% paraformaldehyde in PBS before homogenization and sonication for three 10-sec cycles at 30% power on ice using a Sonics Vibra-Cell sonicator (Sonics & Materials Inc., Newton, CT). Samples were centrifuged at 10,000 × g and the supernatant collected. Chromatin shearing was optimized and analyzed by gel electrophoresis. Aliquots of cross-linked chromatin were incubated at 4 C overnight with 4 μg anti-serum response factor (SRF) (sc-335; Santa Cruz Biotechnology, Santa Cruz, CA) or antirabbit IgG (Jackson ImmunoResearch, Stratech, Newmarket, UK). Thirty microliters of magnetic protein-A beads (Dynabeads, Life Technologies, Paisley, UK) were added to samples 1 h before collection before washes with 150 mm and 500 mm NaCl, 0.25 mm LiCl, and Tris/EDTA buffers. Chromatin was eluted using 1% sodium dodecyl sulfate with 0.1% NaHCO₃ at room temperature before salt precipitation and proteinase K digestion for 1 h at 45 C followed by phenol-chloroform extraction. Primers for chromatin immunoprecipitation-quantitative PCR were designed based on previously published SRF response elements at both mir1 and 133a genomic loci: SRF cis-element 1 on chromosome 2 and SRF cis-element 2 on chromosome 18.

Western blot analysis

Twenty-five milligrams of ventricular tissue were powdered on dry ice and homogenized in 300 μl lysis buffer [50 mm HEPES (pH 8), 150 mm NaCl, 1% Triton X-100, 1 mm Na₃VO₄, 30 mm NaF, 10 mm Na₄P₂O₇, 10 mm EDTA with protease inhibitors (set III; Calbiochem Millipore, Watford, UK). Total protein concentration of lysates was determined by a copper/bicinchoninic assay (Sigma-Aldrich, Poole, UK). Total protein (20 μg) from each sample (each animal and each litter represented by one sample; n = 7 controls, n = 9 Mat-Ob) was subjected to SDS-PAGE along with protein markers (Fermentas, Thermo Scientific, St. Leon-Rot, Germany). Equal protein loading was confirmed by Coomassie Blue staining. For all subsequent Western analyses, equal loadings of total protein were separated by SDS-PAGE and transferred onto a polyvinyl difluoride membrane (Immobilon-P; Millipore, Billerica, MA). Protein blots were incubated in blocking buffer, before incubation in primary antibodies according to the manufacturer's protocols. Primary antibodies used were against insulin receptor substrate (IRS)-1, phospho-IRS-1 (Ser307), phosphatidylinositol 3-kinase (PI3K) p85α, and catalase (Upstate Biotechnology, Lake Placid, NY); AKT1, phospho-AKT (Ser473), PI3K p110β, phospho-ERK1/2 (Thr202/Tyr204), mammalian target of rapamycin (mTOR), phospho-mTOR (Thr 2448), p38MAPK, phospho-p38MAPK, nuclear factor of activated T cells (NFAT), c-MYC, and phospho-Forkhead box Os (FOXOs; Cell Signaling Technology, Beverly, MA); insulin receptor-β subunit, and ERK 1/2 (Santa Cruz Biotechnology, Santa Cruz, CA); glucose transporter 4 and manganese superoxide dismutase (MnSOD; Abcam, Cambridge, UK); and sirtuin (SIRT)-3 (Millipore, Watford, UK). After primary antibody incubation, blots were incubated with horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch), and immunoreactivity was detected by chemiluminescence (SuperSignal West Pico; Pierce, Fisher Scientific, Loughborough, UK). Antibody dilutions were optimized, and antibody specificity for the molecules studied was assessed using appropriate positive controls before the commencement of the experiments. Autoradiographs were analyzed by spot densitometry (AlphaEase; AlphaInnotech, San Leandro, CA). Signals from 20 μg and 10 μg of a pooled sample confirmed linearity of exposure.

Lipid peroxidation analysis

Lipid peroxidation was detected by 4-hydroxy-2-trans-nonenal (4-HNE) Adduct ELISA (OxiSelect; Cell Biolabs Inc., San Diego, CA) as per the manufacturer's instructions.

Statistical analysis

Data were analyzed from one male per litter with n = 7 controls, n = 9 Mat-Ob for all the variables studied where n refers to the litter number per group. The significance of any differences was examined by the unpaired Student's t test using Prism 5 (GraphPad, La Jolla, CA) unless otherwise stated. Protein levels are presented as mean percentage expression of control offspring ± sem. For all data sets, P < 0.05 was considered statistically significant.

Results

Maternal phenotype

Dams fed the obesogenic diet (Mat-Ob dams) were heavier than the control dams both at the start of pregnancy, 48 h after birth (after pregnancy), and at the end of lactation (Fig. 1A). Increased body weight throughout pregnancy and lactation was attributed to an increase in absolute (control, 3.4 ± 0.3 g vs. Mat-Ob, 14.3 ± 1.6 g) and relative fat mass (control, 12.1 ± 0.7% vs. Mat-Ob, 34.5 ± 2.7%) (P < 0.0001 for both) as measured at the end of lactation.

Fig. 1.

A, Maternal phenotype prepregnancy, postpregnancy, and weaning body weight. B, Offspring weekly body weights. Data are presented as means ± sem (n = 7 control, n = 9 Mat-Ob) and analyzed by unpaired two-tailed Student t test. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Effects of maternal obesity on offspring

Litter size was not different between the two groups (control, 8.4 ± 0.45 vs. Mat-Ob, 7.54 ± 0.44). Body weight of offspring from birth to 8 wk of age was not different between the two groups (Fig. 1B). At 8 wk of age, the percentage fat mass or percentage lean mass of male offspring (Mat-Ob group) compared with offspring of control-fed dams (control group) were also not different (Table 1). However, heart weight was significantly increased in the Mat-Ob group, and this was reflected in absolute cardiac mass (P < 0.05), when expressed as a percentage of body weight (P < 0.01), and as a ratio of heart weight to tibial length (P < 0.05) (Table 1).

Table 1.

Phenotype and cardiac morphometry

	Off-control	Off-obese
Body weight (g)	24.6 ± 0.52	23.56 ± 0.91
Body composition
Fat (%)	16.8 ± 0.46	17.9 ± 0.93
Lean mass (%)	83.2 ± 0.50	82.2 ± 0.91
Heart weight (mg)	127.2 ± 11.0	153.8 ± 5.9a
HW/BW (%)	0.51 ± 0.03	0.66 ± 0.03b
Tibial length (mm)	19.1 ± 0.04	18.7 ± 0.14a
HW/TL (mg/mm)	6.7 ± 0.56	8.2 ± 0.36a

Data are presented as means ± sem. HW/BW, Heart weight to body weight; HW/TL, heart weight to tibial length.

^aP < 0.05.

^bP < 0.01.

Structural and molecular indices of cardiac hypertrophy

The thickness of the left ventricular wall including both the intraventricular septum and free wall was significantly increased in Mat-Ob offspring (P < 0.01) (Fig. 2A). Separating these parameters revealed this difference resulted from an effect on the left ventricular free wall (P < 0.01) rather than the intraventricular septum (P = 0.32) (Fig. 2A). Area (square millimeters) of the left ventricle combining both the intraventricular septum and the free wall was also significantly increased (control, 16.05 ± 0.60 vs. Mat-Ob, 18.47 ± 0.96; P < 0.05). The myocyte width was also significantly higher in the Mat-Ob group (Fig. 2B; P < 0.0001).

Fig. 2.

A, Combined left ventricular wall width (includes both the intraventricular septum and left ventricular free wall), left ventricular free wall only, and intraventricular septum only. Left ventricular free wall data were log transformed and an unpaired Student t test was performed. B, Frozen sections (5 μm) of cardiac tissue were stained with wheat germ agglutinin and imaged for measurements of myocyte width. Consecutive sections of the same representative images were then stained for hematoxylin-and-eosin-stained sections. Scale bar, 50 μm for all images. Only myocytes in longitudinal axis cross-section were measured and widths measured from a total of 80–100 cells from two separate images per sample. Open bars [control (Ctrl)] and filled bars (Mat-Ob). Data are expressed as means ± sem. RNA from ventricular tissue of control and Mat-Ob males at 8 wk of age (n = 7 and 9, respectively, in which one offspring represents one litter) was isolated, purified, and cDNA obtained by reverse transcription. C, Gene expression of Nppb, Nppa, Myh7, and Myh6 was quantified by real-time PCR using primers shown in Supplemental Table 1. All data were normalized to Gapdh as described in Materials and Methods. The ratio of Mhy7 to Myh6 was analyzed by a Mann-Whitney U test and presented as means ± interquartile ranges (n = 7 control and n = 9 Mat-Ob for all analyses). ***, P < 0.001; **, P < 0.01; *, P < 0.05.

The expression of Nppb/BNP but not Nppa/ANF was up-regulated in the Mat-Ob group (P < 0.05; Fig. 2C) and the ratio of Myh7-Myh6 to β-MHC-α-MHC transcripts was also significantly increased in the Mat-Ob group (P < 0.05; Fig. 2C), consistent with these molecular markers of hypertrophy.

Expression of genes implicated in cardiac hypertrophy

We then examined genes that are frequently differentially expressed in cardiomyocyte hypertrophy. The level of miR-133 was significantly increased in ventricular tissue of the Mat-Ob group (Fig. 3A; P < 0.01). MiR-1 expression tended to be increased but this was not significant (Fig. 3B; P = 0.1). p300 levels were unchanged (Fig. 3C), but GATA-4 was significantly down-regulated (Fig. 3D; P < 0.05).

Fig. 3.

Gene expression was quantified by real-time PCR using primers shown in Supplemental Table 1, and all data were normalized to Gapdh. A–D, mir-133a, mir-1, p300, and Gata-4 expression. E, Gene expression of Srf and myocardin. F, Western blotting of SRF. G, Crossed-linked chromatin from ventricular tissue of control and Mat-Ob males was immunoprecipitated with anti-SRF, and enrichment of mir-133a SRF cis elements was quantified by quantitative PCR amplification of an approximately 100 bp amplicon around the two cis-elements. Open bars [control (Ctrl)] and filled bars (Mat-Ob). Data expressed as means ± sem (n = 7 control and n = 9 Mat-Ob for all analyses). **, P < 0.01; *, P < 0.05.

Up-regulated mir-133a expression is not associated with an increased SRF expression or SRF binding to the mir-133a cis-element

Mir-133a expression may be regulated by two SRF-responsive cis-elements in the mir-133a gene locus (20). However, neither SRF nor its coactivator myocardin was altered at the transcript level in the Mat-Ob hearts (Fig. 3E). There was also no difference in SRF protein expression (Fig. 3F) or occupancy at the mir-133a cis-elements (Fig. 3G).

Hyperinsulinemia mediates cardiac AKT1 activation and proliferation signaling

Fasting plasma insulin was significantly increased in offspring of obese dams (Fig. 4A). The fasting glucose to insulin ratio was also significantly reduced, indicating insulin resistance in relation to the regulation of glycemia (Fig. 4B). We then investigated whether levels of molecules involved in the proximal insulin signaling pathway and their activation states in the heart were altered. Insulin receptor expression was reduced in the Mat-Ob group (Fig. 4C; P < 0.01). Protein levels of total IRS-1 (Fig. 4D), Ser307 phosphorylated IRS-1 (Fig. 4E), p110β (Fig. 4F), and p85α (Fig. 4G) were unchanged.

Fig. 4.

A, Fasting insulin concentration of serum. B, Fasting insulin concentration of fasting glucose to insulin ratio of control (Ctrl) and Mat-Ob males (n = 7 and n = 9, respectively). C–G, Protein lysates from ventricular tissue of control and Mat-Ob males (n = 7 and n = 9, respectively) were subject to SDS-PAGE and Western blotted and analyzed by band densitometry. The levels of insulin receptor-β subunit (IRβ), IRS-1, phospho-IRS-1 Ser307, PI3K catalytic subunit p110β, and PI3K regulatory subunit p85α are expressed as a percentage relative to chow-fed control means ± sem (n = 7 control and n = 9 Mat-Ob for all analyses). **, P < 0.01; *, P < 0.05.

Total AKT, ERK1/2, mTOR, and p38MAPK protein levels were comparable between the two groups (Fig. 5, A, D, G, and J). However, AKT1-Ser473 phosphorylation, as well as levels of phospho-ERK1/2, phospho-mTOR, and phospho-p38MAPK, were significantly elevated in the Mat-Ob group (Fig. 5, B, E, H, and K; P < 0.001, P < 0.05, P < 0.01, and P < 0.05, respectively). The phosphoprotein to total protein ratios for all four proteins were therefore significantly increased as a result of maternal obesity (Fig. 5, C, F, I, and L).

Fig. 5.

Protein lysates from ventricular tissue of control and Mat-Ob males (n = 7 and n = 9, respectively) were subject to SDS-PAGE and Western blotted and analyzed by band densitometry. A, D, G, and J, Total AKT, ERK1/2, mTOR, and p38MAPK. B, E, H, and K, Phospho-AKT1 Ser 473, phospho-ERK1/2, phospho-mTOR, and phospho-p38MAPK are expressed as a percentage relative to chow-fed control means ± sem. C, F, I, and L, Ratios of phosphoprotein to total protein (n = 7 control and n = 9 Mat-Ob for all analyses). **, P < 0.01; *, P < 0.05.

Evidence for oxidative damage

Protein levels of MnSOD were significantly attenuated as a consequence of maternal obesity (Fig. 6A; P < 0.01), whereas catalase levels were elevated (Fig. 6B; P < 0.05). SIRT3 protein levels were reduced in the Mat-Ob group (Fig. 6C; P < 0.05). Total FOXO3a protein levels were not different; however, the amount of FOXO3a that was phosphorylated was increased (Fig. 6D; P < 0.01). There was no difference in Ncf1 (p47^phox), Cyba (p22^phox), Cybb (gp91^phox), Nox4, or Rac1 transcript levels. Ncf2 (p67^phox) expression was, however, up-regulated significantly in the cardiac tissue of the Mat-Ob group (Table 2; P < 0.05).

Fig. 6.

A–D, Protein lysates from ventricular tissue of control and Mat-Ob males (n = 7 and n = 9, respectively) were subject to SDS-PAGE and Western blotted and analyzed by band densitometry. The levels of MnSOD, catalase, and SIRT3 are expressed as a percentage relative to chow-fed control means ± sem. Phosphorylation level of FOXO3a is presented as a ratio to total FOXO3a protein levels; means ± sem (n = 7 control and n = 9 Mat-Ob for all analyses. **, P < 0.01; *, P < 0.05.

Table 2.

Expression of NOX enzyme complex

	Control	Mat-Ob
Ncf1 (p47phox)	16.10 ± 1.23	23.33 ± 7.98
Cyba (p22phox)	16.49 ± 2.13	17.37 ± 2.50
Cybb (gp91phox)	18.52 ± 1.94	20.43 ± 3.83
NOX4	20.74 ± 6.93	38.93 ± 17.33
Rac1	94.0 ± 13.61	131.0 ± 17.51
Ncf2 (P67phox)	18.11 ± 2.27	29.67 ± 4.78a

Data are presented as means ± sem.

^aP < 0.05.

Levels of the 4-HNE adduct were significantly increased in the Mat-Ob offspring (Mat-Ob, 1.18 ± 0.09 vs. control, 0.84 ± 0.06, P < 0.01), indicative of increased lipid peroxidation due to oxidative stress.

Discussion

Increased maternal body weight and obesity was confirmed in dams fed the obesogenic diet. The effect of this increased maternal adiposity was increased heart mass in the male offspring of obese dams compared with offspring of control dams without any difference in body weight. This was associated with increased myocyte width, in particular thickening of the left ventricular free wall and accompanied by increased expression of molecular markers of myocyte hypertrophy. Several key fetal genes implicated in pathological cardiac hypertrophy and function were up-regulated in the offspring of obese dams. Although a previous study (11) reported that cardiac weight as a percentage of body weight at 3 and 6 months of age did not differ between the control and Mat-Ob groups, at both these ages, body weight and adiposity was significantly greater in the Mat-Ob group. This implies that heart weight increased in parallel with body weight at those ages. In the current study, we focused on an earlier period, at a time when there is no difference in body weight or adiposity to address effects in the hearts that arise independently of these parameters. Our observation that heart weight is increased at 8 wk of age demonstrates for the first time that cardiac hypertrophy precedes differences in body weight and adiposity in the offspring and that it is therefore not a consequence of those differences but a programmed effect resulting from exposure to a suboptimal environment in early life.

The young offspring of obese mothers were hyperinsulinemic, most likely as a result of peripheral tissue insulin resistance as indicated by a reduced glucose to insulin ratio; however, insulin resistance was not measured directly in this study. This occurred in the absence of increased adiposity, thus suggesting that insulin resistance in relation to glucose homeostasis had arisen in the primary insulin sensitive tissues responsible for the regulation of glycemia, i.e. the liver, muscle, and adipose tissue. Pertinent to this study, cardiac insulin receptor levels were reduced, most likely as a result of increased internalization of the insulin-insulin receptor complex for degradation, a direct response to the hyperinsulinemia. However, despite the reduction in cardiac insulin receptors, there was increased insulin action through both PI3K and MAPK signaling pathways. Hyperinsulinemia has been previously independently associated with cardiac hypertrophy (21) as a consequence of the increased mitogenic actions of insulin in heart tissue (22). Therefore, in the hearts of obese offspring, increased insulin action through the PI3K and MAPK signaling pathways could contribute to the observed cardiac hypertrophy.

We observed a significant increase in ser473 phosphorylation of AKT1, a well-known downstream effector of PI3K activation and an established downstream event after insulin and IGF-I stimulation and an established indicator of insulin action. Importantly, we did not observe increased phosphorylation of AKT in skeletal muscle (23), implying tissue-specific mechanisms in response to hyperinsulinemia. Transgenic mice that overexpress Akt1 show spontaneous physiological cardiac hypertrophy and prolonged AKT1 activation in the myocardium resulted in dilatation and cardiac dysfunction (24). Others have shown that AKT1 is directly involved in physiological adaptive hypertrophy such as in IGF-I and exercise induced hypertrophy (25). The exact differences in AKT1 signaling in physiological vs. pathological hypertrophy are therefore controversial and not well defined.

ERK1/2 are directly regulated and phosphorylated by two kinases, MAPK kinase 1 and MAPK kinase 2, and it has been shown that ERK1/2 signaling functions in cellular proliferation, differentiation, and survival, and importantly, it regulates the induction of cardiac hypertrophy (26). Although cardiac-specific mTOR-deficient mice develop a fatal, dilated cardiomyopathy (27), partial loss of mTOR activity (27) and inhibition of mTOR by rapamycin (28) significantly impair hypertrophic cardiac growth. The increased relative phosphorylation of mTOR is therefore suggestive of an activation of protein translation to support proliferation in the context of cardiac hypertrophy.

We therefore hypothesize that activation of the AKT-ERK-mTOR pathway plays an important role in the initiation of hypertrophy and that p38MAPK activation is likely to promote pathologic cardiac remodeling (29).

In recent years numerous miRs have been shown to regulate cardiomyocyte development, structure and function in both healthy and diseased tissue. MiR-133, is down-regulated in some models of hypertrophy (14); however, in diabetic cardiomyopathy, it is up-regulated, in which it contributes to electrophysiological abnormalities such as a prolonged interval of electrical activity between the Q and T waves in human and animal diabetic hearts (18). GATA-4, which is up-regulated in many models of hypertrophy, is conversely diminished in the hearts of streptozotocin and db/db diabetic mice (30). Here we found that cardiac mir-133 and GATA-4 transcripts are up-regulated and down-regulated respectively, further supporting the hypothesis that the cardiac hypertrophy in this model is most likely a consequence of selective peripheral tissue insulin resistance, and because cardiac insulin sensitivity is preserved, systemic hyperinsulinemia is proposed as the mechanism driving cardiac hypertrophy.

Mir-133 expression has also been shown to be induced by oxidative stress (16) with suggested roles in the pathogenesis of heart failure (31) and in the transition from hypertrophy to heart failure (32). Reactive oxygen species, which are generated during normal cellular activity, are normally metabolized by antioxidant systems. The mitochondrial superoxide dismutase MnSOD dismutates superoxide anions generated within the mitochondrial membrane into hydrogen peroxide, which is then immediately degraded by catalase and other peroxidases. MnSOD deficiency has been shown to result in oxidative stress and heart failure (33), whereas a compensatory elevation in catalase levels has been observed in failing human hearts (34). Thus, reduced MnSOD levels in the hypertrophic hearts, with an opposing and compensatory increase in catalase levels, suggests a state of oxidative stress. Furthermore, the increased AKT-mediated phosphorylation of FOXO-3a, which promotes its nuclear exclusion and a transcriptional block of Foxo-dependent genes such as MnSOD, may also contribute to the observed reduction in MnSOD. Functionally, recent studies have shown that SIRT3, the main mitochondrial deacetylase, binds, deacetylates, and activates MnSOD (35, 36). A diminished SIRT3 in the hypertrophic cardiac tissue may therefore contribute to reduced MnSOD activity and a lower antioxidant capacity.

Of the seven known members of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family of NADPH oxidases, NOX2 (gp91^phox) and NOX4 are the most abundant in cardiomyocytes. NOX2/gp91^phox activation occurs through a series of complex protein-protein interactions between p22^phox (the constitutively active subunit), the organizer subunit p47^phox, the activator subunit p67^phox, p40^phox, and the GTPase Rac protein. Cell stimulation leads to the translocation of p47^phox and p67^phox to the membrane in which p67^phox directly interacts with and activates NOX2 (37). The final interaction with Rac1 GTPase activates the complex to generate superoxide by transferring an electron from NADPH in the cytosol to oxygen on the lumenal or extracellular space. The p67^phox up-regulation in the cardiac tissue of Mat-Ob offspring suggests a greater capacity to generate superoxides, contributing to oxidative stress, which in turn may induce mir-133 expression. Levels of 4-HNE adduct, a product of lipid peroxidation, were elevated, providing direct evidence for increased oxidative stress, which has a role in cardiac remodeling and dysfunction by activating apoptosis and mitochondrial dysfunction. 4-HNE has been shown to activate p38MAPK in both RL34 epithelial cells (38) and 3T3L1 adipose cells (39), and p38MAPK activation has also been demonstrated in response to hydrogen peroxide treatment in neonatal rat ventricular myocytes in vitro (40). Thus, the presence of cardiac oxidative stress may be partly responsible for the activation of intracellular signaling pathways AKT, ERK, and p38MAPK (41).

SRF and its coactivator myocardin are key positive regulators of mir-133a expression in cardiac development and hypertrophy and are up-regulated in diabetic hearts (18). Because we did not observe differences in SRF and myocardin expression or in the enrichment of SRF responsive mir-133a cis elements, we propose alternative mechanisms for regulating cardiac hypertrophy, driven by hyperinsulinemia and/or oxidative stress, which may involve mir-133.

In conclusion, maternal diet-induced obesity during pregnancy and lactation induces cardiac hypertrophy in offspring associated with hyperinsulinemia and activation of insulin signaling pathways before differences in offspring body weight and adiposity. Further experiments will determine whether this progresses to dilated cardiomyopathy or contractile dysfunction in later life and whether it arises before the development of hypertension. This model provides an opportunity to gain mechanistic insight into the role of insulin resistance in mediating developmentally programmed cardiovascular dysfunction and to establish its potential as a target of therapeutic intervention.