Coenzyme Q₁₀ prevents accelerated cardiac aging in a rat model of poor maternal nutrition and accelerated postnatal growth

Abstract

Studies in human and animals have demonstrated that nutritionally induced low birth-weight followed by rapid postnatal growth increases the risk of metabolic syndrome and cardiovascular disease. Although the mechanisms underlying such nutritional programming are not clearly defined, increased oxidative-stress leading to accelerated cellular aging has been proposed to play an important role. Using an established rodent model of low birth-weight and catch-up growth, we show here that post-weaning dietary supplementation with coenzyme Q₁₀, a key component of the electron transport chain and a potent antioxidant rescued many of the detrimental effects of nutritional programming on cardiac aging. This included a reduction in nitrosative and oxidative-stress, telomere shortening, DNA damage, cellular senescence and apoptosis. These findings demonstrate the potential for postnatal antioxidant intervention to reverse deleterious phenotypes of developmental programming and therefore provide insight into a potential translatable therapy to prevent cardiovascular disease in at risk humans.

Graphical abstract

1. Introduction

It has been known for several years that low birth weight babies have significantly increased risk of developing the metabolic syndrome and cardiovascular disease (CVD) in later life [1–4]. These findings led to the proposal of the ‘Thrifty Phenotype Hypothesis’ [5], which postulates that under conditions of sub-optimal in-utero nutrition, the fetus permanently alters its organ structure and function to ensure immediate survival of the organism. Although beneficial in continued conditions of poor postnatal nutrition, such programming is proposed to be detrimental in postnatal conditions of adequate or over nutrition. This hypothesis is supported by both human [6,7] and animal studies [8,9], which have demonstrated that accelerated postnatal growth exaggerates the detrimental effect of low birth weight.

The concept of developmental programming has been demonstrated in a diverse range of animal models including rodents [8,9], sheep [10] and non-human primates [11,12]. Such models have been particularly important for dissecting the underlying mechanisms linking suboptimal early nutrition and long-term health. Emerging mechanisms mediating the effects of a suboptimal early environment on cardiovascular disease include a reduction in cardiomyocyte number at birth [13], structural changes in the aorta [14,15], increased cardiac oxidative-stress [16] and accelerated telomere shortening [17]; a robust measure of cellular aging [18]. A growing body of evidence suggests that telomere shortening plays a pivotal role in vascular senescence and CVD risk [19,20].

Reactive oxidative species (ROS) and reactive nitrogen species (RNS) are collectively known as reactive inflammatory species (RIS) [21]. These can accumulate during cardiovascular aging [22,23] and can cause damage to DNA, protein and lipids. Additionally, impairment in the antioxidant defense network can contribute to the age-associated increase in RIS. Telomeric DNA is particularly susceptible to RIS damage [24,25] and this damage can lead to accelerated telomere shortening. Critically short telomeres are known to activate a series of DNA damage checkpoint proteins including p53, p21 and p16^INK, which can induce cellular senescence and apoptosis [26]. In the heart, one pathological consequence of increased RIS is congestive heart failure [27].

Oxidative-stress has been suggested to play a pivotal role in developmental programming [28–31]. We recently reported that animals that are born small as a result of maternal protein restriction, but grow rapidly during lactation, demonstrate increased nitrosative and oxidative-stress, up-regulation of the base excision repair (BER) machinery and increased DNA damage in heart tissue by weaning [16]. These observations are all consistent with an accelerated cardiac cellular aging phenotype. Furthermore, they suggest that increased oxidative stress leading to DNA damage is a very early consequence of suboptimal nutrition in-utero and accelerated postnatal growth and therefore may be an important therapeutic target.

Maternal antioxidant supplementation can reduce some of the effects of adverse conditions in-utero. Sen and Simmons [32] reported a reduction in adiposity and improved glucose tolerance in offspring from high-fat fed pregnancies, supplemented with a mixture of vitamins A, C, E and Selenium. Giussani et al. [33] demonstrated that maternal treatment with vitamin C protects against programmed cardiac and peripheral vascular endothelial dysfunction in the adult offspring of hypoxic pregnancy. Additionally, hypertension, vascular dysfunction and microvascular rarefaction, in a rat protein restriction model can be prevented by antenatal treatment with the antioxidant Lazaroid [34].

Although these studies show proof of principle that maternal antioxidants can prevent detrimental programming effects, the doses used to achieve these effects are not recommended for use in humans and therefore unlikely to ever be feasible to administer to pregnant women. Importantly, these studies focus on interventions to the mother. In many cases, evidence is not present for suboptimal in-utero exposure until at the time of, or just after delivery. Therefore it is also important to address the potential beneficial effects of targeted postnatal antioxidant supplementation.

Ubiquinone or Coenzyme Q (CoQ) encompasses a group of homologous molecules that are present in almost all tissues. They consist of a benzoquinone ring linked to an isoprenoid side chain, the length of which varies between species. In rodents, the most common form is CoQ₉, containing 9 isoprenoid units, whereas CoQ₁₀ (containing 10 isoprenoid units) is the most common isoform in humans. It is known that rodents can convert dietary CoQ₁₀ into CoQ₉. CoQ has been shown in humans to be a safe supplement with minimal side effects [35–37]. In its quinone form, CoQ acts as an electron carrier, transferring electrons in the mitochondrial electron transport chain (ETC) between complexes I and III and complexes II and III. In its quinol form (the reduced form of quinone), it acts as the most abundant and efficient antioxidant in the body [38]. CoQ has a pivotal role as a redox link between flavoproteins and cytochromes in the mitochondrial respiratory chain. CoQ₁₀ at high doses has also been shown to act as a regulator of gene transcription in vivo, including those genes important in PPARα signaling and lipid metabolism and it has been hypothesized that CoQ₁₀ can trigger ROS-sensitive intracellular pathways that regulate the induction of specific genes [39].

In-vitro, mitoQ a modified form of CoQ, prevented telomere shortening and increased the lifespan of fibroblasts under conditions of mild oxidative-stress [40]. In-vivo supplementation of CoQ₁₀ to rats fed a high-fat diet reduced DNA double stranded breaks in peripheral blood mononuclear cells and increased lifespan [41], protected against age-related oxidative-stress and improved mitochondrial function in cardiac tissue [42], liver and skeletal muscle [43]. Furthermore, MitoQ supplementation has recently been shown to decrease features of the metabolic syndrome in ATM+/− and ApoE−/− mice [44]. In humans, several meta-analyses have demonstrated that CoQ₁₀ supplementation improves clinical outcome in patients with heart failure [45–47]. Therefore there is good evidence from studies in humans, animal models and cell culture systems, that CoQ is an effective antioxidant.

The current study therefore tested the hypothesis that supplementation with a clinically relevant dose of CoQ₁₀ would prevent the detrimental effects of nutritional programming on cardiac aging. We used model of nutritionally induced low birth-weight and accelerated postnatal growth (recuperated animals) that demonstrates cardiac mitochondrial dysfunction, increased oxidative-stress and accelerated cellular aging. We assessed the potential of postnatal CoQ₁₀ supplementation to prevent the molecular and structural cardiac defects in both young and old offspring.

2. Materials and methods

2.1. Animal experimental groups

All procedures involving animals were conducted under the British Animals (Scientific Procedures) Act (1986). Pregnant Wistar rats were maintained on a 20% protein diet (control) or, an isocaloric low protein (LP) (8%) diet fed ad libitum, as previously described [48]. Both diets were purchased from Arie Blok, The Netherlands. The day of birth was recorded as day 1 of postnatal life. Pups, born to LP diet-fed dams were cross fostered to control-fed mothers on postnatal day 3, in order to create a recuperated litter. Each recuperated litter (R) was culled to 4 male pups at random to maximize their plane of nutrition. The control (C) group was the offspring of mothers fed the 20% protein diet and suckled by 20% protein fed dams. Each control litter was culled to 8 pups as a standard. To prevent any stress or rejection to the animals when cross fostered, pups were transferred with some of their own bedding. Body weights were recorded at postnatal days 3, 7, 14, 21, 3 months and 12 months. At 21 days, 2 males per litter were weaned onto standard laboratory chow (SDS) and the other 2 littermates were weaned onto the same diet supplemented with CoQ₁₀ to give a dose of 1 mg/kg/body weight/day (group abbreviations; CQ and RQ). Animals were maintained on these diets until 3 or 12 months of age. All animals were killed by CO₂ asphyxiation. At post mortem, hearts were either removed, weighed, and snap frozen in liquid nitrogen and then stored at −80°C or fixed in formaldehyde until analysis. For all measurements, one pup per litter was used, thus N indicated throughout represents number of litters.

2.2. CoQ₁₀ dosage methodology

In order to determine the effectiveness of supplementation, and to test palatability of the supplemented diet, a 4-week pilot study was performed, using 1 mg/kg of body weight per day CoQ₁₀ a dose that is tolerated by humans without any side effects [49–51]. This dose was achieved by appropriate CoQ₁₀ supplementation of lab chow based on calculations of weekly food intake in relation to body weight. No significant difference in body weight gain (%) (13.9; 13.4; 14.3) or food intake (224.9 g±13.7; 214.9 g±12.9; 212.0 g±11.2) was observed between the CoQ₁₀ treated, acetone only or control groups, respectively. Aortic CoQ₉ was increased in the CoQ₁₀ treated group compared to the control and acetone treated groups (approximately 50%), and serum CoQ₁₀ was increased (approximately 200%) in the CoQ₁₀ treated group compared to the control and acetone treated groups. CoQ₁₀ was impregnated into the diet pellets by dissolving CoQ₁₀ in acetone and mixing this with the diet pellets [52]. This mix was left in a fume hood overnight to allow evaporation of acetone. Diet was prepared twice a week throughout the study.

2.3. Telomere length analysis

High molecular weight DNA was extracted using The Wizard Genomic DNA Isolation kit (Promega) according to the manufacturer's instructions. DNA quantity and purity were determined using a Nanodrop spectrophotometer (Nanodrop Technologies) [17]. DNA (1.2 µg) was digested by Hinf1 and RSA1 restriction enzymes at 37 °C for 2 h and separated by pulsed field gel electrophoresis (PFGE) and transferred to nylon membranes by Southern Blotting [17]. Standard undigested and digested genomic DNA from a 3-month control animal was also included on each gel to verify digestion efficiency [17]. Telomere length was measured using Telo TAGGG telomere length assays (Roche Diagnostics). Telomere signals were analyzed using Adobe Photoshop (Adobe Systems Inc) and MacBas software (Fujifilm UK) [17]. Telomere length was quantified where the percentage intensity (% telomere length) of the telomeric signal was determined in 4 molecular size regions, as defined by molecular weight markers [17].

2.4. Nitrosative and oxidative-stress analysis

Protein oxidation was assayed using a Nitrotyrosine ELISA kit (MitoSciences), according to the manufacturer's instructions. Lipid peroxidation was analyzed using an OxiSelect HNE Adduct ELISA kit (Cambridge Biosciences), according to the manufacturer's instructions.

2.5. Total CoQ₉ and CoQ₁₀ measurements

Total tissue CoQ₉ and CoQ₁₀ status was quantified by reverse phase HPLC with UV detection at 275 nm according to the method of Duncan et al. [53]. CoQ₁₀ was separated on a HPLC column (Techsphere ODS 5 µ, 150×4.6 mm²). The mobile phase consisted of ethanol:methanol:60% (v/v) perchloric acid; 700:300:1.2 (v:v) to which is added 7 g of sodium perchlorate [54]. The flow rate was maintained at 0.7 ml/min.

2.6. Gene expression analysis

RNA was extracted using the TRIzol kit (Sigma) following manufacturers' instructions. An additional DNaseI digestion step was carried out during the clean-up stage to ensure no genomic DNA contamination of the samples. RNA quantification was performed using a NanoDrop spectrophotometer (Nanodrop Technologies) and RNA integrity was assessed using an Agilent Bioanalyser (Agilent Technologies). One microgram RNA was used to synthesize cDNA using oligo-dT primers and M-MLV reverse transcriptase (Promega). Gene expression was determined using custom designed primers (Sigma) and SYBR Green reagents (Applied Biosystems). Primer sequences are presented in Supplemental Table 1A. Quantification of gene expression was performed using the Step One Plus RT-PCR machine (Applied Biosystems). Equal efficiency of the reverse transcription of RNA from all groups was confirmed through quantification of expression of the house-keeping gene ppia. Expression did not differ between groups at either 3 or 12 months, or with aging, (Supplemental Table 1B).

2.7. Protein analysis

Western blotting analysis was performed to determine protein expression of OGG-1, NTHL-1, NEIL-1, p47^phox, p67^phox, XO, MnSOD, CuZnSOD, catalase, HO-1, PRDX-3, GPx1, GR, p53, p21, p16^INK, Bax, Bcl2 and caspase 3. Protein was extracted and assayed as described previously [16]. Protein (20 μg) was loaded onto polyacrylamide gels, and was electrophoresed and transferred to polyvinylidene fluoride membranes. OGG-1, NTHL-1, PRDX-3, HO-1, catalase, GPx1, GR, Bax, Bcl2, p21 (Abcam), XO (Santa Cruz), p67^phox (Cell Signaling), MnSOD (Merck Millipore) and caspase-3 (Stressgen Biotechnologies) were all detected using anti-rabbit IgGs. p47^phox, NEIL-1, p16^INK (Santa Cruz) and CuZnSOD (R & D Systems) were measured using anti-goat IgGs. p53 (Abcam) was analyzed using anti-mouse IgGs. All IgGs were purchased from Jackson ImmunoResearch. Equal protein loading was confirmed by staining electrophoresed gels with Coomassie Blue to visualize total protein.

2.8. Stereological analysis

Twelve-month hearts were fixed in 10% neutral buffered formalin, processed, embedded in paraffin wax and cut exhaustively with all sections collected. Slides were stained with haematoxylin and eosin. Analysis was performed at 1.25× magnification using Computer Assisted Stereology Toolbox (CAST) software to quantify wall and lumen width and area. The observer was blinded to the treatment groups during analysis. Wall and lumen width was measured where the superimposed diagonal lines crossed the wall or lumen under analysis. Area was calculated using the Cavalieri principle.

2.9. Statistical analysis

All data were analyzed using a 3-way ANOVA with maternal diet, CoQ₁₀ supplementation and age as the independent variables. Data are represented as mean±S.E.M. A value of p<0.05 was considered statistically significant. All statistical analyses were performed using Statistica 7 software (Statsoft Inc). In all cases, N refers to the number of litters.

3. Results

3.1. Recuperated animals are born small and undergo rapid postnatal catch-up growth

Recuperated animals were significantly (p<0.001) lighter compared to controls on postnatal day 3 [6.1±0.1 g vs 7.3±0.1 g] and day 7 [13.4±0.6 g vs 16.2±0.8 g]. However, by day 14, this group had undergone accelerated postnatal growth hence the body weights were similar between groups [32.5±1.3 g vs 32.0±1.3 g]. Body weight (Supplementary Table 2A), cardiac weight and cardiac wall width; measured using stereology (Supplementary Table 2B), were similar between groups at both 3 and 12 months of age. However, there was a significant (p<0.001) effect of age upon body weight (Supplementary Table 2A) and absolute cardiac weight (Supplementary Table 2B), with an increase in weight observed between 3 and 12 months of age.

3.2. Dietary CoQ₁₀ supplementation prevents CoQ₁₀ deficits in the recuperated offspring

Dietary supplementation with CoQ₁₀ [1 mg/kg of body weight/day] led to an approximate doubling of plasma CoQ₁₀ at both 3 (p<0.05) and 12 months (p<0.01) (Figure 1A). No effect of maternal diet was observed on plasma CoQ₁₀ levels (Figure 1A); however plasma CoQ₉ (p<0.001) and CoQ₁₀ (p<0.001) levels increased with age in both groups (Supplementary Figure 1 and Figure 1A). No significant effect of maternal diet was observed on plasma CoQ₉ at 3 months (Supplementary Figure 1). At 12 months, cardiac CoQ₉ and CoQ₁₀ were reduced (p<0.05) in the recuperated group compared to controls, however no significant effect of maternal diet was observed upon cardiac CoQ₉ or CoQ₁₀ at 3 months (Figure 1B). Dietary CoQ₁₀ supplementation increased cardiac CoQ₁₀ (p<0.05) and CoQ₉ (p=0.06) in recuperated animals at 12 months (Figure 1B), but supplementation did not alter cardiac CoQ₁₀ levels in control animals (Figure 1B). Consistent with the plasma data, cardiac CoQ₁₀ and CoQ₉ levels increased with age (Figure 1B).

Figure 1.

The effect of in-utero protein restriction and accelerated postnatal growth upon cardiac and plasma CoQ₉ and CoQ₁₀ levels in 3 and 12 month rat hearts. CoQ₉ and CoQ₁₀ levels were measured using HPLC. Results are expressed as mean±S.E.M. * p<0.05 and ** p<0.01 compared to control/recuperated CoQ. (N=10 per group). C=control; CQ=control CoQ; R=recuperated and RQ=recuperated CoQ. (See also Supplementary Figure 1). (A) Plasma CoQ₁₀, (B) Cardiac CoQ₉ and CoQ₁₀.

3.3. CoQ₁₀ supplementation prevents the effects of maternal diet on telomere shortening

Telomere length was assessed as a marker of cardiovascular aging. At 3 months, shorter cardiac telomeres were observed in the recuperated group compared to controls, as reflected by fewer long telomeres [145–48.5 kb and 48.5–8.6 kb] (p<0.05 and p<0.01 respectively) and more short [4.2–1.3 kb] telomeres (p<0.01). CoQ₁₀ supplementation prevented these changes by increasing the percentage of long telomeres [145–48.5 kb and 48.5–8.6 kb] (p<0.01 and p<0.05 respectively) and reducing (p<0.01) the number of short telomeres [4.2–1.3 kb], (Figure 2A). At 12 months, again fewer long [145–48.5 kb] (p<0.05) and more short (p<0.05) [4.2–1.3 kb] telomeres were observed in the recuperated group compared to controls (Figure 2B). CoQ₁₀ supplementation ameliorated this shortening. Age-dependent telomere shortening was apparent in both groups with fewer long [145–48.5 kb] (p<0.001) and more short [4.2–1.3 kb] (p<0.01) telomeres at 12 months compared to 3 months (Figure 2A and B).

Figure 2.

The effect of in-utero protein restriction and accelerated postnatal growth upon telomere length in 3 and 12 month rat hearts. Telomere length was measured using Pulsed Field Gel Electrophoresis (PGFE) and Southern Blotting. Results are expressed as mean±S.E.M. * p<0.05 and ** p<0.01 compared to control/recuperated CoQ. N=6 per group; C=control; CQ=control CoQ; R=recuperated and RQ=recuperated CoQ. (A) 3 months and (B) 12 months.

3.4. CoQ₁₀ supplementation prevents the effects of maternal diet on 3-NT levels

ROS and RNS accumulation is a major mechanism that leads to accelerated telomere shortening [23,24]. We therefore investigated the effects of early nutrition upon indices of cellular stress. Levels of 4-HNE (a marker of lipid peroxidation) did not differ between any of the groups (Supplementary Figure 2A). In contrast, 3-NT levels (a marker of peroxynitrite-mediated nitration) were significantly (p<0.05) increased in recuperated animals at both 3 and 12 months, with the effect becoming more prominent with age. This damage was prevented by CoQ₁₀ supplementation at 3 months of age (Figure 3A).

Figure 3.

The effect of in-utero protein restriction and accelerated postnatal growth upon 3-NT, XO and p47^phox levels in 3 and 12 month rat hearts. 3-NT and 4-HNE levels were measured using ELISA. XO and p47^phox protein expressions were analyzed using Western blotting. Results are expressed as mean±S.E.M. * p<0.05 and *** p<0.001 compared to control/recuperated CoQ. N=10 per group (3-NT and 4-HNE levels). N=6 per group (XO protein expression). C=control; CQ=control CoQ; R=recuperated and RQ=recuperated CoQ. (See also Supplementary Figure 2). (A) 3-NT, (B) XO and (C) p47^phox.

3.5. CoQ₁₀ supplementation prevents the effects of maternal diet on xanthine oxidase

Xanthine oxidase (XO) and NADPH oxidase-2 (NOX-2) are well-established sources of cardiac ROS generation [55]. To investigate the potential sources of the increased cellular stress in the recuperated group, we measured protein levels of XO and NOX-2. XO expression was increased at 3 months (p<0.01) and 12 months (p<0.001) in the recuperated group compared to controls (Figure 3B) and therefore may contribute to the increased 3-NT levels. This increase was prevented by CoQ₁₀ supplementation (Figure 3B). p67^phox and p47^phox were the only components of NOX-2 that could be detected by Western blotting. p67^phox protein expression was not different between any of the groups (Supplementary Figure 2B). p47^phox protein levels were increased (p<0.05) in recuperated animals, however, this effect was not prevented by CoQ₁₀ supplementation (Figure 3C).

3.6. CoQ₁₀ supplementation prevents the effects of maternal diet on base excision repair enzymes

As a response to oxidative damage to DNA, enzymes in the base excision repair (BER) DNA damage pathway can be activated [56]. Therefore, we investigated the potential consequences of increased cellular stress on DNA damage repair. Protein levels of the DNA glycosylases OGG-1, NEIL-1 and NTHL-1 were measured. Protein levels of OGG-1 were increased (p<0.001) in the recuperated group at 3 months (Figure 4A). CoQ₁₀ intervention reduced (p<0.001) OGG-1 expression in the recuperated group at this age, restoring it back to the levels observed in the control group (Figure 4A). Protein levels of NTHL-1 were increased (p<0.01) in recuperated animals, however CoQ₁₀ supplementation did not alter NTHL-1 levels (Figure 4B). No effect of maternal diet or CoQ₁₀ intervention upon NEIL-1 protein expression was observed (Supplementary Figure 3).

Figure 4.

The effect of in-utero protein restriction and accelerated postnatal growth upon expression of DNA repair enzymes in 3 and 12 month rat hearts. Protein expression was analyzed using Western blotting. Results are expressed as mean±S.E.M. * p<0.05 and *** p<0.001 compared to control/recuperated CoQ. N=6 per group. C=control; CQ=control CoQ; R=recuperated and RQ=recuperated CoQ. (See also Supplementary Figure 3). (A) OGG1 and (B) NTHL1.

3.7. CoQ₁₀ supplementation prevents the effects of maternal diet on antioxidant defense capacity

DNA can be damaged if there is an imbalance between cellular RIS levels and antioxidant defenses. We investigated whether increased cellular stress in recuperated animals was accompanied by differences in antioxidant defense capacity, by measuring protein expression of antioxidant enzymes. These included the superoxide dismutase (SOD) enzymes [manganese superoxide-dismutase (MnSOD), and copper-zinc superoxide dismutase (CuZnSOD)] and the peroxidases [catalase, glutathione peroxidases (GPxs), glutathione reductase (GR) and the peroxiredoxins (PRDXs)]. MnSOD (p<0.001), CuZnSOD (p<0.01) and catalase (p<0.001) were all increased in recuperated animals at 12 months (Figure 5A–C). CoQ₁₀ supplementation prevented the increase in MnSOD at 3 months and CuZnSOD and catalase levels at 12 months (Figure 5A–C). Heme oxygenase-1 (HO-1), an inducer of MnSOD, was also increased in recuperated animals (p<0.001) at 12 months, however CoQ₁₀ supplementation did not alter HO-1 levels (Figure 5D). Peroxiredoxin-3 (PRDX-3) levels were lower in the recuperated group compared to controls at 12 months, an effect that was prevented by CoQ₁₀ supplementation (Figure 5E). GPx1 levels were increased (p<0.05) in the recuperated group compared to controls at 12 months and CoQ₁₀ intervention increased (p<0.05) GPx1 levels in the control group (Figure 5F). No statistically significant effect of maternal diet or CoQ₁₀ intervention upon HO-1 [100±17; 96±6; 93±9; 89±6], CuZnSOD [100±6; 107±8; 77±5; 152±10], PRDX-3 [100±11; 90±10; 99±8; 126±13], or GPx1 [100±8; 133±19; 76±12; 166±10] protein levels was observed at 3 months [C vs R vs CQ vs RQ]. No difference in GR protein expression was observed at either 3 months; [100±5; 88±5; 89±6; 88±4] or 12 months [113±6; 131±10; 121±6; 125±7]; [C vs R vs CQ vs RQ].

Figure 5.

The effect of in-utero protein restriction and accelerated postnatal growth upon antioxidant protein expression in 3 and 12 month rat hearts. Protein expression was analyzed using Western blotting. Results are expressed as mean±S.E.M. * p<0.05, ** p<0.01 and *** p<0.001 compared to control/recuperated CoQ. N=6 per group. C=control; CQ=control CoQ; R=recuperated and RQ=recuperated CoQ. (A) MnSOD, (B) CuZnSOD (12 months), (C) Catalase, (D) HO-1 (12 months), (E) PRDX3 (12 months) and (F) GPx1 (12 months).

3.8. CoQ₁₀ supplementation prevents the effects of maternal diet on markers of cellular senescence and apoptosis

Telomeric DNA which has been exposed to high levels of cellular stress and/or is critically short, can activate a series of DNA damage checkpoint proteins including p53, p21 and p16^INK which can induce cellular senescence and/or apoptosis [26]. Therefore, cellular senescence and apoptosis markers were measured in order to investigate consequences of accelerated cardiac telomere shortening in the recuperated group. p53 and p21 protein levels were substantially increased (p<0.001) in recuperated animals compared to controls at 12 months. This difference was prevented by CoQ₁₀ supplementation [Figure 6A (i) and (ii)]. No significant effect of maternal diet or CoQ₁₀ supplementation upon p53 [100±12.8; 101.7±13.0; 93.6±5.2; 98.9±6.6] or p21 [100±22.7; 149.1±22.8; 89.3±8.8; 148.14.0] protein levels was observed at 3 months of age. p16^INK levels increased modestly in the recuperated group compared to controls at 3 months (Supplementary Figure 4A). p16^INK levels were similar between groups at 12 months of age. Bcl2-associated X protein (Bax) is a transcriptional target of p53 and a major pro-apoptotic molecule, conversely Bcl2 is a major anti-apoptotic protein; hence the ratio of Bax/Bcl2 is a well used measure of apoptosis [57]. This ratio was increased (p<0.01) in recuperated animals compared to controls and CoQ₁₀ intervention abolished the increase at both ages (Figure 6B). This was primarily driven by an up-regulation of Bax in recuperated offspring (Supplementary Figure 4B). Under cellular stress, Bax promotes a major apoptotic molecule; caspase-3. This was increased (p<0.001) in the recuperated group compared to controls and was attenuated by CoQ₁₀ supplementation [Figure 6A (iii)]. No effect of maternal diet or CoQ₁₀ supplementation upon caspase-3 was observed at 3 months [100±2.5; 109.9±5.4; 89.3±3.6; 97.6±1.9]; [C vs R vs CQ vs RQ].

Figure 6.

The effect of in-utero protein restriction and accelerated postnatal growth upon apoptosis and senescence marker protein expression in 3 and 12 month rat hearts. Protein expression was analyzed using Western blotting. Results are expressed as mean±S.E.M. * p<0.05, ** p<0.01 and *** p<0.001 compared to control/recuperated CoQ. N=6 per group. C=control; CQ=control CoQ; R=recuperated and RQ=recuperated CoQ. (See also Supplementary Figure 4). (A) (i) p53 (12 months), (ii) p21 (12 months), Caspase 3 (12 months) and (B) Bax /Bcl2.

3.9. CoQ₁₀ supplementation prevents the effects of maternal diet upon cardiac fetal gene expression

The cardiac fetal genes such as natriuretic peptide A (nppa), natriuretic peptide B (nppb), actin, alpha-1 (acta-1) and sarco endoplasmic reticulum Ca(2+) ATPase (atp2a2) are re-expressed when the heart is under pathological stress. The sarcomere proteins (myosin heavy chains), undergo major isoform switches at birth. In postnatal healthy hearts of rodents, the ‘fast twitch’ myosin heavy chain 6 (myh6) isoform is predominant over the ‘slow twitch’ myosin heavy chain 7 (myh7). However, in failing hearts, a switch occurs, so that the myh7 isoform is predominant. In recuperated animals, the ratio of (myh7/myh6) expression was increased (p<0.05) compared to controls. Nppb was also increased (p<0.05) in the recuperated group. CoQ₁₀ supplementation restored both levels to that of controls (Figure 7A and B). These data may suggest increased cardiac stress in the recuperated group, which is normalized by CoQ₁₀ supplementation. GATA-4, a transcription factor which can induce multiple fetal cardiac genes, was reduced (p<0.05) in the recuperated group compared to controls (Figure 7C). No significant effect of maternal diet or CoQ₁₀ intervention upon nppa [438.3±101.7; 433.6±94.9; 339.4±73.7; 240.1±34.4], acta-1 [10588.4±1379.2; 9063.1±1313.6; 8069.2±1313.6; 11059.6±2127.2] or atp2a2 [42256.6±4034.7; 39197.4±6698; 53012.9±6595.6; 43390.1±3778.2] expression was observed; [C vs R vs CQ vs RQ].

Figure 7.

The effect of in-utero protein restriction and accelerated postnatal growth upon fetal cardiac genes in 3 and 12 month rat hearts. Fetal cardiac genes were measured by RT-PCR. Results are expressed as mean±S.E.M. * p<0.05 and ** p<0.01 compared to control/recuperated CoQ. N=10 per group. C=control; CQ=control CoQ; R=recuperated and RQ=recuperated CoQ. (A) Myh7/Myh6, (B) Nppb and (C) GATA4.

All results are summarized in Supplementary Table 3.

4. Discussion

Oxidative-stress is emerging as a common mechanism underlying the effects of a suboptimal environment in early life on long-term metabolic health. It is therefore becoming increasingly apparent that antioxidants may represent potential therapeutic agents to prevent the detrimental effects of suboptimal early life exposures. Although in experimental animals, maternal antioxidant supplementation in adverse pregnancy has helped to define the causal effects of oxidative-stress on programmed outcomes in the offspring, the challenge has been to identify suitable antioxidants translatable to human treatment, in terms of appropriate dosing regimens and treatment periods.

CoQ is an important inhibitor of oxidative damage [58] and has been shown to be a potential therapy for heart failure [45–47]. In addition to the cardioprotective effects, CoQ has also been widely reported have beneficial effects on aging [59,60]. Therefore, the current study aimed to test the hypothesis that poor maternal nutrition followed by accelerated postnatal growth increases RIS resulting in accelerated cardiac cellular aging during postnatal development and that dietary CoQ₁₀ supplementation of the offspring postnatal diet would prevent this effect, thus ameliorating programmed premature cardiac aging, including accelerated cardiac telomere shortening, cellular senescence and apoptosis.

Deficits in cardiac CoQ₉ and CoQ₁₀ levels were observed in the recuperated animals compared to controls, reflecting our previous findings in the kidney [9]. Administration of a clinically relevant dose of CoQ₁₀ restored cardiac CoQ₉ levels in recuperated animals, but had no effect on these levels in the control group. This suggests that when no deficit in CoQ exists, the heart does not accumulate further CoQ₁₀. Consistent with this observation CoQ₁₀ supplementation had little effect on most of the parameters measured in control animals. These data support previous findings that cardiac CoQ did not increase in control rats after 13 weeks of dietary supplementation [59]. Plasma levels of CoQ₉ and CoQ₁₀ were, as expected, highly elevated in the CoQ₁₀ treated groups, and it is known that administration of CoQ₁₀ to patients with chronic heart failure increases plasma CoQ₁₀ levels and improved the contractility of dysfunctional myocardium [61]. This may suggest that some of the current observed beneficial effects of CoQ₁₀ treatment could partially stem from the increased plasma CoQ₁₀ levels.

We have previously reported significantly shortened aortic telomere length at 12 months in recuperated offspring [17]. Here we demonstrate that recuperated offspring have shortened cardiac telomeres both at 3 and 12 months of age compared to controls, indicative of accelerated cardiac aging. Interestingly, this attrition occurs much earlier than in the aorta, despite their close proximity. Cardiac telomere length is related to increased CVD risk in humans [19,20]; therefore accelerated cardiac telomere shortening may link low birth-weight and catch-up growth to increased risk of CVD. DNA damage, such as single strand breaks (SSBs) is a major mechanism of telomere shortening [62]. We have previously demonstrated significantly increased DNA SSBs in the recuperated animals at 22 days of age [16], which may contribute to accelerated telomere shortening later in life. CoQ₁₀ supplementation prevented accelerated telomere shortening in the recuperated group, suggesting that this intervention prevents accelerated cardiac cellular aging in recuperated animals.

The recuperated group had significantly increased 3-nitrotyrosine (3-NT) levels, which are generated by the accumulation of peroxynitrite, a highly reactive oxidant, formed by the combination of nitric oxide (NO) and superoxide (^•O₂⁻). This suggests that hearts of recuperated animals have permanently increased RIS. Dietary CoQ₁₀ supplementation normalized 3-NT levels in recuperated animals, thus providing a mechanism by which it may prevent the accelerated telomere shortening. XO, the enzymatic form of xanthine oxidioreductase (XOR) activity is associated with the synthesis of large amounts of ROS and RNS. XO was increased in the recuperated animals and again CoQ₁₀ supplementation prevented this. Additionally, p47^phox protein expression was significantly higher in the recuperated group compared to controls, but this was not prevented by CoQ₁₀ supplementation. These data suggest that XO generation, and to a lesser extent, NOX2; may be sources of cellular stress in recuperated cardiac tissue and this, in the case of XO, is corrected by CoQ₁₀ administration. It has been shown that XO, but not NOX enzymes largely mediates O₂⁻ production after myocardial infarction in rats [63].

At 3 months of age, OGG-1, which repairs 8-hydroxy-deoxyguanine (8-OH-dG) DNA lesions, was markedly increased in the recuperated group compared to controls and CoQ₁₀ supplementation restored these levels to that of the controls. Interestingly, incubation of isolated mitochondrion in the presence of succinate and antimycin (which maximize the reduced CoQ pool), has been shown to eliminate 8-OH-dG oxidative damage [58]. Additionally, as site-specific DNA damage at guanine rich sequences by oxidative-stress can accelerate telomere shortening, the increase in OGG-1 in the recuperated group may provide a partial mechanism underlying their accelerated telomere shortening. No difference in OGG-1 levels was observed between groups at 12 months that may suggest that the aging process has blunted up-regulation of some of the BER machinery. NTHL-1, which excises oxidized pyrimidines, was significantly up-regulated in recuperated animals compared to controls at both ages. CoQ₁₀ supplementation did not alter NTHL-1 levels. These data suggest that both purine and pyrimidine-based lesions are increased in the recuperated group. However, CoQ₁₀ supplementation is more effective in decreasing purine-based DNA lesions.

MnSOD and catalase protein levels were significantly increased in recuperated animals. These data are consistent with our previous findings at 22 days of age [16] and may suggest a compensatory up-regulation in response to the observed increased RIS. Others have shown that insults such as abdominal aortic coarctation in rats [64] and high-fat feeding in mice [65] causes a compensatory up-regulation of MnSOD and catalase to deal with excess ROS in the cardiovascular system. Additionally, peroxynitrite-mediated tyrosine nitration can cause post-translational modifications to human MnSOD whereby total protein increased after nitration [66]. CoQ₁₀ supplementation reduced MnSOD and catalase protein levels at 3 months, and catalase at 12 months of age. There were also significant increases in CuZnSOD and GPx1 levels in the recuperated group compared to controls at 12 months. Again, these increases may be compensatory responses to increased RIS. CoQ₁₀ supplementation significantly reduced CuZnSOD levels in the recuperated group, again indicating that CoQ₁₀ may be ameliorating cellular stress in recuperated offspring. HO-1 was significantly increased in recuperated animals at 12 months; however CoQ₁₀ intervention did not alter HO-1 levels. HO-1 is up-regulated under conditions of nitrosative stress [67] and can be induced by, and acts against oxidant-induced tissue injury [68]. There was a significant decrease in PRDX-3 levels in the recuperated group compared to controls at 12 months of age. PRDX-3 deficiency can increase susceptibility to oxidative-stress [69,70]; therefore this reduction may make recuperated animals even more vulnerable to oxidative-stress. CoQ₁₀ supplementation restored these levels to those of the control group. Taken together these data suggest that CoQ₁₀, through its own potent antioxidant properties, ameliorates RIS by altering antioxidant defense capacity in recuperated animals. The antioxidant properties of CoQ₁₀ occur as a result of its redox active quinoid moiety that can relatively easily accept and donate electrons. Its hydrophobic tail means that it is soluble and mobile in lipid membranes, including the mitochondrial inner membrane, where it effectively protects the cell from phospholipid peroxidation and free-radical induced oxidative damage.

At 12 months of age, p53 and p21 protein levels were significantly increased in the recuperated group and CoQ₁₀ supplementation normalized these levels. These data suggest that recuperated animals have increased cardiac cellular senescence that can be prevented by CoQ₁₀ supplementation. There was only a modest effect of maternal diet upon p16^INK protein levels; which may suggest that the mechanism of cellular senescence/apoptosis is mediated via a p53/p21 dependent pathway. Bax and caspase-3 were also significantly increased in cardiac tissue of the recuperated group and CoQ₁₀ supplementation reduced both caspase-3 and Bax levels to that of controls. These data suggest that hearts of recuperated animals have an increased pro-apoptotic phenotype and CoQ₁₀ supplementation reduces apoptosis levels to that of controls. In addition, GATA-4 was significantly down-regulated in the recuperated group. It is known that apoptosis, induced by cellular stress, decreases the expression of GATA-4 and restoration of GATA-4 via ectopic expression attenuates apoptosis [71,72]. These data suggest that the observed decrease in GATA-4 mRNA in recuperated animals may be associated with the observed cellular stress-induced apoptosis.

CoQ₁₀ supplementation also prevented the observed increases in the ratio of myh7/myh6 and nppb in recuperated offspring. Reactivation of these cardiac fetal genes is initially an adaptive process to increase the contractility, excitability and plasticity of cardiomyocytes in response to pathological stress, which if sustained, will lead to heart failure. Therefore, the reactivation of cardiac fetal genes observed in the current study may suggest an increased risk of heart failure in later life.

It remains to be established if supplementation with CoQ₁₀ prevents any of the other detrimental effects of low birth weight and catch up growth, such as a reduction in lifespan. The potential role of ROS in determination of lifespan is debated [73]. Initial evidence suggested that that increased ROS was important in the pathogenesis of aging [74–76]. However other studies have shown that antioxidant supplementation did not alter [77] or actually shortened lifespan [78]. The reasons for this are unclear but it could relate to the poor mitochondrial penetrance (a major site of ROS production) or lack of effectiveness of certain anti-oxidants in vivo.

5. Conclusions

Nutritionally induced in-utero growth restriction followed by accelerated postnatal growth results in increased oxidative and nitrosative stress, accelerated telomere shortening, altered DNA repair and antioxidant defense capacity, in addition to increased cellular senescence and apoptosis in hearts of adult offspring. These are all indicative of a premature cardiac aging phenotype. CoQ₁₀ supplementation of the offspring postnatal diet corrected the cardiac cellular stress, telomere shortening, antioxidant defense alterations, and cellular senescence and apoptosis, thereby protecting against premature cardiovascular aging (please see Graphical Abstract). An important next step will be to establish if other suboptimal in utero exposures, such as fetal hypoxia lead to a similar phenotypic outcome in relation to accelerated cardiac aging. Such information will provide insight as to whether CoQ₁₀ supplementation is likely to represent a widely applicable intervention strategy that is likely to prevent the increased risk of cardiovascular disease resulting from an array of different in utero insults. These findings would have important implications for translatable therapies targeted towards at-risk human populations.

Conflict of interest

The authors wish to declare that there are no conflicts of interest associated with this manuscript.

Appendix A. Supplementary materials

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.molmet.2013.09.004.

Appendix A. Supplementary materials

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