Prenatal hypoxia plus postnatal high‐fat diet exacerbated vascular dysfunction via up‐regulated vascular Cav1.2 channels in offspring rats

Abstract Background This study aimed to examine whether and how postnatal high‐fat diet had additional impact on promoting vascular dysfunction in the offspring exposed to prenatal hypoxia. Methods and Results Pregnant Sprague‐Dawley rats were randomly assigned to hypoxia (10.5% oxygen) or normoxia (21% O2) groups from gestation days 5‐21. A subset of male offspring was placed on a high‐fat diet (HF, 45% fat) from 4‐16 weeks of age. Prenatal hypoxia induced a decrease in birth weight. In offspring‐fed HF diet, prenatal hypoxia was associated with increased fasting plasma triglyceride, total cholesterol, free fatty acids, and low‐density lipoprotein‐cholesterol. Compared with the other three groups, prenatal hypoxic offspring with high‐fat diet showed a significant increase in blood pressure, phenylephrine‐mediated vasoconstrictions, L‐type voltage‐gated Ca2+ (Cav1.2) channel currents, and elevated mRNA and protein expression of Cav1.2 α1 subunit in mesenteric arteries or myocytes. The large‐conductance Ca2+‐activated K+ (BK) channels currents and the BK channel units (β1, not α‐subunits) were significantly increased in mesenteric arteries or myocytes in HF offspring independent of prenatal hypoxia factor. Conclusion The results demonstrated that prenatal hypoxia followed by postnatal HF caused vascular dysfunction through ion channel remodelling in myocytes.

initiates the centralisation of blood flow to vital organs, such as the heart and brain. 5,8 Although this adaptation is necessary for critical organs, the decreased peripheral blood flow can impair the development of other tissues and results in intrauterine growth retardation (IUGR). Numerous retrospective and prospective studies have revealed that low birth weight is associated with the development of CVD in later life. 3,4,9 Unhealthy living habits after birth could exacerbate the impact of adverse exposure in utero. 2 For example, postnatal high salt intake could exacerbate blood pressure (BP) and dysfunction of ion channels in vascular smooth muscle cells (VSMCs) in the offspring exposed to prenatal hypoxia. 10 Prenatally programmed vulnerability to CVD could be exacerbated by high-salt diets in postnatal life. 11 It is well known that high fat intake plays an important role in the pathogenesis of CVD. Previous studies demonstrated that a combination of prenatal hypoxic insult and postnatal high-fat diets could increase the susceptibility to cardiovascular dysfunction, 12 the mechanisms of which are still unknown.
Vascular dysfunction with increased arterial tone is a major contributing factor for hypertension. Mesenteric arteries (MA) are typical peripheral resistance vessels. They play an important role in regulating BP based on functions of VSMCs. 13 In general, large-conductance Ca 2+ -activated K + (BK) channels in VSMCs can be activated by membrane depolarisation and intracellular local Ca 2+ release. Activation of BK channels increases K + efflux, causing cell membrane hyper-polarisation, and deactivation of voltage-dependent Ca 2+ channels, ultimately resulting in vascular relaxation. 14 Therefore, the coordination of BK and voltage-dependent Ca 2+ channels in VSMCs are critical in regulating vascular tone. However, whether and how those two channels may contribute to mesenteric artery dysfunctions in the offspring exposed to prenatal hypoxia and postnatal high-fat diet is largely unknown.
The present study established a rat model: following prenatal hypoxia, the offsprings were given either high fat or normal diets during early postnatal life. Then, we assessed the impact of chronic prenatal hypoxia or/and postnatal high fat intake on arterial blood pressure, functional resistance vessels, and ion channels of mesenteric artery smooth muscle cells (MASMCs), including BK and Cav1.2 channels in male young adult offspring. We suggested that vascular or cellular functions, as well as ion channel activities, would be affected by prenatal hypoxia, which would be further exacerbated

| Animals
Sprague-Dawley rats (Su Pusi Biotech., Suzhou, China), 240-270 g, were allowed access to standard food and tap water ad libitum and housed under a 12 hours light-dark cycle. After acclimatisation for a week, female rats were mated and pregnancy was confirmed by the presence of vaginal plug observed the following day, which was designated as gestational day (GD) 0. On GD 5, pregnant rats were randomly assigned to normoxia control (Con) and prenatal hypoxia (PH) group. From GD 5 to GD 20, the control rats were housed in a chamber filled with room air, while the hypoxia group was treated with the same chambers infused with nitrogen to maintain the oxygen concentration at 10.5%. On GD 21, all dams were moved out from chambers for natural delivery.
A subset of male offspring aged 4 weeks (n = 26 offspring from 13 L, each group) was randomly allocated to receive either a high-fat (HF) diet (45% fat) or a normal diet with low fat (LF) (5% fat, Slacom, Shanghai, China). Then, four groups were created: normoxia control offspring with the LF diet (CLF; n = 13 from 13 L), normoxia control offspring with the HF diet (CHF; n = 13 from 13 L), prenatal hypoxia offspring with the LF diet (HLF; n = 13 from 13 L), and prenatal hypoxia offspring with the HF diet (HHF; n = 13 from 13 L). Those feeding were provided for 12 weeks before testing.

| Measurement of blood pressure
Male young adult offspring rats (n = 7-10/group) were anaesthetized with sodium pentobarbitone (50 mg/kg, IP) for measurement of BP as described. 15 Polyethylene catheters filled with heparin were implanted in the femoral artery and were tunnelled subcutaneously, externalized at the nape of the neck. Two days after surgical recovery, BP was recorded in conscious, freely moving rats at the same time for all rats (4:00 PM). The baseline BP was monitored for 1 hour using the Power-Lab system and software (AD Instruments, Bella Vista, NSW, Australia).

| Plasma analyses
After an overnight fast from 8:00 PM to 8:00 AM, offspring rats were killed using sodium pentobarbital (100 mg/kg, i.p.). Blood samples were collected from the abdominal aorta with heparin sodium. After being centrifuged at 2000 g for 10 minutes, plasma was collected for the determination of triglyceride (TG), total cholesterol (TC), lowdensity lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), and free fatty acid (FFA). TG, TC, FFA, LDL-C, and HDL-C were determined by an automatic spectrophotometer according to the manufacturer's protocols, the testing kits were purchased from Nanjing Jiancheng Bioengineering Institute.
The mesenteric arteries were given a testing tension of 0.9 of L13.3 kPa by using a normalisation software package 10

| Electrophysiology
where V 1/2 is the voltage of half-maximal channel activation. The Ca 2+sensitivity data were fitted with the Hill equation: where η H is the Hill co-efficient, and K d is the dissociation constant defined as [Ca 2+ ] i required for half activation.

| Real-time quantitative PCR (RT-qPCR)
Total RNA was extracted from freshly isolated MA with RNAiso Plus Trizol (Takara, Japan). RNA was then reverse transcribed into cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo LI ET AL.

| 1185
Scientific, USA). The reference primer sequences for qPCR assays were acquired from previous the study. 17,18 The qPCR was performed with a SYBR ® Premix Ex Taq ™ mix (TaKaRa, Japan). Data were normalized against β-actin as internal control and calibrated with a normal control cDNA. The relative expression ratio was calculated with the 2 −ΔΔCt method.

| Western blotting
Mesenteric arteries were homogenized in lysis buffer containing a cocktail of protease inhibitors. After incubation on ice for 30 minutes, the homogenate was centrifuged at 13 800 g (30 min, 4°C).
Then the supernatant was collected and protein concentration was measured using the Bradford protein assay. Equal amount of protein (50 μg) from each group was loaded for gel electrophoresis and then electrophoretically transferred to polyvinylidene fluoride membrane. After blocked with 5% bovine serum albumin prepared in Tris-buffered saline containing 0.2% Tween-20 (TBST), the membranes were incubated with different subunit-specific primary antibodies overnight at 4°C. The antibodies included specific polyclonal antibodies (Cav1.2α1C, 1:200; BKα, 1:500; BK β1,1:500; Alomone, Jerusalem, Israel; β-actin,1:2,000; Beyotime Biotech, Shanghai, China). After three washes with TBST, the membrane was incubated with secondary horseradish peroxidase-conjugated goat antirabbit antibody (1: 4000) for 2 hours at room temperature. The immunoreactive bands were identified using enhanced chemiluminescence, and signals were recorded using an Imaging System (Tanon, Shanghai). Protein bands were quantified using Quantity One software (Bio-Rad).

| Statistical analysis
All data are presented as the mean ± SEM and analysed using GraphPad Prism, version 5.0 (GraphPad Software, San Diego CA).
Data were analysed with one-way or two-way ANOVA followed by Bonferroni post hoc tests. P < 0.05 was considered statistically significant.
However, at 4 weeks of age, there was no difference in the body weight between PH and control pups ( Figure 1A). After 12 weeks of feeding, the HF-fed offspring gained more weight than that of LF offspring ( Figure 1A). Compared with the offspring on LF diet, in HF-fed offspring, the ratio of energy intake to body weight was decreased, while the total energy intake per animal was significantly increased ( Figure 1B, C).

| Effect of prenatal hypoxia and postnatal HF diet on lipid profiles and blood pressure
High-fat diet increased plasma lipid concentrations, including TG, TC, FFA, and LDL-C, and decreased HDL-C in both control and PH-exposed rats (Table 1). PH-exposed and HF-fed rats exhibited higher concentrations of TG, TC, LDL-C, and FFA than HF-fed control offspring, and an interaction of PH and HF in elevated plasma FFA was observed (Table 1). Besides, PH alone also increased plasma TC, LDL-C, and FFA compared to the control (Table 1). After 12 weeks of the feeding, the baseline blood pressure, including SBP and DBP were increased in HF-fed rats (Table 1).
With LF diet, there was no difference in SBP and DBP between the PH-exposed and control rats. However, with HF diet, SBP and DBP were significantly increased in PH-exposed rats compared to the control (Table 1). Heart rate was increased in HF-fed rats independently from prenatal hypoxia factor (Table 1).
The effect of prenatal hypoxia and postnatal high-fat (HF) diets on body weight (A), energy intake (B), and absolute energy intake adjusted by body weight (C). LF: Low fat (n = 13 by group). CLF, control offspring with the LF diet; CHF, control offspring with the HF diet; HLF, prenatal hypoxia offspring with the LF diet; HHF, prenatal hypoxia offspring with the HF diet; PH, prenatal hypoxia.**P < 0.001, two-way ANOVA. #P < 0.05, Bonferroni post hoc test, comparing between the PH and control offspring receiving the same diet   Figure 5A). Furthermore, the Δ phenylephrine-induced vasoconstrictions and Δ AUC following Nife were significantly increased in HF-fed offspring ( Figure 5A). Importantly, a further significant increase of Δ phenylephrine-induced vasoconstrictions was observed in HHF compared with CHF, while no difference was observed between CLF and HLF ( Figure 5A).
Voltage-dependent Cav1.2 channels were further investigated using conventional whole-cell patch clamping. Figure 5B shows the T A B L E 1 Lipid profiles, blood pressure, and heart rate in a 4-month-old male offspring   Table 2). Furthermore, the peak baseline current in HHF was significantly higher than that in CHF, whereas no difference was found between CLF and HLF ( Figure 5B, Table 2). In the presence of BayK 8644 (5 μmol/L), the peak inward current densities were significantly increased and shifted to the left in MASMCs of all groups ( Figure 5B, Table 2). The peak current densities following BayK 8644 in HF-fed offspring were significantly higher than that in LF-fed offspring. Besides, prenatal hypoxia resulted in a further significant increase of peak currents HF caused up-regulated BK β1-subunits, leading to increased BK channel currents, independent of prenatal hypoxia ( Figure 6).
Mounting evidence suggests that IUGR and the following accelerated growth after birth are predictors of adult-onset diseases, such as hypertension. [19][20][21] The present study found that the birth weight of rats exposed to chronic prenatal hypoxia was decreased. Through the catch-up growth, the prenatal hypoxia offspring had comparable weights to controls after 1 month old. Consistent with previous reports, 22,23 the present study showed that more weight gain was found in the HF-exposed offspring, which might be due to significantly increased caloric intake. In addition, the offspring with both  half-maximal voltages in voltage-dependent inactivation. *P < 0.05 for the respective sources of variation (PH, diet, or their interaction) using two-way ANOVA (Con with LF diet, n = 6; PH with LF diet, n = 7; Con with HF diet, n = 9; PH with HF diet, n = 8). † P < 0.05 (Bonferroni post hoc test compared to the control offspring fed with the same diet).
F I G U R E 6 A model for a mechanistic explanation of the effect of prenatal hypoxia and postnatal HF diets on blood pressure (BP) in young adult offspring. Prenatal hypoxia increased the susceptibility to postnatal high-fat diets, which exacerbated dyslipidemia. Higher plasma FFA may contribute to elevation of Cav1.2 currents in MASMC, increasing vasoconstriction in MA, and resulting in higher BP in HHF group. The up-regulated β1-subunits of BK channels contributed to increased BK channel activities in MASMC of rats exposed HF diets as a temporary protective and compensatory measure for the augmented calcium currents that led to the increased vasoconstriction and B includes: to those with history of prenatal insults, special attention should be given to avoid high fat in later life.
The present study demonstrated that prenatal hypoxia alone did not significantly change BP in offspring at 4-month-old if postnatal diets were normal or healthy. However, the baseline BP was significantly increased in the offspring following feeding high fat for 12 weeks, which was exacerbated by prenatal hypoxia. One previous report suggested that postnatal HF diets did not alter the baseline BP in the rat offspring either born from control dams or dams exposed to hypoxia during late gestation. 24 The discrepancy may be due to the differences in experimental conditions, such as the duration of hypoxia and feeding of high fat. It has been demonstrated that high-fat diets could elevate BP in both human and animal models. [25][26][27] The novel information in our results suggests that the offspring exposed to prenatal hypoxia was more susceptible to hypertension when postnatal high-fat diet was offered.
It is known that vascular tone in peripheral resistance arteries plays a dominant role in regulating BP, and vascular tone mainly depends on constrictor state of vascular smooth muscle cells. 28 To determine possible mechanisms involved in the elevated BP in the offspring exposed to prenatal hypoxia and postnatal HF, peripheral resistance vessels were investigated in the present study. Contrary to the results reported by others using the model of hypoxia during late gestation, 23 we found that chronic foetal hypoxia significantly exacerbated the PE-mediated MA constriction following postnatal HF. This discrepancy may be due to the differences in hypoxic conditions and experimental protocols used, such as a longer period of hypoxia and treatments of high-fat diet in the present study. In addition, we also found that foetal hypoxia alone was able to increase the sensitivity of PE-induced vasoconstrictions as the EC 50 values (performance as pD2) were significantly shifted to the left in the offspring compared with controls. The endothelium plays an important role in vascular regulations. In blood vessels, acetylcholine (ACh) acts on cholinergic receptors in the endothelium to produce NO that causes vascular relaxation. In the present study, NO mediation of endothelial dependent relaxation was evaluated using ACh. We found that ACh-mediated dosedependent vasodilatation was reduced in HF-exposed offspring, independent from prenatal hypoxia factor. These results demonstrated that prenatal hypoxia plus postnatal HF could significantly increase vasoconstrictions in MA, contributing to the elevated BP in HHF offspring.
To determine the mechanisms underlying the increased vascular tone, next of our experiments focused on ion channels on smooth muscle cells of resistance arteries. BK channels in VSMC usually serve as a negative feedback mechanism to counteract membrane depolarisation and vasoconstrictions. 29,30 Numerous studies showed that dysfunction of BK channels contributed to vascular disorders.
For example, impaired BK channel functions in arteriolar smooth muscle cells presented in models of insulin resistance, 31 diabetes, 32 , genetic obesity 33 and hypertension. 34 Therefore, our initial ideas proposed that prenatal hypoxia plus postnatal high-fat diets may result in a down-regulation of BK channels in MASMC of young adult offspring, as a mechanism for increased BP and PE-mediated constriction of MA. However, in the present study, an interesting finding revealed a significant increase rather than the anticipated decrease in BK channel activities such as whole-cell BK current density was observed in the MA of offspring exposed to postnatal HF, regardless of prenatally treated with normoxia or hypoxia. Previous studies also demonstrated that high-fat diets could increase BK channel activities in middle cerebral arteries, 35 aorta, 36 and coronary arteries. 37 It is well known that activation of BK channels induces vascular cellular membrane hyper-polarisation,and then counteract vascular constrictions via reducing Cav1.2 channel activity. 38  Early studies demonstrated that FFAs could increase L-type Ca 2+ channel currents in cardiac myocytes, probably through modification of physicochemical properties of the protein or lipid interface. 47 Plasma higher fatty acids also have been reported to contribute to increased BP via elevation of Ca 2+ current density in the VSMC. 25 Uteroplacental insufficiency resulted in alterations in gene expression of hepatic fatty acid-metabolising enzymes in the foetal, juvenile, and adult male rats, which may contribute to dyslipidemia. 48 In the present study, elevated plasma free fatty acid was found in postnatal HF offspring, and exacerbated by prenatal hypoxia. Based on the above findings, a model for a mechanistic explanation of abnormal vessel tone and hypertension caused by prenatal hypoxia plus postnatal HF was proposed. Briefly, although prenatal hypoxia alone could not significantly affect the vessel tone and BP in young adult offspring rats, it decreased the physiological reserve as hepatic fatty acid metabolism was altered, which may increase the susceptibility to postnatal high-fat diets and then result in dyslipidemia (especially the higher FFA) in later life. The increased plasma FFA may contribute to the elevation of Cav1.2 currents in MASMC, increasing vasoconstriction in MA and BP in HHF group. However, more evidence should be obtained through further investigation. In addition, the up-regulated expression of β1-subunits of BK channels contributed to increased BK channel activities in MASMC of rats exposed to HF diets, which may be a temporary protective and compensatory measure for the augmented calcium currents that lead to the increased vasoconstriction and BP. We also realize the limitations of the present study. For example, if foetal data under conditions of prenatal hypoxia could be obtained, that will be helpful for further understanding the acute influence on foetal vascular systems following prenatal insults.
In conclusion, the present study demonstrated that the offspring with prenatal hypoxia was more susceptible to hypertension when high-fat diets were introduced after birth.