Parental obesity alters offspring blood pressure regulation and cardiovascular responses to stress: role of P2X7R and sex differences

Abstract

We examined the impact of parental obesity on offspring blood pressure (BP) regulation and cardiovascular responses to stress. Offspring from normal (N) diet-fed C57BL/6J parents were fed either N (NN) or a high-fat (H) diet (NH) from weaning until adulthood. Offspring from obese H diet-fed parents were also fed N (HN) or H diet (HH). Body weight, calorie intake, and fat mass were measured at 22 wk of age when cardiovascular phenotyping was performed. Male and female HH offspring were 15% heavier than NH and 70% heavier than NN offspring. Male HH and HN offspring had elevated BP (121 ± 2 and 115 ± 1 mmHg, by telemetry) compared with male NH and NN offspring (108 ± 6 and 107 ± 3 mmHg, respectively) and augmented BP responses to angiotensin II, losartan, and hexamethonium. Male HH and HN offspring also showed increased BP responses to air-jet stress (37 ± 2 and 38 ± 2 mmHg) compared with only 24 ± 3 and 25 ± 3 mmHg in NH and NN offspring. Baseline heart rate (HR) and HR responses to air-jet stress were similar among groups. In females, BP and cardiovascular responses to stress were similar among all offspring. Male H diet-fed offspring from obese H diet-fed purinoreceptor 7-deficient (HH-P2X7R-KO) parents had normal BP that was similar to control NN-P2X7R-KO offspring from lean parents. These results indicate that parental obesity leads to increased BP and augmented BP responses to stress in their offspring in a sex-dependent manner, and the impact of parental obesity on male offspring BP regulation is markedly attenuated in P2X7R-KO mice.

INTRODUCTION

The prevalence of obesity has increased dramatically in the United States during the past three decades. This increase has occurred in children, adolescents, and adults of both sexes and all ethnic groups (1, 2). Such rapid increases in obesity and related cardiovascular (CV) and metabolic disorders cannot be simply explained by changes in genotype but may result from interactions of environmental and genetic/epigenetic influences. Obesity is a major independent risk factor for hypertension (HTN) and diabetes which, in turn, are leading causes of chronic CV and renal diseases (3–5). Uncontrolled HTN occurs in ∼58.1% and 45.5% of hypertensive men and women aged 20 and over with a higher incidence in subjects with overweight and obesity (6).

Maternal obesity is associated with a greater risk of obesity and associated metabolic disorders in their offspring (7). Although considerable effort has been devoted to investigating the genetic bases of metabolic diseases, limited evidence is available on intergenerational nongenomic causes of CV diseases. In addition, the mechanisms by which parental obesity may program CV dysfunction in their offspring are still unclear and remain largely unexplored. For example, an important unanswered question is whether parental obesity programs their offspring to not only develop increased adiposity but to also develop HTN even when the offspring consume a healthy diet and remain lean. Therefore, an important goal of the present study was to examine if parental obesity leads to greater adiposity and increased blood pressure (BP) in their offspring even when the offspring are fed a normal diet and do not become obese.

We also investigated potential mechanisms responsible for parental obesity-induced HTN in their offspring, including the potential role of excess purinoreceptor 7 (P2X7R) activation in contributing to parental-obesity-induced BP elevation in offspring. P2X7R is a purinergic receptor for ATP, and its activation triggers calcium influx, cytosolic calcium overload, endoplasmic reticulum (ER) stress, and cytotoxicity (8). Genetic variation in the region of the P2X7R gene is associated with elevated BP in Caucasians (9), and increased expression of P2X7R occurs in glomeruli of diabetic and hypertensive rats (10). P2X7R antagonism attenuates salt-sensitive HTN and renal injury in Dahl SS rats (11), which is consistent with the possibility that excessive activation of P2X7R may be a driver for kidney dysfunction and HTN. In addition, P2X7R expression is increased in kidneys from individuals with metabolic syndrome and type 2 diabetes (12). In the present study, we also investigated potential sex differences regarding the impact of parental obesity on offspring BP regulation.

METHODS

All experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee (IACUC: Protocols No. 1154 D and 0967 F) of the University of Mississippi Medical Center, Jackson, Mississippi. Mice were placed in a 12-h dark (6:00 PM to 6:00 AM) and light (6:00 AM to 6:00 PM) cycle and given free access to food and water throughout the study.

Animals

Male and female offspring from C57BL/6J obese and lean parents from our colony were weaned at 21 days and fed a normal diet (N) or high-fat (H) diet. Parental obesity was induced by feeding sires and dams an H diet (Western diet, TD.08811, 45% fat, 15% protein, and 41% carbohydrate: 4.7 kcal/g) with mating starting at 9 wk of age. Lean control parents were fed an N diet (Teklad 8640, 17% fat; 29% protein, and 54% carbohydrate: 3.0 kcal/g) and mated at the same age as obese breeding pairs. We used 5–7 breeder pairs with litter sizes of 6–8 pups/mother. After weaning, offspring were fed either the N or H diet, thus totaling four groups: 1) NN: offspring from lean (N) parents that were also fed N diet; 2) NH: offspring from lean (N) parents that were fed H diet; 3) HN: offspring from obese (H) parents that were fed N diet; and 4) HH: offspring from obese (H) parents that were also fed H diet.

Male and female P2X7R-KO mice purchased from Jackson Laboratories (B6.129P2-P2RX7^tm1Gab/J, Stock No. 005576; C57BL/6J are controls for P2RX7^tm1Gab/J mice; https://www.jax.org/strain/005576) were fed N or H diets and bred in our colony. After being weaned, offspring from obese H diet-fed parents were also fed an H diet (HH-P2X7R-KO), whereas offspring from lean N diet-fed parents were fed an N diet (NN-P2X7R-KO) and served as controls. The number of males and females ranged from 40% to 60%, and since we used a large number of breeders, we excluded any potential role for the number of pups/gender/parents.

Body Weight, Body Composition, and Food Intake Measurements

At 22 wk of age, male and female offspring from all groups were housed individually for determination of daily food intake and body weight measurements for five consecutive days. Body composition was also analyzed on the 5th day using magnetic resonance (4 in 1 EchoMRI-900, Echo Medical System, Houston, TX). To minimize potential stress caused by a single housing, we used environmental enrichment in each cage.

Blood Pressure and Heart Rate Measurements

After determination of food intake and body composition, the mice were anesthetized with 2% isoflurane and, under aseptic conditions, a telemetry probe (TA11PA-C10, Data Science, MN) was implanted in the left carotid artery and advanced into the aorta. Ten days after recovery from surgery, mean arterial pressure (MAP) and HR were measured 24 h/day for five consecutive days using computerized methods for data collection as previously described (13, 14). Daily MAP and HR were obtained from the average of 12:12 h light:dark recording using a sampling rate of 1,000 Hz during 10 s every 10-min period. We examined BP lability by averaging hourly BP over a 24-h period. To analyze the dipping pattern of BP in mice, we also compared inactive/active phase differences in MAP in male and female offspring from lean and obese parents.

Ovariectomy

At 3 wk of age, female mice were weaned and fed an H diet. Three weeks later, under 2% isoflurane anesthesia, the mice were ovariectomized (OXV) bilaterally, and MAP was measured at 22 wk of age.

Air-Jet Stress Test

To determine whether parental obesity alters offspring MAP and HR responses to stress, mice implanted with BP telemeters were placed in special cages and subjected to an air-jet stress test as previously described (13). Mice were allowed to acclimate to the cages for at least 4 h and then MAP and HR were continuously measured for 30 min. The air-jet stressor was then turned on and consisted of 5-s pulses of compressed air directed at the animal’s forehead every 10 s for 5 min. After the 5-min air-jet stress, MAP and HR measurements were continued for an additional 30-min recovery period. Changes in BP and HR to this acute stress were measured by subtracting average baseline values from the average values recorded during the stressor phase. Areas under the MAP curve (AUC) during the air-jet stress and recovery periods were calculated using the following parameters: average change in MAP for each minute during the 5-min air-jet stress and for each 5 min during the 30-min recovery period.

Spontaneous Baroreflex Sensitivity and Heart Rate Variability

To assess the impact of parental obesity on offspring reflex control of CV function, we measured spontaneous baroreflex sensitivity (BRS) by the sequence method as previously described (15). We also computed power spectral densities of systolic arterial pressure (SAP) and RR interval (RRI) oscillations by 512-point fast Fourier transform integrated over the specific frequency range (low frequency: LF, 0.25–0.75 Hz; high frequency: HF, 0.75–5.00 Hz) using Nevrokard SA-BRS software (Medistar, Ljubljana, Slovenia). To examine the impact of parental obesity on offspring heart rate variability (HRV), RRIs were extracted using a detection algorithm based on peak-picking of SAP continuously measured for 2 h (10:00–12:00 AM). We excluded one animal with extreme RRI values (<70 and >500 ms) and other outliers based on the means and standard deviations (SD) of nearby RR values. Time and frequency domain measures were calculated for each second of normal RR interval data using Kubios HRV software (version 2.1). In the time domain, the mean RRI, standard deviation of normal RRI (SDNN), and root mean square of the successive differences (RMSSDs) were calculated. The coefficient of variance (CV%), defined as the ratio of SDNN to mean RR, which reflects the variability due to the combination of long, intermediate, and short-term components, was also calculated. In the frequency domain, the power spectral density of RRI time series was computed, and two different frequency-domain measures of HRV were calculated, low-frequency (LF) 0.4–1.5 Hz and high-frequency range (HF) 1.5–4.0 Hz, with data expressed as absolute and arbitrary units. These measurements were performed at 23 wk of age.

Blood Pressure Responses to Losartan, Angiotensin II, and Hexamethonium

To examine if overactivation of the renin-angiotensin system (RAS) or increased sensitivity to angiotensin II contributes to increased BP in offspring from obese parents, losartan (AT1R antagonist) was injected (bolus intraperitoneal injection of 5 mg/kg) in male mice (n = 5–6/group), and BP was continuously measured 30 min before and 60 min after injection. To further test if offspring from obese parents are more sensitive to the pressor effects of angiotensin II compared with offspring from lean parents, we injected angiotensin II (bolus intraperitoneal injection of 10 ng) and measured BP continuously for at least 30 min before and 60 min after injection.

We also examined the contribution of increased sympathetic activation to increased BP in offspring from obese parents by a bolus intraperitoneal injection of hexamethonium (30 mg/kg) following the same BP recording protocol used for losartan and angiotensin II.

Western Blotting

Kidney samples were homogenized in lysis buffer (KPO4, pH 7.4), sonicated and cleaned by centrifugation (3,500 g for 5 min, 4°C). The supernatant protein concentration was determined as previously described (14). Forty micrograms of protein were separated in a 4%–15% precast linear gradient polyacrylamide gel (Bio-Rad). After being transferred to nitrocellulose membranes, blots were rinsed with PBS and blocked in SuperBlock blocking buffer (Thermo Scientific) for 1 h at room temperature and incubated with rabbit monoclonal anti P2X7R (1:200, Cat. No. APR004, Alomone) overnight at 4°C. We used a positive control provided by Alomone laboratory (APR004 and AG1940) before performing Western blots in kidney samples (Supplemental Fig. S1; see https://doi.org/10.5281/zenodo.6334835). Total protein was used as a loading control. Membranes were incubated with IR700-conjugated donkey anti-rabbit (1:1,000 Rockland Immunochemicals). Antibody labeling was visualized using Odyssey infrared scanner (LI-COR) for detection of fluoroprobes and fluorescence intensity analysis was performed using Odyssey software (LI-COR). All proteins measured were normalized for total protein in each Western blot sample (Supplemental Fig. S1).

Statistical Analyses

Data are expressed as means ± SE. The Shapiro–Wilk test was used to confirm normal distribution of all data collected. Significant differences between the two groups were determined by unpaired Student’s t test or two-way ANOVA followed by Bonferroni’s post hoc test when appropriate. One-way ANOVA with repeated measures followed by Dunnett’s post hoc test was used for comparisons between control and experimental groups when appropriate. A P value of <0.05 indicates a significant difference.

RESULTS

Parental Mice Body Weight, Body Composition, and Plasma Hormones

Paternal and maternal obese mice were 32% and 23% heavier, respectively, compared with lean parents (Table 1). The increased body weight was due to higher fat mass and not lean mass, and was associated with increased plasma glucose, insulin, and leptin concentrations (Table 1).

Table 1.

Metabolic phenotyping of male and female breeders

	BW, g	Lean Mass, g	Fat Mass, g	Blood Glucose, mg/dL	Plasma Leptin, ng/mL	Plasma Insulin, ng/mL
C57/BL6J: Males
NN	24.5 ± 0.4	23.1 ± 0.3	1.3 ± 0.1	105.3 ± 12.1	1.6 ± 0.6	0. 8 ± 0.1
NH	32.5 ± 1.6*	24.2 ± 0.5	9.1 ± 1.4*	192.7 ± 10.7*	46.9 ± 2.0*	7.9 ± 0.8*
C57/BL6J: Females
NN	21.7 ± 0.1	19.2 ± 0.1	2.0 ± 0.2	111.5 ± 8.2	3.0 ± 0.8	1.1 ± 0.1
NH	27.5 ± 1.7*	20.6 ± 0.5	6.8 ± 1.4*	156.7 ± 17.4*	27.8 ± 2.2*	4.3 ± 1.0*
P2X7R-KO: Males
NN	28.1 ± 0.6	25.8 ± 0.7	2.3 ± 0.2	122.3 ± 8.7	1.9 ± 0.9	1.1 ± 12.1
NH	37.1 ± 1.7*	26.6 ± 1.0	11.3 ± 0.9*	173.8 ± 7.5*	37.1 ± 2.5*	6.6 ± 0.6*
P2X7R-KO: Females
NN	23.0 ± 0.4	20.2 ± 0.4	2.0 ± 0.1	111.0 ± 5.3	1.7 ± 0.8	0.5 ± 0.1
NH	28.6 ± 0.8*	20.7 ± 0.4	8.0 ± 0.7*	144.8 ± 15.4*	25.9 ± 1.1*	1.7 ± 0.1*

Values are means ± SE and represent measurements at 12 wk of age. *P < 0.05 compared with respective NN. N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning.

Body Weight and Body Composition in Adult Offspring from Lean and Obese Parents

Parental obesity led to greater body weight and fat mass in male and female offspring that were also fed a high-fat diet when compared with high-fat diet-fed offspring from lean parents (Fig. 1, A, C, D, and F), despite similar calorie intake (Fig. 1, B and E). We did not observe significant differences in fat mass in male and female offspring fed N diet from obese parents compared with offspring fed N diet from lean parents (Fig. 1, C and F).

Figure 1.

Metabolic phenotype of male and female offspring from lean and obese parents. Body weight (A), calorie intake (B), and fat mass in male offspring (C); body weight (D), calorie intake (E), and fat mass in female offspring (F) from lean and obese parents. [n = 5–8 offspring from 5 to 7 dams/group, *P < 0.05 vs. NN offspring (one-way ANOVA), #P < 0.05 vs. NH offspring (one-way ANOVA)]. H, high-fat; HH, offspring from obese (H) parents that were also fed an H diet after weaning; HN, offspring from obese (H) parents that were fed N diet after weaning; N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning.

Impact of Parental Obesity on Blood Pressure and Heart Rate Regulation in Their Offspring

Compared with offspring from lean parents, male offspring from obese parents exhibited higher BP and greater lability of BP, monitored 24 hr/day for five consecutive days, independent of the diet consumed by the offspring after weaning (Fig. 2, A–C). In female offspring, however, we observed no differences in BP among groups (Fig. 2, D–F). We also observed that MAP frequency distribution was wider and shifted to the right in male offspring from obese parents, independent of the diet consumed, compared with offspring from lean parents (Fig. 2C). We did not observe significant differences among female offspring from lean or obese parents (Fig. 2F).

Figure 2.

Cardiovascular phenotype of male and female offspring from lean and obese parents. Circadian variation in mean arterial pressure (MAP; A), average MAP (B), and frequency distribution of systolic blood pressure (BP; C) in male offspring; circadian variation in MAP (D), average MAP (E), and frequency distribution of systolic BP (F) in female offspring; % dipping (G), heart rate (HR; H), and spontaneous baroreflex sensitivity (sBRS; I) in male offspring; % dipping (J), HR (K), and sBRS (L) in female offspring from lean and obese parents. [n = 5–9 offspring from 5 to 7 dams/group, *P < 0.05 vs. NN offspring (one-way ANOVA), #P < 0.05 vs. NH offspring (one-way ANOVA)]. H, high-fat; HH, offspring from obese (H) parents that were also fed an H diet after weaning; HN, offspring from obese (H) parents that were fed N diet after weaning; N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning.

To examine if ovarian hormones play an important role in BP control in obese female offspring from obese parents, we performed bilateral ovariectomy in mice at 6 wk of age. We found that BP in OVX mice was similar to sham female siblings from obese parents, suggesting that ovarian hormones may not play an important role in BP control in our parental obesity model (Supplemental Fig. S2; see https://doi.org/10.5281/zenodo.6334835).

The increased MAP and BP lability were associated with a nondipping BP phenotype in male offspring from obese parents, which also occurred independent of the diet consumed by these offspring (Fig. 2G). Conversely, female offspring did not exhibit altered BP dipping patterns (Fig. 2J), suggesting an important sex-dependent influence on sleep/awake BP regulation.

Average HR was not significantly different among groups for male and female offspring from lean and obese parents (Fig. 2, H and K).

Spontaneous BRS (sBRS) was analyzed by time and frequency domains (alpha indexes) and showed that male, but not female, offspring fed an H diet born from obese parents had reduced sBRS (Fig. 2, I and L). Spectral analyses data of HR and BP oscillations between 0.2 and 0.7 Hz (low-frequency region, LF) were used to assess sympathetic modulation on HR and BP, whereas oscillations of HR in the high-frequency (HF) region between 0.7 and 2.0 Hz were used to assess parasympathetic tone. The HF range of HR, reflecting parasympathetic tone to the heart, was slightly higher in male offspring from obese parents, although the difference did not reach statistical significance (Table 2). We also did not observe significant differences in the HF or LF range of HR among female offspring groups (Table 2). However, we found enhancement of the power of SAP spectra in the LF range in H diet-fed male offspring from obese parents, suggesting increased sympathetic modulation of BP compared with N diet-fed or H diet-fed male offspring from lean parents (Table 2).

Table 2.

Spectral analysis data of RR interval and systolic arterial pressure in male and female offspring from lean and obese parents

	RRI				SAP
	LF, Hz	HF, Hz	LF, nu	HF, nu	LF, Hz	HF, Hz	LF, nu	HF, nu
Male
NN	0.27 ± 0.01	1.34 ± 0.03	22.4 ± 2.1	21.0 ± 3.9	0.29 ± 0.02	1.30 ± 0.2	18.5 ± 2.5	18.1 ± 2.3
NH	0.34 ± 0.06	1.19 ± 0.37	20.8 ± 3.7	21.9 ± 4.3	0.28 ± 0.02	1.30 ± 0.2	20.1 ± 1.3	16.2 ± 3.6
HN	0.28 ± 0.01	2.10 ± 0.30	16.2 ± 2.0	32.0 ± 6.3	0.27 ± 0.01	1.88 ± 0.3	21.7 ± 1.6	16.1 ± 3.7
HH	0.40 ± 0.08	1.4 ± 0.30	17.3 ± 2.6	33.2 ± 9.5	0.34 ± 0.02	1.23 ± 0.3	25.9 ± 2.1*#	15.0 ± 2.2
Female
NN	0.31 ± 0.01	1.18 ± 0.25	24.7 ± 3.4	22.8 ± 5.7	0.31 ± 0.02	1.45 ± 0.2	27.8 ± 2.4	21.1 ± 1.7
NH	0.27 ± 0.01	1.29 ± 0.40	26.1 ± 2.7	20.2 ± 2.6	0.31 ± 0.02	1.27 ± 0.3	22.8 ± 3.2	14.2 ± 4.9
HN	0.27 ± 0.01	2.16 ± 0.49	16.8 ± 3.2	38.8 ± 10.6	0.28 ± 0.02	1.85 ± 0.6	23.1 ± 1.5	19.1 ± 2.0
HH	0.27 ± 0.02	1.56 ± 0.45	17.8 ± 1.0	23.5 ± 7.4	0.27 ± 0.02	1.36 ± 0.1	23.5 ± 0.1	16.6 ± 2.3

Values are means ± SE. *P < 0.05 compared with NN; #P < 0.05 compared with NH. HF, high frequency; LF, low frequency; nu, normalized units (represent the relative value of each power component in proportion to the total power minus VLF (very low frequency) component; N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning; RRI, RR interval; SAP, systolic arterial pressure.

To determine whether parental obesity alters HRV in their offspring, we analyzed time and frequency domain measures for each second of normal RR intervals. We found that the root mean square of successive differences in R-R intervals (RMSSDs), a measure of parasympathetic nervous system activity (PSNA), were significantly reduced in male offspring from obese parents when compared with male offspring from lean parents irrespective of offspring diet (Table 3). No significant differences among male offspring groups were observed for standard deviation of normal-to-normal intervals (SDNN). We also did not observe differences in HRV in female offspring from lean and obese parents (Table 3). We did not find significant differences in LF (which reflects cardiac sympathetic activity) and HF (which reflects cardiac parasympathetic activity) components of HR variability in male and female offspring from lean and obese parents (Table 4).

Table 3.

Heart rate variability in male and female offspring mice from lean and obese parents

Measure	NN Male	NH Male	HN Male	HH Male	NN Female	NH Female	HN Female	HH Female
RR, ms	119.0 ± 1.4	129.4 ± 8.4	119.0 ± 1.4	119.2 ± 5.6	109.5 ± 3.4	106.8 ± 4.7	115.2 ± 3.0	109.2 ± 5.2
SDNN, ms	17.7 ± 1.0	16.3 ± 1.6	14.0 ± 1.5	13.1 ± 1.0	10.9 ± 2.3	119.0 ± 1.4	10.5 ± 1.6	13.2 ± 2.7
RMSSD, ms	9.8 ± 1.4	11.0 ± 1.9	5.8 ± 1.0*#	5.9 ± 1.0*#	4.9 ± 1.0	3.3 ± 1.0	5.5 ± 2.0	5.3 ± 1.0
CV%	14.9 ± 0.6	14.5 ± 2.4	11.9 ± 1.3	11.8 ± 0.9	9.7 ± 1.7	11.0 ± 2.1	14.1 ± 1.7	11.7 ± 1.9

Data are means ± SE. *P < 0.05 compared with NN; #P < 0.05 compared with NH. CV%, coefficient of variance (100 × (SDNN/mean RR)); HRV, heart rate variability; N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning; RMSSD, root mean square of successive difference; RR, the intervals from systolic peak; SDNN, standard deviation of all R-R intervals.

Table 4.

Frequency domain analysis on heart rate variability in male and female offspring from lean and obese parents

Groups	Power, ms		Power, %		Power, nu		LF/HF
	LF (0.04–0.15 Hz)	HF (0.15–0.40 Hz)	LF (0.04–0.15 Hz)	HF (0.15–0.40 Hz)	LF (0.04–0.15 Hz)	HF (0.15–0.40 Hz)
Male
NN	15.4 ± 1.6	12.4 ± 3.8	11.1 ± 2.0	8.4 ± 2.2	58.0 ± 4.5	41.8 ± 4.5	1.5 ± 0.2
NH	21.8 ± 2.7	24.3 ± 3.6	14.4 ± 4.7	15.6 ± 4.9	53.4 ± 4.6	46.4 ± 4.5	1.2 ± 0.1
HN	20.9 ± 6.2	14.0 ± 4.3	12.1 ± 2.1	8.5 ± 2.0	59.8 ± 3.8	40.0 ± 3.8	1.7 ± 0.1
HH	12.7 ± 1.3	11.1 ± 1.1	12.0 ± 2.1	10.3 ± 1.3	54.1 ± 1.0	45.6 ± 1.0	1.2 ± 0.1
Female
NN	9.4 ± 2.0	7.4 ± 1.3	12.8 ± 1.7	10.7 ± 1.8	55.3 ± 1.6	44.5 ± 1.6	1.3 ± 0.1
NH	11.8 ± 3.7	7.2 ± 3.5	9.9 ± 1.0	5.8 ± 0.2	62.2 ± 2.4	37.6 ± 2.4	1.7 ± 0.1
HN	20.5 ± 3.8	23.3 ± 8.6	7.7 ± 1.2	8.7 ± 2.8	51.19 ± 4.4	48.7 ± 4.5	1.1 ± 0.1
HH	13.8 ± 4.0	8.8 ± 3.7	10.8 ± 2.4	7.5 ± 2.5	61.9 ± 3.6	37.9 ± 3.6	1.7 ± 0.2

Values are means ± SE. HF, high frequency; HRV, heart rate variability; LF, low frequency; nu, normalized units (represent the relative value of each power component); N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning.

Impact of Parental Obesity on BP and HR Responses to Acute Air-Jet Stress and BP Responses to Losartan, Angiotensin II, and Hexamethonium

Acute air-jet stress significantly increased MAP by 38 ± 6 and 37 ± 5 mmHg in male HN and HH mice compared with only 24 ± 3 and 25 ± 3 mmHg in male NH and control NN mice, respectively (Fig. 3, A–D). MAP remained significantly higher poststress in male HH mice (Fig. 3A). HR responses to air-jet stress were similar among male groups (Fig. 3I). In females, MAP and HR responses to air-jet stress test were similar among groups (Fig. 3, E–H, and J).

Figure 3.

Mean arterial pressure (MAP) and heart rate (HR) responses to acute stress in male and female offspring from lean and obese parents. Δ MAP (A), peak MAP (B), area under curve (AUC) MAP during stress (C), AUC MAP poststress (D) in male offspring; Δ MAP (E), peak MAP (F), AUC MAP during stress (G), and AUC MAP poststress (H) in female offspring; Δ HR in male offspring (I) and Δ HR in female offspring (J) from lean and obese parents. [n = 5 or 6 offspring from 5 to 6 dams/group, *P < 0.05 vs. NN offspring (one-way ANOVA)]. H, high-fat; HH, offspring from obese (H) parents that were also fed an H diet after weaning; HN, offspring from obese (H) parents that were fed N diet after weaning; N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning.

Acute AT1R blockade with losartan significantly reduced MAP in offspring from obese parents, regardless of offspring diet, when compared with offspring from lean parents (Fig. 4A). Lean and obese offspring from obese parents also showed increased sensitivity to the pressor effects of angiotensin II (Fig. 4B) compared with offspring from lean parents. Ganglionic blockade with hexamethonium evoked more pronounced reductions in BP in lean and obese offspring from obese parents than in lean and obese offspring from lean parents (Fig. 4C). These findings suggest that overactivation of the RAS and the sympathetic nervous system may be responsible, at least in part, for increased BP in offspring from obese parents.

Figure 4.

Change in mean arterial pressure (MAP) induced by intraperitoneal administration of losartan (5 mg/kg), angiotensin II (10 ng), and hexamethonium (30 mg/kg) in male offspring from lean and obese parents. Delta and percentage changed in MAP in response to losartan (A), angiotensin II (B), and hexamethonium (C). [n = 5 or 6 offspring from 5 to 6 dams/group, *P < 0.05 vs. NN offspring (one-way ANOVA) and #P < 0.05 vs. NH offspring (one-way ANOVA)]. H, high-fat; HH, offspring from obese (H) parents that were also fed an H diet after weaning; HN, offspring from obese (H) parents that were fed N diet after weaning; N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning.

Body Weight, Body Composition, and Fat Mass Content in Offspring with P2X7R Deficiency from Lean and Obese Parents

We observed increased P2X7R protein expression in kidneys of obese and lean male offspring from obese parents (Fig. 5A). However, no differences in P2X7R protein expression in kidneys were observed in female offspring from lean and obese parents (Fig. 5E). To examine if P2X7R deficiency alters the impact of parental obesity on body weight and adiposity in their offspring, we measured body weight, calorie intake, and fat mass content in adult offspring from lean and obese P2X7R-deficient parents. We found that P2X7R deficiency did not alter the consequences of parental obesity to increase body weight and adiposity in male or female offspring fed an H diet compared with offspring from lean parents (Fig. 5, B–D, F, and H). Calorie intake was slightly increased in male, but not female, P2X7R-deficient mice from obese parents compared with offspring from lean parents (Fig. 5, C and G).

Figure 5.

Expression of P2X7R in the kidneys and metabolic phenotype of male and female P2X7R-deficient (P2X7R-KO) offspring from lean and obese P2X7R-KO parents. P2X7R protein expression (A), body weight (B), calorie intake (C), and fat mass (D) in male offspring; P2X7R protein expression (E), body weight (F), calorie intake (G), and fat mass (H) in female offspring from lean and obese parents. [n = 5 offspring from 5 dams/group, #P < 0.05 vs. NN and NH offspring (one-way ANOVA), *P < 0.05 vs. NN-P2X7R-KO offspring (unpaired two-tailed t test)]. H, high-fat; HH, offspring from obese (H) parents that were also fed an H diet after weaning; HN, offspring from obese (H) parents that were fed N diet after weaning; N, normal; NH, offspring from lean (N) parents that were fed high-fat (H) diet after weaning; NN, offspring from lean normal (N) diet-fed parents that were also fed an N diet after weaning.

Impact of P2X7R Deficiency on Blood Pressure Regulation and Responses to Acute Stress in Offspring from Obese Parents

Since P2X7R has been implicated in the regulation of chronic inflammation and BP, we also examined whether P2X7R contributes to the effects of parental obesity on CV regulation in their offspring. Thus, we compared BP, 24-h BP lability, BP frequency distribution, and BP and HR responses to acute stress in P2X7R-deficient offspring fed an N diet from lean parents (NN-P2XR7-KO) to similar variables measured in P2X7R-deficient offspring fed an H diet from obese parents (HH-P2XR7-KO). We found that P2X7R deficiency prevented the increase in BP and reduced BP lability in male HH-P2XR7-KO offspring from obese parents (Fig. 6, A–C). P2X7R deficiency also abolished the augmented BP response to acute stress in male HH-P2XR7-KO when compared with male NN-P2XR7-KO control mice (Fig. 7, A–D). No major changes were observed in baseline 24-h HR in male NN-P2XR7-KO or HH-P2XR7-KO offspring (Fig. 6G). We also did not observe any significant changes in 24-h BP or HR in female P2X7R-KO offspring from lean or obese parents (Fig. 6, D–F and H). However, lean NN female P2X7R-KO offspring showed a fast recovery from the air-jet stress test compared with obese HH female P2X7R-KO offspring, whereas BP and HR responses during air-jet were similar between groups (Fig. 7, E–H, and J).

Figure 6.

Cardiovascular phenotype of male and female P2X7R-deficient (P2X7R-KO) offspring from lean and obese P2X7R-KO parents. Circadian variation in mean arterial pressure (MAP; A), average MAP (B), and frequency distribution of systolic blood pressure (BP; C) in male offspring; circadian variation in MAP (D), average MAP (E), and frequency distribution of systolic BP (F) in female offspring; heart rate (HR) in male (G), and HR in female (H) offspring from lean and obese P2X7R-KO parents. (n = 5 offspring from 5 dams/group). H, high-fat; HH-P2X7R-KO, offspring from obese high-fat (H) diet-fed P2X7R-KO parents that were also fed an H diet after weaning; NN-P2X7R-KO, offspring from lean normal (N) diet-fed P2X7R-KO parents that were also fed an N diet after weaning.

Figure 7.

Mean arterial pressure (MAP) and heart rate (HR) responses to acute stress in male and female P2X7R-deficient (P2X7R-KO) offspring from lean and obese P2X7R-KO parents. Δ MAP (A), peak MAP (B), area under curve (AUC) MAP during stress (C), and AUC MAP poststress (D) in male offspring; Δ MAP (E), peak MAP (F), AUC MAP during stress (G), and AUC MAP poststress (H) in female offspring; Δ HR in male offspring (I) and Δ HR in female offspring (J) from lean and obese P2X7R-KO parents. (n = 5 offspring/group). HH-P2X7R-KO, offspring from obese high-fat (H) diet-fed P2X7R-KO parents that were also fed an H diet after weaning; NN-P2X7R-KO, offspring from lean normal (N) diet-fed P2X7R-KO parents that were also fed an N diet after weaning.

DISCUSSION

An important goal of this study was to test the hypothesis that parental obesity (paternal + maternal obesity) programs their offspring to greater adiposity, elevated BP, and augmented BP response to acute stress even when the offspring are fed a normal diet and remain lean. Sex differences were investigated to determine whether male and female offspring were differentially affected by parental obesity. We also determined the contribution of RAS and sympathetic nervous system activation to BP regulation of offspring from obese and lean parents. In addition, we examined if P2X7R overactivation could be a potential mechanistic link between parental obesity and BP dysregulation in their offspring.

Our most important findings are that parental obesity programs their offspring to greater adiposity in both sexes and altered BP regulation in male offspring, even when offspring are fed a normal diet and remain lean. We also observed in male offspring from obese parents enhanced BP responses to acute stress that were independent of the diet consumed by the offspring after weaning. In contrast, female offspring did not exhibit altered baseline BP or augmented BP responses to acute stress despite having similar adiposity as their male siblings. Furthermore, we show that increased RAS and sympathetic nervous system activation contribute to increased BP in lean and obese male offspring from obese parents. Another important finding of our study is that P2X7R deficiency completely prevented increased BP, increased BP lability, and exaggerated BP responses to acute stress in male offspring from obese parents. These findings suggest that attenuating P2X7R activity may provide CV protection from parental obesity-induced increases in BP, especially in obese male offspring.

The proportion of women and men of reproductive age who are overweight and obese has been steadily increasing, and parental obesity and excessive gestational weight gain may predispose their offspring to health-related consequences during childhood and adulthood (16, 17). Preventing obesity and its adverse consequences in future generations may depend on ensuring that parents are healthy before they conceive. However, there is still limited understanding of the mechanisms that contribute to the transmission of obesity and cardiovascular dysfunction from one generation to the next.

Our results indicate that male and female offspring from obese parents have increased adiposity and greater body weight gain despite similar calorie intake compared with obese offspring from lean parents. Previous epidemiological studies showed that parental overweight was associated with increased body mass index (BMI) of their offspring (18). In fact, children with two obese parents are 10 to 12 times more likely to become obese and develop metabolic derangements compared with children with parents who have normal body weight (19, 20). The Trøndelag Health Study (HUNT study) also demonstrated that when both parents are overweight, the effect on BMI in their offspring was approximately doubled compared with only one parent being overweight (21). Other studies found that overweight in daughters was strongly associated with maternal overweight, whereas overweight in sons was associated with maternal and paternal overweight or only paternal overweight (21, 22).

The association between parental and offspring adiposity may persist into midadulthood (23) when risks for CV diseases are increasing. Excessive weight gain and adiposity are major causes of HTN and metabolic abnormalities, and these metabolic abnormalities may interact to potentiate their individual effect on CV risks (7, 17, 24). Although male and female offspring from obese parents had greater weight gain and adiposity, we found that male offspring were more susceptible to the effects of parental obesity to induce increased BP. In fact, male offspring from obese parents exhibited elevated BP even when fed a normal diet and remaining lean, suggesting important sex differences in the impact of parental obesity on offspring BP regulation.

Previous studies suggest that ovarian hormones (especially estrogens) may protect, whereas androgens exacerbate, the impact of obesity on BP regulation (25, 26). However, BP in ovariectomized female offspring from obese parents was similar to BP in sham female siblings. This finding indicates that ovarian hormones may not play a major role in exacerbating the impact of parental obesity on BP in our studies.

Increased BP lability and impaired baroreflex control are associated with target organ injury (27, 28). Lability of BP often increases with aging and is usually lower in young and middle-aged females than in males (29). Our results showed that male, but not female, offspring from obese parents have increased BP lability, and BP responses to acute stress independent of the diet consumed by the offspring. We also found a higher LF component of systolic arterial pressure oscillation, suggesting increased sympathetic tone in obese male offspring from obese parents. This finding is consistent with the possibility that the increased BP and greater BP lability were due, at least in part, to increased sympathetic tone. Furthermore, male offspring from obese parents also had increased BP response to acute stress and their BP remained elevated for a longer period poststress. Despite these observations in male offspring from obese parents, we found no evidence that parental obesity alters HRV or BP response to acute stress in female mice, suggesting important sex differences of the impact of parental obesity on cardiovascular regulation of their young adult offspring.

Although BP normally decreases during sleep (dipping of BP), obesity attenuates normal circadian variation of BP (30, 31). Previous studies showed that the prevalence of nondipping BP in obese patients with essential HTN may be as high as 67% (32). Although obesity may partially explain the nondipping pattern of BP in offspring from obese parents, we found that lean male offspring from obese parents also had attenuated BP dipping during daytime, the period that rodents normally sleep. Since nondipping BP pattern during sleep and elevated BP during sleep (daytime BP in rodents) are important risk factors for CV diseases (30), our results suggest that the adverse impact of parental obesity may be exacerbated in male offspring.

Mechanisms linking parental obesity to adverse CV outcomes in their offspring are still poorly understood. One potential mechanism for parental obesity-induced developmental programming of cardiorenal function is excessive activation of P2X7R. Glomerular expression of P2X7R is scarce in normal kidneys, but is upregulated in chronic inflammatory conditions, suggesting a role for P2X7R in the inflammatory response or in tissue repair and remodeling in these settings (11, 33). In the present study, we observed that mice with P2X7R deficiency are protected from parental obesity-induced HTN and augmented BP responses to stress in their offspring. These observations suggest that P2X7R activation may contribute to the developmental programming of HTN and associated CV disorders in offspring of obese parents. However, further studies are needed over longer periods of time to assess the impact of P2X7R on development of cardiorenal injury in offspring of obese parents.

P2X7R activation triggers several signaling cascades that may contribute to the phenotype we observed in the offspring from obese parents. For instance, P2X7R activation stimulates caspase 3 activation and cytokine release, plasma membrane reorganization, ectodomain shedding, and cell death among other effects (34). The majority of these P2X7R effects occur because of P2X7-dependent modulation of Ca²⁺ influx and/or K⁺ efflux (35). However, we did not investigate the various molecular interactions of P2X7R with signaling complexes in the present study. Another important unanswered question that requires additional studies is whether excessive P2X7R activation in offspring of lean parents results in a similar phenotype as observed in obese offspring from obese parents.

The fact that lean and obese offspring from obese parents exhibited more pronounced BP responses to losartan, angiotensin II, and hexamethonium strongly suggests an important contribution of the sympathetic nervous system and the RAS in parental obesity-induced elevation in BP in their offspring. Previous studies have highlighted the importance of the RAS and sympathetic nervous system in obesity-associated hypertension (3, 5); however, our observations indicate that these systems are even further activated in lean offspring from obese parents that were fed a normal diet. Previous studies showed that angiotensin II infusion for 2 wk markedly increased P2X7R expression in renal tubular cells and infiltrating B and T lymphocytes and macrophages (34), and increased P2X7R activation contributes to angiotensin II-induced HTN (36). These observations may help to explain why parental obesity appears to influence their offspring’s P2X7R, RAS, and autonomic nervous system beyond what is commonly observed in diet-induced obesity not associated with parental obesity.

One potential limitation of our study is the lack of data from P2X7R-KO mice fed a normal diet from obese P2X7R-KO parents. We did not include this group mainly because obese P2X7R-KO offspring fed a high-fat diet were obese and yet were protected from parental obesity-induced HTN and the greater responses to acute stress. Thus, our findings strongly suggest that overactivation of P2X7R in the kidneys may play a critical role in linking parental obesity with increased BP and hypersensitivity to angiotensin II and AT1R antagonism in their offspring, especially when the offspring are consuming the same obesogenic diet as their parents.

In the present study, we focused on phenotyping the offspring of lean and obese parents and did not investigate metabolic and cardiovascular function in the parents. However, others have reported the cardiometabolic changes in mice fed high-fat diet (HFD) similar to that used in the present study (37). Although we did not measure BP during the breeding period and subsequent pregnancy and lactation, we anticipate no major alterations in BP, particularly in the mothers, since they were young (9–12-wk old), and we did not find increased BP in obese male or female offspring from lean parents even at a much older age of 22 wk and were fed the same HFD. Thus, feeding a HFD did not raise BP at 22 wk of age if the mice were the offspring from lean parents.

Another limitation of the present study is that we did not quantify P2X7R expression in other tissues besides the kidney. Thus, additional studies are needed to determine if parental obesity alters P2X7R expression in other tissues that are also important for cardiovascular regulation and whether increased renal P2X7R expression is accompanied by increased P2X7R activity.

Perspectives and Significance

Overall, our results suggest that parental obesity programs their offspring to greater adiposity and increased body weight gain, increased BP, nondipping of BP, and exaggerated BP responses to acute stress. In fact, these effects of parental obesity on offspring CV function are observed in male offspring even when these mice are fed a normal diet and remain lean. Our findings also suggest that parental obesity exacerbates the increased adiposity and associated adverse metabolic effects induced by consumption of a high-fat diet. Although our observations indicate that male, but not female, offspring from obese parents have major BP dysregulation, global P2X7R deficiency appears to prevent these alterations via mechanisms that are still unclear. The adverse cardiometabolic effects of parental obesity on their offspring highlight the importance of developing more effective therapeutic approaches for the prevention and treatment of obesity and associated abnormalities. Further studies are needed to determine the relevance of these findings to humans and whether therapeutic strategies may be developed to avoid the adverse metabolic and CV effects of parental obesity on the health of their offspring.

SUPPLEMENTAL DATA

Supplemental Figs. S1 and S2: https://doi.org/10.5281/zenodo.6334835.

GRANTS

The authors were supported by the National Heart, Lung, and Blood Institute Grant P01 HL51971 (to J. E. Hall), the National Institute of General Medical Sciences Grants P20 GM104357 and U54 GM115428 (to J. E. Hall), and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK121411 (to J. M. do Carmo).

DISCLAIMERS

J. M. do Carmo is the guarantor of this work and had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.A.d., J.E.H., and J.M.d conceived and designed research; A.A.d., S.P.M., X.D., G.C.B., A.C.M.O., and J.M.d. performed experiments; A.A.d., G.C.B., and J.M.d. analyzed data; A.A.d., G.C.B., A.C.M.O., Z.W., X.L., A.J.M., J.E.H., and J.M.d. interpreted results of experiments; A.A.d. and J.M.d. prepared figures; A.A.d. and J.M.d. drafted manuscript; A.A.d., S.P.M., X.D., A.C.M.O., Z.W., X.L., A.J.M., J.E.H., and J.M.d. edited and revised manuscript; A.A.d., S.P.M., X.D., G.C.B., A.C.M.O., Z.W., X.L., A.J.M., J.E.H., and J.M.d. approved final version of manuscript.