Prenatal Morphine Exposure Increases Cardiovascular Disease Risk and Programs Neurogenic Hypertension in the Adult Offspring

Abstract

Background: The opioid overdose and opioid use disorder epidemics are concomitant with increased metabolic and CVD risk. Although opioid use disorder causes adverse pregnancy outcomes, the offspring's cardiovascular health is understudied. We hypothesized that offspring exposed to in utero morphine exposure (IUME) would show increased CVD risk factors and endogenous opioid system dysregulation.

Methods: Sprague Dawley dams were treated with saline (vehicle, n=10) or escalating doses of morphine (5-20 mg/kg per day, SC, n=10) during gestation. Cardiovascular and metabolic parameters were assessed in adult offspring.

Results: Litter size and pups' birth weight were not different in response to IUME. Female and male IUME offspring showed reduced body length at birth (P<0.05) and body weight from weeks 1 to 3 of life (P<0.05), followed by a catch-up growth effect. By week 16, female and male IUME rats showed reduced tibia length (P<0.05) and fat mass. IUME increases the mean arterial pressure and the depressor response to mecamylamine (5 mg/kg per day, IP) induced by IUME were abolished by a chronic treatment with an alpha-adrenergic receptor blocker (prazosin; 1 mg/kg per day, IP). Although circulating levels of angiotensin peptides were similar between groups, IUME exacerbated maximal ex vivo Ang (angiotensin) II-induced vasoconstriction (P<0.05) and induced endothelial dysfunction in a sex-specific manner (P<0.05). Proenkephalin, an endogenous opioid peptide that lowers blood pressure and sympathetic-mediated vasoconstriction, showed reduced mRNA expression in the heart, aorta, and kidneys from morphine versus vehicle group (P<0.05).

Conclusions: Among the effects of IUME, neurogenic hypertension, vascular dysfunction, and metabolic dysfunction could be associated with the dysregulation of the endogenous opioid system.

Keywords: body weight; heroin; morphine; opioid peptide; prazosin.

Figure 1.. Morphine exposure does not affect pregnancy outcomes however results in catch-up growth in the in utero morphine exposed offspring:

A) total litter size B) female and male neonates birth weight, C) morphometric measurements in female neonates, D) morphometric measurements in male neonates, E) body weight trajectory from weeks 1-16 in female offspring, F) body weight trajectory from weeks 1-16 in male offspring. Data were averaged by litter, analyzed by t-test and reported as mean ± SEM. Data were analyzed by 2-Way ANOVA repeated measures followed by Tuckey multiple comparisons post-hoc test for body composition trajectories; n=9-10/group; * p<0.05 vs. control. VEH=vehicle, MOR=morphine, F=females, M=males, PD=postnatal day, AGD=anogenital distance, WC=waist circumference, HC=Head circumference, TL=tail length FL=full length, CI=cephalization index

Figure 2.. In utero morphine exposure reduces fat mass in adult offspring:

A) tissue and organ weights and tibia length (inset graph) in adult female rats, B) tissue and organs weights and tibia length (inset graph) in adult male rats, C) representative MRI scan of adult female rats, D) representative MRI scan of adult male rats, E) quantification of the subcutaneous and visceral fat in female rats, F) quantification of the subcutaneous and visceral fat in male rats. Data were averaged by litter, analyzed by t-test and reported as mean ± SEM; n=6-8/group for tissue, organs weight and tibia length and MRI analysis; * p<0.05 vs. control. VEH=vehicle, MOR=Morphine, F=females, M=males, gWAT=perigonadal white adipose tissue, scWAT= subcutaneous white adipose tissue, periWAT=perirenal white adipose tissue, SC=subcutaneous, VISC=visceral, MESE=mesenteric white adipose tissue, BAT=brown adipose tissue.

Figure 3.. In utero morphine exposure increases plasma and urine biomarkers of cardiometabolic disease risk factors:

A) FBG, B) insulin, C) leptin, D) FPLC analysis of plasma male and female rats, E) ACR and F) urinary NE. Data were analyzed by 2-Way ANOVA followed by Tuckey multiple comparisons post-hoc test and reported as mean ± SEM; n=8-10/group, Fig 3A–F,F, n=6-8/group, Fig. 3G–H;H; * p<0.05 vs. control. VEH=vehicle, MOR=morphine, F=females, M=males, FBG=fasting blood glucose, ACR= albumin creatinine ratio, NE= norepinephrine.

Figure 4.. In utero morphine exposure increases blood pressure and sympathetic activation:

A) Mean arterial pressure in female rats untreated and treated with prazosin, B) acute blood pressure response to mecamylamine in female rats untreated and treated with prazosin, C) mean arterial pressure in male rats untreated and treated with prazosin D) acute blood pressure response to mecamylamine in female rats untreated and treated with prazosin E) cardiovascular tissue adrenergic receptor expression in female rats, and F) cardiovascular tissue adrenergic receptor expression in male rats. Data were analyzed by 2-Way ANOVA followed by Tuckey multiple comparisons post-hoc test for blood pressure in A and B. For AngII-induced pressor response, data were analyzed using a 2-way ANOVA repeated measures, while RAS components were analyzed by t-test. Data were reported as mean ± SEM; n=7-9/group * p<0.05 vs. control. VEH=vehicle, MOR=Morphine, F=females, M=males, MAP=mean arterial pressure, Meca=mecamylamine, PRAZ=prazosin.

Figure 5.. In utero morphine exposure promotes sex-specific impairments of the vascular function:

AngII-induced constriction with and without naloxone in rings from A) female and B) male rats, C) PE-induced constriction in male and female rats, D) KCl-induced constriction in male and female rats, Ach and SNP-induced relaxation in rings from E) female and F) male rats. Data were reported as mean ± SEM; n=7-9/group * p<0.05 vs. control. VEH=vehicle, MOR=Morphine, F=females, M=males, MAP=mean arterial pressure, AngII=angiotensin II, PE=phenylephrine, Ach=acetylcholine, SNP=sodium nitroprusside.

Figure 6.. In utero morphine (MOR) exposure reduces proenkephalin (PENK) expression in the heart, aorta, and kidney and delays developing tolerance to morphine-induced locomotor hypoactivity:

A) PENK mRNA expression in female rats, B) PENK mRNA expression in male rats, C) distance traveled by female rats after 1 and 6 days of MOR injections, D) distance traveled by male rats after 1 and 6 days of MOR injections. Data were analyzed by t-test for gene expression and paired t-test for locomotor activity and reported as mean ± SEM; n=6-8/group for gene expression and n=8 per group for locomotor activity test; * p<0.05 vs. control. VEH=vehicle, MOR=morphine, F=females, M=males, PENK=proenkephalin.