The Effect Of Maternal Obesity On Fetal Programming Of Cardiac Dysfunction In Offspring And Underlying Mechanisms

Part oneThe Effect of Maternal Obesity on Fetal Cardiac Structure andCardiomyocyte Proliferation and Underlying MechanismsObjectiveTo investigate the effect of maternal obesity on cardiac structure and cardiomyocyteproliferation in fetus and underlying mechanisms.MethodsTime-dated pregnant Sprague Dawley rats were randomly divided into the controlgroup and the obesity group (high fat diet from day1to21of gestation). Themyocardial cells from fetus were cultured and the cell cyle of cardiomyocyts wasdetermined by flow cytometry. The ultramicrostructure of fetal hearts were observedby Transmission Electron Microscope. The protein expression of AT1R and AT2Rwere measured by Western Blot. AT1R and AT2R mRNA abundance was determinedby RT-PCR. Chromatin Immunoprecipitation assays were performed for GRE at theAT1aR or AT2R promoter in DNA sequences pulled down by GR antibodies.Results1. Compared to control group, at the day1of gestation, the body weight of pregnantrats were no significant difference in obesity group, but at the day21of gestation,the body weight of pregnant rats were significantly increased in obesity group(P<0.05). 2. There were no difference in fetal heart weight and body weight between controland obesity group. But the heart to body weight ratio of obesity group was obviouslyhigher than that of control group (P<0.05).3. In cultured primary cardiac cells, G1phase percentage was significantly increased,whereas S and G2phase percentage was decreased in the primary heart cells fromthe obesity group compared with those from the control（P<0.05）.4. Maternal obesity induced ultrastructure changes in the fetal heart includingdisorganized myofibrillae, swlloen mitochondria, cristae loss, and vacuolization.5. Compared to control group, AT1R protein in the fetal heart was significantlydecreased while AT2R protein was obviously increased in obesity group (P<0.05).6. Compared to control group, AT1aR mRNA in the fetal heart was significantlydecreased while AT2R mRNA was obviously increased in obesity group (P<0.05).7. Maternal obesity decreased total GR protein, nuclear GR protein and mRNAabundance in the fetal hearts（P<0.05）.8. ChIP assays showed that maternal obesity decreased the binding of GR to GRE atthe AT1aR and AT2R promoters.Summary1. Maternal obesity induced ultrastructure changes in the fetal heart includingdisorganized myofibrillae, swollen mitochondria, cristae loss, and vacuolization andinhibited cardiomyocyte proliferation, which may be related to alteration ofangiotensin II receptor.2. Decreased binding of GR and GRE at AT1aR and AT2R promoter may contributeto maternal obesity-induced alteration of AngII receptors in the fetal heart.Part twoThe Effect of Maternal Obesity on Fetal Programming of CardiacDysfunction in Adult Offspring and Underlying MechanismsObjective To investigate the effect of maternal obesity on fetal programming of cardiacdysfunction in adult offspring and underlying mechanisms.MethodsTime-dated pregnant Sprague Dawley rats were randomly divided into the controlgroup and the obesity group (high fat diet from day1to21of gestation). Theoffsprings were raised under the same condition. The heart function was measuredby high-resolution ultrasound imaging system. Isolated hearts from3-month-oldmale and female offspring were subjected to ischemia and reperfusion injury in alangendorff preparation. The infarct size was assessed by triphenyl tetrazoliumchloride (TTC) staining. LDH activity was measured by specific LDH kit. Ang II inthe serum was measured by EIA kit. Cardiac AT1R and AT2R protein expression inadult male and female offspring was measured by Western Blot. AT1R and AT2RmRNA abundance was determined by RT-PCR. Chromatin Immunoprecipitationassays were performed for GRE at the AT1R or AT2R promoter in DNA sequencespulled down by GR antibodies.Results1. There were no difference in body weight between control and obesity group inadult male offspring. But the heart weight and the heart to body weight ratio ofobesity group were obviously higher than those of control group (P<0.05). Incontrast to male offspring, maternal obesity had no effect on female heart weight,body weght and the heart weight to body weight ratio.2. M-mode images showed that compared to the control group, the LVAWd,LVAWs, LVPWd, LVPWs, EF, FS were increased and the LVIDd, LVIDs weredecreased in the obese male group (P<0.05). However, maternal obesity had noeffect on wall thickness and systolic function in female offspring.3. Echocardiographic data showed that maternal obesity had no effect on diastolicfunction in both male and female offspring. Parameters inflecting diastolic functionare MVA, MVE, E/A ratio, DT, IVRT, and ET.4. Maternal obesity increased heart susceptibility to ischemia-reperfusion injury, infarct size and LDH activity in adult male offspring（P<0.05）, but not femaleoffspring.5. Maternal obesity increased plasma AngII in male（P<0.05）, but not femaleoffspring. There was an increase in mRNA and protein abundance of AT2R in male（P<0.05）, but not female offspring. However, maternal obesity had no effect onAT1R expression in both male and female offspring.6. Inhibition of AT2R abrogated maternal obesity-induced increase in cardiacischemic vulnerability in male adult rats.7. Maternal obesity resulted in decreased glucocorticoid receptors (GRs) binding tothe glucocorticoid response element (GREs) at the AT2R promoter（P<0.05）, whichwas due to decreased glucocorticoid receptors (GRs) and binding affinity of nuclearextract to GREs in the heart of adult male offspring.Summary1. Maternal obesity induced cardiac hypertrophy in a sex-dependent manner withoutchange of systolic and diastolic function.2. In utero obesity causes programming of increased AT2R gene expression in theheart by downregulation of GR, contributing to the heightened heart vulnerability toischemic injury in adult male offspring in a sex-dependent manner.ConclusionIn the present study, we demonstrated that Ang II receptors plays an important role inmaternal obesity-related cardiac dysfunction. The present investigation offerssupporting evidence that maternal obesity had effect on the fetal cardiac structure andcardiomyocytes proliferation, which may be related to the AT2R/AT1R ratio in thefetal heart. Maternal obesity causes programming of increased AT2R gene expressionin the heart by downregulation of GR, contributing to the heightened heartvulnerability to ischemic injury in adult male offspring in a sex-dependent manner.