Prenatal Inflammation Stimulus Predisposes Off Spring To Myocardial Injury Induced By Cardiovascular Risk Factors And Mechanisms Study

Cardiovascular disease(CVD) is one of the most important factors that threat the health of human beings. Nowadays, CVD has been beyond cancer and other chronic non communicable diseases and becomes the leading global cause of death, accounting for 31% of deaths worldwide. According to the report of World Health Organization in 2014, approximately 17.5 million patients dead from CVD every year with the number being expected to increase to over 23.6 million by 2030. Although the cost associated with the treatment and prevention of CVD is recognized as one of the most costly diseases to the health care system in relation to all the chronic non-infectious diseases, the corresponding continual rise in incidence and mortality associated with CVD, suggest that there are other hidden factors that might be overlooked. In CVD, ischemic heart disease causes the death account for nearly half of its total deaths. A large number of clinical and animal experimental data suggest that prenatal exposure to adverse environment, such as malnutrition, hypoxia et al, is an acceptable cause predisposing to CVD risk factors in adult offspring. Our lab constructed rat offspring model of prenatal programmed hypertention successfully by prenatal stimulation with lipopolysaccharide(LPS) or zymosan. Furthermore, we also found that those offspring from maternal LPS exposure developed moderate cardiac remodeling and fibrosis with decreased diastolic function when elder than 8 months old. The changes of myocardial pathological morphology and function in offspring of prenatal inflammation stimulation may be caused by abnormal activation of the NF-κB pathway and local RAAS in heart, but the detailed mechanisms remain unclear. Accordingly, we speculate that there may be an important link between cardiac remodeling caused by prenatal inflammation and higher susceptibility of cardiac disease in adult offspring, and higher risk of cardiac disease in adult offspring may be originated from inflammation exposure during pregnancy. At present, the study of the causes and mechanisms of cardiac disease susceptibility mainly concentrated in the adult individuals. Therefore, the theory of our study may extend the time window for prevention and treatment of cardiac disease, the time of preventing and curing cardiac disease is back to early life.Methods:1.The procedure of pregnant rats treatment: time-dated pregnant SD rats(250 g to 300 g) were randomly divided into two groups(n=9-10), and were intraperitoneally(i.p.) administered with saline(Control group) and LPS(0.79 mg/kg/day)(LPS group), respectively, on gestation days 8, 10 and 12. Pups were weaned at the age of 3 weeks, and then ad libitum feeded until 20 weeks of age.2. Treatment scheme of offspring postnatally: Pups from the above-mentioned LPS group at the age of 20 weeks were randomly divided into four groups(n=6), and were treated with vehicle(subcutaneous injections), 2 weeks of ISO(5 mg/kg/day, subcutaneous injections), 2 weeks of both ISO(5 mg/kg/day, subcutaneous injections) and SB202190 daily(0.4 mg/kg/day, i.p.), 2 weeks of both ISO(5 mg/kg/day, subcutaneous injections) and NAC(2.5 g/L, estimated oral intake of 500 mg/kg/d daily) for 2 weeks, defined as LPS+Ve, LPS+ISO, LPS+ISO+SB202190 and LPS+ISO+NAC group, respectively. Offspring of control were treated with the same as that of prenatal LPS exposure, defined as Con+Ve, Con+ISO, Con+ISO+SB202190 and Con+ISO+NAC groups. In ISO acute experiment, 20-week-old offspring from both control and LPS group(n=6) were treated with vehicle and ISO(5 mg/kg, subcutaneous injections) for 30 min one time.3. High resolution small animal ultrasound imaging system and echocardiography was performed to determine the cardiac function and hemodynamics in rat offspring.4. The overall morphology and cell size of cardiomyocytes were evaluated by Hematoxylin-Eosin staining, myocardial cell size. Masson staining was used to evaluate myocardial fibrosis and collagen deposition.5. To evaluate the production of tissue reactive oxygen species, fresh unfixed left ventricular tissue samples were stained with dihydroethidium(DHE) fluorescent dye and examed with confocal microscope.6. q RT-PCR was performed to analysis gene expression referred to ROS generation and eliminaion, such as Nox2、Nox4、Sod1、Sod2 et al and other cardiac hypertrophy and fibrosis relative genes, such as α-MHC、β-MHC、ANP、BNP、Col1a1、Col3a1、Mmp2、Mmp9.7. Protein expression in left ventricle was determined by immunoblotting, such as the expression levels of phosphorylation of p38、JNK MAPK in MAPK cascade pathway, NAPDH oxidase NOX2, antioxidant enzyme SOD1, SOD2, and cardiac remodeling related protein expression, such as α-MHC, β-MHC.Results:1. Effects of prenatal inflammatory stimulation on cardiac morphology and function in adult rat offspring.1.1 Compared to control group, offspring of prenatal LPS exposure displayed left ventricular remodeling with increased IVST and decreased LVIDS significantly(P < 0.05), left ventricular collagen volume fraction(CFV%). The m RNA expression of Col1a1 and enzymatic degradation Mmp9 were also increased in offspring of prenatal LPS exposure(P < 0.05). These indicated significant cardiac remodeling exists in offspring of prenatal inflammation exposure.1.2 Compared with control group, offspring of prenatal LPS exposure display increased LVEF% and LVFS%, significantly(P < 0.05), which suggested change of cardiac function in offspring of prenatal inflammation exposure.2. Prenatal inflammatory stimulation predisposes to myocardial injury induced by ISO treatment in offspring.2.1 After ISO stimulation for two weeks, heart weight index group(HMI) and left ventricular mass index(LVMI) were increased significantly in both offspring of control(P < 0.001) and prenatal LPS exposure. However, the increased ratio was greater in offspring of prenatal exposure than that in control offspring(P < 0.001).2.2 After 2 weeks of ISO stimulation, the m RNA expression of cardiac hypertrophy related embryonic gene, such as β-MHC was significantly decreased(P < 0.05), whereas ANP was significantly increased(P < 0.01) in control offspring. The m RNA expression of BNP and non-embryonic gene, α-MHC, showed no significant changes(P > 0.05) in control offspring. In addition, the ratio of α-MHC/β-MHC mRNA expression was significantly increased(P < 0.01) in offspring of control. However, in offspring of prenatal LPS exposure, cardiac hypertrophy related embryonic genes, such as β-MHC, ANP, BNP were increased significantly in mRNA levels(P < 0.01), whereas non-embryonic gene, such as α-MHC was significantly decreased in m RNA level. Of note, the ratio of α-MHC/β-MHC m RNA expression was decreased both in m RNA and protein level(P < 0.01), which was also significantly lower than the offspring of control with the same stimulation(P < 0.05). This indicated that the sensitivity of ISO induced myocardial hypertrophy in offspring of inflammatory stimulation during pregnancy is increased.2.3 After 2 weeks of ISO stimulation, CVF%, the m RNA expressions of Col1a1, Col3α1 and enzymatic degradation Mmp2 expression were increased significantly(P < 0.01) in left ventricular in offspring of both control and prenatal LPS exposure. However, it showed greater changes in offspring of prental LPS exposure than those in control offspring(P < 0.05).These data indicated that prenatal inflammation exposure predisposed offspring to myocardial fibrosis induced by ISO treatment.2.4 After 2 weeks of ISO stimulation, LVEF% and LVFS% have no significant change(P > 0.05) in offspring of control, however, both of which were significant decreased by 13% and 23% respectively(P < 0.001) in offspring of prenatal LPS exposure. The results suggested that prenatal inflammation exposure predisposes offspring to cardiac systolic dysfunction after ISO treatment.3. Mechanism study of higher sensitivity to myocardial injury induced by ISO in offspring of prenatal LPS exposure3.1 Imbanlance of ROS generation and elimination attributes to increased sensitivity to ISO induced myocardial injury in offspring of prenatal inflammation exposure.3.1.1 Without ISO stimulation, superoxide anion level deteremined by fluorescence probe DHE staining in heart tissue showed that there was higher level of DHE fluorescence intensity, but no statistic difference in offspring of prenatal LPS exposure as compared to that of control. After 2 weeks of ISO stimulation, DHE fluorescence intensity in heart tissue was significantly increasedin offspring of control(P < 0.01). Furthermore, the DHE fluorescence intensity was further increased in offspring of prenatal inflammation exposure(P < 0.001), which wasgreater than that of control with ISO stimulation(P < 0.001).3.1.2 Compared with offspring of control, the mRNA expressions of ROS generation related genes(NADPH oxidase Nox2, p47phox) and the protein expression of NOX2 were significantly increased(P < 0.05) in left ventricular in offspring of prenatal LPS exposure without ISO treatment, which indicated ROS activation in offspring of prenatal inflammation stimulation. After 2 weeks of ISO stimulation, ROS generation related genes(NADPH oxidase, such as Nox2 and p67phox) mRNA expression in left ventricular were increased significantly(P < 0.01), whereas Nox4 and p22 phox m RNA expression only showed an increasing trend with no statistic difference in control offspring. However, the m RNA expressions of Nox2 and p67 phox, Nox4 and p22 phox were also significantly increased(P < 0.05 or 0.01) in offspring of prenatal LPS exposure after ISO stimulation, but greater than that of control with ISO stimulation(P < 0.05 or 0.01). In addition, the NOX2 expression in protein levels was consistent with mRNA levels.3.1.3 Compared to offspring of control, the mRNA expression of ROS scanvenge related genes(Sod1, Sod2, Sod3, catalase) were significantly increased(P < 0.05 or 0.01) in left ventricular in offspring of prenatal LPS exposure. After 2 weeks of ISO stimulation, Sod1, Sod2, Sod3, catalase mRNA expression in left ventricular significantly decreased in both offspring of control(P < 0.05 or 0.01), and that of prenatal LPS exposure(P < 0.01 or 0.001). Moreover, the decreased trend was greater in offspring of prenatal exposure to LPS than that of the control after ISO stimulation, although without statistic difference. SOD1 and SOD2 expression in protein levels displayed the similar trend with mRNA expression.3.1.4 Antioxidant NAC(GSH precursor) alleviates myocardial fibrosis and improves myocardial contractility in offspring of prenatal inflammation exposure after ISO stimulation.3.2 Activation of p38 MAPK promotes increased sensitivity to ISO induced myocardial injury in offspring of prenatal LPS exposure.3.2.1 Compared with offspring of control, expression of p-p38 in left ventricular was higher in offspring of prenatal LPS exposure(P < 0.05), suggesting p38 MAPK is activated tin offspring of prenatal inflammation exposure.3.2.2 Whatever ISO stimulation or not, expression of p-p38 remained in a higher level in offspring of prenatal LPS exposure than that of control(P < 0.05). After ISO stimulation for 2 weeks, there was no significant change in both offspring of control prenatal inflammation stimulation(P > 0.05).3.2.3 Taking into account the periodic character of p38 activation, we performed an ISO acute stimulation experiment. In ISO acute stimulation test, p-p38 expression in left ventricular was significantly increased in both offspring of control group(P < 0.05) and that of prenatal LPS exposure(P < 0.01). Furthermore, p-p38 expression was remarkably higher in offspring of prenatal LPS exposure than that of control(P < 0.01) in challenge of ISO stimulation.3.2.4 Inhibition of p38 activity by SB202190 alliviates cardiac hypertrophy and myocardial fibrosis and improve the myocardial contractility caused by ISO challenge in offspring of prenatal inflammation exposure.3.3 Positive crosstalk of ROS and p38 MAPK activation aggravates myocardial injury caused by ISO treatment in offspring of prenatal inflammation stimulation.3.3.1 Antioxidant NAC reduced the p-p38 expression in left ventricular of offspring of prenatal inflammation stimulation with ISO challenge(P < 0.05).3.3.2 p38 inhibitor, SB202190, reduced the m RNA expression of ROS generation related genes(NADPH oxidase Nox2, p67 phox, p22phox) in left ventricular of offspring of prenatal LPS exposure with ISO challenge(P < 0.01), suggesting that ROS and p38 MAPK jointly promote myocardial injury via a positive crosstalk caused by ISO in offspring of prenatal inflammation stimulation offspring.Conclusion:1. Prenatal inflammatory stimulation predisposes offspring to ISO induced myocardial injury, characterized by more serious cardiac hypertrophy, myocardial fibrosis, and significant decreased left ventricular systolic function.2. Imbalance of ROS generation and elimination is one of the most important mechanism that predisposes offspring of prenatal inflammation exposure to myocardial injury caused by ISO treatment.3. P38 MAPK activation is another mechanism that predisposes offspring of prenatal inflammation exposure to myocardial injury caused by ISO treatment.4. Postive crosstalk of ROS and p38 MAPK promotes the increased sensitivity of ISO induced myocardial damage in offspring of prenatal inflammation exposure.