| Intrauterine hypoxia can lead to fetal hypoxemia,resulting in adverse maternal and fetal outcomes,and is the most common type of complication during pregnancy.The "fetal origin of adult disease theory" suggests that intrauterine hypoxia and other adverse stresses lead to an increased risk of cardiovascular disease in offspring in adulthood,but the mechanism remains unclear.The heart is a highly metabolic organ and myocardial mitochondrial dysfunction is closely associated with myocardial injury.NMDAR is a glutamate ionotropic receptor,and its activation produces excitotoxicity and can cause mitochondrial damage.NMDAR is also present in peripheral organ systems such as the heart,lungs and pancreas.In our group,we have found that intrauterine hypoxia-induced impairment of lung,pancreas and brain function in rats at birth and after birth may be associated with NMDAR activation.However,it is not clear whether NMDAR is involved in mediating myocardial morphological and functional injury at birth and postnatally in rats due to intrauterine hypoxia.It was found that memantine,an NMDAR antagonist,has potential myocardial protective effects,suggesting that NMDAR may be involved in myocardial injury.There are no studies on the mechanism of myocardial morphological and functional injury at birth and after birth in rats due to intrauterine hypoxia involving NMDAR.OBJECTIVE: The aim of this study was to investigate the role and mechanism of cardiac NMDAR involvement in mediating myocardial injury caused by intrauterine hypoxia in rats.I.Intrauterine hypoxia induced myocardial morphological and functional impairment at birth and after birth in ratsMethod1.Experimental grouping and animal model preparation: The experimental grouping was divided into air control group and intrauterine hypoxia group.The intrauterine hypoxia intervention was 9.5-11.5%hypoxia treatment given to pregnant rats at 19-20 days of gestation.Body weight was measured on the day of birth,7 days,14 days,21 days,28 days and 8 weeks after birth,and the remaining relevant indexes were measured on the day of birth,28 days and 8 weeks,respectively.2.Observation of growth and development: Body weight,heart weight and heart weight/body weight ratio were observed in rats at different postnatal ages.3.Examination of myocardial histomorphology: HE staining,percentage of binucleated cardiomyocytes,and right ventricular structure by echocardiography were observed in rats at different postnatal ages.4.Examination of myocardial hypertrophy and myocardial fibrosis:WGA staining,Masson staining and BNP levels were perfected on the day of birth and at 8 weeks in rats.5.Examination of cardiac function: echocardiographic assessment of left heart systolic function,exhaustion test and non-invasive blood pressure test were perfected in rats at 8 weeks of life.Results:Intrauterine hypoxia causes a decrease in body weight and heart weight at birth and an increase in heart weight/body weight ratio in rats,and the increase in heart weight/body weight ratio persists into adulthood;intrauterine hypoxia causes morphological damage to the myocardium of rats at birth and after birth,as evidenced by loosening of myocardial fiber arrangement,blurring of transverse lines,an increase in the percentage of binucleated cardiomyocytes at birth,an increase in the cross-sectional area of cardiomyocytes,and an increase in the level of BNP,a marker of myocardial hypertrophy.In addition,intrauterine hypoxia caused a decrease in left heart systolic function,a decrease in exercise tolerance and an increase in blood pressure after exhaustive exercise in rats in adulthood.It is suggested that intrauterine hypoxia can cause myocardial morphology and functional impairment in rats.II.Blockade of NMDAR attenuates myocardial morphological and functional impairment at birth and after birth in rats with intrauterine hypoxiaMethod1.Experimental grouping and animal model preparation:experimental grouping was divided into air control group,air +memantine group,intrauterine hypoxia group and hypoxia + memantine group.Intrauterine hypoxia intervention with part 1.Body weight was measured on the day of birth,7 days,14 days,21 days,28 days and 8weeks after birth,and the remaining relevant indexes were measured on the day of birth,28 days and 8 weeks,respectively.2.Expression and changes of NMDA receptor subunits: NMDAR subunits were detected on the day of birth in rats and compared with brain tissue,NR1 protein expression and CTn T and NR1 immunofluorescence.3.Observation of growth and development: same as part 1.4.Examination of myocardial hypertrophy and myocardial fibrosis:same as part 1.5.Examination of cardiac function: same as part 1.6.Exploration of molecular biological mechanisms of myocardial injury due to intrauterine hypoxia: refinement of Tunel staining,MCM2 protein levels and NKX2.5 protein levels in rats on the day of birth and at8 weeks.Results:The mRNA levels of the NMDA receptor subunits NR1,NR2 A,NR2B,NR2 C and NR2 D were detected in brain and myocardial tissues of rats at birth,with NR2 B being the most abundantly expressed in brain tissue and NR2 D in heart tissue.Prenatal blockade of NMDAR improved the impaired growth and development caused by intrauterine hypoxia,as evidenced by improved body weight,decreased heart weight and abnormal increase in heart weight/body weight in rats;prenatal blockade of NMDAR also improved the impaired myocardial morphology at birth and after birth in rats caused by intrauterine hypoxia.In addition,prenatal blockade of NMDAR also ameliorated the impaired myocardial function in adult rats caused by intrauterine hypoxia,as evidenced by improved left heart systolic function,decreased exercise tolerance,and increased blood pressure after exhaustive exercise.Prenatal blockade of NMDAR ameliorated increased apoptosis,impaired proliferation,and decreased NKX2.5 protein levels in rats born with intrauterine hypoxia.It is suggested that blockade of NMDAR reduces myocardial morphological and functional impairment at birth and after birth in rats with intrauterine hypoxia,which may be related to its improvement of myocardial cell apoptosis and proliferation.III.Blockade of NMDAR attenuates intrauterine hypoxia-induced mitochondrial damage in rat myocardiumMethod1.Experimental grouping and animal model preparation: same as part 22.Observation of myocardial mitochondrial morphology: myocardial mitochondrial ultrastructure,mitochondrial lengthening and interconnectivity were observed under transmission electron microscopy at different postnatal ages in rats to assess the degree of mitochondrial fragmentation.3.Assay of myocardial mitochondrial number: measurement of citrate synthase activity was perfected on the day of birth and at 8 weeks in rats.4.Detection of myocardial mitochondrial function: ATP content and ROS levels were perfected on the day of birth and at 8 weeks in rats.5.Exploring the mechanisms of myocardial mitochondrial injury due to intrauterine hypoxia: refinement of mitochondrial copy number to reflect mitochondrial biogenesis,MFN2 level to reflect mitochondrial fusion,and DRP1 level to reflect mitochondrial division in rats on the day of birth and at 8 weeks.Results:Prenatal blockade of NMDAR improved the morphological and structural damage of myocardial mitochondria and reduced myocardial mitochondrial fragmentation in rats induced by intrauterine hypoxia.Prenatal blockade of NMDAR improved the increase of citrate synthase activity in adult rats caused by intrauterine hypoxia,suggesting that prenatal blockade of NMDAR could improve the increase of myocardial mitochondria in adult rats caused by intrauterine hypoxia.Prenatal blockade of NMDAR ameliorated the decrease in ATP content and increase in ROS level in myocardial tissue at birth and in adulthood in rats caused by intrauterine hypoxia.This suggests that blocking NMDAR reduces myocardial mitochondrial damage in rats with intrauterine hypoxia.In addition,prenatal blockade of NMDAR ameliorated the increased mitochondrial copy number in adult rats caused by intrauterine hypoxia,suggesting that blockade of NMDAR ameliorated the increased mitochondrial biogenesis in adult rats caused by intrauterine hypoxia.Prenatal blockade of NMDAR ameliorated the decrease in DRP1 levels at birth and in adulthood in rats caused by intrauterine hypoxia,suggesting that blockade of NMDAR ameliorated the decrease in mitochondrial division in rat heart muscle caused by intrauterine hypoxia.IV.Mechanism of blocking NMDAR to attenuate intrauterine hypoxia-induced cardiomyocyte and mitochondrial damage in ratsMethod1.Hypoxia-induced cardiomyocyte injury is associated with NMDAR: expression of different subunits of NMDAR,NR1 levels and cell supernatant glutamate levels.2.Detection of myocardial cell injury: cell viability was detected by CCK8 method at different hypoxic times(24h,48 h and 72h)and different NMDA treatment concentrations(0.1m M,0.3m M,1m M,3m M,10 m M)and times(24h,48 h and 72h),and cell supernatant lactate dehydrogenase activity reflected cytotoxicity.3.Hypoxia-induced cardiomyocyte and mitochondrial damage is mediated by NMDAR: MK-801(50 μM)and NR1 knockdown followed by hypoxia to detect intracellular calcium ion concentration and mitochondrial ROS.4.Effects of NMDAR activation on mitochondrial morphology and function: After treatment of H9c2 cardiomyocyte cell line with different concentrations of NMDA,transmission electron microscopy was used to observe mitochondrial ultrastructure and mitochondrial extension and interconnectivity,and to detect ATP content,mitochondrial membrane potential,ROS,mitochondrial ROS and intracellular calcium ions.5.Mechanism of mitochondrial damage induced by NMDAR activation: detection of DRP1 protein levels after different concentrations of NMDA treatment and NR1 knockdown intervention.Results:NR1,NR2 A,NR2B,NR2 C and NR2 D were all distributed on H9c2 cardiomyocyte cell lines,with NR2 D being the most abundantly expressed.Different hypoxic times caused a decrease in H9c2 cardiomyocyte viability and an increase in supernatant lactate dehydrogenase levels,resulting in cardiomyocyte injury.NR1 expression was upregulated after 24 hours of hypoxia,and the supernatant glutamate concentration increased;MK-801 and NR1 knockdown reduced the intracellular calcium ion concentration and mitochondrial ROS elevation caused by hypoxia.ion concentration,decreased DRP1 levels,and NR1 knockdown ameliorated the decrease in DRP1 levels due to NMDAR activation.It is suggested that hypoxia causes damage to cardiomyocytes and myocardial mitochondria through activation of NMDAR.Overall conclusion.1.Intrauterine hypoxia causes myocardial morphological damage at birth in rats and can persist into adulthood with impaired cardiac function in adulthood.2.For the first time,prenatal blockade of NMDAR was found to attenuate myocardial morphological and functional impairment at birth and after birth in rats with intrauterine hypoxia.3.For the first time,prenatal blockade of NMDAR was found to attenuate intrauterine hypoxia-induced impairment of myocardial mitochondrial morphology,number and function at birth and postnatally in rats.4.Hypoxia can cause increased glutamate release and elevated NR1 expression levels in cardiomyocytes,and upregulate the glutamate-NMDAR signaling pathway to cause cardiomyocyte injury.For the first time,a decrease in DRP1 levels and reduced mitochondrial division after NMDAR activation was found to be one of the important mechanisms of hypoxia-induced myocardial mitochondrial injury. |
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