1,25-Dihydroxyvitamin-d Treatment Alleviates Heart Damage By Inhibition Of Cardiomyocyte Apoptosis In Reduction In Uteroplacental Perfusion (RUPP) Rats

Preeclampsia is a pregnancy-specific disease affected about 9.4-10.4%of all pregnancies,with variety of complications,such as heart disease,renal failure,DIC,HELLP syndrome and so on.Heart disease is marked by left ventricular dysfunction and heart failure which is a potentially life-threatening pregnancy-associated disease.It remains a main cause of maternal morbidity and mortality which represents a significant global health issue.Despite years of research on preeclampsia complication,the prevention and prediction are still impossible.What’s more,preeclampsia foretells increased rates of cardiovascular disease in later life,which could be pay attention to for education and intervention.The effect of Vitamin D is recognized by means of variety epidem-iological investigations and laboratory studies.Now Vitamin D is regarded as a pleiotropic secosteroid hormone more than a simple vitamin via both genomic response and non-genomic response.The vitamin D endocrine system includes the calcium homeostasis,immune function,disease preven-tion and cancer treatment.Data from observational studies have shown inverse associations between low concentration plasma 25（OH）D with metabolic and cardiovascular disorders,cancer,immune diseases and neurodegenerative diseases.Notably,maternal vitamin D deficiency increases risk of preecla-mpsia.The incidence rate is highest in winter months because the production of vitamin D by sunlight is lowest.A recent animal data demonstrated that low vitamin D status in pregnant mice resulted in symptoms of preeclampsia,as elevated blood pressure and impaired placental development.Further more,vitamin D addition beginning at the early gestation in preeclampsia model rat had various effects on blood pressure,fetus,placenta and metabolism.Gezmish,O.reported young rats exposed to vitamin D deficiency in early life led to left ventricular hypertrophy and subsequent interstitial fibrosis within the myocardium in vitamin D-deficient adult rats.Studies indicated 1,25（OH）₂D treatment reduces cardiac hypertrophy,left ventricular diameter and stroke volume in spontaneously hypertensive heart failure-prone rats.In epidemiological studies,vitamin D deficiency is associated with an increased cardiovascular risk.Therefore,present evidence does not support cardiovascular benefits of vitamin D supplementation,and whether vitamin D has cardiovascular effects in preeclampsia pregnancies remains to be evaluated.The rat model of reduction in uteroplacental perfusion pressure（RUPP）is one of the most well characterized models which can mimic the central role of reduced placental perfusion and express the same syndromes of preeclampsia in human pregnancy,including elevated blood pressure,proteinuria,and heart dysfunction.So we used this rat model to test whether vitamin D treatment could alleviate heart damage in RUPP rats.Our trail would provide laboratory data for clinical interventional of vitamin D in preeclampsia with heart disease.PartⅠ1,25（OH）₂D improved myocardial function of RUPP ratObjective:We surveyed the effect of 1,25（OH）₂D injection in RUPP rats on maternal blood pressure,urine protein and myocardial function.Methods:Pregnant rats were divided into 3 groups:normal pregnant（NP,n=10）,pregnant with reduction in uteroplacental perfusion（RUPP,n=10）,and pregnant RUPP plus 1,25（OH）2D（RUPP+VD,n=10）.On day 1,7 and 14 of pregnancy,RUPP+VD group of rats received 120 ng/100 g body weight of1,25（OH）₂D by subcutaneous injection.The NP group and RUPP group received vitamin D vehicle（saline solution）subcutaneously.On day 14 of pregnancy,RUPP and RUPP+VD group rats were anesthetized to operate vascular stenosis surgery reducing uteroplacental perfusion.Rats in NP group were sham operated.On day 19 of pregnancy,we recorded arterial pressure and heart function,collected 24h urine,serum and heart tissue.Results:1 1,25（OH）₂D decreased systolic blood pressure and urine protein/creatinine of RUPP ratIn rats of RUPP group systolic blood pressure was significantly elevated compared with rats of NP group（164.30±12.60 mmHg vs.134.80±12.60mmHg,P<0.001）;24h urine protein/creatinine increased in rats of RUPP group compared with rats of NP group（1.90±0.67 vs.0.83±0.30,P<0.01）,which were agreed with previous research.As a contrast,there was a significant decrease in systolic blood pressure（148.10±11.40 mmHg vs.164.30±12.60 mmHg,P<0.05）,in 24h urine protein/creatinine（0.85±0.22 vs.1.90±0.67,P<0.01）,in rats of RUPP+VD group compared to rats of RUPP group.There were no differences in rat serum alkaline phosphatase,urea and creatinine among three groups.Rat serum free calcium elevated in RUPP+VD group remarkably than NP group（1.22±0.06 mmol/L vs.1.09±0.05 mmol/L,P<0.01）and RUPP group（1.22±0.06 mmol/L vs.1.14±0.10 mmol/L,P<0.05）.2 1,25（OH）₂D improved myocardial function of RUPP ratEchocardiography results demonstrated impaired myocardial function in rats of RUPP group compared with rats of NP group,including decreased left ventricular volume（diastole）（197.90±50.22 ul vs.284.22±41.83ul,P<0.01）,left ventricular internal diameter（diastole）（6.21±0.75 mm vs.7.32±0.48 mm,P<0.01）and increased left ventricular anterior wall（diastole）corrected by weight（0.48±0.12 mm/100g vs.0.31±0.11 mm/100g,P<0.05）.Data from echocardiography showed improved myocardial function in RUPP+VD rats compared to RUPP rats,including increased left ventricular volume（diastole）（255.49±64.53 ul vs.197.90±50.22 ul,P<0.05）,left ventricular internal diameter（diastole）（6.95±0.82 mm vs.6.21±0.75 mm,P<0.05）and decreased left ventricular anterior wall（diastole）corrected by weight（0.36±0.15 vs.0.48±0.12 mm/100g,P<0.05）.Summary:We proved that 1,25（OH）₂D administration beginning at the early gestation could decrease systolic blood pressure and urine protein/creatinine of RUPP rat,what is more,1,25（OH）₂D could improve myocardial function of RUPP rat.PartⅡEffect of 1,25（OH）₂D on cardiomyocyte structure of RUPP ratObjective:We surveyed the effect of 1,25（OH）₂D addition on cardiomyocyte in RUPP rats.Methods:Rat heart slices were fixed for HE stain and ultrastructural cytology.VDR,CYP24A1 expressions were studied in rat heart.Plasma level of renin and angiotensinⅡwere assayed by radioimmunoassay.RT-PCR was used to analysis ANP,BNP,eNOS and iNOS gene expressions in heart of RUPP rat.Results:1 1,25（OH）₂D improved cardiomyocyte histological structure of RUPP ratIn histological examination,in RUPP group cardiomyocyte cell nucleus arranged in disorder compared to NP group,In RUPP+VD group,cardiom-yocyte nucleus arranged more neatly than in RUPP group.2 1,25（OH）₂D improved cardiomyocyte ultrastructure of RUPP ratTransmission electron microscopy image of rat cardiomyocyte proved mitochondrial crest disappeared,and edema was obviously in perinuclear space in RUPP group.The cardiomyocyte from NP group had organized mitochondria.In RUPP+VD group,mitochondrial damage was reduced and edema was mild in perinuclear space compared to RUPP group.3 1,25（OH）₂D increased VDR expression in heart of RUPP ratVitamin D receptor（VDR）mRNA expression in rat heart had no remarkable differences among the three groups.In cardiomyocytes,immunohistochemistry staining suggested VDR protein expression was higher in RUPP group than NP group,and the highest in RUPP+VD group.4 1,25（OH）₂D increased CYP24A1 gene expression in heart of RUPP ratCYP24A1（the key enzyme of 1,25（OH）₂D degradation）is a VDR-target gene,its gene expression in rat heart was significantly increased in RUPP+VD group compared to NP group（P<0.05）and RUPP group（P<0.01）.It indicated that 1,25（OH）₂D may play a role in attenuating heart damage of RUPP rat probably via its genomic effect.5 1,25（OH）₂D effected plasma level of renin and angiotensinⅡof RUPP ratRadioimmunoassay showed plasma level of renin decreased in RUPP group（P<0.01）compared to NP group,however,it increased in RUPP+VD group compared to RUPP group（P<0.05）.Radioimmunoassay showed there was no difference between RUPP group and RUPP+VD group in plasma level of angiotensinⅡ.The level of angiotensinⅡdecreased in RUPP+VD group compared to NP group（P<0.05）.6 1,25（OH）₂D had no effect on ANP and BNP gene expressions in heart of RUPP ratANP and BNP are cardiomyocytes hypertrophy markers,there were no significant differences among three groups on gene expressions in heart of rat.7 1,25（OH）₂D had no effect on eNOS and iNOS gene expressions in heart of RUPP rateNOS and iNOS are endothelial dysfunction markers,there were no significant differences among three groups on gene expressions in heart of rat.Summary:1 1,25（OH）₂D improved cardiomyocyte histological structure and ultrastructure of RUPP rat.2 1,25（OH）₂D increased VDR and CYP24A1 expressions in heart of RUPP rat.PartⅢ1,25（OH）₂D alleviated heart damage of RUPP rat by inhibitingcardiomyocyte apoptosisObjective:We established 1,25（OH）₂D alleviated cardiomyocyte damage of RUPP rat by decreasing the levels of oxidative stress,endoplasmic reticulum（ER）stress and inhibiting cardiomyocyte apoptosisMethods:The apoptosis level of rat heart was assayed with TUNEL stain.We observed markers expressions of oxidative stress,endoplasmic reticulum stress and apoptosis by RT-PCR and immunohistochemistry staining.Results:1 1,25（OH）₂D decreased oxidative stress level in heart of RUPP ratHO-1,P47-phox,P22-phox,HIF-1αare oxidative stress markers,however,there were no significant differences among three groups.In cardiomyocytes,as oxidative stress markers,the expression of P47-phox and p-P47 phox,at protein levels were increased in RUPP group compared with NP group（P<0.001）,these increases were attenuated in RUPP+VD group（P<0.001）.2 1,25（OH）₂D decreased level of endoplasmic reticulum（ER）stress in heart of RUPP ratBy immunohistochemical analysis,the protein expression of CHOP（GADD153,c/EBP homologous protein/DNA-damage-inducible gene 153,contributing in the apoptosis of ER-stressed cells）and GRP78（glucose-regulated protein,78kDa,contributing in facilitating the assembly of multimeric protein complexes inside the endoplasmic reticulum）,as the ER stress markers,were higher in heart of RUPP group than that in NP group（P<0.001）.Compared with RUPP group,the levels of CHOP and GRP78decreased in RUPP+VD group（P<0.001）.3 1,25（OH）₂D inhibited mitochondria apoptosis in heart of RUPP ratCaspase 9 and Cleaved Caspase 9 are markers of mitochondria apoptosis pathway.The protein expression of Caspase 9（P<0.001）and Cleaved Caspase9（P<0.001）increased significantly in RUPP group compared to NP groups by immunohistochemistry staining.The level of Caspase 9 and Cleaved Caspase9 decreased in RUPP+VD group（P<0.001）compared to RUPP group.4 1,25（OH）₂D decreased Caspase 12 expression in heart of RUPP ratCaspase 12 was involved in apoptosis induced by endoplasmic reticulum stress pathway.The tendency of protein expression among three groups（P<0.001）was similar with that of Caspase 9.5 1,25（OH）₂D decreased Cleaved Caspase 3 expression in heart of RUPP ratIn immunohistochemistry staining,protein expression of Caspase 3decreased significantly in RUPP group and RUPP+VD group compared to NP group（P<0.001）.At the same time,Cleaved Caspase 3 protein expression was low in NP group,while in RUPP group,it expressed obviously higher than NP group（P<0.001）,which leading to more apoptosis.1,25（OH）₂D treatment apparently lowered Cleaved Caspase 3 protein expression compared with RUPP group（P<0.001）.6 1,25（OH）₂D inhibited apoptosis in heart of RUPP ratTUNEL image in confocal laser scanning microscope showed apoptotic cells.In NP group,the apoptosis cells were rare,while in RUPP group,several apoptosis cells were stained in red-fluorescence.Compared with RUPP group,the apoptosis cells decreased in RUPP+VD group.Summary:1,25（OH）₂D alleviated heart damage of RUPP rat by inhibiting cardiomyocyte apoptosis and improving myocardial function.Conclusions:1 1,25（OH）₂D could improved clinical feature and myocardial function of RUPP rat.2 1,25（OH）₂D improved cardiomyocyte histological structure and ultrastructure of RUPP rat.3 1,25（OH）₂D alleviated heart damage of RUPP rat by inhibiting cardiomyocyte apoptosis and improving myocardial function.