Breeding ABR-DRS And Exploring The Phenotype And AMPA Receptors In NA Involve In The Deficiency Of BRS In ABR-DRS

Arterial baroreflex activity regulates cardiovascular function.Baroreflex sensitivity?BRS?,commonly assessed as change of heart rate–heart rate interval in response to change in systolic blood pressure?SBP??ms/mm Hg?,is a well-recognized marker of autonomic activity.Large multicenter prospective studies of subjects with or without cardiovascular diseases show that impaired BRS is associated with mortality as well as development of hypertension,arrhythmias,stroke,heart failure and myocardial infarction.A recent study suggests that BRS may help to identify patients with resistant hypertension who are likely to benefit from renal sympathetic denervation.Impairment in the baroreflex function also has been demonstrated in diabetic patients and habitual smokers.On the other hand,activation of the baroreceptors may be a promising therapy for the treatment of resistant hypertension and heart failure.All these observations collectively suggest a strong link between impaired autonomic nervous system and cardiovascular diseases.However,these data can't answer whether BRS plays a key role in the occurrence of cardiovascular diseases.We made large-scale artificial selection for deficient and normal BRS in SD rats.We defined rats with BRS<0.6 ms/mmHg as arterial baroreflex-deficient rats?ABR-DRs?,and rats with BRS>0.8 ms/mmHg as arterial baroreflex-normal rats?ABR-NRs?.During the breeding process,the BRS of the two animals showed a significant difference.ABR-DRs showed mild hypertension.The mechanism of BRS dysfunction also has been investigated by using the ABR-DRs.ABR-DRs mainly showed the dysfunction of the vagus,while the sympathetic function is normal,so ABR-DRs is a simple BRS dysfunction animal model.The AMPA receptor was found to be involved in the electrical activity abnormality of NA dorsal fissure neurons in vitro for ABR-DRs.But whether AMPA receptors are involved in BRS and which subtypes are still in need of further study,and there is no relevant study.This study mainly focus on the identification of ABR-DRs and the mechansim of BRS dysfunction.We found ABR-DRs show metabolic syndrome,including,overweight,hyperlipemia,and high blood glucose.Compared with ABR-DRs,ABR-DRs did not show cardiac hypertrophy,abnormal heart function,and susceptibility to arrhythmia.The mechanism of BRS dysfuntion:Our former data have found that the glutamte receptor,the AMPA type glutamate receptor may be involved in the dysfuntion of BRS.AMPA type glutamate receptor includes four main subunits,GluR1,GluR2,GluR3 and GluR4.Among them,GluR2 subunit is the key determinant of AMPA receptor function.Subsequent research mainly focused on GluR2.Transfecting GluR2 to clear that suspected GluR2 may be a key protein for BRS dysfunction.Transfection of GluR2 in NA can improve the BRS values and decreased blood pressure in ABR-DRsMethods and results are as followed:1.Identification,Screening and Breeding of Rats Model across 26 to 29 enerationThe BRS,blood pressure and heart rate of each generation of ABR-NRs and ABR-DRs were measured by clear-press technique.The animals and their offspring were excluded from each group of BRS noncompliance or abnormal blood pressure.The coincidence rate of BRS in ABR-DRs was close to 90%,and coincidence rate of BRS in ABR-NRs reached 80%.The blood pressure of ABR-DRs was significantly higher than ABR-NRs?approximately 10 mm Hg?despite strict control of parental blood pressure during screening.More importantly,the incidence of hypertension in ABR-DRs was also significantly higher.The incidence of hypertension in ABR-NRs was 0?n=15?,while the incidence of hypertension in ABR-DRs was 62.5%?n=15?.2.Metabolic syndrome emerges in ABR-DRsThe body weight,random blood glucose,fasting blood glucose,glycosylated serum protein,blood lipid and liver and kidney function metabolic indexes were measured at the 29th generation.The results showed that,compared with ABR-NRs,The levels of fasting blood glucose,glycosylated serum protein,total cholesterol,triglyceride,low density lipoprotein and serum albumin were significantly increased in ABR-DRs,and the blood glucose levels of ABR-DRs were significantly increased at 0,15,30,60 and 120min,and the area under the curve of blood glucose was also significantly increased in glucose tolerance test.The abnormalities of these indicators coincide with the clinical metabolic syndrome.3.Cardiac function and aconitine induced arrhythmia are not different etween ABR-NRs and ABR-DRs.We measured cardiac function by echocardiography in both ABR-NRs and ABR-LRs at 8-month of age.Compared to ABR-NRs,except for the thickness of left ventricle wall,all indexes of cardiac function were similar in ABR-DRs.Except for the indexes of aorta,morphological examination failed to show any differences in the cardiac indexes between ABR-NRs and ABR-DRs.We used aconitine to induce arrhythmia in all animals of the same age by the reported method.The threshold doses of aconitine required for ventricular premature beat?VPB?,ventricular fibrillation?VF?and cardiac arrest?CA?were used to evaluate the sensitivity to develop cardiac arrhythmia?n=6?.Doses required to induce VPB?34.9±4.8?g/kg vs 37.3±6.9?g/kg in ABR-NRs,P>0.05?,VF?60.5±11.8?g/kg vs 61.5±5.8?g/kg in ABR-NRs,P>0.05?or CA?93.2±12.5?g/kg vs 90.9±24.8?g/kg in ABR-NRs,P>0.05?were similar in the two groups.Thus it appears that impaired BRS may be not a factor for the occurrence of cardiac dysfunction and arrhythmia.4.Involvement of AMPA receptor in the dysfunction in NA of ABR-DRsTo further investigate the effect of AMPA receptor on the regulation of BRS,we injected glutamate or miscible liquids of CNQX and glutamate in NA to activate and block the AMPA receptor in vivo,respectively?n=6 both in ABR-DRs and ABR-NRs?.The NA was chemically identified by L-glutamate?1 nM,50 nL?microinjection.At the end of each experiment,100 nL of 2%pontamine sky blue solution was injected into the NA to mark the site.In ABR-NRs,glutamate microinjection?1 nM,50 nL?significantly increased the BRS values by about 51%?1.38±0.39 ms/mm Hg vs 0.92±0.17 ms/mm Hg before adding glutamate,n=6,P<0.01?.In ABR-DRs,glutamate increased the BRS values by about 29%?0.30±0.13 ms/mm Hg vs 0.23±0.10 ms/mm Hg before adding glutamate,n=6,P<0.05,?.Microinjection with miscible liquids of CNQX and glutamate?0.5 nM,50 nL?in NA significantly decreased BRS in ABR-NRs?0.74±0.11 ms/mm Hg vs 1.04±0.22 ms/mm Hg before adding CNQX,n=6,P<0.01?.Although CNQX decreased the BRS in ABR-DRs?0.30±0.11 ms/mm Hg vs 0.39±0.10 ms/mm Hg before adding CNQX,n=6?,the reduction was not so significant?P=0.08?.All these data indicated that AMPA receptor involved in the dysfunction in the NA and the deficiency of BRS of ABR-DRs.5.Glu2R deficiency was detected in NA of ABR-DRsAMPA receptors are widely distributed in the cerebral cortex,the limbic system and the thalamus.AMPA receptors are coupled with calcium channels to form receptor complexes that mediate fast excitatory signaling and are the main receptors in the brain that cause excitatory postsynaptic potentials.It's formed by 4 subunits?GluR1-4?assemble as homo-or heterotetramers.GluR2 submit controls AMPA receptor assembly.It is the key determinant of AMPA receptor function.Without GluR2,AMPA receptors are permeable to Ca²⁺ions and exhibit a high single-channel conductance.These changes in the expression Ca²⁺-permeable AMPARs can alter synaptic properties,Ca²⁺-dependent signaling cascades,or lead to damage of selectively vulnerable neurons and glial cells.So in order to investigate the molecular dysfunction of NA,we mainly detected the expression of GluR2 by immunocytochemistry method using a confocal laser scanning microscope.The amount and average optical density?AOD?of GluR2 in NA were quantified and analyzed with ImageJ software.To identify the NA region,we injected fluorescent microspheres?2%,1?l;catalog no.T8864?into the nodose ganglion to retrograde label motoneurons in the NA.The NA region was identified by green fluorescence.GluR2 mainly expressed in the NA area.Co-expression data showed that GluR2 was located in the cytoplasm of neurons of NA.Compared with ABR-NRs,the amount and AOD of GluR2 were significantly decreased in NA of ABR-DRs?Amount,12%±1.6%in ABR-DRs vs 22%±1.1%in ABR-NRs;AOD,9.5±0.2 in ABR-DRs vs13.4±0.6 in ABR-NRs,P<0.01,n=5?.All these data indicated the deficiency of GluR2in ABR-DRs.6.Transfection of GluR2 in NA improved the BRS values and decreased blood ressure and the indexes related to metabolic syndrome in ABR-DRsWe prepared lentiviral vector LV5 encoding GluR2?LV5-GluR2?and its normal control encoding green fluorescent protein?LV5-GFP?.The transfection efficiency was confirmed by PC12 cells.The transfection efficiency was maintained over 80%as determined by fluorescence microscope,with no detectable cellular toxicity.Transfection with LV5-GluR2 significantly enhanced the expression of GluR2?215±12 vs 121±11in LV5-GFP group,n=3,P<0.01?.Then LV5-GluR2 and its control?LV5-GFP?were stereotaxically injected into the two sides of NA?0.5×10⁵ TU/site,0.5?l,n=5 in each group?in ABR-DRs.The sham control ABR-DRs only performed sham injection operation?n=5?.Three weeks after injection,the expression of GluR2 in NA was detected by immunocytochemistry method using a confocal laser scanning microscope?Fluoview FV1000?.The amount of GluR2 was quantified with ImageJ software?NIH?.Local injection of LV5-GluR2 led to approximately 1-fold?overexpression?change in GluR2 positive cells.GluR2 overexpression in NA significantly improved BRS values of ABR-DRs?0.59±0.20 vs 0.19±0.06 ms/mm Hg in LV5-GFP groups,P<0.01?.The SBP and DBP were significantly decreased by 12 mm Hg and 8 mm Hg,respectively.LV5-GluR2,LV5-GFP injection and their sham operation were performed in another 18 ABR-DRs?4 months old,n=6 in each group?.Four months after injection,LV5-GFP did not change the body weight,lipid and glucose levels in ABR-DRs.Compared with LV5-GFP group,body weight was significantly decreased by 51 g in LV5-GluR2 group.The serum levels of cholesterol,triglyceride,low-density lipoprotein,glucose and leptin were also significantly decreased in LV5-GluR2 animals?P<0.05?.Other indexes,such as alanine transaminase,urea nitrogen and uric acid were also significantly decreased?P<0.05?.Thus it appears that GluR2 overexpression in NA may be an important factor for the regulation of lipid and carbohydrate metabolism.The overexpression of GluR2 might also be involved in the function of liver and kidney.Conclusion1.ABR-DRs mainly showed metabolic syndrome,overweight,high blood pressure,high blood sugar,high blood lipids and other cardiovascular risk factors.But ABR-DRs showed nonsusceptibility of arrhythmia and normal cardiac function.2.ABR-DRs showed AMPA receptor dysfunction and Its subtype GluR2 eficiency may be a critical molecular mechanism of BRS dysfunction.3.Transfection of GluR2 in NA improved the BRS values and decreased blood ressure and the indexes related to metabolic syndrome in ABR-DRs.