| BackgroundThe arterial baroreflex plays an important role in cardiovascular activity.The sensitivity of the baroreflex is attenuated in heart failure(HF)patients and HF animal models,which disturbs autonomic balance,with an increased sympathetic tone and decreased parasympathetic activity,resulting in increased morbidity and mortality.Studying the mechanism of HF in reducing baroreflex has important clinical significance for preventing sudden death caused by HF.It has been reported that reduced baroreflex afferent nerve sensitivity blunted the baroreflex sensitivity in HF.However,the mechanisms responsible for the reduced sensitivity of the afferent nerve to blood pressure have not yet been elucidated.The epithelial sodium channel(ENa C),located in the aortic depressor nerve endings,senses vasodilation at the aortic arch,exciting depressor nerves,thereby transmitting information on changes in blood pressure.The level of ENa C is negatively regulated by the ubiquitin ligase Nedd4-2.Previous studies have found that increasing aldosterone levels in the body can reduce the expression of ENa C channels in depressor nerves and reduce the sensitivity of baroreflex.Both HF patients and HF animal models showed elevated aldosterone levels and decreasedbaroreflex function.ObjectiveIn this experiment,a rat heart failure model was made by ligating the left anterior descending coronary artery to observe changes in 1)aldosterone levels and baroreflex sensitivity;2)ENa C and p-ERK1/2 expression in the nodose ganglion;and 3)interaction between Nedd4-2 and ENa C,to investigate the mechanism by which heart failure reduces ENa C.Methods1.A rat model of heart failure was made by ligating the left anterior descending coronary artery.The terminal experiments were performed on the day of 6-8 weeks after ligation of the artery.2.Healthy male SD rats were randomly divided into 4 groups,(1)normal control group(Ctrl,n=6)(2)sham group(sham,n=6)(3)heart failure group(HF,n=6)(4)heart failure with spironolactone group(HF+SPI,n=6).3.Measurement of blood pressure and heart rate,after anesthesia,femoral artery catheterization measured blood pressure and heart rate.4.Measurement of arterial baroreflex sensitivity.Injection of sodium nitroprusside(SNP)and phenylephrine(PE)induces pressure rise reflex and fall reflex,respectively,and the ratio of the change in systolic pressure before and after the reflex to the value of the cardiac cycle(?SBP)/?CC)is used as an index of pressure rise reflex sensitivity(BRS-SNP)and blood pressure reflex sensitivity(BRS-PE).5.Measurement of plasma aldosterone concentration.Rat arterial blood was taken and the concentration of aldosterone in rat plasma was measured using a rat aldosterone assay kit.6.HE staining,Sirius red staining and Masson trichrome staining techniques were used to detect the histological changes in rat hearts.7.The expression of ENaC-?,ENaC-? and p-ERK1/2 in nodose ganglion(NG)was detected by immunohistochemical technique,and the binding of Nedd4-2 to ENa C was detected by Co-IP technique.8.Statistical analysis.The experimental data were expressed as mean ± SEM,using Graph Pad Prism5.0 statistical software,using one-way analysis of variance or two-factor analysis of variance and t test,P < 0.05 was considered statistically significant.Results1.Histopathological examination of rat heart showed that,after routine coronary artery ligation,the left ventricular anterior myocardial infarction area in rats was thinned,myocardial tissue was loose,myocardial nucleus disappeared,and a large number of inflammatory cells infiltrated.Sirius red and Masson trichrome staining further confirmed the fibrosis of the myocardial infarction site.2.After 8 weeks of model manufacture,the aldosterone concentration in rats was determined to be 362.4+26.01 pg/ml in the Ctrl group,358.8+29.17 pg/ml in the sham group,and 626.2+52.47 pg/ml in the HF group,in the HF+SPI group,600.7+47.88 pg/ml.Aldosterone concentrations in the HF group and the HF+SPI group were significantly higher than those in the Ctrl group(P < 0.005).3.After the model was successfully prepared,the rats' BRS was 1.095+0.025 ms/mm Hg in the Ctrl group,1.065+0.008 ms/mm Hg in the sham group,and 0.405+0.013 ms/mm Hg in the HF group.The HF+SPI group was 0.593+0.037 ms/mm Hg.The BRS in the HF group and the HF+SPI group was significantly lower than that in the Ctrl group and the sham group(P < 0.0001),and the aldosterone receptor blocker SPI attenuated the effect of HF on BRS,making it significantly higher(P<0.001).However,it cannot be restored to normal level.4.The protein expression of ENaC-? and ENaC-? was detected by Western Blotting.Calculated the grayscale relative value of each band.The results showed that the ENa C-? and ENa C-? proteins in the nodose ganglion of the Ctrl group were 1.207+0.217 and 0.6922+0.0363,respectively.The sham group was 1.329+0.165 and 0.7659+0.0352,there was no significant difference between the two groups(P > 0.05);HF group was 0.3309+0.121 and 0.2822+0.025,which was significantly lower than the Ctrl group(P < 0.05);while the HF+SPI group was 0.736+0.0424 and 0.3848+0.008 also showed reduced ENa C expression compared to the Ctrl group.It was shown that HF caused a decrease in the expression of ENa C.The expression of ENa C protein in HF+SPI group was higher than that in HF group(P < 0.05),indicating that SPI can improve the HF-induced changes in ENa C,but it cannot be restored to normal level.5.Co-IP detected the binding between Nedd4-2 and ?/?ENaC.The gray value result for each band showed that the protein was precipitated with Nedd4-2 antibody and detected with ?/?ENa C antibody.The Ctrl group was 338.5+75.67 and 341.2+53.95,respectively.The sham group was 343.2+58.28 and 489.1+61.26,respectively,there was no significant difference between the two groups(P > 0.05);HF group was 1038+162.9 and 919.6+43.9,respectively,significantly higher than the Ctrl group(P < 0.05).The HF+SPI group was 172.9+28.39 and 524.4+58.32,respectively,which were not significantly different from the Ctrl group(P > 0.05),but significantly lower than those of the HF group(P < 0.05).The ?/?ENa C antibody precipitated protein and Nedd4-2 antibody were used for western blot.The Ctrl group was 78.72+36.47 and 349.3+97.85,and the sham group was 223.8+122.5 and 495.9+82.81,respectively.There was no significant difference between the two groups(P > 0.05).HF group were 2000 +355.7 and 1701 +354.8,significantly increased than Ctrl group(P < 0.05);HF + SPI group were 182.6 +73 and 228.4 +89.52,and there was no significant difference from Ctrl group(P > 0.05),significantly lower than HF group(P < 0.05).It was shown that the binding of Nedd4-2 to ENa C was increased in the nodose ganglion of heart failure rats,and the aldosterone receptor blocker SPI reduced their binding.6.The protein expression of ENa C-? and ENa C-? was detected by Western Blotting.Calculated the grayscale relative value of each band.The results showed that the p-ERK1/2 protein expression in the nodose ganglion of the Ctrl group was 1.192+0.0926,1.087+0.302 in the sham group,and 5.210+0.581 in the HF group,which was significantly increased compared to the Ctrl group(P < 0.005),HF+SPI group was 1.021+0.365,which was not significantly different from Ctrl group(P >0.05)and significantly lower than that in HF group(P < 0.005),indicating that p-ERK1/2 protein expression in nodose ganglion of HF rats.The amount was significantly increased,suggesting that ERK1/2 activation is involved in the regulation of ENa C by aldosterone,and aldosterone blocker can block the activation of ERK1/2.ConclusionRats with heart failure increased aldosterone levels,activated the ERK1/2 of nodose ganglion through the receptor pathway,promoted the binding of Nedd4-2 to ENa C,decreased ENa C,and reduced the sensitivity of arterial depressor reflexes. |
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