| Background Heart failure(HF)is a kind of disease that seriously impairs the contraction of myocardium,which causes a decrease in the pumping function of the heart as well as low cardiac output,the condition can significantly affect quality of life or even be life-threatening.The disease is characterized by autonomic imbalance with upregulation of sympathetic outflow and withdrawal of parasympathetic activity.Blunted baroreflex is thought to promote the autonomic imbalance and increase ventricular arrhythmia and sudden cardiac death in HF patients and experimental animal HF models.Arterial baroreflex plays an important role in cardiovascular activity by a homeostasic mechanism that alters heart rate in response to changes of blood pressure tension detected by mechanosensory nerve terminals in arterial wall in the carotid sinus and aortic arch.It has been reported that increasing aldosterone in the blood of normal dogs or humans significantly reduces the arterial baroreflex,while HF significantly increases aldosterone in patients.Another studies have also pointed out that HF causes a decrease in Na+ channel function and thus inhibits the electrical activity of arterial baroreflex.Our recent studies have found that the expression of ENaC protein is significantly reduced in nodose ganglia(NG)from HF rat or by elevated rat plasma aldosterone.Given ENaC is regulated by aldosterone in the distal kidney tubules,therefore,it is speculated that HF increases aldosterone and reduces the arterial baroreflex by affecting the expression of ENaC,but the molecular mechanism of aldosterone reducing ENaC is still unclear.The MAPK/ERK pathway is a highly conserved module that communicates a signal from a receptor to the DNA of the cell and is involved in a wide range of cellular response,among which ERK plays a key role in MAPK cascade.Some studies have shown that ERK1/2 is involved in ENaC-mediated Na+ resorption in renal collecting duct cells,moreover,other studies have indicated that aldosterone can activate ERK1/2 signaling pathway.Objective To explore whether aldosterone reduces the expression and function of ENaC through the ERK1/2 signal pathway in decompression nerve,thereby reducing the sensitivity of arterial baroreflex in the HF rats.Methods 1.The rat model of heart failure was prepared by ligating the left anterior descending branch of the left coronary artery.On the day of the terminal experiment(6-8 weeks after the model was prepared),the required tissues were extracted from the mice.2.The rat model of aldosterone group was prepared by subcutaneously implanting osmotic pump to infuse aldosterone.On the day of the terminal experiment(3 weeks after the model was prepared),the required tissues were extracted from the mice.3.A glass microelectrode connected to a micropump(Picospritzer II)was used to microinject the virus into the NG of rats.4.Male SD rats were randomly divided into two groups,the first group:(1)sham operation group(sham,n = 6):(2)heart failure group(HF,n = 6),(3)heart failure plus AAV empty vector sh RNA(HF + control sh RNA,n = 6)(4)Heart failure plus AAV-mediated ERK1/2 sh RNA group(HF + ERK1/2 sh RNA,n = 6).The second largest group:(1)solvent group(control,n = 6)(2)aldosterone group(Ald,n = 6)(3)aldosterone plus AAV-mediated empty vector sh RNA group(Ald + control sh RNA,n = 6)(4)aldosterone plus AAV Mediated ERK1/2 sh RNA group(Ald + ERK1/2 sh RNA,n = 6).5.Small animal ultrasound was used to detect the physiological changes in the heart of rats with heart failure after 6-8 weeks.6.TTC staining was used to detect the histological changes in the heart of rats with heart failure after 6-8 weeks.7.Measurement of plasma aldosterone concentration in each group of rats: aldosterone kit was used to determine the concentration of aldosterone in fresh arterial blood plasma of rats.8.Measurement of blood pressure and heart rate: The blood pressure and heart rate of rats in each group were measured by femoral artery cannulation.9.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).10.The expression of ENaC-?,ENaC-? and ERK1/2 in nodose ganglion(NG)was detected by Western blot technique.11.Statistical analysis.The experimental data were expressed as Mean ± SD,one-way analysis of variance or two-factor analysis of variance and t test were performed,P < 0.05 in each set of data comparisons was considered statistically significant.All analysis was performed using Graph Pad Prism5.0 statistical software.Results 1.Cardiology tests showed that the left ventricular function of the HF group was lower than that of the sham group,and the infarct area of the left ventricle reached more than 30% of the left ventricle.2.Twenty-one days after virus injection,Western Blotting was used to detect the protein expression of ERK2 and ERK1 in NG,and the relative grayscale values of the bands were calculated.The results showed that the expression of ERK2 and ERK1 in rat NG were down-regulated to 5% and 15% of the control group,respectively.3.After the HF model is successfully prepared.There was no significant difference in the weight of rats in each group.The ratio of rat heart/body weight is: sham group(0.280 ± 0.017),HF group(0.330 ± 0.027),HF + Control sh RNA group(0.334 ± 0.021),HF + ERK1/2 sh RNA group(0.324 ±0.029).The rats in the other three groups were significantly higher than those in the sham group(P <0.05).The thickness of the left ventricle posterior wall in each group was measured: sham group(0.311 ± 0.026 cm),HF group(0.392 ± 0.022 cm),HF + Control sh RNA group(0.402 ± 0.022 cm),HF + ERK1/2 sh RNA group(0.405 ± 0.027 cm).The rats in the other three groups were significantly higher than those in the sham group(P <0.001).4.After successful preparation of the HF model,the arterial baroreflex sensitivity(BRS)of the rats were: sham group(1.147 ± 0.21 ms/mm Hg),HF group(0.330 ± 0.11 ms/mm Hg),HF + Control sh RNA group(0.378 ± 0.08 ms/mm Hg),HF + ERK1/2 sh RNA group(0.665 ± 0.08 ms/mm Hg).HF group,HF + Control sh RNA group and HF + ERK1/2 sh RNA group had significantly lower BRS than sham group(P <0.0001),Reducing the expression of ERK1/2 in NG of HF rats can attenuate the effect of HF on BRS and increase it significantly(P <0.001),but it cannot restore it to normal levels.5.After the HF model was successfully prepared,the expression of ENaC-? and ENaC-? protein in each group of rats was detected by Western Blotting,and the relative values of band grayscale were calculated.The results were: sham group were 1.000 ± 0.259 and 1.000 ± 0.031,HF group were: 0.187 ± 0.083 and 0.061 ± 0.004,HF + Control sh RNA group were 0.152 ± 0.029 and 0.067 ± 0.043,HF + ERK1/2 sh RNA group were 0.428 ± 0.139 and 0.634 ± 0.191,the other three groups were better than sham Both groups were significantly reduced(P <0.05),and the expression of ENaC protein in the HF + ERK1/2 sh RNA group was higher than that in the HF group(P <0.05).6.21 days after the preparation of the rat model in the aldosterone group,the concentration of aldosterone in the rat plasma was detected.The results showed that: Control group(246.6 ± 24.49 pg/ml),Ald group(550.0 ± 76.00 pg/m),Ald + Control sh RNA group(540.3 ± 72.97 pg/m),Ald + ERK1/2 sh RNA group(533.4 ± 81.42 pg/m).The plasma aldosterone concentration in the Ald group and Ald treatment group was more than twice that in the solvent group.7.After the model of aldosterone group was successfully prepared,the protein expression of ERK2 and ERK1 in each group of rats was detected,and the gray value of the band was calculated.The results showed that: Control group was1.000 ± 0.194 and 1.000 ± 0.289,Ald group was 0.758 ± 0.046 and 0.888 ± 0.191,Ald + Control sh RNA group were 0.856 ± 0.302 and 0.731 ± 0.135,Ald + ERK1/2 sh RNA group were 0.227 ± 0.064 and 0.219 ± 0.086,compared with the other three groups Ald + ERK1/2 sh RNA,the ERK1/2 protein in the group was significantly down-regulated.8.After the rat model of aldosterone was successfully prepared,there was no significant difference in the body weight,heart/body weight ratio,and left ventricular posterior wall thickness.9.After the rat model of aldosterone group was successfully prepared,the BRS of each group of rats was: Control group(1.114 ± 0.22 ms/mm Hg),Ald group(0.332 ± 0.09 ms/mm Hg),Ald + Control sh RNA group(0.326 ± 0.09 ms /mm Hg),the Ald + ERK1/2 sh RNA group(0.619 ± 0.09 ms/mm Hg),the BRS of the other three groups was significantly lower than the Control group(P <0.001),the BRS of the Ald + ERK1/2 sh RNA group was compared with the Ald group Significantly increased(P <0.001).10.The aldosterone model was successfully prepared.Western Blotting was used to detect the expression of ENaC-? and ENaC-? protein,and the relative value of the band grayscale was calculated.The results were: ENaC-? and ENaC-? in NG of the Control group were 1.000 ± 0.138 and 1.000 ± 0.190,Ald group were 0.196 ± 0.028 and 0.073 ± 0.021,Ald + Control sh RNA group were 0.211 ± 0.076 and 0.082 ± 0.021,Ald + ERK1/2 sh RNA group were 0.418 ± 0.065 and 0.635 ± 0.195,the other three groups The Control group was significantly reduced(P <0.05),and the expression of ENaC protein was increased in the Ald + ERK1/2 sh RNA group compared with the Ald group(P <0.05).Conclusion 1 ? Ligating the left anterior descending coronary artery causes myocardial hypertrophy and decreased cardiac function,and inhibiting the expression of ERK1/2 in NG cannot improve the pathological changes of myocardium;2?HF and elevated rat aldosterone activate ERK1/2 in NG,reduce the expression of ENaC-? and ENaC-? protein in NG,and weaken the sensitivity of decompression reflex;3?AAV-mediated sh RNA inhibits ERK1/2 expression in NG,partially restores NG ENaC-? and ENaC-? protein expression,and improves decompression reflex;These results indicate Heart failure rats raise aldosterone levels and reduce the expression of ENaC protein by activating ERK1/2 in NG,thereby weakening BRS. |
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