Over-expression of SK2 channel in PVN reduces renal sympathetic nerve activity in chronic heart failure rats

AIM：To investigate the effect of over-expression of small-conductance calcium-activated potassium channel subtype 2 (SK2) in the paraventricular nucleus (PVN) of hypothalamus on heart rate(HR), mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) of the rats with chronic heart failure (CHF). METHODS：Adenovirus vector encoding SK2 (pDC316-mCMV-EGFP-rKCNN2) was constructed.The CHF model of the male Sprague Dawley (SD) rats was established by the ligation of anterior descending coronary artery. Echocardiogram was used at the 6th week after the operation to identify the CHF model. Adenovirus vector encoding SK2 (pDC316-mCMV-EGFP-rKCNN2) or control adenovirus vector (pDC316-mCMV-EGFP) were transfected into the PVN in vivo. The cardiac function was monitored by echocardiogram. The expression of SK2 at mRNA and protein levels was determined by RT-PCR, Western blotting and immunofluorescence. The HR, MAP and RSNA were measured by PowerLab 8/30 in anesthetized rats with bilateral PVN microinjection of SK channel blocker apamin. RESULTS：Treatment with pDC316-mCMV-EGFP-rKCNN2 significantly decreased the renal sympathetic nerve activity in the rat with CHF. Injection of pDC316-mCMV-EGFP did not change the renal sympathetic nerve activity in the rats in sham group. CONCLUSION：The expression and function of SK channels in PVN in the heart failure rats were decreased, suggesting a reduced negative regulation of sympathetic activity in central neural system. Increased expression of SK2 in the PVN leads to a reduction in sympathetic outflow under the condition of CHF, which may provide a new target for the treatment of heart failure.     

Activation of corticotrophin releasing hormone-containing neurons in hypothalamic paraventricular nucleus contributes to sympathoexcitation in rats with congestive heart failure

AIM:To observe the expression of corticotropin releasing hormone (CRH) within the paraventricular nucleus of hypothalamus (PVN) and to explore the relationship between the activated CRH-containing neurons and sympathetic activity in rats with heart failure (HF).METHODS:Healthy male Sprague-Dawley (SD) rats were subjected to coronary artery ligation to induce HF,and chronic intracerebroventricular (ICV) infusion was performed by osmotic pump for 4 weeks.The rats in sham group and HF group were given vehicle (VEH;artificial cerebrospinal fluid 0.25 μL/h).The rats in HF plus treatment group were treated with CRH competitive inhibitor αh-CRH (15 mg/h).Meanwhile,the Lewis rats and Fischer 344 rats for control study also underwent coronary ligation to induce HF or sham surgery.After 4 weeks,left ventricular end-diastolic pressure (LVEDP) and maximum positive/negative change in pressure over time (±dp/dt_max) were determined.The right ventricular-to-body weight (RV/BW) and lung-to-body weight (lung/BW) ratios were calculated.The renal sympathetic nerve activity (RSNA) was recorded and the plasma norepinephrine (NE) level was measured.The expression of CRH in the PVN combined with the plasma adrenocorticotrophic hormone (ACTH) levels were measured.RESULTS:Compared with the sham-SD rats,the HF-SD rats had a greater number of CRH positive neurons in the PVN (accordingly the plasma ACTH levels were increased),accompanied by decreased±dp/dt_max and increased RSNA,plasma NE,LVEDP,lung/BW and RV/BW.However,ICV treatment with αh-CRH attenuated these changes in the HF-SD rats (P<0.05).Compared with the sham-Fisher 344 rats,the HF-Fisher 344 rats also had a greater number of CRH positive neurons in the PVN (accordingly the plasma ACTH levels were increased).In addition,they had significantly increased RSNA and plasma NE level,higher LVEDP,RV/BW and lung/BW,and lower±dp/dt_max(P<0.05).Compared with the SHAM-Lewis rats,the HF-Lewis rats had not significantly changed in the above parameters.CONCLUSION:In CHF,the CRH-containing neurons in PVN are activated,thus aggravating cardiac function by increasing sympathoexcitation.     

Upregulated FAAH expression in PVN contributes to sympathoexcitation in rat with chronic heart failure

AIM: To investigate the expression of fatty-acid amide hydrolase (FAAH) in paraventricular nucleus (PVN) and its contribution to renal sympathetic nerve activity in rats with chronic heart failure (CHF). METHODS: A rat model of CHF was established by ligation of the left coronary artery to induce acute myocardial infarction. Eight weeks after ischemia, the rat model of CHF was identified by echocardiogram and histopathological observation. The plasma level of norepinephrine (NE) was detected by ELISA. The protein expression levels of FAAH in the PVN were determined by Western blot. The N-arachidonoylethanolamide (AEA) generation in PVN was analyzed by high-performance liquid chromatography. After microinjection of AEA, PF3845 (an FAAH inhibitor) or rAAV2-FAAH shRNA virus in PVN, the sympathetic drive indexes were recorded in different experiment groups. RESULTS: Compared with the rats in sham group, the cardiac function and AEA concentration in PVN were significantly reduced, while the plasma NE level and FAAH expression in PVN were obviously increased in the CHF rats (P<0.05). After microinjecion of PF3845, AEA or rAAV2-FAAH shRNA virus in PVN, the sympathetic drive indexes were decreased significantly and the cardiac function were improved in the CHF rats. CONCLUSION: Upregulated FAAH expression in PVN may result in sympathoexcitation in the rat with CHF.     

Effect of arachidonylethanolamide in paraventricular nucleus on cardiac function and sympathetic nerve activity in rats with heart failure

AIM:To determine the roles of the arachidonylethanolamide (AEA) in the paraventricular nucleus (PVN) in cardiac function and sympathetic activity in the rats with chronic heart failure (CHF). METHODS:Chronic heart failure was induced by left coronary ligation in Wistar rats and was confirmed using echocardiography. The rats with CHF and the sham-operated controls (sham group) were treated for 4 weeks with a continuous PVN infusion of AEA, cal-cium-calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) selective inhibitor KN-93, transient receptor potential vanilloid type 1 (TRPV1) channel blocker capsazepine (CPZ), intracellular calcium chelator BAPTA-AM, small-conductance calcium-activated potassium channel (SK channel) blocker apamin and artificial cerebrospinal fluid (vehicle). Sympathetic drive indexes and cardiac function were detected. NG108 cells were incubated with AEA, and then the intracellular cal-cium concentration was measured by fluorometry. The protein expression levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 were determined by Western blot. RESULTS:Compared with sham group, the left ventricular end-diastolic pressure (LVEDP) increased significantly, while peak rate of rise/decline of left ventricular pressure (±dp/dt_max) and ejection fraction (EF) decreased significantly in the CHF group. The concentrations of AEA and intracellular calcium, and the protein levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 in PVN were significantly lower in CHF rats. Compared with the vehicle group, the mortality and sympathetic drive were decreased significantly and cardiac function was improved after treatment with AEA in CHF group. However, PVN perfusion of KN-93, CPZ, BAPTA-AM or apamin contributed to the sympathetic drive and deteriorated the cardiac function. AEA dose-dependently increased intracellular calcium ion concentration, and the protein levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 in NG108 cells. CONCLUSION:AEA in the PVN may be involved in the improvement of cardiac function and sympathetic overdrive via CaMKⅡ/TRPV1/Ca²⁺/SK₂ pathway in rats with CHF.     

Microinjection of AT1 and AT2 receptor antagonists into PVN affects renal sympathetic nerve activity in chronic heart failure rats

AIM: To examine the renal sympathoexcitation affected by microinjection of angiotensin Ⅱ type 1 (AT₁) receptor antagonist L-158809 and angiotensin Ⅱ type 2 (AT₂) receptor antagonist PD123319 into paraventricular nucleus (PVN) in heart failure rats.METHODS: Left anterior descending coronary artery ligation was used to induce rat heart failure (HF) . Four weeks after operation, the left ventricular end-diastolic pressure (LVEDP), the ratios of heart weight/body weight and lung weight/body weight, and the ratio of infarct area of the left ventricle were observed. Under anesthesia, SD rats were fixed into the brain stereo controller to locate PVN for microinjection and the artificial cerebrospinal fluid (ACSF) was used for control. The left kidney was exposed by retroperitoneal approach and the renal sympathetic nerve was separated under surgical microscope. The heart rate, blood pressure and the activity of renal sympathetic nerve discharge (RSNA) were recorded by POWERLAB 8/30 system. RESULTS: Microinjection of AT₁ receptor antagonist into PVN induced a decrease in RSNA in both HF rats and sham rats. The RSNA responses to L-158809 in the HF rats were significantly greater (P<0.05) than those in the sham rats. However, microinjection of AT₂ receptor antagonist and ACSF into PVN induced no change of RSNA in both HF and sham rats. CONCLUSION: There are some differences of sympathetic nerve outputs between using AT₁ receptor antagonist and AT₂ receptor antagonist on PVN, indicating the up-regulation of AT₁ receptors in PVN during HF. The central renin-angiotensin-aldosterone system(RAAS) may be affected by AT₁ receptor, not by AT₂ receptor.     

Increased central reactive oxygen species mediate the attenuated baroreflex function in heart failure

AIM: To determine the effect of reactive oxygen species on the baroreflex and to investigate the intracellular mechanism responsible for baroreflex dysfunction in the heart failure state.METHODS: In the rat model of cardiomyocytes infarct induced heart failure, baroreflex function was evaluated by measuring the relationship between renal sympathetic nerve activity(RSNA)responses and change of blood pressure by intravenous injection of nitroglycerin and phenylephrine. Alteration in baroreflex function was measured under the different reactive oxygen species(ROS)level induced by intracerebroventricular administration of several chemicals. RESULTS: (1)The range of RSNA response, average slope and maximum gain of baroreflex function curve were(92.2±9.9) mmHg,(0.07%±0.01%)/mmHg and(1.20%±0.10%)/mmHg, respectively, in CHF rats, which were significantly lower than those in sham rats(65.6±7.4) mmHg,(0.13%±0.02%)/mmHg and(3.00%± 0.20%)/mmHg(P<0.01).The minimum RSNA of baroreflex curve was higher in CHF rats than that in sham rat［(21.6%±4.8%)vs(7.5%±2.1%), P<0.01］.(2)Intracerebroventricular(icv)infusion of superoxide scavenger tempol and NADPH oxidase inhibitor apocynin significantly improved the blunted baroreflex in CHF rats. On contrast, icv administration of superoxide dismutase inhibitor diethyldithiocarbamate(DETC)decreased baroreflex function in sham rats.(3)The superoxide production in the hypothalamus of CHF rats was higher than that in sham rats［(73.9±9.8)RLU·5min-1·mg-1vs(40.6±7.1)RLU·5min-1·mg-1, P<0.01］.(4)Protein expression of NADPH oxidase subunits gp91phox in the paraventricular nucleus of the hypothalamus were increased by 1.3 fold in CHF rats than that in sham rats. CONCLUSION: Elevated intracellular ROS in the hypothalamus plays an important role in the attenuation of baroreflex function in the heart failure state and results from upregulation of NADPH oxidase protein expression.     

Effects of cardiac contractility modulation on ventricular electrical remodeling in rabbits with chronic heart failure

AIM: To investigate the effects of cardiac contractility modulation (CCM) on ventricular electrical remodeling in a rabbit model of chronic heart failure. METHODS: The rabbits were divided into sham group, heart failure(HF) group and HF+ CCM group. The rabbit model of chronic heart failure was established by ligating the ascending aortic root. Then electrical stimulations during the absolute refractory period were delivered lasting 6 h everyday for 4 weeks in rabbits of HF+ CCM group. The QTc and ventricular effective refrective period (VERP) were recorded. The protein and mRNA levels of Kv1.4, Kv4.3 and connexin 43 (Cx43) were determined by Western blot and RT-qPCR. RESULTS: Compared with sham group, QTc were significantly prolonged in HF rabbits at week 12 (P<0.05). CCM therapy shortened QTc of rabbits with heart failure at week 16 (P<0.05). Compared with sham group, VERP significantly increased in HF group and HF+ CCM group, while CCM therapy shortened VERP of rabbits with heart failure at week 16 (P<0.05). Compared with sham group, the mRNA and protein levels of Kv1.4, Kv4.3 and Cx43 were decreased in HF group and HF+ CCM group (P<0.05). However, CCM therapy restored the mRNA and protein levels of Kv1.4, Kv4.3 and Cx43 of rabbits with heart failure (P<0.05). CONCLUSION: CCM suppresses ventricular electrical remodeling in heart failure and the underlying mechanism may be associated with increasing Kv1.4, Kv4.3 and Cx43 expression.     

Roles of the Kv1.2, Kv1.5, Kv2.1 potassium channels in hypoxia pulmonary vasoconstriction

AIM: To determine the role of Kv1.2, Kv1.5, Kv2.1 in the hypoxia pulmonary vasoconstriction (HPV). METHODS: Male Wistar rats were divided into two groups: normoxic group and hypoxic group. The single smooth muscle cell was obtained from pulmonary artery of Wistar rats with acute enzymatic digestion method. The conventional whole-cell patch clamp technique was used to record the resting membrane potential (Em) and the potassium currents of voltage-gated potassium channel (IKv) in rat pulmonary arterial smooth muscle cells (PASMC). Intracellular application of Kv1.2/Kv1.5/Kv2.1 antibodies (1∶125) was conducted through the whole-cell patch clamp system. RESULTS: ① Em of PASMC was depolarized after 24 h hypoxia compared with that of control cells . IKv of PASMC was decreased after 24 h hypoxia, . ② The mixture of Kv1.2/Kv1.5/Kv2.1 antibodies depolarized Em and inhibited IKv in PASMC from normoxic rat, whereas the mixture of Kir2.1/Kir2.3/Kir4.1 antibodies had no effects on them. ③ The mixture of Kv1.2/Kv1.5/Kv2.1 antibodies and the mixture of Kir2.1/Kir2.3/Kir4.1 antibodies had no effects on IKv and Em from rats hypoxic for 24 h. CONCLUSION: Kv1.2, Kv1.5, Kv2.1 might be oxygen sensitive potassium channels which mediated HPV.     

Effect of chronic cigarette smoking on BKCa and Kv1.5 expression in rat pulmonary arterial smooth muscle cells

AIM:To investigate the role of potassium channel expression alteration in chronic cigarette smoking-induced increase in pulmonary vascular responsiveness, the effect of chronic cigarette smoking on large-conductance calcium-activated potassium channel (BK_Ca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) expression in rat pulmonary smooth muscle cells were investigated in vivo. METHODS:HE staining, immuno-histochemistry and in situ hybridization techniques were used. RESULTS: (1) Chronic cigarette smoking downregulates the protein and mRNA expression of BK_Ca in pulmonary arterial smooth muscles. (2) Chronic cigarette smoking downregulated the protein and mRNA expression of Kv1.5 in pulmonary arterial smooth muscles. (3) In big artery, BK_Ca decreased more makedly than Kv1.5, but in small artery, both of them decreased equally. CONCLUSION:Chronic cigarette smoking downregulates the levels of BK_Ca and Kv1.5 in rat pulmonary arterial smooth muscle cells in vivo, which maybe contribute to the mechanism of cigarette smoking-induced increase in pulmonary vascular responsiveness.     

Role of central PGE2 on sympathetic excitation in chronic heart failure

AIM: To observe the effect of central prostaglandin E₂ (PGE₂) on sympathetic activation in chronic heart failure (CHF) and to explore the underlying mechanism. METHODS: Male SD rats were subjected to coronary artery ligation to induce heart failure (HF), and the intracerebroventricular infusion was performed by osmotic pump continuously. The rats in sham group and HF group were given artificial cerebrospinal fluid (0.25 μL/h). The rats in HF plus treatment group was given celecoxib (CLB; 20 mg/h). After 4 weeks, the levels of PGE₂ in cerebrospinal fluid (CSF), the sympathetic nerve excitability and cardiac function were measured, and the changes of corticotropin-hormone releasing hormone (CRH)-containing neurons activation and neurotransmitter contents in the hypothalamic paraventricular nucleus (PVN) were also determined. RESULTS: Compared with the sham-operated rats, the HF rats had raised level of PGE₂ in CSF, up-regulated renal sympathetic nerve activity and plasma norepinephrine, increased left ventricular end diastolic pressure, lung-to-body weight and right ventricular-to-body weight ratios, and decreased maximal increase and decreased rate of left ventricular pressure (P<0.05). In addition, the number of CRH positive neurons in PVN and the level of plasma adrenocorticotropic hormone were higher in HF rats than those in sham-operated rats (P<0.05). After administration of CLB into the lateral ventricle of HF rats, the contents of PGE₂ in CSF were significantly reduced, the number of activation CRH neurons in PVN was decreased, the excitability of sympathetic nerves was down-regulated and cardiac function was improved (P<0.05). Compared with the sham-operated rats, the content of glutamic acid in PVN of HF rats was increased, the content of γ-aminobutyric acid and the number of glutamate decarboxylase 67-positive neurons were decreased (P<0.05). After the CLB was given, the above indexes were reversed (P<0.05). CONCLUSION: These findings indicate that in CHF, the increased central PGE₂ may activate CRH-containing PVN neurons and contribute to the augmented sympathetic drive possibly by modulating the neurotransmitters within the PVN.     

Role of K+ channels in hypoxic pulmonary vasoconstriction in normal and chronic hypoxic rats

AIM:To investigate the role of K⁺ channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS:Blockers of three kinds of K⁺ channels, 4-AP(voltage dependent K⁺ channel blocker), TEA(Ca²⁺ activated K⁺ channel blocker), GLIB(ATP sensitive K⁺ channel blocker) were used in isolated perfused rat lungs to detect the role of K⁺ channels in HPV. RESULTS:In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION:The opening of Kv, K_Ca, K_ATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.     

Effects of exercise stress on cigarette smoking-induced downregulation of BKCa and Kv1.5 expression in rat bronchial smooth muscle cells

AIM: To investigate the effect of exercise stress on chronic cigarette smoking-induced downregulation of expression of large-conductance calcium-activated potassium channel (BK_Ca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) in rat bronchial smooth muscle cells. METHODS: Rats were divided into 3 groups: normal control, smoking control and smoking plus exercise training group. The alteration of airway responsiveness and plasma cortisol level were detected, and potassium channel expression and pathological changes in lung tissue were determined with HE staining, immunohistochemistry, in situ hybridization and Western blot techniques. RESULTS: (1) Cigarette smoking induced an increase in airway responsiveness, smoking plus exercise lead to a decrease in airway responsiveness in contrast to smoking control group; (2) Plasma level of cortisol determined immediately after exercise was higher than that determined before exercise; (3) HE staining showed that there was severe chronic pulmonary inflammatory response in smoking control group, which was slight in the smoking plus exercise group; (4) The protein and mRNA expression of BK_Ca in cigarette smoking group were less than that in control group in BSMC, the mRNA expression of BK_Ca in exercise group were higher than that in smoking group; (5) The protein and mRNA expression of Kv1.5 in smoking group were less than that in control group in BSMC, and expression of Kv1.5 in exercise group was higher than that in smoking group in bronchioli. CONCLUSION: Proper exercise training can increase the expression of potassium channel BK_Ca and Kv1.5, and increase the cortisol secretion, which may contribute to the decreasing of airway hyperresponsiveness induced by cigarette smoking.     

Urinary concentration of aquaporin-2 water channel protein at different stages of chronic heart failure rats

AIM: To study urinary concentration of aquaporin-2 water channel protein(AQP2) at different stages of chronic heart failure(CHF) rats and its relation to hyponatremia. METHODS: Male Sprague-Dawley rats(200~250 g) underwent either a left coronary artery ligation (a model of CHF) or a sham-operation. At different stage after surgery, urinary AQP2 concentration was measured by Western blot. 24-hours urine volume, serum sodium and urine osmolality were measured at the same time. RESULTS: There were two peaks of urinary excretion of AQP2 in severe heart failure rats model: one was the third day after operation, the other was the 9th week. Serum sodium and urine osmolality were significantly different in CHF rats as compared with sham-operated rats. Seven weeks after surgery, urinary AQP2 concentration was increased significantly insevere CHF rats compared with the mild ones and the control ones(365%±103% vs 179%±81% and 99%±48%, P＜0.01).CONCLUSION: The variation of urinary AQP2 excretion at different stages after ligation showed that the expression of AQP2 gene was increased obviously in CHF rats, and its expression level was higher as the heart failure became more severe.This is the important reason of heart failure with hyponatremia.