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.     

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.     

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.     

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.     

Effects of sodium hydrosulfide on cardiac function and activity of renin-angiotensin-aldosterone system in rats with chronic heart failure

AIM: To investigate the effect of sodium hydrosulfide (NaHS) on cardiac function and activity of renin-angiotensin (Ang)-aldosterone (ALD) system (RAAS) in the rats with chronic heart failure (CHF).METHODS: The CHF rat model was established by abdominal aortic coarctation. SD rats were randomly divided into sham operation group, model group, low dose of NaHS group and high dose of NaHS group (n=6). The left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and left ventricular ejection fraction (LVEF) were measured before and after treatment by echocardiography in each group. The levels of renin, AngⅡ and ALD in the plasma were measured by ELISA. The expression of angiotensin-converting enzyme (ACE) and angiotensin Ⅱ type 1 receptor (AT1R) at mRNA and protein levels in the myocardium tissues was determined by qPCR and Western blot, respectively.RESULTS: After treatment with NaHS, compared with model group and before treatment, LVEDD and LVESD in low dose of NaHS group and high dose of NaHS group were decreased significantly, while LVEF was increased significantly (P<0.05). Compared with low dose of NaHS group, LVEDD and LVESD were decreased, while LVEF was increased in high dose of NaHS group (P<0.05). Compared with sham operation group, the levels of renin, AngⅡ and ALD in the plasma of model group were significantly increased (P<0.05), and the expression of ACE and AT1R at mRNA and protein levels in the myocardium tissues of model group were significantly increased (P<0.05). Compared with model group, the plasma levels of renin, AngⅡ and ALD in low dose of NaHS group and high dose of NaHS group were significantly decreased (P<0.05), and the myocardial expression of ACE and AT1R at mRNA and protein levels was significantly down-regulated (P<0.05). The plasma levels of renin, AngⅡ and ALD, and the myocardial expression of ACE and AT1R at mRNA and protein levels in high dose of NaHS group were significantly lower than those in low dose of NaHS group (P<0.05).CONCLUSION: NaHS inhibits the activation of RAAS, thus improving the cardiac function of CHF rats, and the effect of high-dose NaHS is better than that of low-dose NaHS.     

Inhibitory effect of aerobic exercise on left ventricular remodeling and sympathetic neural remodeling in rats with heart failure after myocardial infarction

AIM: To investigate the effects of long-term aerobic exercise on the heart and sympathetic neural remodeling (structure and function remodeling) in heart failure rats induced by myocardial infarction. METHODS: Heart failure model after myocardial infarction was performed by ligating anterior descending coronary artery in the Wistar rats. Four weeks after operation, the rats were randomly divided into sham operation sedentary (S) group, heart failure sedentary (H) group and heart failure exercise (HE) group. The animals in HE group underwent 10-week treadmill running, while those in S group and H group were sustained in a resting state. The cardiac structure and function including left ventricular internal diameter at diastole (LVIDd), left ventricular internal diameter at systole (LVIDs), left ventricular anterior wall diameter at diastole (LVAWDd), left ventricular anterior wall diameter at systole (LVAWDs), left ventricular posterior wall diameter at diastole (LVPWDd) and left ventricular posterior wall diameter at systole (LVPWDs), and cardiac function parameters including fractional shortening (FS) and left ventricular ejection fraction (LVEF) were measured by echocardiography. The myocardium was collected for histopathological observation with Masson staining, and the collagen volume fraction (CVF) was determined. The concentrations of norepinephrine (NE) in the myocardium and plasma were measured by high-pressure liquid chromatography. The frequency domain analysis was applied for determining the heart rate variability (HRV) via subcutaneous recording electrode involving total power (TP), normalized low power (LFn), normalized high power (HFn) and LF/HF ratio. The mRNA expression of collagen type I (Col-I), collagen type III (Col-III), atrial natriuretic factor (ANF), α-myosin heavy chain (α-MHC), β-myosin heavy chain (β-MHC), sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) was detected by real-time PCR. The protein levels of nerve growth factor (NGF) and its receptor (TrkA), and tyrosine hydroxylase (TH) were measured by Western blotting. RESULTS: (1) Compared with S group, body weight (BW), LVIDd, FS, LVEF, TP, HFn, the mRNA expression of α-MHC and SERCA2a, and the protein levels of NGF, TrkA and TH decreased (P<0.05). Left ventricular weight (LVW), left ventricular mass index (LVMI), LVAWDd, LVAWDs, LVPWDd, LVPWDs, CVF, plasma and myocardial NE content, LFn, LF/HF, and the mRNA expression of ANF, β-MHC, Col-I and Col-III increased (P<0.05) in H group. (2) Compared with H group, LVW, LVMI, LVIDd, FS, LVEF, TP, HFn, the mRNA expression of α-MHC and SERCA2a, and the protein levels of NGF, TrkA and TH were raised (P<0.05), while CVF, plasma and myocardial NE content, LFn, LF/HF, and the mRNA expression of ANF, β-MHC, Col-I and Col-III decreased (P<0.05) in HE group. CONCLUSION: Long-term aerobic exercise training leads to inhibition of heart and sympathetic neural remodeling and improvement of cardiac function and autonomic modulation in the rats after myocardial infarction.     

Xinshuaikang inhibits myocardial autophagy and MAPK/ERK1/2 signaling pathway in rats with chronic heart failure

AIM:To explore the effect of Xinshuaikang on myocardial autophagy in the rats with chronic heart failure and its relationship with the MAPK/ERK1/2 signaling pathway. METHODS:The rats were divided into sham group, model group (rat model of chronic heart failure was established by ligation of anterior descending branch of left coronary artery), low-, middle-, and high-dose Xinshuaikang treatment (TL, TM and TH) groups and captopril group (treated with captopril as positive control), with 12 in each group. Doppler echocardiography was used to evaluate the cardiac function. The morphological changes of the myocardium were observed by HE staining. TUNEL staining was used to detect cardiomyocyte apoptosis. The expression of microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) in the myocardium was detected by immunofluorescence labeling. The protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ, beclin-1 and p62 in the myocardium were determined by Western blot. RESULTS:Compared with sham group, left ventricular end-diastolic dia-meter (LVEDD) and left ventricular end-systolic diameter (LVESD) in model group were increased, while left ventricular posterior wall thickness at end-diastole (LVPWTd), left ventricular posterior wall thickness at end-systole (LVPWTs), left ventricular ejection fraction (LVEF), cardiac output (CO), left ventricular diastolic pressure (LVDP), left ventricular systolic pressure (LVSP) and maximum rate of rise/decrease of left ventricular pressure (+dp/dt_max/-dp/dt_max) were decreased (P<0.05). The myocardial cells were deformed and necrotic, and the myocardial fibers were broken, with inflammatory cell infiltration. The apoptotic rate, the positive rate of LC3-Ⅱ, and the protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ/LC3-I and beclin-1 were increased, and the protein expression of p62 was decreased (P<0.05). Compared with model group, the levels of LVEDD and LVESD were decreased, LVPWTd, LVPWTs, LVEF, CO, LVSP, LVDP, +dp/dt_max and -dp/dt_max were increased in Xinshuaikang groups and captopril group (P<0.05). The morphological changes of myocardial cells were gradually returned to normal, and inflammatory cell infiltration, the apoptotic rate and the positive rate of LC3-Ⅱ were decreased. The protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ/LC3-I and beclin-1 were decreased, and the protein expression of p62 was increased (P<0.05). CONCLUSION:Xinshuaikang inhibits myocardial auto-phagy to play a role of cardiac protection in the rats with chronic heart failure, and its mechanism may be related to inhibition of MAPK/ERK1/2 signaling pathway.     

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.     

Change of Gq-phosphoinositide signaling pathway in left ventricular tissue in rats with chronic heart failure and reverse effect of benazepril on it

AIM: To investigate the changes of Gq-phosphoinositide pathway in left ventricular tissue of rats with chronic heart failure in order to assess the role of this signal pathway in the formation of heart failure. METHODS: Male Sprague-Dawle rats were divided into three groups: control, chronic heart failure and benazepril therapy group. Chronic heart failure was induced with adriamycin. Rats in benazepril group received benazepril 10 mg·kg-1·d-1 and adriamycin at the same time. Hemodynamic measurement was carried out after 4 weeks. The expression of Gα q/11 protein in left ventricle was detected by Western blotting analysis and activity of phospholipase C was measured by the method of hydrolysis of nuclear substrate. RESULTS: Compared with control group, the ±dp/dtmax in chronic heart failure group significantly decreased, and protein Gα q/11 expression, basic and stimulated phospholipase C activity significantly increased (P<0.01). The ±dp/dtmax in benazepril group was significantly lower than that in control but obviously higher than that in chronic heart failure group (P<0.05). Gα q/11 expression, basic and stimulated phospholipase C activity in benazepril group were significantly higher than those in control but obviously lower than those in heart failure group (P<0.01). CONCLUSION: Gq-phosphoinositide signaling pathway may play a role in the formation of chronic heart failure. Benazepril partialy attenuates the activation of phosphoinositide signaling pathway.