Sympathoexcitation In Rats Research Articles

PHARMACOLOGIC NEUROMODULATION TARGETING NEUROINFLAMMATION AS A NOVEL THERAPEUTIC STRATEGY FOR PULMONARY HYPERTENSION AND RIGHT HEART FAILURE

TYPE: Late Breaking Abstract TOPIC: Pulmonary Vascular Disease PURPOSE: Pulmonary hypertension (PH) is associated with aberrant sympathoexcitation leading to right ventricular failure (RVF). Neuroinflammation has been implicated in sympathoexcitation in experimental PH. Here we hypothesize that pharmacologic neuromodulation targeting TSC neuroinflammation may serve as a novel therapy for PH and RVF. METHODS: Adult male Sprague Dawley rats received either Monocrotaline (MCT, 60mg/kg, subcutaneous, n=10) and were followed for 4-weeks or Sugen (SuHx, 20mg/kg, subcutaneous, n=10) and kept in hypoxia (10% O2) for 3-weeks followed by 2-weeks of normoxia. Saline treated rats served as controls (CTRL, n=10). Next, MCT-rats received either daily intrathecal specific NOS1-inhibitor S-Methyl-L-thiocitrulline (SMTC; 0.5mg/kg, n=7) or PBS (n=7) from day 14-28. Echo and RV-catheterization were performed terminally. TSC, lung and RV RNASeq, RT-qPCR, NOS1 activity and immunolocalization, and catecholamine ELISA were performed. RESULTS: MCT and SuHx rats highlighted similar degree of PH, RVF and TSC-specific neuroinflammation and apoptosis. TSC transcriptome showed common genes and pathways linked to neuroinflammation, nitrosative stress and apoptosis. NOS1 was one of the top-most upregulated genes in TSC. NOS1-specific inhibition rescued PH, RV-hypertrophy, and RVF (all p<0.05). SMTC significantly decreased pulmonary arteriolar wall thickness, TSC NOS activity, microglia and astrocyte activation, apoptosis, plasma catecholamines and QTc prolongation (all p<0.05). This rescue was associated with reversal of RV and lung transcriptome and sympathoexcitation. CONCLUSIONS: Pharmacologic neuromodulation via intrathecal NOS1-specific inhibition rescues PH and RVF by reducing TSC neuroinflammation, apoptosis and sympathoexcitation in rats. CLINICAL IMPLICATIONS: Targeting NOS1 in TSC may serve as a novel therapeutic strategy for clinical PH and RVF. DISCLOSURE: No significant relationships. KEYWORD: Pulmonary Hypertension

Role of CNS in the increased sympathoexcitation in rats with femoral artery ligation.

Role of CNS in the increased sympathoexcitation in rats with femoral artery ligation.

Sympathoexcitatory role for neurons projecting from the mesencephalic locomotor region to the rostral ventrolateral medulla in rats

C1 neurons in the rostral ventrolateral medulla (RVLM) are reportedly excited by voluntary exercise. The present study aimed to explore neural pathway(s) upstream to the RVLM that can play a role in sympathetic adjustments to exercise. First, via neuronal tracing experiments, we found that rat mesencephalic locomotor region (MLR), in which cholinergic neurons are abundantly distributed, sends direct projections to the RVLM (MLR‐RVLM neurons) since the MLR contained a number of neuronal cells immunoreactive to cholera toxin subunit B (CTb) that had been injected into the RVLM in advance (n = 8). It is noted that very few neurons immunoreactive to choline acetyltransferase in the MLR were labeled with CTb (n = 3), suggesting that the MLR‐RVLM neurons are not cholinergic. Next, we found that optogenetic stimulation of MLR‐RVLM neurons causes sympathoexcitation in rats. An AAV vector that encodes the channelrhodopsin variant, ChIEF‐tdTomato was bilaterally microinjected into the MLR (n = 7). More than 3 weeks later, bilateral photostimulation (473 nm wavelength, 10 mW, 40 Hz) provided to the RVLM of the anesthetized rats significantly (P &lt; 0.05) elevated blood pressure. In another set of rats (n = 3), a retrograde AAV vector that encodes the channelrhodopsin‐GFP was bilaterally microinjected to the RVLM. In these rats, bilateral photostimulation of the MLR, in which a number of GFP‐labeled cell bodies were confirmed, significantly increased renal sympathetic nerve activity and blood pressure. Finally, confocal imaging revealed that MLR‐derived, tdTomato‐labeled axons containing VGLUT2 were closely associated with tyrosine hydroxylase‐positive neurons in the RVLM, suggesting that MLR‐RVLM glutamatergic neurons form synaptic contacts with RVLM C1 neurons. Based on these observations, we propose that excitation of MLR‐RVLM glutamatergic neurons contribute to sympathetic adjustments to exercise via stimulation of RVLM C1 neurons.Support or Funding InformationThis study was supported by JSPS KAKENHI 15H05367 (SK) &amp; 16K15190 (SK); 16H05128 &amp; 15H05932 (KN).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Downregulation of large conductance calcium-activated potassium channels in paraventricular nucleus contributes to sympathoexcitation in rats with chronic heart failure

Objective: To elucidate the association between large conductance calcium-activated potassium channels (BKCa) in the paraventricular hypothalamic nucleus (PVN) and sympathetic outflow in rats with chronic heart failure (CHF) . Methods: Male Wistar rats (6-7 weeks old) were randomized to sham operated group and CHF group (coronary artery ligation) . Two weeks after operation, BKCa inhibitor Iberiotoxin (IBTX) was infused into PVN by osmotic minipumps, rats were divided into following groups: sham+aCSF, CHF+aCSF, sham+low dose IBTX (0.125 nmol/nl) , CHF+low dose IBTX, sham+moderate dose IBTX (1.25 nmol/nl) , CHF+moderate dose IBTX, sham+ high dose IBTX (12.5 nmol/nl) , and CHF+high dose IBTX (n=6 each) . Additional rats were grouped as follows: sham+vehicle, sham+KCNMB4 knockdown (by rAAV2-KCNMB4 shRNA virus injection in PVN) , CHF+vehicle, CHF+ KCNMB4 knockdown group (n=6 each) . The cardiac function was determined by echocardiography, left ventricular hemodynamics were measured invasively, renal sympathetic nerve activity (RSNA) was recorded at 6 weeks after coronary artery ligation or sham operation. The contents of norepinephrine (NE) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in plasma were determined by enzyme-linked immunosorbent assay. The protein and mRNA expression of KCNMB4 in PVN were measured by immunofluorescence staining, Western blot, and real-time PCR, mRNA expression of BKCa in PVN was detected by real-time PCR. Results: Compared with the sham operation group, the cardiac function of the heart failure group was significantly reduced (P<0.05) , and the plasma NE and the serum NT-proBNP were significantly elevated (P<0.05) . The protein and mRNA expression of KCNMB4 in PVN were obviously down-regulated in CHF rats (P<0.05) . After perfusion of IBTX or KCNMB4 knockdown by microinjection of rAAV2-KCNMB4 shRNA virus,right ventricular weight/body weight and lung weight/body weight ratio as well as left ventricular end-diastolic diameter were increased and left ventricular ejection fraction was decreased (all P<0.05) , the sympathetic driving indexes was increased in sham rats, changes of these parameters further aggravated in CHF rats (P<0.05) . KCNMB4 knockdown further downregulated protein expression in PVN of CHF rats. Conclusion: Downregulation and blunted function of BKCa in PVN may contribute to sympathoexcitation and deterioration of cardiac function in rats with chronic heart failure.

High Salt Intake Augments Excitability of PVN Neurons in Rats: Role of the Endoplasmic Reticulum Ca2+ Store.

High salt (HS) intake sensitizes central autonomic circuitry leading to sympathoexcitation. However, its underlying mechanisms are not fully understood. We hypothesized that inhibition of PVN endoplasmic reticulum (ER) Ca2+ store function would augment PVN neuronal excitability and sympathetic nerve activity (SNA). We further hypothesized that a 2% (NaCl) HS diet for 5 weeks would reduce ER Ca2+ store function and increase excitability of PVN neurons with axon projections to the rostral ventrolateral medulla (PVN-RVLM) identified by retrograde label. PVN microinjection of the ER Ca2+ ATPase inhibitor thapsigargin (TG) increased SNA and mean arterial pressure (MAP) in a dose-dependent manner in rats with a normal salt (NS) diet (0.4%NaCl). In contrast, sympathoexcitatory responses to PVN TG were significantly (p < 0.05) blunted in HS treated rats compared to NS treatment. In whole cell current-clamp recordings from PVN-RVLM neurons, graded current injections evoked graded increases in spike frequency. Maximum discharge was significantly augmented (p < 0.05) by HS diet compared to NS group. Bath application of TG (0.5 μM) increased excitability of PVN-RVLM neurons in NS (p < 0.05), yet had no significant effect in HS rats. Our data indicate that HS intake augments excitability of PVN-RVLM neurons. Inhibition of the ER Ca2+-ATPase and depletion of Ca2+ store likely plays a role in increasing PVN neuronal excitability, which may underlie the mechanisms of sympathoexcitation in rats with chronic HS intake.

Exercise training intolerance in heart failure with preserved ejection fraction is associated with altered chemoreflex gain and sympathoexcitation in rats

Heart failure with preserved ejection fraction (HFpEF) is characterized by cardiac dysfunction and autonomic imbalance. In HF, sympathoexcitation is strongly associated to altered chemoreflex drive. Exercise training (EX) has been demonstrated that normalizes autonomic control and chemoreflex function in HF with reduced ejection fraction (HFrEF). Indeed, in HFrEF patients EX improve survival. Importantly, this effect appears to be related to EX tolerance (EX‐T). In contrast to what is known in HFrEF, much less is known about the effects of EX in HFpEF and the mechanisms underpinning the EX‐T. Then, we determine i) EX tolerance/intolerance in experimental HFpEF and ii) whether EX intolerance is associated with heightened chemoreflex drive in HFpEF. HFpEF was induced by volume overload in male Sprague‐Dawley rats (250±20g). EX (60 min/day, 25 m/min, 10% inclination) was performed for 6 weeks. EX intolerance (CHF+EX‐inT) was defined as the incapacity to complete daily training session (&lt;50% total training time). Rats that accomplished total training sessions were defined as EX tolerant (CHF+EX‐T). Degree of HF was estimated by echocardiography and intraventricular pressure‐volume loops. Arrhythmia index was scored. Sympathetic drive was assessed by ΔHR following 1mg/kg propranolol i.v. infusion. Peripheral and central chemoreflex drive were study using hypoxic (HVR) and hypercapnic ventilatory response (HCVR), respectively. EX intolerance in CHF was ~40% and was independent of the degree of cardiac failure. Compared to sedentary HF rats (CHF‐sed), CHF+EX‐T rats showed a 1.5‐fold improvement in cardiac systolic function without changes in diastolic function. In addition, a significant decrease in the number of arrhythmias (43±33 vs. 196±85, events/hr, CHF+EX‐T vs. CHF‐sed, respectively) and sympathetic drive (−61.3±10.1 vs. −90.0±13.1 bpm, ΔHR, CHF+EX‐T vs. CHF‐Sed, respectively) were found in CHF+EX‐T rats. In contrast, EX severely compromises diastolic dysfunction in CHF+EX‐inT rats (289.5±21.3 vs. 237.8±6.5 μl V0, CHF+EX‐inT vs. CHF‐Sed, respectively). No effects of EX on systolic function, cardiac arrhythmias and sympathetic drive were found in CHF+EX‐inT. In addition, we found that EX normalized the HVR only in CHF+EX‐T rats but not in CHF+EX‐inT rats. Indeed, we found that EX induced a 1.5‐fold increase in HVR in CHF+EX‐inT animals compared to CHF‐Sed animals (respiratory frequency: 232.3±19.8 vs. 152.5±5.2 %bas, CHF+EX‐inT vs. CHF‐Sed, respectively). No effects of EX on HCVR was found in CHF+EX‐T and CHF+EX‐inT vs. CHF+Sed. Importantly, we found that acute hypoxic exposure (FiO2 10%) induced severe lethal cardiac arrhythmias in CHF+EX‐inT rats (~60% survival) but not in CHF+EX‐T rats. CHF‐sed nor CHF+EX‐T groups exhibited mortality events during hypoxia. Our results showed that EX in CHF+EX‐T rats improves cardiac function, arrhythmia incidence and sympathetic drive. In contrast, EX in CHF+EX‐inT rats results in a further deterioration of cardiac function and an increased peripheral chemoreflex drive. Importantly, life‐threating cardiac arrhythmic events triggered by hypoxic exposure lead to sudden death in CHF+EX‐inT rats.Support or Funding InformationFONDECYT 1140275 and 1150040

C-Jun N-terminal Kinase mediates prostaglandin-induced sympathoexcitation in rats with chronic heart failure by reducing GAD1 and GABRA1 expression.

Prostaglandin E2 mediates sympathoexcitation in chronic heart failure (CHF) through EP3 receptors (PTGER3) in the paraventricular nucleus (PVN). The aim of this study was to investigate the role of c-Jun N-terminal kinase (JNK) in expressional regulation of gamma-aminobutyric acid signalling in PVN in CHF rats. Chronic heart failure was induced by left coronary ligation in Wistar rats. Renal sympathetic nerve discharge (RSND) and mean arterial pressure (MAP) responses to the PVN infusion were determined in anaesthetized rats. Osmotic minipumps were used for chronic PVN infusion. PTGER3 expression was examined with immunofluorescence staining, quantitative real-time PCR and Western blot. Chronic heart failure rats had increased JNK activation and decreased glutamate decarboxylase 1 (GAD1) and GABAA receptor alpha 1 subunit (GABRA1) expression in the PVN. PVN infusion of the PTGER3 agonist SC-46275 caused sympathoexcitation in sham-operated control (Sham) rats and increased it further in CHF. The PTGER3 antagonist L798106 reduced sympathoexcitation and cardiac dysfunction in CHF. PVN infusion of EP1 receptor antagonist SC-19220, EP2 receptor antagonist AH6809 or EP4 receptor antagonist L-161982 had no effect on sympathoexcitation. The JNK inhibitor SP600125 normalized sympathoexcitation and GAD1 and GABRA1 expression in PVN in CHF rats. Both the p44/42 and p38 mitogen-activated protein kinase inhibitors PD98059 and SB203580 could not prevent the downregulation of GAD1 and GABRA1 expression in PVN in CHF. PTGER3 agonist activated JNK but downregulated GAD1 and GABRA1 expression in NG108 neuronal cells. Prostaglandin signalling through upregulated PTGER3 activates JNK which reduces GAD1 and GABRA1 expression in the PVN, and contributes to sympathoexcitation in CHF.

Attenuated dopaminergic tone in the paraventricular nucleus contributing to sympathoexcitation in rats with Type 2 diabetes

The study was conducted to investigate the role for dopamine in the centrally mediated sympathoexcitatory response in rats with Type 2 diabetes (T2D). T2D was induced by a combination of high-fat diet (HFD) and low-dose streptozotocin (STZ). HFD/STZ treatment for 12-14 wk resulted in significant increase in the number of FosB-positive cells in the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM). In anesthetized rats, administration of exogenous dopamine (dopamine hydrochloride, 20 mM) in the PVN, but not in the RVLM, elicited decreases in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in control rats and but not in the T2D rats. Blocking the endogenous dopamine with dopamine D1/D5 receptor antagonist SCH39166 (2 mM) in the PVN and RVLM, resulted in increases in RSNA, MAP, and heart rate (HR) in both control and T2D rats. These responses were significantly attenuated in T2D rats compared with control rats (PVN - ΔRSNA: 21 ± 10 vs. 44 ± 2%; ΔMAP: 7 ± 3 vs. 19 ± 6 mmHg, ΔHR: 17 ± 5 vs. 32 ± 4 bpm, P < 0.05). There were no significant increases in response to dopamine D2/D3 receptor antagonist raclopride application in the PVN and RVLM of both control and T2D rats. Furthermore, there were decreased dopamine D1 receptor and D2 receptor expressions in the PVN of T2D rats. Taken together, these data suggest that reduced endogenous dopaminergic tone within the PVN may contribute to the sympathoexcitation in T2D.

Hypothalamic paraventricular nucleus activation contributes to neurohumoral excitation in rats with heart failure.

Heart failure (HF) is a serious cardiovascular disease and is characterized by exaggerated sympathetic activity. In this paper, we review these limited studies, with particular emphasis on examining the role of the paraventricular nucleus (PVN) in the neurohumoral excitation in HF. The PVN is an important neuroendocrine and preautonomic output nucleus, and is considered as the important central site for integration of sympathetic nerve activity. Accumulating evidences demonstrate that a number of neurohumoral processes are involved in the pathophysiology of HF, such as renin-angiotensin system (RAS), proinflammatory cytokines (PICs), neurotransmitters, and reactive oxygen species (ROS). Recent studies about neurohumoral regulation indicate that angiotensin II type1 receptor (AT1-R) is the important product mediated by cytoplasmic nuclear factor-kappa B (NF-κB) which is up-regulated along with elevated PICs and angiotensin II (ANG II) in the PVN of HF rats. These findings suggest that the NF-κB mediates the cross-talk between RAS and PICs in the PVN in HF. The further studies indicate that the interaction between AT1-R and NF-κB in the PVN contributes to oxidative stress and sympathoexcitation by modulating neurotransmitters in heart failure, and the superoxide activates NF-κB in the PVN and contributes to neurohumoral excitation. In conclusion, the neurohumoral excitation in HF is based on the interaction of RAS, PICs, ROS, NF-κB and neurotransmitters in the PVN; and the activated NF-κB in the PVN modulates the neurotransmitters and contributes to sympathoexcitation in rats with heart failure.

Effects of exercise training on SFO-mediated sympathoexcitation during chronic heart failure

Exercise training (ExT) has been shown to reduce sympathetic drive during heart failure (HF). The subfornical organ (SFO) is involved in the neural control of sympathetic drive. We hypothesized that an activated SFO contributes to enhanced sympathetic activity in HF. We also postulated that ExT would reduce the activation of the SFO and its contribution to the sympathetic drive during HF. Sprague-Dawley rats were subjected to coronary artery ligation to induce HF. Rats were assigned to ExT for 3-4 wk. Rats with HF had a 2.5-fold increase in FosB-positive cells in the SFO compared with sham-operated rats, and this was normalized by ExT. Microinjection of ANG II (100 pmol) into the SFO resulted in a greater increase in renal sympathetic nerve activity (RSNA), blood pressure, and heart rate in the HF group than in the sham-operated group. These responses were normalized after ExT (change in RSNA: 23 ± 3% vs. 8 ± 2%). ExT also abolished the decrease in RSNA in HF rats after the microinjection of losartan (200 pmol) into the SFO (-21 ± 4% vs. -2 ± 3%). Finally, there was elevated mRNA (5-fold) and protein expression (43%) of ANG II type 1 receptors in the SFO of rats with HF, which were reversed after ExT. These data suggest that the enhanced activity of the SFO by elevated tonic ANG II contributes to the enhanced sympathoexcitation exhibited in HF. The decrease in ANG II type 1 receptor expression in the SFO by ExT may be responsible for reversing the neuronal activation in the SFO and SFO-mediated sympathoexcitation in rats with HF.

Tiron, a superoxide scavenger, suppresses fictive locomotionevoked sympathoexcitation in rats with heart failure

We have previously reported that fictive locomotion‐evoked sympathoexcitation in rats with heart failure was exaggerated and that this exaggeration was suppressed by Tempol, a superoxide dismutase mimetic, injected bilaterally into the rostral ventrolateral medulla (RVLM). It should be noted that Tempol also possess a property to open ATP‐sensitive and Ca2+‐activated K+ channels. In the present study, we tested if Tiron, which does not chemically relate to Tempol but has a similar superoxide scavenging activity, injected bilaterally into the RVLM of rats with heart failure would reduce fictive locomotion‐evoked sympathoexcitation. Heart failure was induced in rats by coronary artery ligation. Fictive locomotion was evoked in decerebrated and paralyzed rats by electrical stimulation of the mesencephalic locomotor region. In the rats with heart failure (N8, fractional shortening &lt; 35%), Tiron mirrored the effect of Tempol; bilateral injection into the RVLM of Tiron (10 mM, 100 nl) significantly (P&lt;0.05) reduced the renal sympathoexcitatory and pressor responses to fictive locomotion by 42% and 43%, respectively. The present data support the concept that oxidative stress in the medulla of heart failure plays a role in excessively amplifying central command‐mediated sympathoexcitation. Supported by Mitsui Life Social Welfare Foundation Research Grant (to S. Koba).

Oxidative stress exaggerates skeletal muscle contraction-evoked reflex sympathoexcitation in rats with hypertension induced by angiotensin II

Muscle contraction stimulates thin fiber muscle afferents and evokes reflex sympathoexcitation. In hypertension, this reflex is exaggerated. ANG II, which is elevated in hypertension, has been reported to trigger the production of superoxide and other reactive oxygen species. In the present study, we tested the hypothesis that increased ANG II in hypertension exaggerates skeletal muscle contraction-evoked reflex sympathoexcitation by inducing oxidative stress in the muscle. In rats, subcutaneous infusion of ANG II at 450 ng·kg(-1)·min(-1) for 14 days significantly (P < 0.05) elevated blood pressure compared with sham-operated (sham) rats. Electrically induced 30-s hindlimb muscle contraction in decerebrate rats with hypertension evoked larger renal sympathoexcitatory and pressor responses [+1,173 ± 212 arbitrary units (AU) and +35 ± 5 mmHg, n = 10] compared with sham normotensive rats (+419 ± 103 AU and +13 ± 2 mmHg, n = 11). Tempol, a SOD mimetic, injected intra-arterially into the hindlimb circulation significantly reduced responses in hypertensive rats, whereas this compound had no effect on responses in sham rats. Tiron, another SOD mimetic, also significantly reduced reflex renal sympathetic and pressor responses in a subset of hypertensive rats (n = 10). Generation of muscle superoxide, as evaluated by dihydroethidium staining, was increased in hypertensive rats. RT-PCR and immunoblot experiments showed that mRNA and protein for gp91(phox), a NADPH oxidase subunit, in skeletal muscle tissue were upregulated in hypertensive rats. Taken together, hese results suggest that increased ANG II in hypertension induces oxidative stress in skeletal muscle, thereby exaggerating the muscle reflex.

Exercise training suppresses central command‐evoked sympathoexcitation in rats with heart failure

Sympathoexcitation evoked by central command, a parallel activation of the locomotor and autonomic neural circuits in the brain, has been shown to become exaggerated in heart failure (HF). In the present study, we tested whether exercise training (ET) in HF normalizes central command‐evoked sympathoexcitation. HF was induced in rats &gt; 6 wks after coronary artery ligation. A subset of the HF rats was treadmill trained (20 m/min, 5% grade, 60 min/day, 5 days/wk, 8–14 wks) (HF+ET). Central command was activated by fictive locomotion in decerebrate rats. Fictive locomotion induced by 30‐s electrical stimulation at 35 μA of the mesencephalic locomotor region evoked a larger (P&lt;0.05) increase in renal sympathetic nerve activity in HF rats [269 ± 45 arbitrary unit (AU), fractional shortening (FS) = 20 ± 3%, N12] than that in HF+ET (121 ± 33 AU, FS = 20 ± 2%, N10) and sham (111 ± 37 AU, FS=46 ± 3%, N6) rats. The sympathoexcitation in the HF rats was significantly reduced by bilateral injection into the rostral ventrolateral medulla of Tempol (10 mM, 100 nl), a superoxide dismutase mimetic. In the HF+ET and sham rats, Tempol had no effect on the response. These results suggest that ET has a therapeutic effect to suppress sympathoexcitation evoked by central command in HF. Further, the role played by central superoxide in amplifying central command‐evoked sympathoexcitation in HF may be altered by ET. Supported by JSPS Kaken 22790226 (SK).

NF-κB in the paraventricular nucleus modulates neurotransmitters and contributes to sympathoexcitation in heart failure.

Findings from our laboratory indicate that proinflammatory cytokines and their transcription factor, nuclear factor-kappaB (NF-κB), are increased in the hypothalamic paraventricular nucleus (PVN) and contribute towards the progression of heart failure. In this study, we determined whether NF-κB activation within the PVN contributes to sympathoexcitation via interaction with neurotransmitters in the PVN during the pathogenesis of heart failure. Heart failure was induced in rats by left anterior descending coronary artery ligation. Sham-operated control (SHAM) or heart failure rats were treated for 4weeks through bilateral PVN infusion with SN50, SN50M or vehicle via osmotic minipump. Rats with heart failure treated with PVN vehicle or SN50M (inactive peptide for SN50) had increased levels of glutamate, norepinephrine (NE), tyrosine hydroxylase (TH), superoxide, gp91(phox) (a subunit of NAD(P)H oxidase), phosphorylated IKKβ and NF-κB p65 activity, and lower levels of gamma-aminobutyric acid (GABA) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN compared with those of SHAM rats. Plasma levels of cytokines, norepinephrine, epinephrine and angiotensin II, and renal sympathetic nerve activity (RSNA) were increased in heart failure rats. Bilateral PVN infusion of SN50 prevented the decreases in PVN GABA and GAD67, and the increases in RSNA and PVN glutamate, norepinephrine, TH, superoxide, gp91(phox), phosphorylated IKKβ and NF-κB p65 activity observed in vehicle or SN50M-treated heart failure rats. A same dose of SN50 given intraperitoneally did not affect neurotransmitters concentration in the PVN and was similar to vehicle-treated heart failure rats. These findings suggest that NF-κB activation in the PVN modulates neurotransmitters and contributes to sympathoexcitation in rats with ischemia-induced heart failure.

Paraventricular nucleus corticotrophin releasing hormone contributes to sympathoexcitation via interaction with neurotransmitters in heart failure.

Recent studies indicate that systemic administration of tumor necrosis factor (TNF)-α induces increases in corticotrophin releasing hormone (CRH) and CRH type 1 receptors in the hypothalamic paraventricular nucleus (PVN). In this study, we explored the hypothesis that CRH in the PVN contributes to sympathoexcitation via interaction with neurotransmitters in heart failure (HF). Sprague-Dawley rats with HF or sham-operated controls (SHAM) were treated for 4 weeks with a continuous bilateral PVN infusion of the selective CRH-R1 antagonist NBI-27914 or vehicle. Rats with HF had higher levels of glutamate, norepinephrine (NE) and tyrosine hydroxylase (TH), and lower levels of gamma-aminobutyric acid (GABA) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN when compared to SHAM rats. Plasma levels of cytokines, NE, ACTH and renal sympathetic nerve activity (RSNA) were increased in HF rats. Bilateral PVN infusions of NBI-27914 attenuated the decreases in PVN GABA and GAD67, and the increases in RSNA, ACTH and PVN glutamate, NE and TH observed in HF rats. These findings suggest that CRH in the PVN modulates neurotransmitters and contributes to sympathoexcitation in rats with ischemia-induced HF.

Central angiotensin type 2 receptor stimulation reduces blood pressure and norepinephrine excretion in conscious normal rats

In previous studies, we found a significant down regulation of Angiotensin II Type 2 receptors (AT2R) in the rostral ventrolateral medulla (RVLM) and a critical role of the AT2R in sympatho‐excitation in rats with chronic heart failure. In this experiment, we determined the effects of intracerebroventricular (icv) infusion of Compound 21 (C21), the first selective non‐peptide AT2R agonist, on the mean blood pressure (MAP), heart rate (HR), and norepinephrine (NE) excretion in conscious normal rats. C21 was infused by a Micro‐osmotic pump at a rate of 0.5μg/μl/2hrs for 7 days. On day 7 the rats were euthanized and the RVLM was punched out to measure AT1R and nNOS protein expression by western blot. We found that: (1) C21 treatment significantly decreased MAP (81.5 ± 6.2 vs 93.2 ± 5.9 mmHg, P &lt; 0.05), but did not change HR; (2) both NE concentration in urine (104 ± 12.6 vs 366 ± 9.7 ng/ml, P &lt; 0.05) and NE excretion (1.9 ± 0.3 vs 8.7 ± 1.2 μg/day, P &lt; 0.05) were decreased in the C21 treated rats; (3) C21 down‐regulated AT1R protein expression (0.5 ± 0.1 vs 1.2 ± 0.3 AT1R/GAPDH, P &lt; 0.05) and up‐regulated nNOS (0.7 ± 0.1 vs 0.3 ± 0.1 nNOS/GAPDH, P &lt; 0.05) protein expression in the RVLM. These results suggest that activation of central AT2R evoked hypotension through a sympatho‐inhibitory mechanism, in part, by decreasing AT1R expression and activating nNOS signaling.

NF‐κB activation and oxidative stress in the paraventricular nucleus contribute to sympathoexcitation via interaction with neurotransmitters in heart failure

Nuclear factor kappa B (NF‐κB) is a potent inducer of proinflammatory cytokines (PIC) and oxidative stress in cardiovascular disease. In this study, we determined whether NF‐κB activation and oxidative stress in the paraventricular nucleus (PVN) contribute to sympathoexcitation via interaction with neurotransmitters in the PVN in heart failure (HF). Rats with HF induced by coronary ligation and sham‐operated controls (SHAM) were treated for 4 weeks with bilateral PVN infusion of NF‐κB inhibitor SN50, or the superoxide scavenger tempol, or vehicle. Compared with SHAM rats, HF rats had higher levels of norepinephrine (NE) and glutamate and lower levels of gamma‐aminobutyric acid (GABA) in the PVN, along with high levels of Fra‐LI (an indicator of chronic neuronal activation), PIC, superoxide, gp91phox (a subunit of NAD(P)H oxidase), phosphorylated IKKβ (p‐IKKβ) in the PVN, and increased renal sympathetic nerve activity (RSNA). HF rats had higher plasma levels of NE, epinephrine (EPI), angiotensin II and PIC. Treatment with bilateral infusion of SN50 or tempol attenuated these HF‐induced changes. These findings suggest that NF‐κB activation and oxidative stress in the PVN modulate neurotransmitters and contribute to sympathoexcitation in rats with ischemia‐induced HF.

Downregulated Kv4.3 expression in the RVLM as a potential mechanism for sympathoexcitation in rats with chronic heart failure

Elevated central angiotensin II (ANG II) plays a critical role in the sympathoexcitation of chronic heart failure (CHF) by stimulating upregulated ANG II type 1 receptors (AT(1)R) in the rostral ventrolateral medulla (RVLM). However, the link between enhanced ANG II signaling and alterations in the electrophysiological characteristics of neurons in the RVLM remains unclear. In the present experiments, we screened for potentially altered genes in the medulla of rats with CHF that are directly related to neuronal membrane conductance using the Rat Genome 230 2.0 Array GeneChip. We found that CHF rats exhibited a 2.1-fold reduction in Kv4.3 gene expression, one of the main voltage-gated K(+) channels, in the medulla. Real-time RT-PCR and Western blot analysis confirmed the downregulation of Kv4.3 in the RVLM of CHF rats. In intact animals, we found that microinjection of the voltage-gated potassium channel blocker, 4-aminopyridine, into the RVLM evoked a sympathoexcitation and hypertension in both normal and CHF rats. CHF rats exhibited smaller responses to 4-aminopyridine than did normal rats. Finally, we used a neuronal cell line (CATH.a neurons) to explore the effect of ANG II on Kv4.3 expression and function. We found that ANG II treatment significantly downregulated mRNA and protein expression of Kv4.3 and decreased the A-type K(+) current. Employing this cell line, we also found that the ANG II-induced inhibition of Kv4.3 mRNA expression was attenuated by the superoxide scavenger Tempol and the p38 MAPK inhibitor SB-203580. The effects of ANG II were abolished by the AT(1)R antagonist losartan. We conclude that the sympathoexcitation observed in the CHF state may be due, in part, to an ANG II-induced downregulation of Kv4.3 expression and subsequent decrease in K(+) current, thereby increasing the excitability of neurons in the RVLM. The ANG II-induced inhibition of Kv4.3 mRNA expression was mediated by ANG II-AT(1)R-ROS-p38 MAPK signaling.

Subarachnoid haemorrhage-induced sympathoexcitation in rats is reversed by bosentan or sodium nitroprusside.

1. The roles played by nitric oxide (NO) and endothelin (ET) in the genesis of sympathetic nervous activation following experimental subarachnoid haemorrhage was investigated using spectral analysis of blood pressure rhythms. 2. Subarachnoid haemorrhage was induced in conscious rats by injecting 0.3 mL homologous blood via a catheter placed along the surface of the brain and directed towards the circle of Willis. Three hours after the insult and after sympathetic activation was evident, animals received either an acute injection of the ET antagonist bosentan (5 mg/kg, i.v.; n = 7), an infusion of the NO donor sodium nitroprusside (SNP; 18 microg/h; n = 7) or no treatment (n = 7). 3. Three hours following the induction of subarachnoid haemorrhage, the mid-frequency components of systolic blood pressure were markedly elevated, indicating a pronounced sympathoexcitation. However, blood pressure and heart rate levels remained unchanged at this time. In the absence of treatment, the mid-frequency components of blood pressure remained elevated for a subsequent 2 h. Treatment with a non-hypotensive dose of SNP reversed the sympathoexcitation within 1 h. Treatment with bosentan was also effective in reducing the mid-frequency oscillations in blood pressure associated with subarachnoid haemorrhage. 4. Our results indicate that subarachnoid haemorrhage is associated with an acute activation of the sympathetic nervous system. The degree of sympathoexcitation can be reversed by the use of either bosentan or SNP.