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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Free Radic Biol Med. 2019 Jun 7;141:84–92. doi: 10.1016/j.freeradbiomed.2019.06.002

Upregulating Nrf2 in the RVLM ameliorates sympatho-excitation in mice with chronic heart failure

Anyun Ma 1, Juan Hong 1, Julia Shanks 1, Tara Rudebush 1, Li Yu 1, Bryan T Hackfort 1, Hanjun Wang 1, Irving H Zucker 1,*, Lie Gao 1,*
PMCID: PMC6718296  NIHMSID: NIHMS1531676  PMID: 31181253

Abstract

Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor that maintains redox homeostasis by governing a broad array of antioxidant genes in response to oxidant stress. We hypothesized that overexpression of Nrf2 in the rostral ventrolateral medulla (RVLM) ameliorates sympatho-excitation in mice with coronary artery ligation-induced chronic heart failure (CHF). To address this, we overexpressed Nrf2 in the RVLM using an HIV-CamKIIa-Nrf2 lenti virus in C57BL/6 mice. In addition, we used a Lenti-Cre virus in Keap1flox/flox mice to upregulate Nrf2 non-selectively in the RVLM. Arterial blood pressure (AP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded under conscious and anesthetized conditions, respectively. Protein expression was assayed using western blotting and immunofluorescence staining. We found that (1) Nrf2 and two target proteins, NQO1 and HO-1 in the RVLM were significantly lower in CHF compared to Sham mice. Nrf2 viral transfection of the RVLM upregulated Nrf2 protein. (2) Urinary NE excretion in CHF mice was markedly attenuated following Nrf2 upregulation (812 ± 133 vs 1120 ± 271 ng/24hr mean±SE, *p < 0.05, n = 8/group). (3) In the conscious state, CHF mice overexpressing Nrf2 exhibited an enhancement in spontaneous baroreflex gain and in phenylephrine-induced baroreflex control of HR. (4) Acute experiments under anesthetisa revealed a significant decrease in basal RSNA (44.0 ± 6.5 vs 64.7 ± 8.3 % of Max. *P < 0.05 n = 8/group) and enhancement in baroreflex sensitivity (Maximal gain −1.8 ± 0.3 vs 1.1 ± 0.2 of mmHg. **p < 0.01. n = 6/group) in CHF mice that were virally transfected with Nrf2 compared with CHF mice transfected with Lenti-GFP. Finally, Lenti-Cre viral overexpression of Nrf2 in Keap1flox/flox mice reduced Keap1 protein and increased Nrf2, NQO1, and HO-1 in the RVLM of Sham and CHF mice. CHF-Cre mice exhibited a significant decrease in baseline RSNA and plasma NE concentration (8.9 ± 1.1 vs 12.7 ± 0.9 ng/mL. *P < 0.05 n = 6/group) as compared with CHF-GFP mice. Based on the above data, we conclude that upregulating Nrf2 selectively in the RVLM attenuates sympatho-excitation in CHF mice. Nrf2 may be an important central target for autonomic modulation in cardiovascular disease and during stress.

Keywords: chronic heart failure, RVLM, Nrf2/Keap1, oxidative stress, sympathetic regulation, arterial baroreflex

Graphical Abstract

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Introduction

A hallmark of chronic heart failure (CHF) is enhanced sympatho-excitation, which undermines peripheral organ function and exacerbates the failing heart. Reduction in sympathetic activation has become a mainstream therapy for this syndrome. Beta adrenergic-blockers, Angiotensin Converting Enzyme inhibitors and and Angiotensin II Receptor Blockers have all been demonstrated to ameliorate symptoms and improve the prognosis of CHF patients. On the other hand, long-term treatment with beta-blockers increases the risk of withdrawal syndrome, which may result in clinical deterioration[1, 2]. In addition beta-blocker therapy does not attenuate peripheral sympatho-excitation. Accordingly, understanding central mechanisms of sympatho-excitation in CHF is critical to uncover novel therapeutic targets for the development of enhanced pharmacological strategies for patients with CHF.

Sympatho-excitation in CHF can be directly attributed to the dysfunction of central sympathetic regulatory networks, in which the rostral ventrolateral medulla (RVLM) functions as a prime efferent outflow pathway for sympathetic fibers that project to the spinal cord. The RVLM houses pre-sympathetic neurons that send a monosynaptic projection to the intermediolateral nucleus (IML) of the spinal cord to regulate the activity of sympathetic preganglionic neurons[3]. Various studies from our laboratory demonstrated that sympatho-excitation in CHF rabbits was ameliorated either by oral treatment with simvastatin, a 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor[4], or by chronic exercise training[5], in part, by reducing oxidative stress in the RVLM.

We previously demonstrated that enhanced oxidative stress in the RVLM contributes to sympatho-excitation in a rabbit model of pacing-induced CHF[6]. Redox homeostasis in the brain depends on the balance of reactive oxygen spieces (ROS) generation and elimination. At the cell level, ROS are produced, in part, by mitochondria[7] and NAD(P)H oxidases[8] and scavenged by both enzymatic and non-enzymatic mechanisms[9]. In the RVLM of CHF rabbits, we found that the expression and activity of NAD(P)H oxidase (NOX2) was upregulated[6] whereas SODs, were downregulated[5], suggesting that the increased ROS in the RVLM in the setting of CHF is characterized by enhanced pro-oxidant processes and impaired antioxidant defenses. While the upregulation of NOX2 in the RVLM in CHF has been attributed to an activated Angiotensin II system[6], the mechanisms underlying downregulation of SODs and other antioxidant enzymes remain to be elucidated.

The nuclear factor erythroid-derived 2 (Nrf2)/Kelch ECH-associating protein 1 (Keap1) complex is a redox-sensitive transcriptional regulatory system where Keap1 functions as a sensor of ROS and electrophiles, while Nrf2 serves as an effector for the coordinated activation of cytoprotective genes including a battery of antioxidant enzymes[10]. We recently demonstrated that this system plays a critical role in the RVLM to maintain normal sympathetic tone and blood pressure in normal conscious mice[11]. However, the clinical implications of the Nrf2 system in the RVLM remains to be elucidated. The role of RVLM Nrf2 modulation on sympathetic outflow in diseases such as CHF and hypertension has not been characterized in cardiovascular disease. Therefore, in the present experiments, we hypothesized that reduced Nrf2 signaling contributes to the impaired antioxidant defenses in the RVLM in CHF, leading to enhanced oxidative stress and sympathetic dysfunction. We further hypothesized that upregulating Nrf2 will restore redox homeostasis in the RVLM in the CHF state, thus reducing the augmented sympathetic outflow.

Materials and Methods

Fifty four C57BL/6 mice and twenty-four Keap1flox/flox mice (56 male and 22 female) aged between 10 – 12 weeks were used in these experiments. C57BL/6 mice were purchased from Charles River, Inc. (Wilmington, MA). Keap1flox/flox mice were originally obtained from the Johns Hopkins University and bred at The University of Nebraska Medical Center, Department of Comparative Medicine. Keap1flox/flox mice contain LoxP sites flanking exons 2 and 3 of the Keap1 gene[12]. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and were carried out under the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Induction of Heart Failure

The CHF model was produced by permanent coronary artery ligation, as previously described[13]. Briefly, under isoflurane anesthesia (~2% and 98% O2), mice were intubated and ventilated with a mouse ventilator (Mouse Ventilator MiniVent model 845, Hugo Sachs Elektronik; tidal volume: 150 - 250 μL; frequency: 200 breaths/min). The heart was exposed through a left thoracotomy at the fourth intercostal space. The anterior descending branch of the left coronary artery was permanently ligated with a 6-0 suture. Sham mice underwent thoracotomy and manipulation of the heart, but coronary artery ligation was not performed. To reduce the acute mortality in the coronary ligated group during mice received postoperative care by supplying the housing cage with supplemental 100% oxygen and providing a heating pad set at 30-32 °C for 3 days post-surgery or longer as needed to ensure a high survival rate. The survival rate of mice receiving ligation was 78% and the mouse numbers reported above are for surviving for the entire experiment.

Echocardiography

At the fourth week post coronary ligation or sham surgery, mice were echocardiographically imaged on a Visual Sonics Vevo 3100 ultrasound using a 40 MHz probe under light isoflourane anesthesia (0.5-1%). 2D B-mode images were acquired in the long and short parasternal axis. M-Mode images were acquired at the level of the left ventricular papillary muscles. Left ventricular volumes and diameters were measured. Ejection fraction (EF) was calculated by a standard formula ((LVEDV-LVESV)/LVEDV) × 100. Fractional shortening (FS) was calculated as ((LVEDD-LVESD)/LVEDD) × 100. The echocardiographer was blinded to the origin of the animal groups.

RVLM-Specific Nrf2 Overexpression

Nrf2 expression was upregulated employing the following two viral vectors, which were delivered into the RVLM of mice one week after echocardiographic examination.

HIV-CamKIIa-GFP-Nrf2 virus contains the Nrf2 gene driven by the CamKIIa promoter that has been commonly been used to target excitatory glutamatergic neurons[14]. This virus was used to upregulate Nrf2 selectively in gutamatergic neurons of the RVLM of C57BL/6 mice. The CamKIIa-GFP-virus was used as the control.

Lenti-GFP-Cre virus contains both 5’ and 3’ lentiviral long terminal repeats (LTRs) and all necessary elements for effective transduction and expression of GFP and Cre genes, driven by the Cytomegalovirus (CMV) promoter. Virus was injected into the RVLM of Keap1flox/flox mice to express cre recombinase resulting in a loss of most of central intervening region (IVR) domain (four of six of the kelch domains) of Keap1 protein and upregulation of Nrf2[12]. The lenti-GFP virus was used as the control.

Delivery of viruses into bilateral RVLM to upregulate Nrf2 was performed on a mouse stereotaxic apparatus (SR-5M-HT, NARISHIGE International USA, INC, Amityville, NY) where the mice were anesthetized with isoflurane (~2%) and viruses were injected into the RVLM (20 nl HIV-CamKIIa-eGFP-Nrf2 at 1.3×108 TU/mL, Viral Vector Core, University of Iowa, IA or 20 nl lentiviral-Cre-GFP at 1×108 TU/mL, Kerafast, Inc, Boston, MA; the same doses of control GFP viruses were used), according to the following coordinates: 1.2 mm lateral to the midline, 5.3 mm ventral to the dorsal surface of the brain, 1.9 mm caudal to lambda[11].

Chronic Experiments in the Conscious State

Blood Pressure (BP) Telemetry Implantation:

Two weeks after viral injection, the mice were implanted with radiotelemetry units (PA-C10, Data Science International; St Paul, MN) for blood pressure measurement. Briefly, under 2 % isoflurane anesthesia, mice were placed in the supine position. After making a 10-mm incision in right femoral area, the femoral artery was identified, dissected, and separated from the femoral vein and nerve at the proximal location near the groin, followed by placing a cotton patch saturated with 2% lidocaine underneath the artery which was fully dilated during the process. The tip of the telemetry catheter was then inserted into the femoral artery and advanced into the abdominal aorta to the level of the renal arteries.

BP and Heart Rate (HR) Measurements:

Blood pressure and HR were continuously recorded for 48 hours either immediately after telemetry implantation within the period of surgical stress or three weeks after recovery from surgical stress. Recording of BP was performed at a sampling rate of 1 KHz using a PowerLab® data acquisition system (model 8S; ADInstruments; Colorado Springs, CO). Heart rate was derived from the arterial pressure pulse.

Several techniques were used to evaluate the role of Nrf2 overexpression on sympathetic outflow and BP in animals with and without CHF.

Spontaneous Baroreflex Sensitivity (SBRS) Analysis:

SBRS was elicited from the pulsatile BP signals based on the sequence technique[15–17], using HemoLab Software (Version 20.2, courtesy of Dr. Harald M. Stauss, University of Iowa). Individual sequences of increases or decreases in BP in mmHg (x-axis) and pulse interval in msec (y-axis) values were plotted and subjected to linear regression. The average of the slopes of all individual regression lines were then used as an index of baroreceptor-heart rate reflex sensitivity (ms/mmHg).

Induced HR Baroreflex Sensitivity in the Conscious State (IBRS-C):

this parameter was evaluated by calculating the percent of the decline in HR from the maximal value within 30 min after intraperitoneal injection of PE (10 ug in 50 ul).

Restraint-Stress Induced Cardiovascular Responses:

this test was performed by placing the mice in a rodent restrainer (25 mm ID × 100 mm length) for 5 min during continuous recording of BP and HR for 120 min.

After completing the conscious measurements, mice were euthanized with an overdose of pentobarbital sodium (150 mg/kg, ip). The brainstems were removed and stored at −80 °C for subsequent biochemical measurements.

Acute Experiments under Anesthesia

Mice were anesthetized with ip urethane (800 mg/kg) and α-chloralose (40 mg/kg).

BP. HR, and Renal Sympathetic Nerve Activity (RSNA) Recording:

after tracheal cannulation mice were ventilated (tidal volume: 150 μL. frequency: 200 breaths/min) with room air. The right femoral artery and vein were dissected and separated from the nerve. A catheter-tipped transducer (SPR-1000, Millar Instruments. Houston. TX.) was advanced via femoral artery into the abdominal aorta to measure BP and HR. while the femoral vein was cannulated for saline supplementation and administration of vasoactive agents in order to evaluate baroreflex sensitivity. A bundle of the left renal sympathetic nerves was isolated in the retroperitoneal space through a left flank incision and placed on a pair of platinum-iridium electrodes. The nerve-electrode complex was covered with silicone gel (Kwik-Sil. WPI. Sarasota FL). The RSNA signal was amplified (×1000) and filtered (bandwidth: 30 – 3.000 Hz) using a Grass P55C preamplifier. and input into a PowerLab® data-acquisition system. from which the signal was monitored. recorded. and saved using the LabChart® 7 software (AD Instruments. Colorado Sprngs. CO).

Baseline RSNA and Induced Arterial Baroreflex Sensitivity under Anesthesia (IABS-A):

after 20 minutes of stable baseline hemodynamics and RSNA. phenylephrine (PE) was administrated (0.2 μg/μL in 20-40 μL, iv) in order to increase BP. Blood pressure. HR and RSNA were continuously recorded. Mice were then euthanized and the maximum RSNA was obtained within 1–2 min. Background noise was recorded approximately 15–20 min after death. Baseline RSNA was determined as the percent of maximum RSNA activity after the background noise was subtracted[18].

The IBRS-A was analyzed by logistic regression over the entire pressure range after PE administration. The values for BP and RSNA were acquired every 2 seconds from the threshold to the saturation points. A sigmoid logistic regression curve was fit to the data points using the following equation: RSNA = A/{1+exp[B(MAP–C)]}+D; where A is the RSNA range, B is the slope coefficient, MAP is the mean arterial pressure, C is the pressure at the midpoint of the range (BP50), and D is the minimum RSNA[19]. The peak slope (or maximum gain) was determined by taking the first derivative of the baroreflex curve and was calculated with the equation: Gain max = A(1) × A(2) × [1/4], where A(1) is the range and A(2) is the average slope. The mean values for each curve parameter were used to derive composite curves for each experimental group.

Biochemical and Molecular Measurements

Urinary NE:

mice were placed in a metabolic cage (Harvard Apparatus, Holliston, MA), where a timed urine sample was collected under a layer of mineral oil. Urinary NE was measured using a Norepinephrine Enzyme Immunoassay kit (Labor Diagnostika Nord KG, Nordhorn, Germany). A 50-μ1 urine sample was diluted with 950 μl double-distilled H2O to obtain a 20:1 diluted sample, from which 10 μl was used for NE measurements based on the instructions provided by the company. Duplicate measurements were made for each sample. 24-hour NE excretion was calculated by multiplying NE concentration by 24-hour urine volume.

Plasma NE:

Blood of Keap1flox/flox mice was collected by cardiac puncture following acute experiment. Under 2% isoflourane anesthesia, the heart was exposed via a thoracotomy by removing the ventral segment of the 3rd to 6th ribs together with the sternum. A 21G needle connected to 1 mL syringe was inserted into the left ventricle. Blood was withdrawn slowly and transferred into a 1.5 mL Eppendorf tube containing 10% K EDTA (10 ul). Blood was centrifuged at 1000 × g for 10 min at 4 °C. We were able to reliably obtain ~1 mL blood which resulted in ~500 uL of plasma, from which 300 uL was used to measure the NE concentration.

Western Blot Analysis:

Primary antibodies were Nrf2 (ab31163, R-IgG), Keap1 (sc-33569, R-IgG), NQO1 (sc-376023, M-IgG), HO-1 (sc-10789, R-IgG). GAPDH (sc-32233, M-IgG) served as an internal loading control. RVLM tissue punches were homogenized in RIPA buffer, from which the soluble protein was extracted. Protein concentration was measured using a protein assay kit and then adjusted by adding 4% sodium dodecyl sulfate sample buffer to obtain equal concentrations among these samples. The samples were then loaded on a 10% SDS-PAGE gel (15 ug protein per well) and subjected to electrophoresis. The fractionized protein on the gel was electrically transferred onto a polyvinyl difluoride membrane. The membrane was first probed with the primary antibody to the target protein and then with GAPDH primary antibody. After incubation with primary antibodies, the membranes were probed with secondary antibodies followed by treatment with enhanced chemiluminescence substrate (Pierce). The bands on the membrane were visualized and analyzed using a UVP BioImaging System. The final data is expressed as the target protein band densities divided by the GAPDH density.

Immunofluorescence Staining:

Lenti-GFP-Cre viral- induced Nrf2 overexpression in the RVLM of Keap1flox/flox mice was confirmed using immunofluorescence staining. Mice were deeply anesthetized with sodium pentobarbital and perfused transcardially with PBS, followed by 4% paraformaldehyde in PBS. The entire brainstem was removed, mounted on a specimen stage, and sectioned into 40-μm slices in a cryostat. The slices were then washed with PBS 3 times and permeabilized for 30 minutes at room temperature with a solution containing 0.3% Triton X-100 dissolved in PBS, followed by blocking with a solution containing 10% normal goat serum and 0.3% Triton X-100 in PBS at room temperature for 2 hours. The slices were then incubated with Nrf2 antibody (ab31163 R-IgG), in 10% normal goat serum and 0.3% Triton X-100 in PBS at 4°C overnight. After three washes with PBS, the slices were incubated for 2 hours with secondary fluorescent antibody (Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 546; Invitrogen, A-11010). The slices were mounted with an Aqua-Mount Mounting Medium, and then were examined with a laser confocal microscope (Leica TSC STED).

Statistical analyses

All data are expressed as mean ± SE. A two-way repeated measures ANOVA and the Student-Newman-Keuls test was used for analyzing the differences among the four groups, with the aid of SigmaPlot software. A P value of <0.05 was taken as indicative of statistical significance.

Results

Heart failure model

Our intention was to produce mice with severe heart failure. Using our postoperative care protocol, the left coronary artery was ligated as described above and in a recent publication from our laboratory[20]. We obtained several mice with very severe heart failure (EF < 20%), an example of which is shown in Figure 1. Panel A of Figure 1 show markedly dilated hearts with large infarct areas in both GFP and Nrf2 viral treatments. The size and weight of infarted hearts were doubled compared to the sham hearts. The septal and right ventricular free wall were profoundly thickened, whereas the left ventriclur free wall became paper thin and dilated. Panel B of Figure 1 shows the M-mode and long axis 2D echocardiograms, EF, and tracing of left ventricular pressure and dp/dtmax providing evidence of severely impaired cardiac function in these infarcted hearts. Table 1 shows the grouped data demonstrating the reduced cardiac function in the CHF groups. No significant differences in these parameters were noticed between CHF-Nrf2 and CHF-GFP mice.

Figure 1.

Figure 1.

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Images show the A. appearance of the whole heart and sections, B. echocardiograms and hemodynamic measurements in Sham and severe MI hearts treated with RVLM lenti-GFP or Nrf2. The LV in A was filled with blue gel to better accentuate the MI area. HW: heart weight. EF: ejection fraction. The MI hearts are the most severe ones.

Table 1.

Anatomic, hemodynamic, and echocardiographic measurements associated with failing hearts.

Sham-GFP Sham-Nrf2 CHF-GFP CHF-Nrf2
Animal No. 13 13 14 14
Heart Weight, g 0.14 ± 0.01 0.16 ± 0.02 0.25 ± 0.07 0.23 ± 0.06
Infarct Size, % of LV 0 0 59.9 ± 12.4 55.3 ± 15.8
Ejection Fraction, % 63.6 ± 7.4 65.1 ± 6.9 34.3 ± 5.1** 37.1 ± 4.3@@
Fractional Shortening, % 30.7 ± 6.2 33.5 ± 5.7 13.5 ± 3.1** 15.3 ± 2.2@@
LVEDP, mmHg 2.2 ± 2.4 1.9 ± 2.5 18.8 ± 9.1** 17.3 ± 8.2@@
dp/dt max, mmHg/s 12463 ± 1020 13724 ± 1052 3154 ± 655** 3298 ± 703@@
dp/dt min, - mmHg/s 13101 ± 1112 14166 ± 1281 3540 ± 721** 3719 ± 772@@
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**

P < 0.01 vs Sham-GFP;

@@

P < 0.01 vs Sham-Nrf2.

Nrf2 and target proteins in the RVLM

Figure 2 shows Western blot data of RVLM protein for Nrf2 and two key targets, NQO1 and HO-1. Expression of Nrf2, NQO1, and HO-1 were significantly lower in the CHF-GFP group as compared with the Sham-GFP, suggesting impaired Nrf2 signaling and antioxidant defenses in the RVLM of animals with CHF. Nrf2 gene transfer significantly upregulated Nrf2, NQO1, and HO-1 in the RVLM of sham and CHF mice. Likewise both NQO1 and HO-1 were upregulated following Nrf2 gene transfer in CHF rats. In the Sham group, Nrf2 protein was significantly upregulated whereas the changes in NQO1 and HO-1 did not reach statistical significance.

Figure 2.

Figure 2.

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Expression of Nrf2 and two target proteins in the RVLM of Sham and CHF mice transfected with GFP or Nrf2 viruses. GFP: CamKIIa-GFP-virus; Nrf2: CamKIIa-GFP-Nrf2-virus. *P < 0.05; n = 7 in Sham-GFP and Sham-Nrf2 groups; n = 8 in CHF-GFP and CHF-Nrf2 groups.

Cardiovascular and Sympathetic Regulation in Conscious Mice

In order to determine if overexpression of Nrf2 in the RVLM reduced sympathetic outflow in heart failure we measured several indices of sympatho-excitation. Urinary NE concentration and excretion were significantly elevated in the CHF-GFP group compared with the Sham-GFP group (Figure 3, panel A). Total urinary NE excretion was significantly reduced in the CHF-Nrf2 group. These data strongly suggest that Nrf2 overexpression in the RVLM suppressed sympathetic outflow of CHF. Another index of sympatho-excitation is arterial baroreflex sensivity. Panel B of Figure 3 shows spontaneous arterial baroreflex sensitivity (SABS-C), indicating that both up-sequence gain (1.23±0.20 versus 3.71±0.32 ms/mm Hg, *P<0.05, n=8 and 7) and down-sequence gain (0.90±0.21 versus 2.87±0.19 ms/mm Hg, *P<0.05; n=8 and 7) were significantly decreased in the CHF-GFP group as compared with the Sham-GFP. Nrf2 overexpression significantly enhanced baroreflex sensitivity (up-sequence gain: 2.45±0.31 ms/mm Hg, down-sequence gain: 2.30±0.32 ms/mm Hg; †P<0.05 vs CHF-GFP, n=8 for each group). In addition to the SABS-C, Figure 3, Panel C shows the hemodynamic responses to ip injection of PE. There were no differences in blood pressure among these four groups, however the decline in HR was significantly reduced in the CHF-GFP group as compared with the Sham-GFP. This reduced bradycardia was attenuated in CHF-Nrf2 group. These data suggest that RVLM Nrf2 overexpression improved both spontaneous and induced arterial baroreflex sensitivity in conscious CHF in mice.

Figure 3.

Figure 3.

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Sympathetic outflow and cardiovascular regulation in conscious mice. (A) Urinary NE; (B) Spontaneous Baroreflex Sensitivity; (C) Induced Baroreflex Sensitivity. Left panel is a representative tracing of MAP and HR of one mouse after ip PE; Right panel is mean data showing the percent decline of heart rate following PE induced increase in blood pressure. *P < 0.05 and **P < 0.01 vs Sham-GFP; †P < 0.05 vs CHF-GFP. n = 7, 7, 8, and 8 in Sham-GFP, Sham-Nrf2, CHF-GFP, and CHF-Nrf2 groups.

Cardiovascular Responses to Stress

In order to determine if overexpression of Nrf2 in the RVLM reduced sympathetic tone in CHF per se or was a more generalized phenomenon, we evaluated two stress paradigms that have been associated with sympatho-excitation[21, 22]. Figure 4 shows the hemodynamic response associated with these stressors. Panel A shows the blood pressure and heart rate recorded after the first 24 hours of telemetry unit implantation surgery. The BP in Sham-Nrf2 and CHF-Nrf2 groups were significantly lower than their GFP control groups, suggesting that RVLM Nrf2 overexpression attenuated surgery-induced pressor effects. The CHF-GFP group displayed a lower BP than Sham-GFP most likely because of impaired cardiac function. In addition, there were no significant differences in daytime and nighttime BP between groups, suggesting disordered circadian rhythm following surgical stress. Panel B of Figure 4 shows blood pressure and heart rate recorded on the 21st day after telemetry unit implantation surgery when the animals were completely recovered from acute surgical stress. At this point the circadian rhythmn reappeared and there were no significant differences in BP and HR among these four groups. Panel C of Figure 4 shows cardiovascular responses to a 5-min restraint. The BP in Sham-GFP and CHF-GFP groups was significantly elevated during restraint. However, there was no change the BP of Sham-Nrf2 or CHF-Nrf2 groups.

Figure 4.

Figure 4.

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Nrf2 overexpression reduces the hemodynamic response to non-specific stress. MAP and HR tracing of one mouse (left) and group data (right) on the 1st day (A) and 21st day (B) post telemetry implant surgery. (C) cardiovascular responses to 5 min of physical restraint. *P < 0.05 and **P < 0.01 vs Sham-GFP; †P < 0.05 vs Sham-GFP. n = 7, 7, 8, and 8 in Sham-GFP, Sham-Nrf2, CHF-GFP, and CHF-Nrf2 groups.

Cardiovascular and Sympathetic Regulation in the Anesthetized State

The direct recording of sympathetic nerve activity is difficult in conscious mice with viable signals recorded for up to only 4-5 days[23, 24]. We therefore recorded nerve activity under anesthesia in this set of experiments. Figure 5 shows renal sympathetic nerve activity (RSNA) and iv PE-induced arterial baroreflex regulation of RSNA under the anesthesia (IABS-A). Panel A is an original recording of basal RSNA and its response to PE-induced hypertension (baroreflex). This recording clearly shows increased neural discharge at baseline and a blunted sympatho-inhibitory response during hypertension in CHF-GFP as compared with the Sham-GFP. This attenuated sympatho-inhibition was ameliorated in CHF-Nrf2 mice. Panel B displays the mean data of three baseline RSNA parameters, showing that the frequency, integrated nerve activity, and normalized nerve activity were higher in the CHF-GFP group compared to the Sham-GFP. In the CHF-Nrf2 group baseline RSNA was significantly reduced compared to the CHF-GFP group. Panel C shows the composite baroreflex curves/gains (top) and four parameters of reflex sensitivity (bottom). The maximal gain, average slope and range of RSNA response were significantly decreased, whereas the BP50 and minimum RSNA were significantly higher in CHF-GFP compared with Sham-GFP. Nrf2 overexpression shifted these parameter towards those of the sham group.

Figure 5.

Figure 5.

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Renal sympathetic nerve activity and baroreflex sensitivity by iv PE under anesthesia. (A) Original recording; (B) Analysis of basal sympathetic nerve activity; (C) Baroreflex curves and curve parameters. *P < 0.05 and ** P < 0.01 vs Sham-GFP; † P < 0.05 vs CHF-GFP. n = 6/group.

Effects of Keap1-Knockout-Induced Nrf2 Overexpression

In addition to viral overexpression of Nrf2 in the RVLM we also employed Keap1flox/flox mice treated with RVLM microinjection of Lenti-GFP-Cre virus to overexpress Nrf2 as an alterative strategy to address our hypothesis. These data are shown in Figure 6 where Panel A shows immunofluorescence images of GFP expression in the RVLM of Keap1flox/flox following Lenti-GFP-Cre virus administration for three weeks. Neurons that are positive for GFP (green) also express strong Nrf2 immunoreactivity (red and in merged image). Panel B shows western blot data for Keap1, Nrf2, NQO1 and HO1 proteins. Compared with the GFP groups, both Sham-Keap1flox/flox-Cre mice and CHF-Keap1flox/flox-Cre groups displayed a significantly lower Keap1 and higher Nrf2, NQO1, and HO-1 proteins. In addition, CHF-Keap1flox/flox-GFP mice exhibited a lower Nrf2, NQO1, and HO-1 than Sham-Keap1flox/flox-GFP mice, whereas there were no significant differences in Keap1 levels among these groups. Panels C and D show a higher basal RSNA and plasma NE concentration in CHF-Keap1flox/flox-GFP mice as compared with Sham-Keap1flox/flox-GFP. These changes were remarkably ameliorated in CHF-Keap1flox/flox-Cre mice.

Figure 6.

Figure 6.

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Nrf2, Keap1, NQO1, and HO-1 expression in the RVLM of Keap1flox/flox mice following Lenti-GFP-Cre virus transfection, detected by immunofluorescence (A) and western blot (B); (C): RSNA recording (upper) and mean data (lower); (D): plasma NE concentration. *P < 0.05 vs GFP groups; #P < 0.05 vs Sham-GFP group; †P < 0.05 vs CHF-GFP. n = 6/group.

Discussion

Basal sympathetic tone is essential for cardiovascular and endocrine homeostasis. Sympathetic hyperactivity contributes to a variety of diseases including hypertension and CHF. In CHF sympatho-excitation is a critical compensatory mechanism for maintaining hemodynamic stability in response to reduced pump performance. However, if long-lasting, sympatho-excitation eventually leads to a vicious cycle of worsening heart failure. Although the augmented sympathetic tone in the CHF state originates centrally, the therapeutic strategy for CHF has been to focus on the peripheral sympathetic terminal to block the impact of high sympathetic tone on target organs. For example, β adrergic-blockade in CHF patients protects myocytes but leaves the α-receptor vulnerable to sympathetic stimulation and may result in vasoconstriction thus potentially reducing tissue perfusion. The aim of the current study was to elucidate one potential central mechanism of sympathetic overactivity in CHF for which new therapies can be generated. We found that Nrf2 protein and two of its targets, NQO1 and HO-1, in the RVLM of CHF mice was significantly downregulated as compared with Sham mice. Administration of HIV-CamKIIa-eGFP-Nrf2 or lentiviral-Cre-GFP in the RVLM of C57BL/6 or Keap1flox/flox mice with CHF restored the expression of Nrf2, NQO1, and HO-1, accompanied by improved arterial baroreflex function, lower plasma and urinary excretion of NE, and reduced RSNA. Furthermore, we found that overexpression of Nrf2 in the RVLM of both Sham and CHF mice significantly inhibited surgical- and restraint- evoked increases in blood pressure. Taken together these data strongly suggest that impaired antioxidant defense of a key sympathetic nucleus contributes to sympatho-excitation in CHF and that upregulation of Nrf2 ameliorates sympathetic regulation in this syndrome. These data also suggest that Nrf2 and redox status in the RVLM plays a critical role in the pressor response to non-specific stress. Enhancement of antioxidant mechanisms in the RVLM is a potential strategy of treatment of CHF and prevention of hypertension.

The central neuro-circuitry that regulates sympathetic outflow is complex but involves important nuclei in the brainstem. These include the Nucleus of the Solitary Tract (NTS), the caudal ventro-lateral medulla (CVLM), and the RVLM. The NTS integrates afferent inputs from peripherial sensors and also drives GABAergic neurons in the CVLM to tonically suppress pre-sympathetic neuron activity in the RVLM, by which sympathetic outflow is dynamically adjusted in response to changes in the periphery in a negative feedback manner. In a recent study, we found that selective deletion of Nrf2 and the consenquent enhancement of oxidative stress in the RVLM resulted in sympatho-excitation and hypertension, suggesting that redox homeostasis in this component of the sympathetic network is essential for normal function[11]. Indeed, enhanced oxidative stress in the RVLM is believed to be responsible for increased sympathetic tone in both CHF[25] and hypertension[26]. Although it has been demonstrated that antioxidants, such as SODs, are markedly downregulated in the RVLM in CHF[5] and hypertension[27], the underlying mechanisms remain to be elucidated. It is not clear, why endogenous antioxidant defenses fail to be activated by redox challenges in these cardiovascular diseases. In the present study, we found that Nrf2 expression in the RVLM of CHF mice was significantly lower than in Sham animals, suggesting that the impaired antioxidant defense may be attributed, at least in part, to dysfunctional transcription of antioxidant enzyme genes. Indeed, when Nrf2 levels in the RVLM were restored by Nrf2 gene transfer or by selective knockdown of Keap1, the sympatho-excitation in CHF mice was significantly ameliorated. Consistent with our observations, a downregulation of Nrf2 protein was also found in the paraventricular nucleus (PVN) in the spontaneous hypertensive rat (SHR) [28]. In this study blood pressure and sympathetic nerve activity were significantly reduced by administration of tert-butylhydroquinone (tBHQ), a selective Nrf2 activator. In addition, Nrf2 in the RVLM of rats with hypertension induced by Escherichia coli lipopolysaccharide was also found to be downregulated[29]. These studies confirm a critical role for central Nrf2 in sympathetic regulation.

The cellular mechanism by which overexpression of Nrf2 alters RVLM neuronal activity in mice with CHF is not clear. Based on the expression of phenylethanolamine N-methlytransferase (PNMT) presympathetic neurons in the RVLM are categorized as C1 or non-C1 groups, both of which utilize glutamate as the primary excitatory neurotransmitter[30] and therefore can be targeted by a CAMKIIa vector. It was recently reported that in mice, optogenetic activation of C1 cells resulted in a decrease in blood pressure, whereas when C1 and non-C1 cells were stimulated simultaneously, blood pressure was dramatically increased[31] suggesting different functions of C1 and non-C1 cells in the RVLM. In the present study, we were not able to distinguish between these two types of cells because of the employment of an HIV-CamKIIa-GFP-Nrf2 vector. However, we believe that upregulation of Nrf2 in all glutamatergic neurons in the RVLM contributed to the amelioration of sympathetic dysfunction. In addition, the data from the Keap1flox/flox mouse model indicated that overexpressing Nrf2 in a more globally manner in the RVLM, including neurons and non-neuronal cells, also contributed to the suppression of sympathetic nerve activity in CHF, suggesting global oxidative stress in the RVLM. Indeed, increased ROS can markably impact neurons and glia cells by direct oxidation of lipids, proteins, and DNA as well as the induction of mitochondrial injury[32]. Under oxidative stress, neuronal excitability was increased by oxidative modification of potassium channels[33] while glia cells were activated to release pro-inflammatory cyokines[34], both of which may underly the dysfunction in the RVLM in the CHF state.

An important aspect of the current study is that Nrf2 overexpression in the RVLM also modulated stress associated cardiovascular responses. We found that Nrf2 overexpression abolished the increase in blood pressure following surgical or restraint stress. These data suggest that redox imbalance in the RVLM plays a critical role in the sympathetic and pressor responses to a variety of challenges. Stress is a state of mental, physical, or emotional tension in response to noxious stimuli from within and outside of the body that requires the coordination of autonomic, neuroendocrine, and behavioural processes to promote adaptation and survival. The sympatho-adrenomedullary and hypothalamic-pituitary-adrenocortical axes are primary players[35] in the stress response. Acute stress can result in life threatening cardiac dysfunction and sudden cardiac death while chronic stress has been directly linked to the development of hypertension, cardiovascular disease and stroke[36]. While several risk factors contribute to these stress-induced cardiovascular events, activation of the sympathetic nervous system is pivotal in triggering the associated symptoms[37]. Recently, there have been efforts to elucidate the role of the RVLM in the stress-induced sympatho-excitation. In a rat model of neuropathic pain, glutamate concentration in the RVLM was significantly increased accompanied by elevated renal sympathetic nerve activity and hypertension, which were normalized after glutamate receptor blockade[38]. Glutamate has been demonstrated to enhance ROS production in neurons in vitro[39] and in vivo[40]. Therefore, it is likely that a similar scenario exists in the RVLM. High levels of glutamate have been found in the RVLM of rats subjected to electrical foot-shock -induced hypertension, accompanied by increased pro-inflammaotry cytokines, suggesting a role for neuroinflammation in the stress response[41]. Given that Nrf2 positively regulates both antioxidant and anti-inflammatory gene expression [10], we suggest that abolishment of stress-induced hypertension by Nrf2 overexpression in the RVLM is involved in both processes. Interestingly, a recent study found that electic foot shock signicantly downregulated Nrf2 protein in the hippocampus[42], implying the involvement of this antioxidant transcription factor responses to stress globally in the central nervous system.

In summary, we found significantly lower Nrf2 expression in the RVLM of mice with coronary artery ligation-induced CHF and that overexpression of Nrf2 significantly reduced sympathetic nerve activity and improved arterial baroreflex function in CHF mice. This over expression abolished stress-evoked pressor responses in both Sham and CHF mice. Taken together these data suggest that reduced transcriptional regulation of antioxidant genes contribute to oxidative stress in the RVLM in CHF resulting in enhanced oxidative stress and sympathetic overactivity. The data further suggest that upregulating antioxidant enzyme expression through the Nrf2-Keap1 pathway in the RVLM may be a potential new therapeutic strategy to improve autonomic regulation in CHF and prevent the development of stress-associated hypertension.

Hightlights.

  • Nrf2 expression in the RVLM was downregulated in CHF mice, underlying the impared antioxidant defenses and sympatho-excitation.

  • Upregulation of Nrf2 of the RVLM by Nrf2 gene transfer or Keap1 gene deletion ameliorate sympathetic regulation in CHF mice.

  • Overexpression of Nrf2 in the RVLM of mice attenuated the pressor response to both surgical or restraint stresses.

Acknowledgments

The authors acknowledge Dr. Shyam Biswal for providing the Keap1flox/flox mouse strain.

Sources of Funding

This study was supported by National Institutes of Health (NIH) grant P01 HL62222 (IHZ).

Footnotes

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