Male Sprague-Dawley rats (weighing 200–230 g, 6-week-old) were provided by the Animal Center of the People’s Hospital of Wuhan University (animal certificate number: 00270753), and they were housed at the Animal Experiment Center of Renmin Hospital of Wuhan University with a controlled room temperature and free access to food and water under a natural day/night cycle. This study strictly follows the “Guidelines for Animal Protection and Use” formulated by Wuhan University. All animal experiments were approved by the Experimental Animal Ethics Committee of the Renmin Hospital of Wuhan University (Number: 20230901B), and performed in accordance with the Guidelines for the Ethical Review of Laboratory Animal Welfare and was granted on September 4, 2023. The rats were randomly assigned to one of four experimental groups using a random number table: a sham surgery control group (Sham), an abdominal aortic coarctation group (AB), a left stellate ganglionectomy group (LSG), and a right stellate ganglionectomy group (RSG). The AB group underwent isolated abdominal aortic constriction. The LSG group consisted of rats that received a left stellate ganglionectomy combined with abdominal aortic constriction. Similarly, the RSG group included rats that underwent a right stellate ganglionectomy along with the abdominal aortic constriction. There were 5 rats in each group, a total of 20 rats. The discrepancy between the initial number of rats and those ultimately included in the statistical analysis stems from technical difficulties encountered during surgical procedures. To ensure the robust establishment of the experimental model, we supplemented the rats. During the experiment, there were no abnormal deaths of rats, and all rats survived normally until the end of the experiment. At the termination of the experiment, the rats were humanely euthanized using Carbon dioxide (CO₂) inhalation (the CO₂ concentration elevated by 10% to 30% of the chamber volume per minute, continuing this incremental rise until the rodents lose consciousness and ultimately reach a state of euthanized repose).

The surgical procedures were performed based on previously published methodologies [17]. Specifically, the rats were anesthetized following a standardized protocol via an intraperitoneal (IP) injection of 3% sodium pentobarbital at a dose of 50 mg/kg [18]. After skin preparation and disinfection, cut the skin, fascia, muscles, and parietal peritoneum longitudinally along the midline of the abdomen to expose the internal organs. Find the abdominal aorta, abdominal artery, and right renal artery from the abdominal vein upwards, and then passively separate the abdominal aorta between the abdominal artery and right renal artery. Place the 22-G needle parallel to the separated segment of the abdominal aorta, and ligate them. Then, carefully remove the needle to cause partial stenosis of the abdominal aorta, and restore the intestinal canal to its normal position, suturing the muscles, fascia, and skin. After surgery, 10,000 units of penicillin were administered to resist infection.

On the 2nd day after surgery, the rats gradually developed symptoms such as accelerated breathing and heartbeat, fear of cold fatigue, decreased appetite, cyanosis, and subcutaneous edema, indicating that the model was constructed successfully.

A physiological recorder was used to record the electrocardiogram of the rats after successful ligation of the abdominal aorta. Preparing and disinfecting the middle of the neck, and blunt separation of subcutaneous tissue, fascia, and muscle on one side. Find the common carotid artery next to the trachea, and separate the surrounding nerves and blood vessels using a glass dividing needle carefully to get about 2 cm common carotid artery. Cross two 7-0 sutures below the common carotid artery for backup. Before operation, inject 0.3% heparin saline injection into the catheter and pressure transducer until there are no bubbles. Then ligate the distal end of common carotid artery with sutures and clamp the proximal end with arterial clamps to temporarily block this segment of blood vessel. Use ophthalmic scissors to make a small oblique incision on the vascular wall near the ligation at the distal end. Then, insert the prepared catheter into the blood vessel along the incision, after releasing the arterial clamp, push the catheter about 1cm further towards the proximal end to see the blood pressure waveform of the artery. Finally, use the remaining suture to ligate and fix the catheter. After the animal stabilizes for about 5 minutes, data such as heart rate, blood pressure, and electrocardiogram can be recorded.

The stellate ganglionectomy was performed 30 minutes after abdominal aortic constriction surgery. The rats were fixed in a supine position and passively separated subcutaneous tissue, fascia, and muscle at the midpoint incision of the neck. The common carotid artery located at the inner edge of the sternocleidomastoid muscle and near the trachea. The superior cervical ganglia located on the dorsal side of the bifurcation of the common carotid artery. The stellate ganglion was found along the common carotid artery towards the proximal end, and it was severed 3 mm below the superior cervical ganglion. When the rat shows symptoms of Horner’s syndrome such as enophthalmos, pupil narrowing, and ptosis within 5 minutes, it indicates that the stellate ganglionectomy has been successfully completed. Due to the difficulty in survival of rats with both left and right simultaneous stellate ganglionectomy, this experiment will not study on bilateral stellate ganglionectomy.

Echocardiography was performed before and 10 weeks after SG resection for detecting cardiac function using a Mylab30CV ultrasound system (Biosound Esaote, Genoa, Italy) with a 15-MHz transducer. Surgery was performed under isoflurane anesthesia (5% for induction and 1–2% during surgery) (R500, RWD, Shenzhen, China). The ventricular interventricular septal defect (IVSD, mm), left ventricular posterior wall thickness (LVPWD, mm), left ventricular end diastolic diameter and volume (LVEDD, mm), left ventricular end diastolic volume (LVEDV, mL), left ventricular ejection fraction (LVEF, %), and left ventricular short axis shortening fraction (LVFS, %) were measured using animal cardiac ultrasound in three groups of rats. Detect at least 3 or more cardiac cycles for all parameters. The results were analyzed with the Chart 5.0 software (Applied Biosystems, Danvers, MA, USA).

Obtain heart tissue and to homogenize the tissue. Before operation, add sufficient tissue protein extraction reagents and an appropriate amount of protease inhibitors in advance. The thoroughly lysed tissue was centrifuged at 2000 rpm for 5 minutes, carefully collect the centrifuge supernatant as a total protein solution. Firstly, prepare a sufficient amount of BCA working solution, and add a certain concentration of sample and 25 µL standard samples to the micropores respectively. Then add 200 µL BCA working solution in sequence, mix thoroughly, and incubate at 37 °C for 30 minutes. Next, calculate the protein concentration and calculate the total protein (each sample to be 40 µg). Add protein loading buffer to the protein sample and soak in boiling water for 5 minutes. After preparing separation gel and concentration gel, sample loading and electrophoresis are carried out in sequence. After electrophoresis, membrane transfer is carried out. Rinse the PVDF film and place it in a sealing solution. Shake the bed for 2 hours and remove it in TBST for 3 times $\times$ 5 minutes. Incubate overnight in a shaking bed at 4 °C. Then, the membranes were incubated with primary antibodies against atrial natriuretic peptides (ANP) (1:500, PL0403306, PL Laboratories, Beijing, China), growth-associated protein-43 (GAP43) (1:500, 16971-1-AP, proteintech, Rosemont, IL, USA), Tyrosine Hydroxylase (TH) (1:500, 250905, Abbiotec, Danvers, MA, USA), CaMKII (1:5000, BW6769, Abcam, Cambridge, MA, USA), Ryanodine Receptor 2 (RyR2, 1:500, orb794741, Biorbyt, UK), phosphorylated (p) -RyR2 (1:500, BS4358, Bioworld, Danvers, MA ,USA). GAPDH (1:500; ab181602; Abcam, UK) was utilized as an internal reference control. After the completion of the first antibody incubation, wash the membrane 3 times with TBST $\times$ 5 minutes, HRP-linked anti-mouse (1:2000; Applygen, Danvers, MA, USA) or anti-rabbit IgG (1:2000; Applygen, Danvers, MA, USA) was used as the secondary antibody, incubate at room temperature for 1 hour, and perform chemiluminescence detection. Finally, the AlphaEaseFC 3.0 (https://www.genomeweb.com/arrays/alphaeasefc) was used to analyze and calculate the optical density of the target band.

The expression of ANP, growth-associated protein-43 (GAP43), TH, CaMKII, PYR2 in myocardium was measured by RT-qPCR. Total RNA was isolated from ventricular samples with Tripure Extraction Reagent (orb782896, Biorbyt, Danvers, MA, USA) according to the manufacturer’s protocol. cDNA was synthesized using EntiLink™ 1st Strand cDNA Synthesis Kit (EQ003, Yeasen, Shanghai, China). Real-time fluorescent quantitative PCR was performed on the StepOne real-time PCR instrument (4376374, Thermo Fisher, Waltham, MA, USA), and each sample was made into three duplications using EnTurbo™ SYBR Green PCR SuperMix kit (EQ001, Biorbyt, Danvers, MA, USA). GAPDH was used as the internal control (Table 1), and all reactions were performed in triplicate. Relative RNA expression was calculated using the 2^{-Δ⁢Δ⁢Ct} method [15].

The concentration of brain natriuretic peptide (BNP) and norepinephrine (NE) generated from rat serum was detected by ELISA. The BNP concentration in the rat serum (n = 3) in validation cohort was determined by the ELISA BNP Immunoassay (RAB0386-1KT, Sigma, MO, USA) according to the manufacturer’s instructions. The NE levels from rat serum (n = 3) were determined using NA/NE (Noradrenaline/Norepinephrine) ELISA Kit (E-EL-0047, Elabscience, Beijing, China) following the manufacturer’s instructions.

10 weeks after stellate ganglionectomy, rats were first anesthetized with 1.5–2% isoflurane by inhalation firstly, and then were euthanized by cervical dislocation. Take the hearts of rats for pathological examination to observe the degree of myocardial hypertrophy and ventricular remodeling. The heart was fixed in 4% paraformaldehyde buffer (P6148, Sigma, MO, USA) overnight and then transferred to 70% ethanol. Following this, the tissues were embedded in paraffin, sectioned at 30 µm and subjected to hematoxylin-eosin staining (H&E) (PH0516-100, PHYGENE, Fujian, China), Phygene Masson staining (G1340, Solarbio, Beijing, China). H&E staining was performed according to a routine standard operating procedure using a Leica Multistainer (ST5020, Leica, Vizna, Germany). Slides were dewaxed and rehydrated with successive applications of xylene, alcohol 100%, alcohol 70% and tap water. Haematoxylin (Mayer’s, Klinipath, Benelux, USA) was applied for 4 minutes followed by a 20 second differentiation in ammonia after which eosin (Eosin Y A+B, Klinipath, Benelux, USA) was applied for 20 seconds. Masson staining was used to examine the myocardial fibrosis of the heart based on the proportion of myocardial collagen fibers.

Statistical analyses were performed using GraphPad PRISM software v8.4.2 (GraphPad Software Inc, San Diego, CA, USA). As indicated, statistical analysis was performed by calculating mean SEM. Student’s t-test was used when only two groups were analyzed. For comparisons between multiple groups, homo geneity variance was first tested, and the one-way ANOVA (Tukey’s multiple comparisons test) was applied when variance was homogeneous, or non-parametric independent sample t-test (the Mann–Whitney test) was used for inhomogeneity. Paired Student’s t-test or one-way ANOVA was used to analyze the quantitative Western blot as indicated in figure legends. Two-tailed test was used for all t-tests, and p 0.05 was considered statistically significant.

Previous study had demonstrated that inhibiting the activity of the stellate ganglia can effectively treat chronic heart failure [8]. Based on this knowledge, we aimed to further investigate the impact of stellate ganglion activity on cardiac function. To test this hypothesis, we developed a rat model of chronic heart failure (Fig. 1A). The successful establishment of the model was confirmed by comparing the mean arterial pressure and heart rate between the rats in the sham surgery group (Sham) and abdominal aortic coarctation group (AB) (Fig. 1B,C). Next, we surgically removed the left (LSG) or right stellate ganglion (RSG) to further investigate their roles in cardiac hypertrophy and chronic heart failure. Surprisingly, we found that resection of either the left (LSG) or right stellate ganglion (RSG) significantly reduced the mean arterial pressure and heart rate in rats compared with those in the AB group (Fig. 1D,E). These findings suggest that surgical removal of the stellate ganglia can effectively alleviate the elevated mean arterial pressure and tachycardia associated with chronic heart failure.

Fig. 1.

Effective alleviation of cardiac symptoms in chronic heart failure patients through stellate ganglionectomy. (A) Schematic representation of stellate ganglionectomy. (B) The average arterial pressure of each group was tested before and 10 weeks after stellate ganglionectomy. (C) Heart rates of each group were tested before and 10 weeks after stellate ganglionectomy. (D) The average arterial pressure of each group was tested before and 10 weeks after stellate ganglionectomy. (E) Heart rates of each group were tested before and 10 weeks after stellate ganglionectomy. Sham, sham surgery group; AB, abdominal aortic coarctation group; LSG, left ganglionectomy group; RSG, right ganglionectomy group. Error bars, means $\pm$ SEMs (n = 3, each group). p values were determined by one-way ANOVA.

The results of our preliminary experiments demonstrated that stellate ganglionectomy significantly improved abnormal vital signs, such as the mean arterial pressure and heart rate, in rats with chronic heart failure. Chronic overload of mean arterial blood flow and elevated heart rate are known contributors to cardiac overload, leading to heart failure [19]. To investigate the therapeutic potential of stellate ganglionectomy on cardiac function in chronic heart failure patients, we conducted echocardiographic measurements. We assessed interventricular septal defect (IVSD, mm), left ventricular posterior wall thickness (LVPWD, mm), left ventricular end-diastolic diameter (LVEDD, mm), left ventricular end-diastolic volume (LVEDV, mL), left ventricular ejection fraction (LVEF, %), and left ventricular fractional shortening (LVFS, %), among other parameters, to evaluate postsurgical cardiac function across different groups of rats (Fig. 2A). Our findings indicate that stellate ganglionectomy significantly reduces the LVEDV (mL), LVPWD (mm) and LVEDD (mm) (Fig. 2B,E,F) while significantly increasing the LVFS (%) (Fig. 2C) and LVEF (%) (Fig. 2D). Notably, among the treatments considered, solely RSG exerts a significant influence on the interventricular septal diameter (IVSD) (mm) (Fig. 2G). These results suggest that stellate ganglionectomy effectively mitigates cardiac dysfunction associated with chronic heart failure.

Fig. 2.

Stellate ganglionectomy significantly mitigates cardiac dysfunction in chronic heart failure. (A) Representative images of echocardiograms from each group. The scale bars = 100 µm. (B–G) Statistical evaluation of echocardiography data from each group. Left ventricular end-diastolic volume (LVEDV, mL) (B), left ventricular short-axis shortening fraction (LVFS, %) (C), left ventricular ejection fraction (LVEF, %) (D), left ventricular posterior wall thickness (LVPWD, mm) (E), left ventricular end-diastolic diameter and volume (LVEDD, mm) (F), and ventricular interventricular septal defect (IVSD, mm) (G). Sham, sham surgery group; AB, abdominal aortic coarctation group; LSG, left ganglionectomy group; RSG, right ganglionectomy group. Error bars, means $\pm$ SEMs (n = 3, each group). p values were determined by one-way ANOVA.

In addition to investigating the impact of stellate ganglionectomy on cardiac function in chronic heart failure patients, we aimed to further explore whether this procedure can be used to treat structural lesions associated with chronic heart failure. Research has indicated that sustained mean arterial pressure overload and rapid heart rate can lead to heart failure, resulting in compensatory cardiac hypertrophy and ultimately chronic heart failure [20]. Therefore, we utilized the heart-to-body weight ratio and heart size-to-calf length ratio as preliminary indicators of organic cardiac dilation. As anticipated, at 10 weeks post surgery, the rats in the AB group presented significant increases in both the heart-to-body weight ratio and heart size-to-calf length ratio compared with those in the sham group (Fig. 3A,B). Notably, stellate ganglionectomy markedly reduced these ratios (Fig. 3A,B). We further employed H&E staining and Masson staining to assess the pathological changes associated with postoperative cardiac hypertrophy. The results demonstrated that the rats in the AB group presented significant pathological dilation of myocardial fibers, whereas stellate ganglionectomy significantly ameliorated this pathological myocardial fiber dilation (Fig. 3C,D). In conclusion, inhibiting stellate ganglion activity not only effectively mitigates cardiac dysfunction caused by chronic heart failure but also fundamentally addresses the structural lesions associated with chronic heart failure.

Fig. 3.

Stellate ganglionectomy reduces structural cardiac lesions and mitigates dysfunction in chronic heart failure. (A) Statistical evaluation of heart weight-to-body weight (HW/BW) in each group. (B) Statistical evaluation of heart weight to tibia length (HW/TL) in each group. (C) H.&E. Analysis of myocardial fibers in the cardiac tissues of each group. (D) Masson strain analysis of myocardial fibers in the cardiac tissues of each group. Sham, sham surgery group; AB, abdominal aortic coarctation group; LSG, left ganglionectomy group; RSG, right ganglionectomy group. The scale bars = 100 µm for (C) and (D). Error bars, means $\pm$ SEMs (n = 3, each group). p values were determined by one-way ANOVA.

In the early stages of heart failure, a decrease in cardiac output and blood pressure triggers the neurohormonal system, including the renin‒angiotensin‒aldosterone system (RAAS) and sympathetic nervous system (SNS), as a compensatory mechanism to maintain circulatory homeostasis [21]. The stellate ganglia, formed by the fusion of the 7th cervical and 1st thoracic sympathetic ganglia, serve as postganglionic nerves to regulate cardiac function. Prolonged elevation of mean arterial pressure, which leads to cardiac dysfunction, activates the stellate ganglia to secrete neurotransmitters such as norepinephrine, which in turn activate atrial natriuretic peptide (ANP) to provide stress-induced cardiac protection [22]. To further investigate the molecular mechanisms underlying stellate ganglion activity in the context of myocardial fiber dilation and chronic heart failure, we measured the protein expression levels of GAP43 and TH proteins, which are markers of stellate ganglion activity, using protein immunoblotting. Our results indicated that, compared with those in the sham group, GAP43 and TH expression levels were significantly elevated in the AB group. Notably, these elevations were significantly reversed following stellate ganglion resection (Fig. 4A–C). Additionally, we quantified plasma norepinephrine levels using ELISA. Compared with that in the sham group (215.2 $\pm$ 10.64), norepinephrine secretion was significantly increased in the AB (317.0 $\pm$ 4.568) group, and this increase was markedly reduced after stellate ganglionectomy (LSG, 244.6 $\pm$ 7.110, RSG, 257.6 $\pm$ 5.460) (Fig. 4F). Consistent with these findings, protein immunoblotting revealed that ANP protein levels were significantly elevated in the AB group due to chronic heart failure, and stellate ganglion resection significantly decreased this increase (Fig. 4D,E). Therefore, our study demonstrated that inhibiting stellate ganglion activity effectively reduces norepinephrine secretion and mitigates the depletion of cardiac ANP, highlighting a potential therapeutic target for managing chronic heart failure.

Fig. 4.

Modulation of neurohormonal activity by stellate ganglionectomy in chronic heart failure. (A) Western blotting was used to detect the protein expression levels of tyrosine hydroxylase (TH) and growth-associated protein-43 (GAP43) in the cardiac tissues of each group. (B) Statistical analysis of GAP43 expression. (C) Statistical analysis of TH expression.(D) Western blotting was used to detect the protein expression levels of atrial natriuretic peptides (ANP) in the cardiac tissues of each group. (E) Statistical analysis of ANP expression. (F) The concentration of norepinephrine (NE) in serum. Sham, sham surgery group; AB, abdominal aortic coarctation group; LSG, left ganglionectomy group; RSG, right ganglionectomy group. Error bars, mean $\pm$ SEM (n = 3, each group). p values were determined by one-way ANOVA.

Calcium ions play a crucial role in cardiac excitation, with excessive diastolic calcium ion (Ca²⁺) leakage leading to arrhythmia and heart failure. Previous studies have demonstrated that calcium/calmodulin-dependent kinase II (CaMKII) enhances RyR2-mediated Ca²⁺ leakage through the phosphorylation of RyR2 at Ser2814 [23]. However, the precise mechanism by which hyperactivity of the stellate ganglia results in abnormal calcium loading in cardiomyocytes remains unclear. To address this, we assessed the expression levels of CaMKII and RyR2 in different experimental groups by immunoblotting and RT-qPCR. Our findings indicate that chronic heart failure significantly elevates in total CaMKII protein levels, but has no change in the total of RyR2 (Fig. 5A,B). Importantly, stellate ganglionectomy effectively mitigated these increases, reversing the elevated expression levels of total CaMKII protein and the RyR2 phosphorylation associated with chronic heart failure (Fig. 5C,D).

Fig. 5.

Stellate ganglionectomy reverses abnormal calcium regulation in chronic heart failure. (A,B) RT-qPCR was used to measure the gene expression levels of calcium/calmodulin-dependent kinase II (CaMKII) and Ryanodine Receptor 2 (RyR2) in the cardiac tissues of each group. (C) Western blotting was used to detect the protein expression levels of CaMKII, phosphorylated RyR2 and total RyR2 in the cardiac tissues of each group. (D) Statistical analysis of CaMKII and phosphorylated RyR2 expression. Sham, sham surgery group; AB, abdominal aortic coarctation group; LSG, left ganglionectomy group; RSG, right ganglionectomy group. Error bars, mean $\pm$ SEM. (n = 3, each group). p values were determined by one-way ANOVA.