Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Exp Physiol. 2018 Nov 29;104(1):28–38. doi: 10.1113/EP087351

Low-level transcutaneous vagus nerve stimulation attenuates cardiac remodeling in a rat model of heart failure with preserved ejection fraction

Liping Zhou 1, Adrian Filiberti 2, Mary Beth Humphrey 2, Christian D Fleming 2, Benjamin J Scherlag 2,3, Sunny S Po 2,3, Stavros Stavrakis 2,3
PMCID: PMC6312463  NIHMSID: NIHMS996440  PMID: 30398289

Abstract

Inflammation and fibrosis play a central role in the development of heart failure with preserved ejection fraction (HFpEF). We previously showed that low-level, transcutaneous stimulation of the vagus nerve at the tragus (LLTS) is anti-inflammatory. We investigated the effect of chronic intermittent LLTS on cardiac inflammation, fibrosis and diastolic dysfunction in a rat model of HFpEF. Dahl salt-sensitive (DS) rats were randomized in 3 groups: low salt (LS, 0.3% NaCl; n=12; control group without stimulation) and high salt (HS, 4% NaCl) with either active (n=18) or sham (n=18) LLTS at 7 weeks of age. After 6 weeks of diet (baseline), sham or active LLTS (20Hz, 2mA, 0.2ms) was implemented for 30 minutes daily for 4 weeks. Echocardiography was performed at baseline and 4 weeks after treatment (endpoint). At endpoint, left ventricle (LV) histology and gene expression were examined. After 6 weeks of diets, HS rats developed hypertension and LV hypertrophy compared to LS rats. At endpoint, LLTS significantly attenuated blood pressure elevation, prevented the deterioration of diastolic function and improved LV circumferential strain, compared to the HS sham group. LV inflammatory cell infiltration and fibrosis were attenuated in the HS active compared to the HS sham group. Pro-inflammatory and pro-fibrotic genes [tumor necrosis factor, osteopontin, interleukin (IL)-11, IL-18 and IL-23A] were differentially altered in the 2 groups. Chronic intermittent LLTS ameliorates diastolic dysfunction, and attenuates cardiac inflammation and fibrosis in a rat model of HFpEF, suggesting that LLTS may be used clinically as a novel noninvasive neuromodulation therapy in HFpEF.

Keywords: Heart failure with preserved ejection fraction, Neuromodulation, Autonomic Nervous System, Inflammation, Fibrosis

Introduction

Heart failure with preserved ejection fraction (HFpEF) has become a major public health concern, accounting for approximately half of all heart failure patients (Lam et al. 2011, Shah et al. 2017). The hospitalizations related to HFpEF are increasing and outcomes of patients with HFpEF are poor, yet no treatment has been shown to decrease morbidity and mortality (Fonarow et al. 2007, Butler et al. 2014). Recent animal and human studies suggest that a systemic proinflammatory state, produced by comorbidities, including aging and hypertension, plays a central role in the development of HFpEF (Paulus and Tschope 2013, Franssen et al. 2016, Frantz et al. 2018). This proinflammatory state leads to left ventricular (LV) fibrosis, diastolic dysfunction and HFpEF (Paulus and Tschope 2013). Therefore, attenuating the proinflammatory state and suppressing cardiac fibrosis is an attractive therapeutic target for HFpEF. Importantly, reduction of inflammation and fibrosis normalized LV diastolic function and improved survival in a rat model of HFpEF, without attenuation of LV hypertrophy, suggesting that inflammation and fibrosis are not only causative in HFpEF, but also potentially reversible (Gallet et al. 2016).

The anti-inflammatory effects of vagus nerve stimulation (VNS) have been well established (Abboud et al. 2012, Pavlov and Tracey 2015). VNS can be accomplished transcutaneously by stimulating the auricular branch of the vagus nerve (Fallgatter et al. 2003). Using this approach, Wang et al. demonstrated suppression of the inflammatory response, attenuation of cardiac fibrosis and improvement in cardiac function in a canine model of chronic myocardial infarction (Wang et al. 2014). In addition, transcutaneous VNS suppressed lipopolysaccharide-induced serum inflammatory cytokines in rats with endotoxemia (Zhao et al. 2012) and reduced ischemia-induced inflammation and attenuated tissue injury in a rat model of acute ischemic brain injury (Ay et al. 2016). In a recent proof-of-concept study in humans, low-level transcutaneous VNS (LLTS) significantly suppressed systemic inflammatory cytokines in patients with paroxysmal atrial fibrillation (Stavrakis et al. 2015). Collectively, these data suggest that LLTS may be used as a novel non-pharmacological treatment modality for cardiovascular conditions, where inflammation plays a key role, including HFpEF.

The Dahl salt sensitive (DS) rat model is a well-established model of HFpEF and has been widely used to test new treatments for HFpEF (Valero-Munoz et al. 2017). DS rats fed with high salt diet develop progressive hypertension and LV hypertrophy and, eventually, HFpEF (Doi et al. 2000, Valero-Munoz et al. 2017). Importantly, the degree of hypertension depends on the salt amount in the diet and the age of initiation of the high salt-diet (Doi et al. 2000). In the present study, we aimed to investigate the effects of chronic, intermittent LLTS on diastolic dysfunction, inflammation and fibrosis in a DS rat model of HFpEF.

Methods

Ethical approval

The experimental design of all animal experiments was approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Science Center (protocol number 16–106). All the experiments were carried out according to the guidelines laid down by the University of Oklahoma Health Sciences Center animal welfare committee and conform to the previously described principles and regulations for animal experimentation (Grundy 2015), and all steps were taken to minimize the animals’ pain and suffering during the experiments. All animals were obtained from Charles River Laboratories (Wilmington, MA).

Experimental animals

Forty-eight male DS rats were fed with 0.3% NaCl (low salt, LS) diet until 7 weeks of age. As shown in Figure 1A, rats at 7 weeks of age were randomly assigned in a 3:1 to ratio to receive either 4% NaCl diet [high salt (HS) diet] or 0.3% NaCl diet [low salt (LS) diet] for 6 weeks. Twelve DS rats fed LS diet comprised the control group. Water was provided ad libitum. HS diet DS rats were randomized in a 1:1 ratio to either active (n=18) or sham (n=18) LLTS starting at 13 weeks of age. Heart rate, blood pressure (BP) and body weight were measured and echocardiography was performed at 6 weeks after LS/HS diet (baseline) and 4 weeks after treatment (endpoint). BP measurements were obtained non-invasively using the tail-cuff method (AD Instrument, PowerLab Data Acquisition System, New South Wales, Australia). Three animals (1 in each group) died after baseline examination but before 4 weeks endpoint and were not included in the final analysis. At the end of 4 weeks, the animals were euthanized with isoflurane 5% by inhalation and the heart was harvested for analysis (Figure 1A).

Figure 1.

Figure 1.

Open in a new tab

Study protocol, representative images of active LLTS and sham LLTS and ECG tracings before and during active LLTS. (A) Study protocol; (B) Active LLTS; (C) Sham LLTS; (D) Active/sham LLTS area. (E) Representative ECG tracings from one animal at baseline and during active LLTS, indicating a modest prolongation of the RR interval during active stimulation. Note the stimulation artifact in the latter tracing. LLTS, low-level transcutaneous vagus nerve stimulation; BP, blood pressure; HR, heart rhythm; BW, body weight.

LLTS

LLTS (20 Hz frequency, 0.2ms pulse duration, 2mA amplitude) was delivered through a TENS device (InTENSity™ Twin Stim®) for 30 minutes daily over a 4-week period, under 2% isoflurane anesthesia, by placing two oppositely charged magnetic electrodes over the auricular concha region (Figure 1B, D), inside (cathode) and outside (anode), respectively, at each ear, as previously described (Wang et al. 2015). In the HS sham group, the electrodes were placed on the auricular margin (Figure 1C, D), which is devoid of vagus innervation (Peuker and Filler 2002). Consistent with previous transcutaneous VNS studies where sham stimulation was used as the comparison group (Wang et al. 2015, Ay et al. 2016), we did not include a HS diet, no stimulation, control group in this study. The LLTS stimulation parameters were selected empirically, based on the observation that 1 hour of LLTS decreased serum inflammatory cytokines in our previous study in humans (Stavrakis et al. 2015). Furthermore, we elected to stimulate for 30 minutes daily for 4 weeks, given that this regimen normalized glucose levels in diabetic rats in a recent study (Wang et al. 2015). During acute stimulation (averaging all the rats during the baseline and endpoint sessions), active LLTS resulted in a small, but significant drop in heart rate (baseline: 356±15bpm vs. active LLTS: 348±14bpm; p=0.01), suggesting that vagal fibers were stimulated (Figure 1E).

Echocardiography

Echocardiography (Acuson SC2000, Siemens) was performed at 6 weeks after LS/HS diet (baseline) and 4 weeks after treatment (endpoint) to assess diastolic function, under isoflurane 2% anesthesia. Two-dimensional long axis and short axis LV images were obtained using a 10MHz transducer and Pulse-wave Doppler spectra of mitral inflow (E and A waves), as well as mitral annulus tissue Doppler spectra were recorded. The early diastolic mitral annulus velocity (e’) was used to assess diastolic function as previously described (Horgan et al. 2014). Medial and lateral mitral annular e’ velocities were obtained and averaged. In addition, the ratio of early to late mitral inflow Doppler velocity (E/A ratio) and the E/e’ ratio, which correlate with LV diastolic relaxation and stiffness and LV filling pressures, respectively (Horgan et al. 2014), were calculated. LV strain was obtained off-line, in a blinded fashion, using a speckle-tracking algorithm (Acuson SC2000 eSie VVI ™). Due to the poor reproducibility of apical long axis views in rodents, which would render longitudinal strain inaccurate (Bonios et al. 2011), we only analyzed circumferential strain. LV mass was calculated based on M-mode measurements, as previously described (Pawlush et al. 1993). This method demonstrates very good correlation with actual LV mass measurements at necropsy (Pawlush et al. 1993).

Histological measurements

At the end of the experiment, LV tissue was quickly excised and fixed in 4% formalin for histological study. Paraffin blocks were processed and cut into 5μm sections for hematoxylin-eosin (HE) and Masson’s trichrome staining. The number of infiltrating mononuclear cells was counted in a blinded manner by examining 5 fields per section with the use of a microscope (Olympus BX51) at ×400 magnification in each slide, as previously described (Yamamoto et al. 2018). Fibrosis was quantified using the Bioquant software (BIOQUANT Image Analysis, Nashville, TN).

Myocardial gene expression analysis

Resected LV sections were immediately frozen on ice and then stored at −80°C until homogenization. RNA was isolated from homogenized LV samples according to manufacturer’s instructions, using a combined Trizol and RNAqueous 4PCR Kit (Invitrogen) method, with DNase added to remove contaminant DNA. Then, cDNA was generated using a RT2 First Strand Kit (Qiagen Sciences Inc., Germantown, MD) according to manufacturer’s instructions. Gene expression of inflammatory cytokines was quantified, relative to the geometric mean of 5 stable reference genes, using a Qiagen RT2 Profiler PCR Array (PARN-150Z). To evaluate the effect of active relative to sham treatment, fold-change gene expression relative to control (LS group) was calculated using the ΔΔCt method (Yoshida et al. 2014). The investigators performing the gene expression assays were blinded to group assignment. Based on previous investigations showing activation of the cholinergic anti-inflammatory pathway by VNS (Pavlov and Tracey 2015), we hypothesized that LLTS would alter the expression of pro-inflammatory genes related to this pathway.

Statistical analysis

Continuous variables were presented as mean ± standard deviation. Baseline data and histological data at endpoint were compared among groups with one-way analysis of variance (ANOVA). Echocardiographic parameters were compared among groups using repeated measures ANOVA. All pair-wise testing was adjusted for multiple comparisons by Tukey’s method. Gene expression data were analyzed using a Wilcoxon rank-sum test and were adjusted for multiple comparisons using the Benjamini-Hochberg method. Correlations between gene expression data and echocardiographic parameters and BP were examined using Spearman’s rank-order correlation. Statistical significance was set at p<0.05. All analyses were performed using SAS 9.3 software (SAS Institute, Inc., Cary, NC).

Results

Development of hypertension and LV hypertrophy in HS rats

The baseline characteristics measured after 6 weeks of diet in the LS and HS groups are summarized in Table 1. Rats in the HS group developed increased BP (systolic BP: 129.4±14.6 mmHg vs. 114.1±17.4 mmHg, P<0.05; diastolic BP: 89.9±12.7 mmHg vs. 70.1±14.1 mmHg, P<0.05) and LV hypertrophy (interventricular septum: 2.3±0.2 mm vs. 2.0±0.1 mm, P<0.05) compared to LS rats. Baseline body weight, heart rate and LV ejection fraction were similar among the groups. There were no statistically differences between the 2 HS groups at baseline (Table 1).

Table 1.

Baseline vital signs and echocardiographic measurements in the LS and HS group

Variable Group P value
LS HS HS active HS sham LS vs. HS HS active vs. HS sham
Weight (g) 393.5±20.2 393.8±22.9 394.2±28.9 393.3±28.7 0.97 0.93
Systolic BP (mmHg) 114.1±17.4 129.4±14.6 128.6±10.5 130.3±18.5 0.03 0.81
Diastolic BP (mmHg) 70.1±14.1 89.9±12.7 88.7±10.0 91.1±15.4 0.003 0.69
Heart Rate (bpm) 354.0±7.9 357.2±16.0 359.8±14.2 354.6±17.9 0.08 0.64
LV ejection fraction (%) 74.4±6.8 75.9±7.2 75.9±6.5 75.8±8.1 0.55 0.96
LV posterior wall (mm) 2.1±0.2 2.4±0.3 2.4±0.3 2.3±0.3 0.04 0.14
Interventricular septum (mm) 2.0±0.1 2.3±0.2 2.3±0.3 2.3±0.3 0.001 0.73
LV diameter (mm) 5.7±0.8 5.1±0.9 5.2±1.0 5.0±0.6 0.05 0.65
Mitral E/A ratio 1.4±0.1 1.5±0.2 1.5±0.2 1.6±0.3 0.23 0.28
Average e’ (cm/s) 6.6±1.3 7.3±1.3 7.6±1.3 7.0±1.2 0.17 0.26
Average E/e’ ratio 9.0±1.4 9.1±1.5 8.9±1.5 9.3±1.6 0.99 0.42
Circumferential strain (%) −23.9±2.9 −22.2±3.7 −22.5±3.4 −21.8±3.9 0.16 0.58
Open in a new tab

HS = high salt; LS = low salt; BP = blood pressure; LV = left ventricle

LLTS significantly attenuated blood pressure elevation

The results at endpoint (4 weeks after treatment) are summarized in Table 2. Four weeks after treatment, BP in the HS sham group significantly increased compared to the LS group (systolic BP: 158.0±19.6 mmHg vs. 116.8±19.4 mmHg, respectively, P<0.05; diastolic BP: 115.0±21.0 mmHg vs. 79.6±21.4 mmHg, respectively, P<0.05). However, LLTS significantly attenuated the BP elevation in the HS active group compared to HS sham group (systolic BP: 124.1±21.0 mmHg vs. 158.0±19.6 mmHg, respectively, P<0.05; DBP: 86.6±21.8 mmHg vs. 115.0±21.0 mmHg, respectively, P<0.05) (Figure 2A, B).

Table 2.

Endpoint measurements in the, LS, HS active and HS sham groups

Variable Group P value
LS HS active HS sham HS active vs. HS sham
Weight (g) 418.6±28.7 399.7±26.8 406.8±28.8 0.47
Systolic BP (mmHg) 116.8±19.4 124.1±21.0 158.0±19.6 0.0005
Diastolic BP (mmHg) 79.6±21.4 86.6±21.8 115.0±21.0 0.003
Heart Rate (bpm) 350.0±32.5 350.0±23.1 371.0±30.5 0.06
LV ejection fraction (%) 73.1±8.3 75.9±6.6 73.2±9.8 0.79
LV posterior wall (mm) 2.3±0.2 2.3±0.3 2.8±0.3 0.0002
Interventricular septum (mm) 2.1±0.2 2.2±0.2 2.7±0.2 0.0001
LV diameter (mm) 5.3±0.2 5.6±0.2 5.3±0.3 0.21
Mitral E/A ratio 1.4±0.2 1.4±0.2 1.7±0.3 0.04
Average e’ (cm/s) 9.1±0.2 9.0±0.2 7.0±0.1 0.003
Average E/e’ ratio 8.0±1.7 8.1±2.1 11.1±2.3 0.002
Circumferential strain (%) −25.8±4.2 −24.1±4.1 −19.7±4.1 0.009
Fibrosis area (%) 2.3±1.2 2.5±1.5 4.0±2.1 0.02
Inflammatory cell area (%) 6.5±1.7 16.0±2.9 22.7±4.1 0.0001
Open in a new tab

HS = high salt; LS = low salt; BP = blood pressure; LV = left ventricle

Figure 2.

Figure 2.

Open in a new tab

Effects of LLTS on blood pressure and LV hypertrophy at endpoint. After 4 weeks of stimulation, LLTS significantly attenuated the elevation of systolic (A) and diastolic (B) blood pressure induced by HS diet in the HS active group compared to the HS sham group. (C) Representative parasternal long axis M-mode images at endpoint in each group. HS diet induced significant increase in interventricular septum (IVS; D) and LV posterior wall (LVPW; E) thickness in the HS sham group compared to the LS group (#P<0.05). LLTS significantly attenuated LV hypertrophy in the HS active compared to the HS sham group (*P<0.05). # P<0.05 vs. LS group; *P<0.05 vs. HS sham group.

LLTS significantly reduced LV hypertrophy

At endpoint, LV wall thickness in the HS sham group significantly increased compared to the LS group (interventricular septum: 2.7±0.2 mm vs. 2.1±0.2 mm, respectively, P<0.05; LV posterior wall: 2.8±0.3 mm vs. 2.3±0.2 mm, respectively, P<0.05) (Figure 2D, E). Chronic intermittent LLTS prevented further development of LV hypertrophy in the HS active compared to HS sham group (interventricular septum: 2.2±0.2 mm vs. 2.7±0.2 mm, respectively, P<0.05; LV posterior wall: 2.3±0.3 mm vs. 2.8±0.3 mm, respectively, P<0.05) (Figure 2D, E). Representative parasternal long axis M-mode views of the LV at endpoint in each group are shown in Figure 2C. At endpoint, LV mass was significantly increased in the HS sham group compared to LS group (1.2±0.2 g vs. 0.8±0.1 g, respectively; p=0.001), while LLTS prevented this process (0.9±0.1 g vs. 1.2±0.2 g; p=0.001).

LLTS prevented the deterioration of LV diastolic function and improved LV strain

Representative pulse wave Doppler showing E (early filling) and A (late filling) wave in each group at endpoint are shown in Figure 3A. At endpoint, diastolic function in the HS sham group significantly deteriorated compared to the LS group, as indicated by an increase in the E/A ratio (1.7±0.3 vs. 1.4±0.2, respectively; P<0.05), consistent with pseudonormalization pattern as seen with progression of diastolic dysfunction (Maragiannis and Nagueh 2015) (Figure 3B). Likewise, there was an increase in the E/e’ ratio in the HS sham group compared to the LS group (11.1±2.3 vs. 8.0±1.7, respectively; P<0.05), consistent with elevated LA pressures (Maragiannis and Nagueh 2015) (Figure 3C). In addition, LV circumferential strain deteriorated in the HS sham group compared to the LS group (−19.7±4.1% vs. −25.8±4.2%, respectively; P<0.05) (Figure 3D). Chronic intermittent LLTS significantly prevented the deterioration of diastolic function in the HS active group as compared to the HS sham group (E/A ratio: 1.4±0.2 vs. 1.7±0.3, respectively; P<0.05; E/e’ ratio: 8.1±2.1vs. 11.1±2.3, respectively, P<0.05) and improved LV circumferential strain (−24.1±4.1% vs. −19.7±4.1%, respectively; P<0.05) (Figure 3B–D). The LV ejection fraction remained unchanged in both HS groups (Figure 3E).

Figure 3.

Figure 3.

Open in a new tab

Effects of LLTS on LV diastolic function, cardiac mechanics and ejection fraction. (A) Representative pulse wave Doppler showing E (early filling) and A (late filling) wave in each group at endpoint. HS diet induced significant changes in the E/A ratio (B), E/e’ ratio (C) and circumferential strain (D), while LLTS attenuated this effect. There were no changes in LV ejection fraction (E). # P<0.05 vs. LS group; *P<0.05 vs. HS sham group.

LLTS attenuated LV inflammatory cell infiltration and fibrosis

Representative images of the LV inflammatory cell infiltration are shown in Figure 4A. Quantitative analysis revealed that HS diet induced a significant increase of LV inflammatory cell infiltration in the HS sham group compared to the LS group (22.7±4.1/mm2 vs. 6.5±1.7/mm2, respectively, P<0.05) (Figure 4C). However, active LLTS significantly decreased LV inflammatory cell infiltration in the HS active compared to the HS sham group (16.0±2.9/mm2 vs. 22.7±4.1/mm2, respectively, P<0.05) (Figure 4C).

Figure 4.

Figure 4.

Open in a new tab

Effects of LLTS on LV inflammatory cell infiltration and fibrosis. Representative LV inflammatory infiltration (A) and fibrosis (B) in each group. HS diet induced significant increase of LV inflammatory cell infiltration (C) and fibrosis (D) in the HS sham group compared to the LS group, while LLTS attenuated this effect. #P<0.05 vs. LS group; *P<0.05 vs. HS sham group.

Representative images of the fibrosis areas are shown in Figure 4B. Quantitative analysis revealed that active LLTS significantly decreased LV fibrosis in the HS active group compared to the HS sham group (2.5±1.2% vs. 4.0±2.1%, respectively, P<0.05), to the levels seen in the LS group (2.3±1.5%) (Figure 4D).

LLTS induced changes in myocardial gene expression

Gene expression analysis revealed that 4 weeks of LLTS significantly altered the fold-change (relative to LS) in gene expression compared to sham stimulation, with a total of 5 genes encoding for inflammatory cytokines being differentially expressed between the HS active and HS sham groups (Figure 5). Genes that were upregulated in the HS sham group and downregulated in the HS active group, included inflammatory interleukins (IL-11, IL-18 and IL-23A) and immune modulators (SPP1, osteopontin). Overall, these results indicate upregulation of pro-inflammatory and pro-fibrotic genes in the HS sham group, compared to LS rats, an effect which was reversed by LLTS. To explore the extent to which changes in the expression of these pro-inflammatory and pro-fibrotic genes predicted differences in echocardiographic parameters and BP among the groups, we examined the correlation between gene expression relative to control and echocardiographic measures (circumferential strain), as well as systolic BP using Spearman’s rank-order correlation. We found that gene expression of IL-23A (r=0.61, 95% CI 0.24 to 0.82; p=0.003), IL-18 (r=0.65, 95% CI 0.28 to 0.85; p=0.002), IL-11 (r=0.48, 95% CI 0.05 to 0.76; p=0.02) and osteopontin (r=0.43, 95% CI 0.01 to 0.73; p=0.05) correlated positively with circumferential strain (i.e. higher gene expression of these pro-inflammatory and pro-fibrotic genes predicted a poorer circumferential strain). On the contrary, expression of these genes did not correlate with changes in systolic BP, except for IL-11, which showed a modest positive correlation (r=0.45, 95% CI 0.01 to 0.75; p=0.04). These data lend further support to the notion that LLTS prevented the deterioration of LV function by suppressing the myocardial expression of pro-inflammatory and pro-fibrotic genes.

Figure 5.

Figure 5.

Open in a new tab

Effect of LLTS on myocardial RNA gene expression. Median values with interquartile range of statistically significant differences in fold change expression (relative to LS) between HS sham (n=15) and HS active (n=14) groups are shown. Y axis is in Log2 scale. TNF=tumor necrosis factor; IL=interleukin; SPP1=osteopontin.

Discussion

In this study, chronic intermittent LLTS for 4 weeks significantly attenuated cardiac functional and structural remodeling in a rat model of HFpEF. As expected, HS diet induced significant BP elevation, LV hypertrophy and diastolic dysfunction. However, 4-weeks of intermittent daily LLTS significantly attenuated systolic and diastolic BP elevation and improved LV diastolic dysfunction induced by HS diet. Importantly, LLTS suppressed cardiac inflammatory cell infiltration and fibrosis and favorably altered the myocardial expression of key pro-inflammatory and pro-fibrotic genes (tumor necrosis factor, IL-11, IL-18, IL-23A and osteopontin). These data suggest that LLTS may be used as a novel noninvasive neuromodulation therapy to treat HFpEF by suppressing cardiac inflammation and fibrosis.

Inflammation and fibrosis represent the major mechanisms underlying cardiac remodeling in HFpEF (Paulus and Tschope 2013, Franssen et al. 2016, Frantz et al. 2018). Comorbidities, such as aging, obesity, hypertension and diabetes induce a systemic pro-inflammatory state, which leads to recruitment of macrophages and monocytes into the heart. These inflammatory cells contribute to LV fibrosis by promoting the differentiation of fibroblasts into myofibroblasts through the release of transforming growth factor (TGF)-β (Paulus and Tschope 2013, Frantz et al. 2018). The resulting increase in LV collagen content is a major contributor to the increase in passive myocardial fiber stiffness, which eventually leads to diastolic dysfunction and HFpEF. Importantly, it has been recently shown that the number of macrophages in the heart increase both in animal models and in humans with HFpEF (Hulsmans et al. 2018). Collectively, these data indicate that macrophages and their secreted proteins play a central role in cardiomyocyte hypertrophy and HFpEF progression (Frantz et al. 2018). Consistent with these data, we have shown that inflammatory cell infiltration in the heart and LV fibrosis was increased in HS rats compared to LS rats, while LLTS attenuated this effect and ameliorated diastolic dysfunction. Moreover, our data suggest that early intervention targeting inflammation and cardiac fibrosis may prevent the deterioration of heart function and prevent the development of HFpEF.

VNS exerts prominent anti-inflammatory effects (Abboud et al. 2012, Pavlov and Tracey 2015). Current evidence suggests that the vagus nerve provides the efferent and possibly the afferent limb of the cholinergic anti-inflammatory pathway, by which the brain modulates inflammation (Pavlov and Tracey 2015). Consistent with the anti-inflammatory effects of VNS, we have recently shown in a first-in-man clinical trial, that in patients with drug-refractory atrial fibrillation, LLTS for just 1 hour significantly suppressed atrial fibrillation and decreased systemic inflammatory cytokines (Stavrakis et al. 2015). Studies also showed that short-term LLTS significantly suppressed ventricular arrhythmias, decreased cardiac and systemic inflammation, attenuated cardiac fibrosis and improved ventricular function both in a chronic myocardial infarction canine model (Wang et al. 2014) and in patients with myocardial infarction undergoing primary percutaneous coronary intervention (Yu et al. 2017). Importantly, VNS exhibits memory, whereby short periods of stimulation result in long-term anti-inflammatory effects (Koopman et al. 2016). In a recent study in patients with rheumatoid arthritis, VNS for 1 minute up to 4 times a day led to an improvement in disease severity and reduction in inflammatory cytokines (Koopman et al. 2016). In addition, 30 minutes of LLTS per day for 4 weeks normalized glucose levels in diabetic rats (Wang et al. 2015). Notably, the neural processing elements of the cardiac autonomic nervous system also demonstrate memory to VNS (Salavatian et al. 2016), which may also contribute to the cardioprotective effects of LLTS in our study. In the present study, short-term, intermittent LLTS, delivered daily for 4 weeks, significantly suppressed cardiac inflammation and fibrosis, attenuated BP elevation and improved LV hypertrophy and diastolic dysfunction in a rat model of HFpEF. Collectively, these data raise the intriguing possibility that short periods of LLTS may result in a long-lasting anti-inflammatory and anti-fibrotic effect, which in turn may translate into improved clinical outcomes in patients with HFpEF.

HFpEF is characterized by autonomic imbalance with increased sympathetic nerve activity, which is closely associated with diastolic dysfunction (Grassi et al. 2009, Aikawa et al. 2017), while clinical studies have shown that sympathetic hyperactivity contributes to the development of diastolic dysfunction in patients with hypertension and to disease progression in patients with HFpEF (Florea and Cohn 2014). Consistent with our results, a recent study revealed that right cervical VNS decreased sympathetic activity and attenuated the HS diet-induced increase of BP in DS rats (Annoni et al. 2015). The mechanism of this effect involves inhibition of central sympathetic outflow (Annoni et al. 2015). We, and others, have previously shown that VNS, even at subthreshold levels not causing bradycardia, exerted antiadrenergic effects, by injuring the sympathetic neurons in the stellate ganglia (Sha et al. 2011, Chinda et al. 2016). Moreover, VNS attenuated cardiac remodeling and suppressed sympathoexcitation in a guinea-pig model of HFpEF induced by constriction of the thoracic aorta leading to chronic pressure overload (Beaumont et al. 2016). In addition, recent studies in humans have shown that LLTS suppresses sympathetic nerve activity (Clancy et al. 2014, Deuchars et al. 2018). Notably, LLTS inhibited norepinephrine release from cardiac sympathetic nerves, dilated cardiac microcirculatory vessels and increased exercise tolerance in patients with coronary artery disease (Zamotrinsky et al. 1997, Zamotrinsky et al. 2001). Importantly, the anti-adrenergic effects of VNS have been shown to impact basal and reflex function within the intrinsic cardiac nervous system (Beaumont et al. 2016). In light of the autonomic imbalances and impaired autonomic reflexes in HFpEF (Grassi et al. 2009, Deuchars et al. 2018), which in turn are associated with adverse neural remodeling in multiple neural circuits within the neural hierarchy for cardiac control, we speculate that LLTS targets specific elements within the intrinsic cardiac autonomic nervous system to stabilize excessive reflex responses, which are responsible for disease progression (Florea and Cohn 2014), thereby mitigating efferent output. It is also likely that LLTS improves diastolic function by both a direct effect on the myocardium related to inflammation and by reducing BP through its anti-adrenergic effects. The degree to which each of these mechanisms contributes to the improvement in diastolic dysfunction requires further investigation.

The mechanism of the effect of LLTS in our study may be related to changes in myocardial gene expression, insofar as key pro-fibrotic and pro-inflammatory genes were differentially altered. Previously, pressure overload increased IL-18 and osteopontin gene and protein expression in mice, resulting in cardiac fibrosis and diastolic dysfunction (Yu et al. 2009). In another study, IL-18 mediated cardiac hypertrophy in rabbits with pressure overload (Yoshida et al. 2014). Moreover, recent studies in murine models of HFpEF and humans with the disease, identified osteopontin as an important mediator in the interaction between macrophages and fibroblasts, where macrophages activate the proliferation of myofibroblasts and production of collagen, which then leads to fibrosis-mediated cardiac stiffness and eventually HFpEF (Hulsmans et al. 2018). In addition, IL-11 is a crucial determinant of cardiac fibrosis (Schafer et al. 2017). In our study, myocardial gene expression of IL-23A, IL-18, IL-11 and osteopontin correlated positively with circumferential strain (i.e. higher gene expression of these pro-inflammatory and pro-fibrotic genes predicted a poorer circumferential strain). Collectively, these data suggest that LLTS may have attenuated the effects of HS diet and prevented the development of diastolic dysfunction in our study by suppressing the myocardial expression of pro-inflammatory and pro-fibrotic genes. Further studies are warranted to elucidate the exact mechanism of LLTS-mediated amelioration of cardiac fibrosis and hypertrophy.

Study limitations

In the present study, we used 4% NaCl diet, instead of 8%, as was originally described (Doi et al. 2000, Valero-Munoz et al. 2017), and thus the animals did not develop overt signs of heart failure. However, one of the caveats of the DS model for HFpEF is that the BP levels achieved are non-physiologic (>175mmHg), which limits its clinical applicability (Valero-Munoz et al. 2017). Therefore, we opted to feed the HS rats with 4% NaCl diet to achieve BP levels seen in humans with HFpEF and mimic an early stage of HFpEF, as indicated by worsening diastolic function and LV strain, as well as increased cardiac inflammation and fibrosis. In this study, it is not possible to differentiate between the effects of regression of LV hypertrophy and reduction of inflammation and fibrosis on diastolic dysfunction. Nonetheless, it has been previously shown that improvement in diastolic dysfunction can occur without regression of LV hypertrophy in a murine model of HFpEF (Wilson et al. 2009) and that interferon-γ regulates cardiac hypertrophy independent of BP (Garcia et al. 2012), collectively suggesting that improvement in LV inflammation may result in enhanced diastolic function. Further studies are required to investigate the degree to which each of these mechanisms contributes to amelioration of diastolic dysfunction. We did not perform cardiac autonomic activity recordings in this study. Given the role of autonomic dysfunction in HFpEF (Grassi et al. 2009, Aikawa et al. 2017), further studies were warranted to study the effects of LLTS on cardiac autonomic nerve activity in HFpEF models. We only evaluated a fixed set of stimulation parameters for LLTS in our study (frequency 20Hz, pulse duration 0.2ms, amplitude 2mA). These parameters likely activated vagal afferent fibers (Ardell et al. 2017). Future studies should focus on the impact of different stimulation parameters on the observed outcomes. Animal models do not recapitulate all the features of HFpEF, including the integrative complexity of the related comorbidities, such as aging and diabetes (Butler et al. 2014). However, the DS model reproduces some of the typical features of HFpEF, including LV hypertrophy and diastolic dysfunction and is a well-accepted model for pre-clinical testing of new therapies (Valero-Munoz et al. 2017).

Clinical implications

HFpEF represents a therapeutic challenge, as no pharmacologic therapy has been shown to improve outcomes in patients with this condition (Butler et al. 2014). Neuromodulation, by exploiting the plasticity of the neural tissue to obtain therapeutic benefit without damage to the myocardium or nerves, has become an emerging therapeutic modality for the treatment of arrhythmias and other cardiovascular diseases (Hou et al. 2016). VNS through an implantable device has been used successfully in a preliminary clinical trial in patients with rheumatoid arthritis (Koopman et al. 2016). Our proof-of-concept results establish the first evidence of the effects of LLTS in HFpEF and provide the basis for the design of human studies using this modality to target selected populations with HFpEF. Even though a recent large trial using VNS in patients with heart failure with reduced ejection fraction (HFrEF) failed to show a beneficial effect (Gold et al. 2016), another trial in the same patient population, but using different stimulation parameters, demonstrated efficacy (Premchand et al. 2014), highlighting the importance of optimization of the stimulation parameters to obtain a beneficial effect. Specifically, mechanistic studies have suggested that the optimal stimulation parameters for VNS are at the point at which afferent and efferent fibers are activated in a balanced manner, i.e. the afferent-driven decreases in central parasympathetic drive are counteracted by direct activation of the cardiac parasympathetic efferent projections to the intrinsic cardiac autonomic nervous system and the heart, resulting in a relatively neutral heart rate response (Ardell et al. 2017). Consistent with this notion, a recent study of LLTS (30 Hz,10–50 mA, 200 μs, which was slightly below the sensory threshold) in healthy volunteers showed a significant decrease in sympathetic nerve activity without a change in heart rate (Clancy et al. 2014). In addition, IL-1 blockade with anakinra in patients with HFpEF improved peak oxygen consumption in a recent randomized cross-over trial (Van Tassell et al. 2014). In support of the notion that autonomic stimulation may be an attractive non-pharmacologic approach to treat patients with HFpEF, an ongoing clinical study (ANTHEM-HFpEF) is designed to evaluate the feasibility, tolerability and safety of right cervical VNS in patients with symptomatic HFpEF (DiCarlo et al. 2018).

Conclusion

Chronic intermittent LLTS could significantly improve cardiac diastolic dysfunction and attenuate cardiac inflammation and fibrosis in a rat model of HFpEF. These data suggest that LLTS may be used clinically as a novel noninvasive neuromodulation therapy to treat patients with HFpEF. Further studies to examine the efficacy of this novel treatment in patients with HFpEF are warranted.

New findings:

What is the central question of this study?

In this study, we investigated the effect of chronic intermittent low-level transcutaneous vagus nerve stimulation on cardiac inflammation, fibrosis and diastolic dysfunction in a rat model of heart failure with preserved ejection fraction.

What is the main finding and its importance?

In salt-sensitive rats fed with high salt diet, we show that low-level transcutaneous vagus nerve stimulation significantly attenuates blood pressure elevation, ameliorates diastolic function and attenuates left ventricular inflammation and fibrosis compared to the sham group. Further studies to examine the efficacy of this novel treatment in humans are warranted.

Acknowledgments

Funding: Funded by a Presbyterian Health Foundation Seed grant and in part by NIH/NIA R21AG057879 to Stavros Stavrakis

Footnotes

Conflicts of interest: None

References:

  1. Abboud FM, Harwani SC & Chapleau MW (2012). Autonomic neural regulation of the immune system: implications for hypertension and cardiovascular disease. Hypertension. 59, 755–762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aikawa T, Naya M, Obara M, Manabe O, Tomiyama Y, Magota K, … Tsutsui H (2017). Impaired Myocardial Sympathetic Innervation Is Associated with Diastolic Dysfunction in Heart Failure with Preserved Ejection Fraction: (11)C-Hydroxyephedrine PET Study. J Nucl Med. 58, 784–790. [DOI] [PubMed] [Google Scholar]
  3. Annoni EM, Xie X, Lee SW, Libbus I, KenKnight BH, Osborn JW & Tolkacheva EG (2015). Intermittent electrical stimulation of the right cervical vagus nerve in salt-sensitive hypertensive rats: effects on blood pressure, arrhythmias, and ventricular electrophysiology. Physiol Rep. 3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ardell JL, Nier H, Hammer M, Southerland EM, Ardell CL, Beaumont E, … Armour JA (2017). Defining the neural fulcrum for chronic vagus nerve stimulation: implications for integrated cardiac control. J Physiol. 595, 6887–6903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ay I, Nasser R, Simon B & Ay H (2016). Transcutaneous Cervical Vagus Nerve Stimulation Ameliorates Acute Ischemic Injury in Rats. Brain Stimul. 9, 166–173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beaumont E, Wright GL, Southerland EM, Li Y, Chui R, KenKnight BH, … Ardell JL (2016). Vagus nerve stimulation mitigates intrinsic cardiac neuronal remodeling and cardiac hypertrophy induced by chronic pressure overload in guinea pig. Am J Physiol Heart Circ Physiol. 310, H1349–1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bonios M, Chang CY, Pinheiro A, Dimaano VL, Higuchi T, Melexopoulou C, … Abraham MR (2011). Cardiac resynchronization by cardiosphere-derived stem cell transplantation in an experimental model of myocardial infarction. J Am Soc Echocardiogr. 24, 808–814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Butler J, Fonarow GC, Zile MR, Lam CS, Roessig L, Schelbert EB, … Gheorghiade M (2014). Developing therapies for heart failure with preserved ejection fraction: current state and future directions. JACC Heart Fail. 2, 97–112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chinda K, Tsai WC, Chan YH, Lin AY, Patel J, Zhao Y, … Chen PS (2016). Intermittent left cervical vagal nerve stimulation damages the stellate ganglia and reduces the ventricular rate during sustained atrial fibrillation in ambulatory dogs. Heart Rhythm. 13, 771–780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clancy JA, Mary DA, Witte KK, Greenwood JP, Deuchars SA & Deuchars J (2014). Non-invasive Vagus Nerve Stimulation in Healthy Humans Reduces Sympathetic Nerve Activity. Brain Stimul. 7, 871–877. [DOI] [PubMed] [Google Scholar]
  11. Deuchars SA, Lall VK, Clancy J, Mahadi M, Murray A, Peers L & Deuchars J (2018). Mechanisms underpinning sympathetic nervous activity and its modulation using transcutaneous vagus nerve stimulation. Exp Physiol. 103, 326–331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DiCarlo LA, Libbus I, Kumar HU, Mittal S, Premchand RK, Amurthur B, … Anand IS (2018). Autonomic regulation therapy to enhance myocardial function in heart failure patients: the ANTHEM-HFpEF study. ESC Heart Fail. 5, 95–100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Doi R, Masuyama T, Yamamoto K, Doi Y, Mano T, Sakata Y, … Hori M (2000). Development of different phenotypes of hypertensive heart failure: systolic versus diastolic failure in Dahl salt-sensitive rats. J Hypertens. 18, 111–120. [DOI] [PubMed] [Google Scholar]
  14. Fallgatter AJ, Neuhauser B, Herrmann MJ, Ehlis AC, Wagener A, Scheuerpflug P, … Riederer P (2003). Far field potentials from the brain stem after transcutaneous vagus nerve stimulation. J Neural Transm. 110, 1437–1443. [DOI] [PubMed] [Google Scholar]
  15. Florea VG & Cohn JN (2014). The autonomic nervous system and heart failure. Circ Res. 114, 1815–1826. [DOI] [PubMed] [Google Scholar]
  16. Fonarow GC, Stough WG, Abraham WT, Albert NM, Gheorghiade M, Greenberg BH, … Young JB (2007). Characteristics, treatments, and outcomes of patients with preserved systolic function hospitalized for heart failure: a report from the OPTIMIZE-HF Registry. J Am Coll Cardiol. 50, 768–777. [DOI] [PubMed] [Google Scholar]
  17. Franssen C, Chen S, Unger A, Korkmaz HI, De Keulenaer GW, Tschope C, … Hamdani N (2016). Myocardial Microvascular Inflammatory Endothelial Activation in Heart Failure With Preserved Ejection Fraction. JACC Heart Fail. 4, 312–324. [DOI] [PubMed] [Google Scholar]
  18. Frantz S, Falcao-Pires I, Balligand JL, Bauersachs J, Brutsaert D, Ciccarelli M, … Heymans S (2018). The innate immune system in chronic cardiomyopathy: a European Society of Cardiology (ESC) scientific statement from the Working Group on Myocardial Function of the ESC. Eur J Heart Fail. 20, 445–459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gallet R, de Couto G, Simsolo E, Valle J, Sun B, Liu W, … Marban E (2016). Cardiosphere-derived cells reverse heart failure with preserved ejection fraction (HFpEF) in rats by decreasing fibrosis and inflammation. JACC Basic Transl Sci. 1, 14–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garcia AG, Wilson RM, Heo J, Murthy NR, Baid S, Ouchi N & Sam F (2012). Interferon-gamma ablation exacerbates myocardial hypertrophy in diastolic heart failure. Am J Physiol Heart Circ Physiol. 303, H587–596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gold MR, Van Veldhuisen DJ, Hauptman PJ, Borggrefe M, Kubo SH, Lieberman RA, … Mann DL (2016). Vagus Nerve Stimulation for the Treatment of Heart Failure: The INOVATE-HF Trial. J Am Coll Cardiol. 68, 149–158. [DOI] [PubMed] [Google Scholar]
  22. Grassi G, Seravalle G, Quarti-Trevano F, Dell’Oro R, Arenare F, Spaziani D & Mancia G (2009). Sympathetic and baroreflex cardiovascular control in hypertension-related left ventricular dysfunction. Hypertension. 53, 205–209. [DOI] [PubMed] [Google Scholar]
  23. Grundy D (2015). Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology. Exp Physiol. 100, 755–758. [DOI] [PubMed] [Google Scholar]
  24. Horgan S, Watson C, Glezeva N & Baugh J (2014). Murine models of diastolic dysfunction and heart failure with preserved ejection fraction. J Card Fail. 20, 984–995. [DOI] [PubMed] [Google Scholar]
  25. Hou Y, Zhou Q & Po SS (2016). Neuromodulation for cardiac arrhythmia. Heart Rhythm. 13, 584–592. [DOI] [PubMed] [Google Scholar]
  26. Hulsmans M, Sager HB, Roh JD, Valero-Munoz M, Houstis NE, Iwamoto Y, … Nahrendorf M (2018). Cardiac macrophages promote diastolic dysfunction. J Exp Med. 215, 423–440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koopman FA, Chavan SS, Miljko S, Grazio S, Sokolovic S, Schuurman PR, … Tak PP (2016). Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis. Proc Natl Acad Sci U S A. 113, 8284–8289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lam CS, Donal E, Kraigher-Krainer E & Vasan RS (2011). Epidemiology and clinical course of heart failure with preserved ejection fraction. Eur J Heart Fail. 13, 18–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Maragiannis D & Nagueh SF (2015). Echocardiographic evaluation of left ventricular diastolic function: an update. Curr Cardiol Rep. 17, 3. [DOI] [PubMed] [Google Scholar]
  30. Paulus WJ & Tschope C (2013). A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation. J Am Coll Cardiol. 62, 263–271. [DOI] [PubMed] [Google Scholar]
  31. Pavlov VA & Tracey KJ (2015). Neural circuitry and immunity. Immunol Res. 63, 38–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pawlush DG, Moore RL, Musch TI & Davidson WR Jr. (1993). Echocardiographic evaluation of size, function, and mass of normal and hypertrophied rat ventricles. J Appl Physiol (1985). 74, 2598–2605. [DOI] [PubMed] [Google Scholar]
  33. Peuker ET & Filler TJ (2002). The nerve supply of the human auricle. Clin Anat. 15, 35–37. [DOI] [PubMed] [Google Scholar]
  34. Premchand RK, Sharma K, Mittal S, Monteiro R, Dixit S, Libbus I, … Anand IS (2014). Autonomic regulation therapy via left or right cervical vagus nerve stimulation in patients with chronic heart failure: results of the ANTHEM-HF trial. J Card Fail. 20, 808–816. [DOI] [PubMed] [Google Scholar]
  35. Salavatian S, Beaumont E, Longpre JP, Armour JA, Vinet A, Jacquemet V, … Ardell JL (2016). Vagal stimulation targets select populations of intrinsic cardiac neurons to control neurally induced atrial fibrillation. Am J Physiol Heart Circ Physiol. 311, H1311–H1320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schafer S, Viswanathan S, Widjaja AA, Lim WW, Moreno-Moral A, DeLaughter DM, … Cook SA (2017). IL-11 is a crucial determinant of cardiovascular fibrosis. Nature. 552, 110–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sha Y, Scherlag BJ, Yu L, Sheng X, Jackman WM, Lazzara R & Po SS (2011). Low-level right vagal stimulation: anticholinergic and antiadrenergic effects. J Cardiovasc Electrophysiol. 22, 1147–1153. [DOI] [PubMed] [Google Scholar]
  38. Shah KS, Xu H, Matsouaka RA, Bhatt DL, Heidenreich PA, Hernandez AF, … Fonarow GC (2017). Heart Failure With Preserved, Borderline, and Reduced Ejection Fraction: 5-Year Outcomes. J Am Coll Cardiol. 70, 2476–2486. [DOI] [PubMed] [Google Scholar]
  39. Stavrakis S, Humphrey MB, Scherlag BJ, Hu Y, Jackman WM, Nakagawa H, … Po SS (2015). Low-level transcutaneous electrical vagus nerve stimulation suppresses atrial fibrillation. J Am Coll Cardiol. 65, 867–875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Valero-Munoz M, Backman W & Sam F (2017). Murine Models of Heart Failure with Preserved Ejection Fraction: a “Fishing Expedition”. JACC Basic Transl Sci. 2, 770–789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van Tassell BW, Arena R, Biondi-Zoccai G, Canada JM, Oddi C, Abouzaki NA, … Abbate A (2014). Effects of interleukin-1 blockade with anakinra on aerobic exercise capacity in patients with heart failure and preserved ejection fraction (from the D-HART pilot study). Am J Cardiol. 113, 321–327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wang S, Zhai X, Li S, McCabe MF, Wang X & Rong P (2015). Transcutaneous vagus nerve stimulation induces tidal melatonin secretion and has an antidiabetic effect in Zucker fatty rats. PLoS One. 10, e0124195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang Z, Yu L, Wang S, Huang B, Liao K, Saren G, … Jiang H (2014). Chronic intermittent low-level transcutaneous electrical stimulation of auricular branch of vagus nerve improves left ventricular remodeling in conscious dogs with healed myocardial infarction. Circ Heart Fail. 7, 1014–1021. [DOI] [PubMed] [Google Scholar]
  44. Wilson RM, De Silva DS, Sato K, Izumiya Y & Sam F (2009). Effects of fixed-dose isosorbide dinitrate/hydralazine on diastolic function and exercise capacity in hypertension-induced diastolic heart failure. Hypertension. 54, 583–590. [DOI] [PubMed] [Google Scholar]
  45. Yamamoto M, Ishizu T, Seo Y, Suto Y, Sai S, Xu D, … Aonuma K (2018). Teneligliptin Prevents Cardiomyocyte Hypertrophy, Fibrosis, and Development of Hypertensive Heart Failure in Dahl Salt-Sensitive Rats. J Card Fail. 24, 53–60. [DOI] [PubMed] [Google Scholar]
  46. Yoshida T, Friehs I, Mummidi S, del Nido PJ, Addulnour-Nakhoul S, Delafontaine P, … Chandrasekar B (2014). Pressure overload induces IL-18 and IL-18R expression, but markedly suppresses IL-18BP expression in a rabbit model. IL-18 potentiates TNF-alpha-induced cardiomyocyte death. J Mol Cell Cardiol. 75, 141–151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yu L, Huang B, Po SS, Tan T, Wang M, Zhou L, … Jiang H (2017). Low-Level Tragus Stimulation for the Treatment of Ischemia and Reperfusion Injury in Patients With ST-Segment Elevation Myocardial Infarction: A Proof-of-Concept Study. JACC Cardiovasc Interv. 10, 1511–1520. [DOI] [PubMed] [Google Scholar]
  48. Yu Q, Vazquez R, Khojeini EV, Patel C, Venkataramani R & Larson DF (2009). IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice. Am J Physiol Heart Circ Physiol. 297, H76–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zamotrinsky A, Afanasiev S, Karpov RS & Cherniavsky A (1997). Effects of electrostimulation of the vagus afferent endings in patients with coronary artery disease. Coron Artery Dis. 8, 551–557. [PubMed] [Google Scholar]
  50. Zamotrinsky AV, Kondratiev B & de Jong JW (2001). Vagal neurostimulation in patients with coronary artery disease. Auton Neurosci. 88, 109–116. [DOI] [PubMed] [Google Scholar]
  51. Zhao YX, He W, Jing XH, Liu JL, Rong PJ, Ben H, … Zhu B (2012). Transcutaneous auricular vagus nerve stimulation protects endotoxemic rat from lipopolysaccharide-induced inflammation. Evid Based Complement Alternat Med. 2012, 627023. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES