TRanscutaneous Electrical vAgus Nerve sTimulation to Suppress Atrial Fibrillation
Phase: NA
Conditions: Atrial Fibrillation, Inflammation
Sponsor: University of Oklahoma

transcutaneous vagus nerve stimulation (tVNS)

transcutaneous vagus nerve 
tVNS

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improvement in heart failure functional class, quality of life, left ventricular ejection fraction and 
left ventricular end-systolic volume 12,13. We 14-18 and others 19 have recently shown that low level 
vagus nerve stimulation (LLVNS), at voltages substantially below the threshold for slowing the 
sinus rate or AV conduction, suppressed AF inducibility and decreased the duration of AF episodes 
that were induced by strong cholinergic stimulation. In those experiments, LLVNS applied to both 
vagal trunks dissected in the neck 14-16, to the right vagus nerve alone 18, or to the vagal 
preganglionic fibers at the posterior wall of the superior vena cava 17, exerted equally strong anti-
arrhythmic effects.  
The anti-inflammatory properties of the VN have been recently recognized 20,21. The 
cholinergic anti-inflammatory pathway can be activated experimentally by electrical VN 
stimulation to inhibit inflammatory cytokine production (including TNF-Į), prevent tissue injury 
and improve survival in multiple experimental models of systemic inflammation and sepsis 20-24. 
A substantial body of evidence suggests that AF and inflammatory pathways have a bidirectional 
relationship 25. Several studies suggested that inflammation may confer an increased risk of AF. 
In a large population-based study, CRP levels predicted both the presence of AF at baseline and 
the development of AF during follow-up, even after adjustment for other known cardiovascular 
risk factors, and the risk of AF was progressively higher with increasing CRP quartiles 26. In a 
recent experimental study, TNF-Į overexpressing mice developed spontaneous episodes of AF 
and were found to have electrophysiological and structural changes known to promote AF, 
including increased calcium transient and atrial fibrosis 27. Whether AF is the cause or the 
consequence of inflammation cannot be determined on the basis of the available evidence. 
Based on the observation that transcutaneous electrical stimulation of the tragus, the anterior 
protuberance of the outer ear, where the auricular branch of the vagus nerve is located, elicits 
evoked potentials in the brainstem in humans 28, we examined the effects of low-level tragus 
stimulation (LLTS) for inhibiting AF in a canine model of rapid atrial pacing 29. Notably, the anti-
arrhythmic effects of LLTS were similar to those of LLVNS delivered to the cervical vagus nerve 
trunk 29. We recently translated these results into patients with drug-refractory AF undergoing AF 
ablation, and showed that LLTS for just one hour significantly shortened the AF duration, and 
suppressed inflammatory cytokines, indicating that neuromodulation by LLTS is a very promising, 
non-invasive therapy to treat AF and AF-related inflammation 30. Before these results can be 
translated into clinical practice, the long-term clinical efficacy of this approach needs to be shown 
in ambulatory patients with AF. It has been recently shown that transcutaneous VN stimulation 
applied for just 15 minutes in healthy volunteers increases heart rate variability (HRV) and reduces 
sympathetic nerve activity 31. In this study, we hypothesize that intermittent (1 hour daily) LLTS 
for 6 months may decrease AF burden and suppress inflammatory cytokines in patients with 
paroxysmal AF. These investigations will establish the first evidence of the long-term effects of 
LLTS on AF suppression in patients with paroxysmal AF and may provide the basis for a potential 
expansion of the therapeutic targets of this treatment modality beyond AF, into other inflammatory 
conditions, including atherosclerosis 32.  
 
 
C. Preliminary studies 

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LLTS suppresses AF in dogs: We studied the effects of transcutaneous electrical stimulation at 
the tragus for inhibiting AF. In 10 pentobarbital-anesthetized dogs, LLTS (20 Hz) in the right ear 
was accomplished by attaching two alligator clips onto the tragus. The voltage slowing the sinus 
rate was used as the threshold for setting the LLTS at 80% below threshold. The antiarrhythmic 
effects of LLTS were similar to those of LLVNS delivered to the cervical VN trunk (Figure 1A). 
In contrast, LLTS after transection of both VN failed to show any antiarrhythmic effect, indicating 
that the efferent VN are essential for its antiarrhythmic effects (Figure 1B). We concluded that 
LLTS can reverse RAP-induced atrial remodeling and inhibit AF inducibility, suggesting a 
potential non-invasive treatment for AF 29. 
 Neuromodulation suppresses AF and inflammation in humans: We have recently shown that 
transcutaneous electrical stimulation of the auricular branch of the vagus nerve at the tragus (low-
level 
tragus 
stimulation; LLTS) 
in 
humans 
has 
antiarrhythmic and 
anti-inflammatory 
effects 
30. 
Forty 
patients 
with 
paroxysmal 
AF 
who presented in 
sinus rhythm for AF ablation, were randomized to either 1 hour of LLTS (n=20) or control (n=20). 
LLTS in the right ear, 50% lower than the voltage that slowed the sinus rate, was accomplished by 
attaching a flat metal clip onto the tragus. Under general anesthesia, AF was induced at baseline 
and after 1 hour of LLTS or sham stimulation and were analyzed for inflammatory cytokines, 
LQFOXGLQJ71)ĮDQG&53XVLQJDPXOWLSOH[LPPXQRDVVD\3DFLQJ-induced AF duration decreased 
significantly by 6.3±1.9 min compared to baseline in the LLTS group, but not in the control 
(p=0.002 for comparison between groups; Figure 2A). AF cycle length increased significantly 
from baseline by 28.8±6.5ms in the LLTS group, but not in the control (p=0.0002 for comparison 
between groups). Systemic (femoral vein) but not coronary sinus TNFĮ and CRP levels decreased 
significantly only in the LLTS group (Figure 2B). Importantly, the magnitude of decrease in TNF-
Į levels by LLTS was comparable with the difference between patients with active vs. inactive 
inflammatory diseases 33. We concluded that LLTS acutely suppresses AF and decreases 
inflammatory cytokines in patients with paroxysmal AF, supporting the emerging paradigm of 

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neuromodulation to treat AF 30. The results of this study were published in the Journal of the 
American College of Cardiology 30. 
 
D. Research design and Methods 
Specific Aim 1: To examine the effect of intermittent (1 hour daily) LLTS on AF burden in patients 
with paroxysmal AF over a 6-month period. 
This is a prospective double-blind randomized controlled study. Patients with paroxysmal AF, 
documented by ECG, pacemaker/implantable cardioverter defibrillator (ICD) electrogram or 
Holter monitor, within 3 months of randomization on 2 separate occasions, at least 1 day apart, of 
at least 30 seconds duration, will be eligible for inclusion in the study.  
Patients will be excluded if they have any of the following: 
1. Left ventricular dysfunction (Left ventricular ejection fraction <40%) 
2. Significant valvular disorder (i.e., prosthetic valve or hemodynamically relevant valvular 
diseases)  
3. Recent (<6 months) stroke or myocardial infarction  
4. Severe heart failure (NYHA IV) 
5. Left atrial dilatation (>55mm) 
6. Recurrent vaso-vagal syncopal episodes 
7. Unilateral or bilateral vagotomy 
8. Sick sinus syndrome, 2nd or 3rd degree AV block, bifascicular block or prolonged (PR>300ms) 
1st degree AV block 
Patients will be recruited from the inpatient services and outpatient clinics of the University of 
Oklahoma Health Sciences Center. A meeting with all study personnel to review the study protocol 
will be held before initiation of the study, as well as every 3-6 months, depending on study 
progress. Informed consent will be obtained prior to enrollment in the study. The randomization 
schedule will be implemented with the use of concealed envelopes and it will be stratified by sex. 
Blocked randomization will be used to decrease the chance of imbalances in the two groups over 
time. 
After informed consent and screening procedures are completed, all patients will undergo 
noninvasive continuous ECG monitoring for 2 weeks to evaluate their AF burden (the percent of 
time spent in AF) using an adhesive waterproof continuous monitoring patch (Zio® Patch). The 
Zio® Patch (iRhythm Technologies, Inc) is an FDA approved, single-lead, lightweight, 14-day 
ambulatory ECG adhesive patch monitor. The patch is well tolerated and allows significantly 
longer continuous monitoring than a regular Holter monitor, leading to an improved AF detection 
rate 34. After the 2-week monitoring period, patients will be randomly assigned (1:1) to active or 
sham LLTS. Patients will be blinded to the treatment allocation, and every effort will be made to 
conceal from the study personnel the nature of the intervention in each study arm. Blinding of the 
patients will be accomplished by having a sham control arm, in which stimulation will be delivered 
to a different site (the ear lobe), devoid of vagal innervation 35. By doing so, all patients will be 
aware that they are receiving stimulation, but they will not be told which site achieves active VN 

transcutaneous

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Specific Aim 2: To examine the effect of intermittent (1 hour daily) LLTS on inflammatory 
cytokines and their gene expression in patients with paroxysmal AF over a 6-month period. 
The same group assignment described in Aim 1 will be used for Aim 2. The importance of this 
Aim is highlighted by accumulating evidence that inflammation promotes the persistence of AF 
25,41. Blood samples (10ml) will be collected at baseline and at 3 and 6 months for cytokine 
measurement. Samples will be centrifuged (4000g for 10 min), and serum will be stored in 
aliquots at -20°C until assayed. Patients’ serum will be saved frozen and processed in batches of 
10 to 12. The investigators performing the cytokine assays will be blinded to group assignment. 
All assays will be run at the Humphrey Lab at OUHSC. Inflammatory cytokines, including TNF-
Į, and IL-6, will be measured using commercially available immunoassays analyzed on a flow 
cytometer (multiplex assays). In addition, peripheral blood mononuclear cells (PBMCs) will be 
isolated using MACS magnetic bead purification. Inflammatory cytokine gene expression profile 
will be measured at the above time points, using commercially available microarrays. Finally, we 
will perform exploratory analyses in order to determine relationships between clinical and cytokine 
responses to LLTS and identify predictors of response based on baseline cytokine levels. It is 
known that vagus nerve stimulation attenuates inflammation through the cholinergic anti-
inflammatory pathway 20-24. If this pathway is important in AF as well, we expect that the decrease 
in cytokines will be correlated with the anti-arrhythmic effect.  
 
Duration of participation 
Enrolled patients will participate for a maximum of 7 months with 3 outpatient office visits at 1, 3 
and 6 months. 
Patient enrollment 
A maximum of 52 patients meeting the inclusion criteria and not meeting the exclusion criteria 
will be enrolled in the study. The enrollment period is expected to last for at least 17 months, given 
that on average 30 patients with paroxysmal AF are seen in our clinics each month and assuming 
that 1 out of 4 eligible patients would agree to participate in the study.  
 
Data collection and analysis 
The final data will be pooled and analyzed by the investigators. 
  
 
E. Statistical methods 
The primary outcome of this study is the percent of time spent in AF over the monitoring period 
(AF burden) at the 6-month time point. Secondary outcomes include total duration of AF over the 
2-week monitoring period, longest daily duration of AF, measures of HRV and serum cytokine 
levels. Outcomes will be compared between groups using a Generalized Estimating Equations 
modeling approach, with 3 time points (baseline, 3 months, 6 months) and 4 terms included in the 
model (group effect, time effect, group by time interaction, adjustment for baseline measure). 

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Significant interactions will be followed by time trend analyses stratified by intervention group. If 
the interaction is not significant, the interaction term will be dropped from the model and the 
between-group comparisons will be made by combining the 3-month and 6-month data. For all 
pair-wise testing, we will adjust for multiple comparisons using Tukey’s method. The modeling 
assumptions will be evaluated by plotting the residuals by the predicted values (for the constant 
variance assumption) and comparing the normal QQ plot with the QQ plot of the residuals (for the 
normality assumption). If the modeling assumptions are not satisfied, we will perform a natural 
log transformation of the data, to better satisfy these assumptions. For 0 values, a value of 0.01 
will be added to each measure before applying the natural log. Analyses will be based on the 
intention-to-treat principle. In post-hoc exploratory analyses, we will use logistic regression 
analysis to identify predictors of clinical and cytokine response to LLTS, defined as a decrease of 
AF burden and a decrease in cytokines at 6-month follow-up, respectively. Those variables that 
which are associated with response to LLTS in univariate analysis at p<0.1 will be entered into a 
multivariate logistic regression model, using a backward elimination method to identify the most 
parsimonious model. In light of the limited sample size, such analyses will be underpowered and 
will be considered hypothesis-generating only. Statistical significance will be declared at p<0.05.  
Sample size and power calculations: For purposes of sample size calculations we have simplified 
the analysis to focus on the AF burden at the 6-month time point between the 2 groups. Assuming 
a mean±standard deviation AF burden in the control group of 28.4±31.2% 34 and a 87% reduction 
in the LLTS group  to 3.7% (assuming a common standard deviation of 31.2%), 26 patients per 
group would provide 80% power to detect the specified effect sizes at a two-VLGHGVLJQLILFDQFHĮ
level of 0.05. Approximately 30 patients with paroxysmal AF are seen in our clinics each month. 
Assuming that 1 out of 4 eligible patients would agree to participate in the study, we should be 
able to enroll the required number of patients within the proposed 17 months, allowing for 6 
months of additional follow up and a final month for data analysis. 
 
 
F. Gender/Minority/Pediatric Inclusion for Research 
Participants will be age 21 or older. Race, minority status and gender will not affect enrollment. 
G. Human participants 
The study population will include 52 male/female patients with paroxysmal AF, aged 21 or older, 
including all races. Since AF is extremely rare in patients less than 21 years old, this age limit is 
not expected to affect the overall results. 
 
Inclusion Criteria: 
1. Male and female patients older than 21 year old  
2. Paroxysmal AF 
 
Exclusion Criteria: 

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1. Left ventricular dysfunction (Left ventricular ejection fraction <40%) 
2. Significant valvular disorder (i.e., prosthetic valve or hemodynamically relevant valvular 
diseases)  
3. Recent (<6 months) stroke or myocardial infarction  
4. Severe heart failure (NYHA IV) 
5. Left atrial dilatation (>55mm) 
6. Recurrent vaso-vagal syncopal episodes 
7. Unilateral or bilateral vagotomy 
8. Pregnancy or breast feeding 
9. Sick sinus syndrome, 2nd or 3rd degree AV block, bifascicular block or prolonged 
(PR>300ms) 1st degree AV block. 
 
2.  Identify sources of research material in the form of specimens, records or data. 
The research information will consist of the AF burden and serum inflammatory cytokines at each 
time point specified in the protocol (see Figure 4). 
 
3.  Describe plans for recruitment and consent procedures to be followed. 
a.  Describe the location where consent is most likely to take place (will consent be obtained while 
an inpatient, in the ER/ICU, will consents be mailed with follow-up meeting to discuss the 
consent, etc.). 
Patients will be recruited from the inpatient services and outpatient clinics of the University of 
Oklahoma Health Sciences Center. All candidate subjects will have the purpose of the study 
explained to them, including the benefits and risks and options, prior to scheduled ablation 
procedure, will be asked to read the consent form, and after questions have been answered, will be 
asked to participate. Finally, they will be asked to sign the consent form.  
b. Describe provisions for recruiting non-English speaking participants. 
Only English-speaking persons will be recruited. Non-English-speaking adults constitute less than 
1% of the patients referred for ablation. 
c.   Describe measures to decrease coercion of participants (allowing adequate time to review the 
consent, avoid recruitment of your own private patients – or have a qualified assistant assist 
with the consent of these individuals, avoid recruiting employees or staff to serve as controls, 
etc.).  
Patients will have adequate time to review the consent. Recruiting of employees or staff by the 
principal investigator will be avoided. 
 
3. Describe risks and assess likelihood and seriousness. 
 

tVNS
tVNS
tVNS

tVNS

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the poor success and potential side effects of the currently available treatment options, an 
alternative approach such as the one tested in this study (tragus stimulation) may benefit a large 
number of patients in Oklahoma. 
 
H.  Data and Safety Monitoring Plan  
1.  Describe the Data and Safety Monitoring Plan (DSMP) 
An independent Data Safety Monitoring Board (DSMB) committee consisting of 2 cardiologists 
and 1 statistician will monitor the study. All adverse events will be reported to the DSMB 
committee. The committee will review recruitment and retention data, protocol adherence data, 
data quality, and safety data every 6 months 
a. reporting mechanisms for adverse events to the IRB, FDA, and NIH 
Clinical staff will track from patient report, physical assessments and documentation of clinical 
emergencies, adverse events according to federally published grading (0=no adverse event; 
1=mild; 2=moderate; 3=severe and undesirable adverse events; 4=life threatening/disabling; or 
5=death) and attribution (1=unrelated; 2=unlikely; 3=possible; 4=probable; 5=definite) scales. 
Clinical staff will report findings to the Investigator, who will then advise the IRB, NIH and FDA 
as indicated. Given the mild risk nature of the study, composite reports will be reviewed every ten 
patients to assure that untoward events do not occur systematically, and dropout cases will be 
reviewed singly to learn whether further inquiry or modifications should be made to improve study 
implementation. 
 
b. adverse event (AE) grading 
0=no AE; 1=mild; 2=moderate; 3=severe and undesirable AE; 4=life threatening/disabling; or 
5=death 
 
c. plan for unanticipated AE reporting 
Unanticipated AE will be reported to the IRB. 
d. plan for annual reporting of AEs 
Use of IRB forms for reporting. Our data sheet will have a column detaining any AEs that might 
occur. 
e. interim efficacy analysis where appropriate 
Not indicated. 
2. Describe the Data and Safety Monitoring Board (DSMB) that will be responsible for monitoring 
the study. 

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x Institutional studies provide: 
a. Chair, members  
Dr. Ralph Lazzara 
Dr. Nicole Tran 
Dr. Ding Kai 
b. Frequency of safety reviews 
After each study 
     
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