Author + information
- Received January 23, 2017
- Revision received April 11, 2017
- Accepted April 19, 2017
- Published online August 7, 2017.
- Lilei Yu, MD, PhDa,
- Bing Huang, MD, PhDa,
- Sunny S. Po, MD, PhDb,
- Tuantuan Tan, MD, PhDc,
- Menglong Wang, MDa,
- Liping Zhou, MDa,
- Guannan Meng, MDa,
- Shenxu Yuan, MDa,
- Xiaoya Zhou, MD, PhDa,
- Xuefei Li, MDa,
- Zhuo Wang, MDa,
- Songyun Wang, MDa and
- Hong Jiang, MDa,∗ ()
- aDepartment of Cardiology, Renmin Hospital of Wuhan University; Cardiovascular Research Institute, Wuhan University; Hubei Key Laboratory of Cardiology, Wuhan, Hubei, China
- bHeart Rhythm Institute and Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
- cDepartment of Ultrasound Imaging, Renmin Hospital of Wuhan University, Wuhan, Hubei, China
- ↵∗Address for correspondence:
Dr. Hong Jiang, Department of Cardiology, Renmin Hospital of Wuhan University, No. 238 Jiefang Road, Wuchang District, Wuhan City, Hubei Province 430060, China.
Objectives The aim of this study was to investigate whether low-level tragus stimulation (LL-TS) treatment could reduce myocardial ischemia-reperfusion injury in patients with ST-segment elevation myocardial infarction (STEMI).
Background The authors’ previous studies suggested that LL-TS could reduce the size of myocardial injury induced by ischemia.
Methods Patients who presented with STEMI within 12 h of symptom onset, treated with primary percutaneous coronary intervention, were randomized to the LL-TS group (n = 47) or the control group (with sham stimulation [n = 48]). LL-TS, 50% lower than the electric current that slowed the sinus rate, was delivered to the right tragus once the patients arrived in the catheterization room and lasted for 2 h after balloon dilatation (reperfusion). All patients were followed for 7 days. The occurrence of reperfusion-related arrhythmia, blood levels of creatine kinase-MB, myoglobin, N-terminal pro–B-type natriuretic peptide and inflammatory markers, and echocardiographic characteristics were evaluated.
Results The incidence of reperfusion-related ventricular arrhythmia during the first 24 h was significantly attenuated by LL-TS. In addition, the area under the curve for creatine kinase-MB and myoglobin over 72 h was smaller in the LL-TS group than the control group. Furthermore, blood levels of inflammatory markers were decreased by LL-TS. Cardiac function, as demonstrated by the level of N-terminal pro–B-type natriuretic peptide, the left ventricular ejection fraction, and the wall motion index, was markedly improved by LL-TS.
Conclusions LL-TS reduces myocardial ischemia-reperfusion injury in patients with STEMI. This proof-of-concept study raises the possibility that this noninvasive strategy may be used to treat patients with STEMI undergoing primary percutaneous coronary intervention.
- acute myocardial infarction
- ischemia-reperfusion injury
- low-level vagal stimulation
- ventricular arrhythmia
ST-segment elevation myocardial infarction (STEMI) accounts for approximately 25% to 40% of acute myocardial infarction and remains an important cause of disability and mortality throughout the world (1–3). Although reopening the culprit coronary artery via mechanical or pharmacological reperfusion intervention is necessary to rescue the ischemic myocardium and to reduce infarct size in patients with STEMI, paradoxically, reperfusion itself also triggers further injury, which is known as myocardial ischemia-reperfusion injury (MIRI) (4,5). Growing evidence from experimental studies and small-sized proof-of-concept clinical trials shows that MIRI contributes greatly to the final infarct size and cardiac function (6,7). However, there is currently no specific treatment that targets MIRI in patients with STEMI. Thus, new therapies that can decrease MIRI in these patients are needed.
Cervical vagus nerve stimulation (cVNS), which induces significant heart rate reduction (8–10), or with a stimulus strength 50% to 80% below the threshold needed to reduce heart rate (11–13), has been shown to inhibit inflammatory responses, decrease the release of reactive oxygen species, suppress cellular apoptosis, and attenuate MIRI in several myocardial ischemia-reperfusion models. Transcutaneous electric stimulation of the auricular branch of the vagus nerve, located at the tragus, is a noninvasive approach to stimulating the afferent vagal nerve fibers (14). We recently found that low-level tragus stimulation (LL-TS) could decrease inflammatory responses, attenuate cardiac structural and autonomic remodeling, and improve ventricular function in a canine model of chronic myocardial infarction (15,16). LL-TS also has been shown to suppress atrial fibrillation and to decrease levels of inflammatory cytokines in patients with paroxysmal atrial fibrillation (17). In the present study, we investigated whether LL-TS could attenuate MIRI in patients with STEMI undergoing primary percutaneous coronary intervention (PCI).
This was a prospective, single-center, randomized, open-labeled study. The study protocol was approved by the ethics committee of Renmin Hospital of Wuhan University. All patients provided informed consent.
Patients 18 to 80 years of age who were diagnosed with STEMI, presented within 12 h of symptom onset, and underwent primary PCI from November 2015 until September 2016 were enrolled. Major exclusion criteria included a history of prior myocardial infarction, severe heart failure (left ventricular [LV] ejection fraction <30%), cardiogenic shock, ventricular fibrillation or cardiac arrest, previous malignant hematological disease, previous known renal failure (estimated glomerular filtration rate <30 ml/min), and inability or unwillingness to provide informed consent (Figure 1A). In addition, patients with left main or multiple coronary artery disease were also excluded from this study.
The study design is presented in Figure 1B. All patients were randomized to receive either LL-TS and PCI (LL-TS group) or sham LL-TS and PCI treatment (control group). Aspirin (300 mg) and ticagrelor (180 mg) were used once patients were diagnosed with STEMI. Patients underwent PCI according to standard guidelines (1). The use of thrombus aspiration, glycoprotein IIb/IIIa inhibition, and drug-eluting stents during PCI was left to the discretion of the treating physician. Standard medical therapy, including statins, aspirin and ticagrelor or clopidogrel, beta-blockers, and angiotensin-converting enzyme inhibitors or angiotensin receptor blockers at recommended daily doses, was administered for post–myocardial infarction secondary prevention (1) (Table 1). Blood sampling, 24 h Holter monitoring, and echocardiography were conducted at pre-specified time points in the 2 groups.
All patients were conscious and were not given any sedatives during the operation. LL-TS was performed on the tragus in the right ear (Figure 2A). Incremental electric currents were applied to the tragus (20 Hz, 1-ms duration) using a stimulator (S20, Jinjiang, Chengdu City, China) until slowing of the sinus rate was achieved (Figures 2B and 2C). The lowest electric current necessary to slow the sinus rate was defined as the stimulation threshold. LL-TS was set at 50% below the threshold with a duty cycle of 5 s on and 5 s off. LL-TS or sham LL-TS was initiated once the patient arrived in the catheterization room and lasted for 2 h after balloon dilatation (reperfusion) (Figure 2D). The sham stimulation duration in the control group and stimulation duration in the LL-TS group were 156 ± 8 min and 155 ± 6 min, respectively (p = 0.49) (Table 1).
Ischemia-reperfusion related ventricular arrhythmias
In both groups, continuous, digital 12-lead electrocardiographic Holter monitoring (CT-86, Baihui, Hangzhou City, China) was initiated after reperfusion and lasted for 24 h. Holter software was used to analyze the severity of ventricular arrhythmias (VAs), which were classified as isolated ventricular premature beats (VPB), coupled VPBs (2 consecutive VPBs), or ventricular tachycardia (3 or more consecutive VPBs).
Measurements of blood levels of creatine kinase-MB, myoglobin, and N-terminal pro-B-type-natriuretic peptide
Venous blood samples at baseline (on admission) and at 0, 6, 12, 24, 48, and 72 h, as well as 7 days, after reperfusion were obtained from patients in a supine position. For measurements of creatine kinase-MB and myoglobin, samples from 0, 6, 12, 24, 48, and 72 h after reperfusion were evaluated using an ADVIA Centaur XP automatic chemiluminescence immunoassay analyzer (Siemens Healthcare Diagnostics, Munich, Germany). For measurement of N-terminal pro–B-type natriuretic peptide (NT-pro BNP), samples that were obtained at baseline (on admission) and 24 h and 7 days after reperfusion were analyzed using a Dimension EXL with an LM automatic biochemical analyzer (Siemens Healthcare Diagnostics).
Measurement of inflammatory markers
Blood samples that were obtained at baseline (on admission) and 24 h after reperfusion were used to evaluate interleukin-6, interleukin-1β, high-mobility group-box 1 protein, and tumor necrosis factor–α levels with commercially available enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (Human ELISA kit, CUSABIO Science, Wuhan, China). All samples were analyzed in duplicate, and the analysis was repeated if the difference between duplicates was >15%.
All patients underwent conventional 2-dimensional echocardiography on days 5 to 7 after revascularization. Echocardiography was performed using a Philips iE33 equipped with S5-1 transducers (Philips Medical Systems, Andover, Massachusetts) or a GE Vivid 7 model equipped with an M4S transducer (GE Healthcare, Little Chalfont, United Kingdom). All measurements included at least 3 consecutive beats for patients in sinus rhythm. The echocardiographic data were interpreted by 2 experts who were blinded to group information. Ventricular diameters (normalized to body surface area) and interventricular septal and posterior wall thickness were measured. The modified biplane Simpson rule was applied to calculate the LV ejection fraction. The 16-segment model was applied to evaluate the severity of LV regional wall motion abnormality (18). The percentage that was obtained by dividing the number of akinetic and dyskinetic segments by the total number of segments evaluated was used to indicate the extent of LV damage. Early transmitral flow velocity (E), late atrial contraction (A) velocity, and the E/A ratio were used to reflect LV diastolic function.
Preliminary measurements in patients with STEMI at our hospital had an area under the curve (AUC) for creatine kinase-MB in the first 72 h of approximately 6,000 ± 1,000 ng h/ml. It was calculated that to detect a 15% reduction in the AUC for creatine kinase-MB with 95% probability (type II error probability of 0.05), at an alpha level (type I error probability) of 0.05 using a 2-sided test, 40 subjects in each group should be included. Continuous and categorical variables are presented as mean ± SD and as count (percentage), respectively. Independent-sample Student t tests or chi-square tests were used for comparisons of continuous and categorical variables, respectively, for baseline characteristics between the 2 groups. The VAs, the AUCs for creatine kinase-MB and myoglobin during the 72 h after reperfusion, and the echocardiographic characteristics were compared between the 2 groups using independent-sample Student t tests. Levels of NT-proBNP and inflammatory markers were compared between the groups using 2-way analysis of variance followed by the Bonferroni post hoc test. SPSS version 18.0 (SPSS, Chicago, Illinois) was used for data analysis. The differences were considered statistically significant at a 2-sided p value of <0.05.
As shown in Figure 1A, 128 consecutive patients with STEMI were included, and 106 patients were randomized to the LL-TS group (n = 53) or control group (n = 53). The results of coronary angiography showed that 11 higher risk patients for PCI (e.g., left main or multiple coronary artery disease) were excluded from this study, 6 patients in the LL-TS group and 5 patients in the control group. Ultimately, 47 patients in the LL-TS group and 48 patients in the control group were followed in this study (Figure 1A). There were no differences with respect to baseline parameters in the 2 groups, such as the sex ratio, ischemic time, culprit vessel, door-to-balloon time, stimulation threshold, and stimulation or sham stimulation duration (Table 1).
Effect of LL-TS on ischemia-reperfusion-related VAs
The results of Holter analysis showed that the total number of VBPs (154 ± 115 vs. 551 ± 214; p < 0.05), isolated VPBs (133 ± 109 vs. 362 ± 104; p < 0.05), coupled VPBs (3 ± 3 vs. 22 ± 16, p < 0.05), and ventricular tachycardia (2 ± 3 vs. 18 ± 12; p < 0.05) values in the LL-TS group were all significantly lower than the values in the control group (Figure 3).
Effects of LL-TS on creatine kinase-MB and myoglobin levels
The AUCs for creatine kinase-MB and myoglobin during the 72 h after reperfusion were calculated to reflect the myocardial infarction size (Figure 4). The AUC (0 to 72 h) for creatine kinase-MB level was significantly lower in the LL-TS group than in the control group (5,156 ± 782 ng h/ml vs. 7,646 ± 742 ng h/ml; p < 0.05). In addition, the AUC (0 to 72 h) for myoglobin level was also significantly lower in the LL-TS group (8,632 ± 551 μg h/l vs. 10,361 ± 624 μg h/l; p < 0.05).
Effects of LL-TS on NT-PROBNP levels
The blood levels of NT-proBNP in both groups are shown in Figure 5. There was no significant difference in NT-proBNP level between the 2 groups at baseline (317 ± 132 ng/l vs. 365 ± 111 ng/l; p > 0.05). However, LL-TS significantly decreased the blood level of NT-proBNP at the time points of 24 h (905 ± 213 ng/l vs. 1,595 ± 432 ng/l; p < 0.05) and 7 days (548 ± 201 ng/l vs. 1,127 ± 302 ng/l; p < 0.05) after reperfusion.
Effects of LL-TS on inflammatory marker levels
The blood levels of interleukin-6, interleukin-1β, high-mobility group-box 1 protein 1, and tumor necrosis factor-α were evaluated at baseline and 24 h after reperfusion in both groups (Figure 6). There were no significant differences in these inflammatory markers between the 2 groups at baseline. However, a significant improvement in inflammation was observed in the LL-TS group at the time point of 24 h after reperfusion.
As shown in Table 2, there were no differences in LV end-diastolic volume and LV end-systolic volume between the 2 groups. However, a significant improvement in LV ejection fraction was observed in the LL-TS group compared with the control group. The wall motion index, which was calculated using the 16-segment model, was used to evaluate the extent of LV dysfunction. In agreement with the LV ejection fraction, the percentage of LV akinetic and dyskinetic segments was significantly lower in the LL-TS group.
To the best of our knowledge, this study provides the first clinical evidence that LL-TS markedly reduces MIRI as reflected by ischemia-reperfusion-associated VAs, myocardial injury biomarkers, and cardiac function in patients with STEMI undergoing primary PCI. This study also showed that the MIRI reduction is accompanied by an improvement in blood inflammatory cytokine levels. This proof-of-concept study raises the possibility that this noninvasive strategy may be used to treat patients with STEMI undergoing primary PCI.
LL-TS for the treatment of MIRI
Considerable evidence has shown that cVNS with a stimulus strength that was sufficient to reduce heart rate by 20% to 50% is capable of relieving MIRI in different animal species (8,9). However, a reduction in heart rate via cVNS of >20% is not tolerated by most patients, which limits its clinical application. To circumvent this problem, we recently found that low-level cVNS that did not cause heart rate reduction significantly decreased the incidence of VAs and reduced myocardial infarct size in a canine model of acute myocardial ischemia and reperfusion (11). Similar conclusions were reached by Shinlapawittayatorn et al. (12) and by Zhang et al. (13), who also showed that low-level cVNS could attenuate MIRI and improve ventricular function during the ischemia-reperfusion process. During the past few years, stimulation of the auricular branch of the vagus nerve has been developed to overcome the potential barriers of conventional cVNS (19). Anatomic studies suggest that the auricular branch of the vagus nerve is the only peripheral branch of the vagus nerve distribution on the surface of the ear (20). Stimulation of this region on the ear reduces seizure frequency in patients with intractable epilepsy, similar to cVNS (21). Preclinical and clinical studies have also confirmed that LL-TS is a noninvasive alternative to low-level cVNS to treat atrial fibrillation (17,22). Our recent studies showed that LL-TS could attenuate cardiac structural and autonomic remodeling, improve ventricular function, and decrease VA induction in conscious dogs with healed myocardial infarction (15,16). In the present study, we applied LL-TS in patients with STEMI to evaluate whether LL-TS could attenuate MIRI in these patients. MIRI was evaluated via 2 different methods: myocardial injury biomarkers (creatine kinase-MB and myoglobin) and reperfusion-related VAs. The results of both of the methodologies provided evidence of a significant reduction in MIRI that resulted from LL-TS. In addition, LL-TS also significantly decreased the blood level of NT-proBNP and the wall motion index but increased the LV ejection fraction, suggesting a significant improvement in the recovery of cardiac function after reperfusion. Taken together, our data suggest that LL-TS may be a novel, invasive therapy for the treatment of patients with STEMI.
Possible mechanisms underlying the beneficial effects of LL-TS
It is well established that inflammation is critically involved in the pathogenesis of STEMI. After the initial ischemic event, an intense inflammatory response is triggered by myocardial ischemia, characterized mainly by leukocyte infiltration and proinflammatory cytokine production, resulting in cardiomyocyte damage and affecting ventricular remodeling (23). The clinical studies have shown that the levels of inflammation markers can predict ventricular remodeling, cardiac dysfunction, and death in patients with STEMI undergoing primary angioplasty (24). In view of this evidence, the significant inverse correlation of LL-TS treatment with the rise in post-reperfusion inflammatory cytokines suggests a potential mechanism of the observed beneficial effect of LL-TS. Recent studies also have shown that LL-TS could decrease the levels of inflammatory cytokines in post–myocardial infarction canine models (16) and in patients with paroxysmal atrial fibrillation (15). Given that the functional threshold for activation of vagal afferent fibers is lower than that for activation of efferent fibers (25), LL-TS used in the present study probably mainly activated the afferent fibers, and therefore might achieve its anti-inflammatory effects through activation of the hypothalamic-pituitary-adrenal axis pathway (26).
Reperfusion can result in the generation and release of reactive oxygen species, which induces myocardial oxidative stress and ultimately exacerbates myocardial cell apoptosis. A prior experimental study from our group showed that low-level cVNS could attenuate MIRI by suppressing oxidative stress activity and regulating myocardial apoptosis (11). In addition, sympathetic activation has been shown to increase the susceptibility of the heart to ischemia- or reperfusion-related VAs (27). Interventions that decrease sympathetic activity are associated with a lower incidence of VAs in both patients and animal models (27). Low-level cVNS has been shown to suppress the activity of the left stellate ganglion, the gateway of sympathetic innervation to the heart, in ambulatory dogs (28). Taken together, these observations indicate that antioxidative stress, antiapoptosis, and antisympathetic effects produced by low-level cVNS may all contribute to its beneficial effects on MIRI. In the present study, similar mechanisms are likely to underlie the beneficial effects of LL-TS.
Over the past few decades, several advances have been made in the treatment of patients with STEMI; however, there is currently no specific treatment that decreases MIRI, which inevitably occurs as a result of the restoration of coronary patency. The present study indicates that LL-TS is well tolerated and significantly improves inflammation responses, reperfusion-related VAs, blood levels of myocardial injury biomarkers and NT-proBNP, and LV ejection fraction and wall motion index in patients with STEMI. LL-TS may become a novel, noninvasive, and nonpharmacological therapy for the treatment of patients with STEMI undergoing primary PCI if these observations are confirmed in larger randomized trials.
First, cardiac magnetic resonance imaging was not performed to evaluate infarct size and cardiac function during the first 7 days of follow-up. However, echocardiographic methods for the determination of cardiac function have also been widely used (29,30).
Second, we evaluated only the acute (7-day) effects of LL-TS in patients with STEMI. Further studies are necessary to identify whether LL-TS could improve the long-term clinical outcome in these patients.
Third, higher risk patients with left main or multiple coronary artery disease were excluded from this study to avoid introducing confounding factors such as complications caused by high-risk PCI. Therefore, it is unclear whether LL-TS would have similar benefits in these patients.
Finally, the parameters of LL-TS, such as frequency, intensity, and duration, in the present study were based on previous experience with basic and clinical findings, and the optimal parameters of LL-TS were not determined.
LL-TS significantly improves inflammatory responses, reperfusion-related VAs, blood levels of myocardial injury biomarkers and NT-proBNP, and LV ejection fraction and wall motion index in patients with STEMI. This proof-of-concept study raises the possibility that this noninvasive therapy can be used to treat patients with STEMI who are undergoing primary PCI. Future adequately powered prospective multicenter clinical trials are warranted.
WHAT IS KNOWN? Our previous studies suggested that LL-TS could reduce the size of myocardial injury induced by ischemia.
WHAT IS NEW? In this proof-of-concept study, we have demonstrated for the first time that LL-TS markedly reduces myocardial ischemia and reperfusion injury in patients with STEMI undergoing primary PCI, indicating that this novel neuromodulatory approach may be an important step toward a noninvasive and nonpharmacological therapy for the treatment of STEMI.
WHAT IS NEXT? Further adequately powered prospective multicenter clinical trials are warranted.
This work was supported by grants 81530011, 81570463, and 81600395 from the National Nature Science Foundation of China, grants 2016CFA065 and 2016CFA048 from the Natural Science Foundation of Hubei Province, and grant WJ2017C0005 from the Foundation of Health and Family Planning Commission of Hubei Province. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Yu and Huang contributed equally to this work.
- Abbreviations and Acronyms
- area under the curve
- cervical vagus nerve stimulation
- low-level tragus stimulation
- left ventricular
- myocardial ischemia-reperfusion injury
- N-terminal pro–B-type-natriuretic peptide
- percutaneous coronary intervention
- ST-segment elevation myocardial infarction
- ventricular arrhythmia
- ventricular premature beat
- Received January 23, 2017.
- Revision received April 11, 2017.
- Accepted April 19, 2017.
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