Resiniferatoxin reduces ventricular arrhythmias in heart failure via selectively blunting cardiac sympathetic afferent projection into spinal cord in rats

Journal Pre-proof Resiniferatoxin reduces ventricular arrhythmias in heart failure via selectively blunting cardiac sympathetic afferent projection into spinal cord in rats Yong Wu, Zhengtao Hu, Deguo Wang, Kun Lv, Nengwei Hu PII:

S0014-2999(19)30788-5

DOI:

https://doi.org/10.1016/j.ejphar.2019.172836

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EJP 172836

To appear in:

European Journal of Pharmacology

Received Date: 24 August 2019 Revised Date:

25 November 2019

Accepted Date: 29 November 2019

Please cite this article as: Wu, Y., Hu, Z., Wang, D., Lv, K., Hu, N., Resiniferatoxin reduces ventricular arrhythmias in heart failure via selectively blunting cardiac sympathetic afferent projection into spinal cord in rats, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/ j.ejphar.2019.172836. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

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Resiniferatoxin reduces ventricular arrhythmias in heart

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failure via selectively blunting cardiac sympathetic afferent

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projection into spinal cord in rats

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Yong Wu 1,2, Zhengtao Hu1,2，Deguo Wang 1,2, Kun Lv 2, Nengwei Hu1, 3, 4. 1 Department of Gerontology, Yijishan Hospital of Wannan Medical College, Wuhu 241001, PR China. 2

Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher

Education Institution (Wannan Medical College), Wuhu, Anhui 241001, P.R. China. 3 Department of Physiology and Neurobiology, Zhengzhou University School of Medicine, Zhengzhou 450001, China. 4. Department of Pharmacology & Therapeutics and Institute of Neuroscience, Trinity College, Dublin 2, Ireland.

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Correspondence to: Prof. Deguo Wang

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Institutions: Department of Gerontology and Central Laboratory, Yijishan Hospital of

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Wannan Medical College.

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Address: 92ndZheshan Western Road, Wuhu, Anhui 241001, P.R. China

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Corresponding author E-mail: [email protected]

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And co-corresponding author E-mail: [email protected]

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Abstract Excessive sympathetic activity is associated with heart failure and ventricular arrhythmias,

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which regulated by enhanced cardiac sympathetic afferent reflex, which can be blunted by

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resiniferatoxin, a selective receptor agonist of transient vanilloid potential 1 (TRPV1) +

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primary sensory afferents. The present study is aimed to determine whether intrathecal

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resiniferatoxin application affect cardiac sympathetic tone and electrophysiology, furtherly

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create a new effective strategy to prevent lethal arrhythmias in chronic heart failure. Four

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weeks after coronary artery occlusion to induce heart failure in rats, RTX (2µg/10µl) or

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vehicle was injected intrathecally into the T2/T3 interspace. Cardiac sympathetic nerve

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activates (CSNA) and cardiac electrophysiology were evaluated two weeks later. Intrathecal

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resiniferatoxin significantly and selectively abolished the afferent markers expression

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(TRPV1 and calcitonin gene-related peptide) in dorsal horn and reduced overactivated CSNA.

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Electrophysiological studies revealed that resiniferatoxin administration intrathecally

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significantly reversed the prolongation of action potential duration (APD) and APD alternan,

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reduced the inducibilities of ventricular arrhythmias. Moreover, the over-activated calcium

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handling related protein CaMKII and RyR2 in heart failure was reversed by resiniferatoxin

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administration. In conclusion, these results firstly demonstrate that central chemo-ablation of

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the TRPV1+ afferents in spinal cord prevent heart from ventricular arrhythmias in heart

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failure via selectively blunting cardiac sympathetic afferent projection into spinal cord, which

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suggest a novel promising therapeutic method for anti-arrhythmia in heart failure.

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Keywords: cardiac sympathetic nerve activities; myocardial infarction; resiniferatoxin; ventricular arrhythmias; chronic heart failure.

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1. Introduction

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Ventricular arrhythmias causes the most mortality in patients with chronic heart failure

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(Kusumoto et al., 2018). Excessive sympathetic never activation plays an important role in

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the mechanism of heart failure and lethal arrhythmias (Shen and Zipes, 2014). Sympathetic

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activity is regulated by the cardiac sympathetic afferent reflex both in physiological and

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pathophysiological conditions. Under physiological conditions, blocking the reflex can

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weaken normal neurological responses to cardiac stress, which cause abnormal regulation in

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pressure regulation and cardiac function (Lei et al., 2016; Yoshie et al., 2018; Yu et al., 2018).

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In disease states like heart failure, the reflex is over-activated in a positive feedback manner

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and exaggerates cardiac dysfunction(Wang et al., 1999). Inhibition the abnormal reflex might

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protect heart in heart failure because previous study showed that inhibiting cardiac

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sympathetic afferent activity attenuated cardiac remodeling and cardiovascular dysfunction

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(Shanks et al., 2019; Wang et al., 2017; Wang et al., 2014).

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However, it is frustrated that cardiac sympathetic afferent reflex is intricate and difficult to

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precisely intervention. The circuit formed by sympathetic afferent fibers, spinal cord,

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hypothalamic and medullary brain centers, and sympathetic efferent nerves(Chen et al., 2015).

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Cardiac sympathetic afferent terminals contain the transient receptor potential vanilloid 1

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(TRPV1) receptor (Chen et al., 2015), which can be selectively damaged by a TRPV1 agonist

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resiniferatoxin. For example, a single epicardial brushing of resiniferatoxin block the reflex

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and reduce the deleterious cardiac remodeling and autonomic dysfunction in heart failure rats

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because the permissive distribution of TRPV1 receptor in the epicardium (Wang et al., 2017;

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Wang et al., 2014). In another study, resiniferatoxin was delivery to the epicardium via

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percutaneous epicardial puncture and instillation (Yoshie et al., 2018). The two delivery

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routes would be inconvenient in operation if applicated in human being. Recently, a novel

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route via intrathecal resiniferatoxin administration, which central chemo-axotomy of the

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TRPV1+ afferents, had been reported to effectively control pain in human and

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animals(Bishnoi et al., 2011; Leo et al., 2017; Sapio et al., 2018). We also demonstrated that

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intrathecal resiniferatoxin at the levels from T1 to T4 protected the heart from pressure

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overload-induced cardiac remodeling and cardiac dysfunction(Wang et al., 2019).

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Therefore, we deduced that resiniferatoxin application at spinal central terminals can blunt

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the responses of cardiac sympathetic afferent reflex, weaken cardiac sympathetic tone, and

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reduce in the risk of ventricular arrhythmias in heart failure. In the present study, we

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intrathecally injection resiniferatoxin in heart failure models by myocardial infarction and

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found that it is an effective novel route to prevent lethal arrhythmias in chronic heart failure.

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2. Methods and methods

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2.1. Animal groups and drugs

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Fifty male Sprague Dawley rats (weight 200-250g), purchased from experimental animal

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center of Nanjing University, were housed in clear plastic cages, and temperature and light

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periods (12-h light-dark cycle; light on between 6:00 AM and 6:00 PM) were controlled. The

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experimental procedures were approved by Yijishan Hospital Institutional Animal Care and

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Use Ethics Committee (20170033) and carried out according to the National Institutes of

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Health guiding principles.

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Forty animals received coronary artery ligation of the left anterior descending branch to

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induce post-infarcted chronic heart failure (CHF). Four weeks later, the survived animals

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were randomly assigned to two groups. The animals in CHF group were receive 10ul saline

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injection, while CHF+RTX group received 2ug/10µl of resiniferatoxin (RTX) for two weeks

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continuously. Another ten rats underwent sham surgery as control group. Two weeks after

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resiniferatoxin administration, all these rats further underwent cardiac sympathetic nerve

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recording and cardiac electrophysiological studies. After that, rat hearts were subsequently

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harvested, and the ratio of heart weight: body weight (HW:BW) was calculated. Left

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ventricular samples were collected and stored in -80°C for further histological evaluations.

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Resiniferatoxin (Sigma Aldrich, St Louis, MO) was firstly dissolved in 100% ethanol to

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make the stock solution (concentration 1mg/mL), then was further diluted in 20ml saline with

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final concentration of 2ug/10µl when used via intrathecal (T2/T3 intraspinal space) injection.

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2.2. Animal models

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2.2.1. Model of chronic heart failure

Heart failure was created by coronary artery ligation as previously described (Wang et al.,

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2011; Wang et al., 2017; Zhu et al., 2015). Following coronary artery ligation, the thorax at

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the 5thintercostal space was closed progressively from the muscle layer to the skin layer. For

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post-procedure pain management, fentanyl solution (3µg in 0.1ml) was subcutaneously

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injected immediately after surgery and daily for 2 days.

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2.2.2. Intrathecal injection

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Animals received intrathecal injections with resiniferatoxin or saline as previously described with minor modification(Leo et al., 2017; Sapio et al., 2018). Briefly, after

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isoflurane anesthesia (5% isoflurane), intrathecal injections were performed by inserting a

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U-40 insulin syringe with 29G needle, into the T2/T3interspace, perpendicular to the spine.

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As shown in Fig. 1A, a specific long spinous process is the 2nd thoracic vertebrae of the rat.

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Then T2/T3 interspace was selected as injection point after cutting the back skin near

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1stthoracic vertebrae. The needle was carefully and gradually inserted into spinal interspace

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until needle wad reached target nerve. The criterion for the intrathecal placement was

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withdrawal of clear cerebrospinal fluid into the syringe. Then resiniferatoxin (2µg/10µl) or

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vehicle (10ul saline) was injected slowly into intrathecal space. The dose of resiniferatoxin in

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this study was selected according to previous reports (Karai et al., 2004a; Wang et al., 2019).

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Moreover, our pre-experiment had showed that the cardiac sympathetic afferent reflex was

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abolished completely by the present dose (supplementary materials).

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2.3. Electrophysiological studies in vivo

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2.3.1. Cardiac sympathetic nerve activity

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For autonomic nerve activity recording, the right cervical sympathetic nerve were isolated

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through a midline neck incision in anesthetized rats as our previous report (Wang et al., 2019).

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Cervical stellate ganglion was identified behind right carotid artery which shown fleshly

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fusiform-shaped objects attached to the medial aspect of the bifurcation. A branch innervating

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heart was isolated and hooked by a pair of self-made recording tungsten electrodes.

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Recording signals were amplified (×10000), filtered (bandwidth: 100 to 3000 Hz) and stored

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in computer by a biological data acquisition system (RM6240, Chengdu, China) for further

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analysis off-line.

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2.3.2. Electrocardiogram recording and programmed electrical stimulation

In vivo electrophysiological studies were performed two weeks after the first

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resiniferatoxin injection as our previous report(Wang et al., 2016). During the procession of

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nerve activity recording, electrocardiogram was also recorded from three subcutaneous needle

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electrodes, and signals below 10 Hz and above 100 Hz were filtered out.

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To induce ventricular tachycardia, programmed electrical stimulation was performed as

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previously described (Wang et al., 2011). In brief, a pair of hook electrode was fixed into the

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right ventricle for pacing. The threshold potential for stable pacing was assessed at a cycle

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length of 150ms. Stimulation was then initiated twice as much as the threshold. Pacing was

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performed by applying a 2ms pulse pacing with a voltage twice that of the capture threshold.

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As shown in Fig. 6, after a train of 8 electrical stimuli at 150-ms drive cycle length, single,

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double and triple extra stimuli were applied using a standard programmed electrical

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stimulation protocol. The coupling interval of the last extra stimulus was decreased in 2ms

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steps beginning at 100ms and finishing at the ventricular effective refractory period.

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2.3.3. Monophasic action potentials recording

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For monophasic action potentials recording and analyzing, the chest of all rats was opened

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under anesthetized state with endotracheally intubated and mechanically ventilated with room

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air (respiratory rate 60 breaths/min, respiration ratio 1:1, tidal volume 2.5ml). Epicardial

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monophasic action potentials signals were amplified and recorded for subsequent analysis as

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previous report (Wang et al., 2016). A commercially available biological signal analysis

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software (RM6240, Chengdu, China) was used to digitalize, store, and analyze the action

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potential signals. The software was used to analyze the action potential durations (APD90) at

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90% repolarization.

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2.3.4. APD alternans

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APD alternans，an oscillation of APD from beat to beat, which resulted from dynamic

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instability of cardiac repolarization, is a precursor of malignant ventricular arrhythmias(Weiss

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et al., 2011). For determining the APD alternans, paced at right ventricular apex with

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increased frequency and a voltage twice of capture threshold until the occurrence of long and

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short APD from beat to beat. The APD alternans was defined as a variation of >5ms in

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sequential beats with APD， and the threshold was defined as the first CL induced APD

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alternans (Chang et al., 2017).

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2.4. Measurement of noradrenaline.

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Blood samples were obtained from rats 2 weeks after resiniferatoxin injection and stored

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at -70°C until analysis. Noradrenaline levels were determined by enzyme linked

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immunosorbent assay (ELISA) kit (USCN Life Science) according to the manufacturer’s

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instructions(Wang et al., 2019). Briefly, plasma was separated before assaying. Samples and

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different dilution standards were added into testing wells. The wells were shaken and

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incubated 1 h at 37°C. After washed tree times, respective biotinylated Noradrenaline

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antibody was added to each well and incubated 1h at 37°C. The wells were washed five times

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with the washing buffer and streptavidin-peroxidase conjugate was added and washed. After

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that, chromogen substrate solution was added to each well and incubated for 20min at 37°C.

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Stop solution was added and optical density was detected immediately using a microplate

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reader (Bio-Rad, Hercules, CA).

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2.5. Histology

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Animals were killed after completing the electrophysiological studies, and the hearts and

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spinal cord from T1 to T2 were removed. The weight of heart and the body was calculated as

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previously reported (Zhu et al., 2015). The specimens were fixed in 10% buffered formalin,

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embedded in paraffin, and cut into sections (5um thick). The sections were stained with

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hematoxylin-eosin (H&E) and calculated cardiomyocyte size and septal wall thick which

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acted as myocardial hypertrophy(Zhu et al., 2015). Masson’s staining was used to detect

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collagen and infarcted size as previous reported (Wang et al., 2016). The degree of cardiac

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fibrosis was determined based on the area of fibrosis divided by the total area (% cardiac

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fibrosis) by using image proPlus software (Media Cybernetics, Inc.).

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2.6. Immunofluorescence Labelling

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TRPV1 and calcitonin gene-related peptide (CGRP) were used to detect sympathetic

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afferent distribution in T1-4 spinal tissue(Sapio et al., 2018; Wang et al., 2019). Paraffin-fixed

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sections from spine were processed routinely and incubated with mouse monoclonal TRPV1

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(dilution: 1:200; rabbit anti-CGRP: 1:200; Abcam, Cambridge, UK) at 4℃ overnight. A

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rhodamine-conjugated goat anti-mouse IgG (dilution：1:300；Santa Cruz Biotechnology Inc，

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CA) were incubated with the tissue sections at 37℃for 1 h. The immunofluorescence signals

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specific for TRPV1 and CGRP were examined under a confocal immunofluorescence

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microscope (Zeiss LSM510 META, Germany).

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2.7. Western Blot

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Protein expression was assayed by western blot as previous described (Wang et al., 2019).

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The proteins were isolated, and the protein concentrations of the samples were determined by

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bicinchoninic acid protein assay. Total proteins were separated via 5% sodium dodecylsulfate

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polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane.

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The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20

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(10 mM Tris-HCl, 150 mM NaCl, and 0.2% Tween-20, pH 7.6) for 2 h at 37 °C and

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incubated overnight at 4 °C with primary antibodies in certain dilution (Rabbit polyclonal to

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Ryanodine receptor2/RyR-2(phosphor Ser2808),1:1000; Mouse monoclonal [C3-33] to

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Ryanodine Receptor, 1:1000; Rabbit polyclonal to CaMKII (phospho T286), 1:1000; abcam).

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Antibody binding was detected with a horseradish peroxidase-conjugated secondary antibody

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(1:2000; Sigma) and visualized using an ECL kit. The imaging program Quantity One

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(Bio-Rad) was used for quantification.

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2.8. Statistical analysis

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Continuous variables were expressed as mean±S.E.M. Significant differences between

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groups were evaluated using one-way ANOVA, followed by a post-hoc Newman–Keuls

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multiple comparison test. Chi-square test was used to compare the electrophysiological data

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on the inducibility of ventricular arrhythmias. Statistical analyses were performed using SPSS

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19.0, and P < 0.05 was considered significant.

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3. Results

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3.1. Intrathecal resiniferatoxin ablates central TRPV1 afferents in the dorsal horn.

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In normal spinal cord sections, the terminals of afferent nerve express TRPV1 and CGRP,

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which located in the superficial layers of dorsal horn including laminae I and II (Jeffry et al.,

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2009; Sapio et al., 2018). Systematic and focal resiniferatoxin administration can selectively

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abolish TRPV1 positive nerves and exert a strong analgesic action(Bishnoi et al., 2011; Sapio

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et al., 2018). As shown in Fig. 1B and C, there were no evidences of spinal injury and local

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inflammatory responses which related to resiniferatoxin injection. Moreover, no animal

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showed paraplegic performances during the whole experiments. Thoracic sections of spinal

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cords were stained for CGRP and TRPV1 and showed prominent detectable signals in the

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levels from T1 to T4 (Fig. 1D and G). Intrathecal resiniferatoxin prohibited significantly the

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expression of TRPV1 (10.6±3.20 vs 94.6±3.36, P<0.001) and CGRP (17.0±7.70 vs 94.2±4.4,

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P<0.001) immunoreactive signals in dorsal horn from the thoracic spinal cord, which

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suggested a marked loss of the central afferent terminals at the segmental levels

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corresponding to the point of injection (Fig. 1E, F, H and I). We also observed the protein

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expression of TRPV1 and CGRP in heart but no marked difference with or without

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resiniferatoxin injection (supplementary data).

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3.2. Effects of intrathecal resiniferatoxin on cardiac sympathetic activities and

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noradrenaline release.

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Cardiac sympathetic nerve activated in chronic heart failure after myocardial infarction,

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which could be recorded and analyzed through direct or indirect methods(Irie et al., 2017;

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Wang et al., 2017). As shown in Fig. 2A and B, the cardiac sympathetic activities was

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recorded directly in all animals, which shown a shape like action potential when

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magnification. Cardiac sympathetic activities were significantly increased post-myocardial

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infarction (44.7±6.3µV vs 22.0±7.6µV, P<0.01). Intrathecal resiniferatoxin inhibited baseline

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cardiac sympathetic tone, which showing declined frequency and amplitude of sympathetic

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activities (21.1±12.8µV vs 44.7±6.3µV, P<0.01). Noradrenaline levels also were determined

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and showed a significantly increase in blood (148.6±11.4 ng/ml vs 36.3±7.8 ng/ml, P<0.01).

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Intrathecal resiniferatoxin caused a marked declination of noradrenaline in blood which

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suggested that a prohibitive effect on sympathetic neurotransmitter releases (57.1±9.3ng/ml

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vs 148.6±11.4 ng/ml, P<0.05).

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3.3. Effects of intrathecal resiniferatoxin on cardiac remodeling.

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As shown in Fig. 3, myocardial infarction significantly increased heart-to-body weight

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ratios (2.89±0.33 mg/g vs 2.33±0.29 mg/g, P<0.01), cardiomyocyte sizes (15.17±0.89µm

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vs 11.31±1.29µm, P<0.01) and septal wall thickness (3.03±0.29 mm vs 2.53±0.29 mm,

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P<0.01). Two weeks after intrathecal resiniferatoxin administration, no significant differences

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in infarct size (24.2±7.6% vs 25.1±6.64%, P=0.81), myocyte size (15.25±0.8µm vs

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15.17±0.89µm, P=0.884), heart-to-body weight ratios (3.0±0.37mg/g vs 2.89±0.33 mg/g,

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P=0.506) and septal wall thickness (2.81±0.24mm vs 3.02±0.29mm, P=0.141) were

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observed among these myocardial infarcted heart (all P>0.05). Altogether, these data suggest

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that intrathecal resiniferatoxin administration has no significant effect on cardiac remodeling

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after myocardial infarction.

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3.4. Effects of intrathecal resiniferatoxin on action potential durations (APD) and APD

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alternans.

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APD reflects the repolarization of the heart, which can be recorded and analyzed in vivo from epicardial monophasic action potentials (Chang et al., 2017; Wang et al., 2016).

A

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prolonged repolarization is classic electrophysiological feature of heart failure both in human

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and animal (Li et al., 2004; Xiong et al., 2018). As shown in Fig. 4A and B, APD90 was

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significantly prolonged in the post-infarcted heart (93.13±7.59ms vs 65.75±7.26ms, P<0.01).

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The prolongation of APD90 was significantly shortened by intrathecal resiniferatoxin

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administration (72.87±5.14ms vs 93.15±7.59ms, P=0.47). These data indicate that the

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prolonged repolarization might facilitated the inducibilities of ventricular arrhythmias.

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It has been demonstrated that abnormal repolarization or APD alternan is a precursor of

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malignant ventricular arrhythmias(Weiss et al., 2011), and is a highly sensitive marker of

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susceptibility to sudden cardiac death in patients with heart failure (Pastore et al., 1999;

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Wilson et al., 2009). As shown in Fig. 5A and B, some animals showed a visible difference of

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beat to beat in post-infarcted heart. Accelerated pace was performed to evaluate relationship

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between APD alternan and the pacing cycle length in each group (Fig. 5D), there was a

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significantly longer cycle length to induce APD alternan in post-infarcted hearts compared

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with control hearts (7.13±0.83Hz vs 16.75±1.49Hz, P<0.01) (Fig. 5E). Furthermore, there

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was an alternation of long and short QT intervals before the onset of ventricular tachycardia

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or ventricular fibrillation (Fig. 5C). Intrathecal resiniferatoxin significantly shortened cycle

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length to induce APD alternan (9.50±1.19Hz vs 7.13±0.83Hz, P<0.01). These data indicate

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that intrathecal resiniferatoxin administration prevented heart from APD alternan.

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3.5. Effects of intrathecal resiniferatoxin on ventricular arrhythmias.

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Programmed electrical stimulation, an effective method to provoke reentrant arrhythmias,

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has been used widely to evaluated arrhythmic risks(Kuhlmann et al., 2006; Wang et al., 2011).

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As shown in Fig. 6A, B and C, representative electrocardiogram of ventricular tachycardia

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and ventricular fibrillation was induced by electrical stimulation. In control group, ventricular

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tachycardia was induced in one of the 10 animals. The ventricular tachycardia inducibility

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was higher in the post-infarcted heart than that in the control hearts (86.7% vs 10%, P<0.01).

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Intrathecal resiniferatoxin administration significantly decreased the duration and the

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proportion of induced ventricular tachycardia (35.7% vs 86.7%, P<0.05).

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3.6. Effects of intrathecal resiniferatoxin on the expression of RyR2 and CaMKII.

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Abnormal calcium handling and associated dysfunction of ion channels may account for

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ventricular arrhythmias in heart failure (Chang et al., 2017; Pastore et al.,

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1999). Ca2+/calmodulin-dependent protein kinase II (CaMKII) induced robust increases in

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Ca2+ sparks through ryanodine receptor2/RyR-2 activation is associated with susceptibility to

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the arrhythmogenic action potential alternans (Ai et al., 2005; Mitsuyama et al., 2014). As

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shown in Fig. 7, the phosphorylated form of RyR2 in heart homogenates increased

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significantly in rats with heart failure. Treatment with resiniferatoxin reversed the activation

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of RyR2. However, the total RyR2 levels declined significantly in heart failure, which could

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not be reversed by resiniferatoxin administration. In agree with previous studies, CaMKII

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over-activated in heart from heart failure rats, which could be prohibited by resiniferatoxin

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administration. These data indicate that resiniferatoxin administration ameliorated APD

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alternans and the inducibilities of ventricular arrhythmias might be involved in preventing

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heart from abnormal activation of calcium handling ion channels in heart failure.

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4. Discussions

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Resiniferatoxin has long been used to blunt sensory neurons through sustained activation

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of TRPV1 channel, which is involved in calcium cytotoxicity in TRPV1+ primary afferent

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nociceptors due to prolonged opening of this Na+/Ca2+ cation channel(Karai et al., 2004b).

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TRPV1 is expressed in afferent nerve terminals in all species, which being used to achieve

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local selectively, long-term but reversible regional analgesia through regionally application

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resiniferatoxin (Brown et al., 2015; Karai et al., 2004a; Neubert et al., 2008). Different

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application routes allow for targeted pain alleviation with minimal impact on surrounding

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sensory nerve fibers by interrupting spinal cord circuits for transmitting nociceptive signals

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(Iadarola and Gonnella, 2013; Karai et al., 2004a). Recent studies showed that spinal cord

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circuits were also involved in over-activated cardiac sympathetic afferent reflex (Wang et al.,

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2017; Wang et al., 2014). Given the advantages of intrathecal injection over peri-cardiac

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application in operation techniques(Sapio et al., 2018), we try to determine whether

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chemo-axotomy of the centrally projecting axons of TRPV1+neurons at the levels from T1 to

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T4 spinal cord is sufficient for profound depressing reflex. Our findings clarified that

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intrathecal resiniferatoxin application can selectively abolish the spinal terminals of cardiac

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afferent fibers, inhibit cardiac sympathetic nerve sparks, and reduce the risks of ventricular

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arrhythmias in rat with heart failure.

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In agreement with previous studies(Karai et al., 2004a; Sapio et al., 2018; Wang et al., 2019),

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our study showed that resiniferatoxin abolished the neurochemical markers of sensory afferent

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fibers in the rodent dorsal horn through evaluating TRPV1 and CGRP in spinal cord. According to

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previous studies (Sapio et al., 2018; Shanks et al., 2019; Wang et al., 2019), the lesion of afferent

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fibers might be persistent for a long time (more than 8 weeks), and consequently a persistent

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therapeutic action. Moreover, previous study had demonstrated that intrathecal resiniferatoxin

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blocked the transmit signals to the spinal cord accompanied by limited alterations at the level of

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the dorsal root ganglion and preserved the peripheral axon, allowing for retention of interactions

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between primary afferent neurons and sites of peripheral innervation(Sapio et al., 2018). We

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indeed found that TRPV1 and CGRP kept intact in heart after intrathecal resiniferatoxin

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administration (supplementary data). Therefore, the protective effects of CGRP would be

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preserved in myocardial ischemia.

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It was reported that cardiac sympathetic afferent reflex enhanced in heart failure (Wang

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et al., 1999). In order to evaluate cardiac sympathetic responses after loss of central terminals

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of afferent fibers, we measured sympathetic tone by indirect cardiac norepinephrine spill-over

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assaying as well as direct cardiac or cardiac sympathetic nerve activity recording(Chen et al.,

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2015; Wang et al., 2019). Our results revealed that the increased cardiac sympathetic tone in

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heart failure rats reversed by intrathecal resiniferatoxin. The decline of noradrenaline levels in

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blood further confirmed the sympathetic prohibition effects by intrathecal resiniferatoxin

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administration. However, we did not find significant differences of cardiac fibrosis and

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hypertrophy with or without resiniferatoxin administration although previous studies showed

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that enhanced cardiac sympathetic afferent reflex involved in regulation of blood pressure and

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cardiac dysfunction (Wang et al., 2019; Wang et al., 2017; Wang et al., 2014). One

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explanation is that intervention for short term is insufficient to reverse cardiac remodeling.

340

Another explanation is that cardiac remodeling mainly begins at the early stages after

341

myocardial infarction.

342

Enhanced sympathetic nerve tone and sympathetic sparks are involved in ventricular

343

arrhythmias in heart failure (Shen and Zipes, 2014; Zhou et al., 2008). Ablation of cervical

344

stellate ganglion of patients or animals with heart failure could reduce the risk of ventricular

345

tachycardias (Cardona-Guarache et al., 2017; Schwartz, 2014). In the present study, we

346

observed that central chemical ablation TRPV1+ nerve in dorsal horn prohibited the

347

inducibility of ventricular tachycardias. We also found a prolonged APD in heart failure,

348

which is associated with acquired long QT intervals and proarrhythmia risk(Li et al., 2004;

349

Xiong et al., 2018), could be reversed by intrathecal resiniferatoxin.

350

APD alternans have been known as a precursor of malignant ventricular arrhythmias and

351

sudden cardiac death(Pastore et al., 1999; Wilson et al., 2009). In the present study, rats with

352

heart failure showed spontaneous APD alternans or susceptibility to induce APD alternans,

353

which reversed by intrathecal resiniferatoxin. Therefore, intrathecal resiniferatoxin

354

ameliorates the ventricular tachycardias susceptibility and relevant risks. The mechanisms of

355

APD alternans may be involved in large spatial gradients of repolarization and abnormal

356

calcium handling in cardiac myocytes from heart failure models(Chang et al., 2017; Pastore et

357

al., 1999; Wilson et al., 2009). Over activation of CaMKII and RyR-2 in failing heart caused

358

Ca2+ leak from sarcoplasmic reticulum, which associated with the mechanisms of ventricular

359

tachycardias and APD alternans (Ai et al., 2005; Mitsuyama et al., 2014). Our study

360

confirmed that intrathecal resiniferatoxin prevent CaMKII and RyR-2 from over

361

phosphorylation in failing heart. These data indicate that resiniferatoxin administration

362

ameliorated APD alternans and the inducibilities of ventricular arrhythmias might be involved

363

in preventing heart from abnormal activation of calcium handling ion channels in heart

364

failure.

365

Our study firstly confirmed the important role of spinal circuits of cardiac sympathetic

366

afferent reflex in heart failure rats after myocardial infarction. Previous studies had shown

367

that TRPV1+ afferents detected various of noxious stimuli such as temperature(Caterina et al.,

368

1997), pH and some endogenous signaling molecules(Cao et al., 2013; Kaszas et al., 2012;

369

Ross, 2003). The enhanced reflex lead to high cardiac sympathetic nerve efferent activities

370

and noradrenaline spill out, may consequently destroy the electrophysiological stability in

371

heart and facilitate the inducibility of ventricular tachycardias. Most importantly, based on the

372

specific structure of cardiac afferent nerve with one terminal distributed in various sensory

373

organs and the other focused on the dorsal horn, our study firstly created a new method to

374

blunt cardiac sympathetic afferent reflex in heart failure through central selective abolished

375

TRPV1+ afferent nerves and produced a protective effect against cardiac electrical instability.

376

Several limitations should be stated in this study. Firstly, we deduced that the preventive

377

mechanism of resiniferatoxin against ventricular tachycardias occurrence and APD alternans

378

in heart failure may be associated with Ca2+ handling. Because we founded that the

379

abnormality of calcium handling related protein CaMKII and RyR2 was reversed by

380

resiniferatoxin administration although we did not observe the dynamic Ca2+ leak from

381

sarcoplasmic reticulum in heart failure. Secondly, we will acquire certain evidences of

382

improvement of cardiac remodeling or cardiac dysfunction if underwent a long-term

383

observation after resiniferatoxin administration. Finally, we are not sure the occurrence of

384

ventricular tachycardias in non-anesthesia states in heart failure rats because no ECG

385

recording in conscious animal by remote supervision system.

386

In conclusion, even if the existence of some limitations, our study firstly revealed that

387

intrathecal resiniferatoxin administration at levels of T1-T4 is an effective route to blunt the

388

enhanced cardiac sympathetic afferent reflex in heart failure through selective abolished

389

TRPV1+ afferent in dorsal horn.

390

APD alternans and abnormal activation of calcium handling ion channels in heart failure.

391

Unlike the permissive distribution in different organs, TRPV1+ afferents are mainly focus in

392

dorsal horn, convenience to be chemic ablation by resiniferatoxin(Sapio et al., 2018). If

393

guided by computer tomography, intrathecal injection could be applied in large animal and

394

even human. For some means, the mini-invasive procedure may be a safety and repeatable

395

route to selectively block the afferent nerve(Sapio et al., 2018). Therefore, intrathecal

396

resiniferatoxin administration is a potential promising therapeutic strategy for anti-arrhythmia

397

in heart failure.

398

Acknowledgements

399

It may reduce the pro-arrhythmia risk through ameliorating

This work was supported by grants from National Natural Science Foundation of China

400

(Nos. 81670301 to W.D., 81471114 to H.N., 81772180 and 81472017 to L.K..) and the Key

401

projects of Anhui Province University outstanding youth talent support program

402

(gxqZD2016181 to W.D.).

403

Competing financial interests

404

We have no conflict of interest or the appearance of a conflict of interest.

405

The data used to support the findings of this study are included within the article.

406

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Figures and legends

535

Fig. 1. Intrathecal resiniferatoxin ablates central terminals of sympathetic afferent nerves in

536

dorsal horn. (A) Schematic of the experimental intrathecal injection routes in spinal cord. (B) and

537

(C) Representative micrographs showing the cross-section of a spinal cord stained with

538

hematoxylin and eosin (H&E). (D) and (E) Representative immunofluorescence of CGRP and

539

TRPV1 staining (red) in the dorsal horn of lumbar spinal cord sections with or without

540

resiniferatoxin injection. (F) and (G) Relative quantitative analysis CGRP and TRPV1 signals with or

541

without resiniferatoxin injection. Data were analyzed using student t test.

542

** present P<0.01 between two groups（N=5） ；RTX: resiniferatoxin.

*present P<0.05, and

543 544

Fig. 2. Intrathecal resiniferatoxin reduces cardiac sympathetic activities and noradrenaline

545

release in rats with chronic heart failure. (A) Representative trace of cardiac sympathetic nerve

546

activities (CSNA) and a magnified potential from sham control, chronic heart failure (CHF), and

547

CHF plus intrathecal resiniferatoxin (RTX) administration (CHF+RTX). (B) Quantitative analysis of

548

CSNA in rats from different groups. (C) Noradrenaline (NA) levels in plasma from different groups.

549

Data were analyzed using 2-way ANOVA. Means were compared by post hoc multiple-comparison

550

test (Newman-Keuls). * present P<0.05, and ** present P<0.01 between different groups (n=8).

551 552

Fig. 3. Intrathecal resiniferatoxin prevents heart from cardiac remodeling in rats with chronic

553

heart failure. (A) Representative micrographs showing the cross-section of a heart stained with

554

masson’s and hematoxylin and eosin (H&E). (B) in heart from sham control, chronic heart failure

555

(CHF), and CHF plus intrathecal resiniferatoxin (CHF+RTX). (C) Summary data of infarction size (%),

556

(D) myocyte size, heart weight: body weight (HW:BW) ratios and septal wall thickness in heart

557

from different groups. Data were analyzed using 2-way ANOVA. Means were compared by post

558

hoc multiple-comparison test (Newman-Keuls). * present P<0.05, and ** present P<0.01 between

559

different groups (n=8).

560 561

Fig. 4. Intrathecal resiniferatoxin reverses the prolongation of action potential durations (APD)

562

in rats with chronic heart failure. (A) Representative apical action potentials in the apex of the

563

hearts of sham control, chronic heart failure (CHF), and CHF plus intrathecal resiniferatoxin

564

(CHF+RTX). (B) Quantitative analysis the duration of APD90 in control. CHF and CHF+RTX hearts.

565

Data were analyzed using 2-way ANOVA. Means were compared by post hoc multiple-comparison

566

test (Newman-Keuls). * present P<0.05, and ** present P<0.01 between two groups (n=8).

567 568

Fig. 5. Intrathecal resiniferatoxin prevents action potential durations (APD) alternans in rats

569

with chronic heart failure. (A) Representative epicardial monophasic action potentials from

570

control and CHF hearts. (B) Spontaneous APD alternans is seen in some CHF hearts. (C)

571

Representative cardiographic tracings of pacing induced APD alternans and followed ventricular

572

arrhythmias. (D) Representative ECG and APD alternans tracings at 16Hz pacing. (E) Summary

573

data demonstrated that pacing cycle length was prolonged in CHF hearts compared with control

574

hearts, shortened in hearts from CHF plus intrathecal resiniferatoxin (CHF+RTX). Data were

575

analyzed using 2-way ANOVA. Means were compared by post hoc multiple-comparison test

576

(Newman-Keuls). * present P<0.05, and ** present P<0.01 between two groups (n=8).

577 578

Fig. 6. Intrathecal resiniferatoxin reduces ventricular arrhythmias in rats with chronic heart

579

failure. (A) and (B) Representative induced electrocardiogram tracings of programed electrical

580

stimulations (PES), non-sustained ventricular tachycardia (VT), sustained VT and ventricular

581

fibrillations (VF). (C) Summary data of the inducibility of ventricular arrhythmias from the hearts

582

of sham control, chronic heart failure (CHF), and CHF plus intrathecal resiniferatoxin (CHF+RTX).

583

Data were analyzed using 2-way ANOVA. Means were compared by post hoc multiple-comparison

584

test (Newman-Keuls). * present P<0.05 between two groups.

585 586

Fig. 7. Intrathecal resiniferatoxin reverses the phosphorylation of CaMKII and RyR-2 in rats with

587

chronic heart failure. (A) Representative Western blot stain showing the relative protein

588

expression of phosphorylated CaMKII and RyR-2 in heart of sham control, chronic heart failure

589

(CHF), and CHF plus intrathecal resiniferatoxin (CHF+RTX). Quantitative analysis the expression of

590

phosphorylated RyR-2 (B) and CaMKII (D) and total RyR-2 (C) . Data were analyzed using 2-way

591

ANOVA. Means were compared by post hoc multiple-comparison test (Newman-Keuls). * present

592

P<0.05 between two groups (n=5).