- Email: [email protected]
Focal selective chemo-ablation of spinal cardiac afferent nerve by resiniferatoxin protects the heart from pressure overload-induced hypertrophy
Biomedicine & Pharmacotherapy 109 (2019) 377–385
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Focal selective chemo-ablation of spinal cardiac afferent nerve by resiniferatoxin protects the heart from pressure overload-induced hypertrophy
T
⁎
Deguo Wanga,b, , Yong Wua, Yueyun Chena, Ancai Wanga, Kun Lvb, Xiang Kongc, Yang Hed, Nengwei Hue a
Department of Gerontology, Yijishan Hospital of Wannan Medical College, Wuhu, 241001, PR China Non-Coding RNA Research Center of Wannan Medical College, Wuhu, Anhui, 241001, PR China Department of Endocrinology, Yijishan Hospital of Wannan Medical College, Wuhu, 241001, PR China d School of Basic Courses, Wannan Medical College, Wuhu, Anhui, 241001, PR China e Department of Pharmacology and Therapeutics, and Trinity College, Institute of Neuroscience, Biotechnology Building, Trinity College Dublin, Dublin 2, Ireland b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Cardiac sympathetic nerve activities Transverse aortic constriction Resiniferatoxin Cardiac hypertrophy Fibrosis Apoptosis
Resiniferatoxin (RTX), a selective transient receptor potential vanilloid 1 (TRPV1) receptor agonist, can eliminate TRPV1+ primary sensory afferents and blunt cardiac sympathetic afferent reflex for a relatively long period. The present study determined the effects of intrathecal RTX administration on transverse aortic constriction (TAC)-induced cardiac dysfunction and cardiac remodeling in rats. Five days before TAC, RTX (2 μg/ 10 μl) was injected intrathecally into the T2/T3 interspace of rats. Cardiac sympathetic nerve activities (CSNAs) and cardiac structure and function were determined eight weeks after TAC. Intrathecal RTX administration abolished TRPV1 expression in the dorsal horn and reduced over-activated CSNA in the TAC rat model. Hemodynamic analysis revealed that RTX reduced left ventricular end-diastolic pressure, indicating the improvement of cardiac compliance. Histologic analysis, real-time reverse transcription-polymerase chain reaction, and Western blots showed that RTX prevented TAC-induced cardiac hypertrophy, cardiac fibrosis, and cardiac apoptosis and reduced the expression of apoptotic proteins and myocardial mRNAs. In conclusion, these results demonstrate that focal chemo-ablation of TRPV1+ afferents in the spinal cord protects the heart from pressure overload-induced cardiac remodeling and cardiac dysfunction, which suggest a novel promising therapeutic method for cardiac hypertrophy and diastolic dysfunction.
1. Introduction Heart failure (HF) is a common, costly, and potentially fatal condition [1]. It is always preceded by cardiac hypertrophy and diastolic dysfunction [2]. Cardiac hypertrophy is characterized by an increase in heart mass due to cardiomyocyte hypertrophy and proliferation of noncardiomyocyte cells in response to pressure overload [3]. During the process of cardiac hypertrophy, sympathetic activity is always overactivated [4]. Therefore, blocking excessive sympathetic activation would be a promising approach to prevent hypertrophic heart from developing into HF. The sympathetic tone is regulated by the cardiac sympathetic afferent reflex (CSAR), which is formed by sympathetic afferent fibers, dorsal root ganglia (DRG) of the C8–T9 spinal segments, hypothalamic
and medullary brain centers, and sympathetic efferent nerves [5]. Transient receptor potential vanilloid 1 (TRPV1) is expressed in sensory afferent terminals in different organs, including the heart [5]. A previous study found that mice lacking TRPV1 (TRPV1-/-) are resistant to cardiac hypertrophy, which is induced by transverse aortic constriction (TAC) [6]. However, another recent study showed that TRPV1 knockout mice (TRPV1-/-) had excessive inflammation, cardiac hypertrophy, and deteriorated cardiac function after TAC [7]. The completely different results of the two studies illustrated the complex role of TRPV1 in pathophysiological conditions. The results also infer that specifically eliminating TRPV1 in the target location is necessary to avoid systemic damages. Indeed, the inhibition of the cardiac afferent nerve by epicardial capsaicin administration or spinal dorsal horn resection reduced proinflammatory cytokine expression in a mouse TAC model [8].
⁎ Corresponding author at: Department of Gerontology and Non-Coding RNA Research Center of Wannan Medical College, Yijishan Hospital of Wannan Medical College, 92nd Zheshan Western Road, Wuhu, Anhui, 241001, PR China. E-mail address: [email protected] (D. Wang).
https://doi.org/10.1016/j.biopha.2018.10.156 Received 4 August 2018; Received in revised form 25 October 2018; Accepted 25 October 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Sham operation rats underwent the same procedure without ligation of the aorta.
Resiniferatoxin (RTX), a selective TRPV1 agonist, can selectively lesion TRPV1+ primary sensory afferents. Systemic or focal administration of RTX can almost completely abolish pain sensation and inflammation [9]. The application of RTX on the heart surface can blunt CSAR in adult rats, lasting for 10 weeks [10–12]. Obviously, brushing RTX on the heart surface is difficult. Recently, a novel route via intrathecal RTX administration had been reported to reduce pain and inflammation without damaging neurons and peripheral terminals [9,13,14]. Through intrathecal RTX injection, Sapio et al. [15] applied chemo-axotomy of the TRPV1+ afferents for pain control in human and animal models. Therefore, we locally administered RTX at the T1 to T4 spinal cord levels (accepting cardiac afferent fibers) to ablate the cardiac sensory nerve. In the present study, we performed intrathecal RTX application in TAC rats to determine the role of spinal terminals of cardiac afferent fibers in cardiac hypertrophic response to aortic artery stenosis and whether intrathecal RTX application can ameliorate cardiomyocyte apoptosis and interstitial fibrosis.
2.3. Echocardiography assessment Eight weeks after TAC, echocardiography assessment was performed by a tester who was blinded to the experimental groups using an HP5500 cardiac ultrasound system with a 15 MHz linear-array probe. M-mode tracings at the parasternal long- and short-axis views of the LV were used to measure the interventricular septum diameter (IVSd), LV posterior wall diameter (LVPWd), LV end-systolic diameter (LVSd), LV end-diastolic diameter (LVDd), LV percent ejection fraction (LVEF), and LV percent fractional shortening (LVFS) [17]. LVEF = (LVEDVLVESV)/LVEDV × 100%; LVFS = (LVDd-LVDs)/LVDd×100%. 2.4. Physiological information recording and analysis 2.4.1. Cardiac sympathetic nerve activity (CSNA) The rats were anesthetized and fixed in supine position, endotracheally intubated, and mechanically ventilated (respiratory rate 60 breaths/min, respiration ratio 1:1, tidal volume 2.5 ml). Left cervical stellate ganglion was identified behind the left carotid artery. A branch innervating heart was isolated and hooked by a pair of recording electrodes (with 2 mm space). Recording signals were amplified (×10,000) and filtered (bandwidth: 100–3000 Hz) by a biological data acquisition system (RM6240, Chengdu, China) [12].
2. Materials and methods 2.1. Animals and groups Male Sprague–Dawley rats (180–220 g, n = 60) were purchased from the Experimental Animal Center of Nanjing. The experimental procedures were approved by Yijishan Hospital Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health guiding principles. RTX (Sigma Aldrich, St. Louis, MO) was first dissolved in 100% ethanol to make the stock solution (concentration of 2 mg/ml) and then further diluted in 10 ml saline when used for intrathecal (T2/T3 intraspinal space) injection. As shown in Fig. 1A, all animals were randomly assigned to two groups. The RTX group (n = 30) received 2 μg/10 μl of RTX, whereas the control group (n = 30) received 10 μl of vehicle. Then, the rats randomly received TAC or sham surgery operation. Therefore, the animals were divided into four groups: (1) Sham + vehicle, (2) Sham + TAC, (3) TAC + vehicle, and (4) TAC + RTX. All survived animals underwent echocardiography eight weeks post-surgery. Then, they further underwent hemodynamic and cardiac sympathetic nerve recording (Fig. 1C). Afterward, rat hearts were subsequently harvested, and the heart weight:body weight (HW:BW) ratio was calculated. Left ventricular (LV) samples were collected and stored at −80 °C for further molecular biologic and histological evaluations.
2.4.2. Measurement of LV performance LV performance was assessed using a self-made polyethylene catheter inserted via the right carotid artery. Briefly, the catheter, prefilled with fresh heparinized saline (500 U/ml) and connected to a biological data acquisition system (RM6240, Chengdu, China), was inserted into the right carotid artery and advanced into the LV. The pressure signal of LV was recorded continuously and stored in a computer to determine off-line LV end-diastolic pressure (LVEDP), LV systolic pressure (LVSP), maximum first derivative of LV pressure (dp/ dtmax), and minimum first derivative of LV pressure (-dp/dtmax). 2.4.3. Measurement of norepinephrine(NE)in plasma and heart Heart and blood samples were obtained from rats eight weeks after TAC and stored at −70 °C until analysis. NE levels were determined using an ELISA kit (Abnova, Shanghai, China) according to the manufacturer’s instructions. Briefly, LV myocardial samples were homogenized, and plasma was separated before assaying. The samples and different dilution standards were added into testing wells. The wells were shaken and incubated for 1 h at 37 °C. After washing for three times, biotinylated NE antibody was added to each well and incubated at 37 °C for 1 h. The wells were washed five times with the washing buffer, added with streptavidin-peroxidase conjugate, and then washed again. Afterward, chromogen substrate solution was added to each well and incubated at 37 °C for 20 min. Stop solution was added, and optical density was detected immediately using a microplate reader (Bio-Rad, Hercules, CA).
2.2. Animal models 2.2.1. Intrathecal injection The animals received RTX injections as previously described with minor modifications [13]. After isoflurane anesthesia (5% isoflurane), intrathecal injections were performed by inserting a U-40 insulin syringe with 29 G needle into the T2/T3 interspace perpendicular to the spine. The criterion for the intrathecal placement was withdrawal of clear cerebrospinal fluid into the syringe. Then, RTX (2 μg/10 μl) or vehicle (10 μl saline with 10% ethanol) was injected slowly into the intrathecal space.
2.5. Histology 2.5.1. Hematoxylin–eosin (H&E) and Sirius Red stain Hearts from all groups were harvested and weighed. Heart specimens were fixed in 10% buffered formalin, embedded in paraffin, and cut into sections (5 μm thick). The sections were stained with H&E and Sirius Red stain. Cardiomyocyte size was assessed in randomly selected high-power fields in each section. To determine the degree of cardiac fibrosis, images of Sirius Red-stained samples were transferred into a computer. A minimum of 10 fields from each section was scored at 100× magnification. The degree of cardiac fibrosis was determined based on the area of fibrosis divided by the total area (%cardiac
2.2.2. TAC The surgical procedures for the TAC model were performed as previously described with minor modifications [16]. Briefly, the rats were anesthetized with intraperitoneal injection of pentobarbital sodium (60 mg/kg). A 2 cm incision was made along the midline of the abdomen to isolate the abdominal aorta beside the left renal artery. A blunt and bent 21 G needle was placed along the abdominal aorta. A 4-0 ligature was tied twice around the needle and the aorta. Then, the needle was removed, yielding a 0.8 mm-diameter artery constriction. 378
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Fig. 1. Intrathecal RTX administration ablates TRPV1 expression and reduces sympathetic nerve activities. (A) Schematic of the experimental design. (B) Representative immunofluorescence of TRPV1 staining (red) in the dorsal horn of lumbar spinal cord sections. (C) Schematic of spinal elements of cardiac sympathetic afferent nerve (green), sympathetic efferent nerve (yellow), and recording of cardiac sympathetic nerve activities (CSNAs) from cardiac branch of stellate ganglion. (D) CSNAs. (E) Western blot showing the protein expression of TRPV1 in the spinal cord. (F) NE levels in plasma. (G) NE levels in LV myocardial tissue. Values are shown in mean ± SEM; n = 5/each group. Scale bar was 100 μm. *P < 0.05 versus sham + vehicle; # P < 0.05 versus chronic TAC + vehicle. RTX, resiniferatoxin; TAC, transverse aortic constriction; TRPV1, transient receptor potential vanilloid 1 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
fibrosis) by using Image-Pro Plus software.
T1–T4 spinal tissue. Paraffin-fixed sections from the LV and spine were processed routinely and incubated with mouse monoclonal TRPV1 (dilution: 1:200; rabbit anti-tyrosine hydroxylase (TH): 1:200; rabbit anti-NeuN: 1;200; Abcam, Cambridge, UK) at 4 °C overnight. A rhodamine-conjugated goat anti-mouse and an FITC-conjugated goat antirabbit secondary antibody were incubated with the tissue sections at 37 °C for 1 h. The double-stained sections were observed, and the immunofluorescence signals specific for TRPV1 were examined under a confocal immunofluorescence microscope (Zeiss LSM510 META, Germany).
2.5.2. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labeling (TUNEL) assay For the detection of apoptosis, paraffin-fixed LV tissue sections were stained by TUNEL assay using an in situ cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Tissue sections were stained with TUNEL reaction mixture and Converter-POD and observed under a microscope (Eclipse, Nikon, Tokyo, Japan). Apoptotic nuclei were manually identified to ensure that only the apoptotic cardiomyocyte nuclei were included. The number of TUNEL-positive cells was expressed as a percentage of the total number of nuclei.
2.6. Western blot The proteins were isolated, and the protein concentrations of the samples were determined by bicinchoninic acid protein assay. Total proteins were separated via 14% sodium dodecylsulfate polyacrylamide
2.5.3. Immunofluorescence labeling TRPV1 was used to detect sympathetic afferent distribution of 379
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Fig. 2. Effects of intrathecal RTX administration on cardiac hypertrophy in sham or TAC rats. (A) Representative micrographs showing the cross-section of a heart stained with hematoxylin and eosin (H&E) (B) and quantification of cardiomyocyte diameters after intrathecal RTX administration in sham or TAC rats. (C) Heart weight, (D) body weight, (E) and heart weight:body weight (HW:BW) ratios in sham + vehicle, sham + RTX, TAC + vehicle, and TAC + RTX rats. (F) Relative mRNA expression of ANP, (G) BNP, and (H) β-MHC in sham + vehicle, sham + RTX, TAC + vehicle, and TAC + RTX rats. Values are shown in mean ± SEM; n = 8/ each group. *P < 0.05 versus sham + vehicle; # P < 0.05 versus chronic TAC + vehicle. RTX, resiniferatoxin; TAC, transverse aortic constriction; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, beta myosin heavy chain.
reversely transcripted into cDNA using the All-In-One RT MasterMix reagent kit (Applied Biological Materials Inc./G492). Specific products were detected with BrightGreen qPCR MasterMix-Low ROX reaction kit (Applied Biological Materials Inc./MasterMix-S) using the Thermo Fisher 7300 Plus Read-Time PCR System according to the manufacturer’s instructions. The expression of each transcript was normalized to gene of the housekeeping protein GAPDH in the same tissue. The formula used for relative mRNA expression was 2-(OCT sample 2OCT control). The specific forward and reverse primers used are presented in Supplementary Table 2.
gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (10 mM Tris−HCl, 150 mM NaCl, and 0.2% Tween-20, pH 7.6) for 2 h at 37 °C and incubated overnight at 4 °C with primary antibodies in certain dilution. Antibody binding was detected with a horseradish peroxidase-conjugated secondary antibody (1:2000; Sigma) and visualized using an ECL kit. The imaging program Quantity One (Bio-Rad) was used for quantification. 2.7. Real-time reverse transcription-polymerase chain reaction (RT-PCR) RNA levels were detected via real-time RT-PCR. Briefly, the RNA from the LV tissues was isolated with RNAi TRIzol (Invitrogen, CA) and 380
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Fig. 3. Effects of intrathecal RTX administration on cardiac fibrosis in sham or TAC rats. (A) Representative Sirius Red stain (B) and relative area of fibrosis (%) after intrathecal RTX administration in sham or TAC rats. (D) Representative Western blot stain showing the relative protein expression of collagen I (C), α-SMA (E), and TGF-β (F) to GAPDH in LV tissue of sham + vehicle, sham + RTX, TAC + vehicle, and TAC + RTX rats. Values are shown in mean ± SEM; n = 8/each group. *P < 0.05 versus sham + vehicle; # P < 0.05 versus chronic TAC + vehicle. RTX, resiniferatoxin; TAC, transverse aortic constriction; α-SMA, α-smooth muscle actin; and TGF-β, transforming growth factor beta (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
both groups. As shown in Fig. 1D, CSNA was higher in TAC rats than in sham rats. Intrathecal RTX administration inhibited baseline cardiac sympathetic tone. Eight weeks after TAC, NE levels significantly increased in the heart (196 ± 23 ng/ml versus 120 ± 13 ng/ml, P < 0.05) and blood (121 ± 25 ng/g versus 63 ± 14 ng/g, P < 0.05). Intrathecal RTX administration remarkably prevented the rise of NE in the heart (141 ± 17 ng/ml versus 196 ± 23 ng/ml, P < 0.05) and blood (53 ± 18 ng/g versus 121 ± 20 ng/g, P < 0.05). To observe sympathetic efferent fibers in the heart with or without intrathecal RTX administration, we also stained heart slices with anti-TH (green). We found no significant changes of TH (green) in the heart compared with those in control rats (Fig. S2). These results indicated that RTX injection via intrathecal routine in T2 segment can prohibit CSNA and NE release locally and systemically for a relatively long period.
2.8. Statistical analysis The data are expressed as means ± SEM. Two-way analysis of variance and Student’s t-test were performed to analyze statistical differences in each response variable. Statistical significance was considered at P < 0.05. 3. Results 3.1. Intrathecal RTX administration ablates TRPV1 in the dorsal horn and reduces cardiac sympathetic activities In spinal cord sections, TRPV1 was located in the superficial layers of the dorsal horn, including laminae I and II [15,18], which can be selectively abolished by systemic or focal RTX administration [9]. TRPV1 expression significantly increased in the dorsal horn of TAC rats compared with that of sham rats. Intrathecal RTX administration significantly reduced TRPV1 expression of the dorsal horn in the sham and TAC rats (Fig. 1B). Western blot further showed that TRPV1 increased in the thoracic spinal cord of TAC animals and significantly reduced by intrathecal RTX administration in normal and TAC animals (P < 0.05) (Fig. 1E). As shown in the supplementary materials, we also detected the expression of TRPV1 in the DRG and heart with or without intrathecal RTX administration. Two weeks after intrathecal RTX administration, TRPV1-positive signal was reduced in dorsal root ganglia (DRG) from RTX-treated rats compared from control rats (Fig. S1). However, we did not detect any visible TRPV1 signals in the heart from
3.2. Intrathecal RTX administration improved cardiac hypertrophy and cardiac function in TAC rats TAC caused severe LV hypertrophy (HW/BW: 4.43 ± 0.11 g/kg versus 2.89 ± 0.08; cardiomyocyte width: 23.4 ± 0.8 μm versus 13.2 ± 0.4 μm). Intrathecal RTX administration significantly attenuated TAC-induced LV hypertrophy (HW/BW: 3.22 ± 0.12 g/kg versus 4.43 ± 0.11 g/kg; cardiomyocyte width: 17.2 ± 1.1 μm versus 23.4 ± 0.8 μm) (Fig. 2). Moreover, intrathecal RTX administration significantly reduced TAC-induced increases in myocardial mRNA expression of ANP, BNP, and β-MHC. These data suggested that RTX 381
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
nervous system interaction to enhance cardiac sympathetic outflow. However, brushing RTX on the surface of the heart or percutaneous epicardial application of RTX in clinical background is difficult. Therefore, the spinal terminals of cardiac afferent fibers might be a potential target for chemo-ablation. For example, Sapio et al. [15] had successfully controlled pain through chemo-axotomy of the TRPV1+ afferents of spinal segments in human and animal models. Bishnoi et al. [9] also reported that targeting TRPV1-expressing nerve terminals at the spinal cord can selectively abolish inflammatory thermal hypersensitivity without affecting acute thermal sensitivity and preserve the efferent functions of DRG neurons at the peripheral nerve terminals. Given that the dorsal horn of spinal segments T2–T6 is mainly the central projection terminals of cardiac afferent [21], we used intrathecal RTX administration in the T2 segment in this study. Consequently, we observed that intrathecal RTX administration can inhibit TRPV1 expression in the dorsal horn in normal and TAC animals. These results suggest that intrathecal injection is an appropriate route of RTX administration, which might be a suitable method to selectively blunt the cardiac afferent nerve. Previous studies have shown that sympathetic outflow is enhanced in patients with essential or secondary hypertension and a variety of experimental hypertensive models [5]. The elevated sympathetic tone can be measured by indirect cardiac norepinephrine spill-over assaying and direct cardiac or renal SNA recording [5]. Similar to these hypertensive models, TAC also causes high pressure overload to the LV and high norepinephrine spill-over [8]. In the present study, direct CSNA recording revealed that TAC rats had higher basal cardiac sympathetic tone than sham rats. The increased cardiac sympathetic tone in TAC rats can be reduced by intrathecal RTX administration in the T2 segment. Moreover, we did not observe significant changes of TH in the heart even though TRPV1 expression was inhibited in the DRG by intrathecal RTX administration (Fig. S1C and D). These data suggested that focal intrathecal RTX administration in the T2 segment selectively ablated the cardiac afferent nerve but left the cardiac efferent nerve intact in the heart. Therefore, the weakened effects of RTX administration on sympathetic tone in this study may be associated with blunting spinal–heart nervous system interaction. The reduction of NE levels in the blood and heart tissue also confirmed that RTX injection in the T2 segment can inhibit NE release. High sympathetic tone and NE release might be due to continuously activated CSAR, which is known as a positive feedback characteristic in CHF [23] and hypertension [24]. Previous studies had shown that blood pressure and cardiac function were changed by over-activation of CSAR in HF [11,12]. In the present study, TAC rats had significantly reduced cardiac diastolic function but similar systolic function compared with normal rats. Intrathecal RTX administration in the T2 segment could attenuate TAC and caused diastolic dysfunction, which showed a significant improvement of LVEDP. Cardiac compliance is determined by the structural properties of the cardiac muscle and connective tissue [25]. In the present study, intrathecal RTX administration in the T2 segment significantly attenuated the TAC-induced LV hypertrophy, which showed less heart weight and cardiomyocyte width. Importantly, RTX reduced TAC-induced increases in myocardial mRNA expression of ANP, BNP, and β-MHC. These reexpressed embryonic genes are molecular markers of cardiac hypertrophy [26]. These data suggested that cardiac afferent ablation in the spinal cord can protect the heart against TAC-induced LV hypertrophy. The cardiac connective tissue is an important factor of cardiac compliance [27]. In TAC rats, cardiac fibrosis contributes to LV remodeling and cardiac diastolic dysfunction [19]. Our data confirmed that RTX administration in the T2 segment significantly attenuated TAC-induced LV fibrosis and fibrosis-associated proteins TGF-β and αSMA. Cell death is another important factor of cardiac remodeling [20,25]. Our results showed that RTX prevented myocardial apoptosis and abnormal expression of apoptosis-associated proteins in TAC rats. The anti-fibrotic and anti-apoptotic effects might help in reducing the
administration at the spinal cord can protect hearts against TAC-induced LV hypertrophy. Eight weeks after TAC, the rats exhibited comparable systolic parameters, including ejection fraction and fractional shortening with sham rats. However, TAC caused a significant increase in ventricular wall thickness, including LVPWd and IVSd (see Supplementary Table 2). Hemodynamic data further demonstrated significant increases in LVEDP, LVSP, and + dP/dt max in TAC rats compared with those in sham rats, suggesting that TAC caused diastolic dysfunction. Compared with TAC + vehicle rats, TAC + RTX rats showed a significant improvement in LVEDP. These data indicated that intrathecal RTX administration can attenuate TAC, thus causing diastolic dysfunction during the hypertrophic states (see Supplementary Table 3). 3.3. Intrathecal RTX administration improved cardiac fibrosis in TAC rats Cardiac fibrosis exacerbates LV hypertrophy and CHF development, which might also contribute to cardiac diastolic dysfunction in TAC rats [19]. As shown by the Sirius Red stain in Fig. 3, intrathecal RTX administration did not affect LV fibrosis under sham rats. However, RTX administration significantly attenuated TAC-induced LV fibrosis. Western blots further confirmed that the increase in collagen I in TAC rats was attenuated by RTX intervention. Moreover, RTX administration significantly reduced TAC-induced increases in LV TGF-β (a profibrogenic cytokine) and α-SMA (a contractile protein of stress fibers), which is associated with fibrosis development. 3.4. Intrathecal RTX administration improved myocardial apoptosis in TAC rats From adaptive hypertrophy to HF, myocardial apoptosis may play an essential role in pathological cardiac remodeling [20]. In this study, TAC for eight weeks significantly increased the number of apoptotic nuclei in the myocardium (TUNEL staining) compared with the sham rats. Intrathecal RTX administration prevented the pressure overloadinduced apoptosis of cardiomyocytes. Moreover, Western blot showed that activated caspase-3 was remarkably increased in myocardial tissue from TAC rats compared with that from sham rats. Intrathecal RTX administration significantly decreased the activation of cleaved caspases-3. Furthermore, we observed a marked increase in Bax protein and a reduction of Bcl-2 protein in myocardial tissue from TAC rats compared with that from sham rats. Intrathecal RTX administration significantly reversed these TAC-induced changes (Fig. 4). 4. Discussions The study revealed the following findings: Intrathecal application of RTX in the thoracic spinal cord (T1–T4) (1) reduced TRPV1 levels in the dorsal horn and cardiac sympathetic activities in the TAC rat model and (2) prevented TAC-induced cardiac remodeling, including cardiac hypertrophy, cardiac fibrosis, and cardiac apoptosis. To our knowledge, these findings revealed that selective chemo-ablation of TRPV1-containing neural terminals in the thoracic spinal cord (T2) might exert protective effects on the overburdened heart. The cardiac afferent nerve has one neuron located in the DRG and two projection terminals. One is projected close to the epicardial surface, and the other mainly terminates in laminae I, V, VII, and X of spinal segments T2–T6 [21]. Cardiac afferent terminals express TRPV1, which can be ablated for a long period through epicardial brushing of RTX [12]. Brushing with RTX nearly completely ablated TRPV1 expression on the surface of the rat heart for > 10 weeks and provided protective effects against deleterious cardiac remodeling and autonomic dysfunction in myocardial infarction-induced HF model [12]. Recently, Yoshie et al. [22] designed a percutaneous epicardial application of RTX, successfully abolished cardiac TRPV1 expressing nociceptive afferents in pigs, and reorganized the central–peripheral 382
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Fig. 4. Effects of intrathecal RTX administration on cardiac apoptosis in sham or TAC rats. (A) Representative immunofluorescence staining and (C) apoptotic index of TUNEL-positive nuclei in myocardium. (B) Representative Western blot stain showing the relative protein expression of caspase-3 (D), Bax (E), and Bcl-2 (F) to GAPDH in LV tissue from sham + vehicle, sham + RTX, TAC + vehicle, and TAC + RTX rats. Values are shown in mean ± SEM; n = 8/each group. *P < 0.05 versus sham + vehicle; # P < 0.05 versus chronic TAC + vehicle. RTX, resiniferatoxin; TAC, transverse aortic constriction.
of fibrotic and hypertrophic proteins, and prevents the development of congestive HF [32]. In contrast to the above studies, the activation of TRPV1 channels may aggravate congestive HF. TRPV1 knock-out mice (TRPV1-/-) are resistant to cardiac hypertrophy, which is induced by TAC [6]. TRPV1 antagonist had also been reported to improve TACinduced cardiac hypertrophy, inflammation, fibrosis, and apoptosis [33]. Therefore, systemic intervention of TRPV1 channels is inaccurate in efficacy and safety for long-term periods. Targeted precision modulation of local TRPV1 channel is a conceivable choice. In this study, we ablated TRPV1 expression of the dorsal horn through intrathecal RTX administration in the T2 segment, which might blunt the spinal terminal of the cardiac afferent nerve. The cardioprotective effects of RTX in the present study might be due to the focal spinal TRPV1 in the T2 segment rather than from the heart. The presence of an excitatory cardiac chemosensitive sympathetic afferent reflex may provide an explanation for our results. Previous studies had
rigidity of the heart, which leads to improvement of diastolic dysfunction. Cardiac apoptosis is also an important factor of cardiac remodeling under pressure overload [20]. In this study, intrathecal RTX administration significantly ameliorated cardiac apoptosis and prevented the expression of apoptosis-associated proteins. Although the mechanisms underlying the reduction of cardiac apoptosis remain unknown, they may play an important role in RTX-mediated cardioprotective effects. Given that the TRPV1 channels have diverse distribution in various organs and have various roles in pathological conditions, systemic deletion of TRPV1 might be harmful [28]. Previous studies had shown that TRPV1 channel activation produced several beneficial effects on some cardiovascular diseases, such as atherosclerosis, hypertension, and ischemia–reperfusion injury, by modulating NO and substance P production and Ca2+ influx [29–31]. Dietary capsaicin intake activates TRPV1 channels, reduces the level of oxidative stress and the expression 383
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Appendix A. Supplementary data
shown that focal TRPV1 channels, especially in the neutral terminals, have a complex role in modulating sympathetic efferent activities [30,34,35]. RTX-treated animals showed a low expression of TRPV1 in the heart and DRG and weakened sympathoexcitatory responses to capsaicin stimulation [35]. Using coronary artery ligation to induce congestive HF, Wang et al. [12] demonstrated that focal deletion of TRPV1-expressing CSAR afferents via epicardial application of RTX attenuated cardiac remodeling and autonomic dysfunction. Shanks et al. [34] used a new method to chronically ablate thoracic TRPV1expressing afferent soma at the level of T1–T4. They found that epidural application of RTX abolished cardiac and pulmonary sympathoexcitatory responses. Therefore, we deduced that elimination of TRPV1 at the spinal cord end can inhibit CSAR, which was overexcited in aortic constriction conditions.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.10.156. References [1] P. Ponikowski, A.A. Voors, S.D. Anker, H. Bueno, J.G. Cleland, A.J. Coats, et al., ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: the task force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) Developed with the special contribution of the Heart Failure Association (HFA) of the ESC, Eur. Heart J. 37 (2016) 2129–2200. [2] J. Diez, E.D. Frohlich, A translational approach to hypertensive heart disease, Hypertension 55 (2010) 1–8. [3] C.M. Sag, C.X. Santos, A.M. Shah, Redox regulation of cardiac hypertrophy, J. Mol. Cell. Cardiol. 73 (2014) 103–111. [4] F. Triposkiadis, G. Karayannis, G. Giamouzis, J. Skoularigis, G. Louridas, J. Butler, The sympathetic nervous system in heart failure physiology, pathophysiology, and clinical implications, J. Am. Coll. Cardiol. 54 (2009) 1747–1762. [5] W.W. Chen, X.Q. Xiong, Q. Chen, Y.H. Li, Y.M. Kang, G.Q. Zhu, Cardiac sympathetic afferent reflex and its implications for sympathetic activation in chronic heart failure and hypertension, Acta Physiol. 213 (2015) 778–794. [6] C.L. Buckley, A.J. Stokes, Mice lacking functional TRPV1 are protected from pressure overload cardiac hypertrophy, Channels 5 (2011) 367–374. [7] B. Zhong, J. Rubinstein, S. Ma, D.H. Wang, Genetic ablation of TRPV1 exacerbates pressure overload-induced cardiac hypertrophy, Biomed. Pharmacother. 99 (2018) 261–270. [8] K. Fujiu, M. Shibata, Y. Nakayama, F. Ogata, S. Matsumoto, K. Noshita, et al., A heart-brain-kidney network controls adaptation to cardiac stress through tissue macrophage activation, Nat. Med. 23 (2017) 611–622. [9] M. Bishnoi, C.A. Bosgraaf, L.S. Premkumar, Preservation of acute pain and efferent functions following intrathecal resiniferatoxin-induced analgesia in rats, J. Pain 12 (2011) 991–1003. [10] T. Irie, K. Yamakawa, D. Hamon, K. Nakamura, K. Shivkumar, M. Vaseghi, Cardiac sympathetic innervation via middle cervical and stellate ganglia and antiarrhythmic mechanism of bilateral stellectomy, Am. J. Physiol. Heart Circ. Physiol. 312 (2017) H392–H405. [11] H.J. Wang, G.J. Rozanski, I.H. Zucker, Cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states, J. Physiol. 595 (2017) 2519–2534. [12] H.J. Wang, W. Wang, K.G. Cornish, G.J. Rozanski, I.H. Zucker, Cardiac sympathetic afferent denervation attenuates cardiac remodeling and improves cardiovascular dysfunction in rats with heart failure, Hypertension 64 (2014) 745–755. [13] M. Leo, M. Schulte, L.I. Schmitt, M. Schafers, C. Kleinschnitz, T. Hagenacker, Intrathecal resiniferatoxin modulates TRPV1 in DRG neurons and reduces TNF-induced pain-related behavior, Mediat. Inflamm. 2017 (2017) 2786427. [14] L. Karai, D.C. Brown, A.J. Mannes, S.T. Connelly, J. Brown, M. Gandal, et al., Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control, J. Clin. Invest. 113 (2004) 1344–1352. [15] M.R. Sapio, J.K. Neubert, D.M. LaPaglia, D. Maric, J.M. Keller, S.J. Raithel, et al., Pain control through selective chemo-axotomy of centrally projecting TRPV1+ sensory neurons, J. Clin. Invest. 128 (April (4)) (2018) 1657–1670. [16] Z.Z. Li, H. Jiang, D. Chen, Q. Liu, J. Geng, J.Q. Guo, et al., Renal sympathetic denervation improves cardiac dysfunction in rats with chronic pressure overload, Physiol. Res. 64 (2015) 653–662. [17] H. Zhu, X. Sun, D. Wang, N. Hu, Y. Zhang, Doxycycline ameliorates aggregation of collagen and atrial natriuretic peptide in murine post-infarction heart, Eur. J. Pharmacol. 754 (2015) 66–72. [18] J.A. Jeffry, S.Q. Yu, P. Sikand, A. Parihar, M.S. Evans, L.S. Premkumar, Selective targeting of TRPV1 expressing sensory nerve terminals in the spinal cord for long lasting analgesia, PLoS One 4 (2009) e7021. [19] H. Wang, D. Kwak, J. Fassett, L. Hou, X. Xu, B.J. Burbach, et al., CD28/B7 deficiency attenuates systolic overload-induced congestive heart failure, myocardial and pulmonary inflammation, and activated T cell accumulation in the heart and lungs, Hypertension 68 (2016) 688–696. [20] L. Zhao, G. Cheng, R. Jin, M.R. Afzal, A. Samanta, Y.T. Xuan, et al., Deletion of interleukin-6 attenuates pressure overload-induced left ventricular hypertrophy and dysfunction, Circ. Res. 118 (2016) 1918–1929. [21] D.C. Kuo, J.J. Oravitz, W.C. DeGroat, Tracing of afferent and efferent pathways in the left inferior cardiac nerve of the cat using retrograde and transganglionic transport of horseradish peroxidase, Brain Res. 321 (1984) 111–118. [22] K. Yoshie, P.S. Rajendran, L. Massoud, O. Kwon, V. Tadimeti, S. Salavatian, et al., Cardiac vanilloid receptor-1 afferent depletion enhances stellate ganglion neuronal activity and efferent sympathetic response to cardiac stress, Am. J. Physiol. Heart Circ. Physiol. 314 (May (5)) (2018) H954–H966. [23] W. Wang, H.D. Schultz, R. Ma, Cardiac sympathetic afferent sensitivity is enhanced in heart failure, Am. J. Physiol. 277 (1999) H812–7. [24] G.Q. Zhu, Y. Xu, L.M. Zhou, Y.H. Li, L.M. Fan, W. Wang, et al., Enhanced cardiac sympathetic afferent reflex involved in sympathetic overactivity in renovascular hypertensive rats, Exp. Physiol. 94 (2009) 785–794. [25] B. Swynghedauw, Molecular mechanisms of myocardial remodeling, Physiol. Rev. 79 (1999) 215–262. [26] T.M. Marin, K. Keith, B. Davies, D.A. Conner, P. Guha, D. Kalaitzidis, et al., Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD
5. Limitations Several limitations of this study should be stated. First, we used RTX through intrathecal administration in the T2 segment of the spinal cord rather than systemic or epicardial application because these methods resulted in high animal death in rat models. We could not confirm whether all cardiac afferents were abolished; however, a previous study reported that injection dye can gradually diffuse on the surface of the spinal cord [36]. Second, RTX injection was administered five days before TAC operation in this study. Therefore, we could not confirm whether RTX has comparable therapeutic effects on cardiac hypertrophy if RTX was given several days or weeks after TAC surgery. Third, we could not determine the effects of RTX ablation in the T2 segment under stressful moments. Further studies are needed to clarify cardiac adaptability to stress in awake or exercise state after cardiac afferent ablation. Fourth, we could not determine whether RTX injection in the T2 segment causes similar response to TAC in female rats for usage of single gender in this study. However, a previous study had shown that male mice express relatively higher proteins and genes associated with extracellular matrix remodeling than female mice after TAC [37]. Finally, we did not observe these above effects of intrathecal RTX injection for a long time when TAC rat developed into HF. A previous study had demonstrated that intrathecal RTX injection at thoracic segments can ablate cardiac afferent for longer than one year [14]. If sympathetic activity always acts during the process of overburden-induced LV dysfunction, a single intrathecal dose of RTX may offer protective effects on the stressed heart. Further studies in HF models are needed to reveal whether intrathecal RTX injection can prevent TAC-induced HF.
6. Conclusions Altogether, the present study first demonstrated that intrathecal RTX injection at the T2 segment selectively abolished cardiac afferent and prevented cardiac diastolic dysfunction, LV hypertrophy, interstitial fibrosis, and myocardial apoptosis in TAC rats for at least eight weeks. The cardioprotective mechanism of RTX application at the T2 level of the dorsal horn might be associated with the inhibition effects on CSAR. Although further studies are needed to clarify the efficacy of intrathecal RTX injection in HF and stress states, the present study revealed a new method for selective intervention of CSAR.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 81670301 to W.D., 81471114 to H.N., 81772180 and 81472017 to L.V.) and the Key Projects of Anhui Province University Outstanding Youth Talent Support Program (gxqZD2016181 to W.D.). 384
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
syndrome-associated PTPN11 mutation, J. Clin. Invest. 121 (2011) 1026–1043. [27] F.G. Spinale, Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function, Physiol. Rev. 87 (2007) 1285–1342. [28] P.K. Randhawa, A.S. Jaggi, TRPV1 channels in cardiovascular system: a double edged sword? Int. J. Cardiol. 228 (2017) 103–113. [29] S. Xiong, P. Wang, L. Ma, P. Gao, L. Gong, L. Li, et al., Ameliorating endothelial mitochondrial dysfunction restores coronary function via transient receptor potential vanilloid 1-mediated protein kinase A/uncoupling protein 2 pathway, Hypertension 67 (2016) 451–460. [30] H. Sun, D.P. Li, S.R. Chen, W.N. Hittelman, H.L. Pan, Sensing of blood pressure increase by transient receptor potential vanilloid 1 receptors on baroreceptors, J. Pharmacol. Exp. Ther. 331 (2009) 851–859. [31] L. Wang, D.H. Wang, TRPV1 gene knockout impairs postischemic recovery in isolated perfused heart in mice, Circulation 112 (2005) 3617–3623. [32] Q. Wang, S. Ma, D. Li, Y. Zhang, B. Tang, C. Qiu, et al., Dietary capsaicin ameliorates pressure overload-induced cardiac hypertrophy and fibrosis through the
[33] [34]
[35]
[36]
[37]
385
transient receptor potential vanilloid type 1, Am. J. Hypertens. 27 (2014) 1521–1529. J.S. Horton, C.L. Buckley, A.J. Stokes, Successful TRPV1 antagonist treatment for cardiac hypertrophy and heart failure in mice, Channels 7 (2013) 17–22. J. Shanks, Z. Xia, S.J. Lisco, G.J. Rozanski, H.D. Schultz, I.H. Zucker, et al., Sympatho-excitatory response to pulmonary chemosensitive spinal afferent activation in anesthetized, vagotomized rats, Physiol. Rep. 6 (2018) e13742. M.R. Zahner, D.P. Li, S.R. Chen, H.L. Pan, Cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats, J. Physiol. 551 (2003) 515–523. C. Njoo, C. Heinl, R. Kuner, In vivo SiRNA transfection and gene knockdown in spinal cord via rapid noninvasive lumbar intrathecal injections in mice, J. Visualized Exp. 22 (Mar. (85)) (2014). H. Witt, C. Schubert, J. Jaekel, D. Fliegner, A. Penkalla, K. Tiemann, et al., Sexspecific pathways in early cardiac response to pressure overload in mice, J. Mol. Med. 86 (2008) 1013–1024.
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Focal selective chemo-ablation of spinal cardiac afferent nerve by resiniferatoxin protects the heart from pressure overload-induced hypertrophy
T
⁎
Deguo Wanga,b, , Yong Wua, Yueyun Chena, Ancai Wanga, Kun Lvb, Xiang Kongc, Yang Hed, Nengwei Hue a
Department of Gerontology, Yijishan Hospital of Wannan Medical College, Wuhu, 241001, PR China Non-Coding RNA Research Center of Wannan Medical College, Wuhu, Anhui, 241001, PR China Department of Endocrinology, Yijishan Hospital of Wannan Medical College, Wuhu, 241001, PR China d School of Basic Courses, Wannan Medical College, Wuhu, Anhui, 241001, PR China e Department of Pharmacology and Therapeutics, and Trinity College, Institute of Neuroscience, Biotechnology Building, Trinity College Dublin, Dublin 2, Ireland b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Cardiac sympathetic nerve activities Transverse aortic constriction Resiniferatoxin Cardiac hypertrophy Fibrosis Apoptosis
Resiniferatoxin (RTX), a selective transient receptor potential vanilloid 1 (TRPV1) receptor agonist, can eliminate TRPV1+ primary sensory afferents and blunt cardiac sympathetic afferent reflex for a relatively long period. The present study determined the effects of intrathecal RTX administration on transverse aortic constriction (TAC)-induced cardiac dysfunction and cardiac remodeling in rats. Five days before TAC, RTX (2 μg/ 10 μl) was injected intrathecally into the T2/T3 interspace of rats. Cardiac sympathetic nerve activities (CSNAs) and cardiac structure and function were determined eight weeks after TAC. Intrathecal RTX administration abolished TRPV1 expression in the dorsal horn and reduced over-activated CSNA in the TAC rat model. Hemodynamic analysis revealed that RTX reduced left ventricular end-diastolic pressure, indicating the improvement of cardiac compliance. Histologic analysis, real-time reverse transcription-polymerase chain reaction, and Western blots showed that RTX prevented TAC-induced cardiac hypertrophy, cardiac fibrosis, and cardiac apoptosis and reduced the expression of apoptotic proteins and myocardial mRNAs. In conclusion, these results demonstrate that focal chemo-ablation of TRPV1+ afferents in the spinal cord protects the heart from pressure overload-induced cardiac remodeling and cardiac dysfunction, which suggest a novel promising therapeutic method for cardiac hypertrophy and diastolic dysfunction.
1. Introduction Heart failure (HF) is a common, costly, and potentially fatal condition [1]. It is always preceded by cardiac hypertrophy and diastolic dysfunction [2]. Cardiac hypertrophy is characterized by an increase in heart mass due to cardiomyocyte hypertrophy and proliferation of noncardiomyocyte cells in response to pressure overload [3]. During the process of cardiac hypertrophy, sympathetic activity is always overactivated [4]. Therefore, blocking excessive sympathetic activation would be a promising approach to prevent hypertrophic heart from developing into HF. The sympathetic tone is regulated by the cardiac sympathetic afferent reflex (CSAR), which is formed by sympathetic afferent fibers, dorsal root ganglia (DRG) of the C8–T9 spinal segments, hypothalamic
and medullary brain centers, and sympathetic efferent nerves [5]. Transient receptor potential vanilloid 1 (TRPV1) is expressed in sensory afferent terminals in different organs, including the heart [5]. A previous study found that mice lacking TRPV1 (TRPV1-/-) are resistant to cardiac hypertrophy, which is induced by transverse aortic constriction (TAC) [6]. However, another recent study showed that TRPV1 knockout mice (TRPV1-/-) had excessive inflammation, cardiac hypertrophy, and deteriorated cardiac function after TAC [7]. The completely different results of the two studies illustrated the complex role of TRPV1 in pathophysiological conditions. The results also infer that specifically eliminating TRPV1 in the target location is necessary to avoid systemic damages. Indeed, the inhibition of the cardiac afferent nerve by epicardial capsaicin administration or spinal dorsal horn resection reduced proinflammatory cytokine expression in a mouse TAC model [8].
⁎ Corresponding author at: Department of Gerontology and Non-Coding RNA Research Center of Wannan Medical College, Yijishan Hospital of Wannan Medical College, 92nd Zheshan Western Road, Wuhu, Anhui, 241001, PR China. E-mail address: [email protected] (D. Wang).
https://doi.org/10.1016/j.biopha.2018.10.156 Received 4 August 2018; Received in revised form 25 October 2018; Accepted 25 October 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Sham operation rats underwent the same procedure without ligation of the aorta.
Resiniferatoxin (RTX), a selective TRPV1 agonist, can selectively lesion TRPV1+ primary sensory afferents. Systemic or focal administration of RTX can almost completely abolish pain sensation and inflammation [9]. The application of RTX on the heart surface can blunt CSAR in adult rats, lasting for 10 weeks [10–12]. Obviously, brushing RTX on the heart surface is difficult. Recently, a novel route via intrathecal RTX administration had been reported to reduce pain and inflammation without damaging neurons and peripheral terminals [9,13,14]. Through intrathecal RTX injection, Sapio et al. [15] applied chemo-axotomy of the TRPV1+ afferents for pain control in human and animal models. Therefore, we locally administered RTX at the T1 to T4 spinal cord levels (accepting cardiac afferent fibers) to ablate the cardiac sensory nerve. In the present study, we performed intrathecal RTX application in TAC rats to determine the role of spinal terminals of cardiac afferent fibers in cardiac hypertrophic response to aortic artery stenosis and whether intrathecal RTX application can ameliorate cardiomyocyte apoptosis and interstitial fibrosis.
2.3. Echocardiography assessment Eight weeks after TAC, echocardiography assessment was performed by a tester who was blinded to the experimental groups using an HP5500 cardiac ultrasound system with a 15 MHz linear-array probe. M-mode tracings at the parasternal long- and short-axis views of the LV were used to measure the interventricular septum diameter (IVSd), LV posterior wall diameter (LVPWd), LV end-systolic diameter (LVSd), LV end-diastolic diameter (LVDd), LV percent ejection fraction (LVEF), and LV percent fractional shortening (LVFS) [17]. LVEF = (LVEDVLVESV)/LVEDV × 100%; LVFS = (LVDd-LVDs)/LVDd×100%. 2.4. Physiological information recording and analysis 2.4.1. Cardiac sympathetic nerve activity (CSNA) The rats were anesthetized and fixed in supine position, endotracheally intubated, and mechanically ventilated (respiratory rate 60 breaths/min, respiration ratio 1:1, tidal volume 2.5 ml). Left cervical stellate ganglion was identified behind the left carotid artery. A branch innervating heart was isolated and hooked by a pair of recording electrodes (with 2 mm space). Recording signals were amplified (×10,000) and filtered (bandwidth: 100–3000 Hz) by a biological data acquisition system (RM6240, Chengdu, China) [12].
2. Materials and methods 2.1. Animals and groups Male Sprague–Dawley rats (180–220 g, n = 60) were purchased from the Experimental Animal Center of Nanjing. The experimental procedures were approved by Yijishan Hospital Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health guiding principles. RTX (Sigma Aldrich, St. Louis, MO) was first dissolved in 100% ethanol to make the stock solution (concentration of 2 mg/ml) and then further diluted in 10 ml saline when used for intrathecal (T2/T3 intraspinal space) injection. As shown in Fig. 1A, all animals were randomly assigned to two groups. The RTX group (n = 30) received 2 μg/10 μl of RTX, whereas the control group (n = 30) received 10 μl of vehicle. Then, the rats randomly received TAC or sham surgery operation. Therefore, the animals were divided into four groups: (1) Sham + vehicle, (2) Sham + TAC, (3) TAC + vehicle, and (4) TAC + RTX. All survived animals underwent echocardiography eight weeks post-surgery. Then, they further underwent hemodynamic and cardiac sympathetic nerve recording (Fig. 1C). Afterward, rat hearts were subsequently harvested, and the heart weight:body weight (HW:BW) ratio was calculated. Left ventricular (LV) samples were collected and stored at −80 °C for further molecular biologic and histological evaluations.
2.4.2. Measurement of LV performance LV performance was assessed using a self-made polyethylene catheter inserted via the right carotid artery. Briefly, the catheter, prefilled with fresh heparinized saline (500 U/ml) and connected to a biological data acquisition system (RM6240, Chengdu, China), was inserted into the right carotid artery and advanced into the LV. The pressure signal of LV was recorded continuously and stored in a computer to determine off-line LV end-diastolic pressure (LVEDP), LV systolic pressure (LVSP), maximum first derivative of LV pressure (dp/ dtmax), and minimum first derivative of LV pressure (-dp/dtmax). 2.4.3. Measurement of norepinephrine(NE)in plasma and heart Heart and blood samples were obtained from rats eight weeks after TAC and stored at −70 °C until analysis. NE levels were determined using an ELISA kit (Abnova, Shanghai, China) according to the manufacturer’s instructions. Briefly, LV myocardial samples were homogenized, and plasma was separated before assaying. The samples and different dilution standards were added into testing wells. The wells were shaken and incubated for 1 h at 37 °C. After washing for three times, biotinylated NE antibody was added to each well and incubated at 37 °C for 1 h. The wells were washed five times with the washing buffer, added with streptavidin-peroxidase conjugate, and then washed again. Afterward, chromogen substrate solution was added to each well and incubated at 37 °C for 20 min. Stop solution was added, and optical density was detected immediately using a microplate reader (Bio-Rad, Hercules, CA).
2.2. Animal models 2.2.1. Intrathecal injection The animals received RTX injections as previously described with minor modifications [13]. After isoflurane anesthesia (5% isoflurane), intrathecal injections were performed by inserting a U-40 insulin syringe with 29 G needle into the T2/T3 interspace perpendicular to the spine. The criterion for the intrathecal placement was withdrawal of clear cerebrospinal fluid into the syringe. Then, RTX (2 μg/10 μl) or vehicle (10 μl saline with 10% ethanol) was injected slowly into the intrathecal space.
2.5. Histology 2.5.1. Hematoxylin–eosin (H&E) and Sirius Red stain Hearts from all groups were harvested and weighed. Heart specimens were fixed in 10% buffered formalin, embedded in paraffin, and cut into sections (5 μm thick). The sections were stained with H&E and Sirius Red stain. Cardiomyocyte size was assessed in randomly selected high-power fields in each section. To determine the degree of cardiac fibrosis, images of Sirius Red-stained samples were transferred into a computer. A minimum of 10 fields from each section was scored at 100× magnification. The degree of cardiac fibrosis was determined based on the area of fibrosis divided by the total area (%cardiac
2.2.2. TAC The surgical procedures for the TAC model were performed as previously described with minor modifications [16]. Briefly, the rats were anesthetized with intraperitoneal injection of pentobarbital sodium (60 mg/kg). A 2 cm incision was made along the midline of the abdomen to isolate the abdominal aorta beside the left renal artery. A blunt and bent 21 G needle was placed along the abdominal aorta. A 4-0 ligature was tied twice around the needle and the aorta. Then, the needle was removed, yielding a 0.8 mm-diameter artery constriction. 378
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Fig. 1. Intrathecal RTX administration ablates TRPV1 expression and reduces sympathetic nerve activities. (A) Schematic of the experimental design. (B) Representative immunofluorescence of TRPV1 staining (red) in the dorsal horn of lumbar spinal cord sections. (C) Schematic of spinal elements of cardiac sympathetic afferent nerve (green), sympathetic efferent nerve (yellow), and recording of cardiac sympathetic nerve activities (CSNAs) from cardiac branch of stellate ganglion. (D) CSNAs. (E) Western blot showing the protein expression of TRPV1 in the spinal cord. (F) NE levels in plasma. (G) NE levels in LV myocardial tissue. Values are shown in mean ± SEM; n = 5/each group. Scale bar was 100 μm. *P < 0.05 versus sham + vehicle; # P < 0.05 versus chronic TAC + vehicle. RTX, resiniferatoxin; TAC, transverse aortic constriction; TRPV1, transient receptor potential vanilloid 1 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
fibrosis) by using Image-Pro Plus software.
T1–T4 spinal tissue. Paraffin-fixed sections from the LV and spine were processed routinely and incubated with mouse monoclonal TRPV1 (dilution: 1:200; rabbit anti-tyrosine hydroxylase (TH): 1:200; rabbit anti-NeuN: 1;200; Abcam, Cambridge, UK) at 4 °C overnight. A rhodamine-conjugated goat anti-mouse and an FITC-conjugated goat antirabbit secondary antibody were incubated with the tissue sections at 37 °C for 1 h. The double-stained sections were observed, and the immunofluorescence signals specific for TRPV1 were examined under a confocal immunofluorescence microscope (Zeiss LSM510 META, Germany).
2.5.2. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labeling (TUNEL) assay For the detection of apoptosis, paraffin-fixed LV tissue sections were stained by TUNEL assay using an in situ cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Tissue sections were stained with TUNEL reaction mixture and Converter-POD and observed under a microscope (Eclipse, Nikon, Tokyo, Japan). Apoptotic nuclei were manually identified to ensure that only the apoptotic cardiomyocyte nuclei were included. The number of TUNEL-positive cells was expressed as a percentage of the total number of nuclei.
2.6. Western blot The proteins were isolated, and the protein concentrations of the samples were determined by bicinchoninic acid protein assay. Total proteins were separated via 14% sodium dodecylsulfate polyacrylamide
2.5.3. Immunofluorescence labeling TRPV1 was used to detect sympathetic afferent distribution of 379
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Fig. 2. Effects of intrathecal RTX administration on cardiac hypertrophy in sham or TAC rats. (A) Representative micrographs showing the cross-section of a heart stained with hematoxylin and eosin (H&E) (B) and quantification of cardiomyocyte diameters after intrathecal RTX administration in sham or TAC rats. (C) Heart weight, (D) body weight, (E) and heart weight:body weight (HW:BW) ratios in sham + vehicle, sham + RTX, TAC + vehicle, and TAC + RTX rats. (F) Relative mRNA expression of ANP, (G) BNP, and (H) β-MHC in sham + vehicle, sham + RTX, TAC + vehicle, and TAC + RTX rats. Values are shown in mean ± SEM; n = 8/ each group. *P < 0.05 versus sham + vehicle; # P < 0.05 versus chronic TAC + vehicle. RTX, resiniferatoxin; TAC, transverse aortic constriction; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, beta myosin heavy chain.
reversely transcripted into cDNA using the All-In-One RT MasterMix reagent kit (Applied Biological Materials Inc./G492). Specific products were detected with BrightGreen qPCR MasterMix-Low ROX reaction kit (Applied Biological Materials Inc./MasterMix-S) using the Thermo Fisher 7300 Plus Read-Time PCR System according to the manufacturer’s instructions. The expression of each transcript was normalized to gene of the housekeeping protein GAPDH in the same tissue. The formula used for relative mRNA expression was 2-(OCT sample 2OCT control). The specific forward and reverse primers used are presented in Supplementary Table 2.
gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (10 mM Tris−HCl, 150 mM NaCl, and 0.2% Tween-20, pH 7.6) for 2 h at 37 °C and incubated overnight at 4 °C with primary antibodies in certain dilution. Antibody binding was detected with a horseradish peroxidase-conjugated secondary antibody (1:2000; Sigma) and visualized using an ECL kit. The imaging program Quantity One (Bio-Rad) was used for quantification. 2.7. Real-time reverse transcription-polymerase chain reaction (RT-PCR) RNA levels were detected via real-time RT-PCR. Briefly, the RNA from the LV tissues was isolated with RNAi TRIzol (Invitrogen, CA) and 380
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Fig. 3. Effects of intrathecal RTX administration on cardiac fibrosis in sham or TAC rats. (A) Representative Sirius Red stain (B) and relative area of fibrosis (%) after intrathecal RTX administration in sham or TAC rats. (D) Representative Western blot stain showing the relative protein expression of collagen I (C), α-SMA (E), and TGF-β (F) to GAPDH in LV tissue of sham + vehicle, sham + RTX, TAC + vehicle, and TAC + RTX rats. Values are shown in mean ± SEM; n = 8/each group. *P < 0.05 versus sham + vehicle; # P < 0.05 versus chronic TAC + vehicle. RTX, resiniferatoxin; TAC, transverse aortic constriction; α-SMA, α-smooth muscle actin; and TGF-β, transforming growth factor beta (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
both groups. As shown in Fig. 1D, CSNA was higher in TAC rats than in sham rats. Intrathecal RTX administration inhibited baseline cardiac sympathetic tone. Eight weeks after TAC, NE levels significantly increased in the heart (196 ± 23 ng/ml versus 120 ± 13 ng/ml, P < 0.05) and blood (121 ± 25 ng/g versus 63 ± 14 ng/g, P < 0.05). Intrathecal RTX administration remarkably prevented the rise of NE in the heart (141 ± 17 ng/ml versus 196 ± 23 ng/ml, P < 0.05) and blood (53 ± 18 ng/g versus 121 ± 20 ng/g, P < 0.05). To observe sympathetic efferent fibers in the heart with or without intrathecal RTX administration, we also stained heart slices with anti-TH (green). We found no significant changes of TH (green) in the heart compared with those in control rats (Fig. S2). These results indicated that RTX injection via intrathecal routine in T2 segment can prohibit CSNA and NE release locally and systemically for a relatively long period.
2.8. Statistical analysis The data are expressed as means ± SEM. Two-way analysis of variance and Student’s t-test were performed to analyze statistical differences in each response variable. Statistical significance was considered at P < 0.05. 3. Results 3.1. Intrathecal RTX administration ablates TRPV1 in the dorsal horn and reduces cardiac sympathetic activities In spinal cord sections, TRPV1 was located in the superficial layers of the dorsal horn, including laminae I and II [15,18], which can be selectively abolished by systemic or focal RTX administration [9]. TRPV1 expression significantly increased in the dorsal horn of TAC rats compared with that of sham rats. Intrathecal RTX administration significantly reduced TRPV1 expression of the dorsal horn in the sham and TAC rats (Fig. 1B). Western blot further showed that TRPV1 increased in the thoracic spinal cord of TAC animals and significantly reduced by intrathecal RTX administration in normal and TAC animals (P < 0.05) (Fig. 1E). As shown in the supplementary materials, we also detected the expression of TRPV1 in the DRG and heart with or without intrathecal RTX administration. Two weeks after intrathecal RTX administration, TRPV1-positive signal was reduced in dorsal root ganglia (DRG) from RTX-treated rats compared from control rats (Fig. S1). However, we did not detect any visible TRPV1 signals in the heart from
3.2. Intrathecal RTX administration improved cardiac hypertrophy and cardiac function in TAC rats TAC caused severe LV hypertrophy (HW/BW: 4.43 ± 0.11 g/kg versus 2.89 ± 0.08; cardiomyocyte width: 23.4 ± 0.8 μm versus 13.2 ± 0.4 μm). Intrathecal RTX administration significantly attenuated TAC-induced LV hypertrophy (HW/BW: 3.22 ± 0.12 g/kg versus 4.43 ± 0.11 g/kg; cardiomyocyte width: 17.2 ± 1.1 μm versus 23.4 ± 0.8 μm) (Fig. 2). Moreover, intrathecal RTX administration significantly reduced TAC-induced increases in myocardial mRNA expression of ANP, BNP, and β-MHC. These data suggested that RTX 381
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
nervous system interaction to enhance cardiac sympathetic outflow. However, brushing RTX on the surface of the heart or percutaneous epicardial application of RTX in clinical background is difficult. Therefore, the spinal terminals of cardiac afferent fibers might be a potential target for chemo-ablation. For example, Sapio et al. [15] had successfully controlled pain through chemo-axotomy of the TRPV1+ afferents of spinal segments in human and animal models. Bishnoi et al. [9] also reported that targeting TRPV1-expressing nerve terminals at the spinal cord can selectively abolish inflammatory thermal hypersensitivity without affecting acute thermal sensitivity and preserve the efferent functions of DRG neurons at the peripheral nerve terminals. Given that the dorsal horn of spinal segments T2–T6 is mainly the central projection terminals of cardiac afferent [21], we used intrathecal RTX administration in the T2 segment in this study. Consequently, we observed that intrathecal RTX administration can inhibit TRPV1 expression in the dorsal horn in normal and TAC animals. These results suggest that intrathecal injection is an appropriate route of RTX administration, which might be a suitable method to selectively blunt the cardiac afferent nerve. Previous studies have shown that sympathetic outflow is enhanced in patients with essential or secondary hypertension and a variety of experimental hypertensive models [5]. The elevated sympathetic tone can be measured by indirect cardiac norepinephrine spill-over assaying and direct cardiac or renal SNA recording [5]. Similar to these hypertensive models, TAC also causes high pressure overload to the LV and high norepinephrine spill-over [8]. In the present study, direct CSNA recording revealed that TAC rats had higher basal cardiac sympathetic tone than sham rats. The increased cardiac sympathetic tone in TAC rats can be reduced by intrathecal RTX administration in the T2 segment. Moreover, we did not observe significant changes of TH in the heart even though TRPV1 expression was inhibited in the DRG by intrathecal RTX administration (Fig. S1C and D). These data suggested that focal intrathecal RTX administration in the T2 segment selectively ablated the cardiac afferent nerve but left the cardiac efferent nerve intact in the heart. Therefore, the weakened effects of RTX administration on sympathetic tone in this study may be associated with blunting spinal–heart nervous system interaction. The reduction of NE levels in the blood and heart tissue also confirmed that RTX injection in the T2 segment can inhibit NE release. High sympathetic tone and NE release might be due to continuously activated CSAR, which is known as a positive feedback characteristic in CHF [23] and hypertension [24]. Previous studies had shown that blood pressure and cardiac function were changed by over-activation of CSAR in HF [11,12]. In the present study, TAC rats had significantly reduced cardiac diastolic function but similar systolic function compared with normal rats. Intrathecal RTX administration in the T2 segment could attenuate TAC and caused diastolic dysfunction, which showed a significant improvement of LVEDP. Cardiac compliance is determined by the structural properties of the cardiac muscle and connective tissue [25]. In the present study, intrathecal RTX administration in the T2 segment significantly attenuated the TAC-induced LV hypertrophy, which showed less heart weight and cardiomyocyte width. Importantly, RTX reduced TAC-induced increases in myocardial mRNA expression of ANP, BNP, and β-MHC. These reexpressed embryonic genes are molecular markers of cardiac hypertrophy [26]. These data suggested that cardiac afferent ablation in the spinal cord can protect the heart against TAC-induced LV hypertrophy. The cardiac connective tissue is an important factor of cardiac compliance [27]. In TAC rats, cardiac fibrosis contributes to LV remodeling and cardiac diastolic dysfunction [19]. Our data confirmed that RTX administration in the T2 segment significantly attenuated TAC-induced LV fibrosis and fibrosis-associated proteins TGF-β and αSMA. Cell death is another important factor of cardiac remodeling [20,25]. Our results showed that RTX prevented myocardial apoptosis and abnormal expression of apoptosis-associated proteins in TAC rats. The anti-fibrotic and anti-apoptotic effects might help in reducing the
administration at the spinal cord can protect hearts against TAC-induced LV hypertrophy. Eight weeks after TAC, the rats exhibited comparable systolic parameters, including ejection fraction and fractional shortening with sham rats. However, TAC caused a significant increase in ventricular wall thickness, including LVPWd and IVSd (see Supplementary Table 2). Hemodynamic data further demonstrated significant increases in LVEDP, LVSP, and + dP/dt max in TAC rats compared with those in sham rats, suggesting that TAC caused diastolic dysfunction. Compared with TAC + vehicle rats, TAC + RTX rats showed a significant improvement in LVEDP. These data indicated that intrathecal RTX administration can attenuate TAC, thus causing diastolic dysfunction during the hypertrophic states (see Supplementary Table 3). 3.3. Intrathecal RTX administration improved cardiac fibrosis in TAC rats Cardiac fibrosis exacerbates LV hypertrophy and CHF development, which might also contribute to cardiac diastolic dysfunction in TAC rats [19]. As shown by the Sirius Red stain in Fig. 3, intrathecal RTX administration did not affect LV fibrosis under sham rats. However, RTX administration significantly attenuated TAC-induced LV fibrosis. Western blots further confirmed that the increase in collagen I in TAC rats was attenuated by RTX intervention. Moreover, RTX administration significantly reduced TAC-induced increases in LV TGF-β (a profibrogenic cytokine) and α-SMA (a contractile protein of stress fibers), which is associated with fibrosis development. 3.4. Intrathecal RTX administration improved myocardial apoptosis in TAC rats From adaptive hypertrophy to HF, myocardial apoptosis may play an essential role in pathological cardiac remodeling [20]. In this study, TAC for eight weeks significantly increased the number of apoptotic nuclei in the myocardium (TUNEL staining) compared with the sham rats. Intrathecal RTX administration prevented the pressure overloadinduced apoptosis of cardiomyocytes. Moreover, Western blot showed that activated caspase-3 was remarkably increased in myocardial tissue from TAC rats compared with that from sham rats. Intrathecal RTX administration significantly decreased the activation of cleaved caspases-3. Furthermore, we observed a marked increase in Bax protein and a reduction of Bcl-2 protein in myocardial tissue from TAC rats compared with that from sham rats. Intrathecal RTX administration significantly reversed these TAC-induced changes (Fig. 4). 4. Discussions The study revealed the following findings: Intrathecal application of RTX in the thoracic spinal cord (T1–T4) (1) reduced TRPV1 levels in the dorsal horn and cardiac sympathetic activities in the TAC rat model and (2) prevented TAC-induced cardiac remodeling, including cardiac hypertrophy, cardiac fibrosis, and cardiac apoptosis. To our knowledge, these findings revealed that selective chemo-ablation of TRPV1-containing neural terminals in the thoracic spinal cord (T2) might exert protective effects on the overburdened heart. The cardiac afferent nerve has one neuron located in the DRG and two projection terminals. One is projected close to the epicardial surface, and the other mainly terminates in laminae I, V, VII, and X of spinal segments T2–T6 [21]. Cardiac afferent terminals express TRPV1, which can be ablated for a long period through epicardial brushing of RTX [12]. Brushing with RTX nearly completely ablated TRPV1 expression on the surface of the rat heart for > 10 weeks and provided protective effects against deleterious cardiac remodeling and autonomic dysfunction in myocardial infarction-induced HF model [12]. Recently, Yoshie et al. [22] designed a percutaneous epicardial application of RTX, successfully abolished cardiac TRPV1 expressing nociceptive afferents in pigs, and reorganized the central–peripheral 382
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Fig. 4. Effects of intrathecal RTX administration on cardiac apoptosis in sham or TAC rats. (A) Representative immunofluorescence staining and (C) apoptotic index of TUNEL-positive nuclei in myocardium. (B) Representative Western blot stain showing the relative protein expression of caspase-3 (D), Bax (E), and Bcl-2 (F) to GAPDH in LV tissue from sham + vehicle, sham + RTX, TAC + vehicle, and TAC + RTX rats. Values are shown in mean ± SEM; n = 8/each group. *P < 0.05 versus sham + vehicle; # P < 0.05 versus chronic TAC + vehicle. RTX, resiniferatoxin; TAC, transverse aortic constriction.
of fibrotic and hypertrophic proteins, and prevents the development of congestive HF [32]. In contrast to the above studies, the activation of TRPV1 channels may aggravate congestive HF. TRPV1 knock-out mice (TRPV1-/-) are resistant to cardiac hypertrophy, which is induced by TAC [6]. TRPV1 antagonist had also been reported to improve TACinduced cardiac hypertrophy, inflammation, fibrosis, and apoptosis [33]. Therefore, systemic intervention of TRPV1 channels is inaccurate in efficacy and safety for long-term periods. Targeted precision modulation of local TRPV1 channel is a conceivable choice. In this study, we ablated TRPV1 expression of the dorsal horn through intrathecal RTX administration in the T2 segment, which might blunt the spinal terminal of the cardiac afferent nerve. The cardioprotective effects of RTX in the present study might be due to the focal spinal TRPV1 in the T2 segment rather than from the heart. The presence of an excitatory cardiac chemosensitive sympathetic afferent reflex may provide an explanation for our results. Previous studies had
rigidity of the heart, which leads to improvement of diastolic dysfunction. Cardiac apoptosis is also an important factor of cardiac remodeling under pressure overload [20]. In this study, intrathecal RTX administration significantly ameliorated cardiac apoptosis and prevented the expression of apoptosis-associated proteins. Although the mechanisms underlying the reduction of cardiac apoptosis remain unknown, they may play an important role in RTX-mediated cardioprotective effects. Given that the TRPV1 channels have diverse distribution in various organs and have various roles in pathological conditions, systemic deletion of TRPV1 might be harmful [28]. Previous studies had shown that TRPV1 channel activation produced several beneficial effects on some cardiovascular diseases, such as atherosclerosis, hypertension, and ischemia–reperfusion injury, by modulating NO and substance P production and Ca2+ influx [29–31]. Dietary capsaicin intake activates TRPV1 channels, reduces the level of oxidative stress and the expression 383
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
Appendix A. Supplementary data
shown that focal TRPV1 channels, especially in the neutral terminals, have a complex role in modulating sympathetic efferent activities [30,34,35]. RTX-treated animals showed a low expression of TRPV1 in the heart and DRG and weakened sympathoexcitatory responses to capsaicin stimulation [35]. Using coronary artery ligation to induce congestive HF, Wang et al. [12] demonstrated that focal deletion of TRPV1-expressing CSAR afferents via epicardial application of RTX attenuated cardiac remodeling and autonomic dysfunction. Shanks et al. [34] used a new method to chronically ablate thoracic TRPV1expressing afferent soma at the level of T1–T4. They found that epidural application of RTX abolished cardiac and pulmonary sympathoexcitatory responses. Therefore, we deduced that elimination of TRPV1 at the spinal cord end can inhibit CSAR, which was overexcited in aortic constriction conditions.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.10.156. References [1] P. Ponikowski, A.A. Voors, S.D. Anker, H. Bueno, J.G. Cleland, A.J. Coats, et al., ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: the task force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) Developed with the special contribution of the Heart Failure Association (HFA) of the ESC, Eur. Heart J. 37 (2016) 2129–2200. [2] J. Diez, E.D. Frohlich, A translational approach to hypertensive heart disease, Hypertension 55 (2010) 1–8. [3] C.M. Sag, C.X. Santos, A.M. Shah, Redox regulation of cardiac hypertrophy, J. Mol. Cell. Cardiol. 73 (2014) 103–111. [4] F. Triposkiadis, G. Karayannis, G. Giamouzis, J. Skoularigis, G. Louridas, J. Butler, The sympathetic nervous system in heart failure physiology, pathophysiology, and clinical implications, J. Am. Coll. Cardiol. 54 (2009) 1747–1762. [5] W.W. Chen, X.Q. Xiong, Q. Chen, Y.H. Li, Y.M. Kang, G.Q. Zhu, Cardiac sympathetic afferent reflex and its implications for sympathetic activation in chronic heart failure and hypertension, Acta Physiol. 213 (2015) 778–794. [6] C.L. Buckley, A.J. Stokes, Mice lacking functional TRPV1 are protected from pressure overload cardiac hypertrophy, Channels 5 (2011) 367–374. [7] B. Zhong, J. Rubinstein, S. Ma, D.H. Wang, Genetic ablation of TRPV1 exacerbates pressure overload-induced cardiac hypertrophy, Biomed. Pharmacother. 99 (2018) 261–270. [8] K. Fujiu, M. Shibata, Y. Nakayama, F. Ogata, S. Matsumoto, K. Noshita, et al., A heart-brain-kidney network controls adaptation to cardiac stress through tissue macrophage activation, Nat. Med. 23 (2017) 611–622. [9] M. Bishnoi, C.A. Bosgraaf, L.S. Premkumar, Preservation of acute pain and efferent functions following intrathecal resiniferatoxin-induced analgesia in rats, J. Pain 12 (2011) 991–1003. [10] T. Irie, K. Yamakawa, D. Hamon, K. Nakamura, K. Shivkumar, M. Vaseghi, Cardiac sympathetic innervation via middle cervical and stellate ganglia and antiarrhythmic mechanism of bilateral stellectomy, Am. J. Physiol. Heart Circ. Physiol. 312 (2017) H392–H405. [11] H.J. Wang, G.J. Rozanski, I.H. Zucker, Cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states, J. Physiol. 595 (2017) 2519–2534. [12] H.J. Wang, W. Wang, K.G. Cornish, G.J. Rozanski, I.H. Zucker, Cardiac sympathetic afferent denervation attenuates cardiac remodeling and improves cardiovascular dysfunction in rats with heart failure, Hypertension 64 (2014) 745–755. [13] M. Leo, M. Schulte, L.I. Schmitt, M. Schafers, C. Kleinschnitz, T. Hagenacker, Intrathecal resiniferatoxin modulates TRPV1 in DRG neurons and reduces TNF-induced pain-related behavior, Mediat. Inflamm. 2017 (2017) 2786427. [14] L. Karai, D.C. Brown, A.J. Mannes, S.T. Connelly, J. Brown, M. Gandal, et al., Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control, J. Clin. Invest. 113 (2004) 1344–1352. [15] M.R. Sapio, J.K. Neubert, D.M. LaPaglia, D. Maric, J.M. Keller, S.J. Raithel, et al., Pain control through selective chemo-axotomy of centrally projecting TRPV1+ sensory neurons, J. Clin. Invest. 128 (April (4)) (2018) 1657–1670. [16] Z.Z. Li, H. Jiang, D. Chen, Q. Liu, J. Geng, J.Q. Guo, et al., Renal sympathetic denervation improves cardiac dysfunction in rats with chronic pressure overload, Physiol. Res. 64 (2015) 653–662. [17] H. Zhu, X. Sun, D. Wang, N. Hu, Y. Zhang, Doxycycline ameliorates aggregation of collagen and atrial natriuretic peptide in murine post-infarction heart, Eur. J. Pharmacol. 754 (2015) 66–72. [18] J.A. Jeffry, S.Q. Yu, P. Sikand, A. Parihar, M.S. Evans, L.S. Premkumar, Selective targeting of TRPV1 expressing sensory nerve terminals in the spinal cord for long lasting analgesia, PLoS One 4 (2009) e7021. [19] H. Wang, D. Kwak, J. Fassett, L. Hou, X. Xu, B.J. Burbach, et al., CD28/B7 deficiency attenuates systolic overload-induced congestive heart failure, myocardial and pulmonary inflammation, and activated T cell accumulation in the heart and lungs, Hypertension 68 (2016) 688–696. [20] L. Zhao, G. Cheng, R. Jin, M.R. Afzal, A. Samanta, Y.T. Xuan, et al., Deletion of interleukin-6 attenuates pressure overload-induced left ventricular hypertrophy and dysfunction, Circ. Res. 118 (2016) 1918–1929. [21] D.C. Kuo, J.J. Oravitz, W.C. DeGroat, Tracing of afferent and efferent pathways in the left inferior cardiac nerve of the cat using retrograde and transganglionic transport of horseradish peroxidase, Brain Res. 321 (1984) 111–118. [22] K. Yoshie, P.S. Rajendran, L. Massoud, O. Kwon, V. Tadimeti, S. Salavatian, et al., Cardiac vanilloid receptor-1 afferent depletion enhances stellate ganglion neuronal activity and efferent sympathetic response to cardiac stress, Am. J. Physiol. Heart Circ. Physiol. 314 (May (5)) (2018) H954–H966. [23] W. Wang, H.D. Schultz, R. Ma, Cardiac sympathetic afferent sensitivity is enhanced in heart failure, Am. J. Physiol. 277 (1999) H812–7. [24] G.Q. Zhu, Y. Xu, L.M. Zhou, Y.H. Li, L.M. Fan, W. Wang, et al., Enhanced cardiac sympathetic afferent reflex involved in sympathetic overactivity in renovascular hypertensive rats, Exp. Physiol. 94 (2009) 785–794. [25] B. Swynghedauw, Molecular mechanisms of myocardial remodeling, Physiol. Rev. 79 (1999) 215–262. [26] T.M. Marin, K. Keith, B. Davies, D.A. Conner, P. Guha, D. Kalaitzidis, et al., Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD
5. Limitations Several limitations of this study should be stated. First, we used RTX through intrathecal administration in the T2 segment of the spinal cord rather than systemic or epicardial application because these methods resulted in high animal death in rat models. We could not confirm whether all cardiac afferents were abolished; however, a previous study reported that injection dye can gradually diffuse on the surface of the spinal cord [36]. Second, RTX injection was administered five days before TAC operation in this study. Therefore, we could not confirm whether RTX has comparable therapeutic effects on cardiac hypertrophy if RTX was given several days or weeks after TAC surgery. Third, we could not determine the effects of RTX ablation in the T2 segment under stressful moments. Further studies are needed to clarify cardiac adaptability to stress in awake or exercise state after cardiac afferent ablation. Fourth, we could not determine whether RTX injection in the T2 segment causes similar response to TAC in female rats for usage of single gender in this study. However, a previous study had shown that male mice express relatively higher proteins and genes associated with extracellular matrix remodeling than female mice after TAC [37]. Finally, we did not observe these above effects of intrathecal RTX injection for a long time when TAC rat developed into HF. A previous study had demonstrated that intrathecal RTX injection at thoracic segments can ablate cardiac afferent for longer than one year [14]. If sympathetic activity always acts during the process of overburden-induced LV dysfunction, a single intrathecal dose of RTX may offer protective effects on the stressed heart. Further studies in HF models are needed to reveal whether intrathecal RTX injection can prevent TAC-induced HF.
6. Conclusions Altogether, the present study first demonstrated that intrathecal RTX injection at the T2 segment selectively abolished cardiac afferent and prevented cardiac diastolic dysfunction, LV hypertrophy, interstitial fibrosis, and myocardial apoptosis in TAC rats for at least eight weeks. The cardioprotective mechanism of RTX application at the T2 level of the dorsal horn might be associated with the inhibition effects on CSAR. Although further studies are needed to clarify the efficacy of intrathecal RTX injection in HF and stress states, the present study revealed a new method for selective intervention of CSAR.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 81670301 to W.D., 81471114 to H.N., 81772180 and 81472017 to L.V.) and the Key Projects of Anhui Province University Outstanding Youth Talent Support Program (gxqZD2016181 to W.D.). 384
Biomedicine & Pharmacotherapy 109 (2019) 377–385
D. Wang et al.
syndrome-associated PTPN11 mutation, J. Clin. Invest. 121 (2011) 1026–1043. [27] F.G. Spinale, Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function, Physiol. Rev. 87 (2007) 1285–1342. [28] P.K. Randhawa, A.S. Jaggi, TRPV1 channels in cardiovascular system: a double edged sword? Int. J. Cardiol. 228 (2017) 103–113. [29] S. Xiong, P. Wang, L. Ma, P. Gao, L. Gong, L. Li, et al., Ameliorating endothelial mitochondrial dysfunction restores coronary function via transient receptor potential vanilloid 1-mediated protein kinase A/uncoupling protein 2 pathway, Hypertension 67 (2016) 451–460. [30] H. Sun, D.P. Li, S.R. Chen, W.N. Hittelman, H.L. Pan, Sensing of blood pressure increase by transient receptor potential vanilloid 1 receptors on baroreceptors, J. Pharmacol. Exp. Ther. 331 (2009) 851–859. [31] L. Wang, D.H. Wang, TRPV1 gene knockout impairs postischemic recovery in isolated perfused heart in mice, Circulation 112 (2005) 3617–3623. [32] Q. Wang, S. Ma, D. Li, Y. Zhang, B. Tang, C. Qiu, et al., Dietary capsaicin ameliorates pressure overload-induced cardiac hypertrophy and fibrosis through the
[33] [34]
[35]
[36]
[37]
385
transient receptor potential vanilloid type 1, Am. J. Hypertens. 27 (2014) 1521–1529. J.S. Horton, C.L. Buckley, A.J. Stokes, Successful TRPV1 antagonist treatment for cardiac hypertrophy and heart failure in mice, Channels 7 (2013) 17–22. J. Shanks, Z. Xia, S.J. Lisco, G.J. Rozanski, H.D. Schultz, I.H. Zucker, et al., Sympatho-excitatory response to pulmonary chemosensitive spinal afferent activation in anesthetized, vagotomized rats, Physiol. Rep. 6 (2018) e13742. M.R. Zahner, D.P. Li, S.R. Chen, H.L. Pan, Cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats, J. Physiol. 551 (2003) 515–523. C. Njoo, C. Heinl, R. Kuner, In vivo SiRNA transfection and gene knockdown in spinal cord via rapid noninvasive lumbar intrathecal injections in mice, J. Visualized Exp. 22 (Mar. (85)) (2014). H. Witt, C. Schubert, J. Jaekel, D. Fliegner, A. Penkalla, K. Tiemann, et al., Sexspecific pathways in early cardiac response to pressure overload in mice, J. Mol. Med. 86 (2008) 1013–1024.