ORIGINAL ARTICLE
Effects of Nardostachys chinensis on Spontaneous Ventricular Arrhythmias in Rats With Acute Myocardial Infarction Jing Zhang, MD,* Can-Can Qiang, MD,* Wei-Jie Li, MD,* Li-Juan Liu, MD, PhD,* Xiao-Xiong Lin, MD,* Yun-Jiu Cheng, MD,* Kai Tang, MD, PhD,* Feng-Juan Yao, MD,† and Su-Hua Wu, MD, PhD*
Objectives: To investigate the effects and mechanisms of Nardostachys chinensis (NC) on spontaneous ventricular arrhythmias in rats with hyper-acute myocardial infarction (AMI).
Methods: Seventy-two rats were randomly divided into the control group (n = 24), metoprolol group (n = 24), and the NC group (n = 24). Premature ventricular contractions (PVCs), ventricular tachycardias (VTs), ventricular fibrillations (VFs), and blood pressure were monitored for 4 hours after coronary artery ligation. The connexin 43 (Cx43) expression in ventricular myocardium was measured by immunohistochemistry, Western blot, and real-time RT-PCR. Results: Compared with the control, metoprolol and NC decreased the VF incidence (50% vs. 4.2%, P , 0.001, and 50% vs. 12.5%, P = 0.005, respectively). There was a steady decrease in the cumulative number of PVCs and VTs within 4 hours from ligating in 3 groups. Compared with the control, metoprolol and NC reduced the cumulative number of VTs and PVCs. Compared with control, metoprolol and NC decreased the infarct size of the left ventricular tissue (55.98% 6 Received for publication January 15, 2014; accepted March 10, 2014. From the *Department of Cardiovascular Medicine, The First Affiliated Hospital, and the Key Laboratory of Assisted Circulation, Ministry of Health, Sun Yat-Sen University, Guangzhou, China; and †Department of Ultrasonography, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China. J. Zhang, C.-C. Qiang, and W.-J. Li have contributed equally. Study concept and design: S.-H. Wu; acquisition of data: J. Zhang, C.-C. Qiang, W.-J. Li, L.-J. Liu, X.-X. Lin, Y.-J. Cheng, K. Tang, F.-J. Yao; analysis and interpretation of data: J. Zhang, C.-C. Qiang, W.-J. Li, S.-H. Wu, L.-J. Liu, X.-X. Lin, Y.-J. Cheng, K. Tang, F.-J. Yao; drafting of the manuscript: J. Zhang, S.-H. Wu, C.-C. Qiang, W.-J. Li; statistical analysis: J. Zhang, C.-C. Qiang, W.-J. Li. Supported by National Natural Science Foundation of China (No. 81370285), Guangdong Province Natural Science Foundation (No. 06021338); Guangdong Province Science and Technology Program (No. 2012B031800091, 2007B031508003) and National Ministry of Education Scholarly Exchanges Foundation (No. 200724). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors report no conflicts of interest. Reprints: Su-Hua Wu, MD, PhD, Department of Cardiovascular Medicine, The First Affiliated Hospital, Sun Yat-Sen University, No. 58, Zhongshan Rd II, Guangzhou 510080, China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins. This is an open access article distributed under the terms of the Creative Commons AttributionNoncommercial-No Derivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.
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6.20% vs. 39.13% 6 4.53%, P , 0.001, and 55.98% 6 6.20% vs. 42.39% 6 3.44%, P , 0.001, respectively). The results from immunohistochemistry, Western blot, and real-time RT-PCR showed that the protein expression of Cx43 in the control group was significantly lower than that in the metoprolol and NC groups in the infarcted zone.
Conclusions: NC decreased the incidence of spontaneous ventricular arrhythmias (especially VF), reduced Cx43 degradation, and improved Cx43 redistribution in myocardial infarcted zone in rats with hyper-AMI. The data of the present study indicated that NC may be a promising drug in the future to prevent patients with AMI from lethal ventricular arrhythmias in prehospital setting. Key Words: Nardostachys chinensis, ventricular arrhythmias, myocardial infarction, metoprolol, connexin 43 (J Cardiovasc Pharmacol 2014;64:127–133)
INTRODUCTION Eighty percent of the ventricular fibrillation (VF) caused by acute myocardial infarction (AMI) occurs at the hyper-acute stage (within 4 hours after coronary occlusion), and 50% of the deaths because of AMI are caused by the VFs during hyperacute stage (often prehospital).1–4 The decrease in the incidence of hyper-acute VF will reduce the mortality caused by AMI. Gap junctions are low-resistance channels between adjacent myocardial cells that are predominantly composed of connexin 43 (Cx43) in the ventricle. Cx43 has an important role in the propagation of action potential to mediate current flow and to coordinate the spread of excitation and subsequent contraction throughout the myocardium.5,6 Gene-targeting studies demonstrated that reduced expression of Cx43 caused a significant reduction in conduction velocity and increased the incidence of ventricular tachyarrhythmias7 in mice during acute myocardial ischemia.8 These results suggested that the dysfunction of Cx43 in cardiomyocytes could be one of the components of the substrate that promotes lethal ventricular tachyarrhythmias in acute myocardial ischemia. The rhizomes and roots of Nardostachys chinensis (NC) Batalin, members of the family Valerianaceae growing in Himalayan regions to southwest China, have been used as crude drugs in traditional Chinese medicine.9 The rhizomes of NC are also named as “Gansongxiang” or “Gansong” in Chinese. NC contains mainly guaiane, aristolane, www.jcvp.org |
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nardosinane-type sesquiterpenoids, neolignan, and lignans.10 They are used to treat abdominal pain, indigestion, vomiting, diarrhea, epilepsy, hysteria, and mental disorders.11 Recently, NC has been shown to have novel pharmacologic effects, such as cardiac protection (especially protection from arrhythmias), antioxidant properties, and hypolipidemic effect.12–14 However, there is little known about the effect of NC in the prevention and conversion of hyper-acute ventricular arrhythmias (especially VF) after AMI. Beta-receptor blockers (metoprolol) have been recognized as a drug that can effectively reduce malignant ventricular arrhythmias and sudden cardiac death after myocardial infarction.15,16 In the present study, we investigate the anti-ventricular arrhythmia effects of NC, compared with metoprolol, in a rat model of AMI. At the same time, we examine the effects of NC and metoprolol on the levels, localization, and function of the ventricular gap junction protein Cx43.
MATERIALS AND METHODS Animal Experiment All procedures for these experiments have been approved by the Animal Experimentation and Ethics Committee of the Sun Yat-Sen University (protocol numbers 242, 2012). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Seventy-two rats (Sprague–Dawley, SPF class, either sex, 10–12 weeks of age, 280–310 g) were used by the experimental animal center of Sun Yat-Sen University. These rats were randomly divided into control group (n = 24), metoprolol group (n = 24), and the NC group (n = 24), which were housed under standard laboratory conditions at 25 6 28C, relative humidity of 50% 6 15%, and normal photoperiod (12 hours dark and 12 hours light) with free access to food and water. The rats of control group received 2 mL/d saline, metoprolol group received 100 mg$kg21$d21 metoprolol, and NC group received 600 mg$kg21$d21 NC for 1 month. NC was made of paste, 500 mg/mL, provided by the Xi’an sai’ao Biological Technology Co. Ltd. All surgeries were performed under surgical anesthesia level, and all efforts were made to minimize animal stress and suffering. Intensive care and monitoring were given to all animals after surgeries. After 1 month, rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). The animals were restrained and ventilated artificially in a dorsally recumbent position after the surgical anesthesia level was reached, and the right carotid artery was cannulated to assess the changes in blood pressure. Blood pressure and a lead II electrocardiogram were monitored, digitized, and recorded for subsequent analysis. Body temperature was monitored with a rectal temperature probe and maintained at 378C with a heating pad. A left thoracotomy in the fourth intercostal space was performed and the pericardium removed. Myocardial infarctions were created by ligating the left anterior descending coronary arteries (LAD) midway along their lengths using a 7-0 Prolene suture, placed between
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the pulmonary artery cone and the left auricle. Regional ischemia was confirmed by visual inspection for cyanosis and dyskinetic wall motion. AMI model was succeeded by monitoring lead ST segment elevation or QRS-T wave fusion. The premature ventricular contractions (PVCs), ventricular tachycardias (VTs), and VFs were monitored for 4 hours from LAD ligation. The definitions of PVC, VT, and VF were proposed according to the Lambeth Conventions (II).17 We terminated the observation once VF was recorded after the ligation.
Assessment of the Infarct Size and Pathologic Change One percent of 2,3,5-triphenyltetrazolium chloride was injected via the right jugular vein to identify the infarcted area (noninfarcted area was dark purple and infarcted area was gray). The heart was rapidly removed and placed in ice-cold 0.9% saline, and the atria, right ventricle, and great vessels were removed. The infarcted and noninfarcted areas of the left ventricular tissue were separated by careful dissection, and the infarct size was determined as percentage of wet weight. Pathology was performed by sampling full thickness sections of the myocardium from the ligation site to apex. Tissue was fixed in 10% neutral buffered formalin and processed using routine laboratory.
Immunohistochemistry The ventricular myocardial tissues were fixed in 4% formaldehyde, embedded in paraffin, sectioned at a thickness of 5 mm, and mounted on neutral balsam. Then sections were deparaffinized, baked for 2 hours at 608C, placed in citrate buffer, and incubated overnight with primary antibodies directed against Cx43 (Millipore) at 48C overnight. After 15 minutes rinse (3 · 5 minutes, phosphate-buffered saline), slices were incubated with secondary antibodies (Millipore) for 30 minutes at 378C. Then slices were rinsed for 50 minutes (5 · 10 minutes), mounted on neutral balsam, and stored at room temperature. Cx43 was examined with the use of a CX31 laser scanning confocal microscope (OLYMPUS). High-intensity Cx43 signals were measured and analyzed with the use of Image-Pro Plus Version 6.0 for Windows software (Media Cybernetics Inc, Silver Spring, MD).
Western Blot Ventricular myocyte tissues were lysed in complete lysis buffer (1.0 mmol/L Tris–HCl, pH 7.4, 10% sodium dodecyl sulfate, 150 mmol NaCl, 0.02% sodium azide, 10 mmol/L phenylmethanesulfonyl fluoride, 10% ammonium persulfate, 1% Triton X-100, 50 mmol sodium fluoride, and 1 mmol Na3VO4). Lysates were triturated and spun at 10,000 rpm for 10 minutes. After removal of the pellet, protein levels were tested using bicinchoninic acid protein Assay Kit (BioRad). Total proteins/samples were loaded onto an sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and electrophoresed. The protein gels were transferred onto polyvinylidene difluoride membranes for 45 minutes at 48C (110 V). After blocking in 5% nonfat milk for 2 hours at room temperature, the membranes were treated with anti-Cx43 (1:1500) overnight. 2014 Lippincott Williams & Wilkins
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Next day, the membranes were washed with blocking buffer and incubated with secondary antibodies directed against the primary and conjugated with horseradish peroxidase. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for equal input of protein samples. Western blot bands were quantified using Odyssey v1.2 software by measuring the band intensity (area · optical density) for each group and normalized by GAPDH. The final results are expressed as fold changes by normalizing the data to the control values.
Real-time PCR Oligonucleotide primer sequences for the RT-PCR assays of murine Cx43 and GAPDH gene expression were as follows: Cx43, forward 5-GCCCTGCCATAAGCCCTC TTGC-3, reverse 5-GTGACC GCCTGCCTACAACTTC-3; GAPDH, forward 5-CCTCGTCTCATAGACAAGATGGT-3, reverse 5-GGGTAGAGTCATACTGGAACATG-3. Total RNA was extracted from ventricular myocyte tissue using TRIzol reagent (TAKARA). An aliquot (2 mg) of RNA was transcribed to complementary DNA (cDNA) using PrimeScript First-Strand cDNA Synthesis Kit (TAKARA) under the following conditions: 378C for 15 minutes and 858C for 5 seconds. The cDNA was diluted 1:10 using sterilized distilled water; the real-time PCR reaction solution consisted of cDNA, SYBR Premix Ex Taq, and forward and reverse primers for a final reaction volume of 25 mL. All results were expressed relative to GAPDH, and a cellular RNA sample without reverse transcription was run as a negative control to test for genomic DNA. Calculated threshold cycle (Ct) values were determined by the apparatus, and the quality of the PCR product was confirmed by analyzing the melt curve.
Statistical Analyses Statistical analysis was performed using SPSS 13.0. Data were expressed as the mean 6 SE. Comparison of the incidence of VF among groups were made by x2 analysis with 3 · 2 contingency table, followed by intergroup comparisons using 2 · 2 table. Changes in continuous variables among
NC on Ventricular Arrhythmias in Rats With AMI
groups were analyzed using analysis of variance. Intergroup comparisons using Dunnett’s test, if analysis of variance showed significance, with 2-tailed P values ,0.05 were regarded as statistically significant.
RESULTS Seventy-eight rats underwent surgery and coronary artery ligation. Of these, 6 rats were excluded from analysis because of early death (2 rats from overdose of anesthesia and 4 rats had excessive blood loss). Of the remaining rats, 24 were included in each group.
The Effects of Metoprolol and NC on Ventricular Arrhythmias Over 4 hours of LAD ligation, spontaneous VF incidence was 50% (12/24) in the control group, 4.2% (1/ 24) in metoprolol group, and 12.5% (3/24) in the NC group. Compared with control, metoprolol and NC decreased the VF incidence (50% vs. 4.2%, P , 0.001, and 50% vs. 12.5%, P = 0.005, respectively) (Fig. 1). There is no statistical difference in spontaneous VF incidence between the groups of metoprolol and NC. The average numbers of arrhythmias for all animals across the PVC, VT, and VF classifications by different time were shown in Figure 1. Compared with control, there was a decrease in PVCs in the metoprolol and NC groups (24.70 6 5.16 vs. 176.08 6 42.50, P , 0.001, and 23.76 6 8.63 vs. 176.08 6 42.50, P , 0.001, respectively) 30 minutes after LAD ligation. Metoprolol and NC reduced the number of PVCs 30–120 minutes (11.61 6 2.56 vs. 110 6 34.96, P , 0.001, and 20.47 6 7.77 vs. 110 6 34.96, P = 0.005, respectively) and 120–240 minutes (13.87 6 3.31 vs. 158 6 50.81, P = 0.007, and 10.1 6 4.20 vs. 158 6 50.81, P = 0.008, respectively) after LAD ligation. Meanwhile, metoprolol and NC reduced the incidence of VTs 30 minutes (2.18 6 1.51 vs. 9.75 6 3.10, P , 0.001, and 3.81 6 1.03 vs. 9.75 6 3.10, P = 0.002, respectively), 30–120 minutes (0.52 6 0.38 vs. 2.33 6 1.64, P = 0.001, and 0.57 6 0.44 vs. 2.33 6 1.64,
FIGURE 1. Ventricular arrhythmias after ligating coronary arteries in 3 groups. Compared with control, NC decreased VF incidence (P = 0.005) and reduced the total numbers of PVCs (P = 0.001) and VTs (P = 0.001) after 4 hours ligation. Meanwhile, compared with control, metoprolol decreased VF incidence (P , 0.001) and reduced the total numbers of PVCs (P , 0.001) and VTs (P , 0.001) after 4 hours ligation. There is no statistical difference in the numbers of ventricular arrhythmias between the groups of metoprolol and NC. 2014 Lippincott Williams & Wilkins
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significantly prolonged after 1 month feed. Compared with NC, metoprolol significantly prolonged P-R interval after 1 month feed. In addition, compared with control, the MAP in groups of metoprolol and NC is significantly increased after ligation (Table 1).
Myocardial Infarct Size and Histology Compared with control, metoprolol and NC decreased the infarct size of the left ventricular tissue (55.98% 6 6.20% vs. 39.13% 6 4.53%, P , 0.001, and 55.98% 6 6.20% vs. 42.39% 6 3.44%, P , 0.001, respectively). Microscopically, the anterior apical region of rats in the control showed extensive interstitial hemorrhage and edema. In contrast, the anterior apical region of rats in the groups of metoprolol and NC showed minimal edema or hemorrhage. Contraction band necrosis was most evident in the control group.
FIGURE 2. Traces of cumulative ventricular arrhythmias in 3 groups. The traces showed reductions in the cumulative average number of PVCs and VTs per animal for the groups of NC and metoprolol.
Cx43 Protein Expression
P = 0.001, respectively), and 120–240 minutes (0.39 6 0.16 vs. 1.75 6 0.86, P , 0.001, and 0.28 6 0.15 vs. 1.75 6 0.86, P , 0.001, respectively) after LAD ligation. The difference of PVC and VT incidence did not reach statistical significance between metoprolol and NC groups 0–240 minutes after LAD ligation. There was a steady decrease in the cumulative number of PVCs and VTs within 4 hours in metoprolol and NC groups. Compared with control, the difference between the cumulative growth in the number of PVCs in metoprolol and NC groups was highly significant (50.17 6 20.56 vs. 443.08 6 35.98, P , 0.001, and 54.33 6 24.87 vs. 443.08 6 35.98, P = 0.001, respectively). Meanwhile, metoprolol and NC reduced the cumulative growth in the number of VTs (3.09 6 1.92 vs. 13.83 6 5.66, P , 0.001, and 5.00 6 3.62 vs. 13.83 6 5.66, P = 0.001, respectively). There was no statistical difference in the cumulative number of PVCs and VTs between groups of metoprolol and NC (Fig. 2).
Cx43 proteins in 3 groups were strong and uniformly distributed in the junctions between the myocardial cells and cells in the immunohistochemical staining under light microscope of noninfarcted zone. They were brownish yellow, most of which exist in the intercalated disk adhesion of myocardial fiber and a few in the long axis joint (Fig. 3). The Cx43 proteins among the 3 groups were not significantly different in noninfarcted zone. In infarcted zone, Cx43 proteins in the 3 groups were significantly less and no rules. Cx43 proteins were weaker and distributed in heterogeneity in the control group, whereas they were enhanced and their distribution was relatively ordered in metoprolol and NC groups. Compared with the control group, the difference between the positive area of Cx43 in metoprolol and NC groups was highly significant (2842.5 6 336.5 vs. 1421.7 6 326.5, P = 0.018, and 2532.7 6 282.7 vs. 1421.7 6 326.5, P = 0.026, respectively). The integrated optical density of Cx43 in the control group was also significantly lower than that in metoprolol and NC groups (223.74 6 42.15 vs. 436.74 6 78.29, P = 0.023, and 223.74 6 42.15 vs. 399.24 6 66.54, P = 0.039, respectively). There was no significant difference in the positive area and integrated optical density of Cx43 between the groups of metoprolol and NC (Fig. 4).
The Effects of Metoprolol and NC on Electrocardiogram and Hemodynamics Metoprolol significantly prolonged the P-R interval (P , 0.001) and decreased the heart rate (HR) (P , 0.001) and mean arterial pressure (MAP) (P , 0.001). However, it produced no significant changes in QRS and Q-T intervals. NC also decreased HR (P = 0.037), but it produced no significant changes in MAP, P-R interval, QRS interval, and Q-T interval (Table 1). Compared with control, the HR and MAP in metoprolol group were significantly decreased and P-R interval was
Western Blot and RT-PCR Analysis Results from Western blot showed that the Cx43 protein expression levels were not significantly different among the 3 groups in noninfarcted zone. However, in infarcted zone, the protein expression of Cx43 in the control
TABLE 1. The Parameters of 3 Groups Before and After Administrating Agent Control (n = 24) Baseline HR (beats/min) MAP (mm Hg) P-R (ms) QRS (ms) Q-T (ms)
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390 102.68 57.04 51.63 87.88
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6 6 6 6 6
20 12.64 9.00 8.89 6.94
Metoprolol (n = 24)
1 mo
P
6 6 6 6 6
0.831 0.971 0.961 0.986 0.881
391 102.19 56.92 51.58 87.58
29 11.78 8.64 7.83 6.50
Baseline 393 102.38 56.25 50.79 87.87
6 6 6 6 6
10 7.49 7.53 3.45 3.91
NC (n = 24)
1 mo
P
6 6 6 6 6
,0.001 ,0.001 ,0.001 0.092 0.076
372 91.29 65.08 52.46 89.58
28 6.22 3.92 3.26 4.63
Baseline 392 98.77 58.83 51.0 87.92
6 6 6 6 6
15 6.81 6.13 3.59 4.32
1 mo
P
6 6 6 6 6
0.037 0.146 0.102 0.231 0.073
381 95.84 61.45 52.25 88.21
19 6.92 4.66 3.54 2.89
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FIGURE 3. Immunohistochemistry: Cx43 expressions in noninfarcted and infarcted zones. In noninfarcted zone, Cx43 proteins in 3 groups were strong and uniformly distributed in the junctions between the myocardial cells and cells in the immunohistochemical staining under light microscope. They were brownish yellow, most of which exist in the intercalated disk adhesion of myocardial fiber and a few in the long axis joint. There is no significant difference among the groups. In infarcted zone, Cx43 proteins in the 3 groups were significantly less and no rules. In control group, Cx43 proteins were more weak and distributed in heterogeneity, whereas they were enhanced and their distribution was relatively ordered in metoprolol and NC groups.
group was significantly lower than that in the metoprolol and NC groups (0.3 6 0.12 vs. 1.39 6 0.16 units, P , 0.05, and 0.3 6 012 vs. 1.50 6 0.21 units, P , 0.05, respectively) (Fig. 5). The results from RT-PCR analysis were consistent with the results of Western blotting. In noninfarcted zone, the Cx43 protein expression level was not significantly different
among the 3 groups. In infarcted zone, the protein expression of Cx43 in the control group was significantly lower than that in the metoprolol and NC groups (0.29 6 0.09 vs. 0.57 6 0.14 units, P , 0.05, and 0.29 6 0.09 vs. 0.56 6 0.12 units, P , 0.05, respectively). There was no significant difference between the groups of metoprolol and NC (Fig. 5).
FIGURE 4. Area and integrated optical density value of Cx43 in noninfarcted and infarcted zones. Compared with the control group, the difference between the positive area of Cx43 in metoprolol and NC groups was highly significant (2842.5 6 336.5 vs. 1421.7 6 326.5, P = 0.018, and 2532.7 6 282.7 vs. 1421.7 6 326.5, P = 0.026, respectively). The integrated optical density of Cx43 in the control group was also significantly lower than that in metoprolol and NC groups (223.74 6 42.15 vs. 436.74 6 78.29, P = 0.023, and 223.74 6 42.15 vs. 399.24 6 66.54, P = 0.039, respectively). There was no significant difference in the positive area and integrated optical density of Cx43 between the groups of metoprolol and NC. 2014 Lippincott Williams & Wilkins
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FIGURE 5. Cx43 protein expression (A, B) and Cx43 messenger RNA expression (C) in 3 groups detected by Western blotting and real-time PCR. Results of the Western blot showed that the Cx43 protein expression levels were not significantly different among the 3 groups in noninfarcted zone. But in the infarcted zone, the protein expression of Cx43 in the control group was significantly lower than that in the metoprolol and NC groups (0.3 6 0.12 vs. 1.39 6 0.16 units, P , 0.05, and 0.3 6 0.12 vs. 1.50 6 0.21 units, P , 0.05, respectively). Results of RT-PCR showed that the Cx43 protein expression levels were not significantly different among the 3 groups. In the infarcted zone, the protein expression of Cx43 in the control group was significantly lower than that in the metoprolol and NC groups (0.29 6 0.09 vs. 0.57 6 0.14 units, P , 0.05, and 0.29 6 0.09 vs. 0.56 6 0.12 units, P , 0.05, respectively). There was no significant difference between the groups of metoprolol and NC.
DISCUSSION The major findings of the present study were that like metoprolol, NC decreased the incidence of spontaneous ventricular arrhythmias (especially VF) and myocardial ischemic size, reduced degradation of Cx43, and improved Cx43 redistribution in myocardial infarcted zone in rats with hyper-AMI. AMI in rats promotes an evolving series of rhythm disturbances with spontaneous ventricular arrhythmias developing postcoronary occlusion in 2 major phases.2,18–20 The hyper-acute phase occurs in 30 minutes postcoronary occlusion and has been attributed to both reentrant and nonentrant mechanisms.18,21–23 The later and longer lasting period of spontaneous arrhythmias is thought to be caused by an increase in the automaticity of surviving Purkinje fibers in the infarct area and/or triggered activity because of delayed afterdepolarizations in Purkinje fibers.24 In our study, we found that most spontaneous VF occurred within 30 minutes postligation in all 3 groups. Metoprolol, a class II antiarrhythmic drug, exerts antiarrhythmic effects by blocking beta-adrenergic receptors. Beta-receptor blockers have been recognized as a drug that can effectively reduce malignant ventricular arrhythmias and sudden cardiac death after myocardial infarction.15,16 Some studies have confirmed that metoprolol can effectively restrain degradation of Cx43 and improve sympathetic remodeling after myocardial infarction. This ability might improve gap junction communication, increase conduction velocity, reduce action potential duration heterogeneity, increase electrical homogeneity, and decrease vulnerability to ventricular arrhythmia and VF.25,26 Recently, some studies on the antiarrhythmic function of NC and its effective components have made a great
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progress.27 The mechanism for antiarrhythmic function of NC is considered to be prolonging the action potential duration by blocking myocardial membrane ion channels, including INa, ICa-l, IK, and Ito.28–30 However, the results from our studies may imply other possible antiarrhythmic mechanisms of NC. First, Cx43 is the major gap junction protein in cardiomyocytes that mediates the function of cell coupling and leads to orderly electrical conduction,31 the quantity alteration or redistribution of Cx43 contributes to various arrhythmias in myocardial infarction. Our results showed that NC and metoprolol could reduce degradation of protein and messenger RNA levels of Cx43 in myocardial infarcted zone. We also noticed that Cx43 protein scattered and lost orderly distribution between intercalary discs in infarcted zone rat hearts. So, we speculated the possible antiarrhythmia mechanism was that NC may reduce degradation of Cx43 and improve Cx43 redistribution as well as metoprolol. However, the mechanism for NC leading to cardiac Cx43 remodeling in this study is not clear, and we will investigate it in the future. Second, another possible antiarrhythmic mechanism of NC might be related to the decrease in the myocardial ischemic size. Such properties of NC have an important clinical significance. If NC tablets or aerosols are available in the future, it perhaps will become a new convenient drug to prevent patients with AMI from lethal ventricular arrhythmias in hyper-acute stage in prehospital setting.
CONCLUSIONS NC could decrease the incidence of spontaneous ventricular arrhythmias (especially VF) in rats with hyperAMI. The possible antiarrhythmic mechanisms of NC might relate to reduce degradation of Cx43 and improve Cx43 2014 Lippincott Williams & Wilkins
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redistribution in myocardial infarcted zone. Our study indicated that NC may become a promising drug in the future to prevent patients with AMI from lethal ventricular arrhythmias in prehospital setting. REFERENCES 1. Clements-Jewery H, Hearse DJ, Curtis MJ. Phase 2 ventricular arrhythmias in acute myocardial infarction: a neglected target for therapeutic antiarrhythmic drug development and for safety pharmacology evaluation. Br J Pharmacol. 2005;145:551–564. 2. Harris AS. Delayed development of ventricular ectopic rhythms following experimental coronary occlusion. Circulation. 1950;1:1318–1328. 3. John RM, Tedrow UB, Koplan BA, et al. Ventricular arrhythmias and sudden cardiac death. Lancet. 2012;380:1520–1529. 4. Fishman GI, Chugh SS, Dimarco JP, et al. Sudden cardiac death prediction and prevention: report from a National Heart, Lung, and Blood Institute and Heart Rhythm Society Workshop. Circulation. 2010;122: 2335–2348. 5. Van Kempen MJ, Fromaget C, Gros D, et al. Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart. Circ Res. 1991;68:1638–1651. 6. Jalife J, Morley GE, Vaidya D. Connexins and impulse propagation in the mouse heart. J Cardiovasc Electrophysiol. 1999;10:1649–1663. 7. Ando M, Katare RG, Kakinuma Y, et al. Efferent vagal nerve stimulation protects heart against ischemia-induced arrhythmias by preserving connexin43 protein. Circulation. 2005;112:164–170. 8. Camelliti P, Devlin GP, Matthews KG, et al. Spatially and temporally distinct expression of fibroblast connexins after sheep ventricular infarction. Cardiovasc Res. 2004;62:415–425. 9. Liu CL, Duan YH, Dai Y, et al. Study on the chemical constituents of roots and stems of NC [in Chinese]. Zhong Yao Cai. 2011;34:1216–1219. 10. Zhang X, Lan Z, Dong XP, et al. Study on the active components of NC [in Chinese]. Zhong Yao Cai. 2007;30:38–41. 11. Qian XZ. The Pharmacopoeia of the People’s Republic of China. Pt 1. 1978:120–130. 12. Wang J, Zhao J, Liu H, et al. Chemical analysis and biological activity of the essential oils of two valerianaceous species from China: NC and Valeriana officinalis. Molecules. 2010;15:6411–6422. 13. Tanitsu MA, Takaya Y, Akasaka M, et al. Guaiane- and aristolane-type sesquiterpenoids of NC roots. Phytochemistry. 2002;59:845–849. 14. Tanaka K, Komatsu K. Comparative study on volatile components of Nardostachys rhizome. J Nat Med. 2008;62:112–116. 15. Sherman L, Niemann J, Youngquist ST, et al. Beta-blockade causes a reduction in the frequency spectrum of VF but improves resuscitation outcome: a potential limitation of quantitative waveform measures. Resuscitation. 2012;83:511–516.
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NC on Ventricular Arrhythmias in Rats With AMI 16. Ibanez B, Fuster V, Macaya C, et al. Study design for the “effect of metoprolol in cardioprotection during an acute myocardial infarction” (metocard-cnin): a randomized, controlled parallel-group, observerblinded clinical trial of early pre-reperfusion metoprolol administration in ST-segment elevation myocardial infarction. Am Heart J. 2012;164: 473–480. 17. Curtis MJ, Hancox JC, Farkas A, et al. The Lambeth Conventions (II): guidelines for the study of animal and human ventricular and supraventricular arrhythmias. Pharmacol Ther. 2013;139:213–248. 18. Weiss JN, Chen PS, Qu Z, et al. Ventricular fibrillation: how do we stop the waves from breaking? Circ Res. 2000;87:1103–1107. 19. Gray RA, Jalife J, Panfilov AV, et al. Mechanisms of cardiac fibrillation. Science. 1995;270:1222–1223. 20. Berenfeld O, Jalife J. Purkinje-muscle reentry as a mechanism of polymorphic ventricular arrhythmias in a 3-dimensional model of the ventricles. Circ Res. 1998;82:1063–1077. 21. Jalife J. Ventricular fibrillation: mechanisms of initiation and maintenance. Annu Rev Physiol. 2000;62:25–50. 22. Huelsing DJ, Spitzer KW, Pollard AE. Electrotonic suppression of early afterdepolarizations in isolated rabbit Purkinje myocytes. Am J Physiol Heart Circ Physiol. 2000;279:H250–H259. 23. Pak HN, Hong SJ, Hwang GS, et al. Spatial dispersion of action potential duration restitution kinetics is associated with induction of ventricular tachycardia/fibrillation in humans. J Cardiovasc Electrophysiol. 2004;15: 1357–1363. 24. Wu TJ, Lin SF, Weiss JN, et al. Two types of ventricular fibrillation in isolated rabbit hearts: importance of excitability and action potential duration restitution. Circulation. 2002;106:1859–1866. 25. Wen H, Jiang H, Hu X, et al. Effect of metoprolol on connexin43 expression in infarcted border zone of rat myocardium. Med J Wuhan Univ. 2010;31:170–173. 26. Jiang H, Lu Z, Yu Y, et al. Effects of metoprolol on sympathetic remodeling and electrical remodeling at infarcted border zone after myocardial infarction in rabbits. Cardiology. 2007;108:176–182. 27. Yang T, Hu LJ. Study on anti-arrhythmic effects and electrophysiological mechanism of NC. Mod Diagn Treat. 2008;19:276–278. 28. Yang T, Ge YZ, Luo J, et al. Effects of volatile oil of NC on sodium channel in isolated ventricular myocytes in rats. Li Shi Zhen Med Mater Med Res. 2010;21:284–286. 29. Cao M, Ge YZ, Luo J, et al. Effects of the volatile oil of NC on L-type calcium channel in isolated ventricular myocytes in rats. Li Shi Zhen Med Mater Med Res. 2010;21:2264–2266. 30. Tang QZ, Huang ZR, Shi XT, et al. Effects of NC on sodium channel and calcium channel in isolated ventricular myocytes in rabbits. Chin J Cardiol. 2004;32:267–270. 31. Kleber AG, Rudy Y. Basic mechanisms of cardiac impulse propagation and associated arrhythmias. Physiol Rev. 2004;84:431–488.
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Effects of Nardostachys chinensis on Spontaneous Ventricular Arrhythmias in Rats With Acute Myocardial Infarction Jing Zhang, MD,* Can-Can Qiang, MD,* Wei-Jie Li, MD,* Li-Juan Liu, MD, PhD,* Xiao-Xiong Lin, MD,* Yun-Jiu Cheng, MD,* Kai Tang, MD, PhD,* Feng-Juan Yao, MD,† and Su-Hua Wu, MD, PhD*
Objectives: To investigate the effects and mechanisms of Nardostachys chinensis (NC) on spontaneous ventricular arrhythmias in rats with hyper-acute myocardial infarction (AMI).
Methods: Seventy-two rats were randomly divided into the control group (n = 24), metoprolol group (n = 24), and the NC group (n = 24). Premature ventricular contractions (PVCs), ventricular tachycardias (VTs), ventricular fibrillations (VFs), and blood pressure were monitored for 4 hours after coronary artery ligation. The connexin 43 (Cx43) expression in ventricular myocardium was measured by immunohistochemistry, Western blot, and real-time RT-PCR. Results: Compared with the control, metoprolol and NC decreased the VF incidence (50% vs. 4.2%, P , 0.001, and 50% vs. 12.5%, P = 0.005, respectively). There was a steady decrease in the cumulative number of PVCs and VTs within 4 hours from ligating in 3 groups. Compared with the control, metoprolol and NC reduced the cumulative number of VTs and PVCs. Compared with control, metoprolol and NC decreased the infarct size of the left ventricular tissue (55.98% 6 Received for publication January 15, 2014; accepted March 10, 2014. From the *Department of Cardiovascular Medicine, The First Affiliated Hospital, and the Key Laboratory of Assisted Circulation, Ministry of Health, Sun Yat-Sen University, Guangzhou, China; and †Department of Ultrasonography, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China. J. Zhang, C.-C. Qiang, and W.-J. Li have contributed equally. Study concept and design: S.-H. Wu; acquisition of data: J. Zhang, C.-C. Qiang, W.-J. Li, L.-J. Liu, X.-X. Lin, Y.-J. Cheng, K. Tang, F.-J. Yao; analysis and interpretation of data: J. Zhang, C.-C. Qiang, W.-J. Li, S.-H. Wu, L.-J. Liu, X.-X. Lin, Y.-J. Cheng, K. Tang, F.-J. Yao; drafting of the manuscript: J. Zhang, S.-H. Wu, C.-C. Qiang, W.-J. Li; statistical analysis: J. Zhang, C.-C. Qiang, W.-J. Li. Supported by National Natural Science Foundation of China (No. 81370285), Guangdong Province Natural Science Foundation (No. 06021338); Guangdong Province Science and Technology Program (No. 2012B031800091, 2007B031508003) and National Ministry of Education Scholarly Exchanges Foundation (No. 200724). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors report no conflicts of interest. Reprints: Su-Hua Wu, MD, PhD, Department of Cardiovascular Medicine, The First Affiliated Hospital, Sun Yat-Sen University, No. 58, Zhongshan Rd II, Guangzhou 510080, China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins. This is an open access article distributed under the terms of the Creative Commons AttributionNoncommercial-No Derivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.
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6.20% vs. 39.13% 6 4.53%, P , 0.001, and 55.98% 6 6.20% vs. 42.39% 6 3.44%, P , 0.001, respectively). The results from immunohistochemistry, Western blot, and real-time RT-PCR showed that the protein expression of Cx43 in the control group was significantly lower than that in the metoprolol and NC groups in the infarcted zone.
Conclusions: NC decreased the incidence of spontaneous ventricular arrhythmias (especially VF), reduced Cx43 degradation, and improved Cx43 redistribution in myocardial infarcted zone in rats with hyper-AMI. The data of the present study indicated that NC may be a promising drug in the future to prevent patients with AMI from lethal ventricular arrhythmias in prehospital setting. Key Words: Nardostachys chinensis, ventricular arrhythmias, myocardial infarction, metoprolol, connexin 43 (J Cardiovasc Pharmacol 2014;64:127–133)
INTRODUCTION Eighty percent of the ventricular fibrillation (VF) caused by acute myocardial infarction (AMI) occurs at the hyper-acute stage (within 4 hours after coronary occlusion), and 50% of the deaths because of AMI are caused by the VFs during hyperacute stage (often prehospital).1–4 The decrease in the incidence of hyper-acute VF will reduce the mortality caused by AMI. Gap junctions are low-resistance channels between adjacent myocardial cells that are predominantly composed of connexin 43 (Cx43) in the ventricle. Cx43 has an important role in the propagation of action potential to mediate current flow and to coordinate the spread of excitation and subsequent contraction throughout the myocardium.5,6 Gene-targeting studies demonstrated that reduced expression of Cx43 caused a significant reduction in conduction velocity and increased the incidence of ventricular tachyarrhythmias7 in mice during acute myocardial ischemia.8 These results suggested that the dysfunction of Cx43 in cardiomyocytes could be one of the components of the substrate that promotes lethal ventricular tachyarrhythmias in acute myocardial ischemia. The rhizomes and roots of Nardostachys chinensis (NC) Batalin, members of the family Valerianaceae growing in Himalayan regions to southwest China, have been used as crude drugs in traditional Chinese medicine.9 The rhizomes of NC are also named as “Gansongxiang” or “Gansong” in Chinese. NC contains mainly guaiane, aristolane, www.jcvp.org |
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nardosinane-type sesquiterpenoids, neolignan, and lignans.10 They are used to treat abdominal pain, indigestion, vomiting, diarrhea, epilepsy, hysteria, and mental disorders.11 Recently, NC has been shown to have novel pharmacologic effects, such as cardiac protection (especially protection from arrhythmias), antioxidant properties, and hypolipidemic effect.12–14 However, there is little known about the effect of NC in the prevention and conversion of hyper-acute ventricular arrhythmias (especially VF) after AMI. Beta-receptor blockers (metoprolol) have been recognized as a drug that can effectively reduce malignant ventricular arrhythmias and sudden cardiac death after myocardial infarction.15,16 In the present study, we investigate the anti-ventricular arrhythmia effects of NC, compared with metoprolol, in a rat model of AMI. At the same time, we examine the effects of NC and metoprolol on the levels, localization, and function of the ventricular gap junction protein Cx43.
MATERIALS AND METHODS Animal Experiment All procedures for these experiments have been approved by the Animal Experimentation and Ethics Committee of the Sun Yat-Sen University (protocol numbers 242, 2012). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Seventy-two rats (Sprague–Dawley, SPF class, either sex, 10–12 weeks of age, 280–310 g) were used by the experimental animal center of Sun Yat-Sen University. These rats were randomly divided into control group (n = 24), metoprolol group (n = 24), and the NC group (n = 24), which were housed under standard laboratory conditions at 25 6 28C, relative humidity of 50% 6 15%, and normal photoperiod (12 hours dark and 12 hours light) with free access to food and water. The rats of control group received 2 mL/d saline, metoprolol group received 100 mg$kg21$d21 metoprolol, and NC group received 600 mg$kg21$d21 NC for 1 month. NC was made of paste, 500 mg/mL, provided by the Xi’an sai’ao Biological Technology Co. Ltd. All surgeries were performed under surgical anesthesia level, and all efforts were made to minimize animal stress and suffering. Intensive care and monitoring were given to all animals after surgeries. After 1 month, rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). The animals were restrained and ventilated artificially in a dorsally recumbent position after the surgical anesthesia level was reached, and the right carotid artery was cannulated to assess the changes in blood pressure. Blood pressure and a lead II electrocardiogram were monitored, digitized, and recorded for subsequent analysis. Body temperature was monitored with a rectal temperature probe and maintained at 378C with a heating pad. A left thoracotomy in the fourth intercostal space was performed and the pericardium removed. Myocardial infarctions were created by ligating the left anterior descending coronary arteries (LAD) midway along their lengths using a 7-0 Prolene suture, placed between
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the pulmonary artery cone and the left auricle. Regional ischemia was confirmed by visual inspection for cyanosis and dyskinetic wall motion. AMI model was succeeded by monitoring lead ST segment elevation or QRS-T wave fusion. The premature ventricular contractions (PVCs), ventricular tachycardias (VTs), and VFs were monitored for 4 hours from LAD ligation. The definitions of PVC, VT, and VF were proposed according to the Lambeth Conventions (II).17 We terminated the observation once VF was recorded after the ligation.
Assessment of the Infarct Size and Pathologic Change One percent of 2,3,5-triphenyltetrazolium chloride was injected via the right jugular vein to identify the infarcted area (noninfarcted area was dark purple and infarcted area was gray). The heart was rapidly removed and placed in ice-cold 0.9% saline, and the atria, right ventricle, and great vessels were removed. The infarcted and noninfarcted areas of the left ventricular tissue were separated by careful dissection, and the infarct size was determined as percentage of wet weight. Pathology was performed by sampling full thickness sections of the myocardium from the ligation site to apex. Tissue was fixed in 10% neutral buffered formalin and processed using routine laboratory.
Immunohistochemistry The ventricular myocardial tissues were fixed in 4% formaldehyde, embedded in paraffin, sectioned at a thickness of 5 mm, and mounted on neutral balsam. Then sections were deparaffinized, baked for 2 hours at 608C, placed in citrate buffer, and incubated overnight with primary antibodies directed against Cx43 (Millipore) at 48C overnight. After 15 minutes rinse (3 · 5 minutes, phosphate-buffered saline), slices were incubated with secondary antibodies (Millipore) for 30 minutes at 378C. Then slices were rinsed for 50 minutes (5 · 10 minutes), mounted on neutral balsam, and stored at room temperature. Cx43 was examined with the use of a CX31 laser scanning confocal microscope (OLYMPUS). High-intensity Cx43 signals were measured and analyzed with the use of Image-Pro Plus Version 6.0 for Windows software (Media Cybernetics Inc, Silver Spring, MD).
Western Blot Ventricular myocyte tissues were lysed in complete lysis buffer (1.0 mmol/L Tris–HCl, pH 7.4, 10% sodium dodecyl sulfate, 150 mmol NaCl, 0.02% sodium azide, 10 mmol/L phenylmethanesulfonyl fluoride, 10% ammonium persulfate, 1% Triton X-100, 50 mmol sodium fluoride, and 1 mmol Na3VO4). Lysates were triturated and spun at 10,000 rpm for 10 minutes. After removal of the pellet, protein levels were tested using bicinchoninic acid protein Assay Kit (BioRad). Total proteins/samples were loaded onto an sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and electrophoresed. The protein gels were transferred onto polyvinylidene difluoride membranes for 45 minutes at 48C (110 V). After blocking in 5% nonfat milk for 2 hours at room temperature, the membranes were treated with anti-Cx43 (1:1500) overnight. 2014 Lippincott Williams & Wilkins
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Next day, the membranes were washed with blocking buffer and incubated with secondary antibodies directed against the primary and conjugated with horseradish peroxidase. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for equal input of protein samples. Western blot bands were quantified using Odyssey v1.2 software by measuring the band intensity (area · optical density) for each group and normalized by GAPDH. The final results are expressed as fold changes by normalizing the data to the control values.
Real-time PCR Oligonucleotide primer sequences for the RT-PCR assays of murine Cx43 and GAPDH gene expression were as follows: Cx43, forward 5-GCCCTGCCATAAGCCCTC TTGC-3, reverse 5-GTGACC GCCTGCCTACAACTTC-3; GAPDH, forward 5-CCTCGTCTCATAGACAAGATGGT-3, reverse 5-GGGTAGAGTCATACTGGAACATG-3. Total RNA was extracted from ventricular myocyte tissue using TRIzol reagent (TAKARA). An aliquot (2 mg) of RNA was transcribed to complementary DNA (cDNA) using PrimeScript First-Strand cDNA Synthesis Kit (TAKARA) under the following conditions: 378C for 15 minutes and 858C for 5 seconds. The cDNA was diluted 1:10 using sterilized distilled water; the real-time PCR reaction solution consisted of cDNA, SYBR Premix Ex Taq, and forward and reverse primers for a final reaction volume of 25 mL. All results were expressed relative to GAPDH, and a cellular RNA sample without reverse transcription was run as a negative control to test for genomic DNA. Calculated threshold cycle (Ct) values were determined by the apparatus, and the quality of the PCR product was confirmed by analyzing the melt curve.
Statistical Analyses Statistical analysis was performed using SPSS 13.0. Data were expressed as the mean 6 SE. Comparison of the incidence of VF among groups were made by x2 analysis with 3 · 2 contingency table, followed by intergroup comparisons using 2 · 2 table. Changes in continuous variables among
NC on Ventricular Arrhythmias in Rats With AMI
groups were analyzed using analysis of variance. Intergroup comparisons using Dunnett’s test, if analysis of variance showed significance, with 2-tailed P values ,0.05 were regarded as statistically significant.
RESULTS Seventy-eight rats underwent surgery and coronary artery ligation. Of these, 6 rats were excluded from analysis because of early death (2 rats from overdose of anesthesia and 4 rats had excessive blood loss). Of the remaining rats, 24 were included in each group.
The Effects of Metoprolol and NC on Ventricular Arrhythmias Over 4 hours of LAD ligation, spontaneous VF incidence was 50% (12/24) in the control group, 4.2% (1/ 24) in metoprolol group, and 12.5% (3/24) in the NC group. Compared with control, metoprolol and NC decreased the VF incidence (50% vs. 4.2%, P , 0.001, and 50% vs. 12.5%, P = 0.005, respectively) (Fig. 1). There is no statistical difference in spontaneous VF incidence between the groups of metoprolol and NC. The average numbers of arrhythmias for all animals across the PVC, VT, and VF classifications by different time were shown in Figure 1. Compared with control, there was a decrease in PVCs in the metoprolol and NC groups (24.70 6 5.16 vs. 176.08 6 42.50, P , 0.001, and 23.76 6 8.63 vs. 176.08 6 42.50, P , 0.001, respectively) 30 minutes after LAD ligation. Metoprolol and NC reduced the number of PVCs 30–120 minutes (11.61 6 2.56 vs. 110 6 34.96, P , 0.001, and 20.47 6 7.77 vs. 110 6 34.96, P = 0.005, respectively) and 120–240 minutes (13.87 6 3.31 vs. 158 6 50.81, P = 0.007, and 10.1 6 4.20 vs. 158 6 50.81, P = 0.008, respectively) after LAD ligation. Meanwhile, metoprolol and NC reduced the incidence of VTs 30 minutes (2.18 6 1.51 vs. 9.75 6 3.10, P , 0.001, and 3.81 6 1.03 vs. 9.75 6 3.10, P = 0.002, respectively), 30–120 minutes (0.52 6 0.38 vs. 2.33 6 1.64, P = 0.001, and 0.57 6 0.44 vs. 2.33 6 1.64,
FIGURE 1. Ventricular arrhythmias after ligating coronary arteries in 3 groups. Compared with control, NC decreased VF incidence (P = 0.005) and reduced the total numbers of PVCs (P = 0.001) and VTs (P = 0.001) after 4 hours ligation. Meanwhile, compared with control, metoprolol decreased VF incidence (P , 0.001) and reduced the total numbers of PVCs (P , 0.001) and VTs (P , 0.001) after 4 hours ligation. There is no statistical difference in the numbers of ventricular arrhythmias between the groups of metoprolol and NC. 2014 Lippincott Williams & Wilkins
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significantly prolonged after 1 month feed. Compared with NC, metoprolol significantly prolonged P-R interval after 1 month feed. In addition, compared with control, the MAP in groups of metoprolol and NC is significantly increased after ligation (Table 1).
Myocardial Infarct Size and Histology Compared with control, metoprolol and NC decreased the infarct size of the left ventricular tissue (55.98% 6 6.20% vs. 39.13% 6 4.53%, P , 0.001, and 55.98% 6 6.20% vs. 42.39% 6 3.44%, P , 0.001, respectively). Microscopically, the anterior apical region of rats in the control showed extensive interstitial hemorrhage and edema. In contrast, the anterior apical region of rats in the groups of metoprolol and NC showed minimal edema or hemorrhage. Contraction band necrosis was most evident in the control group.
FIGURE 2. Traces of cumulative ventricular arrhythmias in 3 groups. The traces showed reductions in the cumulative average number of PVCs and VTs per animal for the groups of NC and metoprolol.
Cx43 Protein Expression
P = 0.001, respectively), and 120–240 minutes (0.39 6 0.16 vs. 1.75 6 0.86, P , 0.001, and 0.28 6 0.15 vs. 1.75 6 0.86, P , 0.001, respectively) after LAD ligation. The difference of PVC and VT incidence did not reach statistical significance between metoprolol and NC groups 0–240 minutes after LAD ligation. There was a steady decrease in the cumulative number of PVCs and VTs within 4 hours in metoprolol and NC groups. Compared with control, the difference between the cumulative growth in the number of PVCs in metoprolol and NC groups was highly significant (50.17 6 20.56 vs. 443.08 6 35.98, P , 0.001, and 54.33 6 24.87 vs. 443.08 6 35.98, P = 0.001, respectively). Meanwhile, metoprolol and NC reduced the cumulative growth in the number of VTs (3.09 6 1.92 vs. 13.83 6 5.66, P , 0.001, and 5.00 6 3.62 vs. 13.83 6 5.66, P = 0.001, respectively). There was no statistical difference in the cumulative number of PVCs and VTs between groups of metoprolol and NC (Fig. 2).
Cx43 proteins in 3 groups were strong and uniformly distributed in the junctions between the myocardial cells and cells in the immunohistochemical staining under light microscope of noninfarcted zone. They were brownish yellow, most of which exist in the intercalated disk adhesion of myocardial fiber and a few in the long axis joint (Fig. 3). The Cx43 proteins among the 3 groups were not significantly different in noninfarcted zone. In infarcted zone, Cx43 proteins in the 3 groups were significantly less and no rules. Cx43 proteins were weaker and distributed in heterogeneity in the control group, whereas they were enhanced and their distribution was relatively ordered in metoprolol and NC groups. Compared with the control group, the difference between the positive area of Cx43 in metoprolol and NC groups was highly significant (2842.5 6 336.5 vs. 1421.7 6 326.5, P = 0.018, and 2532.7 6 282.7 vs. 1421.7 6 326.5, P = 0.026, respectively). The integrated optical density of Cx43 in the control group was also significantly lower than that in metoprolol and NC groups (223.74 6 42.15 vs. 436.74 6 78.29, P = 0.023, and 223.74 6 42.15 vs. 399.24 6 66.54, P = 0.039, respectively). There was no significant difference in the positive area and integrated optical density of Cx43 between the groups of metoprolol and NC (Fig. 4).
The Effects of Metoprolol and NC on Electrocardiogram and Hemodynamics Metoprolol significantly prolonged the P-R interval (P , 0.001) and decreased the heart rate (HR) (P , 0.001) and mean arterial pressure (MAP) (P , 0.001). However, it produced no significant changes in QRS and Q-T intervals. NC also decreased HR (P = 0.037), but it produced no significant changes in MAP, P-R interval, QRS interval, and Q-T interval (Table 1). Compared with control, the HR and MAP in metoprolol group were significantly decreased and P-R interval was
Western Blot and RT-PCR Analysis Results from Western blot showed that the Cx43 protein expression levels were not significantly different among the 3 groups in noninfarcted zone. However, in infarcted zone, the protein expression of Cx43 in the control
TABLE 1. The Parameters of 3 Groups Before and After Administrating Agent Control (n = 24) Baseline HR (beats/min) MAP (mm Hg) P-R (ms) QRS (ms) Q-T (ms)
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390 102.68 57.04 51.63 87.88
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6 6 6 6 6
20 12.64 9.00 8.89 6.94
Metoprolol (n = 24)
1 mo
P
6 6 6 6 6
0.831 0.971 0.961 0.986 0.881
391 102.19 56.92 51.58 87.58
29 11.78 8.64 7.83 6.50
Baseline 393 102.38 56.25 50.79 87.87
6 6 6 6 6
10 7.49 7.53 3.45 3.91
NC (n = 24)
1 mo
P
6 6 6 6 6
,0.001 ,0.001 ,0.001 0.092 0.076
372 91.29 65.08 52.46 89.58
28 6.22 3.92 3.26 4.63
Baseline 392 98.77 58.83 51.0 87.92
6 6 6 6 6
15 6.81 6.13 3.59 4.32
1 mo
P
6 6 6 6 6
0.037 0.146 0.102 0.231 0.073
381 95.84 61.45 52.25 88.21
19 6.92 4.66 3.54 2.89
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FIGURE 3. Immunohistochemistry: Cx43 expressions in noninfarcted and infarcted zones. In noninfarcted zone, Cx43 proteins in 3 groups were strong and uniformly distributed in the junctions between the myocardial cells and cells in the immunohistochemical staining under light microscope. They were brownish yellow, most of which exist in the intercalated disk adhesion of myocardial fiber and a few in the long axis joint. There is no significant difference among the groups. In infarcted zone, Cx43 proteins in the 3 groups were significantly less and no rules. In control group, Cx43 proteins were more weak and distributed in heterogeneity, whereas they were enhanced and their distribution was relatively ordered in metoprolol and NC groups.
group was significantly lower than that in the metoprolol and NC groups (0.3 6 0.12 vs. 1.39 6 0.16 units, P , 0.05, and 0.3 6 012 vs. 1.50 6 0.21 units, P , 0.05, respectively) (Fig. 5). The results from RT-PCR analysis were consistent with the results of Western blotting. In noninfarcted zone, the Cx43 protein expression level was not significantly different
among the 3 groups. In infarcted zone, the protein expression of Cx43 in the control group was significantly lower than that in the metoprolol and NC groups (0.29 6 0.09 vs. 0.57 6 0.14 units, P , 0.05, and 0.29 6 0.09 vs. 0.56 6 0.12 units, P , 0.05, respectively). There was no significant difference between the groups of metoprolol and NC (Fig. 5).
FIGURE 4. Area and integrated optical density value of Cx43 in noninfarcted and infarcted zones. Compared with the control group, the difference between the positive area of Cx43 in metoprolol and NC groups was highly significant (2842.5 6 336.5 vs. 1421.7 6 326.5, P = 0.018, and 2532.7 6 282.7 vs. 1421.7 6 326.5, P = 0.026, respectively). The integrated optical density of Cx43 in the control group was also significantly lower than that in metoprolol and NC groups (223.74 6 42.15 vs. 436.74 6 78.29, P = 0.023, and 223.74 6 42.15 vs. 399.24 6 66.54, P = 0.039, respectively). There was no significant difference in the positive area and integrated optical density of Cx43 between the groups of metoprolol and NC. 2014 Lippincott Williams & Wilkins
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FIGURE 5. Cx43 protein expression (A, B) and Cx43 messenger RNA expression (C) in 3 groups detected by Western blotting and real-time PCR. Results of the Western blot showed that the Cx43 protein expression levels were not significantly different among the 3 groups in noninfarcted zone. But in the infarcted zone, the protein expression of Cx43 in the control group was significantly lower than that in the metoprolol and NC groups (0.3 6 0.12 vs. 1.39 6 0.16 units, P , 0.05, and 0.3 6 0.12 vs. 1.50 6 0.21 units, P , 0.05, respectively). Results of RT-PCR showed that the Cx43 protein expression levels were not significantly different among the 3 groups. In the infarcted zone, the protein expression of Cx43 in the control group was significantly lower than that in the metoprolol and NC groups (0.29 6 0.09 vs. 0.57 6 0.14 units, P , 0.05, and 0.29 6 0.09 vs. 0.56 6 0.12 units, P , 0.05, respectively). There was no significant difference between the groups of metoprolol and NC.
DISCUSSION The major findings of the present study were that like metoprolol, NC decreased the incidence of spontaneous ventricular arrhythmias (especially VF) and myocardial ischemic size, reduced degradation of Cx43, and improved Cx43 redistribution in myocardial infarcted zone in rats with hyper-AMI. AMI in rats promotes an evolving series of rhythm disturbances with spontaneous ventricular arrhythmias developing postcoronary occlusion in 2 major phases.2,18–20 The hyper-acute phase occurs in 30 minutes postcoronary occlusion and has been attributed to both reentrant and nonentrant mechanisms.18,21–23 The later and longer lasting period of spontaneous arrhythmias is thought to be caused by an increase in the automaticity of surviving Purkinje fibers in the infarct area and/or triggered activity because of delayed afterdepolarizations in Purkinje fibers.24 In our study, we found that most spontaneous VF occurred within 30 minutes postligation in all 3 groups. Metoprolol, a class II antiarrhythmic drug, exerts antiarrhythmic effects by blocking beta-adrenergic receptors. Beta-receptor blockers have been recognized as a drug that can effectively reduce malignant ventricular arrhythmias and sudden cardiac death after myocardial infarction.15,16 Some studies have confirmed that metoprolol can effectively restrain degradation of Cx43 and improve sympathetic remodeling after myocardial infarction. This ability might improve gap junction communication, increase conduction velocity, reduce action potential duration heterogeneity, increase electrical homogeneity, and decrease vulnerability to ventricular arrhythmia and VF.25,26 Recently, some studies on the antiarrhythmic function of NC and its effective components have made a great
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progress.27 The mechanism for antiarrhythmic function of NC is considered to be prolonging the action potential duration by blocking myocardial membrane ion channels, including INa, ICa-l, IK, and Ito.28–30 However, the results from our studies may imply other possible antiarrhythmic mechanisms of NC. First, Cx43 is the major gap junction protein in cardiomyocytes that mediates the function of cell coupling and leads to orderly electrical conduction,31 the quantity alteration or redistribution of Cx43 contributes to various arrhythmias in myocardial infarction. Our results showed that NC and metoprolol could reduce degradation of protein and messenger RNA levels of Cx43 in myocardial infarcted zone. We also noticed that Cx43 protein scattered and lost orderly distribution between intercalary discs in infarcted zone rat hearts. So, we speculated the possible antiarrhythmia mechanism was that NC may reduce degradation of Cx43 and improve Cx43 redistribution as well as metoprolol. However, the mechanism for NC leading to cardiac Cx43 remodeling in this study is not clear, and we will investigate it in the future. Second, another possible antiarrhythmic mechanism of NC might be related to the decrease in the myocardial ischemic size. Such properties of NC have an important clinical significance. If NC tablets or aerosols are available in the future, it perhaps will become a new convenient drug to prevent patients with AMI from lethal ventricular arrhythmias in hyper-acute stage in prehospital setting.
CONCLUSIONS NC could decrease the incidence of spontaneous ventricular arrhythmias (especially VF) in rats with hyperAMI. The possible antiarrhythmic mechanisms of NC might relate to reduce degradation of Cx43 and improve Cx43 2014 Lippincott Williams & Wilkins
J Cardiovasc Pharmacol Volume 64, Number 2, August 2014
redistribution in myocardial infarcted zone. Our study indicated that NC may become a promising drug in the future to prevent patients with AMI from lethal ventricular arrhythmias in prehospital setting. REFERENCES 1. Clements-Jewery H, Hearse DJ, Curtis MJ. Phase 2 ventricular arrhythmias in acute myocardial infarction: a neglected target for therapeutic antiarrhythmic drug development and for safety pharmacology evaluation. Br J Pharmacol. 2005;145:551–564. 2. Harris AS. Delayed development of ventricular ectopic rhythms following experimental coronary occlusion. Circulation. 1950;1:1318–1328. 3. John RM, Tedrow UB, Koplan BA, et al. Ventricular arrhythmias and sudden cardiac death. Lancet. 2012;380:1520–1529. 4. Fishman GI, Chugh SS, Dimarco JP, et al. Sudden cardiac death prediction and prevention: report from a National Heart, Lung, and Blood Institute and Heart Rhythm Society Workshop. Circulation. 2010;122: 2335–2348. 5. Van Kempen MJ, Fromaget C, Gros D, et al. Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart. Circ Res. 1991;68:1638–1651. 6. Jalife J, Morley GE, Vaidya D. Connexins and impulse propagation in the mouse heart. J Cardiovasc Electrophysiol. 1999;10:1649–1663. 7. Ando M, Katare RG, Kakinuma Y, et al. Efferent vagal nerve stimulation protects heart against ischemia-induced arrhythmias by preserving connexin43 protein. Circulation. 2005;112:164–170. 8. Camelliti P, Devlin GP, Matthews KG, et al. Spatially and temporally distinct expression of fibroblast connexins after sheep ventricular infarction. Cardiovasc Res. 2004;62:415–425. 9. Liu CL, Duan YH, Dai Y, et al. Study on the chemical constituents of roots and stems of NC [in Chinese]. Zhong Yao Cai. 2011;34:1216–1219. 10. Zhang X, Lan Z, Dong XP, et al. Study on the active components of NC [in Chinese]. Zhong Yao Cai. 2007;30:38–41. 11. Qian XZ. The Pharmacopoeia of the People’s Republic of China. Pt 1. 1978:120–130. 12. Wang J, Zhao J, Liu H, et al. Chemical analysis and biological activity of the essential oils of two valerianaceous species from China: NC and Valeriana officinalis. Molecules. 2010;15:6411–6422. 13. Tanitsu MA, Takaya Y, Akasaka M, et al. Guaiane- and aristolane-type sesquiterpenoids of NC roots. Phytochemistry. 2002;59:845–849. 14. Tanaka K, Komatsu K. Comparative study on volatile components of Nardostachys rhizome. J Nat Med. 2008;62:112–116. 15. Sherman L, Niemann J, Youngquist ST, et al. Beta-blockade causes a reduction in the frequency spectrum of VF but improves resuscitation outcome: a potential limitation of quantitative waveform measures. Resuscitation. 2012;83:511–516.
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