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 hyper-acute 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, 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
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 ± 2°C, relative humidity of 50% ± 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·kg−1·d−1 metoprolol, and NC group received 600 mg·kg−1·d−1 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 37°C 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 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.
The ventricular myocardial tissues were fixed in 4% formaldehyde, embedded in paraffin, sectioned at a thickness of 5 μm, and mounted on neutral balsam. Then sections were deparaffinized, baked for 2 hours at 60°C, placed in citrate buffer, and incubated overnight with primary antibodies directed against Cx43 (Millipore) at 4°C overnight. After 15 minutes rinse (3 × 5 minutes, phosphate-buffered saline), slices were incubated with secondary antibodies (Millipore) for 30 minutes at 37°C. 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).
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 4°C (110 V). After blocking in 5% nonfat milk for 2 hours at room temperature, the membranes were treated with anti-Cx43 (1:1500) overnight. 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.
Oligonucleotide primer sequences for the RT-PCR assays of murine Cx43 and GAPDH gene expression were as follows: Cx43, forward 5-GCCCTGCCATAAGCCCTCTTGC-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 μg) of RNA was transcribed to complementary DNA (cDNA) using PrimeScript First-Strand cDNA Synthesis Kit (TAKARA) under the following conditions: 37°C for 15 minutes and 85°C 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 μL. 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 analysis was performed using SPSS 13.0. Data were expressed as the mean ± SE. Comparison of the incidence of VF among groups were made by χ2 analysis with 3 × 2 contingency table, followed by intergroup comparisons using 2 × 2 table. Changes in continuous variables among 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.
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 ± 5.16 vs. 176.08 ± 42.50, P < 0.001, and 23.76 ± 8.63 vs. 176.08 ± 42.50, P < 0.001, respectively) 30 minutes after LAD ligation. Metoprolol and NC reduced the number of PVCs 30–120 minutes (11.61 ± 2.56 vs. 110 ± 34.96, P < 0.001, and 20.47 ± 7.77 vs. 110 ± 34.96, P = 0.005, respectively) and 120–240 minutes (13.87 ± 3.31 vs. 158 ± 50.81, P = 0.007, and 10.1 ± 4.20 vs. 158 ± 50.81, P = 0.008, respectively) after LAD ligation. Meanwhile, metoprolol and NC reduced the incidence of VTs 30 minutes (2.18 ± 1.51 vs. 9.75 ± 3.10, P < 0.001, and 3.81 ± 1.03 vs. 9.75 ± 3.10, P = 0.002, respectively), 30–120 minutes (0.52 ± 0.38 vs. 2.33 ± 1.64, P = 0.001, and 0.57 ± 0.44 vs. 2.33 ± 1.64, P = 0.001, respectively), and 120–240 minutes (0.39 ± 0.16 vs. 1.75 ± 0.86, P < 0.001, and 0.28 ± 0.15 vs. 1.75 ± 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 ± 20.56 vs. 443.08 ± 35.98, P < 0.001, and 54.33 ± 24.87 vs. 443.08 ± 35.98, P = 0.001, respectively). Meanwhile, metoprolol and NC reduced the cumulative growth in the number of VTs (3.09 ± 1.92 vs. 13.83 ± 5.66, P < 0.001, and 5.00 ± 3.62 vs. 13.83 ± 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).
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 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% ± 6.20% vs. 39.13% ± 4.53%, P < 0.001, and 55.98% ± 6.20% vs. 42.39% ± 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.
Cx43 Protein Expression
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 ± 336.5 vs. 1421.7 ± 326.5, P = 0.018, and 2532.7 ± 282.7 vs. 1421.7 ± 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 ± 42.15 vs. 436.74 ± 78.29, P = 0.023, and 223.74 ± 42.15 vs. 399.24 ± 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).
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 group was significantly lower than that in the metoprolol and NC groups (0.3 ± 0.12 vs. 1.39 ± 0.16 units, P < 0.05, and 0.3 ± 012 vs. 1.50 ± 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 ± 0.09 vs. 0.57 ± 0.14 units, P < 0.05, and 0.29 ± 0.09 vs. 0.56 ± 0.12 units, P < 0.05, respectively). There was no significant difference between the groups of metoprolol and NC (Fig. 5).
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 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.
NC could decrease the incidence of spontaneous ventricular arrhythmias (especially VF) in rats with hyper-AMI. The possible antiarrhythmic mechanisms of NC might relate to reduce degradation of Cx43 and improve Cx43 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.
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