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Nitrite Reduces Ischemia-Induced Ventricular Arrhythmias by Attenuating Connexin 43 Dephosphorylation in Rats

Maruyama, Daisuke MD; Hirata, Naoyuki MD, PhD; Tokinaga, Yasuyuki MD, PhD; Kawaguchi, Ryoichi MD; Yamakage, Michiaki MD, PhD

doi: 10.1213/ANE.0000000000001063
Anesthetic Pharmacology: Research Report

BACKGROUND: Ventricular arrhythmias induced by ischemic heart disease are the main cause of sudden cardiac death. Ischemia can cause life-threatening arrhythmias by modulating connexin 43 (Cx43), a principal cardiac gap junction channel protein. The present study investigates whether nitrite can attenuate ischemia-induced ventricular arrhythmias and dephosphorylation of Cx43 in a rat model.

METHODS: Rats were medicated with normal saline (control, n = 10), nitrite (0.015, 0.15, and 1.5 mg/kg, n = 9 or 10 each), and 0.15 mg/kg nitrite with either the nitric oxide scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (cPTIO; n = 9) or allopurinol (xanthine oxidoreductase inhibitor, n = 9). We determined the severity of ventricular arrhythmias based on arrhythmia scores and levels of phosphorylated Cx43.

RESULTS: The median arrhythmia score may have been lower in the group given 0.15 mg/kg nitrite (4 [interquartile range {IQR}, 4–5]) than that in the control group (7.5 [IQR, 5.25–8]; P = 0.013). There was no difference among the control, the given 0.015 mg/kg nitrite (7 [IQR, 5–8]), and 1.5 mg/kg nitrite (7 [IQR, 5.5–7.75]; P = 0.95). The arrhythmia scores in the cPTIO (6 [IQR, 5–8]; P = 0.030) and allopurinol (7 [IQR, 5–8]; P = 0.005) groups may have been higher than that in 0.15 mg/kg nitrite group. Immunoblotting revealed that the level of phosphorylated Cx43 in the group given 0.15 mg/kg nitrite, but not in the other treated groups, was significantly higher compared with the control group (P = 0.007).

CONCLUSIONS: Nitrite may have attenuated acute ischemia-induced ventricular arrhythmias and Cx43 dephosphorylation in rats. Nitric oxide, which might be generated by xanthine oxidoreductase via nitrite reduction, appears to play a crucial role in this antiarrhythmic effect.

Published ahead of print October 29, 2015

From the Department of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan.

Accepted for publication September 2, 2015.

Published ahead of print October 29, 2015

Funding: This study was supported by the Japan Society for the Promotion of Science (JSPS; Tokyo, Japan); grant numbers 00438045 and 26861525.

Conflict of Interest: See Disclosures at the end of the article.

This report was previously presented, in part, at the annual meetings of the American Society of Anesthesiologists (ASA) October 12–16, 2013, San Francisco, and October 11–15, 2014, New Orleans.

Reprints will not be available from the authors.

Address correspondence to Naoyuki Hirata, MD, PhD, Department of Anesthesiology, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo, Hokkaido 060-8543, Japan. Address e-mail to naohirata@mac.com.

Surgical patients with ischemic heart disease are becoming more prevalent worldwide, and they are at high risk for perioperative myocardial ischemia (MI), which can induce lethal ventricular arrhythmias considered to be the main cause of sudden cardiac death.1,2

Previous studies3–5 have demonstrated that gap junction connexin 43 (Cx43) was involved in ischemia-induced ventricular arrhythmias. Gap junctions that consist of connexin proteins provide the intercellular coupling necessary for rapid action potential propagation through the myocardium, triggering synchronized heart contraction.6 Cx43 is a 43-kDa protein that is expressed primarily within mammalian ventricles7 at intercalated disks.8 Changes in Cx43 have been implicated in ventricular remodeling and the development of arrhythmias in several cardiac diseases,9,10 including MI.3–5 Furthermore, dysfunctional Cx43 contributes to ventricular arrhythmias.11–13

In the perioperative period, volatile anesthetics are widely used in patients who undergo cardiac surgery because these agents have cardioprotective effects.14–18 However, the effects of volatile anesthetics on ischemia-induced ventricular arrhythmias and Cx43 remain controversial.5 Moreover, volatile anesthetics5 and perioperative antiarrhythmic agents19 may depress myocardial function and produce proarrhythmic effects. Therefore, additional strategies are worth exploring to prevent lethal arrhythmias caused by ischemia in the perioperative period.

The dietary constituent and nitric oxide (NO) oxidation product, nitrite, has emerged as an inherent signaling molecule that mediates cytoprotection during ischemia/reperfusion (I/R) in several organ systems.20–24 In the heart, nanomolar increases in circulating nitrite concentrations significantly decrease infarct size in murine and canine models of myocardial infarction.25,26 This protection might be dependent on the reduction of nitrite to bioactive NO by deoxyhemoglobin27,28 and xanthine oxidoreductase (XOR).29 Characteristically, this reduction is optimized under the ischemic conditions of hypoxia and acidosis.30 However, the effects of nitrite on ischemia-induced ventricular arrhythmias and Cx43 remain unclear.

In the present study, we investigated the dose-dependent effects of nitrite on ischemia-induced ventricular arrhythmias and the dephosphorylation of Cx43 protein in rats with acute MI.

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METHODS

Experimental Preparation In Vivo

The Animal Care and Use Committee at Sapporo Medical University (Sapporo, Hokkaido, Japan) approved the animal protocol, and all experiments were conducted in accordance with institutional guidelines and regulations for animal experimentation. The number of experimental animals was determined on the basis of our previous experiment.5 Studies were analyzed at every experiment and stopped to avoid the needless death of animals if there was a clear outcome.

The animals were prepared as described.5 In brief, healthy male Wistar rats (weight, 250–300 g; age, 7–9 weeks) were intraperitoneally anesthetized with 50 mg/kg sodium pentobarbital, and the depth of anesthesia was monitored using the tail-clamp test. The rats were intubated and mechanically ventilated using a volume-controlled, Model 683 rodent respirator (Harvard Apparatus, Holliston, MA) at 65 to 80 strokes/min to maintain the pH, as well as normal PaO2 and PaCO2. The animals were placed supine on a heating pad, and body temperature was monitored using a rectal thermometer and maintained at 37°C using a heat lamp. The left femoral artery was catheterized for direct measurement of blood pressure and arterial blood gas analyses using an ABL700 radiometer (Radiometer Danmark, Copenhagen, Denmark). A thoracotomy was positioned horizontally in the fourth intercostal space, and a 7-0 prolene suture was loosely tied around the left anterior descending (LAD) coronary artery. Electrocardiography proceeded using PowerLab (AD Instruments, Mountain View, CA), which is a computer-based electrical physiology system. Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded throughout each experiment.

Sodium nitrite (NaNO2; 0.015, 0.15, and 1.5 mg/kg; Sigma-Aldrich, St. Louis, MO) was dissolved in normal saline (NS) to a final volume of 500 μL. The NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (cPTIO; 2 mg/kg; Dojindo Laboratories, Kumamoto, Japan)24 and the XOR inhibitor, allopurinol (50 mg/kg; Sigma-Aldrich),31 were also dissolved in a final volume of 500 μL of NS.

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Experimental Design

Experiment 1

Figure 1

Figure 1

We determined whether nitrite dose dependently attenuates ischemia-induced ventricular arrhythmia as follows: The rats were randomly assigned to a sham-operated group (n = 5) that underwent surgery without LAD coronary artery ligation, a control group (n = 10) that underwent 30 minutes of LAD coronary artery ligation and received intraperitoneal (IP) NS, and groups that received 0.015 (n = 9), 0.15 (n = 10), or 1.5 (n = 10) mg/kg of nitrite IP 15 minutes before LAD coronary artery ligation. The rats were stabilized for 15 minutes after the administration of all compounds, and then MI was induced by 30 minutes of LAD coronary artery ligation in all except the sham group. Figure 1A describes the protocol for experiment 1.

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Experiment 2

To determine whether the antiarrhythmic effects of nitrite are mediated by NO, and whether XOR is responsible for converting nitrite to NO, the rats were randomly assigned to groups that received 0.15 mg/kg nitrite with NS (n = 9), 0.15 mg/kg nitrite + cPTIO (n = 9), or 0.15 mg/kg nitrite + allopurinol (n = 9). All agents were administered IP 15 minutes before nitrite. The rats were stabilized for 15 minutes after nitrite administration, and then MI was induced by 30 minutes of LAD ligation. Figure 1B describes the protocol for experiment 2.

MI in each ligated group was visually confirmed as the appearance of focal cyanosis and by ST segment changes in electrocardiography.

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Arrhythmia Study In Vivo

Ischemia-induced ventricular arrhythmias were classified according the Lambeth Conventions.32 Ventricular ectopic beats (VEBs) were defined as identifiable premature QRS complexes. Ventricular tachycardia (VT) was defined as at least 4 consecutive VEBs that were faster than the resting sinus rate. Ventricular fibrillation (VF) was defined as unidentifiable and low-voltage QRS complexes.33

The severity of arrhythmias was quantified using the following scoring system34: 0, no arrhythmia; 1, occasional VEBs; 2, frequent VEBs when 3 or more occurred within 1 minute; 3, VT (1 or 2 episodes); 4, VT (3–5 episodes); 5, VT (>5 episodes); 6, VF (1 or 2 episodes); 7, VF (3–5 episodes); 8, VF (>5 episodes); 9, death induced by arrhythmia or VF sustained for >2 minutes. If VF did not spontaneously revert to sinus rhythm within 10 seconds, we attempted to restore sinus rhythm with precordial taps. The highest arrhythmia score was taken for each 5-minute period during 30 minutes of LAD ligation.

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Determinations of Area at Risk

To confirm the absence of ischemic perfusion, 2 mL of 2% Evans blue dye was injected via the femoral artery at the end of the 30-minute LAD ligation. The hearts were removed, frozen, and sliced into 2-mm-thick transverse sections at the midpoint of the left ventricle. Areas at risk (AAR) were determined by negative staining with Evans blue dye and measured using Image J (National Institutes of Health, Bethesda, MD), a computer-assisted image analysis system. The volume of the AAR was calculated by multiplying the AAR by the slice thickness and is expressed as a ratio (%) of the left ventricular volume.

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Western Blotting for Cx43

This experiment proceeded separately from the arrhythmia study in vivo because the procedure for assessing risk areas did not allow immunoblotting of the excised hearts. Left ventricular Cx43 levels were determined in a separate group of rats (n = 6) from each of the sham, control, and groups given 0.15 mg/kg nitrite, without or with either 2 mg/kg cPTIO or 50 mg/kg allopurinol.

Ischemic sections of myocardial tissue were individually homogenized on ice in homogenizing buffer (50 mM Tris, 1 mM EDTA, and 0.1 M sucrose) supplemented with protease inhibitor cocktail (Sigma-Aldrich). The homogenates were separated by centrifugation at 10,000g for 10 minutes at 4°C, and clear supernatants were Western blotted. The protein concentration in each sample was determined using a detergent-compatible protein assay (Thermo Fisher Scientific Inc., Waltham, MA), with bovine serum albumin as the standard.

Protein extracts (30 μg) suspended in 2× sample buffer (0.125 M Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate, 20% glycerol, and 0.08% bromophenol blue) were loaded onto 15% polyacrylamide gels and resolved by electrophoresis at 30 mA/gel for 60 minutes with running buffer (25 mM Tris containing192 mM glycine and 0.1% sodium dodecyl sulfate). Molecular weight markers (Watson Co. Ltd., Tokyo, Japan) were also applied to each gel. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories Inc., Hercules, CA) at 50 V for 1 hour using transfer buffer comprising 25 mM Tris containing 192 mM glycine and 20% methanol. The blots were incubated with 3% bovine serum albumin in Tris-buffered saline (TBS; pH 7.5) for 2 hours at room temperature to block nonspecific antibody binding. The membrane was then immersed in rabbit polyclonal anti-Cx43 antibody (1:1000 dilution; Sigma-Aldrich) for 2 hours. The concentration of the housekeeping protein, actin, was also measured using rabbit anti-actin antibody (1:1000 dilution; Cell Signaling Technology Inc., Danvers, MA). Blots were washed for 40 minutes with 0.1% Tween-20 in TBS, followed by incubation with a goat anti-rabbit immunoglobulin G secondary antibody conjugated to horseradish peroxidase (1:1000 dilution; Cell Signaling Technology Inc.) for 1 hour at room temperature. The membrane was washed once again for 40 minutes with 0.1% Tween-20 in TBS. Immunoreactive bands detected using chemiluminescence (GE Healthcare UK Ltd., Buckinghamshire, England) were assessed using ATTO Densitograph Software Library CS Analyzer 3 (ATTO Instruments, Tokyo, Japan). Proteins were quantified by densitometry to determine the Cx43/actin ratio for both phosphorylated Cx43 (44–46 kDa; P-Cx43) and nonphosphorylated Cx43 (41 kDa; NP-Cx43).

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Statistical Analysis

Arrhythmia scores are shown as medians with interquartile range (IQR), and the groups were compared using the Kruskal-Wallis test followed by the Dunn post hoc test. The incidence of VEBs, VT, and VF was compared using the Fisher exact test. MAP, heart rate (HR), blood gas analysis data, ischemic areas, and the Cx43/actin ratio are shown as means ± SD. Differences in MAP, HR, pH, the arterial levels of PaO2, PaCO2 before and after 30 minutes of LAD ligation, and among the groups were compared by 2-way analysis of variance with the Tukey multiple comparisons test. Differences in ischemic areas and tissue concentration of Cx43 were compared using 1-way analysis of variance with the Tukey test. On the basis of our sample size35 decision-making process (see earlier), P < 0.01 was considered as statistically significant and P > 0.15 was considered as indicating “not different.” Data were statistically analyzed using Prism 6.0 for Mac (GraphPad Software Inc., La Jolla, CA) with a 2-tailed hypothesis.

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RESULTS

Hemodynamic Parameters and Blood Gas Analyses

Table 1

Table 1

Table 2

Table 2

Table 1 shows MAP and HR in all groups in experiments 1 and 2. There was no difference between before and after ischemia (MAP; P = 0.20 and P = 0.21, HR; P = 0.41 and P = 0.65 in experiments 1 and 2, respectively) and among the groups (MAP; P = 0.59 and P = 0.99, HR; P = 0.78 and P = 0.99 in experiments 1 and 2, respectively). Table 2 shows blood gas analysis before and after 30 minutes of LAD ligation. There was no difference in the pH (P = 0.75 and P = 0.22 in experiments 1 and 2, respectively) and arterial levels of PaO2 (P = 0.52 and P = 0.99 in experiments 1 and 2, respectively) and PaCO2 (P = 0.39 and P = 0.99 in experiments 1 and 2, respectively) before and after LAD ligation. There was also no difference in the pH (P = 0.50 and P = 0.29 in experiments 1 and 2, respectively) and arterial levels of PaO2 (P = 0.40 and P = 0.68 in experiments 1 and 2, respectively) and PaCO2 (P = 0.32 and P = 0.93 in experiments 1 and 2, respectively) among the groups.

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Assessment of Risk Areas

Table 1 also shows AAR after 30 minutes of LAD ligation. There was no difference among the groups in experiment 1 administered with NS, 0.015, 0.15, or 1.5 mg/kg nitrite (50% ± 13%, 51% ± 7%, 51% ± 9% and 50% ± 10%, respectively, P = 0.99) and in experiment 2 administered with nitrite plus NS, cPTIO, or allopurinol (52% ± 5%, 51% ± 9%, and 52% ± 8%, respectively, P = 0.95).

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Effect of Nitrite on Ischemia-Induced Ventricular Arrhythmias

Figure 2

Figure 2

Figure 2 shows arrhythmia scores during 30 minutes of LAD ligation (data not shown for the sham group). The scores peaked between 5 and 10 minutes after MI in all groups except the sham group.

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Experiment 1

Arrhythmias did not arise in the sham group. Figure 2A shows that 0.15 mg/kg nitrite administered 30 minutes before LAD ligation alleviated the severity of ventricular arrhythmias. Arrhythmia scores may have been lower in the group given 0.15 mg/kg nitrite (4 [IQR, 4–5]) than in the control group (7.5 [IQR, 5.25–8.0]; P = 0.013). There were no difference among the control, 0.015, and 1.5 mg/kg nitrite groups (7 [IQR, 5–8] and 7 [IQR, 5.5–7.75]; P = 0.95). These results indicated that the antiarrhythmic effects of nitrite might be associated with dose.

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Experiment 2

Figure 2B shows that the arrhythmia score may have been higher for the 0.15 mg/kg nitrite + cPTIO (6 [IQR, 5–8]) than + NS (4 [IQR, 4–5]; P = 0.030). The score was also significantly higher for the group given 0.15 mg/kg nitrite + allopurinol (7 [IQR, 5–8]) than + NS (4 [IQR, 4–5]; P = 0.005).

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Incidence of VEBs, VT, and VF

Figure 3

Figure 3

Figure 3 shows the incidence of VF induced by MI in the control and the group given 0.15 mg/kg nitrite. Nitrite may have decreased the incidence of VF from 70% (7/10) to 10% (1/10; P = 0.02). However, there was no difference in the incidence of VEBs and VT between the control group (100% and 90%, respectively) and the 0.15 mg/kg nitrite group (90%, P = 0.30, and 90%).

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Effect of Nitrite on Cx43 Protein Expression After Myocardial Ischemia

Figure 4

Figure 4

We measured protein levels of P-Cx43 after 30 minutes of LAD ligation to determine whether one of the antiarrhythmic effects of nitrite is caused by preventing Cx43 dephosphorylation. A polyclonal anti-Cx43 antibody detected one band each at 44 to 46 and at 41 kDa on Western blots. The bands with a lower and higher molecular mass represent NP-Cx43 and P-Cx43, respectively. The loading control was β-actin. Figure 4A shows a representative immunoblot using polyclonal anti-Cx43 and anti-actin antibodies. Figure 4B shows the optical density of the Cx43/actin ratio. The amount of P-Cx43 was significantly decreased after 30 minutes of LAD ligation in the control compared with the sham group (P = 0.001). Some dephosphorylation had occurred at 30 minutes after MI in the group given 0.15 mg/kg nitrite, but the amount of P-Cx43 in the 0.15 mg/kg nitrite group was significantly higher than that in the control group at 30 minutes after the onset of ischemia (P = 0.007). There was no difference in the amount of P-Cx43 among the cPTIO (P = 0.92 versus control), allopurinol (P = 0.96 versus control), and control groups.

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DISCUSSION

In this study, moderate-dose nitrite may have attenuated ventricular arrhythmias induced by acute ischemia in rats via an NO-dependent mechanism, and XOR acted as a functional nitrite reductase. This antiarrhythmic effect of nitrite might, in part, be mediated via the modulation of Cx43, which is a principal ventricular gap junction protein.

Arrhythmias occurring in response to acute MI are primarily caused by reentry.36 Gap junction channels close during early ischemia, which causes neighboring cardiomyocytes to uncouple from each other.37 Such uncoupling is associated with Cx43 dephosphorylation, which can be detected after 15 minutes of ischemia.38 Yao et al.39 reported that NO is involved in the control of Cx43 expression through the activation of protein kinase A. Hence, nitrite-derived NO can preserve cardiomyocytes from uncoupling and reentry caused by MI via Cx43 modulation and thus attenuate ventricular arrhythmias induced by ischemia. Gönczi et al.40 showed that antiarrhythmic protection resulting from an infusion of the NO donor sodium nitroprusside is associated with the modulation of Cx43, which suggests that nitrite, another donor of NO, attenuates ischemia-induced ventricular arrhythmias by preserving Cx43.

The administration of 0.15 mg/kg nitrite before 30 minutes of LAD ligation alleviated the severity of ventricular arrhythmias. In contrast, 0.015 and 1.5 mg/kg nitrite did not suppress ischemia-induced ventricular arrhythmias. The curve of the dose-response relationship between nitrite and its antiarrhythmic effects was not linear but rather U-shaped. These results are similar to previous findings of hepatic,20,21 kidney,22 brain,23 and pulmonary24 injuries using comparable dosages. Although several studies20,23,24 have found that various nitrite doses confer beneficial effects in experimental animals and targeted organs or pathology, almost all of them found that nanomolar increases in the circulating nitrite concentration conferred such effects. Increased nitrite concentrations can cause nitrite-derived NO to become an essential source of harmful peroxynitrite, causing oxidant damage.29 Calvert and Lefer41 reported that a supraphysiological concentration of nitrite might cause cellular necrosis and apoptosis. Hence, nitrite exerts antiarrhythmic effects at the prevailing physiologic concentration, whereas higher concentrations might negate these therapeutic effects.

We administered allopurinol with nitrite to determine the effect of XOR on a model of ischemia-induced ventricular arrhythmias and found that allopurinol blocked the antiarrhythmic effects of nitrite. Others have also shown that nitrite-derived protection is partly mediated by XOR acting as a nitrite reductase in rat models of kidney I/R injury22 or ventilator-induced lung injury.24 Webb et al.42 have shown that XOR can pH and concentration dependently catalyze the formation of NO from nitrite and that this NO could protect the heart against I/R injury. Thus, nitrite might have been reduced to NO by XOR during ischemia in the present study, and bioactive NO might have regulated ischemia-induced arrhythmias via Cx43 modulation.

The NO donors nitroglycerin and nitroprusside also have hypotensive effects that prevent their routine application, particularly under critical conditions, even though they have advantages against ischemia-induced ventricular arrhythmias.39,43 In the experimental setting, morphine can exert significant cardiovascular effects,44 including antiarrhythmia, via activation of some opioids receptors.45 Nevertheless, it is rarely applied in the clinical setting because the required doses are extremely high. We found that the antiarrhythmic concentration of nitrite had little effect on hemodynamics. Others also have shown that the doses of nitrite required for cytoprotection have little effect on general blood pressure.24 Therefore, nitrite can be a novel cytoprotective and antiarrhythmic agent not only during the perioperative period but also when patients are in critical care units.30

The present study has some limitations. First, we focused on NO and Cx43 as pivotal mediators of the antiarrhythmic effects of nitrite. However, this was only one of the possible pathways of nitrite protection in this setting. For example, calcium overload after ischemia is also involved in the development of ischemia-induced ventricular arrhythmias.35 Although Pavlovic et al.46 reported that NO activates Na/K-ATPase and thereby limits calcium overload and arrhythmias, further study is required to elucidate detailed interactions among the nitrite-NO pathway, Cx43, and calcium overload. Second, the detailed mechanisms through which nitrite and/or NO attenuate ischemia-induced ventricular arrhythmias and preserve Cx43 from dephosphorylation remain unclear. As one possibility, the antioxidative stress effects of nitrite23,47 might prevent dysregulation of the cardiac connexon connection induced by oxidant stress.48 Third, we did not assess levels of deoxyhemoglobin, which is another possible nitrite reductase in red blood cells. Hemoglobin is an essential factor in physiologically regulating NO bioactivity in mammals, and it might be important in the pathophysiology of I/R injury.49 We cannot exclude the possible contribution of this mechanism to nitrite-derived antiarrhythmic effects. Fourth, it should be considered that there are specific differences in the electrophysiologic properties between rats and human patients. In particular, rat cardiomyocytes are known to have a much briefer action potential. Despite species differences, knowledge obtained from the study of antiarrhythmic agents in mouse, rat, dog, or other laboratory animals, and their underlying electrophysiologic and metabolic mechanisms of action, has been available in the development of strategies for attenuating ischemia-induced ventricular arrhythmias in humans. Finally, although we performed experiments in a blinded and randomized fashion to avoid type I and II errors, these errors might have arisen in our sample size.

In summary, the present findings showed that nitrite might have attenuated the severity of ischemia-induced ventricular arrhythmias via an NO-dependent mechanism. In addition, NO from nitrite via the XOR pathway reduced the extent of Cx43 dephosphorylation during MI. These results suggest that preserving the function of ventricular gap junctions, presumably via NO-mediated Cx43 phosphorylation, is one mechanism through which nitrite can alleviate ventricular arrhythmias induced by ischemia.

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DISCLOSURES

Name: Daisuke Maruyama, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Conflicts of Interest: Daisuke Maruyama reported no conflicts of interest.

Attestation: Daisuke Maruyama has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Naoyuki Hirata, MD, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Conflicts of Interest: Naoyuki Hirata reported no conflicts of interest.

Attestation: Naoyuki Hirata has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Yasuyuki Tokinaga, MD, PhD.

Contribution: This author helped conduct the study.

Conflicts of Interest: Yasuyuki Tokinaga reported no conflicts of interest.

Attestation: Yasuyuki Tokinaga has seen the original study data and approved the final manuscript.

Name: Ryoichi Kawaguchi, MD.

Contribution: This author helped conduct the study.

Conflicts of Interest: Ryoichi Kawaguchi reported no conflicts of interest.

Attestation: Ryoichi Kawaguchi has seen the original study data and approved the final manuscript.

Name: Michiaki Yamakage, MD, PhD.

Contribution: This author helped analyze the data and write the manuscript.

Conflicts of Interest: Michiaki Yamakage participated in the study entitled, “Optimization of Desflurane in Elderly Patients” sponsored by Baxter Healthcare.

Attestation: Michiaki Yamakage reviewed the analysis of the data and approved the final manuscript.

This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.

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ACKNOWLEDGMENTS

We thank Ryo Miyashita, MD, Yusuke Yoshikawa, MD, and Shunsuke Hayashi, Research Technologist, for valuable technical assistance (Department of Anesthesiology, Sapporo Medical University School of Medicine).

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